Adult Worker Honey Bees Research Articles

PCR Diagnosis of Small Hive Beetles.

Small hive beetles (SHBs), Aethina tumida, are parasites of social bee colonies native to sub-Saharan Africa and have become an invasive species at a global scale. Reliable Polymerase Chain Reaction (PCR) diagnosis of this mandatory pest is required to limit its further spread and impact. Here, we have developed SHB primers, which amplify for 10 native African locations and 10 reported introductions, but not for three closely related species (Aethina concolor, Aethina flavicollis, and Aethina inconspicua). We also show that adult honey bee workers can be used as matrices for PCR-based detection of SHBs. The sensitivity of this novel method appears to be 100%, which is identical to conventional visual screenings. Furthermore, the specificity of this novel approach was also high (90.91%). Since both sensitivity and specificity are high, we recommend this novel PCR method and the new primers for routine surveillance of hives in high-risk areas.

The role of the gut microbiome in health and disease of adult honey bee workers.

The role of the gut microbiome in animal health has become increasingly evident. Unlike most other insects, honey bees possess a highly conserved and specialized core gut microbiome, which consists of nine bacterial species and is acquired mostly through social transmission. Five of these species are ubiquitous in honey bees and are also present in bumble bees. Recent studies have shown that the bee gut microbiome plays a role in metabolism, immune function, growth and development, and protection against pathogens. Disruption of the gut microbiome has also been shown to have detrimental effects on bee health. Overall, evidence suggests that the gut microbiome plays an important role in bee health and disease.

Queen mandibular pheromone modulates hemolymph ecdysteroid titers in adult Apis mellifera workers

To test the hypothesis that exposure to queen mandibular pheromone (QMP) modulates ecdysteroid production in adult worker honey bees, ecdysteroids were measured in hemolymph and other tissues of individual adult worker honey bees reared with or without QMP in cages and field colonies. Ecdysteroid titers were higher in caged workers exposed to QMP continuously from the first day of adult life than in workers reared without QMP. Statistical cluster analysis suggested the possibility that a subgroup of workers (“responders”) is more sensitive to QMP in this regard than other workers. In 12-day-old workers, ecdysteroid titers in workers reared in queenright (QR) colonies were similar to those observed in cages with QMP, but lower than those in queenless (QL) colonies. Differences in number of ovarioles or degree of ovarian activation did not correlate with hemolymph ecdysteroids. Previous studies have demonstrated ecdysteroids in hemolymph in very young adult workers and in workers in QL colonies; the present study indicates that production of ecdysteroids occurs in older adult worker honey bees in the absence of morphological signs of ovarian activation, with cage studies revealing a modulatory role for QMP masked in the complex environment of the hive.

The Effects of Starvation of Honey Bee Larvae on Reproductive Quality and Wing Asymmetry of Honey Bee Drones

Summary Starvation during larval development has a negative effect on adult worker honey bees (Apis mellifera L.), but much less is known about the quality of drones starved during their development. We verified how starvation on the second day (early starvation) or the sixth day (late starvation) of larval development affects body mass, ejaculated semen volume and forewing size, shape, size asymmetry and shape asymmetry in drones after emergence. The larvae were starved for ten hours by being separated from nursing bees with a wire mash for 10 hours either early or late during larval development. Drones starved both early and late were smaller (254.1 ± 1.97 mg and 239.4 ± 2.12 mg, respectively) than the control regularly fed individuals (260.9 ± 2.01 mg), and their wing size changed as well (control: 889.76 ± 1.06; early: 880.9 ± 1.17; late: 868.05 ± 1.48). Starvation at a later phase of larval development caused more pronounced effects than at an earlier phase. On the other hand, ejaculated semen volume (control: 0.7 ± 0.043 μl; early: 0.88 ± 0.040 μl; late: 1.08 ± 0.031 μl), wing size asymmetry (control: 0.49 ± 0.025; early: 0.51 ± 0.026; late: 0.52 ± 0.03) and wing shape asymmetry (control: 17.4 ± 0.47 x 10-3; early: 16.9 ± 0.41 x 10-3; late: 17.6 ± 0.43 x 10-3) were not affected by starvation. This suggests that drones attempt to preserve characters which are important for their future reproduction.

