Diverse origins of tetracycline resistance in the honey bee bacterial pathogen Paenibacillus larvae

Paenibacillus larvae is the causative agent of the important honey bee larval disease American Foulbrood (AFB). This pathogen has been treated in bee colonies by a single registered antibiotic, oxytetracycline (OTC), for fifty years. Recently, widespread resistance to OTC has been reported. In this study, the degree of antibiotic resistance was contrasted with DNA sequence variation for 125 P. larvae isolates collected in North America. Resistance was uncorrelated with bacterial haplotype, suggesting either that resistance has evolved multiple times in P. larvae or that resistance involves recent horizontal transfer via a non-genomic (e.g., plasmid or conjugal transposon) route. The recency of OTC resistance in P. larvae across this broad survey area underscores the need to manage foulbrood infections carefully and to monitor populations for resistance.     

Evolutionary history of the honey bee Apis mellifera inferred from mitochondrial DNA analysis

Variability of mitochondrial DNA (mtDNA) of the honey bee Apis mellifera L. has been investigated by restriction and sequence analyses on a sample of 68 colonies from ten different subspecies. The 19 mtDNA types detected are clustered in three major phylogenetic lineages. These clades correspond well to three groups of populations with distinct geographical distributions: branch A for African subspecies (intermissa, monticola, scutellata, andansonii and capensis), branch C for North Mediterranean subspecies (caucasica, carnica and ligustica) and branch M for the West European populations (mellifera subspecies). These results partially confirm previous hypotheses based on morphometrical and allozymic studies, the main difference concerning North African populations, now assigned to branch A instead of branch M. The pattern of spatial structuring suggests the Middle East as the centre of dispersion of the species, in accordance with the geographic areas of the other species of the same genus. Based on a conservative 2% divergence rate per Myr, the separation of the three branches has been dated at about 1 Myr BP.     

Mitochondrial DNA variation in Moroccan and Spanish honey bee populations

The mitochondrial DNAs of 192 Moroccan and 173 Spanish honey bee colonies were characterized by a rapid test involving the restriction by DraI of a PCR-fragment of the COI-COII region. In Morocco, we found eight haplotypes, all characteristic of the African (A) lineage, suggesting that most if not all the maternal lineages of the colonies repeatedly imported from Europe over the last 150 years have not contributed mitochondrial genomes to the local population. Using two new genetic distances analogous to the shared allele distance defined for nuclear genes, we showed that Morocco was most probably colonized by two sublineages, one from the north-east and the other one from the south of the country and that the contact zone between them extends along both sides of the Atlas range. In Spain, we found eight haplotypes characteristic of lineage A (six in common with Morocco) and four of lineage M (the West European lineage). The distribution of haplotypes of both lineages forms a gradient with c. 10% of lineage M in the south of Spain (Seville) and up to 100% in the north (San Sebastian). Three hypotheses are presented to explain the large differences of haplotype frequencies between Moroccan and lineage A Spanish colonies: a non-Moroccan origin of lineage A in Spain, an ancient Moroccan origin or a recent Moroccan origin with a rapid shift of haplotype frequencies due to a founder effect.     

Micro-CT imaging of denatured chitin by silver to explore honey bee and insect pathologies

Background

Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term “denatured chitin” calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished.

Methodology/Principal Findings

A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT). Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi), at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment.

Conclusions/Significance

The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may provide an additional tool for research on CCD.     

Multiple virus infections in the honey bee and genome divergence of honey bee viruses

Using uniplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV), and described the detection of mixed virus infections in bees from these colonies. We report for the first time that individual bees can harbor four viruses simultaneously. We also developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses. The feasibility and specificity of the multiplex RT-PCR assay suggests that this assay is an effective tool for simultaneous examination of mixed virus infections in bee colonies and would be useful for the diagnosis and surveillance of honey bee viral diseases in the field and laboratory. Phylogenetic analysis of putative helicase and RNA-dependent RNA polymerase (RdRp) encoded by viruses reveal that DWV and SBV fall into a same clade, whereas KBV and BQCV belong to a distinct lineage with other picorna-like viruses that infect plants, insects and vertebrates. Results from field surveys of these viruses indicate that mixed infections of BQCV, DWV, KBV, and SBV in the honey bee probably arise due to broad geographic distribution of viruses.     

