François Cousserans

Prevalence and Seasonal Variations of Six Bee Viruses in Apis mellifera L. and Varroa destructor Mite Populations in France

ABSTRACT A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.

An Expressed Sequence Tag collection from the male antennae of the Noctuid moth Spodoptera littoralis : a resource for olfactory and pheromone detection research

BackgroundNocturnal insects such as moths are ideal models to study the molecular bases of olfaction that they use, among examples, for the detection of mating partners and host plants. Knowing how an odour generates a neuronal signal in insect antennae is crucial for understanding the physiological bases of olfaction, and also could lead to the identification of original targets for the development of olfactory-based control strategies against herbivorous moth pests. Here, we describe an Expressed Sequence Tag (EST) project to characterize the antennal transcriptome of the noctuid pest model, Spodoptera littoralis, and to identify candidate genes involved in odour/pheromone detection.ResultsBy targeting cDNAs from male antennae, we biased gene discovery towards genes potentially involved in male olfaction, including pheromone reception. A total of 20760 ESTs were obtained from a normalized library and were assembled in 9033 unigenes. 6530 were annotated based on BLAST analyses and gene prediction software identified 6738 ORFs. The unigenes were compared to the Bombyx mori proteome and to ESTs derived from Lepidoptera transcriptome projects. We identified a large number of candidate genes involved in odour and pheromone detection and turnover, including 31 candidate chemosensory receptor genes, but also genes potentially involved in olfactory modulation.ConclusionsOur project has generated a large collection of antennal transcripts from a Lepidoptera. The normalization process, allowing enrichment in low abundant genes, proved to be particularly relevant to identify chemosensory receptors in a species for which no genomic data are available. Our results also suggest that olfactory modulation can take place at the level of the antennae itself. These EST resources will be invaluable for exploring the mechanisms of olfaction and pheromone detection in S. littoralis, and for ultimately identifying original targets to fight against moth herbivorous pests.

Extensive synteny conservation of holocentric chromosomes in Lepidoptera despite high rates of local genome rearrangements

The recent assembly of the silkworm Bombyx mori genome with 432 Mb on 28 holocentric chromosomes has become a reference in the genomic analysis of the very diverse Order of Lepidoptera. We sequenced BACs from two major pests, the noctuid moths Helicoverpa armigera and Spodoptera frugiperda, corresponding to 15 regions distributed on 11 B. mori chromosomes, each BAC/region being anchored by known orthologous gene(s) to analyze syntenic relationships and genome rearrangements among the three species. Nearly 300 genes and numerous transposable elements were identified, with long interspersed nuclear elements and terminal inverted repeats the most abundant transposable element classes. There was a high degree of synteny conservation between B. mori and the two noctuid species. Conserved syntenic blocks of identified genes were very small, however, approximately 1.3 genes per block between B. mori and the two noctuid species and 2.0 genes per block between S. frugiperda and H. armigera. This corresponds to approximately two chromosome breaks per Mb DNA per My. This is a much higher evolution rate than among species of the Drosophila genus and may be related to the holocentric nature of the lepidopteran genomes. We report a large cluster of eight members of the aminopeptidase N gene family that we estimate to have been present since the Jurassic. In contrast, several clusters of cytochrome P450 genes showed multiple lineage-specific duplication events, in particular in the lepidopteran CYP9A subfamily. Our study highlights the value of the silkworm genome as a reference in lepidopteran comparative genomics.

Analysis of virion structural components reveals vestiges of the ancestral ichnovirus genome.

