The Effect of Temperature and Moisture Content on Population Growth of Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

Round 1

Reviewer 1 Report

First of all, I would like to thank the authors for such an interesting study. In this context, authors have carried out research to determine the effects of temperature and humidity interactions required for the control of this pest, as well as its progeny production. The subject is important and instructive for researchers working in this field. The materials and methods used are appropriate and the findings are presented in a simple, understandable, and reproducible manner. Statistical analysis is appropriate and the figures are clear. In the Discussion section, a comprehensive discussion covering a wide period (1930-2022) is provided with detailed citations.

This study explores how temperature and moisture levels in the rearing environment influence the population growth of the lesser mealworm, Alphitobius diaperinus. The research reveals that temperature significantly affects adult mortality, with optimal larval development and adult survival occurring at 25 to 32°C. Additionally, higher moisture content in the rearing wheat is linked to a gradual reduction in larval numbers. These insights provide valuable data for enhancing pest control methods in poultry farms and establishing effective standards for large-scale rearing of A. diaperinus as a valuable food and feed resource.

Temperature and Moisture Range: The authors should consider expanding the range of temperature and moisture levels tested in the experiments. This would provide a more comprehensive understanding of the optimal conditions for the growth and progeny production of A. diaperinus.
Replication: It would be beneficial for the authors to increase the number of replicates for each experimental condition. This would enhance the statistical robustness of the results and improve the reliability of the findings.
Control Group: The authors should include a control group in their experimental design. This would allow for a comparison between the treatment groups and the control group, providing a baseline for evaluating the effects of temperature and moisture on the development and progeny production of A. diaperinus.
Further Controls to Consider:
Substrate Quality: It would be important for the authors to ensure consistent substrate quality throughout the experiments. Variations in substrate quality could potentially confound the results and lead to inaccurate conclusions.
Environmental Conditions: The authors should carefully monitor and control other environmental factors such as light intensity, photoperiod, and air circulation. These factors can influence the growth and development of insects and should be kept constant to minimize their potential effects on the experimental outcomes.
Data Collection and Analysis: The authors should establish clear protocols for data collection and analysis to ensure accuracy and consistency. This would involve standardizing methods for measuring variables such as progeny production, larval growth, and adult survival.
Long-term Observations: It would be valuable for the authors to conduct long-term observations to assess the effects of temperature and moisture on the population dynamics of A. diaperinus. This would provide insights into the species' ability to adapt and thrive under different conditions over extended periods of time.
Overall, by implementing these improvements in methodology and considering the suggested further controls, the authors can enhance the reliability and validity of their findings, leading to a more comprehensive understanding of the growth and reproductive capabilities of A. diaperinus.
I find it more appropriate in my own studies that the results obtained are given in tables instead of figures. During the presentation of the data, especially in the data related to mortality, I think that although the column bars give the necessary effect on the reader at first glance, they are insufficient in detail. therefore, it would be more accurate to give the figures as a table.

 

Author Response

Comments and Suggestions for Authors

Reviewer 1

Comment: First of all, I would like to thank the authors for such an interesting study. In this context, authors have carried out research to determine the effects of temperature and humidity interactions required for the control of this pest, as well as its progeny production. The subject is important and instructive for researchers working in this field. The materials and methods used are appropriate and the findings are presented in a simple, understandable, and reproducible manner. Statistical analysis is appropriate and the figures are clear. In the Discussion section, a comprehensive discussion covering a wide period (1930-2022) is provided with detailed citations.

This study explores how temperature and moisture levels in the rearing environment influence the population growth of the lesser mealworm, Alphitobius diaperinus. The research reveals that temperature significantly affects adult mortality, with optimal larval development and adult survival occurring at 25 to 32°C. Additionally, higher moisture content in the rearing wheat is linked to a gradual reduction in larval numbers. These insights provide valuable data for enhancing pest control methods in poultry farms and establishing effective standards for large-scale rearing of A. diaperinus as a valuable food and feed resource.

