Review Reports - Preliminary Investigation into the Predation of <i>Pomacea canaliculata</i> by <i>Aquatica leii</i> Larvae

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Good work, with well-designed experiments and important results for the management of this species in biological control; furthermore, it contributes to the knowledge of Lampyridae biology. Please consider the minor corrections I noted in the text. I would be great if you could include photographs of the experiments.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer1,

We would like to express our sincere gratitude for your thorough and insightful review of our manuscript (ID: insects-4141305). Your valuable comments and suggestions have helped us to significantly improve the clarity and quality of our work. We have carefully considered each of your points and have revised the manuscript accordingly. Below is a point-by-point response to your comments, detailing the modifications we have made.

1.Regarding your suggestions regarding the formatting of the entire text:

We are extremely grateful for your valuable suggestions. We fully agree and accept this suggestion. In fact, other reviewers also made similar suggestions. Therefore, based on your and other reviewers' suggestions, we have made the corresponding modifications.

Regarding the issue you pointed out in the original text L195:

Regarding the repetition issue you pointed out in the paragraph on L218-223 of the original text:

Thank you very much for your valuable suggestions. We fully agree with your viewpoint, so we have removed the repetitive content in this section.

Regarding the suggestion you made at lines 225-226 of the original text regarding the ambiguity:

We are extremely grateful for your valuable suggestions. We fully agree with and adopt your suggestions. It is worth noting that other review experts also pointed out the problems here. Therefore, we have made modifications to this part based on your and their suggestions.(L281-282)

Regarding the issue you mentioned in the original text at L227 about the need to add references:

We sincerely hope that these revisions address all of your concerns and improve the manuscript's clarity, rigor, and reproducibility. We are very grateful for your time and expertise, which have undoubtedly strengthened our work.

Thank you once again for your valuable input.

sincerely,

The Authors

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript attempts to evaluate the biocontrol potential of the native Chinese aquatic firefly Aquatica leii against the invasive apple snail Pomacea canaliculata. While the subject is of interest, several major concernsregarding methodological transparency, statistical reporting, mechanistic depth, and practical applicability need to be addressed before publication.

Lines 94–96: the Aquatica leii larvae were purchased from two different resources (two places in China). Additional information about the firefly is required, including genetic background (same genetic population?), breeding methods (laboratory-reared or wild-collected, rearing history and instar determination criteria), etc., to ensure the physiological uniformity of experimental individuals and the reproducibility of results.

Lines 100–103: (1) The quarantine or health screening procedure of the two snails, Pomacea canaliculate and Cipangopaludina chinensis is required to be described. It is necessary to explain the quarantine, purification and acclimatization breeding conditions after collection, to exclude the potential interference of parasites, pathogens or environmental stress on the experimental results. (2) And the reasons why to choose C. chinensis as the reference prey should be given.

Line 118-119: Clarify whether the 36 repetitions are the total number or the number of repetitions for each instar group to avoid ambiguity.

Section 2.2.3-2.2.4: (1) Clarify whether the two experiments are independent sequences or the same group of individuals were observed successively. Also, clarify the specific meaning of "three replicate treatment groups", i.e., whether the 15 larvae of each instar are divided into 3 subgroups (5 larvae per subgroup) for parallel experiments, or the whole experiment was repeated 3 times (using 15 new larvae each time). The statistical unit must be clear. (2) And how to confirm the death of the snail?

Section 2.2.4: A blank without larvae should be provided to calculate the weight change of P. canaliculata tissue in a week because the water content of the tissue would be changed.

Section 2.2.5: (1) Provide a brief description of the dissection protocol (e.g., anatomical landmarks) and method of crude extract. (2) Provide the final concentration of the supernatant after mixing to facilitate comparison between different studies. (3) Explain the rationale for the 10 µL injection dose and whether a dose-effect pre-experiment was conducted. (4) It is recommended to add a damage control group, such as injecting an equal volume of sterile water or performing a sham injection (inserting the needle but not injecting), to distinguish the physical damage of injection from the chemical effect of the extract.

