Molecular characterization of Nosema bombi (Microsporidia : Nosematidae) and a note on its sites of infection in Bombus terrestris (Hymenoptera : Apoidea)

Investigations of queen, worker and male bumble bees (Bombus terrestris) showed that all individuals became infected with Nosema bombi. Infections were found in Malpighian tubules, thorax muscles, fat body tissue and nerve tissue, including the brain. Ultrastructural studies revealed thin walled emptied spores in host cell cytoplasm interpreted as autoinfective spores, besides normal spores (environmental spores) intended for parasite transmission between hosts. The nucleotide sequence of the gene coding for the small subunit rRNA (SSU-rRNA) from Microsporidia isolated from B. terrestris, B. lucorum, and B. hortorum were identical, providing evidence that N. bombi infects multiple hosts. The sequence presented here (GenBank Accession no AY008373) is different from an earlier submission to GenBank (Accession no U26158) of a partial sequence of the same gene based on material collected from B. terrestris. It still remains to be investigated if there is species diversity among Microsporidia found in bumble bees.

A sting in the spit: widespread cross-infection of multiple RNA viruses across wild and managed bees

Declining populations of bee pollinators are a cause of concern, with major repercussions for biodiversity loss and food security. RNA viruses associated with honeybees represent a potential threat to other insect pollinators, but the extent of this threat is poorly understood. This study aims to attain a detailed understanding of the current and ongoing risk of emerging infectious disease (EID) transmission between managed and wild pollinator species across a wide range of RNA viruses. Within a structured large-scale national survey across 26 independent sites, we quantify the prevalence and pathogen loads of multiple RNA viruses in co-occurring managed honeybee (Apis mellifera) and wild bumblebee (Bombus spp.) populations. We then construct models that compare virus prevalence between wild and managed pollinators. Multiple RNA viruses associated with honeybees are widespread in sympatric wild bumblebee populations. Virus prevalence in honeybees is a significant predictor of virus prevalence in bumblebees, but we remain cautious in speculating over the principle direction of pathogen transmission. We demonstrate species-specific differences in prevalence, indicating significant variation in disease susceptibility or tolerance. Pathogen loads within individual bumblebees may be high and in the case of at least one RNA virus, prevalence is higher in wild bumblebees than in managed honeybee populations. Our findings indicate widespread transmission of RNA viruses between managed and wild bee pollinators, pointing to an interconnected network of potential disease pressures within and among pollinator species. In the context of the biodiversity crisis, our study emphasizes the importance of targeting a wide range of pathogens and defining host associations when considering potential drivers of population decline.

Queen-worker conflict over male production and sex ratio in a facultatively polyandrous bumble bee, Bombus hypnorum: the consequences of nest usurpation.

Evolutionary conflicts among social hymenopteran nestmates are theoretically likely to arise over the production of males and the sex ratio. Analysis of these conflicts has become an important focus of research into the role of kin selection in shaping social traits of hymenopteran colonies. We employ microsatellite analysis of nestmates of one social hymenopteran, the primitively eusocial and monogynous bumblebee Bombus hypnorum, to evaluate these conflicts. In our 14 study colonies, B. hypnorum queens mated between one and six times (arithmetic mean 2.5). One male generally predominated, fathering most of the offspring, thus the effective number of matings was substantially lower (1–3.13; harmonic mean 1.26). In addition, microsatellite analysis allowed the detection of alien workers, those who could not have been the offspring of the queen, in approximately half the colonies. Alien workers within the same colony were probably sisters. Polyandry and alien workers resulted in high variation among colonies in their sociogenetic organization. Genetic data were consistent with the view that all males (n = 233 examined) were produced by a colony’s queen. Male parentage was therefore independent of the sociogenetic organization of the colony, suggesting that the queen, and not the workers, was in control of the laying of male-destined eggs. The population-wide sex ratio (fresh weight investment ratio) was weakly female biased. No evidence for colony-level adaptive sex ratio biasing could be detected.

Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs

Cryptic species diversity is thought to be common within the class Insecta, posing problems for basic ecological and population genetic studies and conservation management. Within the temperate bumble bee (Bombus spp.) fauna, members of the subgenus Bombus sensu stricto are amongst the most abundant and widespread. However, their species diversity is controversial due to the extreme difficulty or inability morphologically to identify the majority of individuals to species. Our character-based phylogenetic analyses of partial CO1 (700 bp) from 39 individuals spread across their sympatric European ranges provided unequivocal support for five taxa (3-22 diagnostic DNA base pair sites per species). Inclusion of 20 Irish specimens to the dataset revealed >= 2.3% sequence divergence between taxa and 200 m) whilst B. cryptarum was relatively more abundant at higher altitudes. Bombus magnus was rarely encountered at urban sites. Both B. lucorum and B. terrestris are nowadays reared commercially for pollination and transported globally. Our RFLP approach to identify native fauna can underpin ecological studies of these important cryptic species as well as the impact of commercial bumble bees on them.

