Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have reported on the hemolymph extraction method from the yellow mealworm, Tenebrio molitor.  This method will be useful for the future scientists on the mealworm. However, I recommend the authors to add one Table for the hemolymph extraction method by comparing the old methods and newly suggested methods. In addition, please provide the flow chart of the optimized hemolymph extraction method (protocol) that the authors have developed in this study. In doing so, the authors can emphasize the most important findings in this study.

Author Response

Reviewer 1

Comments: The authors have reported on the hemolymph extraction method from the yellow mealworm, Tenebrio molitor.  This method will be useful for the future scientists on the mealworm. However, I recommend the authors to add one Table for the hemolymph extraction method by comparing the old methods and newly suggested methods. In addition, please provide the flow chart of the optimized hemolymph extraction method (protocol) that the authors have developed in this study. In doing so, the authors can emphasize the most important findings in this study.

Answer: The authors appreciate your comments, which enrich this work. In this new version, the authors have added comparative information on Table 1 on the most common methods for extracting hemolymph in insects and the protocol developed in this work, which consists of a double mesothoracic puncture. This is intended to highlight the advantages of the proposed new methodology. Lines 202-203, and Table 1.

We have also added a more concise version of the protocol as supplementary file (Quick protocol), with implementation recommendations, including a flowchart for greater clarity, as suggested by the reviewer.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a protocol for high-yield hemolymph extraction from adult Tenebrio molitor using a double mesothoracic puncture plus anticoagulant buffer injection, claiming increased volume and hemocyte yield/viability compared with a reference puncture method. The topic is relevant and the protocol is described in considerable operational detail, which is valuable for the community.

However, the manuscript currently has major weaknesses in experimental design clarity, quantitative reporting, and figure caption completeness, which prevent full evaluation of the claims. In particular, the paired-design comparison is insufficiently described (order/randomization/carry-over), and the results lack essential descriptive statistics and data visualization

Author Response

Reviewer 2

Comments: This manuscript presents a protocol for high-yield hemolymph extraction from adult Tenebrio molitor using a double mesothoracic puncture plus anticoagulant buffer injection, claiming increased volume and hemocyte yield/viability compared with a reference puncture method. The topic is relevant and the protocol is described in considerable operational detail, which is valuable for the community.

However, the manuscript currently has major weaknesses in experimental design clarity, quantitative reporting, and figure caption completeness, which prevent full evaluation of the claims. In particular, the paired-design comparison is insufficiently described (order/randomization/carry-over), and the results lack essential descriptive statistics and data visualization

Answer: The authors thank the reviewer for the comments that improve this manuscript. We added information in the figure captions for higher clarity. We also include a wider description of the paired-design experiment and the larvae culture. Lines 84-87; 91-93; 104-108.

Additionally, the authors present the results of the statistical analysis in a new Figure and an explanation in Results section (Figure 2). Lines 224-237.

Reviewer 3 Report

Comments and Suggestions for Authors

Del Razo-Moreno et al. provide valuable insights on T. molitor adult hemolymph sample extraction. The presented protocol highlights its potential from the large volume, which can be used in cellular, biochemical, and immunological research. The method is clearly described, and the anesthesia issues were properly addressed in the discussion section. The whole manuscript is a clear and nice piece of work; the protocol is extremely well presented, supported with video.

1. The title has to mention the adult/adult stage for the presented protocol.
2. The link between market demand for T. molitor powder (lines 45-47) and the protocol described could be connected to the automatization processes. This point could also be addressed in the Discussion section, where the potential for scaling up and automating the protocol for large-scale production may be further explored.
3. It would be nice to see the biotechnological and immunological introductory aspects of the protocol expanded based on the title.
4. Line 122 may require a reference to support the statement regarding osmotic pressure. Similarly for lines 108-109 realted to hemolymph qualitative characteristics (or rephrase it as authors' choice for the protocol execution). 
5. Lines 137-139 sounds like results coming from the protocol execution.

 

Author Response

Reviewer 3

Comments: Del Razo-Moreno et al. provide valuable insights on T. molitor adult hemolymph sample extraction. The presented protocol highlights its potential from the large volume, which can be used in cellular, biochemical, and immunological research. The method is clearly described, and the anesthesia issues were properly addressed in the discussion section. The whole manuscript is a clear and nice piece of work; the protocol is extremely well presented, supported with video.

1. The title has to mention the adult/adult stage for the presented protocol.
2. The link between market demand for T. molitor powder (lines 45-47) and the protocol described could be connected to the automatization processes. This point could also be addressed in the Discussion section, where the potential for scaling up and automating the protocol for large-scale production may be further explored.
3. It would be nice to see the biotechnological and immunological introductory aspects of the protocol expanded based on the title.
4. Line 122 may require a reference to support the statement regarding osmotic pressure.

