Little is known about the combined effects of stressors on social immunity of honey bees (Apis mellifera) and related gene expression. The interaction between sublethal doses of a neurotoxin, clothianidin, and the ectoparasite, Varroa destructor, was examined by measuring differentially expressed genes (DEGs) in brains, deformed wing virus (DWV) and the proportion and intensity of self-grooming. Evidence for an interaction was observed between the stressors in a reduction in the proportion of intense groomers. Only the lowest dose of clothianidin alone reduced the proportion of self-groomers and increased DWV levels. V. destructor shared a higher proportion of DEGs with the combined stressors compared to clothianidin, indicating that the effects of V. destructor were more pervasive than those of clothianidin when they were combined. The number of up-regulated DEGs were reduced with the combined stressors compared to clothianidin alone, suggesting an interference with the impacts of clothianidin. Clothianidin and V. destructor affected DEGs from different biological pathways but shared impacts on pathways related to neurodegenerative disorders, like Alzheimer’s, which could be related to neurological dysfunction and may explain their negative impacts on grooming. This study shows that the combination of clothianidin and V. destructor resulted in a complex and non-additive interaction.

Nosema ceranae is an emerging pathogen of the western honey bee (Apis mellifera L.), and thus its seasonality and impact on bee colonies is not sufficiently documented for North America. This study was conducted to determine the infection intensity, prevalence, and viability of N. ceranae in >200 honey bee colonies during spring, summer, and fall, in a North American region. We also determined the relationship of N. ceranae infections with colony populations, food stores, bee survivorship, and overwinter colony mortality. The highest rates of N. ceranae infection, prevalence, and spore viability were found in the spring and summer, while the lowest were recorded in the fall. N. ceranae spore viability was significantly correlated with its prevalence and infection intensity in bees. Threshold to high levels of N. ceranae infections (>1,000,000 spores/bee) were significantly associated with reduced bee populations and food stores in colonies. Furthermore, worker bee survivorship was significantly reduced by N. ceranae infections, although no association between N. ceranae and winter colony mortality was found. It is concluded that N. ceranae infections are highest in spring and summer and may be detrimental to honey bee populations and colony productivity. Our results support the notion that treatment is justified when infections of N. ceranae exceed 1,000,000 spores/bee.

While many studies have examined the effects of neonicotinoid insecticides and the parasitic mite Varroa destructor on honey bees (Apis mellifera), more information on the combined effects of such stressors on gene expression, including neural related genes, and their impact on biological pathways is needed. This study analyzed the effects of field realistic concentrations of the neonicotinoid clothianidin on adult bees infested and not infested with V. destructor over 21 consecutive days and then determined bee survivorship, weight, deformed wing virus (DWV) levels and gene expression. V. destructor parasitism with or without clothianidin exposure was significantly associated with decreased survivorship, weight loss and higher DWV levels, while clothianidin exposure was only associated with higher levels of DWV. Expression analysis of the neural genes AmNlg-1, BlCh and AmAChE-2 showed that V. destructor caused a significant down-regulation of all of them, whereas clothianidin caused a significant down-regulation of only AmNrx-1 and BlCh. An interaction was only detected for AmNrx-1 expression. RNAseq analysis showed that clothianidin exposure resulted in 6.5 times more up-regulated differentially expressed genes (DEGs) than V. destructor alone and 123 times more than clothianidin combined with V. destructor. Similar results were obtained with down-regulated DEGs, except for a higher number of DEGs shared between V. destructor and the combined stressors. KEGG (Kyoto Encyclopedia of Genes and Genomes) biological pathway analysis of the DEGs showed that the stressor linked to the highest number of KEGG pathways was clothianidin, followed by V. destructor, and then considerably fewer number of KEGG pathways with the combined stressors. The reduced numbers of DEGs and KEGG pathways associated with the DEGs for the combined stressors compared to the stressors alone indicates that the interaction of the stressors is not additive or synergistic, but antagonistic. The possible implications of the antagonistic effect on the number of DEGs are discussed.

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This study was conducted to evaluate the Indiana "mite-biter" honey bee stock, which has been selected for increased mutilation of Varroa destructor mites ("mite biting" behavior). A comparison between colonies of the selected stock and colonies of unselected Italian bees showed that the proportion of mutilated mites, the severity of mutilations, and winter colony survival were higher in Indiana mite-biter colonies. Additionally, the number of fallen mites and the rate of mite population growth were lower in the colonies of the selected genotype than in those of the unselected genotype. The expression of a gene associated with grooming behavior, AmNrx-1 (neurexin), was significantly higher in the selected stock. Moreover, AmNrx-1 expression was positively correlated with the proportion of mutilated mites but not with mite population growth. AmNrx-1 may have the potential to be used for marker-assisted selection. This study provides evidence that selection for mite-biting behavior reduces V. destructor infestations, increases colony survival and increases the expression of a grooming behaviorassociated gene.

Most studies on the effects of neonicotinoid insecticides on neural processes of honey bees are based on behaviours performed by adult bees exposed as adults. It is unclear how the developing brains of honey bee larvae are affected by sublethal doses of neonicotinoid insecticides when measuring neural processes through behavioural performance in adult bees. In this study, larvae were exposed to three sublethal doses of clothianidin and evaluated 25-36 days later for hygienic and foraging behaviours as adult bees. The medium and highest sublethal doses of clothianidin significantly reduced hygienic and foraging activity. The greatest effects were on the proportion of adult bees foraging and carrying pollen. These results show that exposure of larvae to clothianidin results in negative effects extending into the adulthood of bees, possibly compromising the colony's fitness by impairing pathogen control mechanisms and by reducing pollen collection.

Honey bees (Apis mellifera) are exposed to sublethal doses of insecticides, but little is known about insecticide effects on their survivorship associated to health-related gene expression. To test the effect of sublethal doses of clothianidin, imidacloprid and carbaryl on the lifespan and health of honey bees, workers were orally and topically exposed to LD5 doses of these insecticides. The survivorship of treated bees was monitored and the expression of three immune-related genes, hymenoptaecin (AmHym), basket (AmBask) and lysozyme (AmLyso2) was analyzed at 24 and 72 hours post treatment (hpt), as well as that of the antioxidant-related gene vitellogenin (AmVit2), the poly-U binding factor (AmPuf68), and the detoxification gene cytochrome P450 (AmCYP9Q3). The three insecticides significantly reduced the length of life of bees but the mode of application did not affect survivorship. AmHym, AmBask and AmVit2 expression was significantly down-regulated at 72 hpt in bees treated with clothianidin and imidacloprid, indicating immunosuppression. However, AmLyso2, AmCYP9Q3 and AmPuf68 were significantly up-regulated. The down-regulation of AmVit2 could have caused decreased resistance to oxidative stress. AmPuf68 expression could be associated with increased protection against xenobionts. AmCYP9Q3 was up-regulated at 24 and 72 hpt H Tarek, et al. 2 Genetics and Molecular Research 17 (2): gmr16039908 in oral exposures, but only until 72 hpt in topical exposures, indicating faster sensitivity towards detoxification mechanisms in oral treatments. This study demonstrated detrimental effects of sublethal doses of clothianidin, imidacloprid and carbaryl on honey bee survivorship, immunity and antioxidant mechanisms, and an induction of defense and detoxification responses that could be physiologically costly to the bees.

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