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The effects of queen mandibular pheromone on nurse-aged honey bee (Apis mellifera) hypopharyngeal gland size and lipid metabolism
Queen mandibular pheromone and its effects on honey bee hypopharyngeal gland size and lipid metabolism
https://orcid.org/0009-0007-7164-8081
Oreshkova Angela Conceptualization Data curation Formal analysis Funding acquisition Investigation Project administration Visualization Writing – original draft Writing – review & editing 1 *
https://orcid.org/0009-0005-5169-473X
Scofield Sebastian Conceptualization Data curation Formal analysis Investigation Methodology Writing – review & editing 1
Amdam Gro V. Conceptualization Project administration Resources Supervision Validation Writing – review & editing 1 2
1 School of Life Sciences, Arizona State University, Tempe, AZ, United States of America
2 Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, Aas, Norway
Rueppell Olav Editor
University of Alberta, CANADA
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: aoreshko@asu.edu
6 9 2024
2024
19 9 e029250021 9 2023
5 7 2024
© 2024 Oreshkova et al
2024
Oreshkova et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Queen honey bees (Apis mellifera) release Queen Mandibular Pheromone (QMP) to regulate traits in the caste of female helpers called workers. QMP signals the queen’s presence and suppresses worker reproduction. In the absence of reproduction, young workers take care of the queen and her larvae (nurse tasks), while older workers forage. In nurses, QMP increases lipid stores in abdominal fat tissue (fat body) and protein content in hypopharyngeal glands (HPG). HPG are worker-specific head glands that can synthesize proteinaceous jelly used in colony nourishment. Larger HPG signifies ability to secrete proteinaceous jelly, while shrunken glands characterize foragers that do not make jelly. While it is known that QMP increases abdominal lipid stores, the mechanism is unclear: Does QMP make workers consume more pollen which provides lipids, or does QMP increase lipogenic capacity? Here, we measure abdominal lipogenic capacity as fatty acid synthase (FAS) activity while monitoring abdominal protein content and HPG size in caged workers. Cages allow us to rigorously control worker age, pheromone exposure, and diet. In our 2-factorial design, 3- vs. 8-day-old workers (age factor) were exposed to synthetic QMP or not (pheromone factor) while consuming a lipid deficient diet. We found that QMP did not influence abdominal FAS activity or protein content, but QMP still increased HPG size in the absence of dietary lipids. Our data revealed a positive correlation between abdominal protein content and HPG size. Our findings show that QMP is not a strong modulator of lipogenic capacity in caged worker bees. However, our data may reflect that QMP mobilizes abdominal protein for production of jelly, in line with previous findings on effects of honey bee Brood Pheromone. Overall, our study expands the understanding of how QMP can affect honey bee workers. Such insights are important beyond regulatory biology, as QMP is used in various aspects of beekeeping.

http://dx.doi.org/10.13039/100007482 Arizona State University TPF-FY23-3170019968 https://orcid.org/0009-0007-7164-8081
Oreshkova Angela This project was supported by the Barrett Honors College at Arizona State University. URL: https://barretthonors.asu.edu/. The Honors Thesis and Honors Project Funding grant (TPF-FY23-3170019968) was awarded to AO. The funders did not and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availabilitye have deposited our raw data to Figshare and down below we have provided links to each dataset/figure associated with the manuscript. 1. Supplementary Information Fig 2 (doi: https://doi.org/10.6084/m9.figshare.26280910.v1) 2. Supplementary Information Fig 1 (doi: https://doi.org/10.6084/m9.figshare.26263769.v2) 3. Supplementary Information File (doi: https://doi.org/10.6084/m9.figshare.26263250.v1) 4. Average mortality rate measured in percentage (%) in each cage (doi: https://doi.org/10.6084/m9.figshare.24848235.v1) 5. Hypopharyngeal Gland Photos (doi: https://doi.org/10.6084/m9.figshare.24164346.v1) 6. R code script for the analysis of the manuscript (doi: https://doi.org/10.6084/m9.figshare.24164343.v2) 7. R code history (doi: https://doi.org/10.6084/m9.figshare.24164331.v1) 8. Abdominal metrics dataset (abdominal fatty acid synthase values in nmol/min, abdominal protein values in mg, and normalized fatty acid synthase values in nmol/min/mg) (doi: https://doi.org/10.6084/m9.figshare.24164316.v1) 9. Average hypopharyngeal acini area measured in mm2 (doi: https://doi.org/10.6084/m9.figshare.24164265.v1) 10. Average protein paste and sucrose solution consumption measured in mg/bee/day (doi: https://doi.org/10.6084/m9.figshare.24158433.v1).
Data Availability

e have deposited our raw data to Figshare and down below we have provided links to each dataset/figure associated with the manuscript. 1. Supplementary Information Fig 2 (doi: https://doi.org/10.6084/m9.figshare.26280910.v1) 2. Supplementary Information Fig 1 (doi: https://doi.org/10.6084/m9.figshare.26263769.v2) 3. Supplementary Information File (doi: https://doi.org/10.6084/m9.figshare.26263250.v1) 4. Average mortality rate measured in percentage (%) in each cage (doi: https://doi.org/10.6084/m9.figshare.24848235.v1) 5. Hypopharyngeal Gland Photos (doi: https://doi.org/10.6084/m9.figshare.24164346.v1) 6. R code script for the analysis of the manuscript (doi: https://doi.org/10.6084/m9.figshare.24164343.v2) 7. R code history (doi: https://doi.org/10.6084/m9.figshare.24164331.v1) 8. Abdominal metrics dataset (abdominal fatty acid synthase values in nmol/min, abdominal protein values in mg, and normalized fatty acid synthase values in nmol/min/mg) (doi: https://doi.org/10.6084/m9.figshare.24164316.v1) 9. Average hypopharyngeal acini area measured in mm2 (doi: https://doi.org/10.6084/m9.figshare.24164265.v1) 10. Average protein paste and sucrose solution consumption measured in mg/bee/day (doi: https://doi.org/10.6084/m9.figshare.24158433.v1).
==== Body
pmc1. Introduction

Pheromones are chemical signals used for communication between members of the same species [1]. In insects, some of the extensively studied roles of pheromones include attraction of mates, signaling of reproductive status, maintenance of social hierarchy, and recognition of kin [1]. Insect pheromones are categorized as releaser and/or primer pheromones. Releasers elicit immediate behavioral responses within seconds or minutes, while primers produce long-term endocrine or reproductive changes that may take days to operate in full effect [2, 3]. The long-term physiological effects that primer pheromones exhibit largely influence insect colony organization, caste structure, and the division of labor [4]. One of the most intricate and highly studied insect pheromonal systems is that of honey bees (A. mellifera), with approximately 50 substances that have a biologically relevant role in colony life [5].

Each honey bee colony consists of a reproductive queen, non-reproductive females known as workers, and males known as drones [6]. Adult worker honey bees show a division of labor called age polyethism, in which individuals perform different social tasks as they age. Young workers (first 2–3 weeks after emerging from pupation) work inside the nest to groom and nourish all colony members, while older workers venture outside to collect pollen, nectar, water and propolis [7, 8]. This age polyethism is accompanied by physiological specializations, such as the young workers (nurses) having larger abdominal lipid stores and hypopharyngeal glands (HPGs) to support their role in colony nourishment, while foragers are leaner and rely on sugars to fuel their flights [7–9].

Queen mandibular pheromone (QMP), released by the queen bee, influences worker division of labor and physiology [4]. QMP signals the presence of a queen and is categorized as both a releaser and a primer. As a releaser, QMP prompts young worker bees to groom the queen through a retinue response and further spread the pheromone in the colony [3, 5]. As a primer, QMP suppresses genes associated with foraging while activating genes associated with nursing [10]. These are a few examples of the well-known functions of QMP. Past studies show that QMP can increase abdominal lipid stores and the size of HPGs in nurse-aged workers [11–14]. HPGs are paired head glands that can produce proteinaceous jelly [4]. They contain a higher concentration of lipids than other tissues in the head, and bees fed diets high in lipids gain abdominal fat and develop larger HPGs [15, 16]. Furthermore, there has been research that exposure to QMP increases both pollen and sucrose solution consumption in nurse-aged bees [13]. The variable macronutrient composition (lipids and amino acids) of pollen as well as the potential contamination of pesticides makes it difficult to tease apart how QMP acts to influence physiology in these studies [13, 15, 16]. Are the QMP-induced increases in lipid stores and HPG size behaviorally modulated by increasing consumption of lipid-containing pollen, or are they due to effects on metabolic pathways regulating lipid and protein synthesis and storage?

