Comparative genomics of Lactobacillaceae from the gut of honey bees, Apis mellifera, from the Eastern United States.

Lactobacillaceae are an important family of lactic acid bacteria that play key roles in the gut microbiome of many animal species. In the honey bee (Apis mellifera) gut microbiome, many species of Lactobacillaceae are found, and there is functionally important strain-level variation in the bacteria. In this study, we completed whole-genome sequencing of 3 unique Lactobacillaceae isolates collected from hives in Virginia, USA. Using 107 genomes of known bee-associated Lactobacillaceae and Limosilactobacillus reuteri as an outgroup, the phylogenetics of the 3 isolates was assessed, and these isolates were identified as novel strains of Apilactobacillus kunkeei, Lactobacillus kullabergensis, and Bombilactobacillus mellis. Genome rearrangements, conserved orthologous genes (COG) categories and potential prophage regions were identified across the 3 novel strains. The new A. kunkeei strain was enriched in genes related to replication, recombination and repair, the L. kullabergensis strain was enriched for carbohydrate transport, and the B. mellis strain was enriched in transcription or transcriptional regulation and in some genes with unknown functions. Prophage regions were identified in the A. kunkeei and L. kullabergensis isolates. These new bee-associated strains add to our growing knowledge of the honey bee gut microbiome, and to Lactobacillaceae genomics more broadly.

Classic Hoarding Cages Increase Gut Bacterial Abundance and Reduce the Individual Immune Response of Honey Bee (Apis mellifera) Workers.

Laboratory experiments have advanced our understanding of honey bee (Apis mellifera) responses to environmental factors, but removal from the hive environment may also impact physiology. To examine whether the laboratory environment alters the honey bee gut bacterial community and immune responses, we compared bacterial community structure (based on amplicon sequence variant relative abundance), total bacterial abundance, and immune enzyme (phenoloxidase and glucose oxidase) activity of cohort honey bee workers kept under laboratory and hive conditions. Workers housed in the laboratory showed differences in the relative abundance of their core gut taxa, an increase in total gut bacterial abundance, and reduced phenoloxidase activity, compared to bees housed in hives.

Chlorothalonil Exposure Alters Virus Susceptibility and Markers of Immunity, Nutrition, and Development in Honey Bees.

Chlorothalonil is a broad spectrum chloronitrile fungicide that has been identified as one of the most common pesticide contaminants found in managed honey bees (Hymenoptera: Apidae: Apis mellifera L.), their food stores, and the hive environment. While not acutely toxic to honey bees, several studies have identified potential sublethal effects, especially in larvae, but comprehensive information regarding the impact of chlorothalonil on adults is lacking. The goal of this study was to investigate the effects of exposure to a field relevant level of chlorothalonil on honey bee antiviral immunity and biochemical markers of general and social immunity, as well as macronutrient markers of nutrition and morphological markers of growth and development. Chlorothalonil exposure was found to have an effect on 1) honey bee resistance and/or tolerance to viral infection by decreasing the survival of bees following a viral challenge, 2) social immunity, by increasing the level of glucose oxidase activity, 3) nutrition, by decreasing levels of total carbohydrate and protein, and 4) development, by decreasing the total body weight, head width, and wing length of adult nurse and forager bees. Although more research is required to better understand how chlorothalonil interacts with bee physiology to increase mortality associated with viral infections, this study clearly illustrates the sublethal effects of chlorothalonil exposure on bee immunity, nutrition, and development.

An assessment of pesticide exposures and land use of honey bees in Virginia.

Large-scale honey bee colony loss threatens pollination services throughout the United States. An increase in anthropogenic pressure may influence the exposure of hives to household and agricultural pesticides. The objective of this survey was to provide an assessment of the risk of exposure to commonly used pesticides to honey bee colonies in Virginia in relation to land use. Adult honey bee, pollen, and wax samples from colonies throughout Virginia were evaluated for pyrethroid, organophosphate, organochlorine, and triazine pesticides using gas chromatography-mass spectrometry analysis. Of the 11 pesticides analyzed, nine were detected in one or more hive matrices. The probability of detecting a pesticide in pollen was less in forests than in pasture, agriculture, or urban landscapes. Coumaphos and fluvalinate were significantly more likely to be detected across all matrices with concentrations in wax as high as 15500 and 6970 ng/g (dry weight), respectively, indicating the need for further research on the potential effects of miticide accumulation in wax to larval and adult bees.

