Behavioral disruption in honey bees (Apis mellifera) exposed to isolated and combined insecticides.

Ever since the seminal papers of Hamilton (1964), a common focus of sociobiology has been the genetic relationships among individuals of insect societies. At issue are agents that reduce the average relatedness among colony members since they are difficult to interpret in light of the evolution of sociality by kin selection. One such factor is polyandry or female multiple mating. Low levels of polyandry are common among the social Hymenoptera, occurring across a broad range of taxa and social systems (Page and Metcalf 1982; Page 1986; Crozier and Pamilo 1996). In spite of that, high mating frequencies (‚2 mates/ female) are relatively rare among the highly eusocial Hymenoptera, limited to only one or a few genera in each of the ants, bees, and wasps (Boomsma and Ratnieks 1996). These extraordinary levels of polyandry have been of particular interest among students of social insect research since their adaptive significance is still in question and highly debated (Kraus and Page 1998; Sherman et al. 1998). The genus Apis has been studied in depth for both queen reproductive behavior and mating frequency, facts that allow insight into the evolution of extreme polyandry in these species. The reproductive behavior of honey bee queens (Apis mellifera) occurs early in their lifetimes. Virgin queens initiate mating behavior when they are about 1 wk old by briefly exiting the colony and orienting to local landmarks (Ruttner 1956). After such orientation * To whom correspondence should be addressed; e-mail: drtarpy@

Worker honeybees (Apis mellifera) usually only lay eggs when their colony is queenless. However, an extremely rare 'anarchistic' phenotype occurs, in which workers develop functional ovaries and lay large numbers of haploid eggs which develop into adult drones despite the presence of the queen. Studies of such colonies can give important insights into the mechanisms by which worker sterility is maintained in normal colonies. Here we report on the results of a breeding programme which enhanced the frequency of the anarchistic phenotype. Colonies derived from queens inseminated only by worker-laid males showed up to 9% of workers with highly developed ovaries. In these colonies a large proportion of males arose from worker-laid eggs. Colonies headed by queens inseminated with 50% worker-laid drones and 50% queen-laid drones showed variable phenotypes. In most such colonies there was no worker reproduction. In some, many workers had highly developed ovaries, but no worker-laid eggs were reared. In one colony, many worker-laid eggs were reared to maturity. The results suggest that the anarchy phenotype results from a complex interaction of queen genotype, the worker genotype of subfamilies that successfully reproduce and of those that do not, and the external environment.

Honey bees are both important pollinators and model insects due to their highly developed sociality and colony management. To better understand the molecular mechanisms underlying honey bee colony management, it is important to investigate the expression of genes putatively involved in colony physiology. Although quantitative real-time PCR (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different conditions) must first be identified to ensure accurate normalisation of target genes. To identify reliable reference genes in honey bee (Apis mellifera) colonies, therefore, we evaluated seven candidate genes (ACT, EIF, EF1, RPN2, RPS5, RPS18 and GAPDH) in samples collected from three honey bee tissue types (head, thorax and abdomen) across all four seasons using three analysis programmes (NormFinder, BestKeeper and geNorm). Subsequently, we validated various normalisation methods using each of the seven reference genes and a combination of multiple genes by calculating the expression of catalase (CAT). Although the genes ranked as the most stable gene were slightly different on conditions and analysis methods, our results suggest that RPS5, RPS18 and GAPDH represent optimal honey bee reference genes for target gene normalisation in qRT-PCR analysis of various honey bee tissue samples collected across seasons.

The use antimicrobials for therapeutic and metaphylactic purpose in humans and agriculture exerts selective pressure on animal and environmental microbiota resulting in the survival and spread of antimicrobial resistance genes among bacteria and subsequent development of resistance in bacteria. Previous studies have shown that honey bees' microbiota (Apis mellifera) can accumulate antimicrobial resistance genes in their microbiome and act as collectors and disseminators of resistance genes. The aim of this study was to investigate to what extent honey bees act as reservoir of select antimicrobial resistance genes. This study was conducted on 35 groups of bees. Bees were collected from 35 sites in Umbria, Italy. PCR was used to screen pooled ground bees' specimens for genes that code for resistance against antimicrobials that are commonly used in humans and in veterinary medicine including aminoglycosides (aph), beta-lactams (blaZ), tetracycline (tetM) and sulphonamides (sul1 and sul2). Twenty-four samples out of 35 (68.57%) were positive for at least one antimicrobial resistance gene. Two samples were positive for the aph, 5.71%; eight for blaZ, 22.86%; three for tetM, 8.57%; ten for sul1, 28.57% and eighteen for sul2, 51.43%. Positivity to more than one antimicrobial resistance gene was observed in nine samples, 25.71%. The multivariate analysis identified "presence of farms nearby" as the factor most closely related to PCR positivity. Honey bees (Apis mellifera) from Umbria, Italy, carry antimicrobial resistance genes and can be used as indicators of the presence of resistance genes in the environment.

