Physico-Chemical and Mineral Variability of Apis mellifera Bee Venom Across Seasons and Feeding Regimes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper presents the results of several physicochemical analyses on honey bee venom samples from Timiș County, Romania. The subject is interesting and the analytical data provided add some information on a somehow neglected bee product, but the work has various serious flaws.

The title is misleading since it implies that the observed variability influences venom quality, but such a statement is not supported by the data provided. A less ambitious title simply referring to bee venom variability should be adopted.

Moisture and dry matter data refers to the venom as it is recovered from the collecting device and not to the venom as it is in the honey bee venom sac. Since the venom undergoes a relevant dehydration in the collecting device no inference can be drawn on the nutritional and/or physiological aspects.

Macroelement content variability cannot be inferred from data range, but a different variability index, like standard deviation or confidence interval, should be used.

In Table 1 caption the order “moisture content, dry matter, ash, pH, total amino acids” reported, but table columns are ordered “moisture content, dry matter, pH and total amino acids, ash” and in the text paragraph sequence is “dry matter and moisture, pH, ash, total amino acids”; please uniform and adopt the same order also in the Materials and methods section

In the Materials and methods section procedures to determine impurities and nickel are provided but no results on such parameters appear in the Result and discussion section.

Figure 5 graphs should be carefully checked and merged in a single diagram from April 1^rst to July 31^th by reducing the space between days.

Author Response

Journal: Molecules (ISSN 1420-3049)

Manuscript ID: molecules-4268198

Title: Physico-Chemical and Mineral Variability of Apis mellifera Bee Venom Across Seasons and Feeding Regimes

Authors: Adrian-Dan Rășinar , Isidora Radulov * , Adina Berbecea * , Silvia Pătruică

Section: Natural Products Chemistry

Special Issue: Bee Product Chemistry: Identification and Characterization of Natural Compounds, 2nd Edition

Dear Reviewer,

            Thank you for taking the time to review our manuscript. We greatly appreciate the suggestions made to improve our paper.

We have addressed your comments as follows:

The paper presents the results of several physicochemical analyses on honey bee venom samples from Timiș County, Romania. The subject is interesting and the analytical data provided add some information on a somehow neglected bee product, but the work has various serious flaws.

The title is misleading since it implies that the observed variability influences venom quality, but such a statement is not supported by the data provided. A less ambitious title simply referring to bee venom variability should be adopted.

A: We have revised the title to “Physico-Chemical and Mineral Variability of Apis mellifera Bee Venom Across Seasons and Feeding Regimes.” The new title no longer implies that the observed variability determines venom quality; instead, it neutrally reflects that variability exists. It also accurately represents the content of the manuscript, which focuses on the variation in moisture, pH, amino acids, macro- and microelements, and heavy metals.

Moisture and dry matter data refers to the venom as it is recovered from the collecting device and not to the venom as it is in the honey bee venom sac. Since the venom undergoes a relevant dehydration in the collecting device no inference can be drawn on the nutritional and/or physiological aspects.

A: We thank the reviewer for this observation; however, we disagree with the conclusion that moisture and dry matter values cannot reflect physiological or nutritional influences. In our study, all venom samples were collected using the same device, under identical exposure times and environmental conditions. Therefore, the dehydration process was uniform across treatments and seasons. Under such controlled and standardized conditions, any systematic differences observed between samples cannot be attributed to the collection device but must originate from the initial composition of the venom at the time of secretion. Published research supports that venom water content, secretion rate, and viscosity vary with season, food availability, and the physiological state of the colony prior to dehydration. Dotimas & Hider (1987) describe seasonal changes in venom gland activity and secretion characteristics, while Ali (2012) and Bogdanov (2016) report that environmental and nutritional factors influence the physicochemical properties of venom even before collection. These studies demonstrate that biological variability in venom composition is present at secretion and is not eliminated by subsequent dehydration. Furthermore, dehydration does not erase relative differences between samples. This principle is well established in the analysis of other dried bee products - such as honey, pollen and royal jelly - where moisture and dry matter remain valid indicators of seasonal, botanical and physiological influences even after drying (Bogdanov 2016; Habib et al. 2014). The same is true for venom: although absolute humidity decreases during drying, relative variability between samples is preserved and reflects underlying biological factors. For these reasons, we consider that humidity and dry matter remain significant parameters for characterizing venom variability in the context of our standardized experimental design.

Macroelement content variability cannot be inferred from data range, but a different variability index, like standard deviation or confidence interval, should be used.

