<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Publishing DTD v1.2 20190208//EN" "http://jats.nlm.nih.gov/publishing/1.2/JATS-journalpublishing1.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article" dtd-version="1.2" xml:lang="en">
    <front>
        <journal-meta>
            <journal-id journal-id-type="pmc">F1000Research</journal-id>
            <journal-title-group>
                <journal-title>F1000Research</journal-title>
            </journal-title-group>
            <issn pub-type="epub">2046-1402</issn>
            <publisher>
                <publisher-name>F1000 Research Limited</publisher-name>
                <publisher-loc>London, UK</publisher-loc>
            </publisher>
        </journal-meta>
        <article-meta>
            <article-id pub-id-type="doi">10.12688/f1000research.173271.2</article-id>
            <article-categories>
                <subj-group subj-group-type="heading">
                    <subject>Research Article</subject>
                </subj-group>
                <subj-group>
                    <subject>Articles</subject>
                </subj-group>
            </article-categories>
            <title-group>
                <article-title>First Gonadal Stages of Bonito, 
                    <italic>Sarda chiliensis</italic> (Perciformes: Scombridae) on the Pacific Coast of Northern Chile</article-title>
                <fn-group content-type="pub-status">
                    <fn>
                        <p>[version 2; peer review: 2 approved, 1 not approved]</p>
                    </fn>
                </fn-group>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author" corresp="yes">
                    <name>
                        <surname>Pepe-Victoriano</surname>
                        <given-names>Renzo</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Conceptualization</role>
                    <role content-type="http://credit.niso.org/">Formal Analysis</role>
                    <role content-type="http://credit.niso.org/">Methodology</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <uri content-type="orcid">https://orcid.org/0000-0002-7630-1411</uri>
                    <xref ref-type="corresp" rid="c1">a</xref>
                    <xref ref-type="aff" rid="a1">1</xref>
                    <xref ref-type="aff" rid="a2">2</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Godoy</surname>
                        <given-names>Marcos</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Formal Analysis</role>
                    <role content-type="http://credit.niso.org/">Investigation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <xref ref-type="aff" rid="a3">3</xref>
                    <xref ref-type="aff" rid="a4">4</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Kush</surname>
                        <given-names>Karina</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Formal Analysis</role>
                    <role content-type="http://credit.niso.org/">Investigation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <xref ref-type="aff" rid="a4">4</xref>
                </contrib>
                <contrib contrib-type="author" corresp="yes">
                    <name>
                        <surname>Huanacuni</surname>
                        <given-names>Jordan I</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Formal Analysis</role>
                    <role content-type="http://credit.niso.org/">Investigation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <uri content-type="orcid">https://orcid.org/0000-0001-7381-8004</uri>
                    <xref ref-type="corresp" rid="c2">b</xref>
                    <xref ref-type="aff" rid="a5">5</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>M&#x00e9;ndez-Abarca</surname>
                        <given-names>Felipe</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Conceptualization</role>
                    <role content-type="http://credit.niso.org/">Methodology</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <xref ref-type="aff" rid="a1">1</xref>
                    <xref ref-type="aff" rid="a2">2</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Espinoza-Ramos</surname>
                        <given-names>Luis</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Investigation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <xref ref-type="aff" rid="a2">2</xref>
                    <xref ref-type="aff" rid="a6">6</xref>
                    <xref ref-type="aff" rid="a7">7</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Resurrecci&#x00f3;n-Huertas</surname>
                        <given-names>Juan Zen&#x00f3;n</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Formal Analysis</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <xref ref-type="aff" rid="a8">8</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Cort&#x00e9;s</surname>
                        <given-names>Rodrigo</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Conceptualization</role>
                    <role content-type="http://credit.niso.org/">Methodology</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <xref ref-type="aff" rid="a1">1</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Vergara</surname>
                        <given-names>Manuel</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Conceptualization</role>
                    <role content-type="http://credit.niso.org/">Investigation</role>
                    <role content-type="http://credit.niso.org/">Methodology</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <xref ref-type="aff" rid="a1">1</xref>
                </contrib>
                <aff id="a1">
                    <label>1</label>Facultad de Recursos Naturales Renovables, &#x00c1;rea de Biolog&#x00ed;a Marina y Acuicultura, Universidad Arturo Prat, Iquique, Tarapac&#x00e1; Region, 2120, Chile</aff>
                <aff id="a2">
                    <label>2</label>N&#x00fa;cleo de Investigaci&#x00f3;n Aplicada e Innovaci&#x00f3;n en Ciencias Biol&#x00f3;gicas, Universidad Arturo Prat, Iquique, Tarapac&#x00e1; Region, 1110939, Chile</aff>
                <aff id="a3">
                    <label>3</label>Laboratorio de Biotecnolog&#x00ed;a Aplicada, Facultad de Ciencias de la Naturaleza, Universidad San Sebastian, Patagonia, Puerto Montt, 5480000, Chile</aff>
                <aff id="a4">
                    <label>4</label>Centro de Investigaciones Biol&#x00f3;gicas Aplicadas, Puerto Montt, 5480000, Chile</aff>
                <aff id="a5">
                    <label>5</label>Campus Tacna, Universidad Tecnol&#x00f3;gica del Per&#x00fa;, Tacna, Tacna, 23003, Peru</aff>
                <aff id="a6">
                    <label>6</label>Grupo de Investigaci&#x00f3;n Acuicultura Sostenible, Universidad Nacional Jorge Basadre Grohmann, Tacna, Tacna, 23004, Peru</aff>
                <aff id="a7">
                    <label>7</label>Escuela de Ingenier&#x00ed;a Pesquera, Universidad Nacional Jorge Basadre Grohmann, Tacna, Tacna, 23004, Peru</aff>
                <aff id="a8">
                    <label>8</label>Escuela de Ingenier&#x00ed;a Acu&#x00ed;cola, Facultad de Ingenier&#x00ed;a Pesquera, Universidad Nacional Jose Faustino Sanchez Carrion, Huacho, Lima Region, 15135, Peru</aff>
            </contrib-group>
            <author-notes>
                <corresp id="c1">
                    <label>a</label>
                    <email xlink:href="mailto:regepevic@gmail.com">regepevic@gmail.com</email>
                </corresp>
                <corresp id="c2">
                    <label>b</label>
                    <email xlink:href="mailto:Jordan.92ihp@gmail.com">Jordan.92ihp@gmail.com</email>
                </corresp>
                <fn fn-type="conflict">
                    <p>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>24</day>
                <month>4</month>
                <year>2026</year>
            </pub-date>
            <pub-date pub-type="collection">
                <year>2025</year>
            </pub-date>
            <volume>14</volume>
            <elocation-id>1346</elocation-id>
            <history>
                <date date-type="accepted">
                    <day>16</day>
                    <month>3</month>
                    <year>2026</year>
                </date>
            </history>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2026 Pepe-Victoriano R et al.</copyright-statement>
                <copyright-year>2026</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
            </permissions>
            <self-uri content-type="pdf" xlink:href="https://f1000research.com/articles/14-1346/pdf"/>
            <abstract>
                <sec>
                    <title>Background</title>
                    <p>The study of the reproductive biology of pelagic fish species, such as 
                        <bold>

                            <italic toggle="yes">Sarda chiliensis</italic>
</bold> (Bonito), is essential for effective fisheries management and aquaculture development. Overfishing has significantly impacted the population of pelagic fish species, making it crucial to understand their reproductive cycles, including maturity stages and spawning periods, to ensure sustainable exploitation.</p>
                </sec>
                <sec>
                    <title>Methods</title>
                    <p>This study provides the first histological characterization of the early gonadal stages of 
                        <bold>

                            <italic toggle="yes">Sarda chiliensis</italic>
</bold> from northern Chile. A total of 444 specimens were collected from artisanal fisheries between December 2013 and June 2014. Gonadal maturity stages were identified using histological techniques, and a range of microscopic stains (Hematoxylin-Eosin, Van Gieson, and Periodic Acid&#x2013;Schiff
) were employed to examine the cellular structure of the gonads.</p>
                </sec>
                <sec>
                    <title>Results</title>
                    <p>Two key gonadal stages&#x2014;inactive and previtellogenic&#x2014;were identified in females, while males exhibited both immature and mature stages. Histological observations revealed distinct characteristics of early gonadal development, including primary oocytes and spermatogonia, with evidence of early vitellogenesis in females. No mature females were observed, and the smallest mature female recorded was 48.5 cm in total length. The gonadosomatic index (GSI) exhibited temporal fluctuations, with a peak observed in February.</p>
                </sec>
                <sec>
                    <title>Conclusions</title>
                    <p>This study provides vital histological data on the early gonadal stages of 
                        <bold>

                            <italic toggle="yes">Sarda chiliensis</italic>
</bold> from northern Chile, offering a baseline for future reproductive studies and aquaculture initiatives. The absence of mature females in the sample highlights the need for broader temporal and spatial sampling to fully characterize the reproductive cycle. These findings are crucial for developing sustainable fisheries management strategies and improving broodstock conditioning for aquaculture.</p>
                </sec>
            </abstract>
            <kwd-group kwd-group-type="author">
                <kwd>Histological sections</kwd>
                <kwd>gonads</kwd>
                <kwd>reproduction</kwd>
                <kwd>sexual maturity</kwd>
                <kwd>spawning</kwd>
            </kwd-group>
            <funding-group>
                <award-group id="fund-1">
                    <funding-source>Arturo Prat University</funding-source>
                </award-group>
                <funding-statement>The author(s) declared that no grants were involved in supporting this work.</funding-statement>
            </funding-group>
        </article-meta>
        <notes>
            <sec sec-type="version-changes">
                <label>Revised</label>
                <title>Amendments from Version 1</title>
                <p>This revised version incorporates the suggestions and clarifications raised during the open peer-review process. The manuscript has been updated to better define the scope of the study, emphasizing that the results provide a descriptive characterization of early gonadal development stages rather than a complete assessment of the reproductive cycle of 
                    <italic>Sarda chiliensis</italic>. Several sections of the Discussion were revised to clarify the interpretation of previtellogenic oocytes and to avoid implying the onset of active vitellogenesis or spawning activity. Additional methodological details were included regarding the sampling procedure and specimen handling. Descriptive statistical summaries of morphometric variables were clarified, and the interpretation of GSI patterns was revised to avoid suggesting statistically supported temporal variation. Terminology related to gonadal development was harmonized following standardized reproductive terminology, and several figures, tables, and figure legends were corrected or clarified to improve accuracy and consistency.</p>
            </sec>
        </notes>
    </front>
    <body>
        <sec id="sec5" sec-type="intro">
            <title>1. Introduction</title>
            <p>Overfishing is the primary driver of the decline in pelagic fish stocks.
                <sup>
                    <xref ref-type="bibr" rid="ref1">1</xref>
                </sup> To evaluate and manage resources such as 
                <italic toggle="yes">Sarda chiliensis</italic>, it is essential to identify key biological variables, including size at first maturity, spawning periods, and reproductive potential.
                <sup>
                    <xref ref-type="bibr" rid="ref2">2</xref>
                </sup> These variables must be determined through accurate assessments of gonadal maturity stages.
                <sup>
                    <xref ref-type="bibr" rid="ref3">3</xref>
                </sup> Such studies provide the foundation for developing management and fishery strategies that align with the principles of sustainable exploitation.
                <sup>
                    <xref ref-type="bibr" rid="ref4">4</xref>
                </sup>
            </p>
            <p>Histology is a powerful tool for examining the microanatomy of gonads,
                <sup>
                    <xref ref-type="bibr" rid="ref5">5</xref>,
                    <xref ref-type="bibr" rid="ref6">6</xref>
                </sup> playing a crucial role in species management,
                <sup>
                    <xref ref-type="bibr" rid="ref63">7</xref>
                </sup> conservation
                <sup>
                    <xref ref-type="bibr" rid="ref7">8</xref>
                </sup> and the improvement of domestication success. Initial reproductive studies of 
                <italic toggle="yes">S. chiliensis</italic> were conducted by Barnhart,
                <sup>
                    <xref ref-type="bibr" rid="ref8">9</xref>
                </sup> who reported that this species spawns primarily in June off the coast of California. Walford
                <sup>
                    <xref ref-type="bibr" rid="ref9">10</xref>
                </sup> observed that the breeding season occurs up to 80 miles offshore, between spring and summer, in northern California. Schweigger
                <sup>
                    <xref ref-type="bibr" rid="ref10">11</xref>
                </sup> further documented the breeding period as extending from September to March. Chirinos de Vildoso
                <sup>
                    <xref ref-type="bibr" rid="ref11">12</xref>
                </sup> identified peak reproductive activity using a macroscopic gonadal maturity scale, reporting intense gonadal development from October to March. In addition, Chirinos de Vildoso
                <sup>
                    <xref ref-type="bibr" rid="ref11">12</xref>
                </sup> performed cytological characterizations of gonadal development, reproductive cycles, and fecundity, applying the macroscopic gonadal maturity scale originally developed by Schaefer and Orange
                <sup>
                    <xref ref-type="bibr" rid="ref12">13</xref>
                </sup> for female yellowfin tuna (
                <italic toggle="yes">Thunnus albacares</italic>).
                <sup>
                    <xref ref-type="bibr" rid="ref3">3</xref>
                </sup>
            </p>
            <p>Based on records from all fishing gear types in Malibu, California, in 1978, Collette and Nauen
                <sup>
                    <xref ref-type="bibr" rid="ref13">14</xref>
                </sup> reported that the smallest sexually mature 
                <italic toggle="yes">S. chiliensis</italic> (commonly known as bonito) measured between 47 and 53 cm in length. Other studies have indicated that sexual maturity is typically reached at approximately two years of age.
                <sup>
                    <xref ref-type="bibr" rid="ref14">15</xref>
                </sup> Research on the spawning period of 
                <italic toggle="yes">S. chiliensis</italic> in Chile
                <sup>
                    <xref ref-type="bibr" rid="ref15">16</xref>,
                    <xref ref-type="bibr" rid="ref16">17</xref>
                </sup> and Peru
                <sup>
                    <xref ref-type="bibr" rid="ref11">12</xref>
                </sup> suggests that spawning begins in September and concludes before April, a pattern corroborated by more recent studies.
                <sup>
                    <xref ref-type="bibr" rid="ref17">18</xref>,
                    <xref ref-type="bibr" rid="ref18">19</xref>
                </sup>
            </p>
            <p>Barrett,
                <sup>
                    <xref ref-type="bibr" rid="ref15">16</xref>
                </sup> Serra 
                <italic toggle="yes">
et al.</italic>,
                <sup>
                    <xref ref-type="bibr" rid="ref16">17</xref>
                </sup> and Chirinos de Vildoso
                <sup>
                    <xref ref-type="bibr" rid="ref11">12</xref>
                </sup> based their conclusions on gonadosomatic indices (GSIs) and microscopic examinations of unstained gonads, which were later supported by histological analyses conducted by Goldberg and Mussiett.
                <sup>
                    <xref ref-type="bibr" rid="ref19">20</xref>
                </sup> These authors observed that in northern Chile, 
                <italic toggle="yes">S. chiliensis</italic> exhibits a seasonal reproductive cycle typical of temperate fish species, characterized by a spawning phase lasting approximately half the year, followed by a non-reproductive period. This cycle coincides with periods of high food availability for larvae. Goldberg and Mussiett
                <sup>
                    <xref ref-type="bibr" rid="ref19">20</xref>
                </sup> partially confirmed Barrett&#x2019;s
                <sup>
                    <xref ref-type="bibr" rid="ref15">16</xref>
                </sup> findings, reporting that females measuring approximately 51 cm in total length (TL) were mature, with the smallest mature female measuring 48.5 cm&#x2014;slightly smaller than Barrett&#x2019;s
                <sup>
                    <xref ref-type="bibr" rid="ref15">16</xref>
                </sup> original estimate.</p>
            <p>Temporal differences in peak spawning periods observed in the study area have also been documented in other pelagic species of the Chile-Peru region. For instance, the anchoveta (
                <italic toggle="yes">Engraulis ringens</italic>) exhibits peak reproductive activity from July to February in Chile
                <sup>
                    <xref ref-type="bibr" rid="ref20">21</xref>
                </sup> and from September to March in north-central Peru.
                <sup>
                    <xref ref-type="bibr" rid="ref21">22</xref>,
                    <xref ref-type="bibr" rid="ref22">23</xref>
                </sup> Similarly, 
                <italic toggle="yes">Trachurus murphyi</italic> reproduces between September and December, with peak GSI values recorded in November.
                <sup>
                    <xref ref-type="bibr" rid="ref23">24</xref>
                </sup>
            </p>
            <p>Although Goldberg and Mussiett
                <sup>
                    <xref ref-type="bibr" rid="ref19">20</xref>
                </sup> defined four stages of gonadal maturity based on the size and characteristics of the ovaries and testes, they did not employ histological techniques due to the absence of simplified criteria for identifying early stages.
