Molecular Identification of Palmistichus elaeisis, Tetrastichus howardi, Trichospilus diatraeae and Trichogramma pretiosum (Hymenoptera: Chalcidoidea)—Important Biocontrol Agents

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper titled as “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichogramma pretiosum and Trichospilus diatraeae (Hymenoptera: Chalcidoidea), important biocontrol agents” sequenced those genome of four species  to discuss SNP difference. The introduction fails to effectively present the core scientific problem, the results are disconnected from the introduction, and the discussion is diffuse, not tightly focused on the specific findings of the study. The research aims to develop a new molecular identification method, but this core value is diluted throughout the paper. The discussion section is excessively long and digressive, while the results section, the core of the research, is described too briefly with insufficient presentation of key data. 
The lack of a detailed bioinformatics pipeline (e.g., software for alignment, variant calling, filtering parameters) renders the experiment irreproducible. It is not specified whether the "50 individuals" were pooled for sequencing or used as biological replicates, which directly affects the assessment of data quality and conclusion reliability.  The rationale for choosing A. certus ("genomic stability") is subjective and vague. No objective assembly quality metrics are provided, nor are the potential issues of using a distantly related species discussed. The use of a "call rate > 0.5" standard for PCA is too lenient and not conventional in genomics.  The genome size of four species is not described in the whole paper. It is very important to analyze SNPs. The lack of estimated genome size and heterozygosity means metrics like sequencing depth lose their frame of reference, and an opportunity to contribute baseline data is missed. Only the number of SNPs is reported, with no information on their genomic position, flanking sequences, sequencing depth, quality scores, or genomic annotation. The interpretation of results like PCA remains descriptive (e.g., this point is close to that one) without statistical support (e.g., Fst calculation) or deep biological insight.  
The discussion does not begin by summarizing the most important findings of the study in one or two sentences. There is no honest discussion of the study's limitations (e.g., single source of samples, distantly related reference genome, lack of validation in complex field samples), undermining its rigor. It fails to objectively compare this method with traditional techniques like DNA barcoding in terms of cost, efficiency, and applicable scenarios, resulting in unclear positioning. 

Comments on the Quality of English Language

The English language usage requires refinement to meet the standards of formal scientific communication.

Author Response

We would like to thank the Reviewer for their careful evaluation of our manuscript and for their constructive comments and valuable suggestions. Their feedback has helped us to improve the clarity, methodological description, and overall quality of the manuscript.

The text was reviewed carefully and accuses the changes as follows
Modified as suggested by reviewer