To assess a possible increase in lipid synthesis in the fat body of workers exposed to QMP, we measured the activity of fatty acid synthase (FAS), an enzyme that catalyzes a rate-limiting step of de novo lipogenic capacity that combines malonyl-CoA with acetyl-CoA to produce long-chain fatty acids, thus serving as a quantitative metric of lipogenic capacity, the capacity of a tissue to synthesize lipids de novo [17]. These long-chain fatty acids are stored as triglycerides in honey bees and most other animals [18]. Thus, in this study we use a FAS activity assay to test whether QMP influences lipogenic capacity in nurse-aged worker bees. Additionally, since nurse bees have high fat body lipids and lipoproteins synthesized there are transported to the HPGs to be used for jelly synthesis [19], we were interested in measuring if there was any relationship between fat body lipogenic capacity and HPG development. Since past studies have shown lipogenesis is more quantitatively important when bees are not fed lipids [20] and that HPG size strongly responds to dietary lipids [16], we fed bees an artificial, lipid-deficient diet, eliminating the possibility that any effect of QMP is due to increased lipid consumption. We hypothesized 1) that QMP increases lipogenic capacity in nurses’ abdominal fat bodies (functionally homologous to liver and white adipose tissue), explaining the larger lipid stores reported previously [11, 12], and 2) that this increased fat body lipogenesis functions to support HPG development so that nurses can fulfill their social role within the colony.

Our specific design was a 2 factorial cage experiment with an age factor and a pheromone factor: 3-day-old (representing developing bees not yet physiologically competent to become nurses or foragers) or 8-day-old (representing bees at peak nursing age) were exposed to synthetic QMP, or not. We chose to measure these two age groups since factors that affect nurse-bee physiology like QMP sometimes have different effects on 3 and 8-day old bees [11] and measuring both timepoints thus gives a better chance of identifying a possible effect. FAS activity in the fat body was normalized by measuring the amount of abdominal protein per nurse-aged bee. We monitored HPG size by measuring the area of the glands’ acini. For each cage, we also monitored the depletion of the lipid-deficient diet as a function of worker mass, to control for whether QMP influenced food consumption.

2. Materials and methods

2.1 Honey bees

The experiments were performed in September through November of 2022 at Arizona State University Campus, Tempe. To start, three frames from 3 different hives were collected, sealed in a mesh cage, placed in an incubator at 33°C, and kept humidified using an open dish of water at the bottom of the incubator. After 24 h, newly emerged bees were collected and placed in small (16 × 12 × 9 cm) Plexiglass and mesh cages (30 bees/cage). Bees were fed 30% w/v sucrose solution ad libitum in 20 ml syringes with their tips cut off and placed at the top of the cage as described previously [21]. To feed the bees sufficient protein for development of HPGs without feeding them lipids, we also fed the bees artificial diets using soy as a protein source as described previously [22]. We chose to use artificial diets because many studies have shown strong effects of dietary pollen on worker physiology [11], but pollen composition is highly variable and difficult to standardize [23] and pollen may be contaminated with pesticides [24]. To eliminate these issues and provide greater insight into specific nutrients in pollen, a number of recent studies have used artificial diets [16, 25, 26]. A paste containing 20% total protein was made with 21.47% soy protein isolate (MP Biomedical) and 78.26% honey and fed to the bees using 1.5 mL Eppendorf feeders placed at the bottom of each cage (Fig 1). Consumption of sucrose solution and protein paste was calculated by weighing the feeders every 24 hours. Cage experiments using cohorts of approximately 30 bees per cage maintained in an incubator are standard practice when investigating the role of pheromones and diet on physiological parameters and have been used in a number of different studies [12, 27, 28].

10.1371/journal.pone.0292500.g001 Fig 1 Cage design used for the queen mandibular pheromone treatments.

The dimensions measured were 16 cm × 12 cm × 9 cm. Because approximately half the cage was used, the effective depth was 6 cm.

A 2 × 2 factorial experimental design was used, and each cage was assigned to an age-group (3 and 8-day-old bees) and a treatment group of QMP+ or QMP−. Each combination of age and presence/absence of QMP was assigned to one cage and this was replicated 3 times, yielding a total of 12 cages (1 cage × 4 treatment groups × 3 replicates; Fig 2). This level of replication has been used for other cage experiments (for example [29]) and is sufficient for a study in which the focus is not on cage level metrics but on sampling bees within each cage. To produce bees of specific ages, cages were set up either 3 or 8 days prior to each sampling day. The cages were maintained at 33°C in a dark, humidified incubator and mortality was recorded every day in each cage. Cages also received the QMP treatment for the duration of those 3 or 8 days. Synthetic QMP (“TempQueen”; Betterbee Inc.) was presented as a slow-release strip placed at the bottom of the QMP+ cages, while QMP− cages did not receive a QMP strip, as in an earlier study [30]. This dose and application of QMP is standard practice for cage experiments examining the role of QMP [11, 31–33]. The strip was used according to the manufacture instructions and is equal to 10 queen equivalents of the 5-component blend of QMP. It mimics exposure to natural QMP, a mixture of 9-keto-2-(E)-decenoic acid (ODA), the enantiomers of 9-hydroxy-2-(E)-decenoic acid (9-HDA)(88% R-(–) and 12% S-(+)), methyl p-hydroxybenzoate (HOB), and 4-hydroxy-3-methoxyphenylethanol (HVA) [2]. One queen equivalent represents the amount of QMP a mated queen will produce in a 24-hour period and contains 200 mg of ODA, 80 mg of 9-HDA, 20 mg of HOB, and 2 mg of HVA [34]. QMP is not volatile as it is spread by honey bee workers via trophallaxis, antennation, and cuticular contact [30]. Thus, all cages were kept in the same incubator. During sampling, bees were anesthetized on ice, euthanized, and dissected for subsequent analyses.

10.1371/journal.pone.0292500.g002 Fig 2 Pictorial depiction of the 2-factorial design used in the study.

The 2-factorial design that represented all combinations of the presence vs absence of synthetic queen mandibular pheromone (‘QMP’) and 3 vs 8-day-old nurse-aged honey bees (‘age’). This resulted in 4 experimental treatments, each applied to one cage of bees. The design was replicated three times, resulting in three replicates. The small boxes labelled inside each large box represent the cage identity (12 total cages used).

2.2 Fatty acid synthase (FAS) activity measurements

Fat body fatty acid synthase (FAS) activity was measured using a previously described method with minor modifications [35]. Briefly, the abdominal carcass (complete abdominal cuticle with adhering fat body tissue minus the stinger, ovaries, gut, and crop) was isolated from workers, pooled in pairs of two, homogenized in phosphate-buffered saline containing protease inhibitors (11697498001; Roche Applied Science; Indianapolis; IN; USA), sonicated for 30 seconds, and centrifuged at 10,000 × g for 5 minutes at 4°C. The resulting supernatant was collected and assayed immediately in a 96-well microplate. In each well, 33.3 μL of supernatant was mixed with 163.3 μL of 2.0 M potassium phosphate buffer, pH 7.1, 16.7 μl of 20 mM dithiothreitol, 20 μl of 0.25 mM acetyl-CoA, 16.7 μl of 60 mM EDTA. To initiate the reaction, 33.3 μl of 0.39 mM malonyl-CoA was added to each well. FAS activity was measured as the oxidation of NADPH at 340 nm and 37°C using a UV/VIS spectrophotometer (Synergy H1 Multimode Reader, BioTek). A background correction was made for the oxidation of NADPH in the absence of malonyl-CoA. Background and sample wells were both measured in duplicate. This assay was replicated 3 times. In each replicate, 2 bees were pooled for each biological sample and 6 samples totaling 12 bees were collected from each cage for all four treatment conditions. This totaled 144 abdomens sampled across three replicates. FAS activity was calculated as nmol of NADPH oxidized/min/abdomen. Additionally, FAS activity was normalized by the amount of total soluble protein in each sample, measured with a BCA Assay Kit (Thermo Scientific) according to manufacturer’s instructions, giving FAS activity in nmol/min/mg. For a more complete picture of the metabolic state of the bee, we report total abdominal FAS activity as well as FAS activity normalized to mg of abdominal protein. Because our dissection approach collects the entire abdominal cuticle and attached fat body, it produces a metric of total lipogenic capacity per abdomen, which is similar to past studies that have measured total lipids per abdomen using the same dissection technique [7, 12, 15, 16]. To control for potential differences in protein extraction efficiency, we also measure protein concentration in each sample and normalize FAS activity relative to extracted protein. Comparing results from the two metrics makes it clearer when treatment group differences are due to changes in the quantity of activated FAS or due to changes in the normalization factor itself.

2.3 Hypopharyngeal gland measurements

To determine whether QMP affects HPG acini size, honey bee heads from the same bees used for FAS activity were flash frozen in liquid nitrogen and kept in a -80°C freezer until they were dissected. Per treatment group, 18 heads were dissected, resulting in a total of 72 heads. For each head, the HPGs were first dissected into a glass plate with concave deep wells containing 40 μl of 10x Giemsa for 7 min. They were then transferred into a flat microscope slide that contained 60 μl of 1x PBS buffer (37 mM NaCl, 2.7 mM KCl, and 10 mM PO4, pH 7.4) and visualized at 60 to 80x magnification, as previously described [36]. The glands were visualized under a Leica M205C stereoscope with a Leica DFC450 camera using the Leica Applications Suite v4.5 software. A blind observer was then told to select 10 acini per bee with the criteria that the acini were in focus, had clear attachment points to the collecting duct, and were average relative to all the acini in the photo. The area (mm) of those 10 selected acini per bee was measured using ImageJ by the researcher. The areas were then averaged per bee and analyzed as a pooled sample in which the heads that were pooled together corresponded to the abdomens that were pooled together during the FAS analysis.

