Periodical crossing of the laboratory population with the natural population would improve fitness in S. sunia ( Lepidoptera: Noctuidae)

Background The research focused on evaluating the biological and reproductive parameters of the species Spodoptera sunia with the introduction of field genetic material, in the Noctuid Insect Breeding Laboratory. Methods The experiment consisted of three treatments and three repetitions involving 30 individuals each. Field-collected S. sunia population was reared in the laboratory under semi-controlled conditions of temperature and humidity for three generations. The progeny of the third generation was crossed with the laboratory population and the resulting progeny was observed for growth and reproductive fitness. Results The results revealed that the hybrid progeny (T3) has a sex ratio (F: M) of 0.82, as against 1.22 and 0.76 observed in the field (T1) and lab populations (T2) respectively. The T1 females oviposited 196 egg masses as against 160 and 59 egg masses by T3 and T2 females respectively. The larval growth was more in hybrid progeny with shorter larval duration. However, no variation was observed in the incubation and pupal periods. Conclusions Since the most optimal results were obtained in T2 and T3, it is recommended to introduce genetic material every six months to maintain a good laboratory population of the insect host under study and better breeding of its natural enemies.


Introduction
In agricultural production, producers have been grappling with pest control, with particular emphasis on pests exhibiting high adaptability.Notably, phytophagous insects have proven to be challenging adversaries in this regard (Brunetti et al., 2022;Saldamando and Marquez, 2012;Extremera et al., 2004;Pérez et al., 2003;Armstrong, 1994).It is worth mentioning that the genus Spodoptera sp.(Guenée) (Lepidoptera: Noctuidae) is a prominent member of the Noctuidae family, with geographical presence spanning across North America, Latin America, France, Italy, the Black Sea region, the Balkans, England, North and Southern Africa, the Middle East, the Iberian Peninsula, and Germany (Kergoat et al., 2021;Zelaya et al., 2022).
Due to its omnivorous nature, S. sunia is widespread and affects various crops.Ministry of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA, 2002) mentions in the case of Mexico that S. sunia is considered a pest of economic importance that periodically affects essential crops.To control pest, many laboratories produce and reproduce these organisms in order to reduce and control the population of pests in crops, some genera are Trichogramma, Spalangia Telenomus, Cochliomyia, Ceratitis, Baculovirus and Nomuraea rileyi (predators and parasitoids, viruses and entomopathogenic fungi) (Jiménez, 2022;Badii & Abreu, 2006).
In the National Autonomous University of Nicaragua, Leon (UNAN-Leon), the Insect Breeding Laboratory began the reproduction of insects in the 80s, establishing quality controls in breeding, in such a way that the life tables allow determining the biological parameters and reproduction of the insect's species.
In addition, due of their significance as hosts for the nuclear polyhedrosis virus (NPV), which is considered a biological control agent for pest insects in economically important crops, they play a crucial role in preventing environmental contamination, safeguarding, human health, and protecting other vertebrates (Haase et al., 2015;Romero Gutierrez and Cruz Reyes, 2011;Carrillo et al., 2003;Muentes, 2000).
The study focused on characterizing the biological parameters of the species S. sunia with the introduction of field genetic material (cross-species) at the Noctuid Insect Breeding Laboratory, Biological Control Agent Production and Reproduction Research Center (CIRCB), UNAN-León.
Ultimately, the subject of study becomes important given the need to study these parameters and the performance of the species S. sunia with the introduction of new natural genetic material (cross-species) to improve breeding and maintain optimal parameters (Raj et al., 2022;Rizo, 1994;Rizo et al., 1994;Passoa, 1991).

REVISED Amendments from Version 2
In this revised version of our article, we have implemented several significant improvements based on the valuable feedback from the reviewers.The major differences between this version and the previously published one are as follows:
The effect of crossing specimens of the species S. sunia was characterized in order to strengthen the genetic material of the breeding of S. sunia insects used as a host in the production of the NPV (Vásquez et al., 2002;Salinas-Sánchez et al., 2020).

