The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

Sub-fossil Insects and ecosystem dynamics in wetlands; implications for biodiversity and conservation.

We review the uses of fossil insects, particularly Coleoptera (beetles) and Chironomidae (non-biting midges) from ancient deposits to inform the study of wetland ecosystems and their ecological and restoration processes. In particular, we focus on two contrasting ecosystems, drawing upon research undertaken by us on British raised mire peats and shallow lake systems, one an essentially terrestrial ecosystem, the other aquatic, but in which wetland insects play an important and integral part. The study of raised mire peats suggests that faunal stability is a characteristic of these wetland systems, over what appear to be extensive periods of time (up to several millennia), whilst studies of shallow lake ecosystems over recent timescales indicates that faunal instability appears to be more common, usually driven by increasing eutrophication. Drawing upon a series of fossil Coleoptera records spanning several thousand years from Hatfield Moors, south Yorkshire, we reconstruct in some detail the mire’s ontogeny and fluctuations in site hydrology and vegetation cover, illustrating the intimate association between substrate, topography and peat development. A comparison between fossil and modern beetle populations indicates that the faunal characteristics of this mire and its adjacent neighbour, Thorne Moors, become established during the early phases of peat development, including its rare endemics, and that the faunal biodiversity on the sites today is dictated by complex site histories. The over-riding characteristic of these faunas is of stability over several thousand years, which has important implications for the restoration of degraded sites, especially those where refugial areas are limited. In contrast, analyses of fossil Chironomidae from shallow lakes allow researchers to track changes in limnological status and while attempts have been made to reconstruct changes in nutrient levels quantitatively, the chironomids respond indirectly to such changes, typically mediated through complex ecosystem dynamics such as changes in fish and/or macrophyte communities. These changes are illustrated via historic chironomid stratigraphies and diversity indices from a range of shallow lakes located across Britain: Slapton Ley, Frensham Great Pond, Fleet Pond, Kyre Pool and Barnes Loch. These sites have shown varying degrees of eutrophication over recent timescales which tends to be associated with a decline in chironomid diversity. While complex functional processes exist within these ecosystems, our evidence suggests that one of the key drivers in the loss of shallow lake chironomid diversity appears to be the loss of aquatic macrophytes. Overall, while chironomids do show a clear response to altered nutrient regimes, multi-proxy reconstructions are recommended for a clear interpretation of past change. We conclude that if we are to have a better understanding of biota at the ecosystem level we need to know more of the complex interactions between different insect groups as well as with other animal and plant communities. A palaeoecological approach is thus crucial in order to assess the role of insect groups in ecosystem processes, both in the recent past and over long time scales, and is essential for wetland managers and conservation organisations involved in long term management and restoration of wetland systems.

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling. (C) 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Organisation of the nervous system in the Acoela: an immunocytochemical study

In order to broaden the information about the organisation of the nervous system in taxon Acoela, an immunocytochemical study of an undetermined Acoela from Cape Kartesh, Faerlea glomerata, Avagina incola and Paraphanostoma crassum has been performed. Antibodies to 5-HT and the native flatworm neuropeptide GYIRFamide were used. As in earlier studies, the pattern of 5-HT immunoreactivity revealed an anterior structure composed mainly of commissures, a so-called commissural brain. Three types of brain shapes were observed. No regular orthogon was visualised. GYIRFamide immunoreactive cell clusters were observed peripherally to the 5-HT immunoreactive commissural brain. Staining with anti-GYIRFamide revealed more nerve processes than did staining with anti-FMRFamide. As no synapomorphies were found in the organisation of the nervous system of the Acoela and that of the Platyhelminthes, the results support the view that the Acoela is not a member of the Platyhelminthes. (C) 2001 Harcourt Publishers Ltd.