Authors’ reply: We thank the reviewer for the time taken to carefully read the manuscript, for their kind comments, as well as for their constructive remarks.

Comment: Temperature and Moisture Range: The authors should consider expanding the range of temperature and moisture levels tested in the experiments. This would provide a more comprehensive understanding of the optimal conditions for the growth and progeny production of A. diaperinus.

Authors’ reply: The purpose of this study was to evaluate the development and progeny production capacity of A. diaperinus under a range of temperatures. Since it is known that the exposure to temperatures above 40^oC for prolonged periods are lethal to insects, the examination of higher temperatures would be rather pointless. This is true, given that at 40^oC we found 100% adult mortality and 0 progeny production.

Comment: Replication: It would be beneficial for the authors to increase the number of replicates for each experimental condition. This would enhance the statistical robustness of the results and improve the reliability of the findings.

Authors’ reply: We designed the protocol of this study based on previous experiments conducted with other stored product pests, in which the authors use from 6 to 9 replicates. Some references are provided below. However, we will keep in mind to increase the replicates in our following experiments.

1. Athanassiou, C.G.; Vayias, B.J.; Dimizas, C.B.; Kavallieratos, N.G.; Papagregoriou, A.S.; Buchelos, C.T. Insecticidal efficacy of diatomaceous earth against Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and Tribolium confusum Du Val (Coleoptera: Tenebrionidae) on stored wheat: Influence of dose rate, temperature and exposure interval. J. Stored Prod. Res. 2005, 41, 47–55.
2. Nayak, M.K.; Collins, P.J. Influence of concentration, temperature and humidity on the toxicity of phosphine to the strongly phosphine-resistant psocids Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae). Pest Manag. Sci. 2008, 64, 971–976.
3. Arthur, F.H. Residual efficacy of deltamethrin as assessed by rapidity of knockdown of Tribolium castaneum on a treated surface: Temperature and seasonal effects in field and laboratory settings. J. Stored Prod. Res. 2018, 76, 151–160. [

Comment: Control Group: The authors should include a control group in their experimental design. This would allow for a comparison between the treatment groups and the control group, providing a baseline for evaluating the effects of temperature and moisture on the development and progeny production of A. diaperinus.

Authors’ reply: Since the experiment was conducted on factors that are suitable for the optimal growth and reproduction of A. diaperinus (see references below for commodity and abiotic conditions), we believe that an alternative control (for example to change the commodity from wheat to another cereal) would be misleading to the aim of this study.

1. Kotsou, K.; Rumbos, C.I.; Baliota, G.V.; Gourgouta, M.; Athanassiou, C.G. Influence of Temperature, Relative Humidity and Protein Content on the Growth and Development of Larvae of the Lesser Mealworm, Alphitobius diaperinus (Panzer). Sustainability 2021, 13, 11087. https://doi.org/10.3390/ su131911087
2. Rueda, L.M.; Axtell, R.C. Temperature-Dependent Development and Survival of the Lesser Mealworm, Alphitobius diaperinus. Med. Vet. Entomol. 1996, 10, 80–86.

Comment: Substrate Quality: It would be important for the authors to ensure consistent substrate quality throughout the experiments. Variations in substrate quality could potentially confound the results and lead to inaccurate conclusions.

Authors’ reply: We have carefully prepared the commodity on which the insects were developed, giving special attention to factors such as the particle size (see experimental design in Materials and Methods). The same batch of wheat bran was used for the whole experiment.

Comment: Environmental Conditions: The authors should carefully monitor and control other environmental factors such as light intensity, photoperiod, and air circulation. These factors can influence the growth and development of insects and should be kept constant to minimize their potential effects on the experimental outcomes.

Authors’ reply: The whole experiment conducted to incubators under controlled conditions. The incubators (which were identical regarding their type, model and year of purchase) were in the same room, with a photoperiod of 0: 24h light: dark.