Laboratory protocol
The protocol used began with the collection of Spodoptera sunia in the field.The Sébaco area located in the coordinates (12°52 0 42.4 00 N 86°05 0 29.3 00 W) was selected.The full protocol can be found on protocols.io(Real-Baca and Zuniga-Gonzalez, 2022a).The selection criteria was that it is an area that produces vegetables and where the presence of noctuid insects is more representative, just as the producers in the area demand NPV products for the control of pest insects.After the collection, they were stored in the Noctuid Insect Breeding Laboratory (CIRCB), a quarantine period of up to three generations was spent.The soybean-based diet was prepared.This diet underwent a sterilization process and was supplemented with vitamins and antibiotics.
In the assembly of the bioassays, two stages were considered (adults and larval length).The treatments were: T 1 : S. sunia species brought from the field, T 2 : S. sunia species from the Noctuid Insect Breeding Laboratory (CIRCB), T 3 : S. sunia species Crossing (field-laboratory).250 field larvae were collected, which were subjected to a quarantine process for three generations, this to eliminate any contaminants from the field.Of these 250 larvae, a mortality of 75 larvae (30%) was obtained.This mortality was due to bacterial infection.Of the remaining 175, 10 had a poor pupal formation and 25 failed to exit the capsule, therefore, they did not reach the adult stage, leaving 140 adults at the end.After passing the three generations, approximately 200 pupae were obtained, of which 60 pupae were taken for the Field (T 1 ) and Laboratory (T 2 ) treatments, which were sexed and weighed to start the assembly of the test.Treatments were as follows: • Treatment 1: S. sunia species brought from the field • Treatment 2: Species of S. sunia from the Breeding Laboratory • Treatment 3: S. sunia species Crossing (field-laboratory) In each repetition, 40 larvae were selected for a total of 120 larvae per treatment, resulting in 360 larvae in total.A six-dayold larva was deposited per four-ounce cup with a 2-cm square piece of diet, with the help of a tweezers the larvae were placed on the millimeter sheet to measure the length of each larva, and they were weighed daily on a Denver Instrument Co Serial No 3570 Analytical Balance.Also, the number of males and females per egg laying cage (replication) was considered.The following variables were evaluated: • Larvae length: Each larva was placed on a millimeter sheet and the length measured in mm was observed.
• Hatching percentage: The number of eggs that hatched was counted visually.
• Weight of larvae: Larvae were weighed on a Denver Instrument Co Denver Analytical Balance, Cool, Serial No 3570.
• Weight of pupae: The pupae were weighed on a Denver Instrument Co Denver Analytical Balance, Cool, Serial No 3570.
• Sex of pupae: The abdominal part of the pupae was observed with the help of a Westover Scientific stereoscope (Rizo et al., 1994).
• Life cycle: The days it took in each of the larval stages were counted.
Student's t-test used to compare the two treatments and determine if there are significant differences between the means of these groups.And it was also, used to evaluate whether the samples from two groups are statistically different in terms of their population mean (Raju, 2005;Malais and Ravensberg, 1992).
K-S test was used to verify the normality of the distribution between treatments (bioassays), equation 1 (Massey, 1951).
The W test was used to contrast the normality of a data set of the combined treatments, equation 2 (Shapiro, 1965).
Mann-Whitney U test was used to check the heterogeneity of two ordinal treatments, equation 3. The starting point is: Treatment data is autonomous.
The data are ordinal or continuous variables.
Under H 0 , the initial distribution is the same for both treatments: P(T 1 > T 2 ) = P(T 2 > T 1 ) In the first H 1 , the value of one type of treatment tends to exceed the other: P(T 1 > T 2 ) + 0.5 P(T 1 = T 2 ) > 0.5.
Where T 1 and T 2 are the sizes of each treatment; R 1 and R 2 are the rank sum of bioassay 1 and 2 observations, respectively (the sum of the relative position of each individual in the treatment).The U statistic is defined as the minimum of U 1 and U 2 (Turcios, 2015).
Lilliefors test is a normality test based on the K-S test.It was used to test H 0 that the data come from a normally distributed population (Lilliefors, 1967(Lilliefors, , 1969)).
Table 1 shows the descriptive statistics of the data used at the time of establishing the bioassays.Table 1 provides a concise overview of the final weight (in milligrams) for each of the three treatments in the bioassay.The data is based on a sample size of 60 for each treatment.On average, the final weight for T 1 was 0.21 mg with a standard deviation of 0.04 mg and a mean standard error of 0.01.Similarly, T 2 exhibited an average final weight of 0.23 mg, with a standard deviation of 0.05 mg and a mean standard error of 0.01.T 3 had an average final weight of 0.20 mg, a standard deviation of 0.04 mg, and a mean standard error of 0.01.These statistics offer valuable insights into the variability and central tendency of the final weight data across the different treatments in the study.

