Advancing Research on Overlooked Invertebrates in Biological Control: A Case Study of Local Hoverflies and Wolf Spiders
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsCongratulations to the authors - they have done impressive work on an important topic. I have only one suggestion: In the Discussion, you state:
“To our knowledge, the sensitivity of Melangyna sp. reported here represents the first evidence of Bt var. kurstaki having detrimental impacts on hoverflies.”
This phrasing leaves unclear whether there is existing published evidence of detrimental effects from other Bacillus thuringiensis varieties on hoverflies. If your report is the first for any Bt variety, please state this clearly. If there are previous findings involving other Bt strains, it would be helpful to cite the relevant studies.
The main question addressed is how widely used agricultural chemicals affect two under-researched natural enemies, hoverflies (Melangyna sp.) and wolf spiders (Venatrix spp.), which play important roles in biological control.
The topic is both original and highly relevant. It addresses a significant gap by focusing on locally occurring species that are often overlooked in chemical toxicity studies. Most research has concentrated on commercially cultured natural enemies, leaving a critical need for data on field-relevant, underrepresented groups.
This study adds a new perspective by providing detailed methodologies for rearing and testing local species, along with robust data showing differing chemical tolerances between hoverflies and wolf spiders. It highlights the importance of species-specific assessments in developing effective Integrated Pest Management (IPM) strategies.
The conclusions are well-supported by the evidence. The study clearly demonstrates that local hoverflies are more sensitive to certain chemicals than previously expected, while wolf spiders exhibit a higher tolerance. These findings directly address the main research question and highlight the importance of including overlooked invertebrates in Integrated Pest Management (IPM) planning. The references appear appropriate and relevant to the study’s context. The tables and figures are clear and informative, effectively summarizing the key findings.
The manuscript is considered acceptable as is. However, future studies might expand the range of chemicals tested and include field validation of laboratory findings to strengthen the practical application of the results.
However, I found one part of the Discussion section somewhat unclear. The authors state: “To our knowledge, the sensitivity of Melangyna sp. reported here represents the first evidence of Bt var. kurstaki having detrimental impacts on hoverflies.” This phrasing does not clarify whether there is existing published evidence of detrimental effects from other Bacillus thuringiensis (Bt) varieties on hoverflies. If this manuscript represents the first report of any Bt strain impacting hoverflies, it would be helpful to state this explicitly. Conversely, if there are previous findings involving other Bt varieties, citing the relevant studies would strengthen the context and interpretation of the results.
Author Response
Comments 1: Congratulations to the authors - they have done impressive work on an important topic. I have only one suggestion: In the Discussion, you state:
“To our knowledge, the sensitivity of Melangyna sp. reported here represents the first evidence of Bt var. kurstaki having detrimental impacts on hoverflies.”
This phrasing leaves unclear whether there is existing published evidence of detrimental effects from other Bacillus thuringiensis varieties on hoverflies. If your report is the first for any Bt variety, please state this clearly. If there are previous findings involving other Bt strains, it would be helpful to cite the relevant studies.
Response 1: Thank you for drawing this to our attention. We believe our study is the first to document harmful impacts of any Bt strain on hoverflies. We have therefore updated the sentence mentioned to say "To our knowledge, the sensitivity of Melangyna sp. reported here represents the first evidence of any Bt strain having detrimental impacts on hoverflies" (Discussion, page 10).
Reviewer 2 Report
Comments and Suggestions for Authors
The present manuscript by Knapp et al. describes a series of experiments investigating how different chemicals impact the survival and behavior of important natural enemies, such as hoverflies and wolf spiders. I have a few suggestions regarding the overall quality and clarity of the manuscript.
General comments
The font sizes and styles are inconsistent throughout the document. Please double-check the formatting.
In abstract, the word "Preserving" is unnecessarily bolded. Please correct this formatting.
Introduction
In the third paragraph, the first sentence is too long. It’d be clearer if you broke it into two. Also, “broad-spectrums” doesn’t sound right. Please change it to “broad-spectrum chemicals” or “broad-spectrum insecticides.”