Simple SummaryWing venation traits are used to identify honey bee subspecies. While several wing-based tools are available, they suffer from weaknesses that were addressed by the recently developed software DeepWings©. This software allows fully automated identification of wing images in a friendly, free, and rapid manner. Here, we sought to test DeepWings© on 14,816 wing images representing 2601 colonies sampled in the native areas of three widespread subspecies in Europe: the Iberian honey bee (Apis mellifera iberiensis), the dark honey bee (Apis mellifera mellifera), both belonging to the M lineage, and the Carniolan honey bee (Apis mellifera carnica), belonging to the C lineage. DeepWings© classification of these colonies largely matched the endemic M and C lineages, with proportions of 71.4% and 97.6%, respectively. At the subspecies-level the matching proportions were 89.7% for the Iberian honey bee, 41.1% for the dark honey bee and 88.3% for the Carniolan honey bee, which can be explained by DeepWings© sometimes confounding closely related subspecies and, more importantly, by genetic pollution. A comparison between DeepWings© data and molecular data revealed that the agreement between the two is weaker when there is genetic pollution. Our results suggest that DeepWings© is a valuable tool for honey bee identification, which can be used not only for breeding and conservation but also for research purposes.DeepWings© is a software that uses machine learning to automatically classify honey bee subspecies by wing geometric morphometrics. Here, we tested the five subspecies classifier (A. m. carnica, Apis mellifera caucasia, A. m. iberiensis, Apis mellifera ligustica, and A. m. mellifera) of DeepWings© on 14,816 wing images with variable quality and acquired by different beekeepers and researchers. These images represented 2601 colonies from the native ranges of the M-lineage A. m. iberiensis and A. m. mellifera, and the C-lineage A. m. carnica. In the A. m. iberiensis range, 92.6% of the colonies matched this subspecies, with a high median probability (0.919). In the Azores, where the Iberian subspecies was historically introduced, a lower proportion (85.7%) and probability (0.842) were observed. In the A. m mellifera range, only 41.1 % of the colonies matched this subspecies, which is compatible with a history of C-derived introgression. Yet, these colonies were classified with the highest probability (0.994) of the three subspecies. In the A. m. carnica range, 88.3% of the colonies matched this subspecies, with a probability of 0.984. The association between wing and molecular markers, assessed for 1214 colonies from the M-lineage range, was highly significant but not strong (r = 0.31, p < 0.0001). The agreement between the markers was influenced by C-derived introgression, with the best results obtained for colonies with high genetic integrity. This study indicates the good performance of DeepWings© on a realistic wing image dataset.

Insect pollinators provide an important ecosystem service that supports global biodiversity and environmental health. The study investigates the effects of the environmental matrix on six oxidative stress biomarkers in the honey bee Apis mellifera. Thirty-five apiaries located in urban, forested, and agricultural areas in Central Italy were sampled during the summer season. Enzyme activities in forager bees were analyzed using an artificial neural network, allowing the identification and representation of the apiary patterns in a Self-Organizing Map. The SOM nodes were correlated with the environmental parameters and tissue levels of eight heavy metals. The results indicated that the apiaries were not clustered according to their spatial distribution. Superoxide dismutase expressed a positive correlation with Cr and Mn concentrations; catalase with Zn, Mn, Fe, and daily maximum air temperature; glutathione S-transferase with Cr, Fe, and daily maximal air temperature; and glutathione reductase showed a negative correlation to Ni and Fe exposure. This study highlights the importance of exploring how environmental stressors affect these insects and the role of oxidative stress biomarkers. Artificial neural networks proved to be a powerful approach to untangle the complex relationships between the environment and oxidative stress biomarkers in honey bees. The application of SOM modeling offers a valuable means of assessing the potential effects of environmental pressures on honey bee populations.