Results and discussion
The research set out to study the biological and reproductive characteristics of S. sunia (Malais and Ravensberg, 1992), known as (Guen) = (Xylomige ssunia) called cutworm, tiger worm, leathery worm.The characterized variables were length and weight of larvae, hatching percentage, weight and sex of pupae (Parra et al., 2022;Peterson et al., 2022;Barcellos, 2022;Machado et al., 2020).
Length and weight of larvae Figure 1 (Real-Baca and Zuniga-Gonzalez, 2022b) presents the length (mm) of the larvae in the different treatments.
It was observed that the treatments begin to have changes in their length after day 10.The three treatments reached their greatest length at 13 days, it was noted that T 3 reached the greatest length (30 mm).
Table 2 presents the results of the normality tests for length (mm) and weight (mg) of the larvae during the larval stage across different treatment combinations.These tests are essential to assess whether the data conforms to a normal distribution, which is a crucial assumption for various statistical analyses.The Kolmogorov-Smirnov and Shapiro-Wilk tests were applied, and the obtained p-values for all combinations were extremely low (less than 0.05), leading to the rejection of the null hypothesis and confirming that the data indeed follow a normal distribution.These findings provide a solid foundation for subsequent statistical analyses.
In the Mann-Whitney U test, it was obtained that the significance of the treatments T 2 -T 3 , and T 3 -T 1 was 0.000 less than 0.05, so the H 0 is rejected, that is, there is a significant difference in terms of the length of the larvae, in the case of treatments T 2 -T 3 the significance was 0.057 greater than 0.05, in terms of larval weight the significance was 0.098 greater than 0.05, so we accept the H 0 of equality between the treatments, that is that there is no significant difference in terms of the length and weight of the larvae (Table 3).
After day 13 in all treatments, a decrease in length was observed because in that week from day 16 to day 22 the environmental conditions were not appropriate.Treatments T 2 -T 3 reached their greatest length on day 23 with 31 mm and later descended their length to enter the pre-pupal stage.The best larval development was in the T 3 treatment with 30 mm because this treatment contains genes from Laboratory larvae, being more adapted to the artificial Soya diet.In the Laboratory treatment, larvae with 27 mm are obtained, this is because it is adapted to the conditions and food, continuing the T 3 larvae with 27 mm because it is not adapted to these controlled conditions, but to natural conditions.
Romero Gutierrez and Cruz Reyes (2011) obtained an average length in the first instar of 0.5 cm, reaching its maximum length at approximately 14 days with a length of 2.5 cm (25 mm), this confirms the results obtained with a length of 27 mm for T 1 and T 2 treatment, the T 3 treatment reached a length of 30 mm, this indicates a genetic improvement when introducing field material and mixing it with the laboratory material.For the T 3 treatment, its larval growth ends at 18 days, therefore, its larval development was faster.
Figure 2 presents the weight of the larvae (mg).Treatment T 3 on day 18 reached its highest weight (0.42 mg), this treatment being the one that reached the highest weight, and then began to lose its weight to enter the pre-pupal stage.The T 1 treatment reaches a weight of 0.33 mg at 19 days and 0.30 mg at 20 days in the T2 treatment.
Romero Gutierrez and Cruz Reyes (2011) obtained weights of 0.25 gr (0.250 mg) and in our study it was 0.33 mg in the T 1 and 0.30 mg for T 2 , in the case of T 3 it was 0.43 mg.Thus, confirming that there is a difference in the crossover and field treatment since they obtained a greater weight; as soon as the laboratory treatment was similar to the weight of its study, this is due to the genetic deterioration found in the offspring.

