Comparison of indoor (refrigerated) versus outdoor winter storage of commercial honey bee, Apis mellifera (Hymenoptera: Apidae), colonies in the Western United States.

Honey bees (Apis mellifera L.) are critical to the pollination of many important crops in the United States, and one crop that demands large numbers of colonies early each year is almonds. To provide adequate numbers of colonies for almond pollination, many beekeepers move colonies of bees to high-density holding yards in California in late fall, where the bees can fly and forage, but little natural pollen and nectar is available. In recent years, high colony losses have occurred in some operations following this management strategy, and alternative approaches, including indoor storage of colonies, have become more commonly used. The current study evaluated colonies kept indoors (refrigerated and/or controlled atmosphere) for the winter compared with those kept outdoors in either Washington or California. Colonies were evaluated for strength (frames of bees), brood area, lipid composition of worker bees, colony weight and survival, parasitic mites (Varroa mites, tracheal mites), and pathogens (Nosema spp.). No differences were found in colony weight, survival, parasitic mite levels, or pathogen prevalence among the treatments. Colonies stored indoors and outdoors in WA had significantly more frames of bees and less brood present after the storage period than colonies stored outdoors in CA. Lipid composition of honey bees stored indoors was significantly higher than colonies stored outdoors in WA or CA. The implications of these findings for overall colony health and improved pollination activity are discussed.

Elevated CO2 Increases Overwintering Mortality of Varroa destructor (Mesostigmata: Varroidae) in Honey Bee (Hymenoptera: Apidae) Colonies.

Indoor storage of honey bees (Apis mellifera L.) during winter months has been practiced for decades to protect colonies from the adverse effects of long, harsh winter months. Beekeepers have recently employed indoor storage to reduce labor, feeding costs, theft, and woodenware degradation. Despite the growing number of colonies stored indoors, national survey results still reveal high losses. Varroa mites (Varroa destructor Anderson and Trueman) are the most critical threat to colony winter survival and health of colonies because they contribute to the transmission of viruses and colony mortality. To investigate the effect of high CO2 on varroa mites during the indoor storage of honey bees, 8-frame single deep colonies were stored in two separate environmental chambers at 4°C each. One environmental chamber was set at 8.5% CO2 (high CO2), while the other was set at low CO2 (0.12%). Dead and falling mites were collected and counted from the bottom of individual colonies weekly during the experiment. There was a significant difference in mite mortality of colonies with high CO2 compared to colonies held at low CO2. These results indicated that high CO2 could increase mite mortality during the period of indoor storage, potentially improving honey bee health coming out of the winter months. Our research offers a critical addition to beekeepers' tools for managing varroa mite populations.

Directed evolution of Metarhizium fungus improves its biocontrol efficacy against Varroa mites in honey bee colonies.

Entomopathogenic fungi show great promise as pesticides in terms of their relatively high target specificity, low non-target toxicity, and low residual effects in agricultural fields and the environment. However, they also frequently have characteristics that limit their use, especially concerning tolerances to temperature, ultraviolet radiation, or other abiotic factors. The devastating ectoparasite of honey bees, Varroa destructor, is susceptible to entomopathogenic fungi, but the relatively warm temperatures inside honey bee hives have prevented these fungi from becoming effective control measures. Using a combination of traditional selection and directed evolution techniques developed for this system, new strains of Metarhizium brunneum were created that survived, germinated, and grew better at bee hive temperatures (35 °C). Field tests with full-sized honey bee colonies confirmed that the new strain JH1078 is more virulent against Varroa mites and controls the pest comparable to current treatments. These results indicate that entomopathogenic fungi are evolutionarily labile and capable of playing a larger role in modern pest management practices.

Impacts of Different Winter Storage Conditions on the Physiology of Diutinus Honey Bees (Hymenoptera: Apidae).

Global decline in insect pollinators, especially bees, have resulted in extensive research into understanding the various causative factors and formulating mitigative strategies. For commercial beekeepers in the United States, overwintering honey bee colony losses are significant, requiring tactics to overwinter bees in conditions designed to minimize such losses. This is especially important as overwintered honey bees are responsible for colony expansion each spring, and overwintered bees must survive in sufficient numbers to nurse the spring brood and forage until the new 'replacement' workers become fully functional. In this study, we examined the physiology of overwintered (diutinus) bees following various overwintering storage conditions. Important physiological markers, i.e., head proteins and abdominal lipid contents were higher in honey bees that overwintered in controlled indoor storage facilities, compared with bees held outdoors through the winter months. Our findings provide new insights into the physiology of honey bees overwintered in indoor and outdoor environments and have implications for improved beekeeping management.

