Environmental and Colony-Related Factors Linked to Small Hive Beetle (Aethina tumida) Infestation in Apis mellifera

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I found the work very interesting and original; it explores the relationships
between environmental variables and the presence of the small hive beetle in
the only European populations of this pest. I think you could better explain
in the introduction why the sampling was done in the fall/winter and not during other seasons. I didn't find any notable errors throughout the text,
nor do I have any other comments or suggestions.

Author Response

Comments 1: I found the work very interesting and original; it explores the relationships between environmental variables and the presence of the small hive beetle in the only European populations of this pest. I think you could better explain in the introduction why the sampling was done in the fall/winter and not during other seasons. I didn't find any notable errors throughout the text, nor do I have any other comments or suggestions.

Response 1: Thank you for your kind observation. The sampling was carried out during the autumn and late winter (around the beginning of spring) because the beekeepers we selected have been involved in the fight against Aethina tumida in Calabria region for about a decade. Based on their empirical experience, they reported that autumn and late winter were the seasons with the highest SHB infestation levels. Therefore, we decided to follow their guidance, especially considering that they conducted the inspections (as we followed a citizen science methodology). I have added these information at line 97-101 (Introduction): "Autumn and late winter were selected as the inspection periods based on the empirical observations of the participating beekeepers, who have been engaged in the management of SHB infestations for approximately a decade. According to their empirical experience, these seasons correspond to the highest infestation levels".

Reviewer 2 Report

Comments and Suggestions for Authors

The authors investigates environmental and colony-related factors influencing small hive beetle (Aethina tumida) infestation levels in honey bee colonies. The study has significant value in beekeeping. However, a revision is needed.

Line 12: Add the background of the study, why the study in necessary.

Line 15-27: Need to rewrite compactly with main findings.

line 36: Small Hive Beetle--- why capitalized the words?

Line 79-83: Need to rewrite. Focus on aims instead of detail methodology. Mention the hypothesis or research questions.

Line 105: add a subheading 'Statistical analysis'

Line 105-140: Rewrite compactly.

Line 142-148: It looks like a methodology instead of the results.

Reference list--- need to check journal's abbreviations.

Author Response

Comments 1: Line 12: Add the background of the study, why the study in necessary.

Response 1: Thank you for this comment. To provide the necessary background, I have added the following information: "Small Hive Beetle (SHB) was first detected in Italy in 2014 and remains confined to the regions of Calabria and Sicily (Italy). The environmental and colony-related factors conducive to SHB development have been extensively studied at the laboratory level; however, it remains unclear whether these findings are also applicable under field conditions within apiaries in regions affected by SHB. Line 11 (Abstract)

Comments 2: Line 15-27: Need to rewrite compactly with main findings

Response 2: I concur with your observation and have revised the abstract to present the main findings more concisely as follows: "Our analysis revealed a significant relationship between SHB infestation level and six factors: number of combs covered by adult bees, total number of combs, combs surveillance, previous month's infestation, sun exposure and season. GLM analysis predicted a higher number of SHBs in colonies with fewer combs covered by adult bees (2.543), with a greater number of combs (1.877), with lower comb surveillance (0.935), with higher SHB infestation level in the previous month (1.192), in shaded locations compared to sunny ones (0.207), and in autumn compared to late winter (0.258) with peak infestations in September." Line 19-26 (abstract)

Comments 3: line 36: Small Hive Beetle--- why capitalized the words?

Response 3: Thank you for pointing this out. I have corrected this by using lowercase letters, as the acronym SHB was previously introduced in the abstract.

Comments 4: Line 79-83: Need to rewrite. Focus on aims instead of detail methodology. Mention the hypothesis or research questions.

Response 4: I appreciate your feedback and have refocused this section on the study aims, as suggested. The revised text (lines 79-83) now reads: "To understand the factors influencing SHB infestation, we conducted a study examining the association with nine environmental and colony-related variables. We specifically investigated whether stronger colonies exhibit a greater ability to resist SHB and maintain lower infestation levels, and if brood or stored resources contribute to increased SHB attraction and infestation. We also explored the potential for queenless colonies to be less susceptible to SHB due to the more aggressive behavior of workers in such conditions. Moreover, we aimed to determine if the climatic conditions found to favor SHB development in the laboratory are also relevant in the field across different seasons." Line 77-85 (Introduction).

Comments 5: Line 105: add a subheading 'Statistical analysis'

Response 5: As suggested, I have added two subheadings: "Study Design" and "Statistical Analysis."

Comments 6: Line 105-140: Rewrite compactly.