2.4 Statistical analysis

The effects of age and QMP on abdominal FAS activity, abdominal protein, and HPG acini area were preferentially processed with an ANOVA test using three variables: age, treatment, and replicate, and the interaction effects between all three variables. Datasets that were analyzed with ANOVA adhered to its assumptions of normality, estimated by normal probability plots of the datasets and a Shapiro-Wilks test, and homogeneity of variances, determined by a Levene’s test. The minimum p value for a significant dataset was 0.05. When assumptions were not met, in the case of normalized FAS activity dataset and protein/sucrose consumption, a non-parametric test was used, specifically, a Kruskal-Wallis test to look at differences between the four treatment groups. The non-parametric test was followed by a Dunn’s Post hoc test if needed. Replicates were included in the ANOVA to control for differences between replicates. A total of three replicates were performed for the study and each replicate reflects a combination of cage/day and FAS assay plate variation. For each parameter analyzed with either a parametric or non-parametric test, replicate effects were included in an appropriate model. When using ANOVA, replicate was added as a factor including interaction effects between replicate and other factors. For the normalized FAS data that did not meet the assumptions for ANOVA, a Kruskal-Wallis test was used to determine if there were any significant differences between replicates. Since the pattern was similar across the three replicates (S2C Fig), the data were pooled to look for differences between treatment groups. The relationships between HPG acini size and abdominal protein and abdominal FAS activity were analyzed using linear regression. Because the data deviated significantly from the assumptions of normality, the relationship between abdominal protein and FAS activity was analyzed using a Kendall-Theil Sen Siegel non-parametric linear regression. All statistics were analyzed with R version 4.3.1, Rstudio, and the packages ‘FSA’, ‘performance’, ‘mblm’, and ‘rcompanion’ [37–42]. For all measurements except for consumption, the experimental unit was bees or pooled bees. For consumption metrics, the cage was the experimental unit. A balanced study design was employed for all factors. The sample size for cage level consumption metrics was low at n = 3 cages per treatment as this was not the focus of our study and the metrics describe the physiological parameters of the bees. For measuring abdominal FAS activity, normalized FAS activity, abdominal protein, and HPG acini area, 2 bees were pooled for each biological sample and 6 samples totaling 12 bees were collected from each cage for all four treatment conditions. This totaled 72 biological samples pooled from 144 abdomens as three replicates were performed. Thus, the sample size was n = 18 pooled samples per treatment group for these three parameters. The sample size for the regression analyses was n = 35 pooled samples as there was a dissection issue for one of the bees and only half of the HPGs of the 24 pooled samples per replicate were measured, except for the case of abdominal protein and abdominal FAS which was n = 72 (144 total abdomens).

3. Results

3.1 Food consumption and mortality

Protein paste consumption did not significantly differ between any of the four treatment groups (Kruskal-Wallis, chi-squared = 2.6923, df = 3, P = 0.4415, n = 3 cages; Fig 3). Furthermore, sucrose consumption did not significantly differ between any of the four treatment groups (Kruskal-Wallis, chi-squared = 0.63041, df = 3, P = 0.8894, n = 3 cages; Fig 4). Protein consumption did not differ significantly between replicates (chi-squared = 3.7308, df = 2, P = 0.1548) but sucrose consumption did differ significantly between replicates (chi-squared = 7.4974, df = 2, P = 0.02355). Mortality rates were low (less than 7%) and within accepted ranges for cage experiments [21] for all ages and did not differ by QMP treatment (Kolmgorov-Smirnov, D(10) = 0.167, P = 1; S1 Fig).

10.1371/journal.pone.0292500.g003 Fig 3 The relationship between age(days)/QMP and protein consumption in mg/bee/day.

A non-parametric Kruskal-Wallis test showed that there were no statistical differences between each treatment’s protein consumption (P = 0.4415). Each point represents the average protein paste consumption for one cage (n = 3 cages). The boxplot shows the mean (middle black line of box), the interquartile range (box boundaries), and the minimum and maximum values of the distribution. Outliers are points outside the maximum and minimum of the distribution.

10.1371/journal.pone.0292500.g004 Fig 4 The relationship between age(days)/QMP and sucrose consumption in mg/bee/day.

A non-parametric Kruskal-Wallis test showed that there were no statistical differences between each treatment’s sucrose solution consumption (P = 0.8894). Each point represents the average sucrose consumption for one cage (n = 3 cages). The boxplot shows the mean (middle black line of box), the interquartile range (box boundaries), and the minimum and maximum values of the distribution. Outliers are points outside the maximum and minimum of the distribution.

3.2 Abdominal FAS activity and abdominal protein

Age (F1,60 = 2.010, P = 0.16140) and QMP treatment (F1,60 = 0.698, P = 0.40668) had no significant effect on abdominal FAS activity in nmol NADPH oxidized per minute per abdomen (Fig 5). Furthermore, there was no interaction effect between age × QMP treatment (F1,60 = 0.484, P = 0.48931), QMP treatment × replicate (F2,60 = 0.962, P = 0.29039), or age × QMP treatment × replicate (F2,60 = 0.726, P = 0.48796). However, there were significant differences in abdominal FAS activity between the three replicates (F2,60 = 29.323, P < 0.001; S2A Fig) and a significant interaction effect between age × replicate (F2,60 = 5.152, P = 0.00862). To normalize the FAS activity assay, the abdominal protein (in mg) of each pooled sample was measured. Abdominal protein was significantly higher in 8-day-old than 3-day-old bees (F1,60 = 35.693, P < 0.001; Fig 6) and significantly different between replicates (F2,60 = 36.000 P < 0.001; S2B Fig); it was not significantly affected by QMP treatment (F1,60 = 0.032, P = 0.8596), age × QMP treatment (F1,60 = 1.057, P = 0.3081), age × replicate (F2,60 = 2.809, P = 0.0682), treatment × replicate (F2,60 = 0.717, P = 0.4925), or age × treatment × replicate (F2,60 = 1.240, P = 0.2968).

10.1371/journal.pone.0292500.g005 Fig 5 The relationship between age/QMP treatment and abdominal FAS activity (NADPH oxidized) in nmol/min.

Age (F1,60 = 2.010, P = 0.16140) and QMP treatment (F1,60 = 0.698, P = 0.40668) had no significant effect on abdominal FAS activity in nmol NADPH oxidized per minute per abdomen. Furthermore, there was no interaction effect between age × QMP treatment (F1,60 = 0.484, P = 0.48931), QMP treatment × replicate (F2,60 = 0.962, P = 0.29039), or age × QMP treatment × replicate (F2,60 = 0.726, P = 0.48796). Each point represents the average abdominal FAS activity for each pooled sample of two bees. As there were 6 pools per cage (12 abdomens/bees sampled) and three replicates performed, the sample size is n = 18 pooled samples. The boxplot shows the mean (middle line of box), the interquartile range (box boundaries), and the expected variation of the data (whiskers, calculated as 1.5 times the interquartile range past the top and bottom of the box). Outliers are points outside the expected variation in the data.

10.1371/journal.pone.0292500.g006 Fig 6 The effect of age/QMP treatment and abdominal protein quantity in mg.

Abdominal protein was significantly higher in 8-day-old than 3-day-old bees (F1,60 = 35.693, P < 0.001). It was not significantly affected by QMP treatment (F1,60 = 0.032, P = 0.8596), age × QMP treatment (F1,60 = 1.057, P = 0.3081), age × replicate (F2,60 = 2.809, P = 0.0682), treatment × replicate (F2,60 = 0.717, P = 0.4925), or age × treatment × replicate (F2,60 = 1.240, P = 0.2968). Each point represents the average abdominal protein for each pooled sample of two bees. As there were 6 pools per cage (12 abdomens/bees sampled) and three replicates performed, the sample size is n = 18 pooled samples. The boxplot shows the mean (middle line of box), the interquartile range (box boundaries), and the expected variation of the data (whiskers, calculated as 1.5 times the interquartile range past the top and bottom of the box). Outliers are points outside the expected variation in the data. The asterisk indicates significant differences (*** denotes P < 0.001).

3.3 FAS activity normalized to abdominal protein

Using the abdominal protein quantity, FAS activity was normalized per pooled sample in nmol of NADPH oxidized per min per mg of protein. Normalized FAS activity significantly differed between the three replicates (chi-squared = 23.87, df = 2, P < 0.001; S2C Fig) and the four treatment groups (Kruskal-Wallis, chi-squared = 9.3498, df = 3, P = 0.02498; Fig 7). Comparisons between the four treatment groups showed that only the 3-day-old QMP + and 8-day-old QMP–groups differed significantly from each other (Dunn’s post-hoc test, Padj = 0.0397).

