Temperature-dependent development of the blow fly Chrysomya pinguis and its significance in estimating postmortem interval

Chrysomya pinguis (Walker) (Diptera: Calliphoridae) is an endemic Asiatic blow fly species of forensic importance. Chrysomya pinguis is one of the first species to colonize a corpse, especially in high altitude areas during spring and autumn when the ambient temperature is lower. Despite its potential for forensic investigations to estimate the minimum postmortem interval (PMImin), little is known about the development of C. pinguis. In this study, C. pinguis was collected from the Yangtze River Delta region of China and reared at seven constant temperatures between 16°C and 34°C to investigate the effect of temperature on development duration, accumulated degree hours and larval body length of C. pinguis. Isomorphen and isomegalen diagrams for C. pinguis were generated using the results, and equations describing the variation in larval body length during development and the temperature-induced variation in development time were also obtained. Chrysomya pinguis can complete its life cycle at 16–34°C. The mean (±s.d.) developmental durations of C. pinguis from egg to adult at 16°C, 19°C, 22°C, 25°C, 28°C, 31°C and 34°C were 811.0 ± 3.8, 544.8 ± 2.0, 379.8 ± 1.8, 306.7 ± 2.4, 250.0 ± 2.8, 203.2 ± 2.1 and 185.3 ± 1.6 h, respectively. The mean (±s.e.) developmental threshold temperature D0 and the thermal summation constant K of the whole developmental process of C. pinguis were estimated as 10.88 ± 0.21°C and 4256.50 ± 104.50 degree hours, respectively. This study provides fundamental development data for the use of C. pinguis to estimate PMImin.

If one wants to use one variable to predict another, the relationship between the two must be specified. So to permit the use of instar to predict age, one needs to show the number of individuals in each life stage observed at each age. In other words, for each temperature there should be a table in which the rows are sample ages and the columns are life stages. The values should be counts of individuals observed to be that life stage.
Based on my interactions with other forensic entomologists, I anticipate some resistance to deleting Table 1 (as should be done). I will be satisfied if instead of doing that the authors present their data in this format as a supplementary file. This would be much closer to the raw data, and therefore more appropriate, than what is currently in the supplementary file.
MINOR COMMENTS -The statement "the oldest progeny of Calliphorids on the corpse are often used to obtain a minimum postmortem interval (PMImin)" is misleading. In practice it is the oldest individual that was collected that is used to estimate PMImin. Whether or not the set of insects collected from the corpse includes the earliest individual deposited as an egg or larva on the corpse is difficult to know.
-Given that no randomization method was specified, how is it that "Adults were randomly selected form [sic] each cage" and "eight larvae were randomly selected from the dish?" If the sample was not truly random, it may have been biased. (see International journal of Legal Medicine 129:405-410).
-What is the purpose of comparing C. pinguis to C. megacephala? It does not improve the interpretation of these results.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
This manuscript investigates forensically-relevant development data from the blow fly Chrysomya pinguis, which is an important colonizer of human corpses and animal carcasses throughout Asia. It also represents the first developmental dataset for this species, which will no doubt be vital to forensic casework in the countries where this fly is found. My main concerns were focused on the establishment of a pure colony for experiments as well as the experimental procedure itself. More details in this regard would be very helpful.
Major comments: Lines 66 -77: This method of identification is slightly worrisome. Why weren't the adult flies that were originally collected in Suzhou definitively identified before establishing the laboratory colony? If there was even a single gravid female that wasn't C. pinguis (perhaps C. megacephala) used as G0, the laboratory colony would be mixed and the results unreliable. These flies could have been missed during the random identification of adults emerged from eggs of the wild flies. Please add a justification as to why this identification route was taken as well as how you know for certain that you started with a pure colony.
Lines 79 -83: The colony was maintained for over a year before new genetic material was introduced -why? Were any of the experiments performed BEFORE the May 2017 addition to the colony? This would make a big difference in interpreting the results as the colony would be quite inbred, which could affect the rate at which larvae developed. At the very least this deserves some attention in the discussion. Also, please indicate the generation of the colony for each experiment (e.g. G3 for the first round of experiments, G10 for the second round, etc.) in the methods.
Lines 88 -91: If 1000 eggs divided into 5 petri dishes = 1 replicate, how many replicates were used per temperature per round of the experiment? How did you determine that you had 1000 eggs and what did you use to divide/separate the eggs? Was this one large cluster of eggs made from numerous females ovipositing in the same place, or many separate clutches of eggs dispersed on the pork?
There have been several studies in recent years discussing the relevance of population origin in developmental studies used in forensic entomology casework. Specifically, distinct geographic populations of conspecific blow flies can have significantly different developmental rates. It would be good to see a brief discussion of this phenomenon in the manuscript, potentially in a "future directions" aspect of this work.
Minor comments: Line 37: "decomposition process of the ecosystem" reads strangely; change to "decomposition process of animals in most terrestrial ecosystems" Line 49: change "Asia" to "Asian countries" Line 55: change "regard" to "regarded" Line 74: Remove "of" Line 75: Change "form" to "from" Line 86: How long were females protein-fed before inducing oviposition? Line 96: The order here is confusing. Talk about the eggs and hatching before talking about the larvae in the previous paragraph. Lines 97 -99: Were eggs randomly assigned to each dish in the first place? How do you know that you didn't place a clutch of eggs from a single female in its own dish rather than a random sample of eggs from the initial 1000 eggs? Line 104: Was each round of experiments performed for all temperatures simultaneously? Lines 140: Paragraphs should be > 3 sentences. I would just combine this section into one paragraph.