Comment: Data Collection and Analysis: The authors should establish clear protocols for data collection and analysis to ensure accuracy and consistency. This would involve standardizing methods for measuring variables such as progeny production, larval growth, and adult survival.

Authors’ reply: We added the software that we used for statistical comparisons, see the reference at the end of the statistical analysis paragraph.

Comment: Long-term Observations: It would be valuable for the authors to conduct long-term observations to assess the effects of temperature and moisture on the population dynamics of A. diaperinus. This would provide insights into the species' ability to adapt and thrive under different conditions over extended periods of time. Overall, by implementing these improvements in methodology and considering the suggested further controls, the authors can enhance the reliability and validity of their findings, leading to a more comprehensive understanding of the growth and reproductive capabilities of A. diaperinus.

Authors’ reply: We totally agree with the reviewer. For this reason we have also incorporated the evaluation of the progeny production after 50 d of incubation. These suggestions will be saved for future experimentation.

I find it more appropriate in my own studies that the results obtained are given in tables instead of figures. During the presentation of the data, especially in the data related to mortality, I think that although the column bars give the necessary effect on the reader at first glance, they are insufficient in detail. Therefore, it would be more accurate to give the figures as a table.

Authors’ reply: We appreciate the reviewer's insightful comments. However, we wish to maintain the same format. If the reviewer or editor deems it essential to substitute figures with tables, we will do so.

Reviewer 2 Report

Dear Authors,

The detailed comments are given in MS file. 
Regards

Comments for author File: Comments.pdf

Minor editing of English language is required.

Author Response

Comments and Suggestions for Authors

Reviewer 2

Comment: Add two background lines.  

Authors’ reply: Done. See the first 5 lines in the Abstract.

Comment: What does it mean by rearing media?

Authors’ reply: We change it to commodity for better understanding.

Comment: Source or level?

Authors’ reply: It is moisture source, as the type of the material that will be introduced in the rearing (agar, potato etc) is also significant.

Comment: moisture contents and RH are different and may have different effects? please give clear background about moisture contents?

Authors’ reply: We corrected the phrase.

Comment: Better to give exact age.

Authors’ reply: Adults were collected from the stock cultures of LEAZ over a period of one week (7d). Thus, their age varying from day 1 to day 7.

Comment: What type of brush?

Authors’ reply: We used only a regular brush for painting.

Comment: ratio?

Authors’ reply: We do not know the ratio, since we collected random individuals from the stock colonies.

Comment: was moisture measured during the experiment? it is possible that different T affected the MC of the grains?

Authors’ reply: The MC values were monitored only once, at the start of the experiment, as it has been described in M&M. No further evaluation of the MC inside the vials was took place. Thus, the addition of carrot slices could increase the MC of the wheat, a factor that was not taken into consideration in this study. We have added a short description, see corresponding lines.

Comment: You need a reference here and how much water was sprayed to these Treatments? how did you spray the water? Oven make and model? did you measure the MC% after the spray of water?

Authors’ reply: We completely changed the description of the procedure we followed to obtain the MC values, adding more details according to the reviewer’s comments. See corresponding lines in M&M.

 

Comment: ratio?

Authors’ reply: We do not know the ratio, since we collected random individuals from the stock colonies.

Comment: same weight of carrot? here you are using different MC levels? Ref? why did you use carrot?

Authors’ reply: We corrected the weight of the carrot slices and we added a reference regarding the use of carrot as a moisture source.

Comment: why 9 and 6 replicates ?

Authors’ reply: The protocol for this study was developed by relying upon previous experiments conducted with different stored product pests, wherein the authors employed 9 replicates. The following references are supplied for further consultation. However, during the bioassays involving various MC values, an incident occurred towards the conclusion of the experimentation process, affecting three vials of 5% MC. Consequently, an equal number of treatments were removed for all MC values for the sake of statistical integrity.