Hatching percentage
Table 4 and Table 5 show the results of the number of eggs in the different treatments.In the T 1 treatment they oviposited nine days, obtaining a total of 23,354 eggs that corresponds approximately between 10 to 450 eggs for each mass with a total of 196 egg masses.The T 2 treatment oviposited seven days for a total of 4,456 eggs ranging from four to 400 eggs each mass with a total of 59 egg masses, and the T 3 treatment obtained the highest number of eggs, 18,025 eggs with nine days of oviposition with a number ranging from 15 to 450 eggs per egg mass for a total of 160 egg masses.In the T 3 and T 1 treatment, oviposition began after three days, but not in the T 2 treatment, this was due to the fact that the fertility of the adult drops due to the fact that they are crosses between the same family, as inbreeding increases, insects decrease their performance (Martinez, 1994;Bárcenas, 1997).The most used variables are fecundity, egg viability, development time, body size, survival and efficiency (Huettel, 1976;Moore et al., 1985;Smith et al., 1996).This confirms that it is important to introduce field material to the laboratory to obtain a better fertility and thus obtain a better reproduction in the breeding of insects.
The normality of the data for the final weight in mg was done with the K-S test, the significance was 0.200 greater than 0.05, so H 0 was accepted, likewise the data follow a normal distribution (Table 6), for the different combinations of treatments, the parametric Student's T-test was calculated for independent samples of the treatments (T 2 -T 3 , T 3 -T 1 , T 2 -T 1 ).It was observed that the homogeneity of variance is fulfilled because the significance is greater than 0.05.In the Student's T-test, a significance of 0.03 less than 0.05 was obtained, it was concluded that there is a significant difference between the final weight in mg of S. sunia of T 2 and T 1 (Table 7).In the Student's T-test, a significance of 0.001 less than 0.05 was obtained, it was concluded that there is a significant difference between the final weight in mg of S. sunia in the Laboratory treatment and the Crossing.In the Student's T-test, a significance of 0.190 greater than 0.05 was obtained, it was concluded that there is no significant difference between the final weight in mg of S. sunia of T 3 and T 1 .

Weight and sex of pupae
Table 8 shows the number of pupae of the populations of S. sunia from treatment T 2 , which obtained a total of 60 pupae, of which 34 males and 26 females pupae, for a sex ratio of 0.7:1.Treatment T 1 obtained a total of 60 pupae, 28 females and 32 males for a sex ratio of 0.8:1 and in treatment T 3 a total of 60 pupae, 33 females and 27 males, and a sex ratio of 1.2 were obtained: 1.This indicates that the proportion for T 2 and T 3 was not optimal and that of T 3 was the most optimal, this is favorable considering that more female pupae than male pupae are needed in the laboratory for greater reproduction and better conditions.
With respect to weight, we observed that the female S. sunia pupae in the three treatments were larger than the males.The T 2 treatment had a higher weight (0.2401), but not the T 3 and T 1 treatments (0.2197 and 0.2112), respectively.