19-Jun-2019
Dear Dr Wang, The editors assigned to your paper ("Temperature-dependent development of the blow fly Chrysomya pinguis and its significance in estimating postmortem interval") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper before 12-Jul-2019. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available, we may invite new reviewers.
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• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data have been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that have been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-190003 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) Chrysomya pinguis is a widespread and common blow fly. Published development data for C. pinguis are not available and could be very useful to death investigators. The manuscript is generally well written , and it potentially merits publication if the authors can revise the methods and data presentation.

MAJOR COMMENTS
-The sampling scheme is not clear to me. As I understand it, for a single replicate at one temperature, five petri dishes, each with ~200 eggs, was set up. Eight larvae were sampled "until the larvae were in the wandering stage." This procedure was repeated five times.
Does each point on Figure 5 represent 8*5 = 40 larvae? Please specify the sample size in the caption.
More detail is needed. If 8 larvae were sampled at each of the ages shown in Figure 5 during each of five replicates, that should be stated, as well as the total number of larvae measured for each age by temperature combination. Was one of the five petri dishes randomly selected at each age? Looking at 19 C, >70 ages were sampled, implying that > 500 of the original ~1000 (if no mortality, the proportion removed was probably greater) larvae were removed before the final sample. This could have considerably altered the growing conditions of the larvae, and should be discussed.
-The format of table 1 repeats a common mistake in the forensic entomology literature. In most of science, if an author publishes a mean and standard deviation, the study involved several measurements of that quantity. But in this or any other development study, the duration of a larval instar was not observed for any individual insect. The table deceives the reader. Furthermore, the format is not useful for the main purpose of this research, insect age estimation, because it does not show the proportion of each life stage observed as a function of age.
If one wants to use one variable to predict another, the relationship between the two must be specified. So to permit the use of instar to predict age, one needs to show the number of individuals in each life stage observed at each age. In other words, for each temperature there should be a table in which the rows are sample ages and the columns are life stages. The values should be counts of individuals observed to be that life stage.
Based on my interactions with other forensic entomologists, I anticipate some resistance to deleting Table 1 (as should be done). I will be satisfied if instead of doing that the authors present their data in this format as a supplementary file. This would be much closer to the raw data, and therefore more appropriate, than what is currently in the supplementary file.

MINOR COMMENTS
-The statement "the oldest progeny of Calliphorids on the corpse are often used to obtain a minimum postmortem interval (PMImin)" is misleading. In practice it is the oldest individual that was collected that is used to estimate PMImin. Whether or not the set of insects collected from the corpse includes the earliest individual deposited as an egg or larva on the corpse is difficult to know.
-Given that no randomization method was specified, how is it that "Adults were randomly selected form [sic] each cage" and "eight larvae were randomly selected from the dish?" If the sample was not truly random, it may have been biased. (see International journal of Legal Medicine 129:405-410).
-What is the purpose of comparing C. pinguis to C. megacephala? It does not improve the interpretation of these results.