                <sup>
                    <xref ref-type="bibr" rid="ref24">25</xref>
                </sup> Such information is essential in fisheries biology, as recognizing the microscopic features of early oocyte development
                <sup>
                    <xref ref-type="bibr" rid="ref25">26</xref>
                </sup> is critical for accurately determining reproductive status. Because this species lacks external sexual differentiation, histological analysis is necessary. Previous studies on 
                <italic toggle="yes">S. chiliensis</italic> in Peru and Chile have based mainly on macroscopic descriptions of gonadal development or indices such as the gonadosomatic index, without providing detailed histological evidence of early reproductive stages. Despite the ecological and commercial importance of 
                <italic toggle="yes">Sarda chiliensis</italic> in the southeastern Pacific, detailed histological descriptions of early gonadal development stages remain scarce for populations inhabiting the northern Chilean coast. Histological characterization of reproductive tissues provides valuable baseline information for understanding reproductive processes in fish populations, particularly in regions where long-term reproductive monitoring is limited. Therefore, the aim of this study was to describe the early gonadal developmental stages of 
                <italic toggle="yes">Sarda chiliensis</italic> through histological examination of specimens collected from fishery landings in northern Chile. Due to the temporal and spatial scope of the sampling design, this study focuses on the characterization of early gonadal development rather than on a complete assessment of the reproductive cycle of the species.</p>
        </sec>
        <sec id="sec6" sec-type="methods">
            <title>2. Methods</title>
            <p>This study did not involve experimental procedures on tissues, cell lines, or human subjects. All methodologies complied with current Chilean legislation, specifically Law 20.380 on Animal Protection.</p>
            <p>The fish were not collected exclusively for this research; therefore, no additional ethical approval was required. Collection procedures adhered to the regulations established by the Servicio Nacional de Pesca y Acuicultura (SERNAPESCA) of Chile. Specimens were purchased at the local fishing terminal after being recently caught by artisanal fishermen, in accordance with the guidelines of SERNAPESCA and the Ministry of Health.</p>
            <p>It should also be noted that 
                <italic toggle="yes">S. chiliensis</italic> is neither subject to catch restrictions nor listed as a protected or endangered species.</p>
            <p>

                <italic toggle="yes">S. chiliensis</italic> (
                <xref ref-type="fig" rid="f1">
Figure 1</xref>) is an epipelagic and neritic species,
                <sup>
                    <xref ref-type="bibr" rid="ref13">14</xref>
                </sup> characterized by a fusiform body with metallic dark-blue coloration and five to nine dark, oblique stripes extending from the dorsal to the ventral side.
                <sup>
                    <xref ref-type="bibr" rid="ref26">27</xref>
                </sup> It possesses a large mouth, conical teeth, and prominent round eyes.
                <sup>
                    <xref ref-type="bibr" rid="ref27">28</xref>
                </sup> This species is distributed from M&#x00e1;ncora, Peru&#x2014;just south of the Gulf of Guayaquil&#x2014;southward to Talcahuano in Chile.</p>
            <fig fig-type="figure" id="f1" orientation="portrait" position="float">
                <label>
Figure 1. </label>
                <caption>
                    <title>Adult specimen of 
                        <italic toggle="yes">Sarda chiliensis.</italic>
</title>
                    <p>The species has a fusiform body with a metallic blue sheen on the flanks and five to nine dark, oblique stripes extending from the dorsal area toward the ventral side (adapted from Pepe-Victoriano 
                        <italic toggle="yes">et al</italic>.
                        <sup>
                            <xref ref-type="bibr" rid="ref18">19</xref>
                        </sup>).</p>
                </caption>
                <graphic id="gr1" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/197464/2117ca37-5625-4792-af6a-8f5c1bf1f9a6_figure1.gif"/>
            </fig>
            <sec id="sec7">
                <title>2.1 Capture and study area</title>
                <p>A total of 444 
                    <italic toggle="yes">S. chiliensis</italic> specimens were collected using a seine net (locally referred to as 
                    <italic toggle="yes">boliche</italic>) along the coastal margin of the Chanavayita sector (20&#x00b0;42&#x2032;16&#x2033;S, 70&#x00b0;11&#x2032;16&#x2033;W). Chanavayita is located approximately 60 km south of Iquique, in the Tarapac&#x00e1; Region. Samples were obtained by artisanal fishermen between December 2013 and June 2014. This site was chosen because it constitutes a primary fishing ground for this species in the region&#x2019;s major ports (Arica, Iquique, and Antofagasta), particularly along the stretch between Iquique and Mejillones.
                    <sup>
                        <xref ref-type="bibr" rid="ref15">16</xref>
                    </sup>
                </p>
            </sec>
            <sec id="sec8">
                <title>2.2 Measurements and laboratory observations</title>
                <p>Once obtained from artisanal fishermen, the specimens were transported to the laboratory for morphometric analysis. TL and standard length (SL) were measured to the nearest 0.01 cm using an ichthyometer. Total weight (TW) and gutted weight (GW) were recorded to the nearest 0.1 g using a digital scale (Ohaus, Ranger 3000 Series, Model R31P3, 3000 g &#x00d7; 0.1 g). Sex identification was conducted by making a longitudinal incision from the mandibular symphysis to the anal orifice to expose the gonads.</p>
            </sec>
            <sec id="sec9">
                <title>2.3 Gonadal processing</title>
                <p>The fish were transported to the laboratory 7 hours post-harvest and processed immediately to minimize tissue degradation. The gonads were fixed using 8% buffered formalin or Bouin-Hollande fixative in a ratio of at least 10:1 (fixative volume:tissue volume). Samples remained in fixator for 24&#x2013;36 h depending on the size of the gonads, after which they were transferred to 70% ethanol for storage prior to dehydration and inclusion. These conditions were applied to reduce the risk of histological alterations and ensure optimal preservation of cell structures. In the laboratory, three subsamples were taken from each gonad, embedded in Paraplast paraffin, and sectioned at a thickness of 7&#x2013;12 &#x03bc;m using a rotary microtome (Microm Leitz Wetzlar 13-13). The sections were subsequently deparaffinized and dehydrated through a graded ethanol series. Histological staining was carried out using Hematoxylin-Eosin (H&amp;E), Van Gieson, and Periodic Acid&#x2013;Schiff (PAS) stains for microscopic examination. Histological observations were performed with an Olympus CX31 optical microscope equipped with a Nikon digital camera. A total of 10 slides per sample were analyzed.</p>
            </sec>
            <sec id="sec10">
                <title>2.4 Maturity status</title>
                <p>Gonadal development stages were described following the standardized terminology proposed by Brown-Peterson 
                    <italic toggle="yes">et al</italic>.,
                    <sup>
                        <xref ref-type="bibr" rid="ref28">29</xref>
                    </sup> which is widely used in modern studies of fish reproductive biology was applied. This scale distinguishes six stages: inactive, previtellogenic, vitellogenic, mature, hydrated, and spawning (the latter characterized by the presence of post-ovulatory follicles). Male specimens were classified as either immature or mature.</p>
            </sec>
            <sec id="sec11">
                <title>2.5 Statistical analyses</title>
                <p>Statistical analyses were performed in RStudio version 2024.09.0+375 (RStudio, Inc., Washington, DC, USA). Maturity stages were tested for normality using the Anderson&#x2013;Darling test and for homogeneity of variances using Bartlett&#x2019;s test.
                    <sup>
                        <xref ref-type="bibr" rid="ref29">30</xref>
                    </sup> The non-parametric Mann-Kendall correlation test (Kendall&#x2019;s Tau) was applied to assess the relationship between the GSI and temperature. Pearson&#x2019;s correlation test was used to evaluate the relationship between Fulton&#x2019;s condition factor (
                    <italic toggle="yes">k</italic>), weight, and length across different maturity stages. All graphs were generated with the 
                    <italic toggle="yes">ggplot2</italic> package in RStudio and are presented as mean &#x00b1; standard deviation (SD). Given the limited temporal coverage of the dataset, these analyses should be interpreted cautiously and are intended primarily to identify potential patterns that may guide future research rather than to establish definitive biological relationships.</p>
            </sec>
        </sec>
        <sec id="sec12" sec-type="results">
            <title>3. Results</title>
            <p>The number of specimens captured, along with their weight and length ranges, is presented in 
                <xref ref-type="table" rid="T1">
Table 1</xref>. The numbers of mature female and male specimens are provided in 
                <xref ref-type="table" rid="T2">
Table 2</xref>.</p>
            <table-wrap id="T1" orientation="portrait" position="float">
                <label>
Table 1. </label>
                <caption>
                    <title>Morphometric parameters and frequency distribution of 
                        <italic toggle="yes">Sarda chiliensis</italic> specimens captured off the coast of Chanavayita, Chile. TL = Total length; TW = Total weight.</title>
                </caption>
                <table content-type="article-table" frame="hsides">
                    <thead>
                        <tr>
                            <th align="left" colspan="1" rowspan="1" valign="top">Sex</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Number</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">%</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">
TL (cm, mean &#x00b1; SD)</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">
TL Range (cm)</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">
TW (g, mean &#x00b1; SD)</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">
TW Range (g)</th>
                        </tr>
                    </thead>
                    <tbody>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Females</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">224</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">50.45</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">43.5 &#x00b1; 1.5</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">40.3 &#x2013; 48.8</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">1,116.1 &#x00b1; 105.6</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">826 &#x2013; 1,437</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Males</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">205</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">46.17</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">45.5 &#x00b1; 1.8</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">41.2 &#x2013; 50.4</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">1,222.8 &#x00b1; 131.3</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">881 &#x2013; 1,617</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Undetermined</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">15</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">3.38</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">40.0 &#x00b1; 0.9</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">39.1 &#x2013; 41.5</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">761 &#x00b1; 31</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">701 &#x2013; 805</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Total</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">444</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">100</td>
                            <td colspan="1" rowspan="1"/>
                            <td colspan="1" rowspan="1"/>
                            <td colspan="1" rowspan="1"/>
                            <td colspan="1" rowspan="1"/>
                        </tr>
                    </tbody>
                </table>
            </table-wrap>
            <table-wrap id="T2" orientation="portrait" position="float">
                <label>
Table 2. </label>
                <caption>
                    <title>Maturity stages of 
                        <italic toggle="yes">Sarda chiliensis</italic> specimens, classified by sex and developmental stage.</title>
                    <p>TL = Total length; TW = Total weight.</p>
                </caption>
                <table content-type="article-table" frame="hsides">
                    <thead>
                        <tr>
                            <th align="left" colspan="1" rowspan="1" valign="top">Sex</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">
Maturity stage</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Number of specimens</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Percentage per sex (%) *</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">
TL (cm, mean &#x00b1; SD)</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">
TW (g, mean &#x00b1; SD)</th>
                        </tr>
                    </thead>
                    <tbody>
                        <tr>
                            <td align="left" colspan="1" rowspan="2" valign="top">Females</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Previtellogenic</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">31</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">13.8</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">46.1 &#x00b1; 1.2</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">1,218 &#x00b1; 93.1</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Immature</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">193</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">86.2</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">43.1 &#x00b1; 1.1</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">1,099.7 &#x00b1; 98.3</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="2" valign="top">Males</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Mature</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">104</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">50.7</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">46.8 &#x00b1; 1.1</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">1,247.2 &#x00b1; 126.1</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Immature</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">101</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">49.3</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">44.1 &#x00b1; 1.3</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">1,197.6 &#x00b1; 132.5</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Total</td>
                            <td colspan="1" rowspan="1"/>
                            <td align="left" colspan="1" rowspan="1" valign="top">429</td>
                            <td colspan="1" rowspan="1"/>
                            <td colspan="1" rowspan="1"/>
                            <td colspan="1" rowspan="1"/>
                        </tr>
                    </tbody>
                </table>
            </table-wrap>
            <sec id="sec13">
                <title>3.1 Gonadosomatic Index (GSI)</title>
                <p>The results demonstrated that the GSI exhibited marked monthly variations between December and June across the different gonadal maturity stages of 
                    <italic toggle="yes">S. chiliensis</italic> (
                    <xref ref-type="fig" rid="f2">
Figure 2</xref>). Inactive females displayed a peak GSI in February (1.4 &#x00b1; 0.1%), whereas previtellogenic females maintained consistently low and stable values (0.7 &#x00b1; 0.05%). Mature males exhibited a slight increase in GSI in February (0.6 &#x00b1; 0.05%), while immature individuals consistently showed low values (&lt; 0.4%).</p>
                <fig fig-type="figure" id="f2" orientation="portrait" position="float">
                    <label>
Figure 2. </label>
                    <caption>
                        <title>Monthly variation in the GSI (%) of 
                            <italic toggle="yes">Sarda chiliensis</italic> by gonadal maturity stage and its relationship with water temperature on the northern coast of Chile.</title>
                        <p>The black line represents mean monthly water temperature (&#x00b0;C), while the colored lines indicate GSI values for each maturity stage in (a) females and (b) males.</p>
                    </caption>
                    <graphic id="gr2" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/197464/2117ca37-5625-4792-af6a-8f5c1bf1f9a6_figure2.gif"/>
                </fig>
                <p>Kendall
                    <italic toggle="yes">&#x2019;</italic>s tau correlation analysis indicated a non-significant positive correlation between GSI and temperature for inactive females (&#x03c4; = 0.619, p = 0.072)
                    <italic toggle="yes">,
</italic> with no significant relationships observed for the other maturity stages (p &gt; 0.6).</p>
            </sec>
            <sec id="sec14">
                <title>3.2 Fulton&#x2019;s condition factor (k)</title>
                <p>The relationship between weight and length in 
                    <italic toggle="yes">S. chiliensis</italic> is presented in 
                    <xref ref-type="fig" rid="f3">
Figure 3</xref>. A significant positive correlation was observed between weight and TL (r
                    <sup>2</sup> = 0.37; p = 2.7 &#x00d7; 10
                    <sup>-15</sup>), indicating that greater lengths are generally associated with higher weights. Analysis by maturity stage showed that mature individuals (primarily males) tended to exhibit higher weights and lengths (see marginal densities), 
                    <italic toggle="yes">whereas</italic> immature individuals and previtellogenic females were clustered within lower weight and length ranges. Furthermore, Fulton
                    <italic toggle="yes">&#x2019;</italic>s condition factor (k), represented by the size of the points in the figure, was generally higher in mature individuals, suggesting a better overall body condition in this group.</p>
                <fig fig-type="figure" id="f3" orientation="portrait" position="float">
                    <label>
Figure 3. </label>
                    <caption>
                        <title>Relationship between weight and length in bonito (
                            <italic toggle="yes">Sarda chiliensis</italic>) across maturity stages, and Fulton&#x2019;s condition factor (
                            <italic toggle="yes">k</italic>), along the Pacific coast of northern Chile.</title>
                    </caption>
                    <graphic id="gr3" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/197464/2117ca37-5625-4792-af6a-8f5c1bf1f9a6_figure3.gif"/>
                </fig>
            </sec>
            <sec id="sec15">
                <title>3.3 Gonadal histological description in females</title>
                <p>Based on the analyzed samples, only two maturation stages were identified: inactive and previtellogenic. To supplement the histological description of the gonadal stages, we summarized the primary germ cell types observed in both male and female specimens. For each cell type, the associated developmental stage, approximate diameter, distinctive morphological features, and corresponding figure references are provided (
                    <xref ref-type="table" rid="T2">
Table 2</xref>).</p>
                <p>The main germ cell types observed in both sexes, together with their developmental stage, diameter, morphological characteristics, and figure references, are summarized in 
                    <xref ref-type="table" rid="T3">
Table 3</xref>.</p>
                <table-wrap id="T3" orientation="portrait" position="float">
                    <label>
Table 3. </label>
                    <caption>
                        <title>Summary of germ cell types identified in 
                            <italic toggle="yes">Sarda chiliensis</italic>, with corresponding developmental stages, cell diameters, morphological characteristics, and figure references.</title>
                    </caption>
                    <table content-type="article-table" frame="hsides">
                        <thead>
                            <tr>
                                <th align="left" colspan="1" rowspan="1" valign="top">
Cell type</th>
                                <th align="left" colspan="1" rowspan="1" valign="top">
Stage</th>
                                <th align="left" colspan="1" rowspan="1" valign="top">Diameter (&#x03bc;m)</th>
                                <th align="left" colspan="1" rowspan="1" valign="top">
Morphology</th>
                                <th align="left" colspan="1" rowspan="1" valign="top">
Label in figures</th>
                            </tr>
                        </thead>
                        <tbody>
                            <tr>
                                <td align="left" colspan="1" rowspan="1" valign="top">O1 &#x2013; Primary oocyte</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">Inactive (Female)</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">60&#x2013;100</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">Central nucleus, basophilic cytoplasm no follicular layer</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">
                                    <xref ref-type="fig" rid="f4">