Reviewer 1 - Major points
“The introduction fails to effectively present the core scientific problem, the results are disconnected from the introduction, and the discussion is diffuse, not tightly focused on the specific findings of the study.”
01 (Line 32-95): We rewrote the introduction to more clearly elucidate the main scientific problem addressed in this study, namely the need for robust molecular approaches for the accurate identification of parasitoid wasps used in biological control programs. In this revised version, we explicitly highlight the limitations of morphological identification in Eulophidae and Trichogrammatidae due to their diminutive size, high morphological similarity, and the limited resolution of traditional molecular markers (e.g., COI and ITS2) for distinguishing closely related taxa. This framework leads directly to the rationale of the study, which is based on the use of whole-genome sequencing to identify species-specific genomic variants.
02 (Lines 338-392): The discussion has been restructured with a precise focus on our findings, beginning with a clear summary of the main results and the effectiveness of the molecular approach for identifying species-specific genomic variants. This section addresses the methodological challenge of reference genome selection, presenting objective metrics that justify Aphelinus certus as the most suitable reference material. We provide an honest comparison with DNA barcoding, acknowledging its established value while demonstrating how whole-genome sequencing offers higher resolution for distinguishing closely related taxa and enable the development of cost-effective targeted assays. The limitations of the study are explicitly discussed, including sample origin and the use of a distantly related reference genome. We emphasize the method's importance for quality control in mass-reared populations, detection of cryptic species, and genetic monitoring in biological control programs.
“The lack of a detailed bioinformatic pipeline renders the experiment irreproducible.”
03 (Lines 143-182): A detailed bioinformatic pipeline was included in Section 2.3 (Bioinformatic analysis for variant detection), specifying the software tools used for sequence alignment (BWA), variant calling (SAMtools mpileup), and the filtering criteria applied. 
“The justification for the choice of A. certus is subjective and vague.”
04 (Lines 216-227): The expression “genomic stability” was replaced by a justification based on objective metrics obtained in this study. Specifically, the selection of Aphelinus certus as the reference genome was supported by: (i) the lower number of fixed variants, (ii) a consistent proportion of high-quality variants, (iii) greater concordance of informative variants across different filtering strategies, including PCA-based analyses, and (iv) the higher mean length of overlapping genomic regions shared among the analyzed species. These criteria collectively support the suitability of this reference genome for comparative variant detection.
“The call rate > 0.5 threshold is too permissive; genome size and SNP characterization are insufficiently described”.
05 (Table 2): We acknowledge that a call rate threshold above 0.5 may be considered permissive in conventional genomic studies. However, in the present study this criterion was adopted considering the experimental design based on pooled sequencing (pool-seq) of multiple individuals per species and the need to maximize the number of comparable variants among phylogenetically distinct taxa. To evaluate the robustness of the observed patterns, a second and more restrictive approach was applied, considering only variants shared among all target species. The consistency of the results obtained with both filtering strategies indicates that species differentiation was not critically influenced by the selected threshold.
Regarding genomic information, we agree that estimates such as genome size and heterozygosity are relevant. Nevertheless, as three of the four analyzed species still lack well-established reference genomes, we adopted a comparative strategy based on shared genomic regions and informative variants. Additional sequencing metrics, including total read number, base yield, and sequencing coverage, were incorporated into Table 1 and Supplementary Table S1 to provide further context regarding data quality and depth. We also clarified in the revised text that the primary objective of this study was the identification of discriminant genomic variants among species rather than a comprehensive genomic characterization. The description of filtering criteria and variant attributes was therefore expanded to improve clarity.
“The interpretation of PCA results remains descriptive and lacks statistical support such as Fst.”
06 (Figure 1): We thank the reviewer for this important observation. In this study, PCA was used primarily as an exploratory approach to assist in the identification of informative genomic markers for species discrimination. Its purpose was not to provide standalone statistical inference, but rather to detect patterns of genetic variation that could guide marker selection. To complement this analysis and provide quantitative support, we incorporated pairwise Euclidean distance analysis among samples, visualized through hierarchical clustering heatmaps. This additional approach provides an objective measure of genetic relationships and reinforces the differentiation patterns observed in the PCA.
Regarding the suggestion to calculate Fst, we emphasize that this metric requires robust estimates of allele frequencies derived from multiple independent samples per population. In the present study, each species was represented by a single pooled sample, which prevents reliable estimation of population-level parameters and makes Fst calculation statistically inappropriate. Our objective was therefore not to infer population structure but to identify a reduced set of diagnostic markers capable of distinguishing species. In this context, exploratory multivariate approaches combined with distance-based analyses are widely used to capture and quantify interspecific genetic differentiation.

Minor points
The title was revised from “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichogramma pretiosum and Trichospilus diatraeae (Hymenoptera: Chalcidoidea), important biocontrol agents” to “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichospilus diatraeae and Trichogramma pretiosum (Hymenoptera: Chalcidoidea), important biocontrol agents,” to follow the order in which the species are presented in the manuscript.
Introduction
1st paragraph
(Line 32): The definition of parasitoid wasps was revised to: "Parasitoid wasps act as natural enemies of a wide range of arthropods, being widely used as biological control agents to regulate insects that cause economic damage in agricultural and forestry systems."
2st paragraph
(Line 41): Added: “Accurate identification of these species is fundamental for the success of biological control programs, since incorrect identification can compromise the efficiency of parasitoid releases and the interpretation of ecological interactions."
(Line 35): “minisucle” by “minuscule”.
3st paragraph
(Line 48-62): The third and fourth paragraphs have been grouped together to present the importance of accurate species identification, the limited molecular data, and the rationale for using whole-genome sequencing to identify species-specific genomic variants more clearly.
4st paragraph
(Line 63): Reformulation of “Therefore, molecular approaches have emerged as complementary tools for rapid and precise identification and differentiation of species [14].” by “Therefore, molecular approaches have emerged as complementary tools for rapid and precise identification and differentiation of species [14].”.
5st paragraph
(Lines 69-77): Additional clarification was included regarding the limited molecular data currently available for these species and the advantages of genomic variants as higher-resolution markers for species identification.
6st paragraph
(Lines 78-81): This section was reformulated to improve clarity and to update the references related to previously sequenced molecular regions..
7st paragraph
(Lines 82-90): The paragraph was rewritten to better justify the methodological approach adopted in this study.
8st paragraph
(Line 94): The objective of the study was reformulated to improve precision and scientific clarity.
(Line 95): “genomic sequencing” by “whole-genomic sequencing”.