Section 2.2.6: (1) Clearly state all statistical test methods used in the methods section, including analysis of variance (ANOVA) for lethal time and consumption amount. Also, explain whether the data meet the assumptions of homogeneity of variance and normality, and if necessary, state whether data transformation or non-parametric tests were used. (2) Software (e.g., SPSS, R, GraphPad) and version are required.

Section 3.1: (1) only 36 replicates across all instars combined (not per instar, but section 2.2.2 mentioned four instar), the chi-square test is underpowered to detect meaningful differences. (2) Prey preference indices are known to be density-dependent. Testing only a 1:1 ratio provides no information about actual feeding decisions under natural conditions where prey abundance varies dramatically. It would be better if more results of the choice assay were conducted at different prey densities.

Line 188: p<0.05 should be P>0.05, and the same as line 211.

Line 195: “strongest lethal capacity against ??”

Section 3.3: Firefly Larvae were provisioned with dissected snail tissue rather than live, intact prey. The results may reflected ingestion rate, not the predation capacity.

Line 238: table 4 should be table 2.

Comments on the Quality of English Language

Whole manuscript: (1) Proofread typos and formatting inconsistencies, such as the italicization of scientific name and P, the capitalization of P value, and (2) standardize the reference format according to the journal.

Author Response

Dear Reviewer2,

We would like to express our sincere gratitude for your thorough and constructive review of our manuscript (ID: insects-4141305). Your insightful comments and suggestions have been invaluable in helping us improve the quality and clarity of our work. We have carefully considered each of your points and have revised the manuscript accordingly. Below is a point-by-point response to your comments, detailing the modifications we have made.

Comment 1 (Lines 94–96): The Aquatica leii larvae were purchased from two different resources (two places in China). Additional information about the firefly is required, including genetic background (same genetic population?), breeding methods (laboratory-reared or wild-collected, rearing history and instar determination criteria), etc., to ensure the physiological uniformity of experimental individuals and the reproducibility of results.

Response: We thank the reviewer for this important observation. We agree that providing detailed information about the origin and rearing history of the experimental insects is essential for reproducibility. Accordingly, we have revised Section 2.1.1 to specify that all larvae originated from the same captive-reared population maintained under standardized conditions for multiple generations. We have also added details on rearing history (diet, environmental conditions) and instar determination criteria (head capsule width measurement and molting records, following Fu et al.). These additions ensure clarity regarding the physiological uniformity of the individuals used.(L97-105)

Comment 2 (Lines 100–103): (1) The quarantine or health screening procedure of the two snails, Pomacea canaliculate and Cipangopaludina chinensis is required to be described. It is necessary to explain the quarantine, purification and acclimatization breeding conditions after collection, to exclude the potential interference of parasites, pathogens or environmental stress on the experimental results. (2) And the reasons why to choose C. chinensis as the reference prey should be given.

Response: We thank the reviewer for highlighting the need for more rigorous description of snail handling procedures. In response, we have expanded Section 2.1.2 to include a detailed account of the 7-day quarantine period, visual health inspections, and acclimatization conditions (temperature, water, feeding) applied to both snail species prior to experiments. Furthermore, we have explicitly stated the rationale for selecting C. chinensis as the reference prey: it is a native sympatric snail species commonly found in the same habitats as P. canaliculata in southern China, and it represents a natural prey item for A. leii larvae. This choice allows prey preference to be assessed in an ecologically relevant context.(L111-124)

Comment 3 (Lines 118–119): Clarify whether the 36 repetitions are the total number or the number of repetitions for each instar group to avoid ambiguity.