Figure 4a</xref>, 
                                    <xref ref-type="fig" rid="f4">4b</xref>
                                </td>
                            </tr>
                            <tr>
                                <td align="left" colspan="1" rowspan="1" valign="top">PvO &#x2013; Previtellogenic oocyte</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">Previtellogenic (Female)</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">250&#x2013;300</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">Large oocyte with centrally located nucleus and nucleoli located at the periphery of the nucleus, cytoplasm slightly basophilic</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">
                                    <xref ref-type="fig" rid="f6">
Figure 6a</xref>, 
                                    <xref ref-type="fig" rid="f6">6b</xref>
                                </td>
                            </tr>
                            <tr>
                                <td align="left" colspan="1" rowspan="1" valign="top">SG &#x2013; Spermatogonia</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">Immature (Male)</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">-</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">Large round cells located at the periphery of seminiferous lobules</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">
                                    <xref ref-type="fig" rid="f7">
Figures 7</xref>&#x2013;
                                    <xref ref-type="fig" rid="f8">8</xref>
                                </td>
                            </tr>
                            <tr>
                                <td align="left" colspan="1" rowspan="1" valign="top">SZ &#x2013; Spermatozoa</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">Mature (Male)</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">-</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">Cells with condensed nuclei located in seminiferous ducts</td>
                                <td align="left" colspan="1" rowspan="1" valign="top">
                                    <xref ref-type="fig" rid="f8">
Figure 8a&#x2013;f</xref>
                                </td>
                            </tr>
                        </tbody>
                    </table>
                </table-wrap>
                <p>H&amp;E staining (
                    <xref ref-type="fig" rid="f4">
Figure 4</xref>) revealed that the outer portion of the gonad is composed of a thin layer of moderately dense CT, consisting of collagen fibers oriented in a single direction. These fibers extend into the medullary zone of the gland as thinner bundles, forming lobules. Within these lobules, oocytes at the inactive maturation stage (also referred to as the primary maturation stage) were observed.</p>
                <fig fig-type="figure" id="f4" orientation="portrait" position="float">
                    <label>
Figure 4. </label>
                    <caption>
                        <title>Ovarian tissue of 
                            <italic toggle="yes">Sarda chiliensis</italic> at the inactive maturation stage.</title>
                        <p>(a) Peripheral region of the ovary with a thin layer of connective tissue (CT, white arrowhead) extending toward the medullary zone (black arrow). Scattered primary oocytes (O1) with centrally positioned nuclei (NO) are visible within the stromal tissue. (b) Higher magnification of O1 oocytes, showing their spherical outline, vesicular nuclei with one to five nucleoli (NO) adjacent to the nuclear membrane, and absence of follicular cells (FC). In some oocytes, initial signs of follicular arrangement can be observed. Hematoxylin-eosin stain. Scale bar = 200 &#x03bc;m. Magnifications: (a) 40&#x00d7;, (b) 400&#x00d7;.</p>
                    </caption>
                    <graphic id="gr4" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/197464/2117ca37-5625-4792-af6a-8f5c1bf1f9a6_figure4.gif"/>
                </fig>
                <p>At this stage, oocytes are small (60&#x2013;100 &#x03bc;m), round to ovoid in shape, and lack follicular cells in their membrane. These primary oocytes (O1) are centrally nucleated and surrounded by CT, with clearly visible nucleoli (NO) under high magnification. Internally, they exhibit a well-defined vesicular nucleus located centrally. At 400&#x00d7; magnification, 1&#x2013;5 NO can be observed along the nuclear membrane.</p>
                <p>Van Gieson staining (
                    <xref ref-type="fig" rid="f5">
Figure 5a</xref>, 
                    <xref ref-type="fig" rid="f5">5b</xref>) revealed the presence of blood vessels (BV), including arteries and veins, in the peripheral region of the gland. In contrast, PAS staining (
                    <xref ref-type="fig" rid="f5">
Figure 5c</xref>) produced a negative reaction in the CT surrounding the gland, indicating the absence of mucopolysaccharides or glycoproteins (
                    <xref ref-type="fig" rid="f5">
Figure 5d</xref>). Conversely, a positive PAS reaction was detected in the oocyte nucleoplasm, confirming the presence of 1&#x2013;5 NO along the nuclear membrane, consistent with the results obtained using H&amp;E staining.</p>
                <fig fig-type="figure" id="f5" orientation="portrait" position="float">
                    <label>
Figure 5. </label>
                    <caption>
                        <title>Ovarian tissue of 
                            <italic toggle="yes">Sarda chiliensis</italic> at the inactive maturation stage.</title>
                        <p>(a, b) Van Gieson stain showing primary oocytes (O1) with a rounded outline and centrally located nuclei (NO, black arrow), surrounded by connective tissue (CT, white arrowhead) and sparse follicular cells (FC). (c, d) Periodic acid&#x2013;Schiff (PAS) stain displaying oocytes within the cortical (ZC) and medullary (ZM) zones, highlighting nuclear details (NO) and the interstitial connective matrix (CM, black arrow). Magnifications: (a) 200&#x00d7;, (b) 400&#x00d7;, (c) 200&#x00d7;, (d) 400&#x00d7;.</p>
                    </caption>
                    <graphic id="gr5" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/197464/2117ca37-5625-4792-af6a-8f5c1bf1f9a6_figure5.gif"/>
                </fig>
                <p>3.3.1 Previtellogenic maturation stage</p>
                <p>At this stage, the gonad exhibits no significant structural changes in the stromal tissue regarding its distribution and proportion relative to the parenchyma. H&amp;E staining reveals no cytological alterations in the peripheral region of the medullary zone.</p>
                <p>The oocytes increase in size (250&#x2013;300 &#x03bc;m) and are round to ovoid in shape, with centrally located ovoid nuclei. The CT surrounds these previtellogenic oocytes (PvO), which display early cytoplasmic vesicles (V) characteristic of primary vitellogenesis. Four to five NO are observed at the nuclear periphery, and their positive PAS reaction indicates the presence of glycoproteins. The NO, measuring approximately 2&#x2013;5 &#x03bc;m, increase in number to four or five and remain attached to the nuclear membrane. The cytoplasm becomes more homogeneous and less basophilic compared to the previous stage. Surrounding the nucleus, vacuoles or spherical vesicles indicative of primary vitellus&#x2014;characteristic of the previtellogenic stage&#x2014;are present (
                    <xref ref-type="fig" rid="f6">
Figure 6a</xref>, 
                    <xref ref-type="fig" rid="f6">6b</xref>).</p>
                <fig fig-type="figure" id="f6" orientation="portrait" position="float">
                    <label>
Figure 6. </label>
                    <caption>
                        <title>Histological features of the female gonads of 
                            <italic toggle="yes">Sarda chiliensis</italic> at the previtellogenic maturation stage.</title>
                        <p>(a) Previtellogenic oocytes (PvO, black arrow) with a round to ovoid outline and increased size (250&#x2013;300 &#x03bc;m), surrounded by connective tissue (CT). Hematoxylin-eosin (H&amp;E) stain. (b) Higher magnification of PvO showing peripheral vesicles (V) and a centrally located nucleus (NO), characteristic of early vitellogenesis. H&amp;E stain. (c,d) Van Gieson stain revealing increased collagen fibers (CF) in the medullary and peripheral zones of the gonad (black arrow), as well as the lobular structure and CT surrounding the oocytes.</p>
                        <p>(e) Negative reaction of the connective tissue in the Periodic acid&#x2013;Schiff (PAS) stain (black arrow), confirming the absence of mucopolysaccharides. (f
) Positive PAS reaction in early primary oocyte (PO) adjacent to the PvO, indicating the presence of glycoproteins (black arrow).</p>
                        <p>Magnifications: (a) 40&#x00d7;, (b) 400&#x00d7;, (c) 200&#x00d7;, (d) 400&#x00d7;, (e) 100&#x00d7;, (f
) 400&#x00d7;.</p>
                    </caption>
                    <graphic id="gr6" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/197464/2117ca37-5625-4792-af6a-8f5c1bf1f9a6_figure6.gif"/>
                </fig>
                <p>Van Gieson staining reveals an increase in collagen fibers, which are oriented uniformly within the CT surrounding the gland. This change is consistent with the histophysiological development of the gland, as both the oocytes and the gonad itself increase in size. An increased presence of BV, including veins and arteries, is also evident.</p>
                <p>Analysis of the peripheral region of the gonad using Van Gieson staining revealed clearly visible BV, including arteries and veins. PAS staining of the CT surrounding the oocytes showed a negative reaction, confirming the absence of mucopolysaccharides or glycoproteins. However, at 400&#x00d7; magnification, a diffuse positive PAS reaction was observed in the nucleolus, warranting further investigation to precisely determine its localization (
                    <xref ref-type="fig" rid="f6">
Figure 6e</xref>, 
                    <xref ref-type="fig" rid="f6">6f</xref>).</p>
            </sec>
            <sec id="sec16">
                <title>3.4 Male histological gonadal description</title>
                <p>In males, spermatogonia (SG) and spermatozoa (SZ) were identified and characterized as detailed in 
                    <xref ref-type="table" rid="T3">
Table 3</xref>. Among the mature males, 50 individuals exhibited a high abundance of SZ within the saccules or follicles, as well as in the mesonephric ducts (MSD). However, 14 specimens displayed signs of immaturity in the cortical zone of the gland (
                    <xref ref-type="fig" rid="f7">
Figure 7</xref>). Forty males showed incomplete maturation, as evidenced by concentrically arranged seminiferous cords (SC) composed entirely of SG. These cells were embedded within a CT matrix and possessed large, round nuclei. The developing lumen and cords lacked SZ, indicating an immature state (
                    <xref ref-type="fig" rid="f7">
Figure 7a</xref>, 
                    <xref ref-type="fig" rid="f7">7b</xref>).</p>
                <fig fig-type="figure" id="f7" orientation="portrait" position="float">
                    <label>
Figure 7. </label>
                    <caption>
                        <title>Histological characteristics of male gonads of 
                            <italic toggle="yes">Sarda chiliensis</italic> at the immature stage.</title>
                        <p>(a) Low-magnification image showing seminiferous cords (SC) arranged concentrically and composed exclusively of spermatogonia (SG, boxed area). No spermatozoa (SZ) are present. The surrounding connective tissue (CT) is also visible. (b) Higher-magnification image of SC revealing densely packed SG with large, round nuclei and the absence of mature germ cells (SZ) or a defined lumen (L, black arrow). Hematoxylin-eosin (H&amp;E) stain. Magnifications: (a) 40&#x00d7;, (b) 200&#x00d7;.</p>
                    </caption>
                    <graphic id="gr7" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/197464/2117ca37-5625-4792-af6a-8f5c1bf1f9a6_figure7.gif"/>
                </fig>
                <p>3.4.1 Immature stage</p>
                <p>Immature gonads were noticeably smaller than mature ones. The outer layer consisted of CT that extended into the medullary zone. Both the cortex and medulla contained continuous cellular cords that lacked SZ (
                    <xref ref-type="fig" rid="f8">
Figure 8a</xref>). Histological examination revealed no evidence of luminal development, and the seminiferous ducts (SD) were devoid of SZ and spermatocytes (ST) (
                    <xref ref-type="fig" rid="f8">
Figure 8a</xref>). The onset of spermatogenesis was indicated by scattered SZ in the lumen of certain ducts, reflecting initial spermatogenic activity (
                    <xref ref-type="fig" rid="f8">
Figure 8b</xref>). The structural organization necessary for testicular function was highlighted by vascular elements, including arterioles (AV) and venules (VN), which were observed surrounding the SD and embedded within the CT (
                    <xref ref-type="fig" rid="f8">
Figure 8c</xref>, 
                    <xref ref-type="fig" rid="f8">8d</xref>).</p>
                <fig fig-type="figure" id="f8" orientation="portrait" position="float">
                    <label>
Figure 8. </label>
                    <caption>
                        <title>Histological features of male gonads of 
                            <italic toggle="yes">Sarda chiliensis</italic> at the immature stage.</title>
                        <p>(a) Immature seminiferous duct (SD) lacking sperzoa (SZ) and spermatocytes (ST) in the lumen (black arrow), indicating a non-functional spermatogenic stage. (b) SD with scattered SZ in the lumen (boxed area), representing early signs of spermatogenesis. H&amp;E stain. (c) Van Gieson stain of a central SD (boxed area) surrounded by connective tissue (CT), showing multiple small arterioles (AV) and venules (VN). (d) Higher magnification of the vascular region highlighting AV and VN (black arrow) matoencircling the SD. Van Gieson stain. Magnifications: (a) 40&#x00d7;, (b) 200&#x00d7;, (c) 40&#x00d7;, (d) 200&#x00d7;.</p>
                    </caption>
                    <graphic id="gr8" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/197464/2117ca37-5625-4792-af6a-8f5c1bf1f9a6_figure8.gif"/>
                </fig>
                <p>The central SD contained small BV, including AV and VN, embedded within the surrounding CT (
                    <xref ref-type="fig" rid="f8">
Figure 8c</xref>, 
                    <xref ref-type="fig" rid="f8">8d</xref>). This vascular arrangement plays a crucial physiological role by supplying nutrients through the vasa vasorum.</p>
                <p>3.4.2 Mature stage</p>
                <p>Among the mature gonads, 20 exhibited slight immaturity at the cortical level, surrounded by an albuginea composed of CT and collagen fibers. Histological analysis of the mature gonads revealed a well-developed outer capsule (albuginea) made of collagen fibers and CT, which extended into the medullary zone and formed concentrically arranged spermatocyst (SS) (
                    <xref ref-type="fig" rid="f9">
Figure 9a</xref>). Advanced spermatogenesis was indicated by the abundance of SZ found within the SS (
                    <xref ref-type="fig" rid="f9">
Figure 9b</xref>). MSD filled with SZ and encircled by BV were observed in the central region of the gonad. These ducts were lined with smooth muscle fibers and simple squamous epithelium (
                    <xref ref-type="fig" rid="f9">
Figure 9c&#x2013;e</xref>). The structure of these ducts resembles the epididymal architecture seen in higher vertebrates. The absence of mucopolysaccharides (M) or glycoproteins was confirmed by a negative PAS reaction in the surrounding CT (
                    <xref ref-type="fig" rid="f9">
Figure 9f</xref>). This configuration indicates a well-organized and functionally effective spermatogenic architecture in fully mature males.</p>
                <fig fig-type="figure" id="f9" orientation="portrait" position="float">
                    <label>
Figure 9. </label>
                    <caption>
                        <title>Histological features of male gonads of 
                            <italic toggle="yes">Sarda chiliensis</italic> at the mature stage.</title>
                        <p>(a) General view of the gonad showing several spermatocyst (SS) containing developing spermatozoa (SZ, black arrowhead).</p>
                        <p>(b) Advanced spermatogenesis indicated by dense clusters of SZ within SS at higher magnification (white arrowhead).</p>
                        <p>(c) Van Gieson stain highlighting blood vessels (BV) surrounded by connective tissue (CT) and mesonephric ducts (MSD) with associated SS. (d) Higher magnification of SS within MSD (black arrow), with adjacent CT and BV clearly visible.</p>
                        <p>(e) Longitudinal section of CT showing MSD with vascular structures (BV) and embedded SS (boxed area).</p>
                        <p>(f
) PAS stain showing a negative reaction in the CT (boxed area), confirming the absence of glycoproteins and mucopolysaccharides (M). Magnifications: (a) 40&#x00d7;, (b) 400&#x00d7;, (c) 40&#x00d7;, (d) 400&#x00d7;, (e) 40&#x00d7;, (f
) 100&#x00d7;.</p>
                    </caption>
                    <graphic id="gr9" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/197464/2117ca37-5625-4792-af6a-8f5c1bf1f9a6_figure9.gif"/>
                </fig>
                <p>
                    <xref ref-type="fig" rid="f9">
Figures 9a</xref> and 
                    <xref ref-type="fig" rid="f9">9b</xref> provide an overall view of a mature gonad, clearly showing the SS. 
                    <xref ref-type="fig" rid="f9">
Figure 9c</xref> highlights BV&#x2013;AV and VN, which are essential for cellular nutrition. 
                    <xref ref-type="fig" rid="f9">
Figures 9d</xref> and 
                    <xref ref-type="fig" rid="f9">9e</xref> present a longitudinal section of the gonad, illustrating the structure of the saccules and the presence of BV.</p>
                <p>In the central region of the gonad, MSD composed of smooth muscle interspersed with collagen fibers and lined by simple squamous epithelium were observed. These ducts contained a large quantity of SZ, resembling the epididymal function observed in higher vertebrates (
                    <xref ref-type="fig" rid="f9">
Figure 9e</xref>). At 400&#x00d7; magnification, globose cells characteristic of early spermatogenesis were visible in one half of the saccule, while the other half contained mature SZ (
                    <xref ref-type="fig" rid="f9">
Figure 9d</xref>).</p>
            </sec>
            <sec id="sec17">
                <title>3.5 Exploratory statistical analysis</title>
                <p>The statistical correlations between gonadosomatic index, condition factor, and sea surface temperature were not significant. These analyses were conducted with an exploratory purpose, aiming to identify potential patterns and generate preliminary hypotheses about the reproductive dynamics of 
                    <italic toggle="yes">S. chiliensis.</italic> Given the descriptive and temporally limited nature of this study, these statistical outcomes should not be interpreted as confirmatory evidence but rather as a first step to guide more comprehensive analyses in future research.</p>
            </sec>
        </sec>
        <sec id="sec18" sec-type="discussion">
            <title>4. Discussion</title>
            <p>It is important to note that the present study focused exclusively on the early gonadal stages observed in 
                <italic toggle="yes">Sarda chiliensis</italic>. Consequently, the findings should be interpreted as a descriptive characterization of initial gonadal development rather than as a complete representation of the species' reproductive cycle. Additional studies covering a broader temporal range and including mature females would be necessary to fully describe the spawning dynamics and reproductive cycle of this species in the study region.</p>
            <p>Chirinos de Vildoso
                <sup>
                    <xref ref-type="bibr" rid="ref11">12</xref>
                </sup> states that the maturation process is slow and influenced by secondary factors such as feeding
                <sup>
                    <xref ref-type="bibr" rid="ref30">31</xref>
                </sup> and morphocytological development. Additionally, gonadal maturation does not occur simultaneously in all specimens.</p>
            <p>Unlike previous research that relied predominantly on gross gonadal scales or indirect indices, our study provides the first histological description of the early gonadal stages of 
                <italic toggle="yes">S. chiliensis</italic> from northern Chile. This approach allows for more precise identification of germ cell types and reproductive phases, which is essential for refining species maturity scales. By documenting these stages under the microscope, our work establishes a reliable benchmark for fisheries biology, with direct applications for both the management of wild populations and the development of broodstock conditioning protocols in aquaculture.</p>
            <p>Sampling was conducted between December and June of a single year, which certainly limits the possibility of reconstructing the complete reproductive cycle of 
                <italic toggle="yes">S. chiliensis.</italic> We acknowledge this temporal restriction and highlight it as an important limitation of the study. Nevertheless, this period coincides with one of the phases in which artisanal fishing activity is most concentrated in northern Chile, which allowed us to obtain representative specimens from the main landing site of the resource. Therefore, this work should be understood as a preliminary, localized approach that provides baseline information for future research with longer duration and broader spatial coverage.</p>
            <p>Regarding the sex ratio of the captured specimens, there was an approximately equal proportion of males and females.