Lifecycle
Figure 3 presents the average number of days in each stage of development of the species S. sunia in the three treatments.
In the egg and pupal stages, the three treatments had no difference in days.In the larval stage for treatment T 2 and T 1 there was no difference (24 days), but not for treatment T 3 , which presented a small difference (18 days).In the adult stage, the T 2 treatment obtained 10 days and in the T 1 and T 3 treatments they obtained a difference of two days (12 days).Romero Gutierrez and Cruz Reyes (2011) and Delgado and Hernández (2008) reported that the adult stage lasted 15.56 days.In this research for the T 2 , the adult stage lasted 10 days, while in the T 1 and T 3 treatment it lasted 12 days, less than what they report in their work.
Romero Gutierrez and Cruz Reyes (2011) reported that the duration of the biological cycle, from egg to pupae, was 36.16 days, which was similar to the data obtained in the UNAN-León laboratory under controlled conditions and fed with an artificial soy-based diet.As can be seen, there is an average difference approximately for the T 3 treatment of five more days, for the T 1 treatment of 11 days and for the T 2 treatment it was nine more days.According to Romero Gutierrez and Cruz Reyes (2011), this is due to the type of feeding and conditions in which these insects are raised, which is why the days of their biological cycle increase.

Conclusions
The treatment combinations were statistically validated.Based on the characterization of the periodical crossing of populations S. sunia, it is concluded that the best results are presented in treatments T 1 and T 3 .The considerations of the study were that the biological cycle of the species S. sunia brought from the Field (T 3 ) lasted 47 days, that of the Laboratory (T 2 ) 45 days and that of the Crossing 41 days.The highest average larval weight of S. sunia obtained from T3 was 0.4288 mg.The best sex ratio of the species S. sunia was T 3 (1.22:1).The longest treatment was T 3 (30 mm).T 1 obtained the highest number of eggs (1,744-5,670).
Based on these results, it is recommended to introduce genetic material every six months to maintain a good production of larvae of the species.It is also recommended to maintain stable climatic conditions (temperature and humidity) in the Insect Breeding Laboratory of the populations S. sunia (Montezano et al., 2014;Salinas-Sánchez et al., 2020).When preparing the diet, all the nutritional requirements must be met so that the reproduction of the S. sunia develops well.Finally, after moving to individual vessels, the diet is changed at least twice during the larval stage to prevent drying out and for better larval development.
This project contains the following underlying data: • Data.csv What ever the objective may be 1) Insects come up well only under natural diet the artificial diet is only for our ease of mass producing the insects for a brief period of time.
2) Though artificial diet is a balanced food it only gives required energy to insect for promoting to next stages but it does not activate the real potential of the insect which the insect derives when feeding on live food.As the plants produce certain defense molecules which the insect tries to fight back.
3) The very problem with inbreeding is lack of immunity in the cultures and most of them die due to exposure of the cultures to their own excreta which they release in the same vicinity.
4) The excreta released by the insects after eating the artificial diet is totally different from that released from natural diet.5)These excreta attract large quantum of microbes other than entomopathogens for which the larvae are very susceptible.6) Hence periodical cross will always help in restoring the general vigour and enhances immunity in the insects.
The data on these points will really help in drawing drawing perfect conclusions for the current research.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Host plant resistance, Insect nutrition, Insect Immunity and Insecticide resistance 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.

Introduction
When you first mention the species S. susinia, please provide the complete name of the species: Spodoptera sunia (Guenée) (Lepidoptera: Noctuidae).
○ "To control this pest, many laboratories produce and reproduce these organisms in order to reduce and control the population of pests in crops, some Genera are  and Wilk, 1965;Sánchez Turcios, 2015;Torres et al., 2012)."Please correct the sentence.You say and mention the test and do not finish the sentence correctly.

○
Student t-test is not used to verify normality of data sets.

○
There is no need to provide the formula of neither the statistical tests nor the hypothesis that were tested in material and methods.
○ Table 1 should be presented in results section, and also you should check the information that provides.