Reviewer: 2
Comments to the Author(s) This manuscript investigates forensically-relevant development data from the blow fly Chrysomya pinguis, which is an important colonizer of human corpses and animal carcasses throughout Asia. It also represents the first developmental dataset for this species, which will no doubt be vital to forensic casework in the countries where this fly is found. My main concerns were focused on the establishment of a pure colony for experiments as well as the experimental procedure itself. More details in this regard would be very helpful.
Major comments: Lines 66 -77: This method of identification is slightly worrisome. Why weren't the adult flies that were originally collected in Suzhou definitively identified before establishing the laboratory colony? If there was even a single gravid female that wasn't C. pinguis (perhaps C. megacephala) used as G0, the laboratory colony would be mixed and the results unreliable. These flies could have been missed during the random identification of adults emerged from eggs of the wild flies. Please add a justification as to why this identification route was taken as well as how you know for certain that you started with a pure colony.
Lines 79 -83: The colony was maintained for over a year before new genetic material was introduced -why? Were any of the experiments performed BEFORE the May 2017 addition to the colony? This would make a big difference in interpreting the results as the colony would be quite inbred, which could affect the rate at which larvae developed. At the very least this deserves some attention in the discussion. Also, please indicate the generation of the colony for each experiment (e.g. G3 for the first round of experiments, G10 for the second round, etc.) in the methods.
Lines 88 -91: If 1000 eggs divided into 5 petri dishes = 1 replicate, how many replicates were used per temperature per round of the experiment? How did you determine that you had 1000 eggs and what did you use to divide/separate the eggs? Was this one large cluster of eggs made from numerous females ovipositing in the same place, or many separate clutches of eggs dispersed on the pork?
There have been several studies in recent years discussing the relevance of population origin in developmental studies used in forensic entomology casework. Specifically, distinct geographic populations of conspecific blow flies can have significantly different developmental rates. It would be good to see a brief discussion of this phenomenon in the manuscript, potentially in a "future directions" aspect of this work.
Minor comments: Line 37: "decomposition process of the ecosystem" reads strangely; change to "decomposition process of animals in most terrestrial ecosystems" Line 49: change "Asia" to "Asian countries" Line 55: change "regard" to "regarded" Line 74: Remove "of" Line 75: Change "form" to "from" Line 86: How long were females protein-fed before inducing oviposition? Line 96: The order here is confusing. Talk about the eggs and hatching before talking about the larvae in the previous paragraph. Lines 97 -99: Were eggs randomly assigned to each dish in the first place? How do you know that you didn't place a clutch of eggs from a single female in its own dish rather than a random sample of eggs from the initial 1000 eggs? Line 104: Was each round of experiments performed for all temperatures simultaneously?
Lines 140: Paragraphs should be > 3 sentences. I would just combine this section into one paragraph.

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The authors have adequately addressed my concerns with one exception. They claim to have sampled larvae randomly from a rearing container. When reviewing the original manuscript, I questioned this claim "Given that no randomization method was specified." In the revised manuscript (line 98), the authors again describe sampling as random, but they do not describe any randomization procedure. It seems that sampling of larvae was not random, and because of the profound importance of an unbiased sample in so many aspects of science, the authors should not make this statement.
It is not possible for a human to reach into a petri dish and randomly sample maggots. An explicit randomization procedure would be required, which in this experiment would entail something like removing all larvae from the dish, giving each a computer-generated random number, and selecting the maggots with the 8 highest numbers. The unsampled larvae would be too disturbed to be used for older sample ages.

29-Jul-2019
Dear Dr Wang: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-190003.R1 entitled "Temperature-dependent development of the blow fly Chrysomya pinguis and its significance in estimating postmortem interval" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
• Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-190003.R1 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Please note that we cannot publish your manuscript without these end statements included. We have included a screenshot example of the end statements for reference. If you feel that a given heading is not relevant to your paper, please nevertheless include the heading and explicitly state that it is not relevant to your work.
Because the schedule for publication is very tight, it is a condition of publication that you submit the revised version of your manuscript before 07-Aug-2019. Please note that the revision deadline will expire at 00.00am on this date. If you do not think you will be able to meet this date please let me know immediately.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) The authors have adequately addressed my concerns with one exception. They claim to have sampled larvae randomly from a rearing container. When reviewing the original manuscript, I questioned this claim "Given that no randomization method was specified." In the revised manuscript (line 98), the authors again describe sampling as random, but they do not describe any randomization procedure. It seems that sampling of larvae was not random, and because of the profound importance of an unbiased sample in so many aspects of science, the authors should not make this statement.
It is not possible for a human to reach into a petri dish and randomly sample maggots. An explicit randomization procedure would be required, which in this experiment would entail something like removing all larvae from the dish, giving each a computer-generated random number, and selecting the maggots with the 8 highest numbers. The unsampled larvae would be too disturbed to be used for older sample ages.