A: Thank you for the observation. We have added and indicated in red that all experimental values presented in the tables and figures are expressed as mean ± standard deviation (SD).

In Table 1 caption the order “moisture content, dry matter, ash, pH, total amino acids” reported, but table columns are ordered “moisture content, dry matter, pH and total amino acids, ash” and in the text paragraph sequence is “dry matter and moisture, pH, ash, total amino acids”; please uniform and adopt the same order also in the Materials and methods section

A: Thank you for your comment. We have standardized and adapted the same order throughout the manuscript.

In the Materials and methods section procedures to determine impurities and nickel are provided but no results on such parameters appear in the Result and discussion section.

A: Thank you for your comment. The Materials and Methods section has been revised accordingly.

Figure 5 graphs should be carefully checked and merged in a single diagram from April 1rst to July 31th by reducing the space between days.

A: For greater rigor, we have included the charts generated by the BeeWatch Professional monitoring system; merging the four charts into a single figure would result in a loss of clarity regarding the data trends.

We are grateful for your time and effort to review our paper, and we hope we have successfully addressed all your queries!

Sincerely,

The authors.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper reported seasonal and nutritional determinants of bee venom quality. This is interesting. The paper can be accepted with major revised.

1. The study fully attributes the differences in venom components across different flowering periods to the corresponding nectar sources (rape/acacia/sunflower), yet fails to provide survey data on vegetation coverage during the flowering periods in the experimental area, making it impossible to rule out the interference of other simultaneously blooming wild nectar sources on nutritional input. Meanwhile, the precise matching between the sampling time nodes and the corresponding flowering periods is not clarified. For example, it is not mentioned whether there is an overlap between the late stage of the acacia flowering period and the early stage of the sunflower flowering period, which may lead to misattribution of nectar source contributions. It is recommended to supplement the phenological observation data of flowering periods in the experimental site and the traceability analysis results of nectar pollen to clarify the main nutritional sources in each sampling period.
2. The study concludes that Pb and Cr detected during the natural flowering periods originate from environmental exposure, while Cd detected during the artificial feeding period comes from sugar syrup or the hive environment. However, the study has not carried out synchronous heavy metal testing on the sucrose syrup, supplements, breeding soil, hive building materials used in the experiment, as well as the environmental media (air, soil, nectar) during the corresponding flowering periods. It is recommended to supplement the testing data of heavy metal content in different media to clarify the migration path of pollution sources.

Author Response

Journal: Molecules (ISSN 1420-3049)

Manuscript ID: molecules-4268198

Title: Physico-Chemical and Mineral Variability of Apis mellifera Bee Venom Across Seasons and Feeding Regimes

Authors: Adrian-Dan Rășinar , Isidora Radulov * , Adina Berbecea * , Silvia Pătruică

Section: Natural Products Chemistry

Special Issue: Bee Product Chemistry: Identification and Characterization of Natural Compounds, 2nd Edition

Dear Reviewer,

            Thank you for taking the time to review our manuscript. We greatly appreciate the suggestions made to improve our paper.

We have addressed your comments as follows:

This paper reported seasonal and nutritional determinants of bee venom quality. This is interesting. The paper can be accepted with major revised.

1. The study fully attributes the differences in venom components across different flowering periods to the corresponding nectar sources (rape/acacia/sunflower), yet fails to provide survey data on vegetation coverage during the flowering periods in the experimental area, making it impossible to rule out the interference of other simultaneously blooming wild nectar sources on nutritional input. Meanwhile, the precise matching between the sampling time nodes and the corresponding flowering periods is not clarified. For example, it is not mentioned whether there is an overlap between the late stage of the acacia flowering period and the early stage of the sunflower flowering period, which may lead to misattribution of nectar source contributions. It is recommended to supplement the phenological observation data of flowering periods in the experimental site and the traceability analysis results of nectar pollen to clarify the main nutritional sources in each sampling period.

 A: Thank you for the observation. The only possible overlap period could have been during the harvesting of acacia and rapeseed; however, this was ruled out because, in the area where the hives were located, there was no simultaneous foraging from the two plants. After the rapeseed harvest, the hives were moved to the acacia harvest, and venom collection was carried out after each harvest.

We also note that during the rapeseed and acacia flowering periods, there were no other nectar sources in the area that could provide a production yield. As for the sunflower harvest, venom collection was carried out at the end of the flowering period, with the bee colonies benefiting only from the nectar and pollen of this plant.