In the last paragraph, Venarix should be Venatrix. The citation (Rendon et al., 2015) doesn’t match the numbered citation style used in the rest of the paper. It should be converted to the right format (e.g., [35]).
Experimental Design and Replication
Collection of spiders
In the second paragraph, the sentence with'...eye shine; In wolf spiders, light is reflected...' 'In' should be lowercase after a semicolon. Authors switched between 'Venatrix species' and 'Venatrix spp.' Please pick one format and stick with it throughout.
In the third paragraph, the text with the container dimensions (~126 mm long, 208 mm wide, 178 mm high) has a different font or style. Just double-check that formatting.
Collection and rearing of hoverflies
In the sentence '...absence to gravid females being collected…'. Replace ‘to’ with ‘of’.
While the study aimed for 20 individuals per treatment, some groups had fewer replicates. Authors should briefly justify how this might affect statistical power or interpretation of CI overlaps.
Taxicum spp. should be Taraxacum spp. (Dandelion genus, not Taxicum).
Add space between of and 16:8 in "photoperiod of16:8.
Check the font size and style of ‘(Whattmann, 90 mm diameter)’ information.
Acute Toxicity Bioassays
'dispersal form' should be 'dispersal from' the mother's abdomen.
Statistical Analysis
Why did Abbott’s correction not applied?
Results
Wolf Spiders: 'were the highly toxic' should be 'were highly toxic'.
Discussion
The authors mentioned the increased vulnerability of soft-bodied, legless hoverfly larvae to chemical residues. However, this point is only briefly touched upon. A slightly fuller explanation linking larval behavior (e.g., crawling over treated surfaces) to exposure risk would strengthen the argument.
'Whatever the reason' is too informal and lazy sounding for science writing. It is better to say 'Given these findings' or just skip and go straight to your point.
Author Response
Comments 1: The font sizes and styles are inconsistent throughout the document. Please double-check the formatting.
Response 1: Thank you for picking this up. We agree and therefore refer to the journal's editorial team to format as they see fit for their publication.
Comments 2: In abstract, the word "Preserving" is unnecessarily bolded. Please correct this formatting.
Response 2: The word "Preserving" in the abstract has been un-bolded
Comments 3: In the third paragraph, the first sentence is too long. It’d be clearer if you broke it into two. Also, “broad-spectrums” doesn’t sound right. Please change it to “broad-spectrum chemicals” or “broad-spectrum insecticides.”
Response 3: We agree that the sentence is lengthy and have cut it down to reduce repetition and improve clarity. We have amended the sentence from "While predators contribute significantly to pest control, optimal crop protection often requires incorporating both biological and chemical control within Integrated Pest Management (IPM) frameworks [4]. " to simply "Optimal crop protection often requires incorporating both biological and chemical control within Integrated Pest Management (IPM) frameworks [4]".
We have also replaced the term "broad-spectrums" with "broad-spectrum chemicals" in paragraph 3 of the introduction
Comments 4: In the last paragraph, Venarix should be Venatrix. The citation (Rendon et al., 2015) doesn’t match the numbered citation style used in the rest of the paper. It should be converted to the right format (e.g., [35]).
Response 4: Thank you for identifying these editorial issues. We have amended Venarix to Venatrix in the final paragraph of the discussion and changed the citation to the correct format ([34]).
Comments 5: In the second paragraph (Collection of wolf spiders), the sentence with'...eye shine; In wolf spiders, light is reflected...' 'In' should be lowercase after a semicolon. Authors switched between 'Venatrix species' and 'Venatrix spp.' Please pick one format and stick with it throughout.
Response 5: 'In' has been changed to lowercase. We have updated 'Venatrix species' to 'Venatrix spp.' to be consistent with the rest of the manuscript. Additionally, in the first paragraph of the 'Collection and rearing of hoverflies' section we have amended 'Melangyna species' to 'Melangyna sp.' for consistency with the rest of the manuscript.