Protein nutrition governs within-host race of honey bee pathogens

Multiple infections are common in honey bees, Apis mellifera, but the possible role of nutrition in this regard is poorly understood. Microsporidian infections, which are promoted by protein-fed, can negatively correlate with virus infections, but the role of protein nutrition for the microsporidian-virus interface is unknown. Here, we challenged naturally deformed wing virus - B (DWV-B) infected adult honey bee workers fed with or without pollen ( = protein) in hoarding cages, with the microsporidian Nosema ceranae. Bee mortality was recorded for 14 days and N. ceranae spore loads and DWV-B titers were quantified. Amongst the groups inoculated with N. ceranae, more spores were counted in protein-fed bees. However, N. ceranae infected bees without protein-diet had reduced longevity compared to all other groups. N. ceranae infection had no effect on protein-fed bee’s longevity, whereas bees supplied only with sugar-water showed reduced survival. Our data also support that protein-feeding can have a significant negative impact on virus infections in insects. The negative correlation between N. ceranae spore loads and DWV-B titers was stronger expressed in protein-fed hosts. Proteins not only enhance survival of infected hosts, but also significantly shape the microsporidian-virus interface, probably due to increased spore production and enhanced host immunity.

The ovary and its genes—developmental processes underlying the establishment and function of a highly divergent reproductive system in the female castes of the honey bee, Apis mellifera

The strong dimorphism in ovary phenotype seen between honey bee queens and workers represents the anatomical fixation of reproductive division of labor. We review the developmental processes by which the divergent ovary phenotypes become established, mainly focusing on the massive programmed cell death (PCD) that destroys most of the ovariole primordia in the worker ovary during larval development. Ovary-specific transcriptome analyses revealed a set of differentially expressed genes associated with PCD, including two long noncoding RNAs. PCD also plays a major role regulating ovarian activity in adult honey bee workers, and a major effect candidate gene mediating this process is Anarchy, previously identified through classical genetics in a rebel worker strain. Finally, we ask how the strong ovary phenotype dimorphism in the genus Apis may have evolved, and we discuss this by contrasting honey bees with the equally eusocial stingless bees. Through a comparison of their mating systems (polyandry versus monandry), as well as comparative data on female and male gonad structure across several families of bees, we propose the hypothesis that the exceptional gonad structure in Apis queens and drones evolved via shared developmental pathways. Furthermore, we suggest that selection on massive sperm production in Apis drones may have been a driving force leading to this exaggerated gonad morphology.

Effects of neonicotinoid exposure on molecular and physiological indicators of honey bee immunocompetence

Bee declines have been associated with various stressors including pesticides and pathogens. We separately exposed immune-challenged adult worker honey bees (Apis mellifera L.) to two neonicotinoid pesticides, thiamethoxam (10 ppb) and imidacloprid (102 ppb), by dietary delivery. We found that whereas neonicotinoid exposure weakly affected transcriptional responses of antimicrobial genes, it did not detectably affect the physiological antimicrobial response as measured by a lytic clearance assay of haemolymph. Our findings add to the evidence that transcriptional responses in immune-related genes are not yet reliable indicators of pesticide impacts on bee health, which suggests caution in their future use as biomarkers in pesticide risk assessment.

Hygienic and grooming behaviors in African and European honeybees-New damage categories in Varroa destructor.

Varroa destructor is an ectoparasitic pest of honeybees, and a threat to the survival of the apiculture industry. Several studies have shown that unlike European honeybees, African honeybee populations appear to be minimally affected when attacked by this mite. However, little is known about the underlying drivers contributing to survival of African honeybee populations against the mite. We hypothesized that resistant behavioral defenses are responsible for the survival of African honeybees against the ectoparasite. We tested this hypothesis by comparing grooming and hygienic behaviors in the African savannah honeybee Apis mellifera scutellata in Kenya and A. mellifera hybrids of European origin in Florida, USA against the mite. Grooming behavior was assessed by determining adult mite infestation levels, daily mite fall per colony and percentage mite damage (as an indicator of adult grooming rate), while hygienic behavior was assessed by determining the brood removal rate after freeze killing a section of the brood. Our results identified two additional undescribed damaged mite categories along with the six previously known damage categories associated with the grooming behavior of both honeybee subspecies. Adult mite infestation level was approximately three-fold higher in A. mellifera hybrids of European origin than in A. m. scutellata, however, brood removal rate, adult grooming rate and daily natural mite fall were similar in both honeybee subspecies. Unlike A. mellifera hybrids of European origin, adult grooming rate and brood removal rate did not correlate with mite infestation levels on adult worker honeybee of A. m. scutellata though they were more aggressive towards the mites than their European counterparts. Our results provide valuable insights into the tolerance mechanisms that contribute to the survival of A. m. scutellata against the mite.