In-Hive Acaricides Alter Biochemical and Morphological Indicators of Honey Bee Nutrition, Immunity, and Development.

The honey bee is a widely managed crop pollinator that provides the agricultural industry with the sustainability and economic viability needed to satisfy the food and fiber needs of our society. Excessive exposure to apicultural pesticides is one of many factors that has been implicated in the reduced number of managed bee colonies available for crop pollination services. The goal of this study was to assess the impact of exposure to commonly used, beekeeper-applied apicultural acaricides on established biochemical indicators of bee nutrition and immunity, as well as morphological indicators of growth and development. The results described here demonstrate that exposure to tau-fluvalinate and coumaphos has an impact on 1) macronutrient indicators of bee nutrition by reducing protein and carbohydrate levels, 2) a marker of social immunity, by increasing glucose oxidase activity, and 3) morphological indicators of growth and development, by altering body weight, head width, and wing length. While more work is necessary to fully understand the broader implications of these findings, the results suggest that reduced parasite stress due to chemical interventions may be offset by nutritional and immune stress.

Relationship of Landscape Type on Neonicotinoid Insecticide Exposure Risks to Honey Bee Colonies: A Statewide Survey.

Neonicotinoid insecticide use has been suggested as a cause of honey bee colony decline; however, detection rates and concentrations of neonicotinoid insecticide residues in field-collected honey bees have been low. We collected honey bee and beebread samples from apiaries in agricultural, developed, and undeveloped areas during 2 years in Virginia to assess whether landscape type or county pesticide use was predictive of honey bee colony exposure to neonicotinoid insecticides. Trace concentrations of the neonicotinoid imidacloprid were detected in honey bees (3 of 84 samples, 2.02-3.97 ng/g), whereas higher concentrations were detected in beebread (5 of 84 samples, 4.68-11.5 ng/g) and pollen (three of five pollen trap samples, 7.86-12.6 ng/g). Imidacloprid was only detected in samples collected during July and August and was not detected in honey bees from hives where neonicotinoids were detected in pollen or beebread. The number of hives sampled at a site, county pesticide use, and landscape characteristics were not predictive of neonicotinoid detections in honey bees or beebread (all P > 0.05). Field surveys may underestimate honey bee exposure to field-realistic levels of pesticides or the risk of exposure in different landscapes because of low detection rates. Undetectably low levels of exposure or high levels of exposure that go undetected raise questions with regard to potential threats to honey bees and other pollinators.

Seasonal Effects and the Impact of In-Hive Pesticide Treatments on Parasite, Pathogens, and Health of Honey Bees.

Honey bee, Apis mellifera (L.; Hymenoptera: Apidae), populations are in decline and their losses pose a serious threat for crop pollination and food production. The specific causes of these losses are believed to be multifactorial. Pesticides, parasites and pathogens, and nutritional deficiencies have been implicated in the losses due to their ability to exert energetic stress on bees. While our understanding of the role of these factors in honey bee colony losses has improved, there is still a lack of knowledge of how they impact the immune system of the honey bee. In this study, honey bee colonies were exposed to Fumagilin-B, Apistan (tau-fluvalinate), and chlorothalonil at field realistic levels. No significant effects of the antibiotic and two pesticides were observed on the levels of varroa mite, Nosema ceranae (Fries; Microsporidia: Nosematidae), black queen cell virus, deformed wing virus, or immunity as measured by phenoloxidase and glucose oxidase activity. Any effects on the parasites, pathogens, and immunity we observed appear to be due mainly to seasonal changes within the honey bee colonies. The results suggest that Fumagilin-B, Apistan, and chlorothalonil do not significantly impact the health of honey bee colonies, based on the factors analyzed and the concentration of chemicals tested.

Effects of Pesticide Treatments on Nutrient Levels in Worker Honey Bees (Apis mellifera).