2.The study concludes that Pb and Cr detected during the natural flowering periods originate from environmental exposure, while Cd detected during the artificial feeding period comes from sugar syrup or the hive environment. However, the study has not carried out synchronous heavy metal testing on the sucrose syrup, supplements, breeding soil, hive building materials used in the experiment, as well as the environmental media (air, soil, nectar) during the corresponding flowering periods. It is recommended to supplement the testing data of heavy metal content in different media to clarify the migration path of pollution sources.

A: We thank the reviewer for this important observation. We agree that synchronous testing of all potential environmental and dietary sources would further strengthen the traceability of heavy metal sources. However, our interpretation regarding Pb, Cr, and Cd was based on contextual exclusion supported by site characteristics and experimental conditions. The apiary used in this study is located in a rural, non-industrial area with low pollution, and historical soil data for the region do not indicate any relevant contamination with Pb or Cr. All colonies were housed in identical hive bodies, constructed from the same materials, eliminating differences in hive-derived contamination. Moreover, the essential oils used as supplements do not contain heavy metals, as they are volatile plant-derived compounds without inorganic residues. Under these controlled conditions, the elevated levels of Pb and Cr detected during natural foraging periods are most plausibly attributed to environmental exposure pathways (air deposition or plant uptake), rather than hive materials or supplements. In contrast, the higher Cd levels observed during the artificial feeding period were interpreted as potentially linked to the sucrose syrup, by exclusion of other sources. Although we did not perform direct testing for heavy metals in the syrup, supplements, or environmental matrices, the pattern of their occurrence across the different feeding regimes supports this interpretation.

We are grateful for your time and effort to review our paper, and we hope we have successfully addressed all your queries!

Sincerely,

The authors.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. As shown in Table 3, bees were only fed by three flower nectar and pollens, which were not enough for feeding experiments.
2. In the Introduction, .it was said bee venom quality relates to the accumulation of heavy metals, including Pb, Cd, Cu, Ni, and Zn. Why was the content of Ni not measured?
3. The peptides and enzymes are important chemical constituents of bee venom. However, they were not measured.
4. It was said rapeseed showed lower Ph values 5.6–5.9, which was wrong. The pH values of Rnp2 and Rnp3 were >6.0.
5. For the data 5.5 ± 0.4, 3.1 ± 0.3 and 3.2 ± 0.3, ‘%’ was missing.

Author Response

Journal: Molecules (ISSN 1420-3049)

Manuscript ID: molecules-4268198

Title: Physico-Chemical and Mineral Variability of Apis mellifera Bee Venom Across Seasons and Feeding Regimes

Authors: Adrian-Dan Rășinar , Isidora Radulov * , Adina Berbecea * , Silvia Pătruică

Section: Natural Products Chemistry

Special Issue: Bee Product Chemistry: Identification and Characterization of Natural Compounds, 2nd Edition

Dear Reviewer,

            Thank you for taking the time to review our manuscript. We greatly appreciate the suggestions made to improve our paper.

We have addressed your comments as follows:

1. As shown in Table 3, bees were only fed by three flower nectar and pollens, which were not enough for feeding experiments.

A: We thank the reviewer for this observation. We would like to clarify that the experiment was not based solely on three floral sources for feeding. As shown in Table 3, the study included two distinct feeding phases, each with a different purpose:

Artificial feeding phase (April) – Bees were fed sugar syrup (1:1) supplemented with eight different nutritional treatments (thyme, oregano, basil essential oils; three probiotic formulations; probiotic + oregano; and a control without supplements).

Natural feeding phase (May–July) – During this period, colonies were not artificially fed. Instead, they foraged naturally from three major nectar–pollen sources characteristic of the region: rapeseed (May), acacia (June), and sunflower (July). These three floral sources were not experimental diets, but natural feeding conditions used to evaluate seasonal and botanical effects on venom composition.

These represent the dominant, large-scale nectar flows in the studied region, each lasting several weeks and providing abundant and continuous nutrition to the colonies. Thus, the feeding experiment consisted of eight controlled artificial diets (April), followed by three natural feeding periods, each representing a distinct ecological and nutritional context. This design ensured that the bees received adequate and diverse nutritional input throughout the study and allowed us to separately assess the effects of supplementation (April) and botanical origin/seasonality (May–July).

2. In the Introduction, it was said bee venom quality relates to the accumulation of heavy metals, including Pb, Cd, Cu, Ni, and Zn. Why was the content of Ni not measured?