Response 6: Following your recommendation, as well as that of another reviewer, I have streamlined the writing of the statistical analysis section, primarily focusing on the results of the GLM. The revisions can be found starting from line 123 (Materials and Methods, Statistical Analysis).

Comments 7: Line 142-148: It looks like a methodology instead of the results.

Response 7: I concur with your assessment and have relocated this information to the Methodology section.

Comments 8: Thank you for this important reminder. I checked the reference list.

Reviewer 3 Report

Comments and Suggestions for Authors

I enjoyed reading this manuscript. It was well written and the results and conclusion add what we now know about the Small Hive Beetle in honey bee colonies. I found that the Introduction and Discussion were well written and cited key papers to support their hypotheses and later their findings in the Discussion. The reason that I have suggested that the paper undergo a major revision is the following. 

1) the methods about SHB sampling are not clear enough. While the authors did cite the mobile panel method for sampling SHB, they should still describe exactly how they utilize this protocol. Did they sample all hive bodies and supers in a hive or only a single top super to hive body.

2) The statistical results are somewhat redundant, but also incorrectly applied. The authors first collapsed their data in ordinal rankings of categories and then applied a series of multiple contingency table analyses using Pearson chi square and Cramer's V tests to determine preliminary significance of their hypothesized explanatory variables. Then based upon these results they explore the use of generalized linear models to determine the strength and direction of the linear trends between adult SHB abundance measures and independent independent variables suggested to be significant in the contingency table analyses.

The use of contingency table analyses and Cramer's V were designed for analysis of NOMINAL categorical dependent variables and not ordinal or rank variables such as the categories that the authors constructed for SHB. The authors should delete these analyses and rely just on a series of GLM models that are designed for quantitative continuous or discrete dependent variables. The authors apparently have performed these already although they need to provide much more detail in the methods regarding the error distribution used in their models and the specific structure of the independent variables put into the models. The GLM models can evaluate all the factors that the authors preliminary analyses focused on. A single analysis will be more straight forward.

3. The authors should construct a Table of the GLM results along with graphs suggesting the main effects or interaction effects. They should also delete the Tables with the Chi Square results. The Tables with the raw summaries of data are fine.

4. The authors show the distribution of SHB abundances that were detected in their hive sampling. They stated that the distribution was not normal, however, they did not test this. They should test this assumption and then fit the model to a specific probability density function. I estimated their frequency data from their graph and found that it can be fit very well with a Negative Binomial probability density function (see my comments on manuscript).

If the authors simplify their analyses by relying solely on GLM analyses the results section will be more direct and simplified.

Please look at my other comments and suggestions that are included on the manuscript. I think that the authors can improve this paper if they consider my suggestions. 

Comments for author File: Comments.pdf

Author Response

Comments 1: the methods about SHB sampling are not clear enough. While the authors did cite the mobile panel method for sampling SHB, they should still describe exactly how they utilize this protocol. Did they sample all hive bodies and supers in a hive or only a single top super to hive body.

Response 1: Thank you for your inquiry regarding the SHB sampling methodology. While the Mobile Divider (MD) method was employed, we acknowledge the need for a more detailed explanation of its implementation. Following this protocol, each comb within the hive and also the area behind the Mobile Divider were inspected. The total number of SHBs detected inside the hive was determined by summing the SHB counts observed both behind the Mobile Divider device and within the remaining hive volume. It is important to note that our evaluation was limited to SHB present within the hive body, as neither supers nor bottom boards were utilized in the experimental setup. I added more information at line 119-121 (material and methods). 

Comments 2: The statistical results are somewhat redundant, but also incorrectly applied. The authors first collapsed their data in ordinal rankings of categories and then applied a series of multiple contingency table analyses using Pearson chi square and Cramer's V tests to determine preliminary significance of their hypothesized explanatory variables. Then based upon these results they explore the use of generalized linear models to determine the strength and direction of the linear trends between adult SHB abundance measures and independent independent variables suggested to be significant in the contingency table analyses. The use of contingency table analyses and Cramer's V were designed for analysis of NOMINAL categorical dependent variables and not ordinal or rank variables such as the categories that the authors constructed for SHB. The authors should delete these analyses and rely just on a series of GLM models that are designed for quantitative continuous or discrete dependent variables. The authors apparently have performed these already although they need to provide much more detail in the methods regarding the error distribution used in their models and the specific structure of the independent variables put into the models. The GLM models can evaluate all the factors that the authors preliminary analyses focused on. A single analysis will be more straight forward.

