F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.172783.1 Genome Note Articles Annotated genome of the Eucalyptus snout beetle, Gonipterus sp. n. 2 (Coleoptera, Curculionidae)

[version 1; peer review: 1 approved with reservations, 1 not approved]

 Ashmore Jade S. Data Curation Formal Analysis Investigation Resources Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0001-8334-3989 1 Dittrich-Schröder Gudrun Conceptualization Funding Acquisition Investigation Project Administration Resources Supervision Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0001-5700-6276 a 1 Knoppersen Rosa S. Data Curation Resources Writing – Review & Editing 2 Slippers Bernard Conceptualization Project Administration Supervision Writing – Review & Editing https://orcid.org/0000-0003-1491-3858 2 Hammerbacher Almuth Funding Acquisition Resources Writing – Review & Editing 1 Duong Tuan A. Conceptualization Data Curation Formal Analysis Investigation Project Administration Supervision Validation Writing – Original Draft Preparation Writing – Review & Editing 2 Department of Zoology and Entomology,Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, Gauteng, 0002, South Africa Department of Biochemistry, Genetics and Microbiology,Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, Gauteng, 0002, South Africa gudrun.dittrich@fabi.up.ac.za

No competing interests were disclosed.

 2 3 2026 2026 15 337 18 2 2026 Copyright: © 2026 Ashmore JS et al. 2026 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.

Gonipterus species, or Eucalyptus snout beetles, are defoliators that damage Eucalyptus trees in plantations globally. The Gonipterus sp. n. 2 genome was sequenced using both Oxford Nanopore and Illumina sequencing platforms which produced 76.41 Gb long-read and 57.1 Gb short-read sequence data, respectively. Genome assembly using these data resulted in 1,023 contigs, with an N50 of 2.78 Mb and a genome size of roughly 1.54 Gb. Genome completeness analysis using BUSCO resulted in a score of 98.2%. We used Braker3 to annotate the assembled Gonipterus sp. n. 2 genome using transcriptomic data from gut and reproductive tissues, as well as available protein sequences from selected Coleoptera species. Genome annotation resulted in 42,343 protein coding gene models and a proteome BUSCO completeness score of 99%. Protein clustering with 13 other insect species using OrthoFinder identified 22,245 families with 1,398 families unique to Gonipterus sp. n. 2. The number of cytochrome P450 monooxygenase genes in Gonipterus sp. n. 2 (n = 119) was greater than the other 13 insect species used in comparison. The genome of Gonipterus sp. n. 2 will be a valuable resource to assist in unravelling various aspects of the weevil’s life history, such as the metabolism of xenobiotics or the production of pheromones, and to develop alternative pest control methods.

 Eucalyptus snout beetle Illumina sequencing Oxford nanopore sequencing genome assembly transcriptome proteome prediction Future Leaders—African Independent Research (FLAIR) fellowship FLR\R1\201229 National Research Foundation 137971 Future Leaders—African Independent Research (FLAIR) fellowship [Grant number: FLR\R1\201229]. National Research Foundation [Grant number: 137971] The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1. Introduction

Gonipterus species, or Eucalyptus snout beetles, are coleopterans that damage Eucalyptus trees in plantations globally ( Tooke 1955). International trade has resulted in the introduction of Gonipterus species from their native ranges into other Eucalyptus-growing countries, impacting Eucalyptus plantation forests globally ( Wingfield et al. 2008; Hurley et al. 2016; Schröder et al. 2020). Eucalyptus defoliation by Gonipterus scutellatus ranges between 5% and 80% of plantation trees, depending on the season and the management techniques used ( Loch and Matsuki 2010). Reliable and sustainable management strategies are desperately needed to manage this forestry pest.

With the establishment of next-generation sequencing, the amount of genomic data available for model and non-model organisms, including Coleoptera, has increased exponentially. To date, there are 331 and 840 coleopteran genomes available on InsectBase ( https://www.insect-genome.com/genome) and National Center for Biotechnology Information (NCBI) ( https://www.ncbi.nlm.nih.gov/), respectively, with these coleopterans most commonly classified as pests ( Wang et al. 2025). While InsectBase and NCBI contain 33 and 106 curculionid genomes, respectively, this study represents the first genome of a curculionid from the genus Gonipterus ( Wang et al. 2025). Coleopteran genomes exhibit substantial size variation, ranging from just 18.22 Mb in the scarabid Coptodactyla brooksi, to 2714.43 Mb in the lycid Platerodrilus igneus ( Wang et al. 2025).

The availability of coleopteran genomes has facilitated the exploration of a broad range of research questions. Specifically, Curculionidae genomes have been used to investigate topics such as host adaptation, characterisation of symbiotic relationships, identification of genes involved in environmental tolerance, characterisation of range expansion, microsatellite mining, identification of chemoreceptor genes, and to determine population structure ( Apriyanto and Tambunan 2021; Navarro-Escalante et al. 2021; Keeling et al. 2022; Mohd Rodzik et al. 2023; Wang et al. 2023; Biswas et al. 2024; Chen et al. 2024). From an evolutionary perspective, research has also focused on protein evolution, such as that of luciferin, which is involved in bioluminescence in fireflies ( Zhang et al. 2020). In addition, these coleopteran genomes also allow comparative genomic studies to inform the development of gene-based pest control methods ( Chu et al. 2018).