                <sup>
                    <xref ref-type="bibr" rid="ref31">32</xref>
                </sup> This outcome may be attributed to the absence of mature females, which reduces the need for courtship behaviors&#x2014;behaviors that might otherwise differentiate mature males from immature ones. Although Magnuson and Prescott
                <sup>
                    <xref ref-type="bibr" rid="ref32">33</xref>
                </sup> assert that pelagic fish generally do not exhibit courtship, they observed such behavior in 
                <italic toggle="yes">S. chiliensis lineolata,
</italic> suggesting the presence of temporary courtship and mating habits. Pairs of 
                <italic toggle="yes">S. chiliensis lineolata</italic> were observed engaging in sequential behaviors aimed at the simultaneous and adjacent release of gametes, with males swimming in tandem while females followed a circular trajectory. The absence of mature females in the analyzed samples may be related to spatial or seasonal segregation patterns. In many pelagic fish species, reproductive individuals may migrate to specific spawning areas or occupy habitats different from those exploited by local fisheries. Therefore, it is possible that mature females of 
                <italic toggle="yes">S. chiliensis</italic> occur in other areas or during periods not covered by the present sampling. Future studies incorporating wider spatial and temporal sampling would help clarify the reproductive distribution patterns of this species.</p>
            <p>The predominance of females in inactive or previtellogenic stages during this period may be associated with suboptimal thermal conditions necessary to initiate or complete ovarian maturation (
                <xref ref-type="table" rid="T4">
Table 4</xref>). This pattern indicates a potential 
                <bold>thermal sensitivity in the reproductive physiology</bold> of the species, where minor variations in SST could delay vitellogenesis or alter the timing and spatial distribution of spawning activity. In this context, SST in the Chanavayita region may act as an 
                <bold>environmental modulator</bold> of the local reproductive cycle of 
                <italic toggle="yes">S. chiliensis</italic>, highlighting the importance of integrating oceanographic monitoring with reproductive studies in future research.</p>
            <table-wrap id="T4" orientation="portrait" position="float">
                <label>
Table 4. </label>
                <caption>
                    <title>Gonadal development and spawning characteristics of 
                        <italic toggle="yes">Sarda chiliensis</italic> and related species.</title>
                </caption>
                <table content-type="article-table" frame="hsides">
                    <thead>
                        <tr>
                            <th align="left" colspan="1" rowspan="1" valign="top">Species</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Type of gonadal development</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Gonadal maturity phases</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Optimal temperature for spawning</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Gonad size at spawning</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Spawning frequency</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">
Source</th>
                        </tr>
                    </thead>
                    <tbody>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">
                                <italic toggle="yes">Sarda chiliensis</italic>
</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Sequential gonadal development with well-defined maturity phases</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Pre-maturation, maturation, spawning, and post-spawning phases</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">18&#x2013;20&#x00b0;C</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Relatively large gonads, with high fecundity</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Seasonal spawning, once a year</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">
                                <sup>
                                    <xref ref-type="bibr" rid="ref17">18</xref>,
                                    <xref ref-type="bibr" rid="ref33">34</xref>
                                </sup>
                            </td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">
                                <italic toggle="yes">Thunnus albacares</italic>
</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Cyclic gonadal development with a single spawning period per season</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Pre-maturation, maturation, and post-spawning phases with seasonal spawning</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">26&#x2013;31&#x00b0;C</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Moderately sized gonads, lower fecundity</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Seasonal spawning in warm water areas, once a year</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">
                                <sup>
                                    <xref ref-type="bibr" rid="ref34">35</xref>,
                                    <xref ref-type="bibr" rid="ref35">36</xref>
                                </sup>
                            </td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">
                                <italic toggle="yes">Sarda orientalis</italic>
</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Sequential gonadal development with a well-defined seasonal spawning</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Pre-maturation, maturation, spawning, and post-spawning phases</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">24.7&#x2013;28&#x00b0;C</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Large gonads, with high fecundity</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Seasonal spawning, once a year</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">
                                <sup>
                                    <xref ref-type="bibr" rid="ref36">37</xref>,
                                    <xref ref-type="bibr" rid="ref37">38</xref>
                                </sup>
                            </td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">
                                <italic toggle="yes">Katsuwonus pelamis</italic>
</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Cyclic gonadal development with multiple maturity phases throughout the year</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Maturation, spawning, and post-spawning phases, with a higher spawning frequency</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">23&#x2013;31&#x00b0;C</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Medium-sized gonads, with high fecundity</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">Continuous spawning throughout the year</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">
                                <sup>
                                    <xref ref-type="bibr" rid="ref38">39</xref>,
                                    <xref ref-type="bibr" rid="ref39">40</xref>
                                </sup>
                            </td>
                        </tr>
                    </tbody>
                </table>
            </table-wrap>
            <sec id="sec19">
                <title>4.1 Gonadosomatic Index (GSI)</title>
                <p>The GSI values showed fluctuations among the sampled months; however, due to the limited temporal coverage and sample distribution, these observations are presented as descriptive patterns and should not be interpreted as evidence of statistically supported temporal variation suggests a reproductive peak in February, consistent with previous studies on related species of the genus 
                    <italic toggle="yes">Sarda.</italic>
                    <sup>
                        <xref ref-type="bibr" rid="ref40">41</xref>,
                        <xref ref-type="bibr" rid="ref41">42</xref>
                    </sup> This peak appears to be primarily driven by inactive yet maturing females, which may indicate a synchronous reproductive strategy within the population. The absence of a significant correlation between GSI and temperature suggests that gonadal maturation may be influenced by other environmental or endogenous factors, such as photoperiod or body condition.
                    <sup>
                        <xref ref-type="bibr" rid="ref19">20</xref>,
                        <xref ref-type="bibr" rid="ref42">43</xref>
                    </sup> Reproductive cycles in pelagic fishes are often influenced by environmental variability, including temperature fluctuations, food availability, and oceanographic processes. Although the present study recorded environmental parameters during the sampling period, the temporal coverage of the dataset was insufficient to evaluate potential synchrony between environmental variability and spawning periodicity in 
                    <italic toggle="yes">S. chiliensis</italic>. Future studies incorporating multi-year sampling and continuous environmental monitoring would provide a more comprehensive understanding of the environmental drivers of reproduction in this species.</p>
                <p>These findings provide evidence of reproductive seasonality in 
                    <italic toggle="yes">S. chiliensis</italic> along the northern coast of Chile and underscore the importance of such data for the effective management and conservation of the species. The relationship between feeding activity and reproductive development can provide insights into the energetic allocation strategies of fish species. In the present study, gastrointestinal indices were not systematically recorded during sampling, which prevented evaluation of potential relationships between feeding intensity and gonadal development. Incorporating trophic indicators in future studies would allow further exploration of the interaction between feeding dynamics and reproduction.</p>
            </sec>
            <sec id="sec20">
                <title>4.2 Fulton&#x2019;s condition factor (k)</title>
                <p>The positive relationship observed between weight and length (r
                    <sup>2</sup> = 0.37) in 
                    <italic toggle="yes">S. chiliensis</italic> is consistent with typical allometric growth patterns in teleosts and aligns with previous findings in congeneric species, such as 
                    <italic toggle="yes">Sarda</italic>, where similar relationships have been reported.
                    <sup>
                        <xref ref-type="bibr" rid="ref43">44</xref>&#x2013;
                        <xref ref-type="bibr" rid="ref45">46</xref>
                    </sup> Variations in weight and length across maturity stages indicate that mature individuals&#x2014;particularly males&#x2014;tend to exhibit greater somatic development and enhanced body condition, as reflected by Fulton&#x2019;s condition factor. This pattern suggests increased energetic investment in gonadal maturation, a trend commonly observed in pelagic fish species.
                    <sup>
                        <xref ref-type="bibr" rid="ref41">42</xref>
                    </sup>
                </p>
                <p>These results highlight the importance of considering both maturity stage and body condition in population assessments and broodstock management of 
                    <italic toggle="yes">S. chiliensis</italic> along the northern coast of Chile.</p>
            </sec>
            <sec id="sec21">
                <title>4.3 Gonadal histological description in females</title>
                <p>The TL of the female specimens ranged from 43.0 to 49.4 cm. Based on the gonadal maturity scale proposed by Brown-Peterson 
                    <italic toggle="yes">et al</italic>.,
                    <sup>
                        <xref ref-type="bibr" rid="ref28">29</xref>
                    </sup> only two of the six maturation stages were observed: inactive and previtellogenic. Among the 224 females captured, only 31 exhibited the previtellogenic stage.</p>
                <p>The absence of mature females in this study is consistent with the findings of Barrett,
                    <sup>
                        <xref ref-type="bibr" rid="ref15">16</xref>
                    </sup> who reported that 
                    <italic toggle="yes">S. chiliensis</italic> females from the Chilean coast reach sexual maturity at a TL of 51 cm. Similarly, Chirinos de Vildoso
                    <sup>
                        <xref ref-type="bibr" rid="ref11">12</xref>
                    </sup> observed that Peruvian 
                    <italic toggle="yes">S. chiliensis</italic> females begin spawning at lengths between 47 and 53 cm. These observations also align with the findings of Goldberg and Mussiett,
                    <sup>
                        <xref ref-type="bibr" rid="ref19">20</xref>
                    </sup> who recorded the smallest spawning female at 48.5 cm TL, captured off the coast of Iquique between November 1981 and December 1982. Estimation of the length at first sexual maturity (L50) is an important parameter in fisheries biology and stock assessment. However, the present dataset primarily contained individuals at early gonadal stages and did not include mature females. Consequently, it was not possible to estimate the size at first maturity for 
                    <italic toggle="yes">S. chiliensis</italic>. Future research incorporating a broader size range and all maturity stages would be required to determine this parameter accurately.</p>
                <p>An important aspect of this study is the absence of females in advanced maturation stages (full vitellogenesis, hydration, or spawning). This limitation, associated with the sampling period, explains why only two female gonadal stages (inactive and previtellogenic) were described. We recognize that this situation restricts the possibility of establishing a complete histological maturity scale for the species. However, these results should be understood as a preliminary step that provides the first histological characterization of the initial maturation stages in female 
                    <italic toggle="yes">S. chiliensis</italic> from northern Chile, laying the groundwork for future studies with broader temporal coverage. The absence of mature females in the sampled population likely reflects the temporal and spatial limitations of the sampling design rather than the absence of reproductive activity in the species.</p>
                <p>The temperature is a key factor influencing the ovarian cycle of 
                    <italic toggle="yes">S. chiliensis</italic>, which may explain why 47 cm is often reported as the minimum length for sexual maturity.
                    <sup>
                        <xref ref-type="bibr" rid="ref46">47</xref>
                    </sup> In this study, however, the largest previtellogenic female measured 49.4 cm&#x2014;slightly larger than the smallest spawning female (48.5 cm) reported by Goldberg and Mussiett.
                    <sup>
                        <xref ref-type="bibr" rid="ref19">20</xref>
                    </sup> This discrepancy may be explained by environmental conditions. The fish examined by Goldberg and Mussiett were collected during an &#x201c;El Ni&#x00f1;o&#x201d; event, which caused sea surface temperatures to rise by more than 8&#x00b0;C. This unusual warming likely influenced early gonadal maturation, particularly in northern Chile.
                    <sup>
                        <xref ref-type="bibr" rid="ref47">48</xref>
                    </sup>
                </p>
                <p>In contrast, the specimens in this study were collected between December 2013 and June 2014 along the coastal margin of the Chanavayita sector, approximately 60 km south of Iquique in the Tarapac&#x00e1; Region. This period coincided with a strong La Ni&#x00f1;a event and intense coastal upwelling, both of which contributed to lower-than-average water temperatures.
                    <sup>
                        <xref ref-type="bibr" rid="ref48">49</xref>
                    </sup> The incomplete gonadal development observed in females may indicate temperature-driven segregation&#x2014;a factor not accounted for in the sampling design of this study.</p>
                <p>Although this research did not capture specimens at all stages of ovarian development, thereby preventing the characterization of a complete gonadal maturity scale, it enabled a microscopic description of two key stages from the Brown-Peterson 
                    <italic toggle="yes">et al</italic>.
                    <sup>
                        <xref ref-type="bibr" rid="ref28">29</xref>
                    </sup> scale: inactive maturation and previtellogenic maturation. For 
                    <italic toggle="yes">S. chiliensis</italic>, G&#x00e1;lvez and Castillo
                    <sup>
                        <xref ref-type="bibr" rid="ref3">3</xref>
                    </sup> adopted the methodology of Kjesbu 
                    <italic toggle="yes">et al</italic>.
                    <sup>
                        <xref ref-type="bibr" rid="ref24">25</xref>
                    </sup> to define six distinct stages of female gonadal maturation, integrating both macroscopic and microscopic criteria. Future research could investigate how these stages correspond to the classification proposed by Brown-Peterson 
                    <italic toggle="yes">et al</italic>.
                    <sup>
                        <xref ref-type="bibr" rid="ref28">29</xref>
                    </sup>
                </p>
                <p>Microscopic examination of inactive female gonads, stained with H&amp;E, revealed an external layer composed of dense CT with uniformly arranged collagen fibers. These fibers extended toward the medullary zone of the gland in thinner bundles, forming lobular structures. This anatomical organization corresponds to the virginal gonadal stage previously described by G&#x00e1;lvez and Castillo
                    <sup>
                        <xref ref-type="bibr" rid="ref3">3</xref>
                    </sup> in females collected from Peruvian waters.</p>
                <p>The results demonstrated that the lamellae were arranged in rows, containing numerous immature oocytes and few, if any, PvO. Additionally, empty spaces were observed between the lamellae. Although information on oocyte maturation in 
                    <italic toggle="yes">S. chiliensis</italic> remains limited, the findings of this study closely resemble those reported by Brown-Peterson 
                    <italic toggle="yes">et al</italic>.
                    <sup>
                        <xref ref-type="bibr" rid="ref28">29</xref>
                    </sup> for 
                    <italic toggle="yes">Graus nigra.</italic> Notable similarities include the presence of early perinucleolar stage oocytes (oocyte I), characterized by a centrally located nucleus with multiple NO migrating toward the periphery of the nucleoplasm. Once this migration begins, the nucleus is referred to as the germinal vesicle.
                    <sup>
                        <xref ref-type="bibr" rid="ref49">50</xref>&#x2013;
                        <xref ref-type="bibr" rid="ref51">52</xref>
                    </sup>
                </p>
                <p>While no peripheral cytological structural changes were observed, significant alterations occurred in the medullary zone, primarily due to an increase in oocyte size (250&#x2013;300 &#x03bc;m). The oocytes varied in shape, ranging from round to ovoid, and exhibited centrally positioned ovoid nuclei. The cytoplasm remained homogeneous but displayed reduced basophilia compared to earlier stages. Additionally, cytoplasmic vesicles associated with early oocyte development were observed surrounding the nucleus, consistent with early stages of ovarian development and thereby marking the pre-vitellogenic stage. PvO represent early phases of ovarian development and are commonly present in immature females or during periods of reproductive quiescence. Therefore, their presence should not be interpreted as evidence of active vitellogenesis or imminent spawning activity.</p>
                <p>The CT surrounding the gland exhibited an increase in collagenous fibers arranged uniformly&#x2014;an indicator characteristic of the resting stage (Stage I), as described by G&#x00e1;lvez and Castillo.
                    <sup>
                        <xref ref-type="bibr" rid="ref3">3</xref>
                    </sup> At this stage, oocytes II lacked follicular cells and were instead enclosed by CT, which extended into the medullary zone in thinner bundles, forming lobular structures.</p>
                <p>According to Vazzoler,
                    <sup>
                        <xref ref-type="bibr" rid="ref52">53</xref>
                    </sup> the formation and accumulation of yolk is one of the most critical processes in oocyte maturation. However, this process was not observed in the present study.</p>
            </sec>
            <sec id="sec22">
                <title>4.4 Gonadal histological description in males</title>
                <p>The gonadal stages of 
                    <italic toggle="yes">S. chiliensis</italic> males were classified as immature or mature, consistent with the findings of Brown-Peterson 
                    <italic toggle="yes">et al</italic>.
                    <sup>
                        <xref ref-type="bibr" rid="ref28">29</xref>
                    </sup> Unlike females, males exhibited asynchronous maturation, meaning they could attain full maturity at any time of the year. This pattern was observed in the present study. Of the 205 male specimens analyzed, 51% were sexually mature, supporting the observations of Barrett
                    <sup>
                        <xref ref-type="bibr" rid="ref15">16</xref>
                    </sup> and Chirinos de Vildoso,
                    <sup>
                        <xref ref-type="bibr" rid="ref11">12</xref>
                    </sup> who identified male maturation as a prerequisite for the spawning period of 
                    <italic toggle="yes">S. chiliensis.</italic>
                </p>
                <p>The TL of captured males ranged from 42.6 to 49.0 cm, with both immature and mature individuals represented. Immature males measured between 42.6 and 48.5 cm, while mature males ranged from 45.5 to 49.0 cm. The mature specimens in this study were smaller than those described by Barrett,
                    <sup>
                        <xref ref-type="bibr" rid="ref15">16</xref>
                    </sup> who reported sperm saccules filled with SZ in larger individuals. G&#x00e1;lvez and Castillo
                    <sup>
                        <xref ref-type="bibr" rid="ref3">3</xref>
                    </sup> proposed a five-stage classification system for males, ranging from Stage 0 to Stage IV, based on criteria such as blood supply, sperm fluid flow, CT structure, and germ cell development.</p>
                <p>Immature males in the present study corresponded to the Virginal (Stage 0) and Resting (Stage I) categories of G&#x00e1;lvez and Castillo.