Many thousands of endoparasitic wasp species are known to inject polydnavirus (PDV) particles into their caterpillar host during oviposition, causing immune and developmental dysfunctions that benefit the wasp larva. PDVs associated with braconid and ichneumonid wasps, bracoviruses and ichnoviruses respectively, both deliver multiple circular dsDNA molecules to the caterpillar. These molecules contain virulence genes but lack core genes typically involved in particle production. This is not completely unexpected given that no PDV replication takes place in the caterpillar. Particle production is confined to the wasp ovary where viral DNAs are generated from proviral copies maintained within the wasp genome. We recently showed that the genes involved in bracovirus particle production reside within the wasp genome and are related to nudiviruses. In the present work we characterized genes involved in ichnovirus particle production by analyzing the components of purified Hyposoter didymator Ichnovirus particles by LC-MS/MS and studying their organization in the wasp genome. Their products are conserved among ichnovirus-associated wasps and constitute a specific set of proteins in the virosphere. Strikingly, these genes are clustered in specialized regions of the wasp genome which are amplified along with proviral DNA during virus particle replication, but are not packaged in the particles. Clearly our results show that ichnoviruses and bracoviruses particles originated from different viral entities, thus providing an example of convergent evolution where two groups of wasps have independently domesticated viruses to deliver genes into their hosts.

SPODOBASE : an EST database for the lepidopteran crop pest Spodoptera

BackgroundThe Lepidoptera Spodoptera frugiperda is a pest which causes widespread economic damage on a variety of crop plants. It is also well known through its famous Sf9 cell line which is used for numerous heterologous protein productions. Species of the Spodoptera genus are used as model for pesticide resistance and to study virus host interactions. A genomic approach is now a critical step for further new developments in biology and pathology of these insects, and the results of ESTs sequencing efforts need to be structured into databases providing an integrated set of tools and informations.DescriptionThe ESTs from five independent cDNA libraries, prepared from three different S. frugiperda tissues (hemocytes, midgut and fat body) and from the Sf9 cell line, are deposited in the database. These tissues were chosen because of their importance in biological processes such as immune response, development and plant/insect interaction. So far, the SPODOBASE contains 29,325 ESTs, which are cleaned and clustered into non-redundant sets (2294 clusters and 6103 singletons). The SPODOBASE is constructed in such a way that other ESTs from S. frugiperda or other species may be added. User can retrieve information using text searches, pre-formatted queries, query assistant or blast searches. Annotation is provided against NCBI, UNIPROT or Bombyx mori ESTs databases, and with GO-Slim vocabulary.ConclusionThe SPODOBASE database provides integrated access to expressed sequence tags (EST) from the lepidopteran insect Spodoptera frugiperda. It is a publicly available structured database with insect pest sequences which will allow identification of a number of genes and comprehensive cloning of gene families of interest for scientific community. SPODOBASE is available from URL: http://bioweb.ensam.inra.fr/spodobase

Viral load estimation in asymptomatic honey bee colonies using the quantitative RT-PCR technique