A: We thank the reviewer for this observation. Nickel (Ni) was indeed included in our analytical protocol. However, in all analyzed samples, Ni concentrations were below the detection limit of the ICP-OES method used in this study. As no quantifiable values were obtained, Ni could not be reported in the results tables. To avoid confusion, we have now added a clarification in the Methods/Results section stating that Ni was measured but remained undetectable in all venom samples.

3. The peptides and enzymes are important chemical constituents of bee venom. However, they were not measured.

A: We thank the reviewer for this comment. The present study was intentionally designed to evaluate the physicochemical parameters and the mineral/heavy metal composition of bee venom. For this reason, analyses of peptides or enzymatic components were not included. Our objective was to characterize how seasonal and nutritional factors influence the inorganic and elemental profile of the venom, whereas peptide and enzyme profiling falls outside the analytical scope of this work and is addressed in a separate, dedicated study.

4. It was said rapeseed showed lower Ph values 5.6–5.9, which was wrong. The pH values of Rnp2 and Rnp3 were >6.0.

A: We appreciate the reviewer’s observation. Our statement was not intended to suggest that all rapeseed (Rnp) samples had pH values below 6.0. Rather, we aimed to highlight that the lowest pH values observed across the entire dataset occurred during the rapeseed foraging period, particularly in the V6–Rnp6 variant (pH 5.6). This indicates that rapeseed was the period during which the minimum pH values were recorded, not that every Rnp sample fell within the 5.6–5.9 range. We have revised the wording to avoid this unintended interpretation.

5. For the data 5.5 ± 0.4, 3.1 ± 0.3 and 3.2 ± 0.3, ‘%’ was missing.

A: The changes have been made; thank you for the observation!

We are grateful for your time and effort to review our paper, and we hope we have successfully addressed all your queries!

Sincerely,

The authors.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This paper examines how nutritional supplements and seasonal floral sources affect the physico-chemical and mineral profile of Apis mellifera bee venom. The topic is interesting and the experimental design is reasonably comprehensive. However, several issues need to be addressed before the manuscript can be considered for publication.

Major Issues:

1. There is a clear disconnect between the Introduction and the Results. The Introduction emphasizes the potential of dietary supplements to modulate venom composition (Line 57), but the data consistently show that supplementation has minimal effects. This contradiction should be acknowledged and discussed, why do your results differ from the expectations set by the cited literature?

2. Pb levels up to 36.18 µg/g are reported without any discussion of safety implications. Given that bee venom is used therapeutically, the authors need to address whether such levels pose a health risk, ideally with reference to relevant regulatory limits.

3. The ninhydrin colorimetric method described in Section 3.6 measures free amino acids, not total amino acids. Please correct the terminology or describe the hydrolysis step if one was performed.

4. The Introduction states that antioxidant capacity was evaluated (Line 101), but no such data appear in the paper. Either add the data or remove the claim.

5. Climate data come from a single hive and are discussed only descriptively. Incorporating temperature/humidity into the statistical analyses (e.g., correlation matrix) would substantially strengthen this aspect of the work.

Minor Issues:

1. The last two paragraphs of the Introduction (lines 91-106) are essentially duplicated. Please remove one.

2. Figure captions for Figs. 3 and 4 are too brief, they should be informative enough to stand alone.

3. Reference formatting needs attention: missing page numbers (e.g., 34, 57), apparent errors (e.g., 47, 50), and inconsistent use of DOIs.

Author Response

Journal: Molecules (ISSN 1420-3049)

Manuscript ID: molecules-4268198

Title: Physico-Chemical and Mineral Variability of Apis mellifera Bee Venom Across Seasons and Feeding Regimes

Authors: Adrian-Dan Rășinar , Isidora Radulov * , Adina Berbecea * , Silvia Pătruică

Section: Natural Products Chemistry

Special Issue: Bee Product Chemistry: Identification and Characterization of Natural Compounds, 2nd Edition

Dear Reviewer,

            Thank you for taking the time to review our manuscript. We greatly appreciate the suggestions made to improve our paper.

We have addressed your comments as follows:

This paper examines how nutritional supplements and seasonal floral sources affect the physico-chemical and mineral profile of Apis mellifera bee venom. The topic is interesting and the experimental design is reasonably comprehensive. However, several issues need to be addressed before the manuscript can be considered for publication.