Comments 6: In the third paragraph (collection and rearing of wolf spiders section), the text with the container dimensions (~126 mm long, 208 mm wide, 178 mm high) has a different font or style. Just double-check that formatting.
Response 6: Thank you for raising this. We have updated the font to match the rest of the manuscript and defer to the journal's editorial team for final formatting checks.
Comments 7: In the sentence '...absence to gravid females being collected…'. Replace ‘to’ with ‘of’ (collection and rearing of hoverflies section).
Response 7: 'to' has been replaced with 'of'
Comments 8: While the study aimed for 20 individuals per treatment, some groups had fewer replicates. Authors should briefly justify how this might affect statistical power or interpretation of CI overlaps.
Response 8: Thank you for raising this. We agree that some groups having fewer replicates is worth commenting on in the manuscript with respect to its impact on statistical power. To address this, we have inserted the sentence: "Where fewer than 20 individuals were tested, reduced statistical power is reflected through wider CIs and increased likelihood of overlap." in the Statistical analysis section.
Comments 9: Taxicum spp. should be Taraxacum spp. (Dandelion genus, not Taxicum). (Collection and rearing of hoverflies section)
Response 9: Thank you for alerting us to this. Taxicum has been amended to Taraxacum.
Comments 10: Add space between of and 16:8 in "photoperiod of16:8. (collection and rearing of hoverflies section)
Response 10: A space has been inserted
Comments 11: Check the font size and style of ‘(Whattmann, 90 mm diameter)’ information. (collection and rearing of hoverflies section)
Response 11: Thank you for raising this. We have updated the font to match the rest of the manuscript and defer to the journal's editorial team for final formatting checks.
Comments 12: 'dispersal form' should be 'dispersal from' the mother's abdomen. (Acute toxicity bioassays section)
Response 12: Thank you for highlighting this. 'disperal form' has been updated to 'dispersal from'
Comments 13: Why did Abbott’s correction not applied? (Statistical Analysis)
Response 13: Thank you for your comment. We are aware that applying Abbott's correction is a common practice within the field of pesticide toxicity. In this case, we decided not to apply Abbott's correction for two reasons.
Firstly, as stated in the 'Statistical analysis' section of the manuscript, the mean mortality in negative controls was below 10% (1.36% average for wolf spiders and 7.46% average for hoverflies), which is generally considered sufficiently low mortality to not warrant Abbott's correction. During preliminary analyses we did apply Abbott's correction to the dataset, and it had very minimal impact on treatment % mortalities due to the low negative control mortality. This does not alter the overall conclusions of the paper in any manner.
Secondly, given the above we deemed it to be more informative to simply plot the raw, uncorrected data and focus our analyses on statistical differences between treatment and negative control mortality. Conducting our analyses in this manner accounts for any differences in negative controls between the two species. Further, we only draw statistical significance within species.
Comments 14: Wolf Spiders: 'were the highly toxic' should be 'were highly toxic'. (Results section)
Response 14: 'the' has been removed (Results section, 3.1 wolf spiders)
Comments 15: The authors mentioned the increased vulnerability of soft-bodied, legless hoverfly larvae to chemical residues. However, this point is only briefly touched upon. A slightly fuller explanation linking larval behaviour (e.g., crawling over treated surfaces) to exposure risk would strengthen the argument. (Discussion)
Response 15: Thank you for your suggestion. However, we have deliberately chosen not to labour this argument as it is largely speculative based trends observed in previous work. The focus of our study was simply to test the acute toxicity of insecticides towards hoverflies and spiders, rather than an investigation of the underlying causes of differences in toxicity. To reflect this, we have instead added the phrase "while further studies are needed to confirm this" to the Discussion. The full passage now reads: "As legless, soft-bodied organisms, Melangyna sp. larvae may have greater vulnerability due to increased contact with chemical residues, as suggested for other hoverfly species [24]. While further study is needed to confirm this, our findings indicate that local CBC strategies should be particularly cautious in applying chemicals when hoverfly larvae are seasonally active in crops (e.g., September-October in Victoria, Australia). "
Comments 16: 'Whatever the reason' is too informal and lazy sounding for science writing. It is better to say 'Given these findings' or just skip and go straight to your point. (Discussion)
Response 16: The penultimate paragraph of the discussion has been amended as described above (Response 15)
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
good job with your spiders and flies!