A specific genetic marker for the discrimination of native Bulgarian honey bees (Apis mellifera rodopica): Duplication of coI gene fragment

In this study, we present for the first time SNPs analysis of a coI mitochondrial DNA (mtDNA) gene segment of the local Rhodopa Mountain honey bee, A. m. rodopica, that is well investigated, based on morpho-ethological and biochemical studies. The analysis was performed on adult honey bee workers that were sampled from 29 colonies that are under the control of the Bulgarian National Beekeeping Association. Our data are compared with analogous studies obtained from other A. mellifera subspecies, and we describe a specific characteristic duplication of the coI gene for the Rhodopes honey bee that can be applied as a genetic marker to discriminate the local Bulgarian honey bees and to support the related conservation activities.

The dynamic association between ovariole loss and sterility in adult honeybee workers.

In the social insects, ovary state (the presence or absence of mature oocytes) and ovary size (the number of ovarioles) are often used as proxies for the reproductive capacity of an individual worker. Ovary size is assumed to be fixed post-eclosion whereas ovary state is demonstrably plastic post-eclosion. Here, we show that in fact ovary size declines as honeybee workers age. This finding is robust across two honeybee species: Apis mellifera and A. cerana The ovariole loss is likely to be due to the regression of particular ovarioles via programmed cell death. We also provide further support for the observation that honeybee workers with activated ovaries (mature oocytes present) most commonly have five ovarioles rather than a greater or smaller number. This result suggests that workers with more than five ovarioles are unable to physiologically support more than five activated ovarioles and that workers with fewer than five ovarioles are below a threshold necessary for ovary activation. As a worker's ovariole number declines with age, studies on worker ovariole number need to take this plasticity into account.

Volume and density of microglomeruli in the honey bee mushroom bodies do not predict performance on a foraging task.

The mushroom bodies (MBs) are insect brain regions important for sensory integration, learning, and memory. In adult worker honey bees (Apis mellifera), the volume of neuropil associated with the MBs is larger in experienced foragers compared with hive bees and less experienced foragers. In addition, the characteristic synaptic structures of the calycal neuropils, the microglomeruli, are larger but present at lower density in 35-day-old foragers relative to 1-day-old workers. Age- and experience-based changes in plasticity of the MBs are assumed to support performance of challenging tasks, but the behavioral consequences of brain plasticity in insects are rarely examined. In this study, foragers were recruited from a field hive to a patch comprising two colors of otherwise identical artificial flowers. Flowers of one color contained a sucrose reward mimicking nectar; flowers of the second were empty. Task difficulty was adjusted by changing flower colors according to the principle of honey bee color vision space. Microglomerular volume and density in the lip (olfactory inputs) and collar (visual inputs) compartments of the MB calyces were analyzed using anti-synapsin I immunolabeling and laser scanning confocal microscopy. Foragers displayed significant variation in microglomerular volume and density, but no correlation was found between these synaptic attributes and foraging performance. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1057-1071, 2017.

Innovative Approach for the Use of Huwa-San TR50 in Controlling Cotton Aphids (Aphis gossypii Glover)

Chemical control remains the main method of controlling the cotton aphid (Aphis gossypii Glover). Millions of dollars have been lost due to plant damage which resulted in reduced quality and yield of cotton. Nevertheless, A. gossypii can rapidly develop resistance to different groups of insecticides such as organophosphates, carbamates and pyrethroids. The potential of Huwa-San TR50 in controlling A. gossypii is yet to be tested. Huwa-San TR50 is a formula of hydrogen peroxide which has been stabilized by the addition of a small quantity of silver and has extensively used as a disinfectant. In this study, it was found to be very potent in killing A. gossypii and produced 93.5, 96.5, 97 and 95.5% mortality at 1000, 2000, 3000 and 4000 ppm, respectively, after 48 h of exposure. Furthermore, there was no significant difference between four Huwa-San TR50 concentrations after 48 h of exposure, on the mortality of A. gossypii. Huwa-San TR50 of up to 4000 ppm had no observable effects on the mortality and behavior of adult honeybee workers (Apis mellifera lamarckii) as compared with the control. Also, Huwa-San TR50 concentration of up to 3000 ppm had no observable effect on seven-spot ladybird beetles (Coccinella septempunctata) whereas a concentration of 4000 ppm produced 100% mortality after 24 h of exposure. Huwa-San TR50 concentrations of up to 2000 ppm failed to produce any symptoms on cucumber leaves. The differential effects of Huwa-San TR50 on aphids and beneficial insects, suggest the need for further investigation to understand the effects of Huwa-San TR50 on other host plants of aphids and aphid species.