Detoxification is a crucial adaptive mechanism for insect pests, as it enables them to neutralise harmful substances, such as plant secondary metabolites and insecticides, enhancing their survival, prevalence, and ability to spread. In insects, cytochrome P450 monooxygenases (P450s) play a key role in the metabolic detoxification of xenobiotics, such as plant secondary metabolites and insecticides ( Feyereisen 2012; Cui et al. 2016; Lu et al. 2021). These enzymes reduce the biological activity of toxic compounds by adding an oxygen atom, thereby neutralising their effects. The range of xenobiotics detoxified by insect P450s can vary from narrow to broad, while distantly related P450 enzymes may degrade the same compounds with differing efficiencies ( Cui et al. 2016). Increased gene copy number or enhanced expression of P450 genes is often linked to greater resistance to insecticides ( Feyereisen 2012). As a result, numerous studies have focused on identifying the specific P450 genes and gene superfamilies involved in detoxification, as well as their expression in response to insecticide exposure ( Zhu et al. 2013; Evans et al. 2018; Zhang et al. 2023).

This article presents the first genome of Gonipterus sp. n. 2 (Coleoptera, Curculionidae), an important resource for better understanding the biology of this Eucalyptus pest and enabling further research into alternative pest control mechanisms.

 2. Results and discussion

A total of 57.1 Gb of short-read DNA sequencing data (151 bp paired-end reads) and 23.4 Gb RNA sequencing data were generated with the Illumina HiSeq platform. Long read PromethION sequencing yielded 76.41 Gb data with read N50 of around 22 kb. Genome profiling using short reads with GenomeScope 2.0 resulted in an estimated genome size of 1.8 Gb. The primary NECAT assembly had 2,589 contigs, an N50 of 2.6 Mb and an assembled genome size of 1.76 Mb. After polishing and purging of haplotypes, the final haploid genome assembly had 1,023 contigs, an N50 of 2.78 Mb and a haplotype genome size of 1.54 Gb ( Table 1). BUSCO analysis of the final assembly using the “insecta_odb10” dataset resulted in a completeness score of 98.3% ( Manni et al. 2021).

Statistics of the <italic toggle="yes">Gonipterus</italic> sp. n. 2 genome assembly and annotation.
Genomic features
Genome assembly
 Assembly size (bp) 1 545 062 005
 Number of contigs 1023
 N50 contig length (bp) 2 782 955
 L50 163
 GC content 32.24%
 BUSCO (insecta_odb10, n=1367) 98.3%
Gene models
 Number of gene models 42 343
 Mean gene length (bp) 16 401
 Mean coding sequence length (bp) 972
 Mean number of exons per gene 3.4
 Mean exon length (bp) 325
 Mean intron length (bp) 6239
 BUSCO (insecta_odb10, n=1367) 99%

RepeatModeler identified 4,917 repeat families in the genome, comprising 73.46% of the whole genome when masked with RepeatMasker. The Braker3 pipeline predicted 42,343 protein-coding genes with BUSCO score of 99%, higher than the scores obtained from BUSCO run on the assembly, indicating that the annotation has sufficiently covered the organism’s gene space. Protein clustering of Gonipterus sp. n. 2 predicted proteome with 13 other insect species (11 Coleoptera, one Lepidoptera and one Neuroptera ( Table 2)) using OrthoFinder resulted in 22,245 orthogroups. These species were selected based on the criterion that transcriptomic data were used as gene evidence during genome annotation, suggesting that their proteomes are of high quality. Of the identified orthogroups, 3,770 (16.95%) were shared by Gonipterus sp. n. 2, selected coleopteran species, and the two non-coleopteran species used as the outgroup ( Figure 1). A total of 1,398 (6.28%) orthogroups were unique to Gonipterus sp. n. 2.

A list of the selected coleopteran species and non-coleopteran species used during this study.
Insect species Classification
Anoplophora glabripennis Coleoptera, Cerambycidae
Altica viridicyanea Coleoptera, Chrysomelidae
Bombyx mori a Lepidoptera, Bombycidae
Chrysoperla carnea a Neuroptera, Chrysopidae
Dendroctonus ponderosae Coleoptera, Curculionidae
Gonioctena quinquepunctata Coleoptera, Chrysomelidae
Hypothenemus hampei Coleoptera, Curculionidae
Ips typographus Coleoptera, Curculionidae
Oryctes borbonicus Coleoptera, Scarabaeidae
Protaetia brevitarsis Coleoptera, Scarabaeidae
Rhynchophorus ferrugineus Coleoptera, Curculionidae
Sitophilus oryzae Coleoptera, Curculionidae
Tribolium castaneum Coleoptera, Tenebrionidae

Non-coleopteran insect species used as outgroups.

 Upset-Plot of the number of gene families shared between the species and unique to the species.

There were 3770 genes shared by all the species and the number of genes unique to an individual species was between 1398 and 70 genes by Gonipterus sp. n. 2 and S. oryzae, respectively. The set size for the species was between 5000 and 15000. The abbreviations for the species names are as follows Bmor was B. mori, Ccar was C. carnea, Obor was Oryctes borbonicus (Coleoptera, Scarabaeidae), Pbre was Protaetia brevitarsis (Coleoptera, Scarabaeidae), Tcas was T. castaneum, Agla was Anoplophora glabripennis (Coleoptera, Cerambycidae), Avir was Altica viridicyanea (Coleoptera, Chrysomelidae), Gqui was Gonioctena quinquepunctata (Coleoptera, Chrysomelidae), Dpon was Dendroctonus ponderosae (Coleoptera, Curculionidae), Ityp was Ips typographus (Coleoptera, Curculionidae), Hham was Hypothenemus hampei (Coleoptera, Curculionidae), Gsp2 was Gonipterus sp. n. 2 (Coleoptera, Curculionidae), Rfer was Rhynchophorus ferrugineus (Coleoptera, Curculionidae) and Sory was S. oryzae.