09-Aug-2019
Dear Dr Wang, I am pleased to inform you that your manuscript entitled "Temperature-dependent development of the blow fly Chrysomya pinguis and its significance in estimating postmortem interval" is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published. Revised portion are marked in red in the paper. Our responses to the comments are as following: #Reviewer: 1

Comments to the Author(s):
Chrysomya pinguis is a widespread and common blow fly. Published development data for C. pinguis are not available and could be very useful to death investigators. The manuscript is generally well written, and it potentially merits publication if the authors can revise the methods and data presentation.
Response: Thanks very much for your time on revising our manuscript. We have revised the manuscript according to the recommendation.
MAJOR COMMENTS: 1. The sampling scheme is not clear to me. As I understand it, for a single replicate at one temperature, five petri dishes, each with ~200 eggs, was set up. Eight larvae were sampled "until the larvae were in the wandering stage." This procedure was repeated five times.
Does each point on Figure 5 represent 8*5 = 40 larvae? Please specify the sample size in the caption.
Response: In our study, for a single replicate at one temperature, five petri dishes, each dishes with ~200 eggs, was set up. Eight larvae were sampled from 1000 larvae every 4 hours until the larvae were in the wandering stage. This procedure was repeated five times. The reason why we separated 1000 eggs into 5 portions is because we sampled every 4 hours, so a large numbers of larvae are needed for sampling. In order to reduce the larval mass effect caused by the over-crowded larvae, the 1000 eggs were separated into 5 portions. We have clarified this in the caption of Figure 5 2. More detail is needed. 1) If 8 larvae were sampled at each of the ages shown in Figure 5 during each Appendix A of five replicates, that should be stated, as well as the total number of larvae measured for each age by temperature combination. 2) Was one of the five petri dishes randomly selected at each age? 3) Looking at 19 °C, >70 ages were sampled, implying that > 500 of the original ~1000 (if no mortality, the proportion removed was probably greater) larvae were removed before the final sample. This could have considerably altered the growing conditions of the larvae, and should be discussed. (2) Yes, the dish was randomly selected. The dishes were numbered (marked 1, 2, 3, 4, 5). Before sampling, a series of random numbers were generated by a random number generator. The random numbers will determine which dish will be used for sampling.

Response
(3) We have added a discussion regarding the effect sampling methods on the development, and the details are as follows: In forensic entomology development study, in order to record the body length and/or instar of a species, their larvae were usually reared at different temperatures, and individuals were removed periodically (usually without replacement) [38-41]. There are usually two sampling strategies. One is to select a narrower sampling interval at higher temperatures and a wider sampling interval at lower temperatures. The other is to select fixed sampling interval, just like the sampling method we used in the study. Though the method we used is more convenient as we don't need to change our sampling interval at different temperature, it usually consumes more samples, especially at lower temperature. For example, over half of the larvae (> 500) were sampled from the origin cohort at 16°C in this study, which may have a potential effect on the development of C. pinguis. An evaluation of sampling methods study by Wells et al. [42] revealed that random subsamples would produce a predictive model similar to that of the full data set, while subsampling the largest larvae produced a predictive model that performed poorly.
The larvae were randomly sampled in this study, which means that our sampling method may have little effect on the development. Further studies are needed to verify this.
science, if an author publishes a mean and standard deviation, the study involved several measurements of that quantity. But in this or any other development study, the duration of a larval instar was not observed for any individual insect. The table deceives the reader.
Furthermore, the format is not useful for the main purpose of this research, insect age estimation, because it does not show the proportion of each life stage observed as a function of age.
If one wants to use one variable to predict another, the relationship between the two must be specified.  5. Given that no randomization method was specified, how is it that "Adults were randomly selected form [sic] each cage" and "eight larvae were randomly selected from the dish?" If the sample was not truly random, it may have been biased. (see International journal of Legal Medicine 129:405-410).
Response: For larvae sampling, many authors collected subsamples that were clearly biased in that they targeted the largest individuals in the rearing container. In our study, eight larvae were randomly sampled from larval mass, not considering their size or stage.
For adults sampling, the target adults were male. So the 'randomly' used is not appropriate. We have deleted this word in the revised manuscript. 6. What is the purpose of comparing C. pinguis to C. megacephala? It does not improve the interpretation of these results.
Response: Thanks for your suggestion. We have deleted this confusing section.