1. Athanassiou, C.G.; Vayias, B.J.; Dimizas, C.B.; Kavallieratos, N.G.; Papagregoriou, A.S.; Buchelos, C.T. Insecticidal efficacy of diatomaceous earth against Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and Tribolium confusum Du Val (Coleoptera: Tenebrionidae) on stored wheat: Influence of dose rate, temperature and exposure interval. J. Stored Prod. Res. 2005, 41, 47–55.
2. Nayak, M.K.; Collins, P.J. Influence of concentration, temperature and humidity on the toxicity of phosphine to the strongly phosphine-resistant psocids Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae). Pest Manag. Sci. 2008, 64, 971–976.
3. Arthur, F.H. Residual efficacy of deltamethrin as assessed by rapidity of knockdown of Tribolium castaneum on a treated surface: Temperature and seasonal effects in field and laboratory settings. J. Stored Prod. Res. 2018, 76, 151–160. [

Comment: why did you use the different tests for the same parameters in response to T and MC? can it not be used the same tests?

Authors’ reply: The test was determined after conducting the Shapiro-Wilk test in the data dor each bioassay (temperature or MC). Thus, Kruskal-Wallis was followed for non-parametric comparisons while ANOVA for parametric, as has been indicated by the Shapiro-Wilk test.

Comment: is progeny per vial valid? because there were different mortalities and remaining adults were also different need a literature support?

Authors’ reply: We calculated the mean progeny per vial as it has been done in many other researches of stored product pests, see references below.

1. Lampiri E.; Baliota, G.V.; Morrison, W.R., III.; Domingue M.J.; Athanassiou, C.G.; 2021 Comparative Population Growth of the Khapra Beetle (Coleoptera: Dermestidae) and the Warehouse Beetle (Coleoptera: Dermestidae) on Wheat and Rice. Journal of Economic Entomology 1–9. https://doi.org/10.1093/jee/toab209
2. Gkourgkouta M.; Baliota, G.V.; Morrison, W.R., III.; Domingue M.J.; Athanassiou, C.G.; 2021. Comparative Capture of Trogoderma granarium (Coleoptera: Dermestidae) and T. variabile in Floor Traps in Single Species Releases With Previously Captured Conspecific or Heterospecific Individuals. Journal of Economic Entomology, 114(6), 2021, 2591–2597 https://doi.org/10.1093/jee/toab174

Reviewer 3 Report

M&M

The MC values in the rearing medium in the experiments (MC) I assume are the initial values. Has the loss of MC in the culture medium been monitored during the course of the experiments? In accordance with what is indicated in material and methods, this has not been taken into account.

L119. Indicate the units (ºC) for all temperature values

L128. Indicate units (%) for all relative humidity values

L119, L132, L133. MC values by MC levels (check this throughout the manuscript, as it is maintained even in the conclusions).

Results

L155. (Figure 1) Temperature values per temperature levels. It is not necessary to repeat each and every one of the temperature values in the caption, as they are specified in the graph itself. Likewise with regard to the values of moisture content (%). The same is applicable to Figures 2, 3 and 4.

L162. 25ºC and 32ºC. Analogously in L163

L165 values instead of levels (revise this in the rest of the script)

Discusión

The results indicate that for adults the mortality rate increases above 35ºC, and is minimum between 25ºC and 32ºC (L147). On the other hand, the MC values tested did not seem to affect their mortality (L150-152). They point out something similar for the larvae, indicating similar mortality values in all MC values tested (L164-166). However, apparently this does not seem to agree with what is stated in the abstract and may mislead the reader. Por elli quizás habría que dedicarle mas atención en el texto con objeto de dilucidar posibles errores de comprensión. As stated in the abstract, “the moisture content of the wheat that served as rearing media was also a critical factor, since a gradual decrease in the larval numbers was observed with the increase of the moisture”…If this decrease is not the result, could be caused by an increase in egg mortality? Or, if applicable, a lower adult fecundity rate with the increase in MC? It would be interesting to determine the possible influence of moisture content on adult fecundity, to try to provide an explanation to these results, since larval production decreases with increasing moisture content, but this is nor related whith the larval survival rate. Although all this is discussed in lines 260 and following, it would be convenient for the authors to comment about the research would be appropriate to provide some explanations. I eagree that there must be an interdependence between the environmental humidity and the MC content, and therefore the only determination of the initial MC values would be insufficient to deepen the discussion. According to the authors, it highlights the need for additional studies.