CAFÉ analysis of Gonipterus sp. n. 2 proteome together with those from 11 other coleopteran species, Bombyx mori (Lepidoptera, Bombycidae) and Chrysoperla carnea (Neuroptera, Chrysopidae) identified 437 gene families with significant expansion and 110 gene families with significant contraction ( Figure 2). One hundred and nineteen P450 genes were found in 22 gene families in Gonipterus sp. n. 2, while the remaining 13 species had between 45 and 105 cytochrome P450 monooxygenase genes ( Figure 2). Four cytochrome P450 monooxygenase gene families in Gonipterus sp. n. 2 experienced significant expansion and contained 79 (66.39%) of the identified cytochrome P450 monooxygenase genes ( Figure 3A– 3D). None of the cytochrome P450 monooxygenase gene families in Gonipterus sp. n. 2 experienced significant contraction.

The number of <italic toggle="yes">cytochrome P450 monooxygenase</italic> genes identified and the number of gene families with significant expansion and contraction.

The largest number of cytochrome P450 monooxygenase genes, indicated in black, was identified in Gonipterus sp. n. 2 (n = 119). The number of cytochrome P450 monooxygenase genes identified in selected coleopteran species ranged from 46 to 105 genes. Chrysoperla carnea and B. mori had 85 and 45 cytochrome P450 monooxygenase genes, respectively. The number of gene families with significant expansion and contraction is indicated in red and blue. Gonipterus sp. n. 2 has the largest significant expansion and smallest significant contraction of gene families of the selected coleopteran species and the outgroups.

 Phylogenetic trees illustrating the expansion and contraction of Orthogroups OG0000010 (A), OG0000089 (B), OG0000101 (C) and OG0000244 (D) in <italic toggle="yes">Gonipterus</italic> sp. n. 2, selected coleopteran species and the outgroups.

The cytochrome P450 monooxygenase gene family protein domain was identified in Orthogroups by the Blast2GO PRO plug-in on the CLC Genomics Workbench. The numbers in red and blue at the nodes indicate expansion and contraction of the orthogroups. The numbers in black at the nodes indicate no expansion or contraction occurred. The statistical significance of the expansion or contraction at the individual nodes is indicated with asterisks; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Gonipterus sp. n. 2 had the highest number of cytochrome P450 monooxygenase genes (119 genes) amongst the species assessed. The expansion of cytochrome P450 monooxygenase genes may enable Gonipterus sp. n. 2 to detoxify the secondary metabolites in Eucalyptus leaves. Eucalyptus species have a high diversity of secondary metabolites with different biological activities, including insecticidal, antimicrobial, and antifeedant ( Brezáni and Karel 2013; Danna et al. 2024). Insect-repellent and insecticidal activities of the Eucalyptus secondary metabolites were effective against different coleopterans, including Tribolium castaneum (Coleoptera, Tenebrionidae) and Sitophilus oryzae (Coleoptera, Curculionidae) ( Danna et al. 2024). However, Gonipterus sp. n. 2 was attracted to the volatiles of damaged leaves from Eucalyptus host species ( Bouwer et al. 2014). Increased diversity, copy number and expression of cytochrome P450 monooxygenase genes in insects is associated with increased xenobiotic resistance ( Feyereisen 2012).

The Gonipterus sp. n. 2 genome may enable the development of alternative control methods, such as genetic pest control, to supplement currently applied pest control measures. Other coleopteran genomes have also been utilized to develop alternative pest control strategies. Specifically, these genomes have been leveraged to identify potential target genes for RNAi-based pest control and to facilitate CRISPR/Cas9 genome editing ( Segers et al. 2023; Johny et al. 2024; Zhang et al. 2024). Such research in Coleoptera has primarily focused on model organisms, such as Tribolium castaneum, highly invasive species like Harmonia axyridis and insect pests of economic importance, such as Leptinotarsa decemlineata ( Gui et al. 2020; Wu et al. 2022; Markley et al. 2024). Knowledge gained from the Gonipterus sp. n. 2 genome thus not only provides opportunities to direct future research to understand the beetle’s biology, but potentially also to improve and develop new pest control methods.

 3. Materials and methods 3.1 DNA extraction and genome sequencing

For long-read sequencing, Gonipterus sp. n. 2 adults were collected from Eucalyptus plantations near Greytown (KwaZulu-Natal, South Africa) (coordinates: 29.218415°S and 30.679624°E) during September 2021. The following research was carried out with approval from the University of Pretoria, Natural and Agricultural Sciences Ethics Committee (Reference number: NAS173/2020). The abdominal tissue only from a single adult was used for DNA extraction following the Monarch High Molecular Weight (HMW) DNA Extraction Kit (catalogue number T3060L; New England Biolabs, Ipswich, MA, United States of America) protocol for tissue, with slight modifications to the manufacturer’s instructions. The following modifications were made in Part 1: Tissue lysis. At step 5, the lysate mixture was incubated at 56 °C for 15 minutes with 1,150 rpm agitation, and, subsequently, 30 minutes without agitation to increase the DNA yield. During the 30 minutes incubation step without agitation, the lysate mixture was inverted every 5 minutes to ensure complete lysis. At step 9, the sample was centrifuged at 25 °C at 16,000 rcf for 15 minutes. At step 11, the DNA phase was aliquoted into a clean Eppendorf tube and centrifuged at 25 °C at 16,000 rcf for 15 minutes. After the second centrifugation step, the adipose was pipetted from the solution, and the DNA phase was transferred to a labelled Monarch 2 ml Tube. The DNA was separated from the solution and eluted following the HMW gDNA Binding and Elution procedure of the Monarch HMW DNA Extraction Kit protocol for tissue.