Response: We appreciate the reviewer’s request for clarification. To eliminate any ambiguity, we have revised the sentence in Section 2.2.2 to read: "A total of 36 independent trials were conducted using new larvae and snail individuals for each trial. The larvae used in these trials were drawn from the 3rd to 6th instar groups (15 individuals per instar) described above." This makes it clear that the 36 trials represent the total number across all instars, with larvae randomly selected from each instar group.(L138-141)

Comment 4 (Section 2.2.3-2.2.4): (1) Clarify whether the two experiments are independent sequences or the same group of individuals were observed successively. Also, clarify the specific meaning of "three replicate treatment groups", i.e., whether the 15 larvae of each instar are divided into 3 subgroups (5 larvae per subgroup) for parallel experiments, or the whole experiment was repeated 3 times (using 15 new larvae each time). The statistical unit must be clear. (2) And how to confirm the death of the snail?

Response: We thank the reviewer for these helpful points. We have now clarified in both Sections 2.2.3 and 2.2.4 that the lethal time and consumption experiments were conducted independently, using separate sets of larvae. We have also explicitly defined “three replicate treatment groups” as three parallel subgroups (n = 5 larvae each) derived from the 15 larvae per instar, run simultaneously. The phrase “repeated three times” has been removed to avoid confusion. Additionally, we have added a clear description of how snail death was confirmed: absence of movement, lack of response to gentle probing, and retraction of the foot deep into the shell for more than 5 minutes, with extended observation in uncertain cases.(L148-L167)

Comment 5 (Section 2.2.4): A blank without larvae should be provided to calculate the weight change of P. canaliculata tissue in a week because the water content of the tissue would be changed.

Response: We agree completely with this suggestion. In Section 2.2.4, we have now included a description of three control containers without larvae, set up in parallel to monitor autogenic changes in tissue weight (e.g., water loss). The consumption amount per larva is now calculated as (initial tissue weight – remaining tissue weight) – (mean weight loss in control containers), and we have specified that tissue was gently blotted before weighing to remove surface water.(L159-167)

Comment 6 (Section 2.2.5): (1) Provide a brief description of the dissection protocol (e.g., anatomical landmarks) and method of crude extract. (2) Provide the final concentration of the supernatant after mixing to facilitate comparison between different studies. (3) Explain the rationale for the 10 µL injection dose and whether a dose-effect pre-experiment was conducted. (4) It is recommended to add a damage control group, such as injecting an equal volume of sterile water or performing a sham injection (inserting the needle but not injecting), to distinguish the physical damage of injection from the chemical effect of the extract.

Response: We thank the reviewer for these detailed and valuable suggestions. We have substantially revised Section 2.2.5 to address each point: (1) We have added a step-by-step description of the dissection protocol, including the anatomical landmarks used to separate the buccal region, foregut, midgut, and hindgut. (2) The final concentration of the supernatant is now stated as approximately 0.5 g tissue equivalent per mL (original tissue diluted 1:1 with saline). (3) We have explained that a dose-finding pre-test (5, 10, and 20 µL) was conducted prior to the main experiment, and the 10 µL dose was selected because it induced clear mortality without causing immediate death from injection trauma. (4) We have included three control groups: physiological saline (vehicle control), sterile water (to control for osmotic effects), and sham injection (needle insertion without fluid injection) to account for physical injury. These additions strengthen the rigor of the experimental design.(L170-192)

Comment 7 (Section 2.2.6): (1) Clearly state all statistical test methods used in the methods section, including analysis of variance (ANOVA) for lethal time and consumption amount. Also, explain whether the data meet the assumptions of homogeneity of variance and normality, and if necessary, state whether data transformation or non-parametric tests were used. (2) Software (e.g., SPSS, R, GraphPad) and version are required.

Response: We thank the reviewer for emphasizing the importance of transparent statistical reporting. We have rewritten Section 2.2.6 to include a comprehensive description of all statistical tests used. We now specify that normality (Shapiro–Wilk) and homogeneity of variances (Levene’s test) were checked prior to ANOVA, and as all data met these assumptions (P > 0.05), one-way ANOVA followed by Tukey’s HSD post-hoc test was used for lethal time and consumption data. For the feeding preference assay, chi-square goodness-of-fit test was used; for mortality rates in the injection experiment, Fisher’s exact test was applied. We have also stated that SPSS Statistics version 26.0 was used for all analyses.(L194-203)

Comment 8 (Section 3.1): (1) only 36 replicates across all instars combined (not per instar, but section 2.2.2 mentioned four instar), the chi-square test is underpowered to detect meaningful differences. (2) Prey preference indices are known to be density-dependent. Testing only a 1:1 ratio provides no information about actual feeding decisions under natural conditions where prey abundance varies dramatically. It would be better if more results of the choice assay were conducted at different prey densities.