Discussion

L265-268. Indeed, this interaction determines the real value of the moisture content in the medium, so it would be interesting to determine the relative humidity inside the vial throughout the experiment for an optimal yield of the production method. This should also be underlined as objet pending investigation.

Author Response

Comments and Suggestions for Authors

Reviewer 3

Comment: The MC values in the rearing medium in the experiments (MC) I assume are the initial values. Has the loss of MC in the culture medium been monitored during the course of the experiments? In accordance with what is indicated in material and methods, this has not been taken into account.

Authors’ reply: The MC values were monitored only once, at the start of the experiment. No further evaluation of the MC inside the vials was took place.

Comment: L119. Indicate the units (ºC) for all temperature values

Authors’ reply: We added the units. See corresponding lines.

Comment: L128. Indicate units (%) for all relative humidity values

Authors’ reply: We added the units. See corresponding lines.

Comment: L119, L132, L133. MC values by MC levels (check this throughout the manuscript, as it is maintained even in the conclusions).

Authors’ reply: Done. We changed the references of MC levels to MC values.

Comment: L155. (Figure 1) Temperature values per temperature levels. It is not necessary to repeat each and every one of the temperature values in the caption, as they are specified in the graph itself. Likewise with regard to the values of moisture content (%). The same is applicable to Figures 2, 3 and 4.

Authors’ reply: We agree with the reviewer and we changed the caption of all figures.

Comment: L162. 25ºC and 32ºC. Analogously in L163

Authors’ reply: Done. See corresponding lines.

Comment: L165 values instead of levels (revise this in the rest of the script)

Authors’ reply: Done. Several revisions are within the text.

Comment: The results indicate that for adults the mortality rate increases above 35ºC, and is minimum between 25ºC and 32ºC (L147). On the other hand, the MC values tested did not seem to affect their mortality (L150-152). They point out something similar for the larvae, indicating similar mortality values in all MC values tested (L164-166). However, apparently this does not seem to agree with what is stated in the abstract and may mislead the reader. As stated in the abstract, “the moisture content of the wheat that served as rearing media was also a critical factor, since a gradual decrease in the larval numbers was observed with the increase of the moisture”…If this decrease is not the result, could be caused by an increase in egg mortality? Or, if applicable, a lower adult fecundity rate with the increase in MC? It would be interesting to determine the possible influence of moisture content on adult fecundity, to try to provide an explanation to these results, since larval production decreases with increasing moisture content, but this is nor related whith the larval survival rate. Although all this is discussed in lines 260 and following, it would be convenient for the authors to comment about the research would be appropriate to provide some explanations. I eagree that there must be an interdependence between the environmental humidity and the MC content, and therefore the only determination of the initial MC values would be insufficient to deepen the discussion. According to the authors, it highlights the need for additional studies.

Authors’ reply: We agree with the comments of the reviewer. Indeed, although we did not observed any significant increase in the adult mortality among the MC values according to the statistical analysis, it was a surprise to us that the progeny production in terms of the emerged larvae was drastically decreased with the increase of MC values. This phenomenon is still unclear to the authors, and we intend to further investigate this factor (moisture content of the commodity) through additional experiments in which the MC values will be continuously monitored throughout the incubation period. 

Comment: L265-268. Indeed, this interaction determines the real value of the moisture content in the medium, so it would be interesting to determine the relative humidity inside the vial throughout the experiment for an optimal yield of the production method. This should also be underlined as objet pending investigation.

Authors’ reply: In response to the reviewer's suggestion, we have incorporated a brief paragraph at the conclusion of the discussion, pertaining to the recommended experimental procedures that should be undertaken in order to further comprehension of this phenomenon.