Similarly for lines 108-109 realted to hemolymph qualitative characteristics (or rephrase it as authors' choice for the protocol execution). 

1. Lines 137-139 sounds like results coming from the protocol execution

Answer: The authors appreciate the comments on the work presented, which have contributed to its improvement. In this regard, we have responded to the reviewer's request.

 

1) The authors included the word adult in the title (“Alternative high quality hemolymph extraction from adult Tenebrio molitor, a tool for biotechnological and immunological research”). Lines 1-2.

2) In the Introduction and Discussion sections, the authors included information related to the protocol and the automatization processes from a nutritional perspective. Lines 48-54 and 278-281.

3) In the Introduction section, the authors have included some biotechnological aspects where the protocol could help to delve deeper into diverse processes. Lines 72-75.

4) The authors have included the corresponding reference to the information presented (osmotic pressure and hemolymph qualitative characteristics). Lines 126-126, 138-139.

5) The authors believe this information is more valuable in the protocol presented as supplementary material, as it contains recommendations for optimal protocol implementation and achieving the best results. Therefore, this information has been removed from the manuscript and added to the concise version of the protocol as a supplementary file.

 

Reviewer 4 Report

Comments and Suggestions for Authors

The search for new useful substances with desired properties, both for biotechnological purposes and for the pharmaceutical industry, is a pressing issue in modern entomology. Many laboratory insects are becoming a new source of antibacterial peptides and other valuable secondary metabolites. Therefore, developing methods for their production is an important task in technical entomology. The manuscript under review is devoted to this task, is relevant, and fully complies with the rules and requirements of the Insects.
1. The "Introduction" section contains no comments, complies with the journal's requirements, contains descriptions of the model species, and demonstrates the relevance of the methodological study.
2. The "Materials and Methods" section describes the entire experimental design and sample preparation process.
3. The results section consistently describes the obtained results.
4. A comment is made in the "Discussion" section. The authors have competently discussed the results and presented encouraging data in favor of isoflurane as an anesthetic agent. However, it was necessary to provide data on hemolymph extraction (hemolymph volumes, factors) from other species, such as Zophobas atratus and other darkling beetle species, to strengthen the scientific value of the developed method and the obtained results. It was also necessary to describe whether hemolymph activity is reduced by the use of anesthetics, the larval culture conditions, etc.
5. The authors included a very useful video in the Supplementary demonstrating the process of obtaining hemolymph from Tenebrio molitor. It is extremely useful and can be replicated by other authors in their research.

Author Response

Reviewer 4

Comments: The search for new useful substances with desired properties, both for biotechnological purposes and for the pharmaceutical industry, is a pressing issue in modern entomology. Many laboratory insects are becoming a new source of antibacterial peptides and other valuable secondary metabolites. Therefore, developing methods for their production is an important task in technical entomology. The manuscript under review is devoted to this task, is relevant, and fully complies with the rules and requirements of the Insects.

1. The "Introduction" section contains no comments, complies with the journal's requirements, contains descriptions of the model species, and demonstrates the relevance of the methodological study.
   2. The "Materials and Methods" section describes the entire experimental design and sample preparation process.
   3. The results section consistently describes the obtained results.
   4. A comment is made in the "Discussion" section. The authors have competently discussed the results and presented encouraging data in favor of isoflurane as an anesthetic agent. However, it was necessary to provide data on hemolymph extraction (hemolymph volumes, factors) from other species, such as Zophobas atratus and other darkling beetle species, to strengthen the scientific value of the developed method and the obtained results. It was also necessary to describe whether hemolymph activity is reduced by the use of anesthetics, the larval culture conditions,etc.
   5. The authors included a very useful video in the Supplementary demonstrating the process of obtaining hemolymph from Tenebrio molitor. It is extremely useful and can be replicated by other authors in their research.

 

Answers: The authors appreciate the feedback received on the work presented, which has improved and enriched it. We believe that the reviewer's comments provide valuable information.

1) Thank you for the comment.

2) The authors appreciate the comment.

3) The authors thank the comment.

4) Thank you for the valuable comment. In the Discussion section, the authors have included information as the reviewer recommended. This is information on obtaining hemolymph using traditional methods in larger mealworms, such as Zophobas morio, and comparing the volume obtained with that from the double mesothoracic puncture method, which yields up to 300 microliters. Thus, if this protocol could be implemented in larger beetles, the hemolymph yield could be even greater. Lines 316-320.