Specific and sensitive detection of Nosema bombi (Microsporidia : Nosematidae) in bumble bees (Bombus spp.; Hymenoptera : Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323 bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 107 down to 10 spores diluted in 100 mu l of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of similar to 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to b more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy. (c) 2005 Elsevier Inc. All rights reserved.

Edaphic factors affecting the toxicity and accumulation of arsenate in the earthworm Lumbricus terrestris

The toxicity and accumulation of arsenate was determined in the earthworm Lumbricus terrestris in soil from different layers of a forest profile. Toxicity increased fourfold between 2 and 10 d. Edaphic factors (pH, soil organic matter, and depth in soil profile) also affected toxicity with a three fold decrease in the concentration that causes 50% mortality with increasing depth in soil (from 0-70 mm to 500-700 mm). In a 4-d exposure study, there was no evidence of arsenic bioconcentration in earthworm tissue, although bioaccumulation was occurring. There was a considerable difference in tissue residues between living and dead earthworms, with dead worms having higher concentrations. This difference was dependent on both soil arsenate concentration and on soil type. Over a wide range of soil arsenate concentrations, earthworm arsenic residues are homeostatically maintained in living worms, but this homeostasis breaks down during death. Alternatively, equilibration with soil residues may occur via accumulation after death. In long-term accumulation studies in soils dosed with a sublethal arsenate concentration (40 μg/g dry weight), bioconcentration of arsenate did not occur until day 12, after which earthworm concentrations rose steadily above the soil concentration, with residues in worms three fold higher than soil concentrations by the termination of the study (23 d). This bioconcentration only occurred in depurated worms over the time period of the study. Initially, depurated worms had lower arsenic concentrations than undepurated until tissue concentrations were equivalent to the soil concentration. Once tissue concentration was greater than soil concentration, depurated worms had higher arsenic residues than undepurated.

PEPTIDES RELATED TO THE DIPLOPTERA-PUNCTATA ALLATOSTATINS IN NONARTHROPOD INVERTEBRATES - AN IMMUNOCYTOCHEMICAL SURVEY

The allatostatins are a family of peptides isolated originally from the cockroach, Diploptera punctata. Related peptides have been identified in Periplaneta americana and the blowfly, Calliphora vomitoria. These peptides have been shown to be potent inhibitors of juvenile hormone synthesis in these species. A peptide inhibitor of juvenile hormone biosynthesis has also been isolated from the moth, Manduca sexta; however, this peptide has no structural homology with the D. punctata-type allatostatins. Investigations of the phylogeny of the D. punctata allatostatin peptide family have been started by examining a number of nonarthropod invertebrates for the presence of allatostatin-like molecules using immunocytochemistry with antisera directed against the conserved C-terminal region of this family. Allatostatin-like immunoreactivity (ALIR) was demonstrated in the nervous systems of Hydra oligactis (Hydrozoa), Moniezia expansa (Cestoda), Schistosoma mansoni (Trematoda), Artioposthia triangulata (Turbellaria), Ascaris suum (Nematoda), Lumbricus terrestris (Oligochaeta), Limax pseudoflavus (Gastropoda), and Eledone cirrhosa (Cephalopoda). ALIR could not be demonstrated in Ciona intestinalis (Ascidiacea). These results suggest that molecules related to the allatostatins may play an important role in nervous system function in many invertebrates as well as in insects and that they also have an ancient evolutionary lineage. (C) 1994 Wiley-Liss, Inc.

Degradation of the polycyclic aromatic hydrocarbon (PAH) fluorene is retarded in a Scots pine ectomycorrhizosphere

Polycyclic aromatic hydrocarbons (PAHs) are an important class of persistent organic pollutants (POPs) in the environment and accumulate in forest soils. These soils are often dominated by ectomycorrhizal (EcM) roots, but little is known about how EcM fungi degrade PAHs, or the overall effect of field colonized EcM roots on the fate of PAHs. The ability of eight EcM fungi to degrade PAHs in liquid culture spiked with ¹⁴C labelled PAHs was investigated. Microcosms were used to determine the impact of naturally colonized mycorrhizal pine seedlings on PAH mineralization and volatilization. Only two EcM fungi (Thelephora terrestris and Laccaria laccata) degraded at least one PAH and none were able to mineralize the PAHs in pure culture. Where degradation occurred, the compounds were only mono-oxygenated. EcM pine seedlings did not alter naphthalene mineralization or volatilization but retarded fluorene mineralization by 35% compared with unplanted, ectomycorrhizosphere soil inoculated, microcosms. The EcM fungi possessed limited PAH degrading abilities, which may explain why EcM dominated microcosms retarded fluorene mineralization. This observation is considered in relation to the 'Gadgil-effect', where retarded litter decomposition has been observed in the presence of EcM roots. © New Phytologist (2004).