SummaryNosema apis and Nosema ceranae are causative agents of Nosemosis in the honey bee Apis mellifera, although N. ceranae may cause a more virulent disease. Selection of colonies with high tolerance to N. ceranae could be important for reducing problems caused by this pathogen. The aim of the present work was to evaluate the existence of honey bee colonies with different degrees of N. ceranae infection and test if this difference could be related to the immune response or vitellogenin expression. Healthy honey bee colonies were relocated to a plantation of Eucalyptusgrandisto favour natural infection of N. ceranae. Fifteen and thirty days after relocation, the proportion of infected bees and the number of N. ceranae spores per field were quantified. The colonies with higher and lower levels of infection (HL and LL, respectively) were selected. Newly emerged bees from both colonies were artificially infected with N. ceranaeand seven days after infection the expression of immune related genes and vitellogenin was evaluated by real time PCR. No significant differences were observed in expression of abaecin, hymenoptaecin, defensin, glucose dehydrogenase or lysozyme mRNA levels between infected bees from HL and LL colonies or between control bees from both colonies. Vitellogenin expression was higher in bees from the LL colony than in bees from the HL colony, when infected or control bees were compared between them. This protein possesses pleiotropic effects and is a central element in the life-history of honey bees. For that reason, its differential expression could be associated with resistance to N. ceranae.

The objective of this study was to measure the efficacy of two organic acid treatments, formic acid (FA) and oxalic acid (OA) for the spring control of Varroa destructor (Anderson and Trueman) in honey bee (Apis mellifera L.) colonies. Forty-eight varroa-infested colonies were randomly distributed amongst six experimental groups (n = 8 colonies per group): one control group (G1); two groups tested applications of different dosages of a 40 g OA/l sugar solution 1:1 trickled on bees (G2 and G3); three groups tested different applications of FA: 35 ml of 65% FA in an absorbent Dri-Loc(®) pad (G4); 35 ml of 65% FA poured directly on the hive bottom board (G5) and MiteAwayII™ (G6). The efficacy of treatments (varroa drop), colony development, honey yield and hive survival were monitored from May until September. Five honey bee queens died during this research, all of which were in the FA treated colonies (G4, G5 and G6). G6 colonies had significantly lower brood build-up during the beekeeping season. Brood populations at the end of summer were significantly higher in G2 colonies. Spring honey yield per colony was significantly lower in G6 and higher in G1. Summer honey flow was significantly lower in G6 and higher in G3 and G5. During the treatment period, there was an increase of mite drop in all the treated colonies. Varroa daily drop at the end of the beekeeping season (September) was significantly higher in G1 and significantly lower in G6. The average number of dead bees found in front of hives during treatment was significantly lower in G1, G2 and G3 versus G4, G5 and G6. Results suggest that varroa control is obtained from all spring treatment options. However, all groups treated with FA showed slower summer hive population build-up resulting in reduced honey flow and weaker hives at the end of summer. FA had an immediate toxic effect on bees that resulted in queen death in five colonies. The OA treatments that were tested have minimal toxic impacts on the honey bee colonies.

Abstract. Ibrahim YS, Rosazan MN, Mamat MII, Anuar ST, Azmi WA. 2025. Detection of microplastics in honey of stingless bee (Heterotrigona itama) and honey bee (Apis mellifera) from Malaysia. Biodiversitas 26: 1271-1278. The demand for stingless bee honey and European bee honey has increased rapidly due to its medicinal benefits. Honey of the Indo-Malaya stingless bee, Heterotrigona itama, and European honey bee, Apis mellifera, are among the most popular bee products that Malaysians commonly consume. It has been reported that the contamination of honey with microplastics (MPs) can occur at various stages of production, from bees collecting the contaminated floral sources to the harvesting and packaging processes. With the emerging plastics pollution in the environment and concerns about potential health risks, this study aims to investigate the presence of MPs in honey samples from stingless bees, H. itama, and honey bees, A. mellifera, from Malaysia. Microplastic particles were extracted from 10 g of honey and characterized under a stereomicroscope to determine their color, size, and type. Polymer types were further identified using FTIR analysis. All honey samples from both species were found to be 100% contaminated with microplastics. H. itama honey contained a higher concentration of microplastics (8.18 ± 2.57 MPs/g) compared to A. mellifera's honey (5.52 ± 1.13 MPs/g). The MPs found in honey from both species were predominantly fibers and fragments, mostly transparent in color, with sizes ranging from 0.7 to 1.8 mm. The findings of this preliminary study are intended to provide an awareness of MPs in honey, especially in the food safety aspect, which needs a better understanding of good practices of beekeeping and processing procedures to minimize the contamination of honey.