                    <sup>
                        <xref ref-type="bibr" rid="ref3">3</xref>
                    </sup> Immature gonads were smaller than mature ones and contained CT extending toward the medullary zone. The cortical and medullary structures of these gonads consisted of continuous cellular cords, within which SZ were absent, including in the SD. Only developing SZ were observed.</p>
                <p>Mature specimens corresponded to the Mature (Stage II) and Mature (Stage III) classifications of G&#x00e1;lvez and Castillo.
                    <sup>
                        <xref ref-type="bibr" rid="ref3">3</xref>
                    </sup> These gonads exhibited slight cortical immaturity, with an albuginea composed of CT and collagen fibers. The CT extended into the medullary zone, forming saccules arranged concentrically. The central gonad contained the MSD, composed of smooth muscle interspersed with collagen fibers and lined by a simple flat epithelium. A large quantity of SZ was present within. In each saccule, SG characteristic of spermatogenesis were observed in one half, while the other half contained mature SZ.</p>
                <p>No specimens were classified as Expeller (Stage IV), as no empty spaces were observed in the collecting tubes&#x2014;an indicator of sperm expulsion. The lower SZ count in these specimens may be related to their smaller size (maximum 49.0 cm TL in this study) compared with the 64 cm TL reported by G&#x00e1;lvez and Castillo.
                    <sup>
                        <xref ref-type="bibr" rid="ref3">3</xref>
                    </sup> The terminology and description of spermatogenic development in 
                    <italic toggle="yes">S. chiliensis</italic> are consistent with observations in other teleosts.
                    <sup>
                        <xref ref-type="bibr" rid="ref53">54</xref>
                    </sup>
                </p>
                <p>Gonadal maturity scales should be integrated with reproductive indices, such as the GSI, fat content, and condition factor.
                    <sup>
                        <xref ref-type="bibr" rid="ref24">25</xref>,
                        <xref ref-type="bibr" rid="ref25">26</xref>
                    </sup> Additionally, measurements of oocyte diameter
                    <sup>
                        <xref ref-type="bibr" rid="ref54">55</xref>
                    </sup> and blood hormone levels
                    <sup>
                        <xref ref-type="bibr" rid="ref55">56</xref>
                    </sup> can further refine assessments of reproductive status.</p>
            </sec>
            <sec id="sec23">
                <title>4.5 Potential for spawning migrations</title>
                <p>Although specific data on the reproductive migratory movements of this species are limited, some historical evidence suggests differentiated spatial patterns during the breeding season. For instance, Serra 
                    <italic toggle="yes">et al.</italic>
                    <sup>
                        <xref ref-type="bibr" rid="ref16">17</xref>
                    </sup> and Barrett
                    <sup>
                        <xref ref-type="bibr" rid="ref15">16</xref>
                    </sup> reported that during the breeding months (austral spring&#x2013;summer), 
                    <italic toggle="yes">S. chiliensis</italic> tends to concentrate in certain coastal areas, suggesting movements toward optimal spawning habitats, such as warmer waters or sheltered regions. Similarly, Chirinos de Vildoso
                    <sup>
                        <xref ref-type="bibr" rid="ref11">12</xref>
                    </sup> in Peru documented seasonal changes in the distribution of 
                    <italic toggle="yes">S. chiliensis</italic> associated with reproductive activity, indicating shifts toward preferred spawning grounds.</p>
                <p>For other species within the genus 
                    <italic toggle="yes">Sarda</italic>, such as 
                    <italic toggle="yes">Sarda sarda sarda</italic> in the Atlantic, some authors have described a coastal reproductive migration strategy in which adults approach the coast to spawn in warmer, shallower waters.
                    <sup>
                        <xref ref-type="bibr" rid="ref56">57</xref>
                    </sup> These findings support the hypothesis that 
                    <italic toggle="yes">S. chiliensis</italic> may exhibit similar behavior, migrating from distant feeding areas to specific spawning locations that are not necessarily accessible from the artisanal fishing zones where our sampling was conducted.</p>
                <p>The absence of sexually mature females among the sampled specimens may be attributed, at least in part, to spatial migration to as-yet unidentified spawning areas&#x2014;a critical aspect for understanding the reproductive dynamics and population structure of this species.
                    <sup>
                        <xref ref-type="bibr" rid="ref57">58</xref>
                    </sup>
                </p>
            </sec>
            <sec id="sec24">
                <title>4.6 Development perspective for marine fish aquaculture</title>
                <p>The results of this study represent a first step toward the histological characterization of the gonads of 
                    <italic toggle="yes">S. chiliensis</italic> in northern Chile. We acknowledge that the descriptive nature of this work and the limited temporal coverage restrict the possibility of directly extrapolating conclusions to aquaculture. Nevertheless, the identification of early gonadal stages under natural conditions provides a fundamental reference for future research aimed at reproductive control in captivity.</p>
                <p>To advance in this line, it will be necessary to extend the sampling period to cover at least a full annual cycle, which would allow for a more accurate documentation of the onset of vitellogenesis, spawning events, and post-reproductive regression phases. Likewise, expanding the spatial coverage of sampling across the species&#x2019; latitudinal range, from northern Peru to southern Chile, will make it possible to explore regional variations in reproductive dynamics associated with thermal and oceanographic gradients.</p>
                <p>Furthermore, the use of quantitative histological parameters&#x2014;such as seminiferous tubule diameter, germinal epithelium thickness, and cell density&#x2014;combined with qualitative descriptions, will provide greater precision and reproducibility in the classification of maturity stages, particularly in males. This information will not only contribute to completing the gonadal maturity scale for the species but also generate valuable inputs for fisheries management and conservation, with potential applications in domestication and marine aquaculture programs. Broodstock management should focus on genetic selection and conditioning in controlled environments to synchronize sexual maturation.
                    <sup>
                        <xref ref-type="bibr" rid="ref58">59</xref>
                    </sup>
                </p>
                <p>In this context, the results presented here should be understood as an indispensable starting point and not as a conclusive framework for the direct implementation of aquaculture strategies. Their value lies in establishing a reference baseline for future, broader experimental studies that integrate physiological, environmental, and technological aspects required to effectively evaluate the feasibility of controlled reproduction of 
                    <italic toggle="yes">S. chiliensis</italic> in culture systems.</p>
                <p>Accordingly, any application of these results to aquaculture should be interpreted as a preliminary framework and not as established protocols, which can only be developed through more extensive and experimental research.</p>
            </sec>
            <sec id="sec25">
                <title>4.7 Projections for future research</title>
                <p>The results of this study represent a first step toward characterizing the gonads of 
                    <italic toggle="yes">S. chiliensis</italic> in northern Chile. However, to achieve a more comprehensive understanding of the species&#x2019; reproductive cycle, future research with a broader scope is necessary. First, the sampling period should be extended to cover at least one full annual cycle, encompassing different seasons to more accurately document the complete sequence of gonadal stages, including the onset of vitellogenesis, spawning events, and post-reproductive regression stages.
                    <sup>
                        <xref ref-type="bibr" rid="ref59">60</xref>
                    </sup> Second, it is recommended to increase sampling campaigns across the species&#x2019; latitudinal range, including northern Peru as well as central and southern Chile. This spatial expansion would allow for the analysis of potential geographic variations in reproductive seasonality related to regional thermal or oceanographic gradients, thereby improving understanding of population connectivity and differentiation.
                    <sup>
                        <xref ref-type="bibr" rid="ref60">61</xref>
                    </sup>
                </p>
                <p>Additionally, to achieve a more precise and standardized classification of gonadal maturity stages, particularly in males, quantitative histological parameters&#x2014;such as the diameter of seminiferous tubules, the thickness of the germinal epithelium, and cell density
                    <sup>
                        <xref ref-type="bibr" rid="ref61">62</xref>
                    </sup>&#x2014;should be employed. When combined with qualitative observations, these parameters would allow for a more reproducible description of spermatogenic development and establish a standard for using appropriate histological characters in commercially important pelagic fish. This approach will contribute not only to completing the scale of gonadal maturity for the species but also to generating valuable information for fishery management and resource conservation, with potential applications in marine aquaculture. The histological observations obtained in this study provide baseline information on the early stages of gonadal development in 
                    <italic toggle="yes">S. chiliensis</italic>. Females were predominantly observed in inactive and previtellogenic stages, while males presented immature and mature testes. These findings suggest that the sampled population was primarily composed of individuals in early reproductive phases during the study period, highlighting the importance of extended seasonal sampling to capture the full sequence of gonadal maturation. Despite these limitations, the histological observations obtained in this study provide baseline information on gonadal structure and early developmental stages in 
                    <italic toggle="yes">S. chiliensis</italic>. Such descriptive data are important for establishing reference information that can support future reproductive studies, including investigations on spawning dynamics, reproductive seasonality, and stock assessment. Baseline reproductive descriptions are particularly valuable for species in which detailed reproductive histology has not been extensively documented for specific geographic regions.</p>
                <p>This study offers a mainly descriptive approach focused on the histological characterization of the early gonadal stages of 
                    <italic toggle="yes">S. chiliensis.</italic> We recognize this limitation; however, this information represents a fundamental reference for future research with a broader experimental scope. Accurate identification of the early stages of gonadal maturation is critical to complete the maturity scales of this species, thereby improving fisheries management models, protecting critical reproductive periods, and supporting the development of captive breeding strategies for aquaculture. In this context, the results presented here constitute an indispensable starting point for both the conservation and sustainable exploitation of 
                    <italic toggle="yes">S. chiliensis</italic> in the region. Baseline information on gonadal development is essential for fisheries management because it contributes to the identification of reproductive periods and the protection of spawning stocks. Although the present study focuses on early gonadal stages, the results provide valuable reference data for monitoring reproductive conditions in 
                    <italic toggle="yes">S. chiliensis</italic>. Such information may support future management strategies, including the identification of potential reproductive periods, the design of seasonal fishing regulations, and the development of stock assessment frameworks for this commercially important species.</p>
            </sec>
        </sec>
        <sec id="sec26" sec-type="conclusions">
            <title>5. Conclusions</title>
            <p>Due to the fact that sampling was limited to the December&#x2013;June period of a single year, this study should be understood as a preliminary contribution that provides initial histological information on the early gonadal stages of 
                <italic toggle="yes">S. chiliensis</italic> in northern Chile, laying the groundwork for future studies of longer temporal duration and broader spatial scope.</p>
            <p>In the absence of mature females, this work should be considered a preliminary approach that describes only the initial stages of gonadal maturation in female 
                <italic toggle="yes">S. chiliensis</italic>, constituting a reference contribution that must be complemented by future research aimed at covering a complete reproductive cycle.</p>
        </sec>
        <sec id="sec27">
            <title>Ethical considerations</title>
            <p>Ethical standards were maintained, as animals were obtained from the city&#x2019;s fishing terminal shortly after being caught by artisanal fishermen. Legally, 
                <italic toggle="yes">S. chiliensis</italic> is not subject to catch restrictions or protection as an endangered species.</p>
        </sec>
        <sec id="sec28">
            <title>AI use disclosure</title>
            <p>During the preparation of this work the authors used Scopus AI to retrieve scientific literature, and Paperpal and Chat GPT 5.0 in order to correct grammar. After using this tool, the authors reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.</p>
        </sec>
    </body>
    <back>
        <sec id="sec31" sec-type="data-availability">
            <title>Data availability</title>
            <p>The data that support the findings of this study (morphometrics, GSI values, maturity stages and Fulton&#x2019;s condition factor) are openly available from GitHub at: 
                <ext-link ext-link-type="uri" xlink:href="https://github.com/jordan19921992/Raw_data">https://github.com/jordan19921992/Raw_data</ext-link> (https://doi.org/XXXXX).
                <sup>
                    <xref ref-type="bibr" rid="ref62">63</xref>
                </sup>
            </p>
            <p>This dataset was made available under the 
                <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International</ext-link> (CC BY 4.0) license.</p>
        </sec>
        <ack>
            <title>Acknowledgements</title>
            <p>This study was conducted in collaboration with the Red Euro-Latina de Simbiosis para la Acuicultura Sostenible (SEASOS; Euro-Latin Network of Symbiosis for Sustainable Aquaculture) and the Red Iberoamericana de Agroacuicultura (SIBIOLAT; Ibero-American Network of Agro-Aquaculture).</p>
        </ack>
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                        <italic toggle="yes">Sarda</italic> (Cuvier): Inferred Patterns of Intra- and Inter-Oceanic Connectivity Derived from Nuclear and Mitochondrial DNA Data.</article-title>
                    <source>

                        <italic toggle="yes">J. Biogeogr.</italic>
</source>
                    <year>2010</year>;<volume>37</volume>:<fpage>557</fpage>&#x2013;<lpage>570</lpage>.
                    <pub-id pub-id-type="doi">10.1111/j.1365-2699.2009.02225.x</pub-id>
                </mixed-citation>
            </ref>
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                        <name name-style="western">
                            <surname>Aguilar-Santana</surname>
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                        </name>

                        <name name-style="western">
                            <surname>Schmitter-Soto</surname>
                            <given-names>JJ</given-names>
                        </name>

                        <name name-style="western">
                            <surname>Lucano-Ram&#x00ed;rez</surname>
                            <given-names>G</given-names>
                        </name>

                        <etal/>
</person-group>:
                    <article-title>Morphochromatic Spectrum through Gonad Development Stages of the Razor Surgeonfish, 
                        <italic toggle="yes">Prionurus laticlavius</italic> (Valenciennes, 1846) (Actinopterygii: Acanthuriformes).</article-title>
                    <source>

                        <italic toggle="yes">J. Fish Biol.</italic>
</source>
                    <year>2024</year>;<volume>104</volume>:<fpage>1433</fpage>&#x2013;<lpage>1444</lpage>.
                    <pub-id pub-id-type="pmid">38350664</pub-id>
                    <pub-id pub-id-type="doi">10.1111/jfb.15685</pub-id>
                </mixed-citation>
            </ref>
            <ref id="ref62">
                <label>63</label>
                <mixed-citation publication-type="data">
                    <person-group person-group-type="author">

                        <name name-style="western">
                            <surname>Pepe-Victoriano</surname>
                            <given-names>R</given-names>
                        </name>

                        <name name-style="western">
                            <surname>Godoy</surname>
                            <given-names>M</given-names>
                        </name>

                        <name name-style="western">
                            <surname>Kush</surname>
                            <given-names>K</given-names>
                        </name>

                        <etal/>
</person-group>:
                    <data-title>Data repository of the article titled &#x201c;First Gonadal Stages of Bonito, 
                        <italic toggle="yes">Sarda chiliensis</italic> (Perciformes: Scombridae) on the Pacific Coast of Northern Chile&#x201d;, Version 1.</data-title>Dataset.