Reviewer 4 Report

The study confirms previous studies on the effect of temperature on insect development and mortality but I am concerned about the design of experiment on the effects of moisture. What keep 5, 10, 15 and 20% wheat from equillibrating with 55% rh in incubator and the carrot and all of wheat reaching the same moisture content.

Line 16 says 25 to 32^oC and line 279 says 25 to 35^oC.

Line 26 says secondary pest, but Rumbos et al. 2020 says “Hence, A. diaperinus can be indeed considered as an insect that can infest sound grains, and as such, a primary colonizer.”

Line 123 should nymphs be pupae?

Line 124 production could be produced.

Line 212 How should effect temperature and moisture on development and mortality be considered in pest management or rearing protocol?

Figure 1 and 2 do not have any lower case letters.

Figure 3 and 4 should indicate the instar distribution range of small larvae compared to that of large larvae.

see comments

Author Response

Comments and Suggestions for Authors

Reviewer 4

Comment: The study confirms previous studies on the effect of temperature on insect development and mortality but I am concerned about the design of experiment on the effects of moisture. What keep 5, 10, 15 and 20% wheat from equillibrating with 55% rh in incubator and the carrot and all of wheat reaching the same moisture content.

Authors’ reply: This experiment was designed as a first investigation of the effect the moisture content may have to the proper development of A. diaperinus. The MC values were monitored only once, at the start of the experiment, as it has been described in M&M. No further evaluation of the MC inside the vials was took place. Thus, the addition of carrot slices could increase the MC of the wheat, a factor that was not taken into consideration in this study. However, although we did not observed any significant increase in the adult mortality among the MC values according to the statistical analysis, it was a surprise to us that the progeny production in terms of the emerged larvae was drastically decreased with the increase of MC values. This phenomenon is still unclear to the authors, and we intend to further investigate this factor (moisture content of the commodity) through additional experiments in which the MC values will be continuously monitored throughout the incubation period. In response to this and other reviewers’ suggestion, we have incorporated a brief paragraph at the conclusion of the discussion, pertaining to the recommended experimental procedures that should be undertaken in order to further comprehension of this phenomenon.

Comment: Line 16 says 25 to 32^oC and line 279 says 25 to 35^oC.

Authors’ reply: We thank the reviewer for pointing out this error. We corrected it accordingly.

Line 26 says secondary pest, but Rumbos et al. 2020 says “Hence, A. diaperinus can be indeed considered as an insect that can infest sound grains, and as such, a primary colonizer.”

Authors’ reply: We thank the reviewer for pointing out this error. We corrected it accordingly. 

Comment: Line 123 should nymphs be pupae?

Authors’ reply: We have excluded the nymphs of this study, according to the suggestion of other reviewers.

Comment: Line 124 production could be produced.

Authors’ reply: Done. See corresponding line.

Comment: Line 212 How should effect temperature and moisture on development and mortality be considered in pest management or rearing protocol?

Authors’ reply: The effect of each of these abiotic factors, whether they be in the pest management strategy or the rearing procedure, has been discussed in detail throughout the manuscript based on the results of our study compared with data obtained from the literature.

Comment: Figure 1 and 2 do not have any lower case letters.

Authors’ reply: Yes, as it has been pointed out in the captions below each figure, “where no letter are present no significant differences were recorded”

Comment: Figure 3 and 4 should indicate the instar distribution range of small larvae compared to that of large larvae.

Authors’ reply: Since the analysis was conducted using the total larvae (alive early instar and late instar larvae) found in each vial, we believe that the instar distribution should also correspond to the total number of larvae. Thus, we would like to keep the figures as they are. If the reviewer or editor deems it essential to substitute the data, we will do so.

Reviewer 5 Report

Abstract:

L9:  The title suggests that this study is about the effect of temperature and relative humidity on the insect growth and development, but the first line of abstract mentioned something else for example moisture content of rearing media, which is quite different to the relative humidity.