Honey bee colony loss continues to be an issue and no factor has been singled out as to the cause. In this study, we sought to determine whether two beekeeper-applied pesticide products, tau-fluvalinate and Fumagilin-B(®), and one agrochemical, chlorothalonil, impact the nutrient levels in honey bee workers in a natural colony environment. Treatments were performed in-hive and at three different periods (fall, spring, and summer) over the course of one year. Bees were sampled both at pre-treatment and two and four weeks post-treatment, weighed, and their protein and carbohydrate levels were determined using BCA and anthrone based biochemical assays, respectively. We report that, based on the pesticide concentrations tested, no significant negative impact of the pesticide products was observed on wet weight, protein levels, or carbohydrate levels of bees from treated colonies compared with bees from untreated control colonies.

Three-Photon-Annihilation Contributions to Positronium Energies at Order mα

Positronium spectroscopy (n=1 hyperfine splitting, n=2 fine structure, and the 2S-1S interval) has reached a precision of order 1 MHz. Vigorous ongoing efforts to improve the experimental results motivate the calculation of the positronium energy levels at order mα^{7}. In this Letter, we present the result for a complete class of such contributions-those involving virtual annihilation of positronium to three photons in an intermediate state. We find an energy shift of 2.6216(11)mα^{7}/(nπ)^{3}=11.5/n^{3} kHz for the spin-triplet S state with principal quantum number n. The corresponding energy shift for true muonium (the µ^{+}µ^{-} bound state) is 2.38/n^{3} MHz with an additional -5.33/n^{3} MHz coming from electronic vacuum polarization.

Risks of neonicotinoid insecticides to honeybees.

The European honeybee, Apis mellifera, is an important pollinator of agricultural crops. Since 2006, when unexpectedly high colony losses were first reported, articles have proliferated in the popular press suggesting a range of possible causes and raising alarm over the general decline of bees. Suggested causes include pesticides, genetically modified crops, habitat fragmentation, and introduced diseases and parasites. Scientists have concluded that multiple factors in various combinations-including mites, fungi, viruses, and pesticides, as well as other factors such as reduction in forage, poor nutrition, and queen failure-are the most probable cause of elevated colony loss rates. Investigators and regulators continue to focus on the possible role that insecticides, particularly the neonicotinoids, may play in honeybee health. Neonicotinoid insecticides are insect neurotoxicants with desirable features such as broad-spectrum activity, low application rates, low mammalian toxicity, upward systemic movement in plants, and versatile application methods. Their distribution throughout the plant, including pollen, nectar, and guttation fluids, poses particular concern for exposure to pollinators. The authors describe how neonicotinoids interact with the nervous system of honeybees and affect individual honeybees in laboratory situations. Because honeybees are social insects, colony effects in semifield and field studies are discussed. The authors conclude with a review of current and proposed guidance in the United States and Europe for assessing the risks of pesticides to honeybees.

Low natural levels of Nosema ceranae in Apis mellifera queens.

Queens are the primary female reproductive individuals in honey bee colonies and, while they are generally free from Nosema ceranae infection, they are nevertheless susceptible. We sought to determine whether queens are naturally infected by N. ceranae, as these infections could be a factor in the rapid spread of this parasite. Queens were analyzed using real-time PCR and included larval queens, newly emerged, and older mated queens. Overall, we found that all tissues we examined were infected with N. ceranae at low levels but no samples were infected with Nosema apis. The infection of the ovaries and spermatheca suggests the possibility of vertical transmission of N. ceranae.

Comparison of within hive sampling and seasonal activity of Nosema ceranae in honey bee colonies.

Nosema ceranae is a microsporidian parasite of the European honey bee, Apis mellifera, that is found worldwide and in multiple Apis spp.; however, little is known about the effects of N. ceranae on A. mellifera. Previous studies using spore counts suggest that there is no longer a seasonal cycle for N. ceranae and that it is found year round with little variation in infection intensity among months. Our goal was to determine whether infection levels differ in bees collected from different areas of the hive and if there may be seasonal differences in N. ceranae infections. A multiplex species-specific real-time PCR assay was used for the detection and quantification of N. ceranae. Colonies were sampled monthly from September 2009-2010 by collecting workers from honey supers, the fringe of the brood nest, and the brood nest. We found that all bees sampled were infected with N. ceranae and that there was no significant difference in infection levels among the different groups of bees sampled (P=0.74). However, significant differences in colony infection levels were found at different times of the year (P<0.01) with the highest levels in April-June and lower levels in the fall and winter. While our study was only performed for one year, it sheds light on the fact that there may be a seasonality to N. ceranae infections. Being able to predict future N. ceranae infections can be used to better advise beekeepers on N. ceranae management.