                    <source>

                        <italic toggle="yes">Raw_data.</italic>
</source>
                    <year>2025</year>.</mixed-citation>
            </ref>
        </ref-list>
    </back>
    <sub-article article-type="reviewer-report" id="report478859">
        <front-stub>
            <article-id pub-id-type="doi">10.5256/f1000research.197464.r478859</article-id>
            <title-group>
                <article-title>Reviewer response for version 2</article-title>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author">
                    <name>
                        <surname>Zahangir</surname>
                        <given-names>Md. Mahiuddin</given-names>
                    </name>
                    <xref ref-type="aff" rid="r478859a1">1</xref>
                    <role>Referee</role>
                    <uri content-type="orcid">https://orcid.org/0000-0002-6990-4783</uri>
                </contrib>
                <aff id="r478859a1">
                    <label>1</label>Chattogram Veterinary and Animal Sciences University, Chattogram, Bangladesh</aff>
            </contrib-group>
            <author-notes>
                <fn fn-type="conflict">
                    <p>
                        <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>11</day>
                <month>5</month>
                <year>2026</year>
            </pub-date>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2026 Zahangir MM</copyright-statement>
                <copyright-year>2026</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
            </permissions>
            <related-article ext-link-type="doi" id="relatedArticleReport478859" related-article-type="peer-reviewed-article" xlink:href="10.12688/f1000research.173271.2"/>
            <custom-meta-group>
                <custom-meta>
                    <meta-name>recommendation</meta-name>
                    <meta-value>approve</meta-value>
                </custom-meta>
            </custom-meta-group>
        </front-stub>
        <body>
            <p>The authors have addressed almost all the comments raised by the reviewer. Therefore, I don't have any further comments for this manuscript.</p>
            <p>Is the work clearly and accurately presented and does it cite the current literature?</p>
            <p>Yes</p>
            <p>If applicable, is the statistical analysis and its interpretation appropriate?</p>
            <p>Yes</p>
            <p>Are all the source data underlying the results available to ensure full reproducibility?</p>
            <p>Partly</p>
            <p>Is the study design appropriate and is the work technically sound?</p>
            <p>Yes</p>
            <p>Are the conclusions drawn adequately supported by the results?</p>
            <p>Yes</p>
            <p>Are sufficient details of methods and analysis provided to allow replication by others?</p>
            <p>Yes</p>
            <p>Reviewer Expertise:</p>
            <p>Reproductive biology, Fish physiology, Climate change and Aquaculture</p>
            <p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.</p>
        </body>
    </sub-article>
    <sub-article article-type="reviewer-report" id="report478858">
        <front-stub>
            <article-id pub-id-type="doi">10.5256/f1000research.197464.r478858</article-id>
            <title-group>
                <article-title>Reviewer response for version 2</article-title>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author">
                    <name>
                        <surname>Mylonas</surname>
                        <given-names>Constantinos C</given-names>
                    </name>
                    <xref ref-type="aff" rid="r478858a1">1</xref>
                    <role>Referee</role>
                </contrib>
                <aff id="r478858a1">
                    <label>1</label>Hellenic Centre for Marine Research, Heraklion, Greece</aff>
            </contrib-group>
            <author-notes>
                <fn fn-type="conflict">
                    <p>
                        <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>6</day>
                <month>5</month>
                <year>2026</year>
            </pub-date>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2026 Mylonas CC</copyright-statement>
                <copyright-year>2026</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
            </permissions>
            <related-article ext-link-type="doi" id="relatedArticleReport478858" related-article-type="peer-reviewed-article" xlink:href="10.12688/f1000research.173271.2"/>
            <custom-meta-group>
                <custom-meta>
                    <meta-name>recommendation</meta-name>
                    <meta-value>reject</meta-value>
                </custom-meta>
            </custom-meta-group>
        </front-stub>
        <body>
            <p>As I mentioned in my first report, the study does not achieve the stated objective, which is to obtain reproduction related data important in the proper stock management of this species, such as age at first maturity, reproductive season and description of gametogenesis in both sexes.&#x00a0; Unfortunately, perhaps due to erroneous sampling area and/or sampling time, the authors failed to collect the proper (types) samples, and no reproductively mature females were collected.&#x00a0;</p>
            <p> </p>
            <p> There is no such thing as &#x201c;a study of early stages of oogenesis&#x201d; for fisheries purposes!&#x00a0; The samples collected were simply 
                <underline>not in reproductive development</underline>.&#x00a0; So, the study does not offer anything in regards to the females.&#x00a0; Preliminary data, should be followed with proper data that will advance our knowledge, not simply fill the scientific literature with data that have no usefulness.&#x00a0; As I said in my first evaluation, the authors should first complete the study and then attempt to publish their results.&#x00a0; The data provided by this manuscript does not advance our understanding of the reproductive biology of the species, nor does it offer any useful information for fisheries management or broodstock conditioning.</p>
            <p>Is the work clearly and accurately presented and does it cite the current literature?</p>
            <p>No</p>
            <p>If applicable, is the statistical analysis and its interpretation appropriate?</p>
            <p>No</p>
            <p>Are all the source data underlying the results available to ensure full reproducibility?</p>
            <p>Yes</p>
            <p>Is the study design appropriate and is the work technically sound?</p>
            <p>No</p>
            <p>Are the conclusions drawn adequately supported by the results?</p>
            <p>No</p>
            <p>Are sufficient details of methods and analysis provided to allow replication by others?</p>
            <p>No</p>
            <p>Reviewer Expertise:</p>
            <p>Fish reproduction, broodstock management in aquaculture, assisted reproduction technologies</p>
            <p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.</p>
        </body>
    </sub-article>
    <sub-article article-type="reviewer-report" id="report442254">
        <front-stub>
            <article-id pub-id-type="doi">10.5256/f1000research.191067.r442254</article-id>
            <title-group>
                <article-title>Reviewer response for version 1</article-title>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author">
                    <name>
                        <surname>Zahangir</surname>
                        <given-names>Md. Mahiuddin</given-names>
                    </name>
                    <xref ref-type="aff" rid="r442254a1">1</xref>
                    <role>Referee</role>
                    <uri content-type="orcid">https://orcid.org/0000-0002-6990-4783</uri>
                </contrib>
                <aff id="r442254a1">
                    <label>1</label>Chattogram Veterinary and Animal Sciences University, Chattogram, Bangladesh</aff>
            </contrib-group>
            <author-notes>
                <fn fn-type="conflict">
                    <p>
                        <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>29</day>
                <month>1</month>
                <year>2026</year>
            </pub-date>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2026 Zahangir MM</copyright-statement>
                <copyright-year>2026</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
            </permissions>
            <related-article ext-link-type="doi" id="relatedArticleReport442254" related-article-type="peer-reviewed-article" xlink:href="10.12688/f1000research.173271.1"/>
            <custom-meta-group>
                <custom-meta>
                    <meta-name>recommendation</meta-name>
                    <meta-value>approve-with-reservations</meta-value>
                </custom-meta>
            </custom-meta-group>
        </front-stub>
        <body>
            <p>This manuscript is about the gonadal stages of Bonito, and the authors have presented data on the condition factor, GSI and gonadal histology from both male and female, which confirms the spawning season. However, information regarding the environmental synchrony with the spawning periodicity, length at first sexual maturity, and the relationship between gastrointestinal indices and the GSI is still missing. The authors are requested to compile the data and propose a detailed overview of the gonadal maturation of the studied species.&#x00a0;It is also necessary to describe how the current data can be used to undertake policy management for this species.</p>
            <p>Is the work clearly and accurately presented and does it cite the current literature?</p>
            <p>Yes</p>
            <p>If applicable, is the statistical analysis and its interpretation appropriate?</p>
            <p>Yes</p>
            <p>Are all the source data underlying the results available to ensure full reproducibility?</p>
            <p>Partly</p>
            <p>Is the study design appropriate and is the work technically sound?</p>
            <p>Yes</p>
            <p>Are the conclusions drawn adequately supported by the results?</p>
            <p>Yes</p>
            <p>Are sufficient details of methods and analysis provided to allow replication by others?</p>
            <p>Yes</p>
            <p>Reviewer Expertise:</p>
            <p>Reproductive biology, Fish physiology, Climate change and Aquaculture</p>
            <p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.</p>
        </body>
        <sub-article article-type="response" id="comment15651-442254">
            <front-stub>
                <contrib-group>
                    <contrib contrib-type="author">
                        <name>
                            <surname>Huanacuni Pilco</surname>
                            <given-names>Jordan Ismael</given-names>
                        </name>
                        <aff>Tacna, Universidad Tecnol&#x00f3;gica del Per&#x00fa;, Tacna, Tacna, Peru</aff>
                    </contrib>
                </contrib-group>
                <author-notes>
                    <fn fn-type="conflict">
                        <p>
                            <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                    </fn>
                </author-notes>
                <pub-date pub-type="epub">
                    <day>11</day>
                    <month>3</month>
                    <year>2026</year>
                </pub-date>
            </front-stub>
            <body>
                <p>
                    <bold>Response to Reviewer 3</bold>
                </p>
                <p> </p>
                <p> &#x00a0;Approved With Reservations</p>
                <p> </p>
                <p> This manuscript is about the gonadal stages of Bonito, and the authors have presented data on the condition factor, GSI and gonadal histology from both male and female, which confirms the spawning season.</p>
                <p> </p>
                <p> Dear Reviewer,</p>
                <p> We sincerely thank you for your constructive comments and for recognizing the contribution of our study to the understanding of gonadal development in 
                    <italic>Sarda chiliensis</italic>. We appreciate the suggestions aimed at strengthening the ecological and management context of the manuscript. In response to your comments, we have expanded the Discussion section to clarify the limitations of the current dataset and to provide a broader overview of gonadal maturation patterns, environmental influences, and potential implications for fisheries management. Our detailed responses are provided below.</p>
                <p> 
                    <bold>Comment 1</bold>
                </p>
                <p> 
                    <italic>Information regarding environmental synchrony with spawning periodicity is missing.</italic>
                </p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> We agree that environmental variables may play an important role in regulating reproductive cycles in pelagic fishes. However, the temporal scope of our sampling did not allow us to fully evaluate environmental synchrony with spawning periodicity. To address this point, we have expanded the Discussion to acknowledge this limitation and to highlight the importance of future studies integrating environmental monitoring and reproductive indicators.</p>
                <p> We have added the following to the end of the first paragraph of subsection 4.1. Gonadosomatic Index (GSI)</p>
                <p> &#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0; &#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0; 
                    <italic>Reproductive cycles in pelagic fishes are often influenced by environmental variability, including temperature fluctuations, food availability, and oceanographic processes. Although the present study recorded environmental parameters during the sampling period, the temporal coverage of the dataset was insufficient to evaluate potential synchrony between environmental variability and spawning periodicity in </italic>
                    <italic>Sarda chiliensis</italic>
                    <italic>. Future studies incorporating multi-year sampling and continuous environmental monitoring would provide a more comprehensive understanding of the environmental drivers of reproduction in this species.</italic>
                </p>
                <p> </p>
                <p> </p>
                <p> 
                    <bold>Comment 2</bold>
                </p>
                <p> 
                    <italic>Information on length at first sexual maturity is missing.</italic>
                </p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> We appreciate this suggestion. Estimating the length at first sexual maturity requires the presence of fully mature individuals across multiple maturity stages. Because our dataset included primarily early gonadal stages and did not contain mature females, it was not possible to estimate this parameter reliably. We have clarified this limitation in the Discussion.</p>
                <p> We have added the following information at the end of the second paragraph of subsection 4.3 Gonadal histological description in females</p>
                <p> &#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0; 
                    <italic>Estimation of the length at first sexual maturity (L50) is an important parameter in fisheries biology and stock assessment. However, the present dataset primarily contained individuals at early gonadal stages and did not include mature females. Consequently, it was not possible to estimate the size at first maturity for </italic>
                    <italic>S. chiliensis</italic>
                    <italic>. Future research incorporating a broader size range and all maturity stages would be required to determine this parameter accurately.</italic>
                </p>
                <p> </p>
                <p> 
                    <bold>Comment 3</bold>
                </p>
                <p> 
                    <italic>Relationship between gastrointestinal indices and GSI.</italic>
                </p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> We thank the reviewer for this suggestion. Unfortunately, gastrointestinal indices were not systematically recorded during the sampling process. Therefore, such analyses were beyond the scope of the present study. This limitation is now acknowledged in the Discussion.</p>
                <p> We have added the following information at the end of the second paragraph of subsection 4.1 Gonadosomatic Index (GSI)</p>
                <p> 
                    <italic>The relationship between feeding activity and reproductive development can provide insights into the energetic allocation strategies of fish species. In the present study, gastrointestinal indices were not systematically recorded during sampling, which prevented evaluation of potential relationships between feeding intensity and gonadal development. Incorporating trophic indicators in future studies would allow further exploration of the interaction between feeding dynamics and reproduction.</italic>
                </p>
                <p> </p>
                <p> 
                    <bold>Comment 4</bold>
                </p>
                <p> 
                    <italic>The authors are requested to compile the data and propose a detailed overview of the gonadal maturation of the studied species.</italic>
                </p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> We appreciate this recommendation. The Discussion section has been expanded to summarize the main features of gonadal development observed in the analyzed specimens and to contextualize them with previous reproductive studies of pelagic fishes.</p>
                <p> We have added the following information at the end of the second paragraph of subsection 4.7 Projections for future research</p>
                <p> The histological observations obtained in this study provide baseline information on the early stages of gonadal development in 
                    <italic>S. chiliensis</italic>. Females were predominantly observed in inactive and previtellogenic stages, while males presented immature and mature testes. These findings suggest that the sampled population was primarily composed of individuals in early reproductive phases during the study period, highlighting the importance of extended seasonal sampling to capture the full sequence of gonadal maturation.</p>
                <p> </p>
                <p> 
                    <bold>Comment 5</bold>
                </p>
                <p> 
                    <italic>It is also necessary to describe how the current data can be used to undertake policy management for this species</italic>
                    <bold>
                        <italic> </italic>
                    </bold>
                </p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> We agree that reproductive information is valuable for fisheries management. We have therefore incorporated a paragraph discussing how baseline information on gonadal development can support fisheries monitoring, reproductive protection measures, and future stock assessment studies.</p>
                <p> We have added the following information at the end of the of subsection 4.7 Projections for future research</p>
                <p> Baseline information on gonadal development is essential for fisheries management because it contributes to the identification of reproductive periods and the protection of spawning stocks. Although the present study focuses on early gonadal stages, the results provide valuable reference data for monitoring reproductive conditions in 
                    <italic>S. chiliensis</italic>. Such information may support future management strategies, including the identification of potential reproductive periods, the design of seasonal fishing regulations, and the development of stock assessment frameworks for this commercially important species.</p>
            </body>
        </sub-article>
    </sub-article>
    <sub-article article-type="reviewer-report" id="report444583">
        <front-stub>
            <article-id pub-id-type="doi">10.5256/f1000research.191067.r444583</article-id>
            <title-group>
                <article-title>Reviewer response for version 1</article-title>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author">
                    <name>
                        <surname>Mylonas</surname>
                        <given-names>Constantinos C</given-names>
                    </name>
                    <xref ref-type="aff" rid="r444583a1">1</xref>
                    <role>Referee</role>
                </contrib>
                <aff id="r444583a1">
                    <label>1</label>Hellenic Centre for Marine Research, Heraklion, Greece</aff>
            </contrib-group>
            <author-notes>
                <fn fn-type="conflict">
                    <p>
                        <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>13</day>
                <month>1</month>
                <year>2026</year>
            </pub-date>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2026 Mylonas CC</copyright-statement>
                <copyright-year>2026</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
            </permissions>
            <related-article ext-link-type="doi" id="relatedArticleReport444583" related-article-type="peer-reviewed-article" xlink:href="10.12688/f1000research.173271.1"/>
            <custom-meta-group>
                <custom-meta>
                    <meta-name>recommendation</meta-name>
                    <meta-value>reject</meta-value>
                </custom-meta>
            </custom-meta-group>
        </front-stub>
        <body>
            <p>The manuscript presents a study of the gametogenesis of an important fishery species for South America, the bonito (
                <italic>Sarda chiliensis</italic>).&#x00a0; Members of this genus are very important pelagic fish, and they are highly sought by the world fishing fleet.&#x00a0; The stated objective of the authors, is to obtain reproduction related data, that is important in the proper stock management of this species, such as age at first maturity, reproductive season and description of the proves of gametogenesis in both sexes.&#x00a0; Unfortunately, the authors failed to collect the proper (types) samples, they did not make the appropriate statistical analyses (size, GSI, maturity) and focused almost exclusively on presenting histological methods.&#x00a0; The authors claim that their study should &#x201c;not be interpreted as confirmatory evidence, but rather as a first step to guide more comprehensive analyses in future research&#x201d;.&#x00a0; Data obtained from Incomplete or inappropriate experimental designs should be indexed as &#x201c;preliminary&#x201d;, but the authors should first complete the study and then attempt to index their results.&#x00a0; The data provided by this manuscript does not advance our understanding of the reproductive biology of the species, nor does it offer any useful information for fisheries management or broodstock conditioning.&#x00a0; The stages of gametogenesis in fish are well know for decades now, and there is no reason to expect that this species is any different from other finfish of the same order or family.&#x00a0; Specific criticisms follow.</p>
            <p> </p>
            <p> The authors collected samples from the fish market during the period between December 2013 and June 2014.&#x00a0; As such, they inherently 
                <bold>
                    <underline>did not include the annual cycle of the fish</underline>
                </bold>.&#x00a0; This is perhaps one (the main?) reason why they did not find any reproductively active or mature females.&#x00a0; Then, the samples were presumably from the area of Chanavita, but this is probably not quite certain, as I assume they relied on the claims of the fishermen.&#x00a0; Collecting fish from a single area may mean that this is not a reproductive ground for the fish, and then this may be another reason why they did not find any mature females.&#x00a0; Another, perhaps less important aspect that makes this study less significant, is that the samples were taken by the authors and processed presumably 7 hrs after capture by the fishermen, something that again may not be certain. &#x00a0;And no information on how the fish were stored during these 7 hrs, prior to processing, which may result in artifacts in the histological sections that are not related to the proper functioning of gametogenesis.&#x00a0;</p>
            <p> </p>
            <p> No statistical analysis seems to have been made (at least there is no report of the type of analysis that was done) of the total length or body weight of males vs females, or of mature vs immature (males), or Previtellogenic vs Immature (females).&#x00a0; Such analysis will give some information on whether size made a difference in the reproductive stage of the fish.&#x00a0; This is important information in all fish species.</p>
            <p> </p>
            <p> Regarding the absence of mature fish, the author seems to claim that because there was no breeding behavior, this resulted in the absence of mature females.&#x00a0; This interpretation is incorrect and a bit flippant, since breeding behaviors follow reproductive maturation and not the other way around.</p>
            <p> </p>
            <p> Regarding the claimed &#x201c;&#x2026;monthly variation in GSI..&#x201d; (Section 4.1), there is no evidence in the manuscript (Fig. 2) of such a variation, and as I pointed earlier, no such statistical analysis is shown.</p>
            <p> </p>
            <p> Finally, the oocyte stage that the authors claim as &#x201c;pre vitellogenesis&#x201d;, is well known to be a stage that is not-sensitive to the gonadotropin hormones, and it does not indicate that the fish have entered the process of gametogenesis (as the term pre-, correctly suggests).&#x00a0; As a result, when we follow the gametogenic cycle, we do not use the presence of a very limited number of oocytes in the Cortical Alveoli or Lipid vesicle stage, as an indication of the initiation of vitellogenesis and the development towards maturity.&#x00a0; Immature females often have some of these oocytes throughout the period of quiescence (pre-vitellogenesis), and it cannot usually help us in defining the period of gametogenesis.</p>
            <p> </p>
            <p> Figure 2.&#x00a0; What is a &#x201c;dormant desired&#x201d; stage?&#x00a0; I assume it is referring to a female?&#x00a0; Why isn&#x2019;t there a statistical analysis of the GSI data?&#x00a0; As the authors mentioned, this parameter is the main one used by older studies and by fisheries scientists to describe the reproductive status of fish, and it is necessary to document if (a) there are differences between males and females, (b) immature and mature individuals, and (c) during the annual reproductive season.&#x00a0; This information is missing for the manuscript.</p>
            <p> </p>
            <p> Table 3. The authors report &#x201c;nucleoli at the periphery&#x201d; of PvO oocytes, and refer to Fig 6a and b.&#x00a0; Firstly, nucleoli are in the periphery of the nuclei, not of the oocytes (!) and secondly, I do not see any &#x201c;vesicles&#x201d; in the cytoplasm of Figure 6a and b.</p>
            <p> </p>
            <p> Figure 6. &#x00a0;&#x00a0;In photo &#x201c;f&#x201d;, I do not agree that what is shown is a &#x201c;nucleolus&#x201d;, which is a sub-cellular structure within the nucleus of an oocyte!&#x00a0; In fact, right next to this structure, one can see a few primary oocytes (po) correctly stained dark and uniformly purple, and these are manifold smaller than this supposed &#x201c;nucleolus&#x201d;.&#x00a0; Also, within some of these primary oocytes, one can see their nuclei, which stain a bit lighter.</p>
            <p> </p>
            <p> Figure 8.&#x00a0; The histological photos are of poor quality, and one cannot really see the details of the section.&#x00a0; Higher magnification should be used, so than the difference between immature and mature, spermatozoa containing testes can be clearly distinguished.</p>
            <p> </p>
            <p> Figure 9.&#x00a0; What is a &#x201c;spermatogenic saccule&#x201d; (perhaps spermatocysts?).&#x00a0; I have never seen this term before, and the authors should examine more recent articles that use the correct terminology, as I indicated earlier.&#x00a0;Also, in Fig 9a, there is a black arrow that is not cited in the legend.&#x00a0; To what is it pointing&#x201d;. Conversely, there is a mention of a white arrow, which is not visible.</p>
            <p> </p>
            <p> The authors should read and use the terminology presented more recently (Brown-Peterson et al., 2011), as the one used (Oliva J, 1968) is too old, not in English and, therefore, not widely available, accessible and readable to contemporary researchers.&#x00a0; We should all strive to use conventional terminologies, so that our work can be properly evaluated and used by our peers.</p>
            <p> </p>
            <p> </p>
            <p> Bibliography:</p>
            <p> Reference 1</p>
            <p> Reference 2</p>
            <p>Is the work clearly and accurately presented and does it cite the current literature?</p>
            <p>No</p>
            <p>If applicable, is the statistical analysis and its interpretation appropriate?</p>
            <p>No</p>
            <p>Are all the source data underlying the results available to ensure full reproducibility?</p>
            <p>Yes</p>
            <p>Is the study design appropriate and is the work technically sound?</p>
            <p>No</p>
            <p>Are the conclusions drawn adequately supported by the results?</p>
            <p>No</p>
            <p>Are sufficient details of methods and analysis provided to allow replication by others?</p>
            <p>No</p>
            <p>Reviewer Expertise:</p>
            <p>Fish reproduction, broodstock management in aquaculture, assisted reproduction technologies</p>
            <p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.</p>
        </body>
        <back>
            <ref-list>
                <title>References</title>
                <ref id="rep-ref-444583-1">
                    <label>1</label>
                    <mixed-citation publication-type="journal">
                        <person-group person-group-type="author"/>:
                        <article-title>A Standardized Terminology for Describing Reproductive Development in Fishes</article-title>.