DNA RFLPs at a highly polymorphic locus distinguish European and African subspecies of the honey bee Apis mellifera L. and suggest geographical origins of New World honey bees

A highly polymorphic locus in the honey bee, Apis mellifera L., was detected with genomic probe pB178. Eighty-five alleles, consisting of Msp I and Dde I RFLPs, were found among the Old and New World bees tested. Forty-one Msp I and 43 Dde I restriction fragment patterns, or variants, were identified. Variants and alleles were discontinuously distributed in Old World European and African subspecies. Principal coordinate analysis of the genetic distances between the alleles resulted in the identification of three distinct groups corresponding to three groups of honey bee races with historically different geographical distributions: east European A. m. ligustica and A. m. caucasica ; west European A. m. mellifera ; and South African A. m. scutellata . The clustering of alleles into these groups is consistent with previous honey bee phylogeographic studies, employing other nuclear and mitochondrial DNA markers, which in part support the evolutionary history of the honey bee hypothesized by Ruttner based on morphometric and allozyme data. The majority of alleles in bees from the USA grouped with those found in east European bees, while other alleles grouped with alleles found in A. m. mellifera . While the majority of the alleles in neotropical bees grouped with or were identical to African alleles, other alleles grouped with alleles found in A. m. mellifera, A. m. ligustica , and A. m. caucasica . Clues to the ancestry of neotropical bees may be found in the identification of alleles that were identical or more similar to alleles found in South African and west European bees; evidence for west European ancestry has been suggested using other taxonomic characters that were not unique to west European bees. Both west European and African alleles were found in individual neotropical colonies, which may indicate that honey bee subspecies which evolved allopatrically have hybridized in the human-assisted extension of their original geographical ranges.     

昆虫卵黄原蛋白功能多效性：以蜜蜂为例

卵黄原蛋白是昆虫卵黄发生的关键物质。随着RNA干扰技术在功能基因研究中的应用和发展,昆虫卵黄原蛋白特别是蜜蜂卵黄原蛋白被发现具有气候适应、激活卵巢、生殖竞争、劳动力分化、行为构建、延长寿命、转化食物等多种功能,因此又被称为多效性蛋白。许多针对蜜蜂卵黄原蛋白功能和调控机制的假说也相继提出,如“交互抑制假说”、“循环反馈机制”、“卵黄原蛋白转化蛋白胨机制”,表明蜜蜂卵黄原蛋白已经由雌虫繁殖过程的一个下游因子上升为高等社会性昆虫主要生命周期的调控子,不仅极大地促进了昆虫社会生物学的研究,对其他具有复杂繁殖行为的昆虫的研究也具有重要的参考意义。针对近年来这一领域相继取得的重大突破,本文以蜜蜂为例介绍了昆虫卵黄原蛋白功能多效性的研究进展,包括卵黄原蛋白的理化性质与分子进化,发生与调控,功能多效性及其研究方法等     

Responsiveness of honey bee (Apis mellifera L.) corpora allata to allatoregulatory peptides from four insect species

Five neuropeptides with known allatotropic or allatostatic activity in other insect species were examined for their effects on honey bee corpora allata. Using an in vitro radiochemical assay, we assessed the ability of these peptides to affect the biosynthesis of juvenile hormone III and its immediate precursor methyl farnesoate, as well as their effects on the conversion of methyl farnesoate into juvenile hormone. None of the allatostatins tested affected JH biosynthesis during the last larval instar of honey bee workers. Manduca sexta allatotropin, however, stimulated JH biosynthesis in a stage-specific and dose-dependent manner. Analysis of intraglandular contents of juvenile hormone and its precursor revealed that the allatotropin significantly increased JH precursor but did not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for early fifth-instar worker larvae. Studies also indicated that the allatotropic effect was reversible at the level of methyl farnesoate production.     