Individual Variability of Nosema ceranae Infections in Apis mellifera Colonies.

Since 2006, beekeepers have reported increased losses of Apis mellifera colonies, and one factor that has been potentially implicated in these losses is the microsporidian Nosema ceranae. Since N. ceranae is a fairly recently discovered parasite, there is little knowledge of the variation in infection levels among individual workers within a colony. In this study we examined the levels of infection in individual bees from five colonies over three seasons using both spore counting and quantitative real-time PCR. The results show considerable intra-colony variation in infection intensity among individual workers with a higher percentage of low-level infections detected by PCR than by spore counting. Colonies generally had the highest percentage of infected bees in early summer (June) and the lowest levels in the fall (September). Nosema apis was detected in only 16/705 bees (2.3%) and always as a low-level co-infection with N. ceranae. The results also indicate that intra-colony variation in infection levels could influence the accuracy of Nosema diagnosis.

Nosema ceranae in drone honey bees (Apis mellifera).

Nosema ceranae is a microsporidian intracellular parasite of honey bees, Apis mellifera. Previously Nosema apis was thought to be the only cause of nosemosis, but it has recently been proposed that N. ceranae is displacing N. apis. The rapid spread of N. ceranae could be due to additional transmission mechanisms, as well as higher infectivity. We analyzed drones for N. ceranae infections using duplex qPCR with species specific primers and probes. We found that both immature and mature drones are infected with N. ceranae at low levels. This is the first report detecting N. ceranae in immature bees. Our data suggest that because drones are known to drift from their parent hives to other hives, they could provide a means for disease spread within and between apiaries.

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia.

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C(T) values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.

Miticide residues in Virginia honeys.

Fifty honey samples from Virginia USA were analyzed for the presence of fluvalinate and coumaphos residues. Samples were collected from hives and from bottled honey provided by beekeepers. No coumaphos or fluvalinate residues above the limit of quantification (0.05 mg/kg) were detected in any of the samples, although trace levels (<0.05 mg/kg) of coumaphos were detected in three samples from hives and trace levels of fluvalinate were found in one hive sample. No residues were detected in any of the bottled honey samples and none of the samples exceeded the US EPA tolerance levels for either miticide.

Survival of honey bee (Hymenoptera: Apidae) spermatozoa incubated at room temperature from drones exposed to miticides.

We conducted research to examine the potential impacts ofcoumaphos, fluvalinate, and Apilife VAR (Thymol) on drone honey bee, Apis mellifera L. (Hymenoptera: Apidae), sperm viability over time. Drones were reared in colonies that had been treated with each miticide by using the dose recommended on the label. Drones from each miticide treatment were collected, and semen samples were pooled. The pooled samples from each treatment were subdivided and analyzed for periods of up to 6 wk. Random samples were taken from each treatment (n = 6 pools) over the 6-wk period. Sperm viability was measured using dual-fluorescent staining techniques. The exposure of drones to coumaphos during development and sexual maturation significantly reduced sperm viability for all 6 wk. Sperm viability significantly decreased from the initial sample to week 1 in control colonies, and a significant decrease in sperm viability was observed from week 5 to week 6 in all treatments and control. The potential impacts of these results on queen performance and failure are discussed.

Effects of antemortem ingestion of ethanol on insect successional patterns and development of Phormia regina (Diptera: Calliphoridae).