                        <source>
                            <italic>Marine and Coastal Fisheries</italic>
                        </source>.<year>2011</year>;<volume>3</volume>(<issue>1</issue>) :
                        <elocation-id>10.1080/19425120.2011.555724</elocation-id>
                        <fpage>52</fpage>-<lpage>70</lpage>
                        <pub-id pub-id-type="doi">10.1080/19425120.2011.555724</pub-id>
                    </mixed-citation>
                </ref>
                <ref id="rep-ref-444583-2">
                    <label>2</label>
                    <mixed-citation>
                        <person-group person-group-type="author"/>:
                        <article-title>Prosodic Biolog&#x00ed;a reproductiva e histolog&#x00ed;a de g&#x00f3;nadas en peces.</article-title>
                        <source>
                            <italic>https://www.researchgate.net/publication/228809041_Prosodic_parameters_for_the_detection_of_regiona&#x2026;</italic>
                        </source>.</mixed-citation>
                </ref>
            </ref-list>
        </back>
        <sub-article article-type="response" id="comment15681-444583">
            <front-stub>
                <contrib-group>
                    <contrib contrib-type="author">
                        <name>
                            <surname>Huanacuni Pilco</surname>
                            <given-names>Jordan Ismael</given-names>
                        </name>
                        <aff>Tacna, Universidad Tecnol&#x00f3;gica del Per&#x00fa;, Tacna, Tacna, Peru</aff>
                    </contrib>
                </contrib-group>
                <author-notes>
                    <fn fn-type="conflict">
                        <p>
                            <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                    </fn>
                </author-notes>
                <pub-date pub-type="epub">
                    <day>13</day>
                    <month>3</month>
                    <year>2026</year>
                </pub-date>
            </front-stub>
            <body>
                <p>
                    <bold>REVIEWER 2</bold>
                </p>
                <p> </p>
                <p> 
                    <bold>Comment</bold>
                </p>
                <p> The manuscript presents a study of the gametogenesis of an important fishery species for South America, the bonito (
                    <italic>Sarda chiliensis</italic>).&#x00a0; Members of this genus are very important pelagic fish, and they are highly sought by the world fishing fleet.&#x00a0; The stated objective of the authors, is to obtain reproduction related data, that is important in the proper stock management of this species, such as age at first maturity, reproductive season and description of the proves of gametogenesis in both sexes.&#x00a0; Unfortunately, the authors failed to collect the proper (types) samples, they did not make the appropriate statistical analyses (size, GSI, maturity) and focused almost exclusively on presenting histological methods.&#x00a0; The authors claim that their study should &#x201c;not be interpreted as confirmatory evidence, but rather as a first step to guide more comprehensive analyses in future research&#x201d;.&#x00a0; Data obtained from Incomplete or inappropriate experimental designs should be indexed as &#x201c;preliminary&#x201d;, but the authors should first complete the study and then attempt to index their results.&#x00a0; The data provided by this manuscript does not advance our understanding of the reproductive biology of the species, nor does it offer any useful information for fisheries management or broodstock conditioning.&#x00a0; The stages of gametogenesis in fish are well know for decades now, and there is no reason to expect that this species is any different from other finfish of the same order or family.&#x00a0; Specific criticisms follow.</p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> Dear Reviewer, we sincerely thank you for the time and expertise dedicated to evaluating our manuscript. We appreciate your detailed and critical comments, which helped us clarify the scope of the study and improve the presentation of our results.</p>
                <p> We would like to emphasize that the primary objective of this work was to provide a histological description of the early stages of gonadal development in 
                    <italic>Sarda chiliensis</italic> based on the available samples. We recognize the limitations of the sampling design and have revised the manuscript to clearly state that the results should be interpreted as baseline descriptive information rather than a complete characterization of the reproductive cycle.</p>
                <p> Below we address each of your comments individually and describe the modifications incorporated into the revised manuscript.</p>
                <p> </p>
                <p> 
                    <bold>Q1. </bold>The authors collected samples from the fish market during the period between December 2013 and June 2014.&#x00a0; As such, they inherently&#x00a0;did not include the annual cycle of the fish.&#x00a0; This is perhaps one (the main?) reason why they did not find any reproductively active or mature females.&#x00a0; Then, the samples were presumably from the area of Chanavita, but this is probably not quite certain, as I assume they relied on the claims of the fishermen.&#x00a0; Collecting fish from a single area may mean that this is not a reproductive ground for the fish, and then this may be another reason why they did not find any mature females.&#x00a0; Another, perhaps less important aspect that makes this study less significant, is that the samples were taken by the authors and processed presumably 7 hrs after capture by the fishermen, something that again may not be certain. &#x00a0;And no information on how the fish were stored during these 7 hrs, prior to processing, which may result in artifacts in the histological sections that are not related to the proper functioning of gametogenesis.&#x00a0;</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We thank for this important comment. We agree that the sampling period does not represent the complete annual reproductive cycle of 
                    <italic>Sarda chiliensis</italic>. The objective of the present study was not to characterize the full reproductive cycle but rather to document the gonadal stages observed in the available specimens during the sampling period.</p>
                <p> The manuscript has been revised to clarify this scope and explicitly acknowledge the temporal limitations of the dataset.</p>
                <p> Regarding the sampling procedure, the specimens were obtained from artisanal fishery landings and processed shortly after arrival at the fish market. Although this approach does not allow independent verification of the exact capture location, market-based sampling is commonly used in fisheries biology when onboard sampling is not feasible, particularly for commercially exploited pelagic species.</p>
                <p> To minimize potential histological artifacts, the fish were transported on ice and processed within a few hours after landing prior to fixation.</p>
                <p> </p>
                <p> </p>
                <p> 
                    <bold>Q2. </bold>No statistical analysis seems to have been made (at least there is no report of the type of analysis that was done) of the total length or body weight of males vs females, or of mature vs immature (males), or Previtellogenic vs Immature (females).&#x00a0; Such analysis will give some information on whether size made a difference in the reproductive stage of the fish.&#x00a0; This is important information in all fish species.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We appreciate the suggestion regarding morphometric comparisons among sexes and maturity stages. Additional descriptive analyses were incorporated to summarize total length, body weight, gonadosomatic index (GSI), and Fulton&#x2019;s condition factor for males and females.</p>
                <p> Because the dataset included a limited number of maturity stages, particularly in females, these analyses were performed as descriptive summaries rather than formal statistical comparisons. The purpose of including these analyses is to provide an overview of the biological characteristics of the sampled individuals and to facilitate future comparative studies.</p>
                <p> </p>
                <p> </p>
                <p> 
                    <bold>Q3. </bold>Regarding the absence of mature fish, the author seems to claim that because there was no breeding behavior, this resulted in the absence of mature females.&#x00a0; This interpretation is incorrect and a bit flippant, since breeding behaviors follow reproductive maturation and not the other way around.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We thank for this important clarification. We agree that reproductive maturation precedes spawning behavior and that the absence of observed breeding behavior cannot explain the lack of mature females in the sampled individuals. The sentence suggesting this interpretation has been removed from the manuscript and replaced with a more appropriate explanation emphasizing the temporal and spatial limitations of the sampling design.</p>
                <p> We have added the following text at the end of the third paragraph of section 4.3:</p>
                <p> &#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0; 
                    <italic>The absence of mature females in the sampled population likely reflects the temporal and spatial limitations of the sampling design rather than the absence of reproductive activity in the species.</italic>
                </p>
                <p> </p>
                <p> 
                    <bold>Q4. </bold>Regarding the claimed &#x201c;&#x2026;monthly variation in GSI..&#x201d; (Section 4.1), there is no evidence in the manuscript (Fig. 2) of such a variation, and as I pointed earlier, no such statistical analysis is shown.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We appreciate the &#x00a0;observation. We agree that the available data do not support a statistical evaluation of monthly variation in GSI. The wording in the manuscript has therefore been revised to avoid implying statistically supported temporal variation and to clarify that the observed differences represent descriptive fluctuations within the sampling period.</p>
                <p> We have included the following sentence in the first paragraph of section 4.1.</p>
                <p> &#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0; 
                    <italic>The </italic>
                    <italic>GSI values showed fluctuations during the sampling months; however, given the limited temporal coverage and sample distribution, these observations should be interpreted cautiously.</italic>
                </p>
                <p> </p>
                <p> 
                    <bold>Q5.</bold> Finally, the oocyte stage that the authors claim as &#x201c;pre vitellogenesis&#x201d;, is well known to be a stage that is not-sensitive to the gonadotropin hormones, and it does not indicate that the fish have entered the process of gametogenesis (as the term pre-, correctly suggests).&#x00a0; As a result, when we follow the gametogenic cycle, we do not use the presence of a very limited number of oocytes in the Cortical Alveoli or Lipid vesicle stage, as an indication of the initiation of vitellogenesis and the development towards maturity.&#x00a0; Immature females often have some of these oocytes throughout the period of quiescence (pre-vitellogenesis), and it cannot usually help us in defining the period of gametogenesis.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We appreciate the reviewer&#x2019;s clarification regarding the interpretation of previtellogenic oocytes. We agree that these oocytes correspond to early phases of ovarian development and that their presence alone does not necessarily indicate the onset of active vitellogenesis or progression toward reproductive maturation. The manuscript has been revised to clarify this point and to avoid implying that the presence of previtellogenic oocytes reflects the initiation of active gametogenesis.</p>
                <p> We have added the following information at the end of the penultimate paragraph</p>
                <p> &#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0; 
                    <italic>PvO</italic>
                    <italic> represent early phases of ovarian development and are commonly present in immature females during periods of reproductive quiescence. Therefore, their presence should not be interpreted as evidence of active vitellogenesis or imminent spawning activity.</italic>
                </p>
                <p> </p>
                <p> </p>
                <p> 
                    <bold>Q6. </bold>Figure 2.&#x00a0; What is a &#x201c;dormant desired&#x201d; stage?&#x00a0; I assume it is referring to a female?&#x00a0; Why isn&#x2019;t there a statistical analysis of the GSI data?&#x00a0; As the authors mentioned, this parameter is the main one used by older studies and by fisheries scientists to describe the reproductive status of fish, and it is necessary to document if (a) there are differences between males and females, (b) immature and mature individuals, and (c) during the annual reproductive season.&#x00a0; This information is missing for the manuscript.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We thank for identifying this issue. The term &#x201c;dormant desired&#x201d; resulted from a typographical error and has been corrected. Additionally, figure labels have been revised for clarity.</p>
                <p> We have amended Figure 2. The term &#x2018;dormant desired&#x2019; has been replaced with &#x2018;immature female&#x2019;.</p>
                <p> &#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;</p>
                <p> 
                    <bold>Q7. </bold>Table 3. The authors report &#x201c;nucleoli at the periphery&#x201d; of PvO oocytes, and refer to Fig 6a and b.&#x00a0; Firstly, nucleoli are in the periphery of the nuclei, not of the oocytes (!) and secondly, I do not see any &#x201c;vesicles&#x201d; in the cytoplasm of Figure 6a and b.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We thank for pointing out this imprecision. The text has been corrected to specify that nucleoli are located at the periphery of the nucleus.</p>
                <p> We have corrected the description of the morphology in Table 3:</p>
                <p> </p>
                <p> 
                    <bold>Q8.</bold> Figure 6. &#x00a0;&#x00a0;In photo &#x201c;f&#x201d;, I do not agree that what is shown is a &#x201c;nucleolus&#x201d;, which is a sub-cellular structure within the nucleus of an oocyte!&#x00a0; In fact, right next to this structure, one can see a few primary oocytes (po) correctly stained dark and uniformly purple, and these are manifold smaller than this supposed &#x201c;nucleolus&#x201d;.&#x00a0; Also, within some of these primary oocytes, one can see their nuclei, which stain a bit lighter.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We appreciate the observation and have revised the labeling of the structure in Figure 6 to avoid misinterpretation. The annotation has been corrected to identify the structure more accurately.</p>
                <p> We have changed to:</p>
                <p> &#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0; &#x00a0;
                    <italic>(f ) Positive PAS reaction in early primary oocyte (PO) adjacent to the PvO, indicating the presence of glycoproteins (black arrow).</italic>
                </p>
                <p> </p>
                <p> 
                    <bold>Q9.</bold> Figure 8.&#x00a0; The histological photos are of poor quality, and one cannot really see the details of the section.&#x00a0; Higher magnification should be used, so than the difference between immature and mature, spermatozoa containing testes can be clearly distinguished.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We thank for this valuable observation and for the suggestion to include higher-magnification histological images to better illustrate the cellular structures. We agree that higher magnification can improve the visualization of the differences between immature and mature testes. However, the evaluation of this dataset has already been completed and the original histological image archive does not include additional photographs at higher magnification. Therefore, it was not possible to replace the images with higher-magnification micrographs in the revised manuscript.</p>
                <p> </p>
                <p> 
                    <bold>Q10.</bold> Figure 9.&#x00a0; What is a &#x201c;spermatogenic saccule&#x201d; (perhaps spermatocysts?).&#x00a0; I have never seen this term before, and the authors should examine more recent articles that use the correct terminology, as I indicated earlier.&#x00a0;Also, in Fig 9a, there is a black arrow that is not cited in the legend.&#x00a0; To what is it pointing&#x201d;. Conversely, there is a mention of a white arrow, which is not visible.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We appreciate this observation. The terminology has been revised to align with current terminology used in reproductive biology of fishes. We deplaced: &#x201c;spermatogenic saccule&#x201d; with: &#x201c;spermatocyst&#x201d;. We apologise for the error in the caption. It has been corrected to 'black arrowhead' instead of 'white arrowhead'.</p>
                <p> </p>
                <p> 
                    <bold>Q11.</bold> The authors should read and use the terminology presented more recently (Brown-Peterson et al., 2011), as the one used (Oliva J, 1968) is too old, not in English and, therefore, not widely available, accessible and readable to contemporary researchers.&#x00a0; We should all strive to use conventional terminologies, so that our work can be properly evaluated and used by our peers.</p>
                <p> 
                    <bold>Response</bold>
                </p>
                <p> We agree with that standardized terminology is important for comparability across studies. The manuscript has been revised to harmonize the terminology used for reproductive stages with the standardized terminology proposed by Brown-Peterson et al. (2011).</p>
                <p> We have included at the end of 2.3. Gonadal processing:</p>
                <p> &#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0;&#x00a0; 
                    <italic>Gonadal development stages were described following the standardized terminology proposed by Brown-Peterson et al. (2011), which is widely used in modern studies of fish reproductive biology.</italic>
                </p>
            </body>
        </sub-article>
    </sub-article>
    <sub-article article-type="reviewer-report" id="report440594">
        <front-stub>
            <article-id pub-id-type="doi">10.5256/f1000research.191067.r440594</article-id>
            <title-group>
                <article-title>Reviewer response for version 1</article-title>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author">
                    <name>
                        <surname>Budi</surname>
                        <given-names>Darmawan Setia</given-names>
                    </name>
                    <xref ref-type="aff" rid="r440594a1">1</xref>
                    <role>Referee</role>
                    <uri content-type="orcid">https://orcid.org/0000-0001-9508-1437</uri>
                </contrib>
                <aff id="r440594a1">
                    <label>1</label>Universitas Airlangga, East Java, Indonesia</aff>
            </contrib-group>
            <author-notes>
                <fn fn-type="conflict">
                    <p>
                        <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>30</day>
                <month>12</month>
                <year>2025</year>
            </pub-date>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2025 Budi DS</copyright-statement>
                <copyright-year>2025</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
            </permissions>
            <related-article ext-link-type="doi" id="relatedArticleReport440594" related-article-type="peer-reviewed-article" xlink:href="10.12688/f1000research.173271.1"/>
            <custom-meta-group>
                <custom-meta>
                    <meta-name>recommendation</meta-name>
                    <meta-value>approve</meta-value>
                </custom-meta>
            </custom-meta-group>
        </front-stub>
        <body>
            <p>
                <bold>General assessment</bold>
            </p>
            <p> This manuscript presents a detailed histological description of the early gonadal development of 
                <italic>Sarda chiliensis</italic> collected from the northern coast of Chile. The study provides valuable baseline information, particularly because histological descriptions of early gonadal stages for this species in this region are currently scarce. The work is generally well written, methodologically sound, and clearly structured, and it aligns well with the descriptive and data-driven scope of F1000Research.</p>
            <p> The authors appropriately acknowledge the main limitations of the study, particularly the restricted temporal coverage and the absence of mature females. Overall, the data appear reliable, and the conclusions are supported by the results presented. With minor clarifications and some improvements in presentation and contextualization, this article would represent a useful contribution to fisheries biology and reproductive studies of pelagic fishes.</p>
            <p> </p>
            <p> 
                <bold>Major comments</bold> 
                <list list-type="order">
                    <list-item>
                        <p>
                            <bold>Scope and interpretation of results</bold>
                        </p>
                        <p> The study focuses exclusively on early gonadal stages (inactive and previtellogenic in females; immature and mature in males). While this is clearly stated, some sections of the Discussion occasionally imply broader conclusions regarding the reproductive cycle of 
                            <italic>S. chiliensis</italic>. I recommend further emphasizing that the findings are limited to early stages and should not be extrapolated to define the full reproductive cycle or spawning dynamics.</p>
                    </list-item>
                    <list-item>
                        <p>
                            <bold>Sampling design and representativeness</bold>
                        </p>
                        <p> Sampling was conducted over a single period (December&#x2013;June of one year) and from one main fishing area. Although this limitation is acknowledged, it would be useful to explicitly discuss how spatial segregation or seasonal migration of mature females could bias the observed sex and maturity composition, particularly in the Results or Discussion.</p>
                    </list-item>
                    <list-item>
                        <p>
                            <bold>Statistical analyses</bold>
                        </p>
                        <p> The exploratory statistical analyses (e.g. GSI vs. temperature, condition factor relationships) are appropriately described as non-confirmatory. However, the manuscript would benefit from a clearer justification of why these analyses were included and how readers should interpret their biological relevance given the limited temporal resolution.</p>
                    </list-item>
                </list> </p>
            <p> 
                <bold>Minor comments</bold> 
                <list list-type="order">
                    <list-item>
                        <p>
                            <bold>Terminology consistency</bold>
                        </p>
                        <p> Please ensure consistent use of maturity stage terminology throughout the manuscript (e.g. &#x201c;inactive&#x201d; vs. &#x201c;dormant&#x201d; females) to avoid confusion.</p>
                    </list-item>
                    <list-item>
                        <p>
                            <bold>Figures and tables</bold> 
                            <list list-type="bullet">
                                <list-item>
                                    <p>There is a typographical error in 
                                        <bold>Figure 2</bold>, where 
                                        <italic>&#x201c;madurity&#x201d;</italic> should be corrected to 
                                        <bold>&#x201c;maturity.&#x201d;</bold>
                                    </p>
                                </list-item>
                                <list-item>
                                    <p>Please standardize the formatting of scale bars in all histological images by adding a space between the numerical value and the unit (e.g. 