Reviewer 2 Report

Comments and Suggestions for Authors

Hymenopteran parasitoids are crucial factor of insect population dynamics in nature. They also serve as important biocontrol agents applied against dangerous agricultural and forest pests. Their vast taxonomic diversity, ecological plasticity and minute dimensions (especially in egg parasitoids) make it difficult to perform reliable species identification. Molecular genetic analysis becomes a solution, but it still requires deep investigation to detect specific markers for species differentiation, based upon robust morphological characterization of voucher specimens used for investigation. As indicated by the authors, it is a complimentary tool. The paper is of interest for specialists in technical entomology, insect molecular biology, etc., and fits the journal’s scope. However, it is not clearly written and poses a lot of questions, as outlined below.

The abstract partially describes the molecular detection methodology (though in a strange way, see below), but leaves out the opportunity to understand how the initial species identification of the specimens was done.

The same applies to the Materials and Methods section. Only sample collection and molecular analysis are described.

Lines 4-5: why enumerating two Eulophidae, then one Trichogrammatidae, then again one Eulophidae members?

Lines 6-7: how the “samples of adult specimens” could have been extracted and sequenced? Please be correct and specific.

Lines 8 and 10: “variants” of what?

Lines 17-18: first of all, the parasitic wasps are biotic mortality factor of insects in nature

Line 33 and throughout: This is habitual to start the sentence with a full, and not contracted genus epithet

Line 34: please check the use of the term “caterpillar” in scientific literature

Line 35: “population of insects” – are you sure there’s just one population?

Line 36 and further: the common names preceding the Latin epithets of insects are welcome!

Line 51-52: “a study …. Described the sequencing” – this is very cumbersome way of saying that sequencing was performed

Lines 72-75: this should be clearly indicated, which parasitoid species is reared in which hosts.

Line 80: “subsequently … subsequently” – what is the meaning of this repetition?

Line 81: this is fair to indicate the exact amount of proteinase K added to each samples, but when the volume of lysis buffer is unknown, this information is senseless

Line 83: the centrifuge speed expressed as round per meter is senseless when the centrifuge radius is unknown.

Ibidem: was DNA extracted from supernatant or pellet?

Line 93: “variants” of what?

 Lines 98-100: what is the reason to “illustrate the evolutionary proximity between the target species”, all belonging to different genera, and one to another family?

Lines 102-103: what is the reason of using a reference sequence from another family?

Line 103: “relationship relatively close” – unclear wording

Lines 112-131: methodological references are missing

Line 136 and further: “variants” of what?

Line 141: “sequences” of what?

Line 151: what is implied by “regions”?

Table 2: which units are used to express “length”? what is the meaning of “minimal length” equaled “1”?

Lines 176-177: what does it mean “variants were located in regions flanked by at least 20 bases on both sides”? How come that only a part of “variants” corresponds to this condition? Does it mean that other “variants” are not flanked by nucleotide bases? How could that be?

Line 177: what “applications” are implied?

Lines 185-187: which methodological approach is followed in this section?

What is the usability of Tables 3 and 4? In the Methods, it is indicated that the target species were compared to Genbank-available references of other species for each genus but one. It the table, a comparison is shown for the four species from four different genera (one belonging to another family). To my mind, whole genome sequencing to differentiate species belonging to different genera is useless. This is necessary to outline borders between closely related species, which are poorly differentiated by morphology and DNA barcoding. In particular, it would be great to see how genomic sequencing and robust bioinformatics overcomes the limitations of DNA barcoding in discrimination of different Trichogramma species.

To conclude, the paper needs to be rewritten and the data re-analyzed to demonstrate the practical outcomes of the study. The usability of genome sequencing requires a more detailed substantiation. I doubt that when different parasitoids are reared in different hosts, such as Tenebrio molitor and Ephestia kuhniella, there is a risk of culture cross-contamination explaining the necessity of in-depth bioinformatics study.