For short read sequencing, Gonipterus sp. n. 2 adults were collected from Eucalyptus plantations near Melmoth (KwaZulu-Natal, South Africa) (coordinates: 28.562856 °S, 31.191291 °E) during October 2020. The gut content was removed and DNA extraction was performed following the E.N.Z.A ® Insect DNA Kit (catalogue number D0926-01; Omega Bio-tek, Norcross, GA, United States of America). An individual adult was frozen in liquid nitrogen and ground to a fine powder. Proteinase K (25 μl) and CTL Buffer (350 μl) (catalogue number D0926-01; Omega Bio-tek, Norcross, GA, United States of America) were mixed with the ground samples, and the mixture was incubated overnight at 37 °C. The DNA was isolated, and RNA was degraded following the E.N.Z.A ® Insect DNA Kit protocol.

The Gonipterus sp. n. 2 genome was sequenced with the Illumina and Oxford Nanopore sequencing platforms. For Illumina sequencing, a paired-end library (350 bp median insert size) was prepared using TruSeq PCR-free protocol and sequenced on the HiSeq platform at Macrogen (Seoul, Korea) to obtain 151 bp paired-end reads. Read quality from the Illumina data was assessed with FastQC v0.11.9 ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and low-quality bases and remaining Illumina adaptors were removed with Trimmomatic v0.38 ( Bolger et al. 2014). For Nanopore sequencing, the library was constructed using the ligation sequencing kit (SQK-LSK110) and sequenced on the FLO-PRO002 flow cell (PromethION) at Centre for Genome Innovation at the University of Connecticut. Basecalling was conducted using Guppy v5.1.12.

 3.2 RNA extraction and sequencing

For RNA sequencing, Gonipterus sp. n. 2 adults were collected from Eucalyptus species near Greytown (KwaZulu-Natal, South Africa) (coordinates: 29.1934795° S and 30.6082423° E) during January 2023. RNA extraction was performed using the gut tissue of the Gonipterus sp. n. 2 adults with the InviTrap® Spin Plant RNA Mini Kit (catalogue number 1064100400; Invitek Diagnostics, Germany) following the manufacturer’s instructions. Briefly, 10 samples, each containing the gut tissue from Gonipterus sp. n. 2 adults, were frozen in liquid nitrogen and ground into a fine powder. Cells were lysed by adding 900 μl of Lysis Solution RP (catalogue number 1064100400; Invitek Diagnostics, Germany). The mixture was incubated for 30 minutes at 55 °C and vortexed every five minutes. The DNA was removed by centrifuging the mixture at 16,160 rcf for one minute and filtering the resulting supernatant with a Prefilter. The RNA was bound to an RNA Spin Filter and washed following the manufacturer’s instructions. To elute the RNA, 30 μl of the Elution Buffer R (catalogue number 1064100400; Invitek Diagnostics, Germany) was added to the RNA Spin Filter, incubated for two minutes at 25 °C, and then centrifuged for one minute at 11,000 rcf. The eluted RNA was immediately stored at -80 °C. A paired-end RNA library was constructed using the TruSeq Stranded mRNA Library Prep Kit, and the library was sequenced with Illumina HiSeq sequencing to obtain 151 paired-end reads at Macrogen Europe (The Netherlands). Adaptors and low-quality RNA sequences of reads were removed with Trimmomatic v0.38.

 3.3 Genome assembly and annotation

The genome size and heterozygosity of Gonipterus sp. n. 2 were estimated from the trimmed Illumina data with JELLYFISH v.1.1.12 ( Marçais and Kingsford 2011) and GenomeScope 2.0 ( Ranallo-Benavidez et al. 2020), with a k-mer value of 21. The NECAT pipeline ( Chen et al. 2021) was used to assemble the uncorrected reads from the Nanopore data using the estimated genome size obtained from GenomeScope and a minimum read length of 3 kb. The raw Nanopore data were mapped to NECAT assembly with minimap 2.0 ( Li 2018), and the mapping file was used to polish the assembled genome with racon v1.3.1 ( Vaser et al. 2017) for three iterations. The trimmed Illumina data were mapped to the racon-polished genome with BWA v0.7.17 ( Vasimuddin et al. 2019) and used to further polish the genome with Pilon v1.23 ( Walker et al. 2014) for three iterations. A final round of polishing was conducted with racon using trimmed Illumina data mapped to the Pilon-polished assembly. Haplotypes in the primary polished assembly were removed with Purge Haplotigs ( Roach et al. 2018). The completeness of the polished genome was assessed with the Benchmarking Universal Single-Copy Orthologs (BUSCO) v.4.0.5 utilising the “insecta_odb10” dataset ( Manni et al. 2021).