10.1371/journal.pone.0292500.g007 Fig 7 The effect of age/QMP treatment and normalized FAS activity in nmol/min/mg.

Normalized FAS activity significantly differed between the four treatment groups (Kruskal-Wallis, chi-squared = 9.3498, df = 3, P = 0.02498). Comparisons between the four treatment groups showed that only the 3-day-old QMP + and 8-day-old QMP–groups differed significantly from each other (Dunn’s post-hoc test, Padj = 0.0397). No other treatment groups were significantly different from one another. Each point represents the average normalized FAS activity for each pooled sample of two bees. As there were 6 pools per cage (12 abdomens/bees sampled) and three replicates performed, the sample size is n = 18 pooled samples. The boxplot shows the mean (middle line of box), the interquartile range (box boundaries), and the expected variation of the data (whiskers, calculated as 1.5 times the interquartile range past the top and bottom of the box). Outliers are points outside the expected variation in the data. Significantly different treatment groups are represented by different letters.

3.4 Average HPG acini area

Mean HPG acini area was significantly higher in 8-day-old than 3-day-old bees (F1,60 = 27.780, P < 0.001; Fig 8) and in bees treated with QMP (F1,60 = 29.156, P < 0.001) and was significantly different between replicates (F2,60 = 6.853, P = 0.00209; S2D Fig). The interaction effect age × treatment × replicate was significant (F2,60 = 4.457, P = 0.01568); the other interaction effects of age × QMP treatment (F1,60 = 0.568, P = 0.45392), age × replicate (F2,60 = 1.338, P = 0.27004), and treatment × replicate (F2,60 = 2.369, P = 0.10226) did not significantly affect mean HPG acini area.

10.1371/journal.pone.0292500.g008 Fig 8 The effect of age/QMP treatment and average HPG acini area in mm2.

Mean HPG acini area was significantly higher in 8-day-old than 3-day-old bees (F1,60 = 27.780, P < 0.001) and in bees treated with QMP (F1,60 = 29.156, P < 0.001). The interaction effect age × treatment × replicate was significant (F2,60 = 4.457, P = 0.01568); the other interaction effects of age × QMP treatment (F1,60 = 0.568, P = 0.45392), age × replicate (F2,60 = 1.338, P = 0.27004), and treatment × replicate (F2,60 = 2.369, P = 0.10226) did not significantly affect mean HPG acini area. Each point represents the average HPG acini area for each pooled sample of two bees. As there were 6 pools per cage (12 abdomens/bees sampled) and three replicates performed, the sample size is n = 18 pooled samples. The boxplot shows the mean (middle line of box), the interquartile range (box boundaries), and the expected variation of the data (whiskers, calculated as 1.5 times the interquartile range past the top and bottom of the box). Outliers are points outside the expected variation in the data. The asterisks indicate significant differences (*** denotes P < 0.001).

3.5 Relationship between HPG size and abdominal metrics

Mean HPG acini size was significantly positively predicted by abdominal protein quantity (F1,33 = 12.66, adjusted R2 = 0.2382, P = 0.00116; Fig 9) and by abdominal FAS activity (F1,33 = 5.819, adjusted R2 = 0.1181, P = 0.02123; Fig 10) but not by normalized FAS activity (F1,33 = 0.944, adjusted R2 = -0.001558, P = 0.3379). Abdominal FAS activity significantly increased with an increase in abdominal protein quantity (Kendall-Theil Sen Siegel linear regression: estimate = 1.5049 ± 1.4016, V = 2265, Efron’s pseudo R2 = 0.0498, P < 0.001; Fig 11). Refer to Table 1 for the regression analysis of P values between all combinations of abdominal protein, abdominal FAS activity and HPG acini area.

10.1371/journal.pone.0292500.g009 Fig 9 Regression scatter plot between abdominal protein (mg) and HPG acini area (mm2).

Mean HPG acini size was significantly predicted by abdominal protein quantity (F1,33 = 12.66, adjusted R2 = 0. 2382, P = 0.00116). Each point represents the correlation between the pooled sample’s (2 bees per) abdominal protein and HPG acini area (n = 35 pooled bees). Per treatment, 18 bees were used except for the case of the 3-day-old QMP + treatment group in the first replicate as there was a dissection issue. An additional sample was measured but not included in the regression analysis.

10.1371/journal.pone.0292500.g010 Fig 10 Regression scatter plot between abdominal FAS (nmol NADPH oxidized/min) and HPG acini area (mm2).

Mean HPG acini size was significantly predicted by abdominal FAS activity (F1,33 = 5.819, adjusted R2 = 0.1181, P = 0.02123). Each point represents the correlation between the pooled sample’s (2 bees per) abdominal FAS activity and HPG acini area (n = 35 pooled bees). Per treatment, 18 bees were used except for the case of the 3-day-old QMP + treatment group in the first replicate as there was a dissection issue. An additional sample was measured but not included in the regression analysis.

10.1371/journal.pone.0292500.g011 Fig 11 Non-parametric linear regression scatter plot between abdominal protein (mg) and abdominal FAS (nmol oxidized NADPH/min).

Abdominal FAS activity was significantly predicted by abdominal protein quantity (Kendall-Theil Sen Siegel linear regression: estimate = 1.5049 ± 1.4016, V = 2265, Efron’s pseudo R2 = 0.0498, P < 0.001). Each point represents the correlation between the pooled sample’s (2 bees per) abdominal protein and abdominal FAS. Per treatment, 36 bees were used, resulting in n = 72 pooled bees and a total of 144 abdomens.

10.1371/journal.pone.0292500.t001 Table 1 Regression analysis of P values between all combinations of abdominal protein, abdominal FAS activity and HPG acini area.

	Abdominal protein	Abdominal FAS activity	HPG acini area	
Abdominal protein		9.61 × 10−8 ***	0.00116**	
Abdominal FAS activity	9.61 × 10−8 ***		0.02123*	
HPG acini area	0.00116**	0.02123*		
Asterisks represent significant differences (* denotes P < 0.05

** denotes P < 0.01

*** denotes P < 0.001) for each correlation. The sample size taken for each regression analysis was N = 35, except for abdominal protein and abdominal FAS which was N = 72. The full regression analysis statistics can be found in S1 File. Normalized FAS activity was not included in this matrix as it was used as a summary statistic and will co-vary from the factors that it was calculated from (abdominal protein/abdominal FAS activity).

4. Discussion

In this study, we explored if QMP can increase fat body lipids and HPG size in nurse-aged worker bees fed a lipid-deficient diet. We show that workers exposed to synthetic QMP have larger HPGs on average, but we did not detect a change in the bees’ lipogenic capacity. In interpreting our results, it is important to note that there were several significant differences between experimental replicates. In the case of the sucrose consumption data, this is likely due to our small sample size. There were also significant replicate effects within the abdominal FAS activity data, abdominal protein data, and HPG acini size. Since we sourced bees in the experiment from three different colonies, these replicate effects may have been a result of biological variation among bees with different genetic backgrounds, but we cannot rule out contributions of technical variation as well.

During the experiment, we monitored food consumption to determine whether QMP increases HPG size through affecting worker lipogenic capacity or through simply increasing the workers’ food consumption. Overall, QMP did not influence the depletion of sucrose solution or protein paste in our cages. This finding is consistent with a previous study in which the sucrose and pollen consumption of nurse-aged bees did not differ between queenright-like treatments (QMP) and queenless-like treatments (no QMP) [14]. However, other work has shown that QMP exposure can increase food consumption in nurse-aged bees fed rich diets that contained pollen, as well as in nurse-aged bees fed with poor diets consisting of just sucrose solution [13]. Our results may have differed from these latter results because we used synthetic QMP strips while Ament and colleagues [13] used 0.1 equivalents of QMP dissolved with isopropanol and water on a microscope over slip. There also may be mixed results on whether QMP affects food consumption due to the varying sample sizes and varying diet compositions in all three studies. While our study had a sample size of 3 cages for consumption, Ament and colleagues [13] had a sample size ranging from 6 to 8 cages and Peters and colleagues [14] had a sample size ranging from around 150 to 300 bees, depending on the treatment group. The lack of significant differences in food consumption between our treatments could be due to our small sample size. Thus, our data should be interpreted cautiously. Furthermore, our study used a lipid deficient diet while Ament and colleagues [13] and Peters and colleagues [14] used a combination of diets consisting of only pollen, pollen and sucrose, or only sucrose. A recent study in our lab found that dietary protein but not fat increases FAS activity in 8-day-old bees [43]. There may be some interacting effects between QMP and nutrition which we did not test for.