Author Response

We would like to thank the Reviewer for their careful evaluation of our manuscript and for their constructive comments and valuable suggestions. Their feedback has helped us to improve the clarity, methodological description, and overall quality of the manuscript.

Reviewer 2 - Major points

“The abstract partially describes the molecular detection methodology but does not explain how the initial species identification of the specimens was performed”.

01 – A voucher sample of each parasitoid species was sent to the Department of Biological Sciences at the Federal University of Espírito Santo (UFES) and to the “Oscar Monte” Entomophagous Insect Collection for morphological identification performed by taxonomic specialists using standard identification keys. This information was added to the Abstract and Materials and Methods sections to clarify the initial taxonomic validation of the specimens studied.

“Table 2: which units are used to express ‘length’? What is the meaning of ‘minimum length’ equal to ‘1’?”.

02 – The length of overlapping genomic regions was expressed in base pairs (bp). The minimum value of 1 bp corresponds to cases in which, after the intersection of homologous regions among different species, only a single nucleotide position remained shared. Although minimum length filters were applied during the initial identification of regions within each species, the overlap step among species may reduce these regions to shorter segments, including overlaps consisting of only one base. Furthermore, due to the inclusion of the table summarizing the reading sequences, the former Table 2 was renumbered as Table 3 in the revised manuscript.

“What does it mean that variants were located in regions flanked by at least 20 bases on both sides?”

03 – By definition, all variants are flanked by nucleotide bases within the genomic sequence. However, the criterion adopted in this study refers specifically to the availability of at least 20 bp of high-quality flanking sequence on each side of the variant after filtering and alignment procedures. Variants located near read ends, within low-quality regions, or in areas with alignment gaps may not retain sufficient reliable flanking sequence for downstream analyses and were therefore excluded.

“What is the usefulness of Tables 3 and 4?”

04 (Table 3): Has been renumbered as Table 4 and presents the set of species-specific genomic variants, together with their allele frequency patterns and genotype quality scores. Table 5 summarizes the characteristics of the genomic regions shared among the studied species, including their number and length distribution, providing a quantitative overview of the genomic intervals used for variant detection. These data support the identification of diagnostic markers for each parasitoid species and provide essential information for data deposition and future validation studies.

“Whole-genome sequencing to differentiate species belonging to different genera is unnecessary. It would be more relevant to demonstrate how genomic approaches overcome DNA barcoding limitations, particularly in Trichogramma”.

05 – We thank the reviewer for this valuable comment. Although DNA barcoding approaches based on markers such as COI can be applied to the studied taxa, their effectiveness depends strongly on the availability and reliability of reference sequences in public databases, which remain variable or limited for several parasitoid groups. In addition, single-marker approaches may show restricted resolution in taxa characterized by high genetic similarity, cryptic diversity, or challenging morphological identification, as frequently reported for genera such as Trichogramma.

In this context, the genomic strategy adopted in the present study enables the identification of multiple informative markers distributed across the genome, providing a more robust, reproducible, and scalable framework for species discrimination. This approach complements traditional DNA barcoding by offering higher resolution and greater flexibility for the development of targeted molecular assays.

Minor points

The title was revised from “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichogramma pretiosum and Trichospilus diatraeae (Hymenoptera: Chalcidoidea), important biocontrol agents” to “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichospilus diatraeae and Trichogramma pretiosum (Hymenoptera: Chalcidoidea), important biocontrol agents,” to follow the order in which the species are presented in the manuscript.

Introduction

(Lines 32-34): The text has been revised to clarify that parasitoid wasps act as natural enemies, regulating insect populations in natural ecosystems.

(Lines 36–39): The order of species presentation was revised for clarity, changing from “Palmistichus elaeisis, Tetrastichus howardi, Trichogramma pretiosum and Trichospilus diatraeae” to “Palmistichus elaeisis, Tetrastichus howardi, Trichospilus diatraeae and Trichogramma pretiosum”. Genus names were also written in full at first occurrence, following scientific conventions.

(Lines 48–62): The term “caterpillar” was revised.

(Lines 74–75): The manuscript was revised to clarify that the term refers to genomic variants, particularly single nucleotide polymorphisms (SNPs).