Response: We are grateful to the reviewer for this insightful methodological critique. In response, we have added a paragraph at the end of Section 3.1 acknowledging the limitations of our preference assay. We now note that the total number of replicates (36 across all instars combined) may limit the statistical power of the chi-square test to detect small but biologically meaningful differences. We also explicitly state that experiments were conducted only at a 1:1 prey ratio, which does not capture potential density-dependent shifts in feeding behavior that may occur in natural habitats. We have suggested that future studies should incorporate multiple prey density ratios to better understand foraging decisions under ecologically relevant conditions. We believe this addition appropriately contextualizes our findings and demonstrates awareness of the study’s limitations.(L212-224)

Comment 9 (Line 188): p<0.05 should be P>0.05, and the same as line 211.

Response: We thank the reviewer for catching this typographical error. We have corrected both figure legends (Figure 1 and Figure 2) to read “P > 0.05” where same letters indicate no significant difference, and we have standardized the notation to italicized capital P throughout the manuscript.(L237-239/L259-261)

Comment 10 (Line 195): “strongest lethal capacity against ??”

Response: We thank the reviewer for pointing out this incomplete sentence. It has now been completed to read “strongest lethal capacity against P. canaliculata” in the revised text.(L245)

Comment 11 (Section 3.3): Firefly Larvae were provisioned with dissected snail tissue rather than live, intact prey. The results may reflect ingestion rate, not the predation capacity.

Response: We agree with the reviewer’s interpretation and appreciate this important distinction. At the end of Section 3.3, we have added a paragraph explicitly noting that the experiment used dissected snail tissue, and therefore the measured consumption amount reflects ingestion rate under simplified conditions, not the full predation capacity (which would include search, attack, and handling of live prey). We advise readers to consider this limitation when interpreting the ecological relevance of the consumption values.(L274-L249)

Comment 12 (Line 238): table 4 should be table 2.

Response: We thank the reviewer for identifying this numbering error. The text has been corrected(L293)

Comments on the Quality of English Language: Whole manuscript: (1) Proofread typos and formatting inconsistencies, such as the italicization of scientific name and P, the capitalization of P value, and (2) standardize the reference format according to the journal.

Response: We thank the reviewer for the careful language review. We have thoroughly proofread the entire manuscript to correct typographical errors and ensure consistent formatting. All scientific names are now in italics, and P values are consistently presented as italicized capitals.

We sincerely hope that these revisions address all of your concerns and improve the manuscript’s clarity, rigor, and reproducibility. We are very grateful for your time and expertise, which have undoubtedly strengthened our work.

Thank you once again for your valuable input.

sincerely,

The Authors

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents the experimental work aimed at elucidating the predation possibilities of Aquatica leii larvae on Pomacea canaliculata mollusks, as well as some predation features.

The experimental data obtained does indeed reveal some new results. This concerns the fundamental possibility of predation, the potential use of Pomacea as food in comparison with the native species Cipangopaludina chinensis, and the effectiveness of the elimination of Pomacea mollusks. The work also contains an attempt to demonstrate the effectiveness of the toxic effect of digestive enzymes of Aquatica leii larvae on Pomacea canaliculata mollusks.

The whole manuscript is imbued with the idea of the possibility of using Aquatica leii larvae as a means of biological control of the abundance of Pomacea canaliculata mollusks. This idea is carried out in most sections of the work, that somehow blurred the actual discussion of the results.

There are quite a lot of comments on the manuscript.

Introduction.

line 73-74. Change to Fireflies (Insecta: Coleoptera: Lampyridae) [9-11],…

line 78. Here is the repeat of the beginning of paragraph.