Honey bee (Apis mellifera L.) colonies are subjected to many persistent viral infections that do not exhibit clinical signs. The identification of criteria that could identify persistent or latent infections in bee colonies is a challenging task for field investigators and beekeepers. With this aim in view, we developed a molecular method to estimate the viral loads for six different RNA viruses in bee and mite individuals collected from seemingly healthy colonies (360 colonies). The data showed very large viral titres in some samples (> 109 copies per bee or mite). Discrepancies between adults and pupae viral RNA loads and, in several instances, significant seasonal variations among viruses were observed. The high titres of some RNA viruses recorded in mites confirm that Varroa destructor could promote viral infections in colonies.ZusammenfassungBei den am häufigsten bei Honigbienen diagnostizierten Viren handelt es sich um 30 nm grosse positive RNA-Partikel, die als Picorna-ähnliche Viren bezeichnet werden. In diese Klasse gehören das Sackbrutvirus (SBV), das Verkrüppelte-Flügelvirus (DWV), das Kaschmir-Bienenvirus (KBV), das Akute-Bienenparalysevirus (ABPV), das Schwarze-Königinnenzellenvirus (BQCV) und das Chronische-Bienenparalysevirus (CBPV). Die Genomsequenzen dieser Viren sind jetzt in DNA-Bibliotheken verfügbar, die die Entwicklung auf PCR beruhender molekularer Diagnoseprotokolle erleichtern. In einer früheren Arbeit berichteten wir die Ergebnisse einer Jahresübersichtsstudie zur Prävalenz und zu jahreszeitlichen Schwankungen der Konzentrationen dieser Viren in augenscheinlich gesunden Bienenständen in Frankreich (Tentcheva et al., 2004). Diese Ergebnisse zeigten klar, dass viele persistente Virusinfektionen in Bienenvölkern etabliert sein können (92 % der Bienenstände waren positiv für mindestens drei verschiedene Viren), ohne dass klinische Symptome zu sehen sind. Der weltweite intensive Austausch von Völkern und Bienenmaterial und die durch die ektoparasitische Milbe Varroa destructor, einem wichtigen Vektor und Aktivator verschiedener Bienenviren, hervorgerufenen Epizootien sind vermutlich für das verstärkte Auftreten dieser Viren verantwortlich.Die Ermittlung von Kriterien, die dazu beitragen können, persistente oder latente Infektionen in Bienenvölkern zu erkennen, stellen deshalb eine Herausforderung für die Feldforschung und für Imker dar. Um einen Einblick in den Gesundheitszustand von Bienenvölkern, sowohl für Einzelvölker als auch Bienenstände zu erhalten, entwickelten wir ein Protokoll, das eine zuverlässige Quantifizierung von sechs RNA-Viren ermöglicht. Diese quantitative PCR Methode kann dazu beitragen, die Viruskonzentrationen in Bienenvölkern mit bestimmten Pathologien zu korrelieren und kritische Schwellenwerte für die Haltung von Völkern festzulegen.In der vorliegenden Arbeit stellen wir die Ergebnisse einer statistischen Untersuchung zur Verteilung der Viruskonzentrationen in Proben von Bienen und Milben (V. destructor) aus 360 augenscheinlich gesunden Völkern vor. Die Analyse der Proben von adulten Bienen wies zwei Populationen in Bezug auf Viruskonzentrationen auf. Die erste betraf Proben, die einen mittleren Befall mit AB-PV und BQCV in Konzentrationen von 1,52 × 108 RNA-Kopie-Äquivalenten pro Biene aufwiesen. Die zweite umfasste Proben, die mit DWV, KBV, SBV und CBPV infiziert waren und Viruskonzentrationen von 2,14 × 109 RNA-Kopie-Äquivalenten pro Biene aufwiesen.Die höchsten Virus-RNA-Konzentrationen fanden wir in den für DWV positiven Proben, die, im Gegensatz zu SBV positiven Proben, für Puppen höhere Werte aufwiesen als für adulte Bienen. Bei den DWV-Titern konnten wir einen Anstieg zum Jahresende hin feststellen, während die BQCV- und SBV-Konzentationen zum Herbst hin abnahmen. In Milben waren die DWV-Konzentrationen signifikant höher als die für ABPV und SBV, was darauf hinweist, dass das DWV sich vermutlich in Milben repliziert und dass Varroa der Verbreitung dieses Virus in Völkern förderlich sein kann. Zusammenfassend zeigen unsere Daten dass (i) Bienenviren sich in ihrer Biologie stark unterscheiden, dass (ii) Bienenvölker relativ hohe Viruskonzentrationen tolerieren können, ohne dass sie klinische Symptome zeigen, und dass (iii) das Konzept der Bienenpathologie in seinen Untersuchungsansätzen trennen muss zwischen einzelner Biene und Bienenvolk.

Genome analysis of a Glossina pallidipes salivary gland hypertrophy virus reveals a novel, large, double-stranded circular DNA virus

ABSTRACT Several species of tsetse flies can be infected by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). Infection causes salivary gland hypertrophy and also significantly reduces the fecundity of the infected flies. To better understand the molecular basis underlying the pathogenesis of this unusual virus, we sequenced and analyzed its genome. The GpSGHV genome is a double-stranded circular DNA molecule of 190,032 bp containing 160 nonoverlapping open reading frames (ORFs), which are distributed equally on both strands with a gene density of one per 1.2 kb. It has a high A+T content of 72%. About 3% of the GpSGHV genome is composed of 15 sequence repeats, distributed throughout the genome. Although sharing the same morphological features (enveloped rod-shaped nucleocapsid) as baculoviruses, nudiviruses, and nimaviruses, analysis of its genome revealed that GpSGHV differs significantly from these viruses at the level of its genes. Sequence comparisons indicated that only 23% of GpSGHV genes displayed moderate homologies to genes from other invertebrate viruses, principally baculoviruses and entomopoxviruses. Most strikingly, the GpSGHV genome encodes homologues to the four baculoviral per os infectivity factors (p74 [pif-0], pif-1, pif-2, and pif-3). The DNA polymerase encoded by GpSGHV is of type B and appears to be phylogenetically distant from all DNA polymerases encoded by large double-stranded DNA viruses. The majority of the remaining ORFs could not be assigned by sequence comparison. Furthermore, no homologues to DNA-dependent RNA polymerase subunits were detected. Taken together, these data indicate that GpSGHV is the prototype member of a novel group of insect viruses.