(Lines 78–81): The wording was adjusted to avoid implying the existence of a single population.

Materials and Methods

(Lines 102-109): It is now specified which parasitoid species were reared on the cited hosts in Section 2.1 (Insect collection) of the Materials and Methods.

(Lines 119-132): The methodological description was revised to clarify that pooled samples of adult specimens were used for genomic DNA extraction, quantification, and sequencing. In addition, the section was updated to include the volume of lysis buffer used (400 µL), to report centrifugation conditions in relative centrifugal force (× g), and to specify that genomic DNA was obtained from the supernatant after centrifugation. These modifications were incorporated into the section “DNA extraction, quantification and sequencing.”

(Lines 153-164): The reference to evolutionary proximity between species aimed to assist in the selection of available and conserved reference genomes to improve alignment performance and variant detection, given the absence of species-specific reference genomes.

(Lines 166-172): The methodological description was revised to clarify that the term “regions” refers specifically to overlapping genomic regions shared among the studied species. In addition, relevant methodological references were incorporated to support the bioinformatic procedures described, improving the clarity and reproducibility of the analytical approach.

(Lines 184-200): Given that the samples consisted of pools of individuals and that the selected regions showed high interspecific divergence, a higher density of variants was observed along these sequences. This scenario imposes limitations on primer design, since the presence of polymorphisms within candidate regions can compromise amplification efficiency and specificity (Kwok et al., 1990; Stadhouders et al., 2010).

Thus, the availability of sufficiently long conserved regions suitable for primer placement was restricted. Methodological studies indicate that a minimum of 18–25 bp free of variation is generally sufficient for adequate PCR primer design (Dieffenbach et al., 1993). In this context, the use of approximately 50 bp of flanking sequence on each side of the SNP was considered appropriate for future genotyping approaches, as it provides sufficient sequence context for the selection of primers with suitable physicochemical properties.

Discussion

(Line 380-392): The text was revised to clarify that the implied applications refer to the future use of the identified genomic variants in molecular diagnostics, species discrimination, quality control of mass-reared parasitoid populations, and genetic monitoring in biological control programs.

References

Kwok, S.; Kellogg, DE.; McKinney, N.; Spasic, D.; Goda, L.; Levenson, C.; Sninsky, JJ. Effects of primer–template mismatches on the polymerase chain reaction: Human immunodeficiency virus type 1 model studies. Nucleic Acids Research 1990, 18(4): 999–1005. DOI: 10.1093/nar/18.4.999.

Stadhouders, R.; Pas, SD.; Anber, J.; Voermans, J.; Mes, THM.; Schutten, M. The effect of primer–template mismatches on the detection and quantification of nucleic acids using the 5′ nuclease assay. Journal of Molecular Diagnostics 2010, 12(1): 109–117. DOI: 10.2353/jmoldx.2010.090035.

Dieffenbach, CW.; Lowe, TMJ.; Dveksler, GS. General concepts for PCR primer design. PCR Methods and Applications 1993, 3(3): S30–S37. DOI: 10.1101/gr.3.3.S30.

Reviewer 3 Report

Comments and Suggestions for Authors

Palombo et al., in the manuscript entitled “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichogramma pretiosum and Trichospilus diatraeae (Hymenoptera: Chalcidoidea), important biocontrol agents”, investigated several genomic variants of four important biocontrol agents for Brazil. The manuscript is well written, and the results are relevant and useful for the molecular identification of the target species and their strains for use in biocontrol programmes. I recommend the manuscript for publication. I suggest that the authors more clearly explain the importance of the detected genomic variants or strains of biocontrol agents and discuss this further.

 

Minor points:

Abstract, line 2 — “the high morphological similarity” should be used instead of “the high similarity”.

Please remove the sentence “Samples comprised of adult specimens were preserved in absolute ethanol, extracted with the DNeasy® Blood & Tissue kit (Qiagen), and sequenced on the Illumina Novaseq 6000 platform.” from the abstract. It should be included in Materials and Methods.

 

What are “specific variants”? Please clarify.

 

Author names below species should be written without brackets.

 

Palmistichus elaeisis Delvare & LaSalle, 1993

Trichogramma pretiosum Riley, 1879

Trichospilus diatraeae Cherian & Margabandhu, 1942

Author Response

We would like to thank the Reviewer for their careful evaluation of our manuscript and for their constructive comments and valuable suggestions. Their feedback has helped us to improve the clarity, methodological description, and overall quality of the manuscript.