Honey Bee Exposure to Pesticides: A Four-Year Nationwide Study.

Pollinators, including honey bees, are responsible for the successful reproduction of more than 87% of flowering plant species: they are thus vital to ecosystem health and agricultural services world-wide. To investigate honey bee exposure to pesticides, 168 pollen samples and 142 wax comb samples were collected from colonies within six stationary apiaries in six U.S. states. These samples were analyzed for evidence of pesticides. Samples were taken bi-weekly when each colony was active. Each apiary included thirty colonies, of which five randomly chosen colonies in each apiary were sampled for pollen. The pollen samples were separately pooled by apiary. There were a total of 714 detections in the collected pollen and 1008 detections in collected wax. A total of 91 different compounds were detected: of these, 79 different pesticides and metabolites were observed in the pollen and 56 were observed in the wax. In all years, insecticides were detected more frequently than were fungicides or herbicides: one third of the detected pesticides were found only in pollen. The mean (standard deviation (SD)) number of detections per pooled pollen sample varied by location from 1.1 (1.1) to 8.7 (2.1). Ten different modes of action were found across all four years and nine additional modes of action occurred in only one year. If synergy in toxicological response is a function of simultaneous occurrence of multiple distinct modes of action, then a high frequency of potential synergies was found in pollen and wax-comb samples. Because only pooled pollen samples were obtained from each apiary, and these from only five colonies per apiary per year, more data are needed to adequately evaluate the differences in pesticide exposure risk to honey bees among colonies in the same apiary and by year and location.

Extracts of Polypore Mushroom Mycelia Reduce Viruses in Honey Bees.

Waves of highly infectious viruses sweeping through global honey bee populations have contributed to recent declines in honey bee health. Bees have been observed foraging on mushroom mycelium, suggesting that they may be deriving medicinal or nutritional value from fungi. Fungi are known to produce a wide array of chemicals with antimicrobial activity, including compounds active against bacteria, other fungi, or viruses. We tested extracts from the mycelium of multiple polypore fungal species known to have antiviral properties. Extracts from amadou (Fomes) and reishi (Ganoderma) fungi reduced the levels of honey bee deformed wing virus (DWV) and Lake Sinai virus (LSV) in a dose-dependent manner. In field trials, colonies fed Ganoderma resinaceum extract exhibited a 79-fold reduction in DWV and a 45,000-fold reduction in LSV compared to control colonies. These findings indicate honey bees may gain health benefits from fungi and their antimicrobial compounds.

Effects of diluents and plasma on honey bee (Apis mellifera L.) drone frozen-thawed semen fertility.

Cryopreservation is an advanced method used to protect germplasm in liquid nitrogen. Honey bees are of special interest to protect because of their pollination activity and critical role in agriculture. There has been important progress in the cryopreservation of honey bee germplasm in recent years, leading to practical recovery of genetic material for breeding purposes following freezing. However, there remains room for improvement and the goal of the present study was to evaluate the effect of different "extenders" added post-thaw on the fertilization rate of cryopreserved honey bee semen. The purpose of adding extender post-thaw was to dilute the cryoprotectant to remove chemicals after centrifugation because of potential adverse effects. The control consisted of frozen-thawed semen without the addition of an extender; treatment groups included the addition of one of the following extenders: glucose solution, fresh ram semen plasma, fresh honey bee semen plasma, extender solution. All of the above treatments and frozen-thawed control were compared to fresh semen. For each group, 15 virgin queens were instrumentally inseminated with the semen-diluent solution and introduced into nucleus colonies to determine the brood patterns of the queens. Percentages of worker brood produced in the fresh semen, frozen-thawed semen control, glucose, fresh ram semen plasma, fresh honey bee semen plasma, and extender solution supplemented groups were 98.±1.1%, 47.0 ± 0.9%, 3.0 ± 0.8%, 0.3 ± 0.1%, 48.1 ± 4.1% and 40.3 ± 2.4%, respectively. Similiarly, spermatozoa numbers in the spermathecae of the same treatment groups were 3.6 × 106, 1.6 × 106, 7.3 × 105, 4.7 × 105, 8.1 × 105, and 4.6 × 105 spermatozoa for the same treatment, respectively. The differences in both worker brood percentage and sperm count in the spermatheca were statistically significant (P < 0.01) among all treatment groups, except the frozen-thawed control group and fresh drone semen plasma group. We found a positive correlation between sperm count in the spermatheca and the percentage of worker brood (r = 0.91). With the exception of fresh honey bee semen plasma, the fertility rate was reduced following the addition of various plasmas and diluents post-freezing.