Polymorphic DNA sequences of the fungal honey bee pathogen Ascosphaera apis

The pathogenic fungus Ascosphaera apis is ubiquitous in honey bee populations. We used the draft genome assembly of this pathogen to search for polymorphic intergenic loci that could be used to differentiate haplotypes. Primers were developed for five such loci, and the species specificities were verified using DNA from nine closely related species. The sequence variation was compared among 12 A.?apis isolates at each of these loci, and two additional loci, the internal transcribed spacer of the ribosomal RNA (ITS) and a variable part of the elongation factor 1α (Ef1α). The degree of variation was then compared among the different loci, and three were found to have the greatest detection power for identifying A.?apis haplotypes. The described loci can help to resolve strain differences and population genetic structures, to elucidate host-pathogen interaction and to test evolutionary hypotheses for the world's most important pollinator: the honey bee and one of its most common pathogens.     

Self-assemblage formation in a social insect: the protective curtain of a honey bee swarm

Summary. This paper considers a little-studied topic in the biology of social insects: the formation of self-assemblages. It focuses on the mechanisms whereby the outermost workers in a bivouacked swarm of honey bees, when rained upon, form a water repellent curtain of bees over the swarm cluster. Specifically, we analyzed how the worker bees in the mantle of a swarm cluster adjust their body orientation, wing spread, and inter-individual spacing to form a protective curtain when wetted. When warm and dry, the mantle bees orient their bodies weakly with respect to gravity, do not tuck their heads under adjacent bees, have high variability in wing spread, and space themselves widely. In contrast, when warm and wet, the mantle bees orient uniformly with head upward, tuck their heads beneath the abdomens of bees above, hold their wings together, and press tightly together. This produces a surface that closely resembles a tiled roof. When cool and dry, the mantle bees generally orient their bodies with head upward, press their heads into the interior of the cluster, hold their wings wide apart, and draw close together. We also examined the age distribution of the mantle bees. Older bees are more likely than younger bees to be found in the mantle of a swarm, perhaps because younger bees are more important than older bees to colony survival after swarming and so occupy a more sheltered position in a swarm. Finally, we tested whether swarm clusters that have formed a protective curtain shed water more effectively than ones that have not formed a curtain. We found that this is the case.Received 28 November 2003; revised 29 February 2004; accepted 11 April 2004.     

Metabolomic analysis of pollen from honey bee hives and from canola flowers

Background and aims

Pollen is essential for successful plant reproduction and critical for plant-pollinator mutualisms, as pollen is essential larval nutrition. However, we understand very little about the chemical constituents of pollen leading us to this exploratory study characterizing plant and beehive pollen.

Methods

We performed a metabolomics assay of canola flower pollen and beehive pollen.

Results and discussion

The metabolome of canola pollen is affected by irrigation showing differences in lipids and non-polar secondary metabolites. Metabolome of beehive pollen is affected by plant source showing differences in pentose sugars, myo-inositol and furanose. Further research is needed to document the nutritional bases of plant-pollinator mutualism.

     

Purification and characterization of honey bee vitellogenin

A protocol has been developed for the purification of vitellogenin from the honey bee, Apis mellifera. Purification allows for the first characterization of a vitellogenin from the large order Hymenoptera. Hymenopteran vitellogenins are unusual among insect vitellogenins in that they contain only one type of apoprotein. The honey bee vitellogenin was isolated from hemolymph of honey bee queens by a combination of density gradient ultracentrifugation, ion-exchange chromatography, and affinity chromatography. The native vitellogenin particle is a very high density glycolipoprotein containing approximately 91% protein, 7% lipid, and 2% carbohydrate. Phospholipid and diacylglycerol are the major lipid components. The equilibrium density (1.28 g/ml) is the same as that for Manduca sexta vitellogenin, which contains a much higher proportion of lipid. The covalently bound carbohydrate moiety of the particle is high in mannose. The amino acid composition of vitellogenin is similar to those of vitellogenins from other insect species. The N-terminal amino acid sequence of the apoprotein was determined, the first such sequence for any insect vitellogenin. When analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis, A. mellifera vitellogenin resolved into a single band with an apparent Mr of 180,000. Gel filtration under reducing and native conditions yielded estimated Mr values of about 300,000.     