Nosema ceranae has infected Apis mellifera in Europe since at least 1998 and may be more virulent than Nosema apis

Nosema ceranae, a microsporidian formerly regarded as confined to its Asiatic host Apis cerana, has recently been shown to parasitise Apis mellifera and to have spread throughout most of the world in the past few years. Using a temporal sequence of N = 28 Nosema isolates from Finland from 1986-2006, we now find (i) that N. ceranae has been present in Europe since at least 1998 and (ii) that it has increased in frequency across this time period relative to Nosema apis, possibly leading to higher mean spore loads per bee. We then present results of a single laboratory infection experiment in which we directly compare the virulence of N. apis with N. ceranae. Though lacking replication, our results suggest (iii) that both parasites build up to equal numbers per bee by day 14 post infection but that (iv) N. ceranae induces significantly higher mortality relative to N. apis.

Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera

The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martin, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N = 9 isolates gave N. apis RFLPs and sequences, N = 20 isolates gave N. ceranae RFLPs and sequences; 100%, correct classification). We then employed the method to analyze N = 115 isolates from across the world. Our data, combined with N = 36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping. (c) 2007 Elsevier Inc. All rights reserved.

Morphological and molecular characters reveal differentiation in a Neotropical social bee, Melipona beecheii (Apidae : Meliponini)

Morphometrics and DNA microsatellites were used to analyse the genetic structure of populations of the stingless bee M. beecheii from two extremes of its geographic range. The results showed that populations from Costa Rica and Yucatan exhibit substantial phenotypic and molecular differentiation. Bees from Yucatan were smaller and paler than those from Costa Rica. The value of multilocus F-ST = 0.280 (P <0.001) confirmed that there were significant molecular genetic differences between the two populations. Populations showed significant deviation from Hardy Weinberg equilibrium and the values of FIS (the inbreeding coefficient) were positive for Costa Rica = 0.416 and the Yucatan Peninsula = 0.193, indicating a lack of heterozygotes in both populations possibly due to inbreeding. The DNA sequence of 678 bp of the mitochondrial gene COI differed between populations by 1.2%. The results of this study should be considered in conservation programmes, particularly with regard to the movement of colonies between regions.

An optimized method for the generation of AFLP markers in a stingless bee (Melipona quadrifasciata) reveals a high degree of intracolonial genetic polymorphism

This study describes an optimized protocol for the generation of Amplified Fragment Length Polymorphism (AFLP) markers in a stingless bee. Essential modifications to standard protocols are a restriction enzyme digestion (EcoRI and Tru1I) in a two-step procedure, combined with a touchdown program in the selective PCR amplification step and product labelling by incorporation of alpha[P-33]dATP. In an analysis of 75 workers collected from three colonies of Melipona quadrifasciata we obtained 719 markers. Analysis of genetic variability revealed that on average 32% of the markers were polymorphic within a colony. Compared to the overall percentage of polymorphism (44% of the markers detected in our bee samples), the observed rates of within-colony polymorphism are remarkably high, considering that the workers of each colony were all of spring of a singly mated queen.

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.

Male mating behaviour and mating systems of bees: an overview

Considerable interspecific diversity exists among bees in the rendezvous sites where males search for females and in the behaviours employed by males in their efforts to secure matings. I present an evolutionary framework in which to interpret this variation, and highlight the importance for the framework of (i) the distribution of receptive ( typically immediate post-emergence) females, which ordinarily translates into the distribution of nests, and (ii) the density of competing males. Other than the highly polyandrous honey bees ( Apis), most female bees are thought to be monandrous, though genetic data with which to support this view are generally lacking. Given the opportunity, male bees are typically polygamous. I highlight intraspecific diversity in rendezvous site, male behaviour and mating system, which is in part predicted from the conceptual framework. Finally, I suggest that inbreeding may be far more widespread among bees than has hitherto been considered the case.

Temporal changes in Varroa destructor fertility and haplotype in Brazil

The relationship between fertility and haplotype was studied in Varroa destructor mites sampled from colonies of A. mellifera carnica and Africanized Honeybees ( Apis mellifera) in Germany and Brazil respectively. Both in Germany and in Brazil, only the V. destructor Korea haplotype was found, though the Japan-Thailand haplotype was formerly thought to have been more abundant in Brazil. The fertility of Varroa mites in Brazil has increased since 1998 and is currently ( 2001) at European levels. Temporal changes in mite fertility and haplotype are not fully congruent.