Response 2: Thank you for identifying this methodological concern. In response, I have focused the analysis primarily on the Generalized Linear Models (GLM) as suggested. The revised version of the manuscript, where these modifications have been implemented, is now available for your review. Due to the complexity and extent of these changes, a detailed step-by-step enumeration within this response is impractical. 

Comments 3: The authors should construct a Table of the GLM results along with graphs suggesting the main effects or interaction effects. They should also delete the Tables with the Chi Square results. The Tables with the raw summaries of data are fine.

Response 3: Thank you for this constructive suggestion. In accordance with your feedback, the tables presenting the Chi-Square test results have been removed, and the tables detailing the GLM results have been retained. Should more detailed GLM output be required, the authors would be pleased to make this information available. These extensive and detailed tables were not included in the main text to avoid excessive length and potential disruption to the flow of the manuscript.

Comments 4: The authors show the distribution of SHB abundances that were detected in their hive sampling. They stated that the distribution was not normal, however, they did not test this. They should test this assumption and then fit the model to a specific probability density function. I estimated their frequency data from their graph and found that it can be fit very well with a Negative Binomial probability density function (see my comments on manuscript).

Response 4: Thank you for this insightful comment regarding the distribution of SHB abundance. Following your suggestion, we have incorporeted the results as follows: "The dependent variable, SHB number per hive, exhibited a non-normal distribution, presenting a positive asymmetric distribution with high variability (Kolmogorov-Smirnov test: Statistic = 0.328, df = 431, p < 0.001; Shapiro-Wilk test: Statistic = 0.568, df = 431, p < 0.001)." Line 152-155 (Results). 

Reviewer 4 Report

Comments and Suggestions for Authors

Climate change is indicated as one of the causes of the spread of Aethina tumida (= Small Hive Beetle; SHB) across Europe. The SHB causes a significant threat to honey bee colonies, therefore every study which providing knowledge on the factors that can increase the occurrence of SHB. The study was conducted in Italy. In general, the study protocol was sufficiently described. Environmental variables studied and colony-related factors were properly characterized.

This work contributes to the understanding of factors that may influence the acclimatization and spread of the beetle in apiaries. The most important achievement is that the SHB infestation level is significantly connected with nine factors (i.e. season, total number of combs, number of combs covered by adult bees, comb surveillance, number of combs containing brood, number of storage combs, sun exposure, and queen presence. The study is an introduction to understanding the factors influencing the spread of the beetle and can be a basis for developing pest control protocols

The text is easy to follow, data are clearly presented. In my opinion, only minor changes are required before publication. 

Abstarct

L11 Give the country name in the first sentence ;……… to the regions of Calabria and Sicily

L 85 – describe climatic conditions of the province of Reggio Calabria, as not everyone is familiar with Italy climate.

L 104 - EurBeST protocol . Please describe it in brief, as not every one knows this protocol

Table 3 – Finale average; just "Average"

Author Response

Comments 1: Line 12: Add the background of the study, why the study in necessary.

Response 1: Thank you for your observation. I have addressed this by including "(Italy)" for clarification in the initial sentence: "Small Hive Beetle (SHB) was first detected in Italy in 2014 and remains confined to the regions of Calabria and Sicily (Italy)."

Comments 2: L 85 – describe climatic conditions of the province of Reggio Calabria, as not everyone is familiar with Italy climate.

Response 2: Thank you for this suggestion. I have provided a description of the climatic conditions of the Calabria region as follows: "The Calabria region exhibits higher average temperatures compared to other Italian regions throughout the year, including during the autumn and winter months. This climatic pattern is attributable to its geographical location in southern Italy and the moderating influence of the Mediterranean Sea." This information has been added to the Materials and Methods section, lines 101-104.

Comments 3: L 104 - EurBeST protocol . Please describe it in brief, as not every one knows this protocol

Response 3: Thank you for this point. I have incorporated a brief description of the EurBeST protocol at line 115 as follows "The colonies were opened from above and the number of combs occupied by bees was estimated without the use of smoke. A comb was considered occupied by adult bees if at least 70% of its surface was covered. Furthermore, each comb was inspected to determine whether it contained brood or stored resources. The number of SHBs detected inside the hive was evaluated as the number of SHB present both behind the Mobile Divider device and in the remaining volume of the hive."

Comments 4: Table 3 – Finale average; just "Average"

Response 4: Following your suggestion, I have revised the table as indicated.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version of the manuscript is improved than the earlier version. Now, it is suitable for the journal.

Reviewer 3 Report

Comments and Suggestions for Authors

I have looked over the revised manuscript and I feel that this manuscript is ready for immediate publication. The authors have addressed all of my concerns and the paper is much improved.