Two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica are related and form a distinct phylogenetic clade.

Glossina pallidipes and Musca domestica salivary gland hypertrophy viruses (GpSGHV and MdSGHV) replicate in the nucleus of salivary gland cells causing distinct tissue hypertrophy and reduction of host fertility. They share general characteristics with the non-occluded insect nudiviruses, such as being insect-pathogenic, having enveloped, rod-shaped virions, and large circular double-stranded DNA genomes. MdSGHV measures 65x550 nm and contains a 124 279 bp genome (approximately 44 mol% G+C content) that codes for 108 putative open reading frames (ORFs). GpSGHV, measuring 50x1000 nm, contains a 190 032 bp genome (28 mol% G+C content) with 160 putative ORFs. Comparative genomic analysis demonstrates that 37 MdSGHV ORFs have homology to 42 GpSGHV ORFs, as some MdSGHV ORFs have homology to two different GpSGHV ORFs. Nine genes with known functions (dnapol, ts, pif-1, pif-2, pif-3, mmp, p74, odv-e66 and helicase-2), a homologue of the conserved baculovirus gene Ac81 and at least 13 virion proteins are present in both SGHVs. The amino acid identity ranged from 19 to 39 % among ORFs. An (A/T/G)TAAG motif, similar to the baculovirus late promoter motif, was enriched 100 bp upstream of the ORF transcription initiation sites of both viruses. Six and seven putative microRNA sequences were found in MdSGHV and GpSGHV genomes, respectively. There was genome. Collinearity between the two SGHVs, but not between the SGHVs and the nudiviruses. Phylogenetic analysis of conserved genes clustered both SGHVs in a single clade separated from the nudiviruses and baculoviruses. Although MdSGHV and GpSGHV are different viruses, their pathology, host range and genome composition indicate that they are related.

Viruses associated with ovarian degeneration in Apis mellifera L. queens.

Queen fecundity is a critical issue for the health of honeybee (Apis mellifera L.) colonies, as she is the only reproductive female in the colony and responsible for the constant renewal of the worker bee population. Any factor affecting the queens fecundity will stagnate colony development, increasing its susceptibility to opportunistic pathogens. We discovered a pathology affecting the ovaries, characterized by a yellow discoloration concentrated in the apex of the ovaries resulting from degenerative lesions in the follicles. In extreme cases, marked by intense discoloration, the majority of the ovarioles were affected and these cases were universally associated with egg-laying deficiencies in the queens. Microscopic examination of the degenerated follicles showed extensive paracrystal lattices of 30 nm icosahedral viral particles. A cDNA library from degenerated ovaries contained a high frequency of deformed wing virus (DWV) and Varroa destructor virus 1 (VDV-1) sequences, two common and closely related honeybee Iflaviruses. These could also be identified by in situ hybridization in various parts of the ovary. A large-scale survey for 10 distinct honeybee viruses showed that DWV and VDV-1 were by far the most prevalent honeybee viruses in queen populations, with distinctly higher prevalence in mated queens (100% and 67%, respectively for DWV and VDV-1) than in virgin queens (37% and 0%, respectively). Since very high viral titres could be recorded in the ovaries and abdomens of both functional and deficient queens, no significant correlation could be made between viral titre and ovarian degeneration or egg-laying deficiency among the wider population of queens. Although our data suggest that DWV and VDV-1 have a role in extreme cases of ovarian degeneration, infection of the ovaries by these viruses does not necessarily result in ovarian degeneration, even at high titres, and additional factors are likely to be involved in this pathology.

The Apis mellifera Filamentous Virus Genome

A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family.