Major Issues:

1. There is a clear disconnect between the Introduction and the Results. The Introduction emphasizes the potential of dietary supplements to modulate venom composition (Line 57), but the data consistently show that supplementation has minimal effects. This contradiction should be acknowledged and discussed, why do your results differ from the expectations set by the cited literature?

A: We thank the reviewer for this important observation. We agree that the Introduction highlights findings from previous studies suggesting that essential oils and probiotic supplements may influence certain biochemical characteristics of bee products, including venom. Compared with other studies, our results show that, under the conditions of this experiment, supplementation produced only minor and inconsistent effects, whereas seasonal dynamics and floral origin were the dominant drivers of physico-chemical and mineral variability. We have now clarified this point in the Results and Discussion section and aligned it with the conclusions of the manuscript (marked with blue).

2. Pb levels up to 36.18 µg/g are reported without any discussion of safety implications. Given that bee venom is used therapeutically, the authors need to address whether such levels pose a health risk, ideally with reference to relevant regulatory limits.

A: Thank you for your comment. Given that the permitted daily exposure to lead for oral, parenteral, and inhalation routes is 5 µg/day (according to the ICH Q3D(R2) Guideline for Elemental Impurities), and that the usual maintenance dose in venom immunotherapy is 100 µg of bee venom, the maximum measured concentration of 36.18 µg/g remains well below this limit (36.18 µg/g × 0.0001 g = 0.0036 µg).

3. The ninhydrin colorimetric method described in Section 3.6 measures free amino acids, not total amino acids. Please correct the terminology or describe the hydrolysis step if one was performed.

A: We thank the reviewer for this valuable observation. We agree that the ninhydrin colorimetric assay quantifies free amino acids unless preceded by a hydrolysis step. In our study, the determination of amino acids was performed after acid hydrolysis, which allows quantification of total amino acids. To clarify this point, we have revised Section 3.6 to include a detailed description of the hydrolysis procedure. The updated method now specifies that 3 mg of dried bee venom were subjected to acid hydrolysis with 6 N HCl at 110 °C for 24 h, under nitrogen to prevent oxidative degradation. After hydrolysis, samples were dried to remove excess HCl, reconstituted, and subsequently reacted with ninhydrin for colorimetric quantification at 570 nm using alanine as the calibration standard. This correction ensures that the terminology “total amino acids” is accurate and consistent with the analytical procedure employed. We have updated the manuscript accordingly to avoid ambiguity and to accurately reflect the methodology used.

4. The Introduction states that antioxidant capacity was evaluated (Line 101), but no such data appear in the paper. Either add the data or remove the claim.

A: We thank the reviewer for pointing out this inconsistency. The evaluation of antioxidant capacity was initially considered but was not included in the final experimental design. To avoid misleading the reader, we have removed the reference to antioxidant capacity from the Introduction and ensured that no such claims remain

5. Climate data come from a single hive and are discussed only descriptively. Incorporating temperature/humidity into the statistical analyses (e.g., correlation matrix) would substantially strengthen this aspect of the work.

A: We thank the reviewer for this constructive suggestion. In the revised manuscript, we have clarified the role of temperature and humidity in interpreting seasonal variations in venom composition. Our objective was to assess how these meteorological variables influence colony foraging activity, metabolic intensity, and biochemical processes involved in venom biosynthesis. This ecological interpretation is supported by the observed seasonal dynamics of hive weight variations, as well as by the cited literature. We were unable to integrate temperature and humidity into multivariate statistical analyses for two methodological reasons:

- Climatic data were recorded at the hive level, not at the level of individual venom samples. This resulted in a single pair of temperature–humidity values for each sampling period, insufficient to ensure the replication required for robust statistical models or correlation analyses.

- The number of sampling points is limited. With only a few seasonal intervals, including meteorological variables in correlation matrices or regression models would not produce statistically reliable or interpretable results.

For these reasons, temperature and humidity were used as contextual ecological variables, intended to support the interpretation of seasonal patterns, rather than as independent predictors in statistical models. This has been clarified in the manuscript, and the descriptive link between meteorological conditions, hive weight dynamics, and venom composition has been strengthened.

Minor Issues:

1. The last two paragraphs of the Introduction (lines 91-106) are essentially duplicated. Please remove one.

A:  Thank you for your comment. The paragraph has been removed.

2. Figure captions for Figs. 3 and 4 are too brief, they should be informative enough to stand alone.

A: We have revised the figure captions for Figs. 3 and 4.