Materials and methods.

2.1.1. A description of the life cycle of A. leii is required, including lifespan and number of larval molts.

Line 96. Please indicate the larval instar of the material used.

Line 101. Indicate the size of P. canaliculate.

Line 102. The Latin name is Italic. What is the size/weight of C. chinensis.

Line 103. Please clarify there were samples from one or several ponds? Indicate the concrete places.

2.2.2. It is unclear how the division the insects into 4 groups of 15 individuals reconciles with the individual use of the insects in the experiment. Why then divide the insects into groups?

Line 126. It is not specified how the larval identity of a particular instar was determined.

Line 144. The authors indicate that three replicate treatment groups were established. It is unclear whether the larvae were used individually or in groups during the experiment.

Line 155. Different concentrations of extracts were used for testing. It is not discussed later.

Results.

Figures (1-2): abbreviations on the x-axis need to be corrected; fix "statically" (lines 189 and 210) in the captions.

Line 190. "The data indicate that the lethal time of the sixth-instar larvae was significantly longer than that of all other instars" – is the lethal time of larvae or mollusks?

Lines 219-223. A full repeat of lines 202-207.

Line 225. The study did not show "feeding preference" (see the first part of the results). It did show the possibility of feeding on this species.

Lines 227-228. "Studies have shown that they primarily inject digestive tract fluids rich in various enzymes (such as proteases and lipases)" please provide the reference.

Lines 228-229. If this type of diet is indicated for this group, a reference is required.

Line 230. The same...

Line 230-231. The authors did not study this. References are required.

3.4. To confirm the specific effect of A. leii midgut contents, another control is necessary. Namely - the injections of the gut contents of larvae of species that do not prey on mollusks.

Furthermore, the fact that the midgut contents, rather than foregut or mouthpart extract, exhibit molluscicidal activity seems strange.

Overall, the result of experiment 3.4. does not appear convincing as described by the authors (see above).

Discussion.

Line 248. Italic.

Line 260. It's important to include the size of the mollusks in the discussion.

Conclusions

This section essentially seems like a repetition of everything that's already been said. It would be helpful to summarize the main findings in three or four bullet points. Without discussing the importance of larvae as biological control.

Author Response

Dear Reviewer3,

Comment 1 (line 73–74): Change to Fireflies (Insecta: Coleoptera: Lampyridae) [9-11],…

Response: We thank the reviewer for this helpful suggestion. We have revised the text accordingly. In the Introduction, the phrase has been changed to "Fireflies (Insecta: Coleoptera: Lampyridae) [9-11]".(L75)

Comment 2 (line 78): Here is the repeat of the beginning of paragraph.

Response: We appreciate the reviewer’s careful observation. We agree that the sentence in line 78 was redundant with the opening of the paragraph.(L78)

Comment 3 (2.1.1): A description of the life cycle of A. leii is required, including lifespan and number of larval molts.

Response: We thank the reviewer for this important suggestion. We have added a description of the life cycle of A. leii in Section 2.1.1.(L95-L106)

Comment 4 (Line 96): Please indicate the larval instar of the material used.

Response: We are extremely grateful for your valuable suggestions. Your suggestions are very correct. Another review expert also pointed out the problems here. Therefore, we have made corresponding changes to this part based on the review suggestions from you and the other review expert.

Comment 5 (Line 101): Indicate the size of P. canaliculata.

Response: We thank the reviewer for this suggestion. We have now added the shell height of the snails in addition to their weight.In Section 2.1.2, the description has been revised to:"...P. canaliculata individuals weighing 0.5–1 g (shell height approximately 10–15 mm) were used as the target prey..."(L111-112)

Comment 6 (Line 102): The Latin name is Italic. What is the size/weight of C. chinensis.