Structural annotation was carried out using the Braker3 pipeline ( Stanke et al. 2006; Stanke et al. 2008; Buchfink et al. 2015; Hoff et al. 2019; Brůna et al. 2021), using Augustus as the gene predictor. RepeatModeler v2.0.2 ( http://www.repeatmasker.org/RepeatModeler/) was used to construct a de novo repeat library, which was used to soft-mask the genome with RepeatMasker v4.1.2 ( http://www.repeatmasker.org/RepeatModeler/). RNA sequencing data from Gonipterus sp. n. 2 reproductive tissue (NCBI accession number: SAMN19700001), alimentary tissue (NCBI accession number: SAMN19700000) ( Souza et al. 2022), and the trimmed RNA sequencing data from the gut of Gonipterus sp. n. 2 (PRJNA1189815) were aligned to the masked Gonipterus sp. n. 2 draft genome with HISAT2 v2.2.1 ( Kim et al. 2019). Proteomes of the selected coleopteran species ( Table 2) were mapped to the Gonipterus sp. n. 2 genome with GenomeThreader v1.7.4 ( Gremme et al. 2005). The aligned transcriptomes and proteomes were used as evidences to train the ab initio prediction tool Augustus for the Braker3 pipeline. The Blast2GO PRO plug-in v1.20.14 on the CLC Genomics Workbench v20.0.4 was used to assign functions to the predicted proteins and identify P450 genes using the protein domain (Pfam = 00067).

 3.4. Analysis of the expansion of the cytochrome P450 gene family

Orthologous gene families from the protein sequences of Gonipterus sp. n. 2, selected coleopteran species ( Table 2), Bombyx mori (Lepidoptera, Bombycidae) and Chrysoperla carnea (Neuroptera, Chrysopidae) were determined using OrthoFinder v2.5.5 ( Emms and Kelly 2015), with the Diamond in sensitive mode (-S) and the inflation factor of 1.5. The coleopteran species and outgroups were selected from InsectBase (Accessed in June 2023) ( Mei et al. 2022) based on the availability of genomes annotated with transcriptomic data, which enabled the prediction of a higher quality and more comprehensive protein set ( Chen et al. 2017). To construct a species phylogeny, protein sequences of the single-copy orthologs were aligned with muscle v3.8.31 ( Edgar 2022), and trees were inferred with IQ-TREE v2.1.2 ( Kalyaanamoorthy et al. 2017). A species phylogeny was inferred from individual gene trees with ASTRAL v5.7.7 ( Zhang et al. 2018). The obtained phylogeny was rooted in B. mori and C. carnea, and the branch length was optimised with RAxML v8.2.11 ( Stamatakis 2014) using the concatenate alignment of all single-copy genes. The rooted phylogenetic tree was transformed into an ultrametric tree and used as input in CAFÉ. CAFÉ v5 ( Mendes et al. 2020) was used to assess the expansion and contraction of P450 gene families.

 Data availability

The preprint of the article is available on bioRxiv: Ashmore, J. S., G. Dittrich-Schröder, R. S. Knoppersen, B. Slippers, A. Hammerbacher et al., 2025 Annotated genome of the Eucalyptus snout beetle, Gonipterus sp. n. 2 (Coleoptera, Curculionidae). bioRxiv: 2025.2012.2003.691750.

Underlying data

NCBI: Gonipterus sp. n. 2, Eucalyptus snout beetle, genome sequencing

https://www.ncbi.nlm.nih.gov/bioproject/1203571

The BioProject (Accession number: PRJNA1203571) contains the following underlying data:

Gsp2_table2asn.sqn (The table 2asn (.sqn) file for the assembled and annotated genome of an adult Gonipterus sp. n. 2).

FigShare: Genome assembly and annotation of Gonipterus sp. n. 2

https://figshare.com/articles/dataset/Genome_assembly_and_annotation_of_i_Gonipterus_i_sp_n_2/30760949

The FigShare item contains the following data:

GSP2_HaploCurated.fsa (The assembled genome of an adult Gonipterus sp. n. 2).

Gsp2_CDS_and_AA.gff3 (The CDS and amino acid annotation information for the Gonipterus sp. n. 2 genome).

Gsp2_table2asn_Final.gbf (The final GenBank flatfile created with table 2asn for the Gonipterus sp. n. 2 genome).

ARRIVE checklist

NCBI Sequence Read Archive (SRA): Gonipterus gut transcriptome

The BioProject (accession: PRJNA1189815) contains the following underlying data:

SRR31481664 (raw FASTQ files: RKAH-05_R1.fastq.gz, RKAH-05_R2.fastq.gz)

SRR31481665 (raw FASTQ files: RKAH-04_R1.fastq.gz, RKAH-04_R2.fastq.gz)

SRR31481666 (raw FASTQ files: RKAH-03_R1.fastq.gz, RKAH-03_R2.fastq.gz)

SRR31481667 (raw FASTQ files: RKAH-02_R1.fastq.gz, RKAH-02_R2.fastq.gz)

SRR31481668 (raw FASTQ files: RKAH-10_R1.fastq.gz, RKAH-10_R2.fastq.gz)

SRR31481669 (raw FASTQ files: RKAH-09_R1.fastq.gz, RKAH-09_R2.fastq.gz)

SRR31481670 (raw FASTQ files: RKAH-08_R1.fastq.gz, RKAH-08_R2.fastq.gz)

SRR31481671 (raw FASTQ files: RKAH-07_R1.fastq.gz, RKAH-07_R2.fastq.gz)

SRR31481672 (raw FASTQ files: RKAH-06_R1.fastq.gz, RKAH-06_R2.fastq.gz)

SRR31481673 (raw FASTQ files: RKAH-01_R1.fastq.gz, RKAH-01_R2.fastq.gz)

SRR31481664–SRR31481673: (raw paired-end RNA-seq FASTQ files from Gonipterus gut tissue; file identifiers RKAH-01 to RKAH-10).

Note: As of December 2025, the data for the BioProject (accession: PRJNA1189815) is under embargo pending a separate publication.

 Acknowledgements

We would like to thank Mr Preston L. Shaw (FABI, University of Pretoria) for assisting with the genome submission process.