Figure 3. Hierarchical clustering dendrogram illustrating the similarity relationships among bee venom samples based on their physico-chemical and mineral parameters. The clustering pattern highlights how samples group according to seasonal conditions and floral origin, revealing distinct profiles associated with rapeseed, acacia, and sunflower flows. This visualization provides an integrated overview of multivariate variability and supports the interpretation that environmental and botanical factors are the primary drivers shaping venom composition.

Figure 4. Pearson correlation matrix illustrating the relationships among environmental variables (temperature, humidity), hive-weight dynamics, and the physico-chemical and mineral parameters of bee venom. Positive and negative correlations are represented by color intensity, highlighting how seasonal conditions and forage availability co-vary with venom composition. The matrix reveals clusters of strongly associated variables, supporting the interpretation that natural floral resources and ecological factors are the primary drivers shaping venom quality and colony metabolic activity.

3. Reference formatting needs attention: missing page numbers (e.g., 34, 57), apparent errors (e.g., 47, 50), and inconsistent use of DOIs.

A: We have carefully rechecked the references and made corrections where inconsistencies were identified. Thank you!

We are grateful for your time and effort to review our paper, and we hope we have successfully addressed all your queries!

Sincerely,

The authors.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript greatly improves over the first version, but three of the serious flaws I evidenced were not addressed at all:

• Discussion on moisture/dry matter content (lines 116-140) is meaningless since moisture content of venom depends mostly on venom dehydration in the collecting apparatus (see line 113), which depends on air relative humidity during the collection time much more than on venom moisture in the venom sac; for a reliable discussion on nutritional and environmental conditions, venom should have been extracted directly from the venom sac and immediately analyzed
• It is not clear what Authors intend for variability when discussing on their results; as an example they state that “Calcium concentrations showed moderate variability, ranging from 0.806 ± 0.143 238 mg/g (V7-Snp7) to 3.686 ± 0.276 mg/g (V2-Anp2)” (lines 238-239) and that “Potassium exhibited the highest variability, with values ranging from 3.195 ± 0.251 247 mg/g (V7-Rnp7) to 11.372 ± 0.405 mg/g (V8-Anp8)” (lines 247-248), but the maximum value for Ca is 4.5 times the lower one, while the maximum value of K is only 3.5 times the lower one; similarily, the maximum value for Mg is 9.5 times the lower one and the maximum value for P is six times the lower one; therefore, Mg shows the highest variability, followed by P, Ca and K and a suitable statistical variability index should be adopted to highlight variability
• Figure 5 graphs present various defects: 1) y-axis graduations cover the interval 0-80 in April, May, and July, but only the interval 0-70 in June and May graduations are 0, 20, 40, 60, and 80, while in the other months graduations are 0, 10, 20, 30, and so on; please uniform them; 2) in June, the label 2025-06-30 is under the dot of June 29^th; 3) in June and July are present label pertaining to the next month; please remove them; 4) uniform the grids in the four diagrams. For all these reasons I repeat my suggestion of merging all the data in a single diagram from April 1^rst to July 31^th by reducing the space between days.

Moreover, some minor points should be considered before publication:

Scientific names should be written in italics; see lines 17, 92

Line 20: 344.0 instead of 344 since the maximum shows one decimal digit

Lines 90-91: add brackets “regimes (sugar syrup, essential oils, probiotic supplements, and natural pollen–nectar sources) shape”

Line 217: “phospholipase A₂” (see line 40) and not “phospholipase A2”

Line 415: delete “μg/g)”

Line 421: delete “μg/g”

Line 464: a paragraph title would help in separating the discussion on heavy metals from the general results introduced by the dendrogram

Lines 542-556: were supers added or removed during the observation period? If so, some sharp weigh shifts should be caused by such management practices and not by environmental factors; please check

Line 561: “further” instead of “urther”

Line 663: I do not understand the repletion of the word oven in brackets; may be something was missed

Author Response

Discussion on moisture/dry matter content (lines 116-140) is meaningless since moisture content of venom depends mostly on venom dehydration in the collecting apparatus (see line 113), which depends on air relative humidity during the collection time much more than on venom moisture in the venom sac; for a reliable discussion on nutritional and environmental conditions, venom should have been extracted directly from the venom sac and immediately analyzed.