Age and QMP treatment did not significantly affect FAS activity analyzed per bee, and only age significantly affected FAS activity analyzed per mg of extracted protein. This suggests that QMP does not significantly affect the de novo synthesis of lipids in nurse-aged honey bee fat bodies, but caged bees can increase their abdominal protein content as they age. Our larger sample size for mean HPG acini area (n = 18) makes these results more robust than our consumption data. In contrast, recent data from our group show that FAS activity does not differ significantly between 3d and 8d bees collected from natural colonies [43]. Our results may have differed due to the environment of both treatments. While the past study experimented on bees within natural hives who were receiving optimal nutrition from being fed by other nurse bees, our study experimented on bees within cages that received less nutrition. Contrary to our findings, it was previously reported that QMP can increase abdominal lipid stores in young bees (3, 4, and 5-day old bees) provided pollen-containing diets, as well as diets consisting only of sucrose solution [11–13]. How can this diversity of results be explained? One possibility is that QMP reduces the activity levels of young bees as shown before [44]. The mechanism underlying the larger lipid stores could be reduced energy expenditure by decreasing the catabolism rate of lipids in young bees. Future work could look at the lipid breakdown rate in bees exposed or not exposed to QMP. If the activity levels and lipid catabolism rates are different between our bees and the bees used in the other studies [11–13] due to varying ages, environment, and diet, this could explain the inconsistency of whether QMP affects abdominal lipid stores.

In our experiment, caged 8-day-old worker bees had larger amounts of abdominal protein than 3-day-old bees, while QMP had no effect on this protein level. Protein levels generally increase in young workers after they emerge from pupation [7], suggesting that our caged workers were able to obtain adequate nutrition. More specifically, abdominal protein levels are correlated with the amount of Vitellogenin (Vg) protein in colony-living worker bees [19]. Vg is an important indicator of nutrition and health in honey bees, and the protein influences several aspects of worker physiology and behavior, including the function of the HPGs [19]. We did not measure Vg in our experiment, but we did measure total abdominal protein. HPG size was not associated with measurable changes in food consumption metrics or abdominal FAS activity but was correlated with the abdominal protein content. Consumption of adequate dietary protein is necessary for the development of nurse bee physiology, such as large HPGs [43]. The correlation between abdominal protein and HPG size supports the link between the protein status of the bee and nursing status. This suggests that there is variation between bees in how much protein they consume and thus have available for synthesis of Vg in the fat body which can then be transported to the HPGs to be used for jelly synthesis [19].

Our experiment detected a significantly increased HPG acini size in both 3d and 8d nurse-aged bees exposed to QMP. QMP has been previously shown to suppress levels of circulating juvenile hormone (JH) [4, 45] and treatment with JH analog will reduce HPG size [46, 47]. Vg production is also known to be increased by QMP exposure [12], potentially due to a reduction in the inhibitory signal from JH. Thus, one explanation is that QMP allowed HPGs to develop because JH was suppressed in this treatment group. In addition to this JH effect, there may be other undiscovered mechanisms responsible for the observed pattern in our study.

It is known that HPGs contain a high concentration of lipids [15]. This, combined with nurses having high fat body lipids relative to foragers, suggests that HPG lipids are important for jelly production. Our study aimed to determine if lipogenic capacity is increased in nurses’ abdominal fat bodies in the presence of QMP, if this increased fat body lipogenesis functions to support HPG development, and whether these two pathways are connected. Using a lipid-deficient diet to eliminate the possibility of QMP increasing nurse-aged bees’ consumption of lipids, we found that QMP significantly increased HPG size without affecting nurse-aged lipogenic capacity, suggesting that the fat body lipids levels are not as important for the development of HPGs compared to abdominal protein levels. However, a limitation of our study is that we only assessed HPG size, not content or secretory activity, so it is possible that there is some relationship between fat body FAS activity and HPG function that we did not address here. For example, bees that are fed protein-containing diets develop both large HPGs and high abdominal FAS activity [43]. A recent study showed that treatment with QMP strips causes 8-day-old bees to have higher HPG expression of major royal jelly protein 1, the most abundant protein in royal jelly, supporting the idea that the increased HPG size we found in this study also results in increased jelly production [11]. Overall, this study emphasized the importance of how pheromonal regulation can influence adaptive physiology and nutrient storage in worker honey bees. This knowledge provides further insight into basic bee biology and to queen pheromone, which is commercially used in apiculture.

Supporting information

S1 Fig The relationship between age/QMP treatment and mortality of bees in %.

(DOCX)

S2 Fig The effects of replicates on abdominal FAS activity, abdominal protein, normalized FAS activity, and HPG acini area.

(DOCX)

S1 File The full regression plot analysis statistics between abdominal protein and HPG acini area, abdominal FAS and HPG acini area, and abdominal protein and abdominal FAS.

(DOCX)

We would like to thank Cahit Ozturk for providing us with the honeybees for this experiment, Amalie Strange for assisting us on how to perform the bee head dissections, Jenna Dobson for blindly choosing the acini, and Matthew Prebus for his training on how to use the Leica microscope. This research was supported by the Barrett Thesis Funding Grant from the Barrett College at Arizona State University. Special thanks to Christine Fleetwood from the Barrett College and Maricel Scalzo from the School of Life Sciences Business Office for assisting with the Thesis funding and reimbursement.

10.1371/journal.pone.0292500.r001
Decision Letter 0
Patterson Rosa Laura Academic Editor
© 2024 Laura Patterson Rosa
2024
Laura Patterson Rosa
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
30 Oct 2023

PONE-D-23-30810

The effects of queen mandibular pheromone on nurse-aged honey bee ( Apis mellifera ) hypopharyngeal gland size and lipid metabolism

PLOS ONE

Dear Dr. Oreshkova,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

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It was extremely difficult to find reviewers willing to review the manuscript - please, make sure to suggest reviewers in a future submission. I also agree with the comments from the reviewer, especially that given the modest sample size and study design flaws can impact the findings and conclusions reported. Although this is an interesting topic, the work itself is lacking quality to be published by PLOS ONE.

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Reviewer #1: Partly

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Reviewer #1: Main commentary:

Queen mandibular pheromone (QMP) increases lipids in the abdomen and proteins in hypopharyngeal glands (HPGs). The authors hypothesized that QMP increases lipid synthesis and HPG size. The results suggest that QMP does not increase lipid synthesis but increases HPG size in a free-lipid protein diet. While the positive effect of QMP on HPG size was previously reported, the finding that such effect does not depend on lipid ingestion is novel. However, the study's experimental design has limitations that reduce the scope of the findings.

General comments:

1) While the authors controlled important experimental variables, several important factors are missing in this experiment. One of them is the absence of trophallactic intersections between the caged bees and older bees in the colony. This lack of trophallactic interactions results in altered microbiota and reduced transfer of food (e.g., MRJPs), which significantly affect the nutritional conditions of the bees under study and potentially the results obtained.

2) Studies analyzing complex social interactions among honey bee castes could require a sufficient number of bees to have a better chance of capturing a realistic result. Thirty bees per cage and three replicates per treatment is a modest experimental size for this type of study.

3) Additional controls such as other diets (e.g., pollen) could have benefited the study.

4) What was the mortality rate of the bees under the different treatments? Was this significant?

Specific points:

Methods.

Ln 105. Correct the dates when the experiments were performed.

Ln 164. How many bees per cage/treatment were analyzed?

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10.1371/journal.pone.0292500.r002
Author response to Decision Letter 0
Submission Version1
17 Dec 2023

Dear reviewer/editor,

Thank you for the feedback on our submission to PLOS ONE and thank you for the opportunity to submit

a revised version of our manuscript. We were pleased to receive thoughtful comments that are easy to

address. Down below we have responded to each of your concerns with details on the revisions carried

out on our manuscript since its original submission.

1. Reviewer comment: “While the authors controlled important experimental variables, several

important factors are missing in this experiment … [these] significantly affect the nutritional

conditions of the bees under study and potentially the results obtained.” 

Authors’ reply: Cage experiments that eliminate these factors are standard practice in the field,

specifically when investigating the role of pheromones and diet on physiological parameters, please

see examples: [1]–[3]. We will provide this information in a new sentence on line 116: “To eliminate

these issues and provide greater insight into specific nutrients in pollen, a number of recent studies

have used artificial diets [16, 23, 24].”

2. Reviewer comment: “Thirty bees per cage and three replicates per treatment is a modest

experimental size for this type of study.”

Authors’ reply: While we realize there is discussion about the appropriate sample size for

consumption metrics, the number of bees we used is standard for inferences on physiological

relationships [3]–[5]. We will explain these facts by inserting a clarifying sentence on line 119: “Cage

experiments using cohorts of approximately 30 bees per cage maintained in an incubator are

standard practice when investigating the role of pheromones and diet on physiological parameters

and have been used in a number of different studies [16, 21, 25].”

3. Reviewer comment: “Additional controls such as other diets (e.g., pollen) could have benefited the

study.”

Authors’ reply: Many articles address roles of dietary pollen, e.g. [6]. Our goal was not to re-do these

studies. Instead, we sought to optimize data reliability: Pollen is a natural compound that is highly

variable and difficult to standardize. Our experiment eliminates this variability, thus allowing for

greater reliability of data. We will clarify this context by inserting a sentence on line 113: “We chose

to use artificial diets because many studies have shown strong effects of dietary pollen on worker

physiology [11], but pollen composition is highly variable and difficult to standardize [21] and pollen

may be contaminated with pesticides [22].”