SummaryBiological, historical and archaeological evidence proves that honey bees (Apis mellifera L.) have been present in the British Isles for at least 4000 years, and suggests that they probably entered from southern Europe after the retreat of the last Ice Age. Recent studies show that rather than having been destroyed by disease in the early 20th century, or obliterated by imports of other strains of honey bee, the dark European honey bee Apis mellifera mellifera still exists as genetically distinct populations in various parts of Britain. There is little information available to indicate the extent of any competition between honey bees and other species of bee in Britain, or to quantify the contribution of honey bees to major ecosystems in Britain. There is a need for strategies for conserving rare or endangered bee species to recognise that local strains of honey bee may be equally endangered, and may be equally deserving of conservation effort. All species of bee are worthy of conservation, and man...

SummaryBiological, historical and archaeological evidence proves that honey bees (Apis mellifera L.) have been present in the British Isles for at least 4000 years, and suggests that they probably entered from southern Europe after the retreat of the last Ice Age. Recent studies show that rather than having been destroyed by disease in the early 20th century, or obliterated by imports of other strains of honey bee, the dark European honey bee Apis mellifera mellifera still exists as genetically distinct populations in various parts of Britain. There is little information available to indicate the extent of any competition between honey bees and other species of bee in Britain, or to quantify the contribution of honey bees to major ecosystems in Britain. There is a need for strategies for conserving rare or endangered bee species to recognise that local strains of honey bee may be equally endangered, and may be equally deserving of conservation effort. All species of bee are worthy of conservation, and management decisions need to be made on a case by case basis, and must be based on a sound understanding of the underlying biology of the ecosystems involved.

Simple SummaryThe honey bee (Apis mellifera) is kept all over the world and plays a dominant role in the pollination of crops. Honey bees may be exposed to different levels of cadmium through the collection of contaminated nectar during their foraging activities. In this study, honey bees were chronically exposed to cadmium to investigate the effects of sublethal cadmium doses on the olfactory learning and brain gene expression profiles in honey bees. Honey bees exhibited significantly impaired olfactory learning performances after being chronically exposed to cadmium and had a significantly lower head weight in comparison with control bees. Furthermore, genes involved in oxidative stress response and odor sensing were dysregulated in the brain of cadmium-treated bees. These results suggest that cadmium exposure exerted oxidative stress and decreased gene expression levels of chemoreceptors in honey bees, which probably resulted in the impaired olfactory learning of honey bees.The honey bee (Apis mellifera) plays vital ecological roles in the pollination of crops and the maintenance of ecological balance, and adult honey bees may be exposed to exogenous chemicals including heavy metals during their foraging activities. Cadmium (Cd) is regarded as a nonessential toxic metal and is readily accumulated in plants; honey bees can therefore acquire Cd through the collection of contaminated nectar. In the present study, honey bees were chronically exposed to Cd to investigate the effects of sublethal cadmium doses on the olfactory learning and brain gene expression profiles of honey bees. The results showed that Cd-treated bees exhibited significantly impaired olfactory learning performances in comparison with control bees. Moreover, the head weight was significantly lower in Cd-treated bees than in control bees after chronic exposure to Cd. Gene expression profiles between the Cd treatment and the control revealed that 79 genes were significantly differentially expressed. Genes encoding chemoreceptors and olfactory proteins were downregulated, whereas genes involved in response to oxidative stress were upregulated in Cd-treated bees. The results suggest that Cd exposure exerts oxidative stress in the brain of honey bees, and the dysregulated expression of genes encoding chemoreceptors, olfactory proteins, and cytochrome P450 enzymes is probably associated with impaired olfactory learning in honey bees.