Response: We thank the reviewer for pointing out the need for italicization and for requesting the size of the reference prey. The italicization of scientific names has been checked and corrected throughout the manuscript (as also suggested by Reviewer 1). The size of C. chinensis has now been added. In Section 2.1.2, we have revised to:"...while Cipangopaludina chinensis (weighing 0.8–1.2 g, shell height approximately 15–20 mm) served as the reference prey."(L112-114)

Comment 7 (Line 103): Please clarify there were samples from one or several ponds? Indicate the concrete places.

Response: We thank the reviewer for this request for more detailed collection information. We have now clarified the collection sites. In Section 2.1.2, we have revised to:"All snails were collected from multiple irrigation ditches and ponds within a 5 km radius of Hangzhou Xiaoshan International Airport (30.23°N, 120.26°E), Zhejiang Province, China. To ensure genetic diversity and representativeness, snails were pooled from at least five distinct collection sites."(L114-117)

Comment 8 (2.2.2): It is unclear how the division the insects into 4 groups of 15 individuals reconciles with the individual use of the insects in the experiment. Why then divide the insects into groups?

Response: We thank the reviewer for highlighting this ambiguity. We have now clarified that larvae were maintained in instar-specific groups for convenience but tested individually. This point was also addressed in response to Reviewer 1’s comment on the same section. (L142-145)

Comment 9 (Line 126): It is not specified how the larval identity of a particular instar was determined.

Comment 10 (Line 144): The authors indicate that three replicate treatment groups were established. It is unclear whether the larvae were used individually or in groups during the experiment.

Comment 11 (Line 155): Different concentrations of extracts were used for testing. It is not discussed later.

Response: We thank the reviewer for this insightful observation. In response to another reviewer's suggestion, we had already standardized all extracts to the same concentration (0.5 g tissue equivalent per mL) in Section 2.2.5 and explained the rationale for selecting the 10 µL dose based on a pre-experiment. However, we agree that this point was not discussed in the Discussion section. We have now added a paragraph in the Discussion (following the analysis of midgut extract activity) that addresses.(L343-350)

Comment 12 (Figures 1-2): abbreviations on the x-axis need to be corrected; fix "statically" (lines 189 and 210) in the captions.

Response: We thank the reviewer for these careful observations. Regarding the x-axis abbreviations, we acknowledge that the original figures use "3rd", "4th", etc. as labels. The purpose of doing this is to ensure the cleanliness of the pictures. However, we fully agree with your suggestion. Therefore, to eliminate any ambiguity, we have provided additional explanations in the captions of the pictures. Regarding "statically," in response to another reviewer's comment, we have already corrected this to "significantly" in both figure legends and standardized the P-value format to italicized capital "P > 0.05". These corrections are now reflected in the revised manuscript.(L241-243/L263-266)

Comment 13 (Line 190): "The data indicate that the lethal time of the sixth-instar larvae was significantly longer than that of all other instars" – is the lethal time of larvae or mollusks?

Response: We thank the reviewer for identifying this ambiguity. We have revised the sentence in Section 3.2 to clarify: "The data indicate that the lethal time required by ..."(L244-245)

Comment 14 (Lines 219-223): A full repeat of lines 202-207.

Response: We thank the reviewer for pointing out this duplication. We have removed the repeated paragraph from Section 3.3 to eliminate redundancy.

Comment 15 (Line 225): The study did not show "feeding preference" (see the first part of the results). It did show the possibility of feeding on this species.

Response: We thank the reviewer for this precise and accurate observation. We agree that our results demonstrate acceptance of P. canaliculata as prey rather than a statistically significant preference. We have revised the opening sentence of Section 3.4 accordingly: "This study has confirmed that A. leii larvae are capable of preying upon P. canaliculata and accept it as a food source."(L287-288)

Comment 16 (Lines 227-228): "Studies have shown that they primarily inject digestive tract fluids rich in various enzymes (such as proteases and lipases)" please provide the reference.

Response: Thank you very much for your valuable suggestions. We fully agree with your suggestions. As a result, we have added the appropriate corresponding references.(L290-295)

Comment 17 (Lines 228-229): If this type of diet is indicated for this group, a reference is required.