A: Thank you for this valuable observation. We agree that the absolute moisture content of electrically collected bee venom is influenced by dehydration occurring on the collection plate. This aspect is acknowledged in our manuscript, where we specify that the analyzed samples represent “dehydrated bee venom, indicating an advanced stage of dehydration” and that moisture and dry matter showed the “expected inverse relationship” (lines 116–120). However, we respectfully disagree with the statement that the discussion is irrelevant or that no conclusions regarding seasonal or environmental influences can be drawn. Several studies using the same electrical collection method (Argena et al., 2021; Koç et al., 2025; de Graaf et al., 2021) report moisture and dry matter values and interpret seasonal or nutritional differences despite dehydration occurring on the collection plate. These studies support the concept that, when collection conditions are standardized, relative differences reflect seasonal and ecological influences rather than dehydration artifacts alone.

1. All samples were subjected to identical standardized collection and dehydration conditions.

Bee venom was collected using the same device (BeeWhisper v.5.1), with the same stimulation duration (30 min), identical exposure conditions, and identical post-collection handling for all experimental variants. Because the dehydration process was constant across all treatments, systematic differences between samples cannot be attributed exclusively to the collection plate.

2. Seasonal and botanical patterns were consistent and biologically meaningful.

Despite identical dehydration conditions, moisture/dry matter values exhibited clear and repeatable seasonal patterns across all variants: minimum moisture during the rapeseed flow period (e.g., V6–Rnp6: 11.2%), intermediate values during the acacia period, and higher values during the sunflower flow period (e.g., V1–Snp1: 16.8%). These patterns are consistent with known seasonal changes in venom secretion physiology and colony metabolism influenced by nectar intake, as documented in the literature and discussed in our manuscript. If dehydration had been the sole determining factor, values would have tended to converge among samples collected within the same harvesting session, which was not observed.

3. Moisture/dry matter variability is widely used in bee venom research, even when venom is collected on plates.

Standardized methodologies for Apis mellifera venom research (de Graaf et al., 2021) explicitly recognize that electrically collected venom is dehydrated; nevertheless, moisture and dry matter remain accepted parameters for evaluating venom quality and stability. Our discussion follows this established methodological framework.

4. The reviewer’s proposed method (direct extraction from the venom sac) is not the standard approach for ecological or field-scale studies.

Direct extraction from the venom gland is appropriate for proteomic or mechanistic studies; however, it is not feasible for multi-colony, multi-season field experiments such as ours. The standard method for ecological, nutritional, and environmental studies is electrical collection, which inherently involves venom dehydration. Therefore, our interpretations remain valid within the methodological framework of field-based bee venom research.

5. Our conclusions do not overestimate moisture as a physiological trait.

We explicitly stated that moisture/dry matter reflects both environmental and biological influences and that supplementation effects were limited. Thus, we do not claim that moisture directly reflects the composition of the venom sac itself, but rather that the relative differences observed under identical collection conditions reflect seasonal and ecological variability, as supported by our data.

-  ARGENA, N., TANANAKI, C., THRASYVOULOU, A., Goras, G., KANELIS, D., & LIOLIOS, V. (2021). Seasonal variation on bee venom collection. The impact on some biological aspects on Apis mellifera. Journal of the Hellenic, Veterinary Medical Society, 72(2), 2861–2868. https://doi.org/10.12681/jhvms.27524

- Uçak Koç, A. (2025). Effect of Season and Genotype on Bee Venom Content in Aegean Region Conditions. Turkish Journal of Agriculture - Food Science and Technology, 13(7), 1827–1832. https://doi.org/10.24925/turjaf.v13i7.1827-1832.7598

- de Graaf, D.C.; Brochetto Braga, M.R.; de Abreu, R.M.M.; Blank, S.; Bridts, C.H.; De Clerck, L.S.; Devreese, B.; Ebo, D.G.; Ferris, T.J.; Hagendorens, M.M.; et al. Standard methods for Apis mellifera venom research. Journal of Apicultural Research 2021, 60, 1-31, doi:10.1080/00218839.2020.1801073.