Gel-coated tubes extend above-freezing storage of honey bee (Apis mellifera) semen to 439 days with production of fertilised offspring.

Honey bees are an important agricultural species; however, relatively little work has been done to improve artificial reproductive technologies for this animal. The collection and distribution of germplasm for breeding and conservation is critical for improving managed honey bee populations and conserving threatened subspecies. The most efficient method of controlling breeding in honey bees is by artificial insemination. The collection of semen for insemination requires the use of antibiotics, which is especially critical if semen is to be stored for any length of time. The introduction of antibiotics is normally done through a balanced salt solution. In this study we compare, at two temperatures, the storage of undiluted semen in antibiotic-gel-coated capillary tubes with storage of semen diluted in a balanced salt solution containing antibiotics. Live-dead cell staining and artificial insemination of honey bee queens were performed at 45, 99 and 439 days after collection of the semen. In every case the antibiotic-gel-coated tube storage method at 14°C produced a higher percentage of fertilised offspring. This study demonstrates the longest period of time spermatozoa have been stored above freezing while maintaining fertilisation capacity.

Sequential generations of honey bee (Apis mellifera) queens produced using cryopreserved semen.

Much of the world's food production is dependent on honey bees for pollination, and expanding food production will further increase the demand for managed pollination services. Apiculturists outside the native range of the honey bee, in the Americas, Australia and eastern Asia, have used only a few of the 27 described subspecies of honey bees (Apis mellifera) for beekeeping purposes. Within the endemic ranges of a particular subspecies, hybridisation can threaten native subspecies when local beekeepers import and propagate non-native honey bees. For many threatened species, cryopreserved germplasm can provide a resource for the preservation of diversity and recovery of endangered populations. However, although instrumental insemination of queen honey bees is well established, the absence of an effective means to cryopreserve honey bee semen has limited the success of efforts to preserve genetic diversity within the species or to develop repositories of honey bee germplasm for breeding purposes. Herein we report that some queens inseminated with cryopreserved semen were capable of producing a substantial number of fertilised offspring. These diploid female larvae were used to produce two additional sequential generations of new queens, which were then back-crossed to the same stock of frozen semen. Our results demonstrate the ability to produce queens using cryopreserved honey bee spermatozoa and the potential for the establishment of a honey bee genetic repository.

Honey bees (Apis mellifera) reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema (Microsporidia) infection.

Nosema ceranae and pesticide exposure can contribute to honey bee health decline. Bees reared from brood comb containing high or low levels of pesticide residues were placed in two common colony environments. One colony was inoculated weekly with N. ceranae spores in sugar syrup and the other colony received sugar syrup only. Worker honey bees were sampled weekly from the treatment and control colonies and analyzed for Nosema spore levels. Regardless of the colony environment (spores+syrup added or syrup only added), a higher proportion of bees reared from the high pesticide residue brood comb became infected with N. ceranae, and at a younger age, compared to those reared in low residue brood combs. These data suggest that developmental exposure to pesticides in brood comb increases the susceptibility of bees to N. ceranae infection.

Nosema ceranae in age cohorts of the western honey bee (Apis mellifera).