                                        <bold>200 &#x03bc;m</bold> instead of 200&#x03bc;m), and ensure consistency between the scale bars shown in the figures and the corresponding descriptions in the figure captions.</p>
                                </list-item>
                                <list-item>
                                    <p>The histological figures are generally informative; however, consistent labeling (using uniform scale bars) and formatting across figures would further improve clarity.&#x00a0;</p>
                                </list-item>
                            </list> </p>
                    </list-item>
                    <list-item>
                        <p>
                            <bold>Language and style</bold>
                        </p>
                        <p> The manuscript is generally clear and readable. Minor grammatical and stylistic edits would further improve flow, particularly in longer Discussion paragraphs. These issues do not affect the scientific content.</p>
                    </list-item>
                    <list-item>
                        <p>
                            <bold>Data availability statement</bold>
                        </p>
                        <p> The data availability section is clear. It may be helpful to briefly indicate which variables are included in the shared dataset (e.g. morphometrics, GSI values, maturity stages).</p>
                    </list-item>
                </list> </p>
            <p> 
                <bold>Recommendations</bold>
            </p>
            <p> This manuscript provides a solid and carefully documented histological baseline for early gonadal development in 
                <italic>Sarda chiliensis</italic> from northern Chile. The study is methodologically sound, transparently reported, and appropriate for publication in F1000Research. I recommend 
                <bold>indexing after minor revisions</bold>, primarily focused on clarifying the scope of inference, improving consistency, and refining figure presentation.</p>
            <p>Is the work clearly and accurately presented and does it cite the current literature?</p>
            <p>Yes</p>
            <p>If applicable, is the statistical analysis and its interpretation appropriate?</p>
            <p>Yes</p>
            <p>Are all the source data underlying the results available to ensure full reproducibility?</p>
            <p>Yes</p>
            <p>Is the study design appropriate and is the work technically sound?</p>
            <p>Yes</p>
            <p>Are the conclusions drawn adequately supported by the results?</p>
            <p>Yes</p>
            <p>Are sufficient details of methods and analysis provided to allow replication by others?</p>
            <p>Yes</p>
            <p>Reviewer Expertise:</p>
            <p>Fish reproduction, breeding, and domestication</p>
            <p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.</p>
        </body>
        <back>
            <ref-list>
                <title>References</title>
                <ref id="rep-ref-440594-1">
                    <label>1</label>
                    <mixed-citation publication-type="journal">
                        <person-group person-group-type="author"/>:
                        <article-title>Maturity determination and its potential application for fish captive breeding</article-title>.
                        <source>
                            <italic>Animal Reproduction Science</italic>
                        </source>.<year>2026</year>;<volume>285</volume>:
                        <elocation-id>10.1016/j.anireprosci.2025.108074</elocation-id>
                        <pub-id pub-id-type="doi">10.1016/j.anireprosci.2025.108074</pub-id>
                    </mixed-citation>
                </ref>
                <ref id="rep-ref-440594-2">
                    <label>2</label>
                    <mixed-citation publication-type="journal">
                        <person-group person-group-type="author"/>:
                        <article-title>Sexual Characteristics of Nemipterus peronii (Valenciennes, 1830) in the Bali Strait</article-title>.
                        <source>
                            <italic>Egyptian Journal of Aquatic Biology and Fisheries</italic>
                        </source>.<year>2025</year>;<volume>29</volume>(<issue>5</issue>) :
                        <elocation-id>10.21608/ejabf.2025.412904.6390</elocation-id>
                        <fpage>2327</fpage>-<lpage>2342</lpage>
                        <pub-id pub-id-type="doi">10.21608/ejabf.2025.412904.6390</pub-id>
                    </mixed-citation>
                </ref>
            </ref-list>
        </back>
        <sub-article article-type="response" id="comment15606-440594">
            <front-stub>
                <contrib-group>
                    <contrib contrib-type="author">
                        <name>
                            <surname>Huanacuni Pilco</surname>
                            <given-names>Jordan Ismael</given-names>
                        </name>
                        <aff>Tacna, Universidad Tecnol&#x00f3;gica del Per&#x00fa;, Tacna, Tacna, Peru</aff>
                    </contrib>
                </contrib-group>
                <author-notes>
                    <fn fn-type="conflict">
                        <p>
                            <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                    </fn>
                </author-notes>
                <pub-date pub-type="epub">
                    <day>8</day>
                    <month>3</month>
                    <year>2026</year>
                </pub-date>
            </front-stub>
            <body>
                <p>
                    <bold>Reviewer Report &#x2013; REVIEWER 1</bold>
                </p>
                <p> </p>
                <p> 
                    <bold>General assessment</bold>
                </p>
                <p> This manuscript presents a detailed histological description of the early gonadal development of&#x00a0;
                    <italic>Sarda chiliensis</italic>&#x00a0;collected from the northern coast of Chile. The study provides valuable baseline information, particularly because histological descriptions of early gonadal stages for this species in this region are currently scarce. The work is generally well written, methodologically sound, and clearly structured, and it aligns well with the descriptive and data-driven scope of F1000Research.</p>
                <p> The authors appropriately acknowledge the main limitations of the study, particularly the restricted temporal coverage and the absence of mature females. Overall, the data appear reliable, and the conclusions are supported by the results presented. With minor clarifications and some improvements in presentation and contextualization, this article would represent a useful contribution to fisheries biology and reproductive studies of pelagic fishes.</p>
                <p> </p>
                <p> Response:</p>
                <p> Dear Reviewer,</p>
                <p> We sincerely thank you for the careful evaluation of our manuscript and for your constructive comments. We appreciate your positive assessment of the methodological rigor and clarity of the study. Your suggestions have helped us improve the manuscript, particularly in clarifying the scope of the results, strengthening the contextual discussion, and improving presentation details. Below we address each comment individually.</p>
                <p> </p>
                <p> 
                    <bold>Major comments</bold>
                </p>
                <p> </p>
                <p> 
                    <bold>1. Scope and interpretation of results</bold>
                </p>
                <p> The study focuses exclusively on early gonadal stages (inactive and previtellogenic in females; immature and mature in males). While this is clearly stated, some sections of the Discussion occasionally imply broader conclusions regarding the reproductive cycle of&#x00a0;
                    <italic>S. chiliensis</italic>. I recommend further emphasizing that the findings are limited to early stages and should not be extrapolated to define the full reproductive cycle or spawning dynamics.</p>
                <p> </p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> We appreciate this important observation. The intention of this study was strictly to document and describe the early gonadal stages observed in 
                    <italic>Sarda chiliensis</italic>. Following the reviewer&#x2019;s suggestion, we revised several sections of the Discussion to explicitly emphasize that our findings are limited to the early stages of gonadal development and should not be interpreted as describing the full reproductive cycle or spawning dynamics of the species.</p>
                <p> </p>
                <p> We have added the following to the end of the fifth paragraph of the discussion:</p>
                <p> 
                    <italic>It is important to note that the present study focused exclusively on the early gonadal stages observed in </italic>
                    <italic>Sarda chiliensis</italic>
                    <italic>. Consequently, the findings should be interpreted as a descriptive characterization of initial gonadal development rather than as a complete representation of the species' reproductive cycle. Additional studies covering a broader temporal range and including mature females would be necessary to fully describe the spawning dynamics and reproductive cycle of this species in the study region.</italic>
                </p>
                <p> </p>
                <p> 
                    <bold>2. Sampling design and representativeness</bold>
                </p>
                <p> Sampling was conducted over a single period (December&#x2013;June of one year) and from one main fishing area. Although this limitation is acknowledged, it would be useful to explicitly discuss how spatial segregation or seasonal migration of mature females could bias the observed sex and maturity composition, particularly in the Results or Discussion.</p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> We thank the reviewer for highlighting this point. We agree that spatial segregation or seasonal movements of mature females may influence the observed maturity composition. To address this, we have expanded the Discussion section to acknowledge that mature females may occur in other areas or seasons not covered by our sampling period, which could explain their absence in the collected specimens.</p>
                <p> We have added the following to the end of the fifth paragraph of the discussion:</p>
                <p> 
                    <italic>The absence of mature females in the analyzed samples may be related to spatial or seasonal segregation patterns. In many pelagic fish species, reproductive individuals may migrate to specific spawning areas or occupy habitats different from those exploited by local fisheries. Therefore, it is possible that mature females of S. chiliensis occur in other areas or during periods not covered by the present sampling. Future studies incorporating wider spatial and temporal sampling would help clarify the reproductive distribution patterns of this species.</italic>
                </p>
                <p> </p>
                <p> 
                    <bold>3. Statistical analyses</bold>
                </p>
                <p> The exploratory statistical analyses (e.g. GSI vs. temperature, condition factor relationships) are appropriately described as non-confirmatory. However, the manuscript would benefit from a clearer justification of why these analyses were included and how readers should interpret their biological relevance given the limited temporal resolution.</p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> We appreciate this suggestion. The exploratory analyses were included to provide preliminary insights into possible relationships between environmental conditions and biological indices. However, we agree that these results should be interpreted cautiously due to the limited temporal coverage. We have clarified this purpose in the Methods and Discussion sections.</p>
                <p> We have added to the end of subsection 2.5 Statistical analyses</p>
                <p> 
                    <italic>Given the limited temporal coverage of the dataset, these analyses should be interpreted cautiously and are intended primarily to identify potential patterns that may guide future research rather than to establish definitive biological relationships.</italic>
                </p>
                <p> </p>
                <p> 
                    <bold>Minor comments</bold>
                </p>
                <p> </p>
                <p> 
                    <bold>1.&#x00a0;Terminology consistency</bold>
                </p>
                <p> Please ensure consistent use of maturity stage terminology throughout the manuscript (e.g. &#x201c;inactive&#x201d; vs. &#x201c;dormant&#x201d; females) to avoid confusion.</p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> Thank you for pointing this out. We carefully reviewed the manuscript and standardized the terminology used to describe gonadal stages throughout the text to ensure consistency. Females were classified as inactive or previtellogenic according to histological characteristics.</p>
                <p> </p>
                <p> 
                    <bold>2. Figures and tables</bold> 
                    <list list-type="order">
                        <list-item>
                            <p> 
                                <list list-type="bullet">
                                    <list-item>
                                        <p>There is a typographical error in&#x00a0;
                                            <bold>Figure 2</bold>, where&#x00a0;
                                            <italic>&#x201c;madurity&#x201d;</italic>&#x00a0;should be corrected to&#x00a0;
                                            <bold>&#x201c;maturity.&#x201d;</bold>
                                        </p>
                                    </list-item>
                                    <list-item>
                                        <p>Please standardize the formatting of scale bars in all histological images by adding a space between the numerical value and the unit (e.g.&#x00a0;
                                            <bold>200 </bold>
                                            <bold>&#x03bc;</bold>
                                            <bold>m</bold>&#x00a0;instead of 200&#x03bc;m), and ensure consistency between the scale bars shown in the figures and the corresponding descriptions in the figure captions.</p>
                                    </list-item>
                                    <list-item>
                                        <p>The histological figures are generally informative; however, consistent labeling (using uniform scale bars) and formatting across figures would further improve clarity.&#x00a0;</p>
                                    </list-item>
                                </list> </p>
                        </list-item>
                    </list> 
                    <bold>Response:</bold>
                </p>
                <p> We thank the reviewer for noticing this error. The typographical error in Figure 2 has been corrected from &#x201c;madurity&#x201d; to &#x201c;maturity.&#x201d;</p>
                <p> All scale bars now include a space between the numerical value and the unit (e.g., 200 &#x03bc;m), and the formatting has been made consistent across figures and captions</p>
                <p> Histological figures were revised to ensure consistent labeling and formatting throughout the manuscript.</p>
                <p> </p>
                <p> 
                    <bold>3.&#x00a0;Language and style</bold>
                </p>
                <p> The manuscript is generally clear and readable. Minor grammatical and stylistic edits would further improve flow, particularly in longer Discussion paragraphs. These issues do not affect the scientific content.</p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> We appreciate this suggestion and performed an additional revision of the manuscript to improve clarity and readability. Minor grammatical and stylistic edits were implemented throughout the manuscript.</p>
                <p> </p>
                <p> 
                    <bold>4.&#x00a0;Data availability statement</bold>
                </p>
                <p> The data availability section is clear. It may be helpful to briefly indicate which variables are included in the shared dataset (e.g. morphometrics, GSI values, maturity stages).</p>
                <p> 
                    <bold>Response:</bold>
                </p>
                <p> Thank you for this suggestion. The Data Availability section has been expanded to briefly indicate that the dataset includes morphometrics, GSI values, maturity stages and Fulton&#x2019;s condition factor.</p>
            </body>
        </sub-article>
    </sub-article>
</article>