It is not clear what Authors intend for variability when discussing on their results; as an example they state that “Calcium concentrations showed moderate variability, ranging from 0.806 ± 0.143 238 mg/g (V7-Snp7) to 3.686 ± 0.276 mg/g (V2-Anp2)” (lines 238-239) and that “Potassium exhibited the highest variability, with values ranging from 3.195 ± 0.251 247 mg/g (V7-Rnp7) to 11.372 ± 0.405 mg/g (V8-Anp8)” (lines 247-248), but the maximum value for Ca is 4.5 times the lower one, while the maximum value of K is only 3.5 times the lower one; similarily, the maximum value for Mg is 9.5 times the lower one and the maximum value for P is six times the lower one; therefore, Mg shows the highest variability, followed by P, Ca and K and a suitable statistical variability index should be adopted to highlight variability

A: Thank you for this observation and for requesting clarification. Our intention was to highlight the amplitude of the range of values obtained for the determined chemical indicators. During the English writing process, the use of the term “variability” resulted in wording that was not sufficiently precise from a scientific perspective. To avoid such interpretations, the paragraphs were reformulated in a clearer manner and in a style more appropriate for the scientific context, and the modifications made were highlighted in red in the manuscript. In this context, we consider that the inclusion of additional statistical variability indices is not necessary.

Figure 5 graphs present various defects: 1) y-axis graduations cover the interval 0-80 in April, May, and July, but only the interval 0-70 in June and May graduations are 0, 20, 40, 60, and 80, while in the other months graduations are 0, 10, 20, 30, and so on; please uniform them; 2) in June, the label 2025-06-30 is under the dot of June 29th; 3) in June and July are present label pertaining to the next month; please remove them; 4) uniform the grids in the four diagrams. For all these reasons I repeat my suggestion of merging all the data in a single diagram from April 1rst to July 31th by reducing the space between days.

A: Thank you for the observation. Following your suggestion, we combined the four separate graphs into a single integrated graph.

Moreover, some minor points should be considered before publication:

Scientific names should be written in italics; see lines 17, 92

Line 20: 344.0 instead of 344 since the maximum shows one decimal digit

Lines 90-91: add brackets “regimes (sugar syrup, essential oils, probiotic supplements, and natural pollen–nectar sources) shape”

Line 217: “phospholipase A₂” (see line 40) and not “phospholipase A2”

Line 415: delete “μg/g)”

Line 421: delete “μg/g”

Line 464: a paragraph title would help in separating the discussion on heavy metals from the general results introduced by the dendrogram

Line 561: “further” instead of “urther”

A: Thank you for the suggestion. We have introduced a paragraph under the subtitle: Cluster analysis of physicochemical and mineral parameters.

Lines 542-556: were supers added or removed during the observation period? If so, some sharp weigh shifts should be caused by such management practices and not by environmental factors; please check

A: Thank you for the observation. The only possible overlap period could have been during the harvesting of acacia and rapeseed; however, this was ruled out because, in the area where the hives were located, there was no simultaneous foraging from the two plants. After the rapeseed harvest, the hives were moved to the acacia harvest, and venom collection was carried out after each harvest.

We also note that during the rapeseed and acacia flowering periods, there were no other nectar sources in the area that could provide a production yield. As for the sunflower harvest, venom collection was carried out at the end of the flowering period, with the bee colonies benefiting only from the nectar and pollen of this plant.

Line 663: I do not understand the repletion of the word oven in brackets; may be something was missed

A: Regarding the minor comments, we would like to thank the reviewer and mention that all suggested corrections have been implemented in the manuscript and highlighted in red.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

can be accepted

Author Response

Thank you for taking the time to review our manuscript. We greatly appreciate the suggestions made to improve our paper. We are grateful for your time and effort to review our paper, and we hope we have successfully addressed all your queries!

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The comments were addressed by the authors. And this manuscript could be accepted in the current form.

Author Response

Thank you for taking the time to review our manuscript. We greatly appreciate the suggestions made to improve our paper. We are grateful for your time and effort to review our paper, and we hope we have successfully addressed all your queries!

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have adequately addressed all the concerns raised in the previous review. The only remaining issue is that the revised captions for Figures 3 and 4 contain interpretive and conclusive statements (e.g., supports the interpretation that environmental and botanical factors are the primary drivers shaping venom composition) that belong in the Discussion rather than in figure legends. Please revise the captions to objectively describe what is shown in each figure, removing any interpretive language.

Author Response

The authors have adequately addressed all the concerns raised in the previous review. The only remaining issue is that the revised captions for Figures 3 and 4 contain interpretive and conclusive statements (e.g., supports the interpretation that environmental and botanical factors are the primary drivers shaping venom composition) that belong in the Discussion rather than in figure legends. Please revise the captions to objectively describe what is shown in each figure, removing any interpretive language.

A: Thank you for the observation. We have made the requested modifications in the manuscript.

We are grateful for your time and effort to review our paper, and we hope we have successfully addressed all your queries!

Author Response File: Author Response.pdf