I have only one "objection" - against term "gravid female" in spiders for those females carrying egg-sack of spiderlings on her body. Term "gravid" must be used for female with eggs/embryos INSIDE her body, not outside. So gravid females are OK, but such females are just "preparing" to lay eggs. I do not know short term for your situation, but I think "egg-sack/larvae carrying females" must be used. (or mother-spider ;))
Applied research in agroecosystem conservation needs to test the effect of pesticides on target and non-target organisms. Information on the impact on non-target organisms is usually quite poor; it is in the interest of manufacturers to report on their safety. More detailed studies are therefore needed to verify the promised effect. The main question of the test is to check the effect of specific pesticides on specific beneficial invertebrates that we want to KEEP in the crop (natural enemies = predators of pests).
Two model beneficial species and about 20 pesticides are selected. The study describes how they bred the model species and how they tested for mortality. As I have written several times - this is nothing groundbreaking or surprising, but it is honest useful research. The work is useful in its practical application - how to breed suitable model species and how they tolerate pesticide treatment of the crop. This information is valuable for practice. The methodology is OK, the conclusions are supported by the data - all the experiments were done and the statistical tests are adequately described. The table is formatted very awkwardly - but I assume that further editing of the text in the editorial office will eliminate this. There are a lot of columns, the text doesn't fit in the cells, it is worth considering orienting the table in landscape orientation, or splitting it up (spider and fly separately). The graphs are clear, BUT as I forgot to mention yesterday, they could be improved. Missing from the graph is the indicated mortality of the control group, the variability of which is used to explain the increased mortality for, for example, Bt. I believe that this more detailed description will help you to decide adequately on the further fate of the submitted manuscript.
Author Response
Comments 1: I have only one "objection" - against term "gravid female" in spiders for those females carrying egg-sack of spiderlings on her body. Term "gravid" must be used for female with eggs/embryos INSIDE her body, not outside. So gravid females are OK, but such females are just "preparing" to lay eggs. I do not know short term for your situation, but I think "egg-sack/larvae carrying females" must be used. (or mother-spider ;))
Response 1: Thank you for highlighting this distinction. In our study we reared spiderlings from both gravid females and females carrying external egg-sacs. As there is no obvious catch-all term for this, we have amended paragraph 1 of the 'collection and rearing of wolf spiders' section as follows: "Adult female Venatrix spp. were collected from parks and gardens in Melbourne (Victoria, Australia) between September and January in 2021/22 and 2023/24. During this time, it is common to observe females carrying egg-sacs or hatched spiderlings on their abdomens in clutches of 100-200, and those not visibly harboring offspring often later produce egg-sacs in the laboratory if gravid (R. Knapp, pers. obs.). Laboratory culturing of gravid or egg-sac bearing females therefore provides an effective method of obtaining large numbers of juveniles for laboratory studies."
Comments 2: The table is formatted very awkwardly - but I assume that further editing of the text in the editorial office will eliminate this. There are a lot of columns, the text doesn't fit in the cells, it is worth considering orienting the table in landscape orientation, or splitting it up (spider and fly separately).
Response 2: We agree that the table should have a landscape orientation as in the originally submited documents. We refer to the journal's editorial team on this.
Comments 3: The graphs are clear, BUT as I forgot to mention yesterday, they could be improved. Missing from the graph is the indicated mortality of the control group, the variability of which is used to explain the increased mortality for, for example, Bt.
Response 3: Negative controls (Water) are already displayed in the graphs and Table 1. All statistical tests of treatment mortality have been performed and discussed in relation to negative control mortality. To make this clearer, we have updated the legend of Figure 1 to read: "Percent mortality of hoverflies (Melangyna sp.) and wolf spiders (Venatrix spp.) after 48 h direct exposure to sprayed chemical residues in laboratory bioassays, where water is the negative control. "