4. Reviewer comment: “What was the mortality rate of the bees under the different treatments? Was

this significant?”

Authors’ reply: Mortality was less than 7% over the course of the experiment. This is within the

accepted range for honey bee cage studies [7]. This information was added on line 212: “Mortality

rates were low (less than 7%) and within accepted ranges for cage experiments [19] for all ages and

did not differ by QMP treatment (Kolmgorov-Smirnov, D(10) = 0.167, P = 1; Fig S1).” A new

supplemental figure was added to the manuscript as well.

Thank you for reviewing our revisions. We would be pleased to assist you with securing additional

reviewers for the manuscript if needed,

On behalf of the author team:

Angela Oreshkova (12/14/2023)

[1] D. Stabler, M. Al-Esawy, J. A. Chennells, G. Perri, A. Robinson, and G. A. Wright, “Regulation of dietary intake of protein and lipid by nurse-age

adult worker honeybees,” J. Exp. Biol., p. jeb.230615, Jan. 2020, doi: 10.1242/jeb.230615.

[2] A. M. Trawinski and S. E. Fahrbach, “Queen mandibular pheromone modulates hemolymph ecdysteroid titers in adult Apis mellifera workers,”

Apidologie, vol. 49, no. 3, pp. 346–358, Jun. 2018, doi: 10.1007/s13592-018-0562-6.

[3] P. Fischer and C. M. Grozinger, “Pheromonal regulation of starvation resistance in honey bee workers (Apis mellifera),” Naturwissenschaften, vol. 95, no. 8, pp. 723–729, Aug. 2008, doi: 10.1007/s00114-008-0378-8.

[4] E. Omar, A. A. Abd-Ella, M. M. Khodairy, R. Moosbeckhofer, K. Crailsheim, and R. Brodschneider, “Influence of different pollen diets on the

development of hypopharyngeal glands and size of acid gland sacs in caged honey bees (Apis mellifera),” Apidologie, vol. 48, no. 4, pp.

425–436, Jul. 2017, doi: 10.1007/s13592-016-0487-x.

[5] C. M. Grozinger and G. E. Robinson, “Endocrine modulation of a pheromone-responsive gene in the honey bee brain,” J. Comp. Physiol. A

Neuroethol. Sens. Neural. Behav. Physiol., vol. 193, no. 4, pp. 461–470, Mar. 2007, doi: 10.1007/s00359-006-0202-x.

[6] V. Corby-Harris et al., “Diet and pheromones interact to shape honey bee (Apis mellifera) worker physiology,” J. Insect Physiol., vol. 143, no.

October, p. 104442, 2022, doi: 10.1016/j.jinsphys.2022.104442.

[7] S. K. Huang et al., “Evaluation of cage designs and feeding regimes for honey bee (Hymenoptera: Apidae) laboratory experiments,” J. Econ.

Entomol., vol. 107, no. 1, pp. 54–62, Feb. 2014, doi: 10.1603/EC13213.

Attachment Submitted filename: Response_to_reviewers (1).docx

10.1371/journal.pone.0292500.r003
Decision Letter 1
Rueppell Olav Academic Editor
© 2024 Olav Rueppell
2024
Olav Rueppell
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
3 Apr 2024

PONE-D-23-30810R1The effects of queen mandibular pheromone on nurse-aged honey bee ( Apis mellifera ) hypopharyngeal gland size and lipid metabolismPLOS ONE

Dear Dr. Oreshkova,

Thank you for resubmitting your manuscript to PLOS ONE and I apologize for the performance of the previous editor. After careful consideration and two additional reviews, we feel that your manuscript has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Given the small sample size, a more modest interpretation and deliberate discussion of limitations is warranted. There are numerous specific comments that also need to be addressed. 

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Reviewer #2: This manuscript presents the findings of a small study examining the effects of QMP on lipogenic activity in the fat bodies of bees and on HPG development. The findings are interesting and generally well explored in the discussion section, though I do agree with a previous reviewer that there are some limitations to the interpretation due to experimental design. In my opinion, these limitations do not invalidate the findings, however they do warrant some discussion. I think the sample size used here was rather small. The authors do a good job discussing the implications of their small sample size on the results of their diet consumption analyses, but might this have implications for other aspects of their study? If the authors don’t think so, please add some explanation as to why this was unlikely to have been an issue to the discussion. I also agree that using only one diet is a limitation. It would have been interesting to see how FAS activity differed depending on nutrient availability, and I think some discussion on the subject would make for a more satisfying discussion. Finally, the authors looked at HPG size but not content. Could protein/lipid composition vary in a way that is related to FAS activity? I have a few other specific comments. See below for details:

L36: A couple of points that may seem unimportant but are somewhat fundamental: HPGs secrete worker jelly and royal jelly, which are different. This should be adjusted in your abstract and when it is mentioned again in the text.

Wang, Y., Ma, L., Zhang, W., Cui, X., Wang, H., Xu, B., 2016. Comparison of the nutrient composition of royal jelly and worker jelly of honey bees (Apis mellifera). Apidologie 47, 48–56. https://doi.org/10.1007/s13592-015-0374-x

Likewise, large HPGs do not always signify nurse bee-like physiology. Winter bees also have enlarged HPGs.

L134: What dose (Qeq’s) does this synthetic lure represent? This can make a big difference in your experiment. The authors do acknowledge that the use of synthetic QMP may have affected their results, but could dose have played a role?

L171: Starting sentences with a number should be avoided.

L191: When Kruskal-Wallis tests were used, did you only compare QMP treatment within age groups and age groups within QMP treatment?

L227: It might not be relevant to look at FAS activity without first normalizing it to abdominal protein content. What is the standard practice for this measurement? Consider eliminating the unnormalized data from this ms.

L333: I might not be fully understanding this statement, but I’m not sure that the authors fully examined whether workers increase HPG size through increased food consumption. They only used a suboptimal, low lipid diet. It’s possible that consumption rates and FAS activity might differ depending on diet composition and QMP presence.

L360: I don’t really follow this logic. It seems far more likely that there is some interaction between diet and QMP that isn’t captured by this experiment.

L378: I’m not really seeing the connection between this statement and reference 20.

Reviewer #3: All my comments are in the attached document. Please see "reviewer comments to authors" for my feedack.

**********

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Reviewer #2: No

Reviewer #3: No

**********

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Attachment Submitted filename: PLoS ONE QMP and HPGs Review.docx

10.1371/journal.pone.0292500.r004
Author response to Decision Letter 1
Submission Version2
1 May 2024

Dear reviewers/editor,

Thank you for the feedback on our submission to PLOS ONE and thank you for the opportunity to submit a revised version of our manuscript. We were pleased to receive thoughtful comments that we are able to address. Down below we have responded to each of your concerns with details on the revisions carried out on our manuscript since its original submission (the line numbers refer to the track changes manuscript document):

1. Reviewer #1 comment: “… The authors do a good job discussing the implications of their small sample size on the results of their diet consumption analyses, but might this have implications for other aspects of their study? If the authors don’t think so, please add some explanation as to why this was unlikely to have been an issue to the discussion. … It would have been interesting to see how FAS activity differed depending on nutrient availability… Finally, the authors looked at HPG size but not content. Could protein/lipid composition vary in a way that is related to FAS activity?”

Authors’ reply: We understand that our original submission was unclear in its communication of sample sizes. In contrast to the small sample size for the consumption results, we had a large sample size for the other components of the study, for example 72 total samples for the FAS activity. We added discussion of sample size to L428. We agree that the effects of dietary nutrition on FAS activity are worth exploring though outside the scope of this study. We have addressed this in a separate study and added a statement and citation explaining this on L422. We added a statement addressing the limitation of our study in not measuring FAS content or secretory activity on L485.

2. Reviewer #1 comment: “HPGs secrete worker jelly and royal jelly, which are different. This should be adjusted in your abstract and when it is mentioned again in the text. Likewise, large HPGs do not always signify nurse bee-like physiology. Winter bees also have enlarged HPGs.”

Authors’ reply: It is true that the composition of worker and royal jelly are different, as noted in the reference provided by the reviewer. To avoid confusion, we replaced “royal jelly” by the term “proteinaceous jelly” in lines 35, 36, and 79 as both types of jelly contain protein as the primary macronutrient [1]. To further take action on this important comment, we added a new sentence on line 474: “It is known that HPGs contain a high concentration of lipids [15]. This, combined with nurses having high fat body lipids relative to foragers, suggests that HPG lipids are important for jelly production.”

3. Reviewer #1 comment: “L134: What dose (Qeq’s) does this synthetic lure represent?...The authors do acknowledge that the use of synthetic QMP may have affected their results, but could dose have played a role?”

Authors’ reply: We understand that our original submission was unclear in what dosage of synthetic lure we used and how the dosage could have played a role in our study. Thus, we added details on Qeqs to L157 and justification of the application and dose to L158.

4. Reviewer #1 comment: “L175: Starting sentences with a number should be avoided.”

Authors’ reply: We agree that starting sentences with a number should be avoided, thus, we corrected line 195 to now read: “Per treatment group, 18 heads were dissected, resulting in a total of 72 heads.”