On the other hand, we did not perform any test to measure hemolymph activity; however, the hemocyte viability obtained strongly suggests that, at least, the hemocytes maintain their physiological capacity. Further tests should be performed to elucidate whether the hemolymph preserves its metabolic activity. The aim of this protocol is to use hemocyte,d not hemolymph components.

5) Thank you for the comment.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer Report

1. Introduction

Lines 57: hemolymph plays a crucial role in an insect's immune system – change to hemolymph plays a crucial role in an insect's circulatory and immune system

Lines ~59–60: “hemolymph … homes several types of cells” → replace “homes” with “houses”.

2. Materials and Methods

This is critical because the first procedure could alter hemocoelic pressure, induce clotting/melanization, or deplete hemolymph and hemocytes, thereby biasing the second measurement

Include information about the colony of T. molitor.

Lines ~116; 125-128: Sentences are isolated in the text as a short paragraph consisting of only one sentence, please correct it. Do this correction along the whole text.

Lines ~155–156: If control extraction is “as described [13]”, provide a brief summary here (site, expected volume, anticoagulant use) to aid reproducibility.

Line 182: Correct sentence: Hemocytes were counted as described above. Cellular viability and metabolic activity were assessed using 5(6)-carboxyfluorescein diacetate (CFDA; Thermo Fisher Scientific, USA).

 

3. Results

Lines ~201–202: Provide actual quantitative summary data and variability; “approximately” without distribution limits interpretability.

Line 225: Change the subtitle 3. 2Statistical analysis to 3.2 Hemocyte quantification

Lines ~251- 253: Consider adding the criteria used to identify hemocyte types, or cite a classification reference if relying on established criteria.

Line 227: The abbreviation DMP appears here for the first time without its full name, which I imagine is for the experimental group - double mesothoracic puncture. It would be interesting to include this abbreviation in the material and methods also for the other experimental group - single pro-mesothorax puncture (SPP). And use this abbreviation throughout the text.

Lines 229 - 234: Change sentences to - The optimized method, DMP, yielded higher cell recovery, with a median of 146.19 × 10³ (range: 42.30–248.40), corresponding to approximately 1.5 × 10⁵ hemocytes. In contrast, the single puncture in the pro-mesothorax (SSP) method resulted in a median of 28.09 × 10³ hemocytes (range: 9.66–49.76), equivalent to approximately 0.3 × 10⁵. Thus, the DMP increased recovery by at least ~94.86 × 10³ hemocytes per beetle, as estimated by the Hodges–Lehmann estimator (95% CI; Figure 2). This finding is consistent with the approximately fivefold increase observed relative to the SSP method.

Line 236: This information could be replaced in the figure 2 legend: The Wilcoxon signed-rank test yielded W = 0.000 and P < 0.001. The effect size was 1.000 (95% bootstrap CI: 1.000–1.000; 10,000 resamples), with all 40/40 specimens showing higher total hemocyte counts with DMP.

Lines: 266 -268: hemocyte viability is “90% after 90 min” and “30% after 5 h at room temperature” in anticoagulant buffer. It is not clear how many biological replicates were used, and whether any variability was observed. The data in figure 4 shows only results for the period of 90 min. Therefore, the data of 30% viability was not presented anywhere in the manuscript.

Figure 4 legend: include the time of the images presented, 90 min or 5h? Include if there’s any replicate in this viability counting.

Actionable request:

Present a small plot of viability vs time with error bars (or at minimum provide mean±SD/SEM or median/IQR). Or a table with number of viable hemocytes and total hemocyte in different time.

Author Response

Comments:

Introduction:

Lines 57: hemolymph plays a crucial role in an insect's immune system – change to hemolymph plays a crucial role in an insect's circulatory and immune system.

Answer: Thank you for the observation. The authors have made the changes as the reviewer suggested. Lines 57-58.

 

Lines ~59–60: “hemolymph … homes several types of cells” → replace “homes” with “houses”.

Answer: Thank you for the observation. The authors have made the changes as the reviewer suggested. Line 59.

Comments:

Materials and Methods

This is critical because the first procedure could alter hemocoelic pressure, induce clotting/melanization, or deplete hemolymph and hemocytes, thereby biasing the second measurement

Include information about the colony of T. molitor.

Answer: The authors appreciate the observation. The authors included information about the T. molitor strain. The INIFAP CENID-SAI Conventional strain was used, maintained, and expanded under standard laboratory conditions in open colonies with basic biosafety systems. The health status of the population was monitored periodically, confirming the absence of clinical signs of disease or evident parasitosis. Lines 95-98

Lines ~116; 125-128: Sentences are isolated in the text as a short paragraph consisting of only one sentence, please correct it. Do this correction along the whole text.