Nosemaceranae intensity (mean spores per bee) and prevalence (proportion of bees infected in a sample) were analyzed in honey bees of known ages. Sealed brood combs from five colonies were removed, emerging bees were marked with paint, released back into their colonies of origin, and collected as recently emerged (0-3 days old), as house bees (8-11 days old), and as foragers (22-25 days old). Fifty bees from each of the five colonies were processed individually at each collection date for the intensity and prevalence of N. ceranae infection. Using PCR and specific primers to differentiate Nosema species, N. ceranae was found to be the only species present during the experiment. At each collection age (recent emergence, house, forager) an additional sample from the inner hive cover (background bees=BG) of each colony was collected to compare the N. ceranae results of this sampling method, commonly used for Nosema spore quantification, to the samples comprised of marked bees of known ages. No recently emerged bees exhibited infection with N. ceranae. One house bee out of the 250 individuals analyzed (prevalence=0.4%) tested positive for N. ceranae, at an infection level of 3.35×10(6) spores. Infection levels were not statistically different between the recently emerged (mean=0 spores/bee) and house bees (mean=1.34×10(4) spores/bee) (P=0.99). Foragers exhibited the highest prevalence (8.3%) and infection intensity (mean=2.38×10(6) spores/bee), with a range of 0-8.72×10(7) spores in individual bees. The average infection level across all foragers was significantly higher than that of recently emerged bees (P=0.01) and house bees (P=0.01). Finally, the prevalence of Nosema in infected bees was found to be positively correlated with the infection intensity in the sample.

Sub-lethal effects of pesticide residues in brood comb on worker honey bee (Apis mellifera) development and longevity.

BACKGROUND: Numerous surveys reveal high levels of pesticide residue contamination in honey bee comb. We conducted studies to examine possible direct and indirect effects of pesticide exposure from contaminated brood comb on developing worker bees and adult worker lifespan. METHODOLOGY/PRINCIPAL FINDINGS: Worker bees were reared in brood comb containing high levels of known pesticide residues (treatment) or in relatively uncontaminated brood comb (control). Delayed development was observed in bees reared in treatment combs containing high levels of pesticides particularly in the early stages (day 4 and 8) of worker bee development. Adult longevity was reduced by 4 days in bees exposed to pesticide residues in contaminated brood comb during development. Pesticide residue migration from comb containing high pesticide residues caused contamination of control comb after multiple brood cycles and provided insight on how quickly residues move through wax. Higher brood mortality and delayed adult emergence occurred after multiple brood cycles in contaminated control combs. In contrast, survivability increased in bees reared in treatment comb after multiple brood cycles when pesticide residues had been reduced in treatment combs due to residue migration into uncontaminated control combs, supporting comb replacement efforts. Chemical analysis after the experiment confirmed the migration of pesticide residues from treatment combs into previously uncontaminated control comb. CONCLUSIONS/SIGNIFICANCE: This study is the first to demonstrate sub-lethal effects on worker honey bees from pesticide residue exposure from contaminated brood comb. Sub-lethal effects, including delayed larval development and adult emergence or shortened adult longevity, can have indirect effects on the colony such as premature shifts in hive roles and foraging activity. In addition, longer development time for bees may provide a reproductive advantage for parasitic Varroa destructor mites. The impact of delayed development in bees on Varroa mite fecundity should be examined further.

Genetic stock identification of Russian honey bees.

A genetic stock certification assay was developed to distinguish Russian honey bees from other European (Apis mellifera L.) stocks that are commercially produced in the United States. In total, 11 microsatellite and five single-nucleotide polymorphism loci were used. Loci were selected for relatively high levels of homogeneity within each group and for differences in allele frequencies between groups. A baseline sample consisted of the 18 lines of Russian honey bees released to the Russian Bee Breeders Association and bees from 34 queen breeders representing commercially produced European honey bee stocks. Suitability tests of the baseline sample pool showed high levels of accuracy. The probability of correct assignment was 94.2% for non-Russian bees and 93.3% for Russian bees. A neighbor-joining phenogram representing genetic distance data showed clear distinction of Russian and non-Russian honey bee stocks. Furthermore, a test of appropriate sample size showed a sample of eight bees per colony maximizes accuracy and consistency of the results. An additional 34 samples were tested as blind samples (origin unknown to those collecting data) to determine accuracy of individual assignment tests. Only one of these samples was incorrectly assigned. The 18 current breeding lines were represented among the 2009 blind sampling, demonstrating temporal stability of the genetic stock identification assay. The certification assay will be used through services provided by a service laboratory, by the Russian Bee Breeders Association to genetically certify their stock. The genetic certification will be used in conjunction with continued selection for favorable traits, such as honey production and varroa and tracheal mite resistance.

Thrice out of Africa: ancient and recent expansions of the honey bee, Apis mellifera.