5. Reviewer #1 comment: “When Kruskal-Wallis tests were used, did you only compare QMP treatment within age groups and age groups within QMP treatment?”

Authors’ reply: We used the Kruskal-Wallis test to compare all four treatment groups (combination of QMP and age) rather than comparing QMP and age separately. This was clarified on line 216.

6. Reviewer #1 comment: “L227 … Consider eliminating the unnormalized data from this ms.”

Authors’ reply: While we agree that eliminating redundant figures is important, there are two important reasons to keep the unnormalized data: first is that the normalization factor itself is significantly different between age groups, potentially confounding interpretation. For full transparency, we want to show the unnormalized trend here. Second is that the unnormalized data are themselves meaningful, since we are reporting the total lipogenic capacity in two bee abdomens. Reporting these data (such as total lipids per abdomen) are standard practice in honey bee research and useful for aiding in interpretation of the data.

7. Reviewer #1 comment: “I’m not sure that the authors fully examined whether workers increase HPG size through increased food consumption. They only used a suboptimal, low lipid diet. It’s possible that consumption rates and FAS activity might differ depending on diet composition and QMP presence.”

Authors’ reply: We agree that consumption rates differ depending on diet composition and QMP presence as highlighted in two studies mentioned in our manuscript [2, 3]. We further explained the limitations of our experiment by inserting the following statements on line 420: “Furthermore, our study used a lipid deficient diet while Ament and colleagues [13] and Peters and colleagues [14] used a combination of diets consisting of only pollen, pollen and sucrose, or only sucrose. There may be some interacting effects between QMP and nutrition which we did not test for.”

8. Reviewer #1 comment: “It seems far more likely that there is some interaction between diet and QMP that isn’t captured by this experiment.”

Authors’ reply: We agree that there are limitations to our experiment as we only studied one type of diet. We inserted a statement on line 441 to explain how diet (among other factors) may contribute to the differing results of QMP affecting abdominal lipid stores: “If the activity levels and lipid catabolism rates are different between our bees and the bees used in the other studies [11,12,13] due to varying ages, environment, and diet, this could explain the inconsistency of whether QMP affects abdominal lipid stores.”

9. Reviewer #1 comment: “I’m not really seeing the connection between this statement and reference 20.”

Authors’ reply: We apologize that the incorrect reference was cited in the original submission. The correct reference to the statement on line 472 is reference 44. However, we have decided to remove this statement from the revised manuscript as we felt it did not provide relevant information to the passage.

10. Reviewer #2 comment: “Please include whether experimental units are cages or bees and provide sample sizes in the summary statistics for how many bees of each treatment were used. This experiment has an n=3 cages per treatment. This is fairly low but could perhaps be justified with clear explanation in the methods … The authors should make this clear and provide proper explanation and justification. Yet, some of the results described in this paper are cage-level results.”

Authors’ reply: We understand that our original submission was unclear of what experimental units we used. We rewrote the passage on line 228 to clarify our experimental units and provide justification to our low consumption metric sample size: “For all measurements except for consumption, the experimental unit was bees or pooled bees. For consumption metrics, the cage was the experimental unit. A balanced study design was employed for all factors. The sample size for cage level consumption metrics was low at n = 3 cages per treatment as this was not the focus of our study and the metrics describe the physiological parameters of the bees.”

11. Reviewer #2 comment: “These results (food consumption, mortality) are not the central findings of the paper, but they are still discussed … However, a lack of significance could come from the very low sample size, and is not necessarily evidence that these metrics would not differ between treatments if the sample sizes were larger. This should be mentioned in the discussion/interpretation.”

Authors’ reply: To clarify that a lack of significance between protein/sucrose consumption among the four treatment groups may be due to our low sample size, we revised line 417 to now read: “The lack of significant differences in food consumption between our treatments could be due to our small sample size. Thus, Because our study had the smallest sample size, our data should be interpreted cautiously.”

12. Reviewer #2 comment: “A more appropriate chart type should be used instead [for food consumption data].”

Authors’ reply: We agree that a more appropriate chart type should be used as the sample size for food consumption data was below 5. Thus, we revised figures 3 and 4 to now be dot plots rather than box plots. Please refer to the new attached figures for these changes.

13. Reviewer #2 comment: “Why does the ‘natural food composition make it difficult to tease apart how QMP acts to influence physiology’? It’s not clear what is meant by “natural food composition” or how it relates to QMP.”

Authors’ reply: We understand that we were not clear what ‘natural food consumption’ means. We define natural food consumption as the “variable macronutrient composition (lipids and amino acids) of pollen as well as the potential contamination of pesticides” and this was added to line 83. Furthermore, we clarified how consumption relates to QMP with a reference added to line 81: “Furthermore, there has been research that exposure to QMP increases both pollen and sucrose solution consumption in nurse-aged bees [13].”

14. Reviewer #2 comment: “Line 85: Are there many possibilities? I suggest either outlining some more potential possibilities, or removing this final line of the paragraph.”

Authors’ reply: We understand that ending this passage openly is vague and can confuse the reader of whether there are more possibilities outside of what we mentioned. Therefore, the final sentence of line 89 was removed for concision and we ended the paragraph on line 87 with our question of: “Are the QMP-induced increases in lipid stores and HPG size behaviorally modulated by increasing consumption of lipid-containing pollen, or are they due to effects on metabolic pathways regulating lipid and protein synthesis and storage?”

15. Reviewer #2 comment: “Can you please define lipogenic capacity here?

Authors’ reply: We understand that we did not define lipogenic capacity well in our original submission and thus added a brief definition of lipogenic capacity to L96.

16. Reviewer #2 comment: “Why does your hypothesis make predictions about HPG size? Can you better explain your reasoning for why you hypothesize that increased lipogenic capacity would mean larger HGPs?

Authors’ reply: We understand that our hypothesis reasoning for why increased lipogenic capacity would mean larger HPGs could be explained better. We added additional explanations and citations to L99-105.

17. Reviewer #2 comment: “Why did you use these two different ages? Can you outline your hypothesis/predictions for 3-day vs. 8-day old bees?”

Authors’ reply: We added an explanation of our reasoning for using two age groups on L115.

18. Reviewer #2 comment: “(Fig 1 legend): What cages are these dimensions for? Are they different from the Plexiglas cages described in line 109 of the methods?”

Authors’ reply: We apologize for the confusion in cage dimensions. We remeasured and inputted the correct cage dimensions on line 128 and 145 which now read: “The dimensions measured were 16 cm × 12 cm × 9 cm. Because approximately half the cage was used, the effective depth was 6 cm.”

19. Reviewer #2 comment: “These sample sizes (3 cages per treatment) seem fairly low. Although the authors addressed a previous reviewer’s concerns about the number of bees per cage (line 120), can you also include justification for the small replicate size?”

Authors’ reply: We understand that our original submission did not discuss the justification for our replicate size. We added justification of the level of replication to L150.

20. Reviewer #2 comment: “The sample sizes were n=3. Does this mean 3 per treatment, or 3 total? What is the makeup of these sample sizes (18, 35, and 72)? Are they pooled within cages or across cages? How are cages and treatments represented within these sample sizes? Are they balanced? If not, please give specifics of how many bees were used from each treatment, per cage.”

Authors’ reply: We understand that the sample size section in our original submission was not adequately explained. We rewrote the passage beginning on line 228 to clarify the makeup of our sample sizes and whether they are balanced: “For all measurements except for consumption, the experimental unit was bees or pooled bees. For consumption metrics, the cage was the experimental unit. A balanced study design was employed for all factors. The sample size for cage level consumption metrics was low at n = 3 cages per treatment as this was not the focus of our study and the metrics describe the physiological parameters of the bees. For measuring abdominal FAS activity, normalized FAS activity, abdominal protein, and HPG acini area, 2 bees were pooled for each biological sample and 6 samples totaling 12 bees were collected from each cage for all four treatment conditions. This totaled 72 biological samples pooled from 144 abdomens as three replicates were performed. Thus, the sample size was n = 18 pooled samples per treatment group for these three parameters. The sample size for the regression analyses was n = 35 pooled samples as there was a dissection issue for one of the bees and only half of the HPGs of the 24 pooled samples per replicate were measured, except for the case of abdominal protein and abdominal FAS which was n = 72 pooled samples (144 total abdomens).”

21. Reviewer #2 comment: “Line 206-214: Please include sample sizes for test statistics. Also, please explain ‘between replicates’: Are these stats explanatory for all 4 treatments?” “Line 233: ‘significant differences between the three replicates’: For which treatment? This needs to be clear each time a significant difference is reported between replicates (like in line 237, for example).”

Authors’ reply: We included sample sizes for food and sucrose consumption on lines 245 and 247. We apologize for not outlining our replicate effects clearly in the original manuscript. To address the reviewers' comments to lines 206 - 214 and line 233, we have included a detailed explanation of what the replicate effect is beginning on line 219: “For each parameter analyzed with either a parametric or non-parametric test, replicate effects were tested for. These describe whether there were significant differences between each replicate and if there was an independent effect of doing a replication of the experiment overall.”