 References Apriyanto A Tambunan VB : Draft genome sequence, annotation, and SSR mining data of Elaeidobius kamerunicus Faust., an essential oil palm pollinating weevil. Data Brief. 2021;34:106745. 10.1016/j.dib.2021.106745 Biswas T Vogel H Biedermann PHW : Few chemoreceptor genes in the ambrosia beetle Trypodendron lineatum may reflect its specialized ecology. BioMed. Central Genomics. 2024;25:764. 10.1186/s12864-024-10678-4 Bolger AM Lohse M Usadel B : Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. 24695404 10.1093/bioinformatics/btu170 Bouwer MC Slippers B Wingfield MJ : Chemical signatures affecting host choice in the Eucalyptus herbivore, Gonipterus sp. (Curculionidae: Coleoptera). Arthropod. Plant Interact. 2014;8:439–451. 10.1007/s11829-014-9327-y Brezáni V Karel Š : Secondary metabolites isolated from the genus Eucalyptus. Current Trends in Medicinal Chemistry. 2013;7:65–95. Brůna T Hoff KJ Lomsadze A : BRAKER2: Automatic eukaryotic genome annotation with GeneMark-EP+ and AUGUSTUS supported by a protein database. Nucleic Acids Research Genomics and Bioinformatics. 2021;3:lqaa108. 10.1093/nargab/lqaa108 Buchfink B Xie C Huson DH : Fast and sensitive protein alignment using DIAMOND. Nat. Methods. 2015;12:59–60. 25402007 10.1038/nmeth.3176 Chen G Shi T Shi L : Characterizing and annotating the genome using RNA-seq data. Sci. China Life Sci. 2017;60:116–125. 27294835 10.1007/s11427-015-0349-4 Chen Y Nie F Xie S-Q : Efficient assembly of nanopore reads via highly accurate and intact error correction. Nat. Commun. 2021;12:60. 10.1038/s41467-020-20236-7 Chen Z Ren L Li J : Chromosomal-level genome assembly of Hylurgus ligniperda: insights into host adaptation and environmental tolerance. BioMed. Central Genomics. 2024;25:792. 10.1186/s12864-024-10711-6 Chu F-C Wu P-S Pinzi S : Microinjection of western corn rootworm, Diabrotica virgifera virgifera, embryos for germline transformation, or CRISPR/Cas9 genome editing. J. Vis. Exp. 2018;e57497. 10.3791/57497 Cui S Wang L Ma L : P450-mediated detoxification of botanicals in insects. Phytoparasitica. 2016;44:585–599. 10.1007/s12600-016-0550-1 Danna C Malaspina P Cornara L : Eucalyptus essential oils in pest control: A review of chemical composition and applications against insects and mites. Crop Prot. 2024;176:106319. 10.1016/j.cropro.2023.106319 Edgar RC : Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny. Nat. Commun. 2022;13:6968. 10.1038/s41467-022-34630-w Emms DM Kelly S : OrthoFinder: Solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol. 2015;16:114–157. 26243257 10.1186/s13059-015-0721-2 PMC4531804 Evans JD McKenna D Scully E : Genome of the small hive beetle ( Aethina tumida, Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory. GigaScience. 2018;7:giy138. 30535280 10.1093/gigascience/giy138 PMC6302959 Feyereisen R : Insect CYP genes and P450 enzymes. Insect Molecular Biology and Biochemistry. Gilbert LI , editor. San Diego: Academic Press;2012; pp.236–316. Gremme G Brendel V Sparks ME : Engineering a software tool for gene structure prediction in higher organisms. Inf. Softw. Technol. 2005;47:965–978. 10.1016/j.infsof.2005.09.005 Gui S Taning CNT Wei D : First report on CRISPR/Cas9-targeted mutagenesis in the Colorado potato beetle, Leptinotarsa decemlineata. J. Insect Physiol. 2020;121:104013. 10.1016/j.jinsphys.2020.104013 Hoff KJ Lomsadze A Borodovsky M : Whole-genome annotation with BRAKER. Gene Prediction: Methods and Protocols. 2019;65–95. 10.1007/978-1-4939-9173-0_5 Hurley BP Garnas J Wingfield MJ : Increasing numbers and intercontinental spread of invasive insects on eucalypts. Biol. Invasions. 2016;18:921–933. 10.1007/s10530-016-1081-x Johny J Nihad M Alharbi HA : Silencing sensory neuron membrane protein RferSNMPu1 impairs pheromone detection in the invasive Asian palm weevil. Sci. Rep. 2024;14:16541. Kalyaanamoorthy S Minh BQ Wong TKF : ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods. 2017;14:587–589. 28481363 10.1038/nmeth.4285 Keeling CI Campbell EO Batista PD : Chromosome-level genome assembly reveals genomic architecture of northern range expansion in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Mol. Ecol. Resour. 2022;22:1149–1167. 34637588 10.1111/1755-0998.13528 Kim D Paggi JM Park C : Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 2019;37:907–915. 31375807 10.1038/s41587-019-0201-4 Li H : Minimap2: Pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094–3100. 29750242 10.1093/bioinformatics/bty191 Loch AD Matsuki M : Effects of defoliation by Eucalyptus weevil, Gonipterus scutellatus, and chrysomelid beetles on growth of Eucalyptus globulus in southwestern Australia. For. Ecol. Manag. 2010;260:1324–1332. 10.1016/j.foreco.2010.07.025 Lu K Song Y Zeng R : The role of cytochrome P450-mediated detoxification in insect adaptation to xenobiotics. Current Opinion in Insect Science. 2021;43:103–107. 33387688 10.1016/j.cois.2020.11.004 Manni M Berkeley MR Seppey M : BUSCO: Assessing genomic data quality and beyond. Current Protocols. 2021;1:e323. 10.1002/cpz1.323 Marçais G Kingsford C : A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics. 2011;27:764–770. 