Effects of synthetic acaricides on honey bee grooming behavior against the parasitic Varroa destructor mite

Varroa destructor is currently one of the main threats for western apiculture. Today, synthetic acaricides (specifically coumaphos, amitraz, and tau-fluvalinate) are the most common methods to control Varroa infestations. These compounds, however, are frequently related to a wide range of side effects in the host, as well as a long half-life inside the hive matrices (wax and honey). The western honey bee, Apis mellifera, exhibits natural defense mechanisms against the mite such as grooming behavior, which is a sequence of bodily movements where the host scrapes its legs across its body surface to remove the mite. We tested the effects of synthetic acaricides on the performance of grooming behavior by adult honey bee workers. We found that acaricide exposure prior to grooming delayed grooming and reduced the overall duration of grooming behavior. Our data add to a list of other sublethal behavioral consequences of acaricides that may subvert a comprehensive approach to Varroa control in managed colonies.

Covert deformed wing virus infections have long-term deleterious effects on honeybee foraging and survival.

Several studies have suggested that covert stressors can contribute to bee colony declines. Here we provide a novel case study and show using radiofrequency identification tracking technology that covert deformed wing virus (DWV) infections in adult honeybee workers seriously impact long-term foraging and survival under natural foraging conditions. In particular, our experiments show that adult workers injected with low doses of DWV experienced increased mortality rates, that DWV caused workers to start foraging at a premature age, and that the virus reduced the workers' total activity span as foragers. Altogether, these results demonstrate that covert DWV infections have strongly deleterious effects on honeybee foraging and survival. These results are consistent with previous studies that suggested DWV to be an important contributor to the ongoing bee declines in Europe and the USA. Overall, our study underlines the strong impact that covert pathogen infections can have on individual and group-level performance in bees.

Prevalence of the microsporidian Nosema ceranae in honeybee (Apis mellifera) apiaries in Central Italy.

Nosema ceranae and Nosema apis are microsporidia which play an important role in the epidemiology of honeybee microsporidiosis worldwide. Nosemiasis reduces honeybee population size and causes significant losses in honey production. To the best of our knowledge, limited information is available about the prevalence of nosemiasis in Italy. In this research, we determined the occurrence of Nosema infection in Central Italy. Thirty-eight seemingly healthy apiaries (2 to 4 hives each) were randomly selected and screened from April to September 2014 (n = 11) or from May to September 2015 (n = 27). The apiaries were located in six areas of Central Italy, including Lucca (n = 11), Massa Carrara (n = 9), Pisa (n = 9), Leghorn (n = 7), Florence (n = 1), and Prato (n = 1) provinces. Light microscopy was carried out according to current OIE recommendations to screen the presence of microsporidiosis in adult worker honeybees. Since the morphological characteristics of N. ceranae and N. apis spores are similar and can hardly be distinguished by optical microscopy, all samples were also screened by multiplex polymerase chain reaction (M-PCR) assay based on 16S rRNA-gene-targeted species-specific primers to differentiate N. ceranae from N. apis. Furthermore, PCR-positive samples were also sequenced to confirm the species of amplified Nosema DNA. Notably, Nosema spores were detected in samples from 24 out of 38 (63.2%, 95% CI: 47.8–78.5%) apiaries. Positivity rates in single provinces were 10/11, 8/9, 3/9, 1/7, or 1/1 (n = 2). A full agreement (Cohen's Kappa = 1) was assessed between microscopy and M-PCR. Based on M-PCR and DNA sequencing results, only N. ceranae was found. Overall, our results highlighted that N. ceranae infection occurs frequently in the cohort of honeybee populations that was examined despite the lack of clinical signs. These findings suggest that colony disease outbreaks might result from environmental factors that lead to higher susceptibility of honeybees to this microsporidian.

Notch signalling mediates reproductive constraint in the adult worker honeybee.

The hallmark of eusociality is the reproductive division of labour, in which one female caste reproduces, while reproduction is constrained in the subordinate caste. In adult worker honeybees (Apis mellifera) reproductive constraint is conditional: in the absence of the queen and brood, adult worker honeybees activate their ovaries and lay haploid male eggs. Here, we demonstrate that chemical inhibition of Notch signalling can overcome the repressive effect of queen pheromone and promote ovary activity in adult worker honeybees. We show that Notch signalling acts on the earliest stages of oogenesis and that the removal of the queen corresponds with a loss of Notch protein in the germarium. We conclude that the ancient and pleiotropic Notch signalling pathway has been co-opted into constraining reproduction in worker honeybees and we provide the first molecular mechanism directly linking ovary activity in adult worker bees with the presence of the queen.