L11: Not a first series, always the first series.

L12: Not 40^oC, which should be like 40 °C.

Comments on abstract: There are numerous grammatical errors, for example ‘a (the) second’. The writing should be clearer and articulative. Should have clear idea between moisture and relative humidity. If moisture content of rearing media was taken into account, the title should be amended with moisture content.

 

Introduction:

L26: Not a (the) secondary.

Introduction comments: The introduction reads ambiguous for me. The researchers must be clear whether they want to develop a protocol of A. diaperinus for the mass rearing purpose or identify the strategic thermal range to deter the insect growth and development. Should emphasise more on how the room temperature, and moisture content on food impact the insect egg laying, larval development, reproduction etc. After all, where is the research gap that you wanted to address in this study?

 

Materials and method:

L101: standard rearing, please specify the standard rearing conditions.

Materials and methods: can be improved on the sentence flow, as the clarity is still lacking.

Results:

The results should focus on the total number of adults and/or larval emergence taken place from the culture and making the comparisons among the temperature or moisture content regimes.  The major objective of this study is to identify the target temperature and moisture content points that favour or deter the lesser mealworm growth and development. But the percentage of adult mortality can divert the study into the opposite direction. As the secondary parameters of this study, the percent larval and adult mortalities, and measurement of insect body weight can be considered, but the number of total progeny emergence must be the first answer for this objective.

Discussion and conclusion:

The wrong interpretation of results carried away the discussion and conclusion in different directions from it is supposed to be. Please rectify the result interpretation and accordingly align the discussion and conclusion as you go ahead.

Some significant grammatical errors were found. More importantly, can be improved on the better sentence structure and flow.

Author Response

Comments and Suggestions for Authors

Reviewer 5

Abstract:

Comment: L9:  The title suggests that this study is about the effect of temperature and relative humidity on the insect growth and development, but the first line of abstract mentioned something else for example moisture content of rearing media, which is quite different to the relative humidity.

Authors’ reply: We are grateful to the reviewer for pointing out our error, and we are completely in agreement with them. We updated the title to reflect the change.

Comment: L11: Not a first series, always the first series.

Authors’ reply: Indeed. We corrected the error.

Comment: L12: Not 40^oC, which should be like 40 °C.

Authors’ reply: Done.

Comment: Comments on abstract: There are numerous grammatical errors, for example ‘a (the) second’. The writing should be clearer and articulative. Should have clear idea between moisture and relative humidity. If moisture content of rearing media was taken into account, the title should be amended with moisture content.

Authors’ reply: We made several changes in the text along with the comments of the reviewer.

Comment: L26: Not a (the) secondary.

Authors’ reply: We deleted the secondary due to concerns of other reviewers.

Comment: Introduction comments: The introduction reads ambiguous for me. The researchers must be clear whether they want to develop a protocol of A. diaperinus for the mass rearing purpose or identify the strategic thermal range to deter the insect growth and development. Should emphasise more on how the room temperature, and moisture content on food impact the insect egg laying, larval development, reproduction etc. After all, where is the research gap that you wanted to address in this study?

Authors’ reply: : The purpose of this experiment was to conduct preliminary research into the potential role that factors such as temperature and relative humidity have in the development of A. diaperinus. This species has the potential to become a pest of stored products, yet it is also utilised as a food and feed purposes. However, there is a shortage of data regarding various aspects of this insect's development and reproduction, data that can be used not only for control but also for mass rearing of this insect. Therefore, the authors of this study are of the opinion that discussing the current situation of this species is required in order to fulfil the objectives of this research. In addition, the literature has not been brought up to speed as of yet, and the majority of the published materials are at least twenty years old, thus addressing these things in passing was regarded as improper. Mention of researches for other species might be considered as irrelevant for this work and will increase more the introduction. As far as the authors are aware, there is no published research that addresses the effects that the temperature of the room and the moisture content of the food have on the laying of insect eggs, the growth of larvae, or any other aspect of insect reproduction of this species. Future work will be conducted and published by the authors to point out these aspects in a more focused experimental procedure.