#Reviewer: 2
Comments to the Author(s): This manuscript investigates forensically-relevant development data from the blow fly Chrysomya pinguis, which is an important colonizer of human corpses and animal carcasses throughout Asia. It also represents the first developmental dataset for this species, which will no doubt be vital to forensic casework in the countries where this fly is found. My main concerns were focused on the establishment of a pure colony for experiments as well as the experimental procedure itself. More details in this regard would be very helpful.
Response: Thanks for your time on our manuscript. We have further clarified our colony establish methods in the following response.
Major comments: 1. Lines 66 -77: This method of identification is slightly worrisome. Please add a justification as to why this identification route was taken as well as how you know for certain that you started with a pure colony. 1) Why weren't the adult flies that were originally collected in Suzhou definitively identified before establishing the laboratory colony? 2) If there was even a single gravid female that wasn't C.
pinguis (perhaps C. megacephala) used as G0, the laboratory colony would be mixed and the results unreliable. These flies could have been missed during the random identification of adults emerged from eggs of the wild flies.
Response: We have confidence that the colony is pure, because (i) though adult C. pinguis has a very similar morphology with C. megacephala, they were easy to distinguish as C. pinguis has brilliant blue body color and gray bucca with black hairs. Eight female adults collected in Suzhou were identified as C. pinguis were used to establish colony. The identification conducted subsequently was used to revalidate using the male genitalia as the most reliable identification is based on the male genitalia differences between species. During the identification, all adults are identified as C. pinguis. (ii) The posterior spiracles of larval C. megacephala and C. pinguis are different. The distance between posterior spiracles in C. megacephala were much narrower than C. pinguis (Fig. 1). During the instar determination, all larvae examined are C. pinguis. After a long-term exploration, we found if we used a plastic bottle with pork to induce eggs, the numbers of eggs production will be increased. By this time, the Chinese Spring Festival is coming, so we reared the larvae at 19 °C to maintain the colony. In the second year, larvae were introduced to prevent the effect of inbred on development. After adults reached at a density of approximately 4000 adults, the study was carried out.
3. Lines 88 -91: 1) If 1000 eggs divided into 5 petri dishes = 1 replicate, how many replicates were used per temperature per round of the experiment? 2) How did you determine that you had 1000 eggs and what did you use to divide/separate the eggs? 3) Was this one large cluster of eggs made from numerous females ovipositing in the same place, or many separate clutches of eggs dispersed on the pork?
Response: 1) In the experiment, in order to prevent larval-mass effect, we divided 1000 eggs collected in 1 hours into five groups and placed them in five petri dishes, and then put the five petri dishes into the same breeding box as a repetition. The eggs collected in 1 hours were only used in one repetition of one temperature, with a total of 7 temperatures, and each temperature was repeated 5 times.
2) The egg masses were weighed by an electronic balance with a precision of 0.0001 g, but it is only a rough estimation. The egg masses were divided by banister brush under stereoscopic microscope.
3) Two plastic bottles of 10 cm in height and 8 cm in diameter containing fresh lean pork were placed in the insect-rearing cage to induce oviposition. Eggs were collected in 1h from many separate clutches of eggs dispersed on the pork in the two plastic bottles above. Then, approximately 1000 eggs were estimated and divided by banister brush from these eggs and used as a replicate. 12. Lines 97 -99: 1) Were eggs randomly assigned to each dish in the first place? 2) How do you know that you didn't place a clutch of eggs from a single female in its own dish rather than a random sample of eggs from the initial 1000 eggs?
Response: 1) The egg masses were weighed by an electronic balance with a precision of 0.0001 g, but it is only a rough estimation. The egg masses randomly were divided by banister brush under stereoscopic microscope.
2) It is hard to know whether a clutch of eggs from a single female or not. But during the experiment, the egg masses were mixed before dividing them into different dishes.