Results and discussion
Length and weight of larvae Figure 1: please express correctly the legend in the y axis.There is no need to describe the hypotheses that were tested; you should summarize the information correctly.Reviewer Expertise: Biological control, entomology, micology Figures 2 and 3 were ordered as there was a location problem (they were inverted).In the same way, we have generally reviewed the article to correct the grammar that other reviewers also indicate.

Introduction
When you first mention the species S. susinia, please provide the complete name of the species: Spodoptera sunia (Guenée) (Lepidoptera: Noctuidae)."To control this pest, many laboratories produce and reproduce these organisms in order to reduce and control the population of pests in crops, some Genera are Trichogramma, Spalangia Telenomus, Cochliomyia, Ceratitis, Baculovirus and Nomuraea rileyi (predators and parasitoids, viruses and entomopathogenic fungi) (Jiménez, 2022;Badii & Abreu, 2006)".Please delete the word 'this', you're referring to biocontrol agents not the plague.

Response to reviewer
Dear reviewer has been corrected it as you indicate.
Methods "The research was carried out at the Noctuid Insect Breeding Laboratory (CIRCB), UNAN-León Agricultural Campus, coordinates (12.11743, -86.23600Turcios, 2015;Torres et al., 2012)."Please correct the sentence.You say and mention the test and do not finish the sentence correctly.Student t-test is not used to verify normality of data sets.

Response to reviewer
Dear reviewer has been corrected it as you indicate.The t-test was clarified and corrected.
There is no need to provide the formula of neither the statistical tests nor the hypothesis that were tested in material and methods.

Response to reviewer
Dear reviewer has been corrected it as you indicate.We would like to keeping the formula for academic purposes.
Table 1 should be presented in results section, and also you should check the information that provides.

Response to reviewer
Dear reviewer we thanks for this observation.We has been added it the analysis of the table, but we consider keep in this section because is part of the data.

Results and discussion
Length and weight of larvae Figure 1: please express correctly the legend in the y axis.
Figure 2 does not provide the information on the weight of the different stages.Please place the correct figure.

Response to reviewer
Dear reviewer your observation is correct.It was a problem of confusion.The Figure 3 is in the place of Figure 2 and vice versa.
There is no need to describe the hypotheses that were tested; you should summarize the information correctly.The information provided in table 2 is also redundant since it provides repeated information.Which values does table 3 provide?You should clarify and place the correct values.

Response to reviewer
Dear reviewer the comment of the table 2 was eliminated and change for other that no describe the hypothesis, regarding to table 3, we have left the comments on the comparisons of the treatments aside since you are asking about the applied values.Figure 2 does not present the average weight of larvae Hatching percentage.
Please check the values provided in table 4 and those express in the text.There are several mistakes or at least inconsistencies.If the number of eggs is an average you should also provide standard deviation values.Furthermore, strange values are observed, within the same treatment for example in T1: 1,744 and 665.

Response to reviewer
Dear reviewer thanks for your observations.The data was corrected is 1744 and not 1,744.
Is was a problem of consider a decimal by comma.Regarding to the Figure 2 has been corrected it Weight and sex of pupae Figure 3, colors in the figure does not match those in the legend.Also, in the figure (x axis) you only refer to the larval stage but in the text you mention egg, larval, pupal and adult stage.Furthermore, which are the statistical values for you to say that there are or not significative differences?
Response to reviewer Dear reviewer has been corrected it.
"Methods: The experiment consisted of three treatments and three repetitions involving 30 individuals each.Field-collected S. sunia population was reared in the laboratory under semi-controlled conditions of temperature and humidity for three generations.The progeny of the third generation was crossed with the laboratory population and the resulting progeny was observed for growth and reproductive fitness.

Results:
The results revealed that the hybrid progeny (T3) has a sex ratio (F: M) of 0.82, as against 1.22 and 0.76 observed in the field (T1) and lab populations (T2) respectively.The T1 females oviposited 196 egg masses as against 160 and 59 egg masses by T3 and T2 females respectively.The larval growth was more in hybrid progeny with shorter larval duration.However, no variation was observed in the incubation and pupal periods.

