Decoupling the effects of nutrition, age, and behavioral caste on honey bee physiology, immunity, and colony health.

Nutritional stress, especially a dearth of pollen, has been linked to honey bee colony losses. Colony-level experiments are critical for understanding the mechanisms by which nutritional stress affects individual honey bee physiology and pushes honey bee colonies to collapse. In this study, we investigated the impact of pollen restriction on key markers of honey bee physiology, main elements of the immune system, and predominant honey bee viruses. To achieve this objective, we uncoupled the effects of behavior, age, and nutritional conditions using a new colony establishment technique designed to control size, demography, and genetic background. Our results showed that the expression of storage proteins, including vitellogenin (vg) and royal jelly major protein 1 (mrjp1), were significantly associated with nursing, pollen ingestion, and older age. On the other hand, genes involved in hormonal regulation including insulin-like peptides (ilp1 and ilp2) and methyl farnesoate epoxidase (mfe), exhibited higher expression levels in young foragers from colonies not experiencing pollen restriction. In contrast, pollen restriction induced higher levels of insulin-like peptides in old nurses. On the other hand, we found a strong effect of behavior on the expression of all immune genes, with higher expression levels in foragers. In contrast, the effects of nutrition and age were significant only the expression of the regulatory gene dorsal. We also found multiple interactions of the experimental variables on viral titers, including higher Deformed wing virus (DWV) titers associated with foraging and age-related decline. In addition, nutrition significantly affected DWV titers in young nurses, with higher titers induced by pollen ingestion. In contrast, higher levels of Black queen cell virus (BQCV) were associated with pollen restriction. Finally, correlation, PCA, and NMDS analyses proved that behavior had had the strongest effect on gene expression and viral titers, followed by age and nutrition. These analyses also support multiple interactions among genes and virus analyzed, including negative correlations between the expression of genes encoding storage proteins associated with pollen ingestion and nursing (vg and mrjp1) with the expression of immune genes and DWV titers. Our results provide new insights into the proximal mechanisms by which nutritional stress is associated with changes in honey bee physiology, immunity, and viral titers.

Non-optimal ambient temperatures aggravate insecticide toxicity and affect honey bees Apis mellifera L. gene regulation.

In this study, we conducted a transcriptional analysis of five honey bee genes to examine their functional involvement vis-à-vis ambient temperatures and exposure to imidacloprid. In a 15-day cage experiment, three cohorts of one-day-old sister bees emerged in incubators, were distributed into cages, and maintained at three different temperatures (26 °C, 32 °C, 38 °C). Each cohort was fed a protein patty and three concentrations of imidacloprid-tainted sugar (0 ppb, 5 ppb and 20 ppb) ad libitum. Honey bee mortality, syrup and patty consumption were monitored daily over 15 days. Bees were sampled every three days for a total of five time points. RT-qPCR was used to longitudinally assess gene regulation of Vg, mrjp1, Rsod, AChE-2 and Trx-1 using RNA extracted from whole bee bodies. Kaplan-Meier models show that bees kept at both non-optimal temperatures (26 °C and 38 °C) were more susceptible to imidacloprid, with significantly higher mortality (P < 0.001 and P < 0.01, respectively) compared to the control. At 32 °C, no differences in mortality (P = 0.3) were recorded among treatments. In both imidacloprid treatment groups and the control, the expression of Vg and mrjp1 was significantly downregulated at 26 °C and 38 °C compared to the optimal temperature of 32 °C, indicating major influence of ambient temperature on the regulation of these genes. Within the ambient temperature groups, both imidacloprid treatments exclusively downregulated Vg and mrjp1 at 26 °C. AChE-2 and the poorly characterized Rsod gene were both consistently upregulated at the highest temperature (38 °C) compared to the ideal temperature (32 °C) in all treatment groups. Trx-1 showed no effect to both temperature and imidacloprid treatments and was regulated in an age-related manner. Overall, our results indicate that ambient temperatures amplify imidacloprid toxicity and affect honey bee gene regulation.

Honey bee populations of the USA display restrictions in their mtDNA haplotype diversity.

The genetic diversity of the USA honey bee (Apis mellifera L.) populations was examined through a molecular approach using two mitochondrial DNA (mtDNA) markers. A total of 1,063 samples were analyzed for the mtDNA intergenic region located between the cytochrome c oxidase I and II (COI-COII) and 401 samples were investigated for the NADH dehydrogenase 2 (ND2) coding gene. The samples represented 45 states, the District of Colombia and two territories of the USA. Nationwide, three maternal evolutionary lineages were identified: the North Mediterranean lineage C (93.79%), the West Mediterranean lineage M (3.2%) and the African lineage A (3.01%). A total of 27 haplotypes were identified, 13 of them (95.11%) were already reported and 14 others (4.87%) were found to be novel haplotypes exclusive to the USA. The number of haplotypes per state/territory ranged between two and eight and the haplotype diversity H ranged between 0.236-0.763, with a nationwide haplotype diversity of 0.597. Furthermore, the honey bee populations of the USA were shown to rely heavily (76.64%) on two single haplotypes (C1 = 38.76%, C2j = 37.62%) of the same lineage characterizing A. m. ligustica and A. m. carnica subspecies, respectively. Molecular-variance parsimony in COI-COII and ND2 confirmed this finding and underlined the central and ancestral position of C2d within the C lineage. Moreover, major haplotypes of A. m. mellifera (M3a, M7b, M7c) were recorded in six states (AL, AR, HI, MO, NM and WA). Four classic African haplotypes (A1e, A1v, A4, A4p) were also identified in nine states and Puerto Rico, with higher frequencies in southern states like LA, FL and TX. This data suggests the need to evaluate if a restricted mtDNA haplotype diversity in the US honey bee populations could have negative impacts on the beekeeping sustainability of this country.