21217122 10.1093/bioinformatics/btr011 Markley HC Helms KJ Maar M : Generating and testing the efficacy of reagents for CRISPR/Cas9 homology directed repair-based manipulations in Tribolium. J. Insect Sci. 2024;24:15. 10.1093/jisesa/ieae082 Mei Y Jing D Tang S : InsectBase 2.0: A comprehensive gene resource for insects. Nucleic Acids Res. 2022;50:D1040–D1045. 34792158 10.1093/nar/gkab1090 Mendes FK Vanderpool D Fulton B : CAFE 5 models variation in evolutionary rates among gene families. Bioinformatics. 2020;36:5516–5518. 10.1093/bioinformatics/btaa1022 Mohd Rodzik FF Sudirman NA Teh C-K : Development of nuclear DNA markers for applications in genetic diversity study of oil palm-pollinating weevil populations. Insects. 2023;14:157. 10.3390/insects14020157 Navarro-Escalante L Hernandez-Hernandez EM Nuñez J : A coffee berry borer ( Hypothenemus hampei) genome assembly reveals a reduced chemosensory receptor gene repertoire and male-specific genome sequences. Sci. Rep. 2021;11:4900. 10.1038/s41598-021-84068-1 Ranallo-Benavidez TR Jaron KS Schatz MC : GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes. Nature. Communications. 2020;11:1432. 10.1038/s41467-020-14998-3 Roach MJ Schmidt SA Borneman AR : Purge Haplotigs: Allelic contig reassignment for third-gen diploid genome assemblies. BioMed. Central Bioinformatics. 2018;19:460. 10.1186/s12859-018-2485-7 Schröder ML Slippers B Wingfield MJ : Invasion history and management of Eucalyptus snout beetles in the Gonipterus scutellatus species complex. J. Pest. Sci. 2020;93:11–25. 10.1007/s10340-019-01156-y Segers A Carpentier J Francis F : Gene silencing of laccase 1 Induced by double-stranded RNA in Callosobruchus maculatus (Fabricius 1775) (Coleoptera: Chrysomelidae) suggests RNAi as a potential new biotechnological tool for bruchid’s control. Agriculture. 2023;13:412. 10.3390/agriculture13020412 Souza NM Wang T Suwansa-ard S : Ovi-protective mothers: exploring the proteomic profile of weevil ( Gonipterus) egg capsules.[dataset]. Heliyon. 2022;8:e10516. 10.1016/j.heliyon.2022.e10516 Stamatakis A : RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–1313. 24451623 10.1093/bioinformatics/btu033 Stanke M Diekhans M Baertsch R : Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics. 2008;24:637–644. 18218656 10.1093/bioinformatics/btn013 Stanke M Schöffmann O Morgenstern B : Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BioMed. Central Bioinformatics. 2006;7:1–11. 10.1186/1471-2105-7-62 Tooke FGC : The Eucalyptus snout beetle: a study of its ecology and control by biological means. Department of Agriculture South Africa;1955. Vaser R Sović I Nagarajan N : Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res. 2017;27:737–746. 28100585 10.1101/gr.214270.116 Vasimuddin M Misra S Li H : Efficient architecture-aware acceleration of BWA-MEM for multicore systems. 2019 Institute of Electrical and Electronics Engineers (IEEE) International Parallel and Distributed Processing Symposium (IPDPS). 2019; pp.314–324. Walker BJ Abeel T Shea T : Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement. Public Library of Science One. 2014;9:e112963. 10.1371/journal.pone.0112963 Wang Z Chen H Jin S : InsectBase 3.0: a comprehensive multi-omics resource for insects. Nucleic Acids Res. 2025;54:D1143–D1151. 10.1093/nar/gkaf1248 Wang Z Liu Y Wang H : Genome and transcriptome of Ips nitidus provide insights into high-altitude hypoxia adaptation and symbiosis. iScience. 2023;26:107793. 10.1016/j.isci.2023.107793 Wingfield MJ Slippers B Hurley BP : Eucalypt pests and diseases: Growing threats to plantation productivity. South. For. J. For. Sci. 2008;70:139–144. 10.2989/SOUTH.FOR.2008.70.2.9.537 Wu MM Chen X Xu QX : Melanin synthesis pathway interruption: CRISPR/Cas9-mediated knockout of dopa decarboxylase (DDC) in Harmonia axyridis (Coleoptera: Coccinellidae). J. Insect Sci. 2022;22:1–9. 10.1093/jisesa/ieac048 Zhang C Rabiee M Sayyari E : ASTRAL-III: Polynomial time species tree reconstruction from partially resolved gene trees. BioMed. Central Bioinformatics. 2018;19:153. 10.1186/s12859-018-2129-y Zhang R He J Dong Z : Genomic and experimental data provide new insights into luciferin biosynthesis and bioluminescence evolution in fireflies. Sci. Rep. 2020;10:15882. 10.1038/s41598-020-72900-z Zhang Y Ke Z Xu L : A faster killing effect of plastid-mediated RNA interference on a leaf beetle through induced dysbiosis of the gut bacteria. Plant Communications. 2024;5:100974. 10.1016/j.xplc.2024.100974 Zhang Z Pei P Zhang M : Chromosome-level genome assembly of Dastarcus helophoroides provides insights into CYP450 genes expression upon insecticide exposure. Pest Manag. Sci. 2023;79:1467–1482. 36502364 10.1002/ps.7319 Zhu F Moural TW Shah K : Integrated analysis of cytochrome P450 gene superfamily in the red flour beetle, Tribolium castaneum. BioMed. Central Genomics. 2013;14:174. 10.1186/1471-2164-14-174 10.5256/f1000research.190537.r465994 Reviewer response for version 1 Madrid Restrepo Maria Antonia 1 Referee https://orcid.org/0000-0003-2383-6161 KU Leuven, Leuven, Belgium