Conclusion:
Since the most optimal results were obtained in T2 and T3, it is recommended to introduce genetic material every six months to maintain a good laboratory population of the insect host under study and better breeding of its natural enemies." Pardon me, I can't reframe the MS beyond this point, it is a tedious job.Kindly get the rest edited for English language and grammar, preferably by a researcher.
Also, the authors did not incorporate all the previous suggestions eg. S. siuna is used again in place of S. sunia and limiting two digits after the whole number.eg.0.4288 mg.It should be 0.43 mg.Also am seeing the same wrongly placed graphs.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Insect pest management 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.
Introduction: this section is written well but needs minor corrections as: Rearrange the references in all text with the same format either alphabetical or year.

○
Transfer this sentence to the end of introduction section (it is an appropriate place): "Ultimately, the subject of study becomes important given the need to study these parameters and the performance of the species S. sunia with the introduction of new natural genetic material (cross-species) to improve breeding and maintain optimal parameters (Raj et al., 2022;Rizo, 1994;Rizo et al., 1994;Passoa, 1991)

○
The weight in mg repeated more times in all text of the article and it is mentioned previously in the methodology sections so write only the weight without in mg.
○ weight and sex of pupae section: '34 are male pupae and 26 female pupae' replaced to '34 males and 26 females'.
○ "Treatment T 3 obtained a total of 60 pupae, 28 females and 32 males for a sex ratio of 0.8:1" -This result is related to treatment T1 not T3, correct it.

○
Life cycle: Not Figure 3 but was Figure 2 about average days of each development.

Conclusions:
Replace 'The highest average larval weight obtained from the species S. sunia obtained was T 3 with 0.4288 mg', to 'The highest average larval weight of S. sunia obtained from T3 was 0.4288 mg.' ○ "of the species S. sunia" (delete it).
○ 'the reproduction of the insects of the populations S. siuna' replaced to, 'the reproduction of S. siuna'.

○
Finally, I read and revise this manuscript carefully based on our knowledge and experience and hence i confirm that it is of an acceptable partially, however it need some revision and correction before indexing.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes Table 4 legend: 'in different treatments' not 'by treatment'.The weight in mg repeated more times in all text of the article and it is mentioned previously in the methodology sections so write only the weight without in mg.

Response to reviewer
Ok.However, I would like to keep it because it is an observation from other reviewers.
weight and sex of pupae section: '34 are male pupae and 26 female pupae' replaced to '34 males and 26 females'."Treatment T3 obtained a total of 60 pupae, 28 females and 32 males for a sex ratio of 0.8:1" -This result is related to treatment T1 not T3, correct it.cycle: Not Figure 3 but was Figure 2 about average days of each development.

Response to reviewer
Dear reviewer thanks for this observation.Has been corrected it.The Figure 3 was change for the Figure 2.

Conclusions:
Replace 'The highest average larval weight obtained from the species S. sunia obtained was T3 with 0.43 mg', to 'The highest average larval weight of S. sunia obtained from T3 was 0.4288 mg.' "of the species S. sunia" (delete it).'the reproduction of the insects of the populations S. siuna' replaced to, 'the reproduction of S. suna'.Finally, I read and revise this manuscript carefully based on our knowledge and experience and hence i confirm that it is of an acceptable partially, however it need some revision and correction before indexing.

Response to reviewer
Dear reviewer, I thank you for your great contribution and improvement to the quality of the article.We have made the indicated corrections.

Bst Rgs Authors
Competing Interests: The authors disclose that no have any competing interests.

