Greg J. Hunt

Multiple Routes of Pesticide Exposure for Honey Bees Living Near Agricultural Fields

Populations of honey bees and other pollinators have declined worldwide in recent years. A variety of stressors have been implicated as potential causes, including agricultural pesticides. Neonicotinoid insecticides, which are widely used and highly toxic to honey bees, have been found in previous analyses of honey bee pollen and comb material. However, the routes of exposure have remained largely undefined. We used LC/MS-MS to analyze samples of honey bees, pollen stored in the hive and several potential exposure routes associated with plantings of neonicotinoid treated maize. Our results demonstrate that bees are exposed to these compounds and several other agricultural pesticides in several ways throughout the foraging period. During spring, extremely high levels of clothianidin and thiamethoxam were found in planter exhaust material produced during the planting of treated maize seed. We also found neonicotinoids in the soil of each field we sampled, including unplanted fields. Plants visited by foraging bees (dandelions) growing near these fields were found to contain neonicotinoids as well. This indicates deposition of neonicotinoids on the flowers, uptake by the root system, or both. Dead bees collected near hive entrances during the spring sampling period were found to contain clothianidin as well, although whether exposure was oral (consuming pollen) or by contact (soil/planter dust) is unclear. We also detected the insecticide clothianidin in pollen collected by bees and stored in the hive. When maize plants in our field reached anthesis, maize pollen from treated seed was found to contain clothianidin and other pesticides; and honey bees in our study readily collected maize pollen. These findings clarify some of the mechanisms by which honey bees may be exposed to agricultural pesticides throughout the growing season. These results have implications for a wide range of large-scale annual cropping systems that utilize neonicotinoid seed treatments.

The Making of a Queen: TOR Pathway Is a Key Player in Diphenic Caste Development

Background Honey bees (Apis mellifera) provide a principal example of diphenic development. Excess feeding of female larvae results in queens (large reproductives). Moderate diet yields workers (small helpers). The signaling pathway that links provisioning to female developmental fate is not understood, yet we reasoned that it could include TOR (target of rapamycin), a nutrient- and energy-sensing kinase that controls organismal growth. Methodology/Principal Findings Here, the role of Apis mellifera TOR (amTOR) in caste determination is examined by rapamycin/FK506 pharmacology and RNA interference (RNAi) gene knockdown. We show that in queen-destined larvae, the TOR inhibitor rapamycin induces the development of worker characters that are blocked by the antagonist FK506. Further, queen fate is associated with elevated activity of the Apis mellifera TOR encoding gene, amTOR, and amTOR gene knockdown blocks queen fate and results in individuals with worker morphology. Conclusions/Significance A much-studied insect dimorphism, thereby, can be governed by the TOR pathway. Our results present the first evidence for a role of TOR in diphenic development, and suggest that adoption of this ancestral nutrient-sensing cascade is one evolutionary pathway for morphological caste differentiation in social insects.

Finding the missing honey bee genes: Lessons learned from a genome upgrade

BackgroundThe first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes.ResultsHere, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data.ConclusionsLessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

Honey bee aggression supports a link between gene regulation and behavioral evolution

A prominent theory states that animal phenotypes arise by evolutionary changes in gene regulation, but the extent to which this theory holds true for behavioral evolution is not known. Because “nature and nurture” are now understood to involve hereditary and environmental influences on gene expression, we studied whether environmental influences on a behavioral phenotype, i.e., aggression, could have evolved into inherited differences via changes in gene expression. Here, with microarray analysis of honey bees, we show that aggression-related genes with inherited patterns of brain expression are also environmentally regulated. There were expression differences in the brain for hundreds of genes between the highly aggressive Africanized honey bee compared with European honey bee (EHB) subspecies. Similar results were obtained for EHB in response to exposure to alarm pheromone (which provokes aggression) and when comparing old and young bees (aggressive tendencies increase with age). There was significant overlap of the gene lists generated from these three microarray experiments. Moreover, there was statistical enrichment of several of the same cis regulatory motifs in promoters of genes on all three gene lists. Aggression shows a remarkably robust brain molecular signature regardless of whether it occurs because of inherited, age-related, or environmental (social) factors. It appears that one element in the evolution of different degrees of aggressive behavior in honey bees involved changes in regulation of genes that mediate the response to alarm pheromone.

Behavioral genomics of honeybee foraging and nest defense

The honeybee has been the most important insect species for study of social behavior. The recently released draft genomic sequence for the bee will accelerate honeybee behavioral genetics. Although we lack sufficient tools to manipulate this genome easily, quantitative trait loci (QTLs) that influence natural variation in behavior have been identified and tested for their effects on correlated behavioral traits. We review what is known about the genetics and physiology of two behavioral traits in honeybees, foraging specialization (pollen versus nectar), and defensive behavior, and present evidence that map-based cloning of genes is more feasible in the bee than in other metazoans. We also present bioinformatic analyses of candidate genes within QTL confidence intervals (CIs). The high recombination rate of the bee made it possible to narrow the search to regions containing only 17–61 predicted peptides for each QTL, although CIs covered large genetic distances. Knowledge of correlated behavioral traits, comparative bioinformatics, and expression assays facilitated evaluation of candidate genes. An overrepresentation of genes involved in ovarian development and insulin-like signaling components within pollen foraging QTL regions suggests that an ancestral reproductive gene network was co-opted during the evolution of foraging specialization. The major QTL influencing defensive/aggressive behavior contains orthologs of genes involved in central nervous system activity and neurogenesis. Candidates at the other two defensive-behavior QTLs include modulators of sensory signaling (Am5HT7 serotonin receptor, AmArr4 arrestin, and GABA-B-R1 receptor). These studies are the first step in linking natural variation in honeybee social behavior to the identification of underlying genes.

Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee

SummaryThe polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 1∶1 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.

Genetic determinants of honey bee foraging behaviour

The amount of pollen stored in honey bee, Apis mellifera, colonies is a selectable trait. Five generations of two-way selection resulted in high and low strains that differed more than six-fold in quantities of stored pollen. Comparisons with hybrid crosses suggested that colony-level, high pollen-hoarding behaviour is inherited as a recessive trait. Colony levels of stored honey, however, showed an over-dominant pattern, with hybrid colonies storing significantly more honey than either of the selected strains. Controlled studies of individual foraging behaviour revealed the same patterns of inheritance at the individual level: high-strain workers specialized on pollen foraging, low-strain workers on nectar, and hybrid workers demonstrated a significantlt greater nectar-collecting bias than workers of the low strain. Genomic mapping studies of colony-level pollen hoarding and individual foraging behaviour have revealed two genomic regions of the honey bee that contain major quantitative trait loci that explain a large portion of the observed variance in pollen hoarding and foraging behaviour of the two strains. The effects of major genes on within- and between-colony variation in individual foraging behaviour are discussed in the context of conducting and interpreting empirical tests of foraging theory.

Genomic survey of the ectoparasitic mite Varroa destructor, a major pest of the honey bee Apis mellifera

BackgroundThe ectoparasitic mite Varroa destructor has emerged as the primary pest of domestic honey bees (Apis mellifera). Here we present an initial survey of the V. destructor genome carried out to advance our understanding of Varroa biology and to identify new avenues for mite control. This sequence survey provides immediate resources for molecular and population-genetic analyses of Varroa-Apis interactions and defines the challenges ahead for a comprehensive Varroa genome project.ResultsThe genome size was estimated by flow cytometry to be 565 Mbp, larger than most sequenced insects but modest relative to some other Acari. Genomic DNA pooled from ~1,000 mites was sequenced to 4.3× coverage with 454 pyrosequencing. The 2.4 Gbp of sequencing reads were assembled into 184,094 contigs with an N50 of 2,262 bp, totaling 294 Mbp of sequence after filtering. Genic sequences with homology to other eukaryotic genomes were identified on 13,031 of these contigs, totaling 31.3 Mbp. Alignment of protein sequence blocks conserved among V. destructor and four other arthropod genomes indicated a higher level of sequence divergence within this mite lineage relative to the tick Ixodes scapularis. A number of microbes potentially associated with V. destructor were identified in the sequence survey, including ~300 Kbp of sequence deriving from one or more bacterial species of the Actinomycetales. The presence of this bacterium was confirmed in individual mites by PCR assay, but varied significantly by age and sex of mites. Fragments of a novel virus related to the Baculoviridae were also identified in the survey. The rate of single nucleotide polymorphisms (SNPs) in the pooled mites was estimated to be 6.2 × 10-5per bp, a low rate consistent with the historical demography and life history of the species.ConclusionsThis survey has provided general tools for the research community and novel directions for investigating the biology and control of Varroa mites. Ongoing development of Varroa genomic resources will be a boon for comparative genomics of under-represented arthropods, and will further enhance the honey bee and its associated pathogens as a model system for studying host-pathogen interactions.

LINKAGE ANALYSIS OF SEX DETERMINATION IN THE HONEY BEE (APIS MELLIFERA)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait ‘low brood-viability’ resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.

Confirmation of QTL Effects and Evidence of Genetic Dominance of Honeybee Defensive Behavior: Results of Colony and Individual Behavioral Assays

The stinging and guarding components of the defensive behavior of European, Africanized, hybrid, and backcross honeybees (Apis mellifera L.) were compared and analyzed at both colony and individual levels. Hybrid and Africanized backcross colonies stung as many times as Africanized ones. European backcross colonies stung more than European bees but not as many times as Africanized or Africanized backcross colonies. The degree of dominance for the number of times that worker bees stung a leather patch was estimated to be 84.3%, 200.8%, and 145.8% for hybrid, backcross European, and backcross Africanized colonies, respectively. Additionally, both guards at the colony entrance and fast-stinging workers of one European backcross colony had a significantly higher frequency of an Africanized DNA marker allele, located near “sting1,” a QTL previously implicated in stinging behavior at the colony level. However, guards and fast-stinging bees from a backcross to the Africanized parental colony did not differ from control bees in their frequency for the Africanized and European markers, as would be expected if large genetic dominance effects for sting1 exist. These results support the hypothesis that genetic dominance influences the defensive behavior of honeybees and confirm the effect of sting1 on the defensiveness of individual worker bees.