Competing interests: No competing interests were disclosed.

 20 4 2026 Copyright: © 2026 Madrid Restrepo MA 2026 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. recommendation approve-with-reservations

This genome note reports the assembly and annotation of the first published genome for  Gonipterus sp. n. 2, an important Eucalyptus pest. The authors generated a 1.54 Gb assembly using Oxford Nanopore and Illumina data, with 1,023 contigs, a contig N50 of 2.78 Mb, and high BUSCO completeness for both the assembly (98.3%) and predicted proteome (99%). They annotated 42,343 protein-coding genes and used comparative analyses to highlight expansion of cytochrome P450 gene families, which they suggest may be relevant to detoxification of Eucalyptus secondary metabolites and pest adaptation. Overall, the study provides a potentially valuable genomic resource for future work on pest biology and control.  The rationale for sequencing this species is clearly described. The protocols are generally appropriate and the work appears broadly technically sound. 

However, I would answer “partly” to whether the methods and data presentation are sufficient, because several important details need strengthening. The biggest issue is the annotation. The reported gene count of 42,343 protein-coding genes is unusually high for a beetle genome, and the manuscript does not yet provide enough evidence for readers to judge whether this reflects biology or overprediction. The BUSCO results are presented only as total completeness, without duplicated and fragmented fractions, and that makes it difficult to assess whether residual haplotypic duplication or annotation inflation may be contributing to the large gene number. The authors should report full BUSCO breakdowns for both assembly and annotation, including complete single-copy, complete duplicated, fragmented, and missing categories. A second issue is that the methods should be updated and clarified in several places. BUSCO should be rerun with a newer lineage set, ideally OrthoDB v12 and, if available, a more specific beetle lineage rather than only insecta_odb10, since that would provide a more biologically informative assessment. I would also answer “partly” to whether the datasets are presented in a fully usable and accessible way. The manuscript provides a BioProject and Figshare deposit, but the data availability section appears incomplete for a genome resource paper because it does not clearly list all raw genome sequencing accessions in the same direct way it lists the gut RNA-seq runs. Points that must be addressed to make the article scientifically sound are the annotation support and reporting. Specifically, the authors should provide a full BUSCO breakdown, better justify the unusually high gene count, clarify contamination handling, and improve data deposition details for the core genome resources. The BUSCO lineage update and added citations for comparative proteomes are also important and should be addressed in revision. Until these points are clarified, the assembly itself looks promising, but the annotation-based conclusions are not yet fully supported.

Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Partly

Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Partly

Are the rationale for sequencing the genome and the species significance clearly described?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Eco-evolutionary genomics, population genetics, bioinformatics, genome assembly

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.

 10.5256/f1000research.190537.r464217 Reviewer response for version 1 Cunningham Christopher 1 Referee https://orcid.org/0000-0003-3965-2076 University of Georgia, Athens, USA

Competing interests: No competing interests were disclosed.

 1 4 2026 Copyright: © 2026 Cunningham C 2026 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. recommendation reject

The project reports the genome assembly from a lesser studied beetle group. The methods are sound and relatively well documented. The article needs has enough details to be reasonably reproducible, but should likely have a little more. The data supporting the publication are publicly available.

Abstract. That is a huge beetle genome. And that is also a very high number of of protein coding genes.  

Keywords. Most of these are found in the title or abstract.

Background. Background. The stated motivation for this study in the introduction is fine – this beetle is a pest. I had no problem understanding the information or its justification.

Results. The BUSCO needs to be reported more comprehensively – what level of duplication was seen and fragmentation. That will also help the reader assess the high protein-coding gene number reported. Tribolium has 12.1K protein coding. 1.4K taxon specific orthogroups is very high as well.

            The annotation not something a reader can accept without more justification. I understand that its from a standardized pipeline, but sometimes otherwise reasonable defaults produce poor results for lesser-studied species/groups. A recent study of the genome of Curculio annulus reports 26.4K protein with a duplicated BUSCO of 22% after purging for haplotigs and decontamination (Davis et al., 2026, G3). So this might be a whole genome duplication?

            To very clear, I’m not saying its wrong. The results are just not well supported enough currently for the reader to accept them as stated.

Methods. BUSCO needs to be updated to OrthoDBv12. Its been published for well over a year now. Also, Insecta is too high a phylogenetic level. Coeloptera is well supported within OrthoDB and will give a much more biological meaningful estimate of expected gene content. Citations for the proteomes used need to be given. They are free to use with attribution; it’s not too much to ask. Was a BUSCO analysis run on the annotation? Although, not needed at any truly comprehensive level, was the annotation checked manually in any way, just to see if anything weird was going on? This is especially important because the authors are stating a very, very high protein coding number for a beetle.

            What was done to decontaminate the assembly? I see that it was purged for haplotigs.

            I’m assuming that very few non-default parameters were used, but its better to be explicit and state that if true.

Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Partly

Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Partly

Are the rationale for sequencing the genome and the species significance clearly described?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Insects, Behavior, Reproduction, Genetics, Genomics, Epigenetics

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.