Estimation of intracolonial worker relationship in a honey bee colony (Apis mellifera L.) using DNA fingerprinting

Summary The number and frequencies of subfamilies in a honey bee colony were determined by DNA fingerprinting. Queen and brood samples were taken from three colonies with artificially inseminated queens and from one colony with a naturally mated queen. UsingHae III restriction enzyme and (GATA)₄ oligonucleotide, the number of subfamilies in the colonies with artificially inseminated queens corresponded with the number of drones used for insemination. In the colony with the naturally mated queen, 12 subfamilies were found in a random sample of 104 workers. Considering that subfamily frequencies range from 1 to 26%, introcolonial worker relationship was estimated to be 0.328, corresponding to a genetical effective number of 6.4 matings.     

Queen substances from the abdomen of the honey bee queen

Summary The secretion of the mandibular glands of a honey bee queen enables the worker bees to react to the presence of their queen. Extirpating the mandibular glands of the queen does not prevent that she is accepted by her colony. Hitherto this was attributed to contamination of the queen's body by mandibular gland substances during or preceding the extirpation. When, however, these glands are extirpated before they have secreted any material and the queens are inseminated artificially, the colonies still accept these queens. A normal-sized retinue, the absence of emergency cell building and the absence of activation of the worker's ovaries indicate that such a queen is still able to maintain her social position. This supports Verheijen-Voogd's (1959) conclusion that the queen's influence on her workers has a behavioural basis (chemoreception) rather than a biochemical one.Laboratory experiments reveal that apart from the mandibular gland substances other queen pheromones are produced in glands on the abdomen, most probably in the glands described by Renner and Baumann (1964).     

Ageing in a eusocial insect: molecular and physiological characteristics of life span plasticity in the honey bee

Commonly held views assume that ageing, or senescence, represents an inevitable, passive, and random decline in function that is strongly linked to chronological age. In recent years, genetic intervention of life span regulating pathways, for example, in Drosophila as well as case studies in non-classical animal models, have provided compelling evidence to challenge these views.Rather than comprehensively revisiting studies on the established genetic model systems of ageing, we here focus on an alternative model organism with a wild type (unselected genotype) characterized by a unique diversity in longevity - the honey bee.Honey bee (Apis mellifera) life span varies from a few weeks to more than 2 years. This plasticity is largely controlled by environmental factors. Thereby, although individuals are closely related genetically, distinct life histories can emerge as a function of social environmental change.Another remarkable feature of the honey bee is the occurrence of reverted behavioural ontogeny in the worker (female helper) caste. This behavioural peculiarity is associated with alterations in somatic maintenance functions that are indicative of reverted senescence. Thus, although intraspecific variation in organismal life span is not uncommon, the honey bee holds great promise for gaining insights into regulatory pathways that can shape the time-course of ageing by delaying, halting or even reversing processes of senescence. These aspects provide the setting of our review.We will highlight comparative findings from Drosophila melanogaster and Caenorhabditis elegans in particular, and focus on knowledge spanning from molecular- to behavioural-senescence to elucidate how the honey bee can contribute to novel insights into regulatory mechanisms that underlie plasticity and robustness or irreversibility in ageing.     

Large scale sex typing of ostriches using DNA extracted from feathers

Background  

Ostrich (Struthio camelus) breeds have been gaining increasing significance around the world. The large-scale sex determination of chicks is an important task in the development of these breeds. To date, two PCR-based methods have been established for ostrich sex typing, neither of them intended for large-scale analyses. Here, we report on a protocol adapted to carry out large-scale gender scoring using DNA obtained from chick feathers.