SB Suby
ICAR-Indian Institute of Maize Research, Ludhiana, India The study proved/concluded periodical crossing laboratory population with field population would increase fitness in Spodoptera sunia.
General comment: After first use of Spodoptera sunia, use abbreviated form S. sunia.
Abstract: Three treatments with 30 individuals and three repetitions.Does it mean 'three treatments with and three repetitions involving 30 individuals each'?
Title: The word cross-species literally mean inter-species, thus a misnomer in this study.It shall be replaced with a suitable title like 'periodical crossing of laboratory population with natural population would improve fitness in Spodoptera sunia'.Crossing populations is appropriate in place of 'crossing specimens'.
Delete the phrase 'It is important to clarify that'

Methods:
The term 'pre-experimental' will confuse the reader.It is proper experiment only.
The number of males and females per egg laying cage (replication) should be mentioned in methods.
Results and discussion: Spodoptera siuna (spell check the entire document).leatheryworm donut (donut?) Figure 2 represents duration of stages (Y axis-days in stadium), but it is captioned as weight.Figure 3 is average weight, but titled as number of days.The legend colour given in the graph is different from actual line colour in graph.
Three digits are enough for fractions eg.0.429 mg in place of 0.4288 mg.
The egg mass and no. of eggs per female should also be mentioned.This should be discussed in comparison with fecundity of S.sunia reported earlier.

Conclusion:
Closing of populations, not species.The conclusion must focus on periodical crossing and the other statements given on nutrition and environment must be supported by quoting previous studies.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?
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Figure 1 .
Figure 1.Average length (mm) of S. sunia larvae in the different treatments.

Figure 2 .
Figure 2. Average weight (mg) of S. sunia larvae of the treatments.

Figure 3 .
Figure 3. Number of days in each stage of development of S. sunia in the treatments.

○Figure 2
Figure 2 does not provide the information on the weight of the different stages.Please place the correct figure.

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Weight and sex of pupaeFigure3, colors in the figure does not match those in the legend.Also, in the figure (x axis) you only refer to the larval stage but in the text you mention egg, larval, pupal and adult stage.Furthermore, which are the statistical values for you to say that there are or not significative differences?○Is the work clearly and accurately presented and does it cite the current literature?PartlyIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?NoIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.

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Table 1 .
Descriptive statistics for the final weight (mg) of the treatments.

Table 2 .
Data normality test with the Kolmogorov-Smirnov statistic for length (mm) and weight (mg) during the larval stage of the treatments.
a Lilliefors Significance correction.

Table 3 .
Mann-Whitney U test for length (mm) and weight (mg) during the larval stage for treatments.
Source: Author's Self calculation.a Grouping variable: Treatments.

Table 4 .
Egg laying of S. sunia in different treatments.

Table 5 .
Oviposition range by mass size for each treatment (%).
Source: Author's Self calculation.*This is a lower limit of the true significance.a Lilliefors significance correction.

Table 7 .
Independent samples test for weight (mg).

Table 8 .
Average weight in (mg) and sex of final pupae in the Spodoptera sunia treatments.
The information provided in table 2 is also redundant since it provides repeated information.Which values does table 3 provide?You should clarify and place the correct values.Figure 2 does not present the average weight of larvae Hatching percentage.Please check the values provided in table 4 and those express in the text.There are several mistakes or at least inconsistencies.If the number of eggs is an average you should also provide standard deviation values.Furthermore, strange values are observed, within the same treatment for example in T1: 1,744 and 665.
○ ○ ○ this section is clear and written well.Delete this sentence: The type of research was experimental and the word species repeated more times.The legend of Figures2 and 3is not correct and un-appropriate.Fig.2notaverageweight of larvae?It is developmental duration of each stage of S. sunia.Fig.3isaverage weight not number of days?Please correct it carefully and the legend color in the graph is not the same of actual line color.Lose weight to enter the pre-pupa stage' replaced to, 'lose its weight to enter the pre-pupal stage'.
".○ Replace a biological controller to: a biological control agent.The study to this study.○Methods: ○ ○ ○ '○Table 4 legend: 'in different treatments' not 'by treatment'.