Honey bees gather pollen from flowering plants, using it as a vital protein source and, in turn, acquire pollen-associated microbes that interact with their existing gut microbiota. Despite their ecological importance, limited information exists regarding the gut microbiota of African savannah honey bees (Apis mellifera scutellata Lepeletier) and how diet and its associated microbial community influence this crucial internal ecosystem. This study aimed to investigate the differences in gut microbiota between wild honey bees collected during the flowering season and microbially depleted honey bees reared under semi-sterile conditions and fed various protein diets. To achieve this, freshly hatched worker bees were maintained in hoarding cages and assigned one of four protein diets: fresh sunflower pollen, casein, sterilised casein, or sterilised pollen. High-throughput DNA metabarcoding was then employed to compare the microbial composition of the honey bee gut across these groups. Our findings revealed that the gut of microbially depleted honey bees exhibited higher species diversity and richness. Conversely, the non-core gut microbial community predominated in wild bees and those fed the different protein diets. Specifically, Commensalibacter, Bartonella, and Bifidobacterium were the most dominant bacterial genera across all treatments. Interestingly, Gilliamella, a common core gut bacterium, was undetected, while Apibacter was exclusively found in wild honey bees. Furthermore, pollen-associated microbes such as Devosia and Pedobacter were identified solely in the gut of honey bees fed a pollen diet. Functional predictions of the gut microbial community also indicated the presence of key enzymes such as β-glucosidase, β-galactosidase, pyruvate dehydrogenase and phosphoglycerate mutase, which are crucial for enhancing nutrient absorption, digestion, and carbohydrate metabolism. These results underscore the intricate relationship between honey bees, microbes, and plants, offering valuable insights into how diet and its associated microbial communities could shape the gut microbiota of African honey bees. KEY POINTS: • The non-core gut microbiota dominates the African savannah honey bee •The type of diet influenced the microbial diversity and community abundance in the honey bee gut •Key enzymes involved in digestion, nutrition absorption, and carbohydrate metabolism were enhanced in the gut •Pollen-associated microbes found in the diet present potential avenues for probiotic development to improve honey bee health.

Nutrition is important in honey bee (Apis mellifera L.) colony decline. While the honey bee is incapable of converting dietary plant sterols to cholesterol, the sterol 24-methylenecholesterol has been previously shown to be selectively transferred from worker honey bees to developing brood in both laboratory and field studies. The importance of 24-methylenecholesterol to honey bee development and nutrition is unknown. This selective transfer suggests either (i) honey bees concentrate 24-methylenecholesterol in their tissues by selectively excreting other sterols in their feces (hypothesis 1), or (ii) some other metabolic/physiological mechanism exists to segregate/sequester 24-methylencholesterol for transfer to brood (hypothesis 2). The objective of this study was to determine if any C28 and C29 sterols other than 24-methylenecholesterol were selectively excreted in the feces by the honey bee (hypothesis 1), thereby concentrating the relative percentages of 24-methylenecholesterol in worker bee tissue, and thereby partially explaining the mechanism of selective sterol transfer in honey bees. We conducted the study by confining worker honey bees in a small cage with glass sides to facilitate the collection of honey bee feces. After four days, the neutral sterols were identified and the percentages of these sterols were determined from the pollen the honey bees presumably consumed prior to confinement, from the tissues of the confined worker bees, and from their feces. There was no apparent excessive excretion of other C28 and C29 phytosterols such as campesterol, sitosterol and isofucosterol and stigmasterol in honey bee feces, suggesting that hypothesis 2 (above) needs investigation.

Honey bee (Apis mellifera L.) toxicology research is crucial to understanding the risks of different chemicals and for finding new compounds to decrease pests and pathogens in honey bee colonies. However, the results of toxicological studies are not always consistent. In this study, we tested compounds from 7 chemical classes to compare topical LD50 values for honey bees that were reared in the winter versus honey bees that were reared in the spring. The compounds included acetamiprid, amitraz, coumaphos, fipronil, fluvalinate, indoxacarb, and hydramethylnon, all of which are periodically used by beekeepers to control honey bee pests. Fluvalinate was 4× more toxic in the spring than in the winter. Conversely, acetamiprid, amitraz, coumaphos, fipronil, and indoxacarb were all more toxic in the winter than the spring by 37×, 7×, >87×, 6×, and 4×, respectively. Coumaphos had the highest discrepancy between the seasons, while hydramethylnon was not significantly different between winter and spring. Overall, our results suggest that toxicology research on honey bees should be accounting for seasonal variation in the honey bees they are testing. The honey bee's susceptibility to each compound may change depending on what time of year they are collected and is unique to each chemical class being tested. This work provides evidence that honey bee susceptibility to pesticides needs to be evaluated in the context of season in future research to properly evaluate their impact on honey bee health.