The effects of antemortem ingestion of ethanol by domestic pigs, Sus scrofa L., on postmortem insect successional patterns and the development of Phormia regina (Meigen) were studied during summer 2003 in Blacksburg, VA. Insect samples were collected from the carcasses of ethanol-treated and untreated pigs for 10 d postmortem during two successional studies. In total, 32 insect taxa were collected during the two studies, with 29 and 27 taxa observed on the carcasses of ethanol-treated and untreated pigs, respectively. The earliest arrivers to both carcass types were dipterans. This group was represented by six families, with P. regina and Phaenicia coeruleiviridis (Macquart) being the most common calliphorids. Beetles in six families were collected on the carcasses of ethanol-treated pigs, but only three of the families were collected on carcasses of the untreated pigs. Permutation analyses to test the null hypothesis of no similarity between successional patterns of insect taxa from carcasses of ethanol-treated and untreated pigs showed that the successional patterns were similar between carcass types in the first (P = 0.003) and the second (P = 0.01) studies. The results of the development study of P. regina maggots in the field show that there was a significant difference between the distributions of length for maggots reared on loin tissue from ethanol-treated and untreated pigs. Maggots that fed on tissue from ethanol-treated pigs took approximately 11.9 h longer to reach the pupal stage than maggots that fed on tissue from untreated pigs. The longer developmental time for maggots on tissue from ethanol-treated pigs was due mainly to the longer postfeeding period of the third instar.

Insect fauna visiting carrion in Southwest Virginia.

Successional patterns of insect fauna on pig carcasses were studied in southwest Virginia. The objective was to identify and qualitatively assess the major taxa of forensic importance in this region. Studies were conducted in spring and summer 2001 and 2002, and fall 2002. Over 50 taxa were collected and identified. Phormia regina was the dominant fly species in the spring (>90%) and co-dominant with Phaenicia coeruleiviridis in the summer. Phaenicia sericata, Lucilia illustris, and Calliphora spp. were collected in spring and summer, but less frequently. Eleven species of Sarcophagidae also were collected with Sarcophaga utilis and Helicobia rapax the most common. In the fall, the dominant fly species were Calliphora vomitoria, L. illustris, and P. coeruleiviridis. The primary beetle species collected in spring and summer included three Staphylinidae (Creophilis maxillosus, Platydracus maculosus, and Aleochara lata) and three Silphidae (Oiceoptoma noveboracense, Necrodes surinamensis, and Necrophila americana). No beetles were collected in the fall.

Analysis of the successional patterns of insects on carrion in southwest Virginia.

Studies of carrion-insect succession on domestic pig, Sus scrofa L., were conducted in the spring and summer of 2001 and 2002 in Blacksburg, VA, to identify and analyze the successional patterns of the taxa of forensic importance in southwest Virginia. Forty-seven insect taxa were collected in the spring. These were represented by 11 families (Diptera: Calliphoridae, Sarcophagidae, Muscidae, Sepsidae, Piophilidae; Coleoptera: Staphylinidae, Silphidae, Cleridae, Trogidae, Dermestidae, Histeridae). In the summer, 33 taxa were collected that were represented by all of the families collected in the spring, except Trogidae. The most common flies collected were the calliphorids: Phormia regina (Meigen) and Phaenicia coeruleiviridis (Macquart). The most common beetles were Creophilus maxillosus L. (Staphylinidae), Oiceoptoma noveboracense Forster, Necrophila americana L., Necrodes surinamensis (F.) (Silphidae), Euspilotus assimilis (Paykull), and Hister abbreviatus F. (Histeridae). Occurrence matrices were constructed for the successional patterns of insect taxa during 21 sampling intervals in the spring and 8 intervals in the summer studies. Jackknife estimates (mean+/-95% confidence limits) of overall Jaccard similarity in insect taxa among sampling intervals in the occurrence matrices were 0.213+/-0.081 (spring 2001), 0.194+/-0.043 (summer 2001), 0.257+/-0.068 (spring 2002), and 0.274+/-0.172 (summer 2002). Permutation analyses of the occurrence matrices showed that the patterns of succession of insect taxa were similar between spring 2001 and 2002 (P = 0.001) and between summer 2001 and 2002 (P = 0.007). The successional patterns seem to be typical for the seasonal periods and provide data on baseline fauna for estimating postmortem interval in cases of human death. This study is the first of its kind for southwest Virginia.