We characterized Apis mellifera in both native and introduced ranges using 1136 single-nucleotide polymorphisms genotyped in 341 individuals. Our results indicate that A. mellifera originated in Africa and expanded into Eurasia at least twice, resulting in populations in eastern and western Europe that are geographically close but genetically distant. A third expansion in the New World has involved the near-replacement of previously introduced "European" honey bees by descendants of more recently introduced A. m. scutellata ("African" or "killer" bees). Our analyses of spatial transects and temporal series in the New World revealed differential replacement of alleles derived from eastern versus western Europe, with admixture evident in all individuals.

Hinf-I digestion of cytochrome oxidase I region is not a diagnostic test for A. m. lamarckii

Restriction fragment length polymorphism of whole mitochondrial DNA or PCR amplified mtDNA regions are known to be useful in discriminating among honey bee lineages and also some individual subspecies. In this study, PCR-amplified fragments of cytochrome oxidase I (CO-I) and cytochrome B (Cyt B) of honey bees sampled from different countries (Cyprus, Turkey, Ethiopia, Syria and Egypt) were digested with Hinf I and Bgl II restriction enzymes, respectively. Eastern Europe and Mediterranean honey bee subspecies were separated by the Cyt B/Bgl II analysis, although Hinf I digestion of the CO-I region yielded much finer resolution within different honey bee lineages. Here we report that CO-I/Hinf-I is a discriminative test for the mitochondrial "O" lineage, rather than a diagnostic site for A. m. lamarckii.

Phylogenetic relationships of honey bees (Hymenoptera:Apinae:Apini) inferred from nuclear and mitochondrial DNA sequence data.

Two different genomic regions (ND2 mitochondrial gene and EF1-alpha intron) were PCR amplified, cloned and sequenced for the ten known honey bee species collected within their natural range distribution. DNA sequences were analyzed using parsimony, distance and maximum likelihood methods to investigate phylogenetic relationships within Apis. The phylogenetic analyses strongly supported the basic topology recoverable from morphometric analysis, grouping the honey bees into three major clusters: giant bees (A. dorsata, A. binghami, and A. laboriosa), dwarf bees (A. andreniformis and A. florea), and cavity-nesting bees (A. mellifera, A. cerana, A. koschevnikovi, A. nuluensis, and A. nigrocincta). However, the clade of Asian cavity-nesting bees included paraphyletic taxa. Exemplars of Apis cerana collected from divergent portions of its range were less related to each other than were sympatric A. cerana, A. nuluensis, and A. nigrocincta taxa. Nucleotide sequence divergence between allopatrically distributed western (A. mellifera) and eastern (A. cerana, A. koschevnikovi, A. nigrocincta, and A. nuluensis) cavity-nesting species, around 18% for the mitochondrial gene and 10-15% for the nuclear intron, suggested an earlier divergence for these groups than previously estimated from morphometric and behavioral studies. This latter finding neccessitates reevaluation of the hypothesized origin of extant European, African, and west Asian Apis mellifera. Sequence divergence between A. laboriosa and A. dorsata was consistent with behavioral data and supports the species status of A. laboriosa.

Recent Mediterranean fruit fly (Diptera: Tephritidae) infestations in Florida--a genetic perspective.

Microsatellite and mitochondrial DNA (mtDNA) variability data were used to study infestations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) in Florida in 1997 and 1998. A total of 132 flies collected in monitoring traps or as larvae removed from fruit were examined at three polymorphic mtDNA restriction sites and two microsatellite loci. All of the flies sampled in Florida in 1997 displayed the mitochondrial AAB haplotype and represent a novel introduction of Mediterranean fruit flies into the state. All flies collected in central Florida in 1998 also displayed the AAB haplotype. Microsatellite analysis of these specimens from 1998 detected only alleles that were present in 1997. These results strongly indicate that the 1998 Florida outbreaks were derived from the Florida populations from the previous year. According to our analyses, the Mediterranean region is the most likely source for the 1997 Florida infestation. Flies from a small outbreak in Miami Springs, Dade County, FL, early in 1998 had a different mtDNA haplotype, characterized by the AAC restriction pattern. Microsatellites of these specimens showed significant differences in their allelic distribution from AAB flies, indicating an origin from a separate source population. South America is the most likely source for the Miami Springs flies.