Response: Thank you very much for your valuable suggestions. We fully agree with your suggestions. As a result, we have added the appropriate corresponding references.(L290-295)

Comment 18 (Line 230): The same...

Response: Thank you very much for your valuable suggestions. We fully agree with your suggestions. As a result, we have added the appropriate corresponding references.(L290-295)

Comment 19 (Line 230-231): The authors did not study this. References are required.

Response: Thank you very much for your valuable suggestions. We fully agree with your suggestions. As a result, we have added the appropriate corresponding references.(L290-295)

Comment 20 (3.4): To confirm the specific effect of A. leii midgut contents, another control is necessary. Namely - the injections of the gut contents of larvae of species that do not prey on mollusks. Furthermore, the fact that the midgut contents, rather than foregut or mouthpart extract, exhibit molluscicidal activity seems strange. Overall, the result of experiment 3.4. does not appear convincing as described by the authors (see above).

Response: We thank the reviewer for this thoughtful critique. We acknowledge the value of including an additional control (gut contents from a non-molluscivorous insect) to further demonstrate the specificity of the observed activity. In response to your comment, we have added a paragraph in the Discussion section that addresses this limitation while also providing plausible explanations for the observed midgut activity(L353-359)

Comment 21 (Line 248): Italic.

Response: We are extremely grateful for your valuable suggestions. Your suggestions are very correct. Another review expert also pointed out the problems here. Therefore we have thoroughly checked the entire manuscript and ensured that all scientific names are properly italicized, including those in the Discussion section.

Comment 22 (Line 260): It's important to include the size of the mollusks in the discussion.

Response: We thank the reviewer for this suggestion. We fully agree that including the size of the mollusks helps readers understand the context of the study. But we would like to note that the size range of P. canaliculata used in this study (0.5–1.0 g, shell height approximately 10–15 mm) was already provided in Section 2.1.2 (Materials and Methods). Regarding the Discussion section, our primary aim was to emphasize the overall predatory capability of A. leii against P. canaliculata rather than to focus on specific size classes. However, we recognize that mentioning the size in the Discussion could be helpful for readers. If you strongly believe this addition is necessary, we would be happy to incorporate it. Please let us know your preference, and we will make the corresponding revision promptly.

Comment 23 (Conclusions): This section essentially seems like a repetition of everything that's already been said. It would be helpful to summarize the main findings in three or four bullet points. Without discussing the importance of larvae as biological control.

Response: We thank the reviewer for this constructive suggestion. We have revised the Conclusions section to adopt a clearer. The revised section now presents the main findings as three distinct points: (1) predatory capability and instar-specific efficacy, (2) physiological mechanism of lethality, and (3) implications for biocontrol development. (L361-383)

Response: We thank the reviewer for these important language and formatting observations. In response to both your comment and a similar suggestion from another reviewer, we have thoroughly proofread the entire manuscript to correct typographical errors and ensure consistent formatting. All scientific names are now properly italicized, and P values are consistently presented as italicized capitals.

Thank you once again for your valuable input.

sincerely,

The Authors

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has become more specific; the authors have clarified many ambiguities in Materials and methods and Results sections. A Conclusions section has been added, which also adds clarity to the work.

Nevertheless, in my opinion, the manuscript is not yet ready for publication.

General comments:

Not all species names are given in italic.

Throughout the text, references are provided without space.

The toxicity of midgut extracts is presented as one of the main evidence for the effect of larvae on mollusks (“the most striking discovery”, see summary and abstract), although, strictly speaking, this is only an observation that requires further verification. This should be discussed more carefully. An example of this is the text added by the authors in the final section of the discussion.

The first sentences of the "Introduction" should be changed like this:

"Pomacea canaliculata (Gastropoda: Mesogastropoda: Ampullariidae) is a widespread South American freshwater mollusk." The species' range is rapidly expanding and is now becoming a common component of the fauna of many countries in Southeast Asia (…). P. canaliculata is listed among China's first cohort of invasive alien species…”

Line 110: First mention of Cipangopaludina… - no indication of the species' taxonomic position.