22. Reviewer #2 comment: “Figures 3 and 4, and their legends: include ‘(days)’ in x-axis labels; These figure legends … Either a simple point plot or bar charts would be more honest visual representations of the data.”

Authors’ reply: We included ‘days’ in the x-axis labels for figures 3 and 4 (lines 253 and 259). We agree that a more appropriate chart type should be used as the sample size for food consumption data was below 5. Thus, we revised figures 3 and 4 to now be dot plots rather than box plots. Please refer to the new attached figures for these changes.

23. Reviewer #2 comment: “Were pooled bees from the same cage? Were there 6 pools per cage? Figures 5, 6, 7, and 8 legends: ‘Outliers are points outside the maximum and minimum distribution.’] How do you have points outside the max and min of the distribution? Do these fall outside the expected variation of the data?”

Authors’ reply: We agree that sample sizes should better be explained for figure 5,6,7, and 8 legends. We clarified how the sample size was calculated for these figures with the following sentence added to lines 285, 298, 321, and 343: “As there were 6 pools per cage (12 abdomens/bees sampled) and three replicates performed, the sample size is n = 18 pooled samples.” We amended the figure legends to explain that whiskers are calculated as 1.5 times the interquartile range and outliers are data points outside the whiskers that depict the expected variation of the data.

24. Reviewer #2 comment: “Fig 10/11 legend: Provide sample sizes in the summary

Attachment Submitted filename: Response to Reviewers.docx

10.1371/journal.pone.0292500.r005
Decision Letter 2
Rueppell Olav Academic Editor
© 2024 Olav Rueppell
2024
Olav Rueppell
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
13 May 2024

PONE-D-23-30810R2The effects of queen mandibular pheromone on nurse-aged honey bee ( Apis mellifera ) hypopharyngeal gland size and lipid metabolismPLOS ONE

Dear Dr. Oreshkova,

Thank you for submitting your manuscript to PLOS ONE. Both reviewers find your manuscript much improved and thus, it is almost ready for acceptance. However, I would like to give you an additional opportunity to take the comments of the reviewers into account for improving your manuscript further.

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We look forward to receiving your revised manuscript.

Kind regards,

Olav Rueppell

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

Reviewer #3: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I appreciate that the authors have addressed most of my concerns. This is a much improved manuscript in terms of clarity, however I have several more comments I feel should be addressed before the manuscript is suitable.

Please note, line numbers refer to the tracked changes version of the manuscript revision.

Please represent significant differences somehow in your figures.

Standard practice for analyzing FAS activity is to normalize it to protein content largely because protein content will vary between samples. You may be seeing a relationship between FAS activity and abdominal protein content because FAS is partially dependent on amino acids and carbohydrates as substrates. Why wouldn’t you depict it the way you analyzed it in your figures? And why isn’t it represented this way in your table?

N=18 is a reasonable sample size for FAS analysis but it’s small for HPGs. The authors continually state that 18 is a relatively large sample size. Relative to what?

I pointed out in my last review that it isn’t grammatically correct to begin a sentence with a number. There are several new instances of this error in this version that need to be addressed.

L216-222: I'm still unclear on some aspects of the stats. I see that you used KW or anova depending on whether the data satified assumptions. How were replicate effects evaluated for KW models? Did you just run separate KW tests for each rep? For ANOVA models, was replicate treated as a factor? Did you do anything to account for possible interaction between factors for ANOVA and KW models? I imagine there were limitations associated with using the KW models since you can't run the equivalent of a 2-way anova. How did you try to address those?

L233-238: All of this should perhaps be described earlier, when discussing FAS activity assay methods.

Reviewer #3: Thank you for addressing my comments. One final note, please add a definition of "queen equivalent" (line 157).

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

Reviewer #3: No

**********

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10.1371/journal.pone.0292500.r006
Author response to Decision Letter 2
Submission Version3
4 Jul 2024

Dear reviewers/editor,

Thank you for the additional feedback on our submission to PLOS ONE and thank you for the opportunity to submit a second revised version of our manuscript. We were pleased to receive additional thoughtful comments that we are able to address. Down below we have responded to each of your new concerns with details on the revisions carried out on our manuscript since its original submission (the line numbers refer to the new track changes manuscript document):

1. Reviewer #2 comment: “Please represent significant differences somehow in your figures.”

Authors’ reply: We agree that significant differences should be represented in our figures. Please refer to attached figures 6, 7, and 8 to view these changes. Furthermore, we adjusted the legends of these figures (L310, L330 and L351) to make note of the symbols we used in the figures and what they signify.

2. Reviewer #2 comment: “Standard practice for analyzing FAS activity is to normalize it to protein content largely because protein content will vary between samples. You may be seeing a relationship between FAS activity and abdominal protein content because FAS is partially dependent on amino acids and carbohydrates as substrates. Why wouldn’t you depict it the way you analyzed it in your figures? And why isn’t it represented this way in your table?”

Authors’ reply: We agree that standard practice for analyzing FAS activity is to normalize it to protein content. However, we did choose to report both normalized and non-normalized data and have added a clarification to L188 to explain why: “For a more complete picture of the metabolic state of the bee, we report total abdominal FAS activity as well as FAS activity normalized to mg of abdominal protein. Because our dissection approach collects the entire abdominal cuticle and attached fat body, it produces a metric of total lipogenic capacity per abdomen, similar to past studies that have measured total lipids per abdomen using the same dissection technique [7, 12, 15, 16]. To control for potential differences in protein extraction efficiency, we also measure protein concentration in each sample and normalize FAS activity relative to extracted protein. Comparing results from the two metrics makes it clearer when treatment group differences are due to changes in the quantity of activated FAS or due to changes in the normalization factor itself.” We apologize if we were not clear as to why we reported both data sets.

3. Reviewer #2 comment: “N=18 is a reasonable sample size for FAS analysis but it’s small for HPGs. The authors continually state that 18 is a relatively large sample size. Relative to what?”

Authors’ reply: We have removed the reference to large sample size on L398 and altered the wording on L425-426 to make it clear that our HPG sample size is large relative to our cage-level consumption data.

4. Reviewer #2 comment: “I pointed out in my last review that it isn’t grammatically correct to begin a sentence with a number. There are several new instances of this error in this version that need to be addressed.”

Authors’ reply: We apologize that there were several more instances of beginning our sentences with a number. We corrected this on the following lines: L118, L141, L223, L364, L372, and L379.

5. Reviewer #2 comment: “L216-222: I'm still unclear on some aspects of the stats. I see that you used KW or anova depending on whether the data satisfied assumptions. How were replicate effects evaluated for KW models? Did you just run separate KW tests for each rep? For ANOVA models, was replicate treated as a factor? Did you do anything to account for possible interaction between factors for ANOVA and KW models? I imagine there were limitations associated with using the KW models since you can't run the equivalent of a 2-way anova. How did you try to address those?”

Authors’ reply: We added information on L215 and L227 about how we addressed replicate effects and interactions between factors for ANOVA. We added two sentences on L229-L232 outlining our strategy for using a Kruskal-Wallis effect to test for replicate and treatment effects in the normalized FAS data.

6. Reviewer #2 comment: “L233-238: All of this should perhaps be described earlier, when discussing FAS activity assay methods.”

Authors’ reply: We agree that there should be a description of how 144 bee abdomens were sampled across three replicates in our FAS activity assay methods and apologize if there was any confusion surrounding this. We added a section on L182 to better describe our sampling methods: “This assay was replicated 3 times. In each replicate, 2 bees were pooled for each biological sample and 6 samples totaling 12 bees were collected from each cage for all four treatment conditions. This totaled with a total of 144 abdomens sampled across three replicates.”

7. Reviewer #3 comment: ‘Thank you for addressing my comments. One final note, please add a definition of "queen equivalent" (line 157).’

Authors’ reply: We agree that the term ‘queen equivalent’ should be defined and therefore, we clarified this on L156: “One queen equivalent represents the amount of QMP a mated queen will produce in a 24-hour period and contains 200 mg of ODA, 80 mg of 9-HAD, 20 mg of HOB, and 2 mg of HVA [34]”.

Attachment Submitted filename: Response to Reviewers (6.28.24).docx

10.1371/journal.pone.0292500.r007
Decision Letter 3
Rueppell Olav Academic Editor
© 2024 Olav Rueppell
2024
Olav Rueppell
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version3
9 Jul 2024

The effects of queen mandibular pheromone on nurse-aged honey bee ( Apis mellifera ) hypopharyngeal gland size and lipid metabolism

PONE-D-23-30810R3

Dear Dr. Oreshkova,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Olav Rueppell

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

10.1371/journal.pone.0292500.r008
Acceptance letter
Rueppell Olav Academic Editor
© 2024 Olav Rueppell
2024
Olav Rueppell
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
17 Jul 2024

PONE-D-23-30810R3

PLOS ONE

Dear Dr. Oreshkova,

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