Comment: L101: standard rearing, please specify the standard rearing conditions.

Authors’ reply: We have specified the conditions right after the first sentence. However, since this will be misleading we changed the description. See the corresponding lines in the “Insects” section.

Comment: Materials and methods: can be improved on the sentence flow, as the clarity is still lacking.

Authors’ reply: We made several changes throughout the manuscript, see the corresponding lines in the “Results” section.

Comment: The results should focus on the total number of adults and/or larval emergence taken place from the culture and making the comparisons among the temperature or moisture content regimes.  The major objective of this study is to identify the target temperature and moisture content points that favour or deter the lesser mealworm growth and development. But the percentage of adult mortality can divert the study into the opposite direction. As the secondary parameters of this study, the percent larval and adult mortalities, and measurement of insect body weight can be considered, but the number of total progeny emergence must be the first answer for this objective.

Authors’ reply: Since no nymphs or newly emerged adults were discovered by the time the trial came to a close, the only progeny that can be accounted for in this study are the larvae. As a result, all of the progeny generation that was discovered in each vial has been depicted in Figures 3 and 4. In addition, the statistical analysis was carried out based on the total number of larvae that were discovered in each temperature/MC value. These numbers, which therefore indicate the total number of progeny that were discovered, were used.

Comment: The wrong interpretation of results carried away the discussion and conclusion in different directions from it is supposed to be. Please rectify the result interpretation and accordingly align the discussion and conclusion as you go ahead.

Authors’ reply: We are grateful to the reviewer for taking the time to read and thoroughly evaluate our material. We have made a number of alterations to the document, and we are hoping that the reviewer will find the updated edition satisfactory.

Comment: Some significant grammatical errors were found. More importantly, can be improved on the better sentence structure and flow.

Authors’ reply: We made several modification throughout the discussion section.

 

 

Round 2

Reviewer 2 Report

NA

Author Response

We thank the reviewer for their suggestions. 

Reviewer 4 Report

I do not find the changes made by the authors satisfactory. Reviewer 3 also asks “Has the loss of MC in the culture medium been monitored during the course of the experiments?” The authors replied to me “We intend to further investigate this factor (moisture content of the commodity) through additional experiments in which the MC values will be continuously monitored throughout the incubation period.” This should be done before the paper is published because no significant change in the adult mortality among the MC values is exactly what would be expected if wheat keep 5, 10, 15 and 20% was equillibrating with 55% rh in incubator and the carrot and all of wheat reached the same moisture content. Also, instar distribution is important in defining what is meant by small and large larvae.

None

Author Response

Authors’ reply: We would like to apology to the reviewer for this inconvenience. Here is our answer regarding the issue.

This experiment was designed as a first investigation of the effect the moisture content may have to the proper development of A. diaperinus. The MC values were monitored only once, at the start of the experiment, as it has been described in M&M. No further evaluation of the MC inside the vials was took place. Thus, the addition of carrot slices could increase the MC of the wheat, a factor that was not taken into consideration in this study. However, although we did not observed any significant increase in the adult mortality among the MC values according to the statistical analysis, it was a surprise to us that the progeny production in terms of the emerged larvae was drastically decreased with the increase of MC values. In response to this and other reviewers’ suggestion, we have added few lines addressing this issue in the materials and methods (see lines 155-156) and we have incorporated a brief paragraph at the conclusion of the discussion (see lines 294-297), pertaining to the recommended experimental procedures that should be undertaken in order to further comprehension of this phenomenon. We replied to the reviewer 3 the following: “The MC values were monitored only once, at the start of the experiment. No further evaluation of the MC inside the vials was took place”.

Moreover, we have completely changed the way in which we separated the larvae and this is apparent in both the manuscript and in the figures. In lines 135-142, we describe the procedure in which we classified the larvae in two groups based on their instar.

Author Response File: Author Response.pdf