Line 211: A line break is needed after the table.

Lines 215-222: This text is more appropriate for discussing the results. It should be moved to the Discussion. Furthermore, the lack of significance in the statistical comparison implies the absence of differences. It is unlikely that trends can be discussed here and this result can be used as an argument for dietary preferences. See Line 295 (Discussion).

Line 266: Correct the capitalization.

Author Response

Dear Reviewers3,

We would like to express our sincere gratitude to the reviewer for the thoughtful and constructive comments on our manuscript entitled "Preliminary Investigation into the Predation of Pomacea canaliculata by Aquatica leii Larvae" (Manuscript ID: insects-4141305). These comments have been very helpful in improving the clarity and quality of our work.

We have carefully considered all the suggestions and have revised the manuscript accordingly. Below is a point-by-point response to the reviewer's comments. All modifications in the manuscript have been highlighted for your convenience.

Reviewer Comment 1: "Not all species names are given in italic."

Response: Thank you for pointing this out. We have carefully reviewed the entire manuscript and have italicized all scientific names of genera and species (e.g., Pomacea canaliculata, Aquatica leii, Cipangopaludina chinensis) throughout the text, including in the main body, figures, and tables.

Reviewer Comment 2: "Throughout the text, references are provided without space."

Response: We agree with this suggestion. We have added a space before each citation throughout the manuscript to ensure proper formatting.

Reviewer Comment 3: "The toxicity of midgut extracts is presented as one of the main evidence for the effect of larvae on mollusks ('the most striking discovery', see summary and abstract), although, strictly speaking, this is only an observation that requires further verification. This should be discussed more carefully. An example of this is the text added by the authors in the final section of the discussion."

Response: We sincerely thank the reviewer for this important nuance. We fully agree that our findings on midgut extract toxicity, while promising, should be presented as an observation requiring further validation rather than as a definitive conclusion. Accordingly, we have revised the language in the Simple Summary, Abstract, and Conclusions sections to reflect a more cautious and balanced tone.(L16-20/L43-45/L372-374)

Reviewer Comment 4: "The first sentences of the 'Introduction' should be changed like this: 'Pomacea canaliculata (Gastropoda: Mesogastropoda: Ampullariidae) is a widespread South American freshwater mollusk.' The species' range is rapidly expanding and is now becoming a common component of the fauna of many countries in Southeast Asia (...). P. canaliculata is listed among China's first cohort of invasive alien species..."

Response: We appreciate this excellent suggestion to improve the introduction. We have revised the opening sentences accordingly.(L55-58)

Reviewer Comment 5: "Line 110: First mention of Cipangopaludina... - no indication of the species' taxonomic position."

Response: Thank you for this observation. We have added the taxonomic information at the first mention of Cipangopaludina chinensis in the Materials and Methods section(L117-119).

Reviewer Comment 6: "Line 211: A line break is needed after the table."

Response: We have followed this suggestion. A line break has been inserted after Table 1 to ensure proper separation between the table and the subsequent text.(L222)

Reviewer Comment 7: "Lines 215-222: This text is more appropriate for discussing the results. It should be moved to the Discussion. Furthermore, the lack of significance in the statistical comparison implies the absence of differences. It is unlikely that trends can be discussed here and this result can be used as an argument for dietary preferences. See Line 295 (Discussion)."

Response: We fully agree with this suggestion. The text discussing the limitations of the feeding preference assay has been removed from the Results section and relocated to the Discussion. It now appears after the sentence discussing the ecological adaptability of A. leii.(L311-316)

Reviewer Comment 8: "Line 266: Correct the capitalization."

Response: Thank you for catching this formatting error. We have already changed "x" to "X".L249

We sincerely thank the reviewer again for the time and effort invested in evaluating our manuscript. We have carefully revised the manuscript according to all the suggestions, and we hope that these modifications have improved the quality of our work in the reviewer's view. We would be honored to invite the reviewer to re-evaluate our manuscript and provide further comments for improvement.

Thank you for your consideration.

Sincerely,

The Authors