Reviewer 3 - Major points

“I suggest that the authors explain more clearly the importance of the detected genomic variants or strains of the biocontrol agents and discuss this topic in greater depth.”

01 – We thank the reviewer for this valuable suggestion. The Discussion section was revised to more clearly highlight the relevance of the identified genomic variants for species discrimination, detection of cryptic diversity, and quality control in biological control programs. Additional clarification was included to emphasize the practical implications of these markers for molecular diagnostics and genetic monitoring.

Minor points 

The title was revised from “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichogramma pretiosum and Trichospilus diatraeae (Hymenoptera: Chalcidoidea), important biocontrol agents” to “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichospilus diatraeae and Trichogramma pretiosum (Hymenoptera: Chalcidoidea), important biocontrol agents,” to follow the order in which the species are presented in the manuscript.

Abstrat

(Line 14): The expression “high similarity” was replaced by “high morphological similarity” to improve precision.

(Lines 21–25): The sentence referring to sample preservation, DNA extraction, and sequencing procedures was removed from the Abstract and incorporated into the Materials and Methods section, as suggested.

Introduction

(Line 36-39): Taxonomic writing conventions were reviewed, and the names of the studied species were reformatted according to scientific standards.

(Line 69): The term “specific variants” was clarified in the revised manuscript to indicate genomic variants, particularly single nucleotide polymorphisms (SNPs) and related sequence differences used for species discrimination.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Dr. Pereira,

I have carefully read your manuscript entitled "Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichogramma pretiosum and Trichospilus diatraeae (Hymenoptera: Chalcidoidea), important biocontrol agents". The paper lists specific genomic variants for the differentiation of these species, which resulted from genomic sequencing. The paper is fairly well-written, scientifically sound and concise. I believe it provides a useful contribution to the process of molecular identification of economically important parasitoids, and the manuscript could therefore be published in Insects after a minor revision. Specifically, there are some overlapping symbols on Fig. 2, which make it less readable. In addition, I found a number of technical errors in the text (please see the attached pdf file).

Comments for author File: Comments.pdf

Author Response

We would like to thank the Reviewer for their careful evaluation of our manuscript and for their constructive comments and valuable suggestions. Their feedback has helped us to improve the clarity, methodological description, and overall quality of the manuscript.

Reviewer 4

Major points

The title was revised from “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichogramma pretiosum and Trichospilus diatraeae (Hymenoptera: Chalcidoidea), important biocontrol agents” to “Molecular identification of Palmistichus elaeisis, Tetrastichus howardi, Trichospilus diatraeae and Trichogramma pretiosum (Hymenoptera: Chalcidoidea), important biocontrol agents,” to follow the order in which the species are presented in the manuscript.

“There are overlapping symbols in Figure 2 that make it difficult to read. In addition, some technical errors were found in the text”.

01 – We thank the reviewer for this observation. Figure 2 was reformatted to improve clarity and readability, including adjustments in symbol positioning and graphical layout.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been thoughtfully revised, thank you for your careful attention to the feedback. Regarding lines 306–308, I kindly suggest removing the species data information, as it is already presented the Materials and Methods section in the manuscript. With this minor revision, the manuscript can be accepted. 

Author Response

We thank the reviewers for their careful evaluation of the manuscript and their constructive comments.

Introduction

2^nd paragraph (Line 47): Following a careful revision of the manuscript, we have updated the expression “the scarcity of taxonomic specialists” to “the high demand for taxonomic expertise,” as we believe this wording more accurately reflects the current context.

Results

10^th paragraph (Lines 306-308): The referenced information has been removed, as it was already presented in the Materials and Methods section.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is revised accordingly

Author Response

We thank the reviewer for the careful evaluation of the manuscript.

We would like to inform you of minor revisions made during the final revision of the manuscript:

Introduction

2^nd paragraph (Line 47): Following a careful revision of the manuscript, we have updated the expression “the scarcity of taxonomic specialists” to “the high demand for taxonomic expertise,” as we believe this wording more accurately reflects the current context.

Results

10^th paragraph (Lines 306-308): The referenced information has been removed, as it was already presented in the Materials and Methods section.