Africanized honey bees in Colombia exhibit high prevalence but low level of infestation of Varroa mites and low prevalence of pathogenic viruses.

The global spread of the ectoparasitic mite Varroa destructor has promoted the spread and virulence of highly infectious honey bee viruses. This phenomenon is considered the leading cause for the increased number of colony losses experienced by the mite-susceptible European honey bee populations in the Northern hemisphere. Most of the honey bee populations in Central and South America are Africanized honey bees (AHBs), which are considered more resistant to Varroa compared to European honey bees. However, the relationship between Varroa levels and the spread of honey bee viruses in AHBs remains unknown. In this study, we determined Varroa prevalence and infestation levels as well as the prevalence of seven major honey bee viruses in AHBs from three regions of Colombia. We found that although Varroa exhibited high prevalence (92%), its infestation levels were low (4.5%) considering that these populations never received acaricide treatments. We also detected four viruses in the three regions analyzed, but all colonies were asymptomatic, and virus prevalence was considerably lower than those found in other countries with higher rates of mite-associated colony loss (DWV 19.88%, BQCV 17.39%, SBV 23.4%, ABPV 10.56%). Our findings indicate that AHBs possess a natural resistance to Varroa that does not prevent the spread of this parasite among their population, but restrains mite population growth and suppresses the prevalence and pathogenicity of mite-associated viruses.

Pesticides in honey bee colonies: Establishing a baseline for real world exposure over seven years in the USA.

Honey bees Apis mellifera forage in a wide radius around their colony, bringing back contaminated food resources that can function as terrestrial bioindicators of environmental pesticide exposure. Evaluating pesticide exposure risk to pollinators is an ongoing problem. Here we apply five metrics for pesticide exposure risk (prevalence, diversity, concentration, significant pesticide prevalence, and hazard quotient (HQ)) to a nation-wide field study of honey bees, Apis mellifera in the United States. We examined samples from 1055 apiaries over seven years for 218 different pesticide residues and metabolites, determining that bees were exposed to 120 different pesticide products with a mean of 2.78 per sample. Pesticides in pollen were highly prevalent and variable across states. While pesticide diversity increased over time, most detections occurred at levels predicted to be of low risk to colonies. Varroacides contributed most to concentration, followed by fungicides, while insecticides contributed most to diversity above a toxicity threshold. High risk samples contained one of 12 different insecticides or varroacides. Exposures predicted to be low-risk were nevertheless associated with colony morbidity, and low-level fungicide exposures were tied to queen loss, Nosema infection, and brood diseases.

Non-Invasive Genotyping of Honey Bee Queens Apis mellifera L.: Transition of the DraI mtDNA COI-COII Test to In Silico.

The honey bee Apis mellifera L. colony is headed by a single and indispensable queen, whose duty it is to ensure brood production and provide pheromonal stability within the colony. This study presents a non-invasive method that allows the identification of the queen maternal lineage and subspecies using the remaining tissue of her clipped wing. The DraI mtDNA COI-COII (DmCC) test was applied to various sizes of queen and worker wings and the results were compared with data obtained from other bee tissues. Furthermore, we propose a new method allowing in silico transition of the DmCC test and haplotype identification based on extended sequencing of the tRNAleu and COII genes. Our results show that DNA extracted by Chelex 10% from one-third of a queen's wing is deemed adequate for a successful identification of her maternal evolutionary lineage, haplotype and subspecies. The in silico method proposed in this study fully adheres to the established guidelines of the DmCC, provides a universal standard for haplotype identification, and offers faster and more precise results by reconciling both cleaved amplified polymorphic sequences (CAPS) and Sanger sequencing approaches.

Recent spread of Varroa destructor virus-1, a honey bee pathogen, in the United States.

RNA viruses impact honey bee health and contribute to elevated colony loss rates worldwide. Deformed wing virus (DWV) and the closely related Varroa destructor virus-1 (VDV1), are the most widespread honey bee viruses. VDV1 is known to cause high rates of overwintering colony losses in Europe, however it was unknown in the United States (US). Using next generation sequencing, we identified VDV1 in honey bee pupae in the US. We tested 603 apiaries the US in 2016 and found that VDV1 was present in 66.0% of them, making it the second most prevalent virus after DWV, which was present in 89.4% of the colonies. VDV1 had the highest load in infected bees (7.45*1012 ± 1.62*1012 average copy number ± standard error) compared to other tested viruses, with DWV second (1.04*1012 ± 0.53*1012). Analysis of 75 colonies sourced in 2010 revealed that VDV1 was present in only 2 colonies (2.7%), suggesting its recent spread. We also detected newly emerged recombinants between the US strains of VDV1 and DWV. The presence of these recombinants poses additional risk, because similar VDV1-DWV recombinants constitute the most virulent honeybee viruses in the UK.