Protein profiling of Apis species (Hymenoptera: Apidae) adult worker honey bees from North-western region of India

In Apis, biodiversity studies at genetic and molecular level were done to identify the proteins responsible for polymorphism in different species of Apis viz. Apis florea F., Apis dorsata F., Apis cerana F. and Apis mellifera L. from north-west region of India using SDS-Polyacrylamide gel electrophoresis (PAGE). Some species specific protein bands were obtained by SDS-PAGE technique followed by Commassie BB staining and silver staining. The present results revealed that the total content of proteins was highest in A. dorsata with highest number of protein bands unique to species followed by A. mellifera, A. florea and A. cerana. Most prominent were at 38.7 kDa and 35.3 kDa in A. dorsata; 46.4 kDa and 29.0 kDa in A. florea; 83.9 kDa and 40.4 kDa in A. mellifera and 61.0 kDa and 27.9 kDa in A. cerana respectively. Some species specific protein bands with silver staining were as 33.6 and 59.2 kDa in Apis florea; 57.2 kDa in A. dorsata; 49.6 and 63.6 kDa in A. mellifera and 36.1 and 38.8 kDa in A. cerana. Based on identification of these unique bands, it is concluded that the species with wide geographic distribution have large number of protein bands as compared to species with least distribution area.

No evidence that DNA methylation is associated with the regulation of fertility in the adult honey bee Apis mellifera (Hymenoptera: Apidae) worker ovary

Epigenetic modifications, mediated by DNA methylation, are involved in caste differentiation between queen and worker honey bees, Apis mellifera. This study uses methylation sensitive amplified fragment length polymorphism (MS-AFLP) to investigate whether genome-wide DNA methylation is implicated in the regulation of honey bee worker sterility and whether CO2 narcosis, which suppresses ovary activation in queenless workers, influences DNA methylation. Queenless workers were divided into control and experimental (CO2 narcosis) cages. We compared MS-AFLP patterns in CO2-treated and non-treated workers with and without activated ovaries at 4 h, 2 and 7 days after treatment. While there was evidence of DNA methylation in all samples, there was no detectable difference in genome-wide methylation patterns between any treatment comparisons. This suggests that mechanisms that regulate worker sterility in the adult honey bee worker are not related to genome-wide methylation, and that CO2 narcosis does not significantly affect DNA methylation patterns in the worker ovary. However, because DNA methylation is sparse in the honey bee, and because MS-AFLP detects only a proportion of methylated sites, it remains possible that a subset of genes that are responsible for the regulation of worker sterility are differentially methylated, but that we failed to pick these up in our genome-wide scan.

Starvation stress during larval development facilitates an adaptive response in adult worker honey bees (Apis mellifera L.).

Most organisms are constantly faced with environmental changes and stressors. In diverse organisms, there is an anticipatory mechanism during development that can program adult phenotypes. The adult phenotype would be adapted to the predicted environment that occurred during organism maturation. However, whether this anticipatory mechanism is present in eusocial species is questionable because eusocial organisms are largely shielded from exogenous conditions by their stable nest environment. In this study, we tested whether food deprivation during development of the honey bee (Apis mellifera), a eusocial insect model, can shift adult phenotypes to better cope with nutritional stress. After subjecting fifth instar worker larvae to short-term starvation, we measured nutrition-related morphology, starvation resistance, physiology, endocrinology and behavior in the adults. We found that the larval starvation caused adult honey bees to become more resilient toward starvation. Moreover, the adult bees were characterized by reduced ovary size, elevated glycogen stores and juvenile hormone (JH) titers, and decreased sugar sensitivity. These changes, in general, can help adult insects survive and reproduce in food-poor environments. Overall, we found for the first time support for an anticipatory mechanism in a eusocial species, the honey bee. Our results suggest that this mechanism may play a role in honey bee queen-worker differentiation and worker division of labor, both of which are related to the responses to nutritional stress.

DNA base modifications in honey bee and fruit fly genomes suggest an active demethylation machinery with species- and tissue-specific turnover rates

Well-known epigenetic DNA modifications in mammals include the addition of a methyl group and a hydroxyl group to cytosine, resulting in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) respectively. In contrast, the abundance and the functional implications of these modifications in invertebrate model organisms such as the honey bee (Apis mellifera) and the fruit fly (Drosophila melanogaster) are not well understood. Here we show that both adult honey bees and fruit flies contain 5mC and also 5hmC. Using a highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) technique, we quantified 5mC and 5hmC in different tissues of adult honey bee worker castes and in adult fruit flies. A comparison of our data with reports from human and mouse shed light on notable differences in 5mC and 5hmC levels between tissues and species.