Answer: The authors appreciate the comment. The authors integrated the information into the paragraph to improve the protocol's performance. Lines 111-113, Lines 127-129, and Lines 173-174. 

Lines ~155–156: If control extraction is “as described [13]”, provide a brief summary here (site, expected volume, anticoagulant use) to aid reproducibility.

Answer: The authors appreciate the observation. The authors integrated the information from the reference Vommaro et al., 2021 [13] in the paragraph. Lines 160-163.

Line 182: Correct sentence: Hemocytes were counted as described above. Cellular viability and metabolic activity were assessed using 5(6)-carboxyfluorescein diacetate (CFDA; Thermo Fisher Scientific, USA).

Answer: The authors appreciate the comment. The change was made as the reviewer suggested. Lines 189-191.

3.            Results

Lines ~201–202: Provide actual quantitative summary data and variability; “approximately” without distribution limits interpretability.

Answer: Thank you for the observation. The authors have provided Table 1, which contains the requested data, based on the coefficient of variation as a recurrent tool in viability data analysis. Lines 221-224.

Line 225: Change the subtitle 3. 2Statistical analysis to 3.2 Hemocyte quantification.

Answer: Thank you for the observation. The authors have made the changes as the reviewer suggested. Line 247.

Lines ~251- 253: Consider adding the criteria used to identify hemocyte types, or cite a classification reference if relying on established criteria.

Answer: The authors thank the comment. In this version, the authors included the reference (Vommaro et al., 2021 [13]), which we used to describe the identity of the hemocytes. Lines 269-271.

Line 227: The abbreviation DMP appears here for the first time without its full name, which I imagine is for the experimental group - double mesothoracic puncture. It would be interesting to include this abbreviation in the material and methods also for the other experimental group - single pro-mesothorax puncture (SPP). And use this abbreviation throughout the text.

Answer: The authors appreciate the contribution. In this reviewed version, the authors described the double mesothoracic puncture as DMP and the single pro-mesothorax puncture as SPP throughout the entire manuscript.

Lines 229 - 234: Change sentences to - The optimized method, DMP, yielded higher cell recovery, with a median of 146.19 × 10³ (range: 42.30–248.40), corresponding to approximately 1.5 × 10⁵ hemocytes. In contrast, the single puncture in the pro-mesothorax (SSP) method resulted in a median of 28.09 × 10³ hemocytes (range: 9.66–49.76), equivalent to approximately 0.3 × 10⁵. Thus, the DMP increased recovery by at least ~94.86 × 10³ hemocytes per beetle, as estimated by the Hodges–Lehmann estimator (95% CI; Figure 2). This finding is consistent with the approximately fivefold increase observed relative to the SSP method.

Answer: Thank you for the observation. The authors have made the changes as the reviewer suggested. Lines 248-257.

Line 236: This information could be replaced in the figure 2 legend: The Wilcoxon signed-rank test yielded W = 0.000 and P < 0.001. The effect size was 1.000 (95% bootstrap CI: 1.000–1.000; 10,000 resamples), with all 40/40 specimens showing higher total hemocyte counts with DMP.

Answer: Thank you for the recommendation. The authors have made the changes as the reviewer suggested, and this information is now in the Figure 2 legend. Lines 263-266.

Lines: 266 -268: hemocyte viability is “90% after 90 min” and “30% after 5 h at room temperature” in anticoagulant buffer. It is not clear how many biological replicates were used, and whether any variability was observed. The data in figure 4 shows only results for the period of 90 min. Therefore, the data of 30% viability was not presented anywhere in the manuscript.

Figure 4 legend: include the time of the images presented, 90 min or 5h? Include if there’s any replicate in this viability counting.

Answer: The authors thank the commenter and appreciate the comment. In this new version, we include micrographs showing the viability of hemocytes after 90 min and 5 h at room temperature in anticoagulant buffer. Lines 290-299.

Actionable request:

Present a small plot of viability vs time with error bars (or at minimum provide mean±SD/SEM or median/IQR). Or a table with number of viable hemocytes and total hemocyte in different time.

Answer: The authors thank the contribution. We included Table 3, which contains the requested data. Definitely, this contribution enriches this work. Lines 300-303.

Time	Mean total hemocytes	SD	Coefficient of variation (%)	Mean viable hemocytes	SD	Coefficient of variation (%)	Viability percentage (%)
90 min	148,796	7,895	0.053 (5.3%)	133,916	8,231	0.061 (6.1%)	90%
5 h	136,520	8,486	0.062 (6.2%)	40,956	2,957	0.072 (7.2%)	30%