Transfer of a chromosomal Maverick to endogenous bracovirus in a parasitoid wasp.

Bracoviruses are used by parasitoid wasps to allow development of their progeny within the body of lepidopteran hosts. In parasitoid wasps, the bracovirus exists as a provirus, integrated in a wasp chromosome. Viral replication occurs in wasp ovaries and leads to formation of particles containing dsDNA circles (segments) that are injected into the host body during wasp oviposition. We identified a large DNA transposon Maverick in a parasitoid wasp bracovirus. Closely related elements are present in parasitoid wasp genomes indicating that the element in CcBV corresponds to the insertion of an endogenous wasp Maverick in CcBV provirus. The presence of the Maverick in a bracovirus genome suggests the possibility of transposon transfers from parasitoids to lepidoptera via bracoviruses.

Mapping candidate genes for Drosophila melanogaster resistance to the parasitoid wasp Leptopilina boulardi.

Drosophila melanogaster resistance against the parasitoid wasp Leptopilina boulardi is under the control of a single gene (Rlb), with two alleles, the resistant one being dominant. Using strains bearing deletions, we previously demonstrated that the 55E2-E6; 55F3 region on chromosome 2R is involved in the resistance phenomenon. In this paper, we first restricted the Rlb containing region by mapping at the molecular level the breakpoints of the Df(2R)Pc66, Df(2R)P34 and Df(2R)Pc4 deficiencies, using both chromosomal in situ hybridization and Southern analyses. The resistance gene was localized in a 100 kb fragment, predicted to contain about 10 different genes. Male recombination genetic experiments were then performed, leading to identification of two possible candidates for the Rlb gene. Potential involvement of one of this genes, edl/mae, is discussed.

The few virus-like genes of Cotesia congregata bracovirus.

The origin of the symbiotic association between parasitoid wasps and bracoviruses is still unknown. From phylogenetic analyses, bracovirus-associated wasp species constitute a monophyletic group, the microgastroid complex. Thus all wasp-bracovirus associations could have originated from the integration of an ancestral virus in the genome of the ancestor of the microgastroids. In an effort to identify a set of virus genes that would give clues on the nature of the ancestral virus, we have recently performed the complete sequencing of the genome of CcBV, the bracovirus of the wasp Cotesia congregata. We describe here the putative proteins encoded by CcBV genome having significant similarities with sequences from known viruses and mobile elements. The analysis of CcBV gene content does not lend support to the hypothesis that bracoviruses originated from a baculovirus. Moreover, no consistent homology was found between CcBV genes and any set of genes constituting the core genome of a known free-living virus. We discuss the significance of the scarce homology found between proteins from CcBV and other viruses or mobile elements.

Maintenance of a large pericentric inversion generated by the hobo transposable element in a transgenic line of Drosophila melanogaster.

The impact of the hobo transposable element in the global reorganization of the Drosophila melanogaster genome has been investigated in transgenic lines generated by the injection of hobo elements into the Hikone strain, which lacked them previously. Extensive surveys of transgenic lines followed for 250 generations have identified 13 inversions with hobo inserts at most breakpoints. One of these inversions is pericentric on chromosome 2. It has been maintained in the line where it was discovered and in several sublines at frequencies from 0.19 to 0.45, generating stable chromosomal polymorphisms, similar to cosmopolitan paracentric inversions in natural populations. Individuals homozygous for this inversion were viable and fertile, allowing the creation of a new homozygous strain.

Polydnavirus genome: integrated vs. free virus.

Polydnaviruses are unique because of their obligatory association with thousands of parasitoid wasp species from the braconid and ichneumonid families of hymenopterans. PDVs are injected into the parasitized hosts and are essential for parasitism success. However, polydnaviruses are also unique because of their genome composed of multiple dsDNA segments. Cytological evidence has recently confirmed the results of genetic and molecular analyses indicating that PDV segments were integrated in the wasp genome. Moreover a phylogenetic study performed using the age of available fossils to calibrate the molecular clock indicated that the polydnaviruses harboured by braconid wasps have resided within the wasp genome for approximately 70 million years. In the absence of horizontal transmission, the evolution of the PDV genomes has been driven exclusively by the reproductive success they have offered the wasps. The consequences of this particular selection pressure can be observed in the gene content of certain PDV genomes from which increasing sequence data are available. Molecular mechanisms already identified could be involved in the acquisition and loss of genes by the PDV genomes and lead us to speculate on the definition of the virus genome.

Genomic organization and transcription of satellite DNA in the ant Aphaenogaster subterranea (Hymenoptera, Formicidae).

A satellite DNA family (APSU) was isolated and characterized in the ant Aphaenogaster subterranea. This satellite DNA is organized in tandem repeats of 162 bp and is relatively AT rich (51.9%). Sequence analysis showed a high level of homogeneity between monomers. Loss of satellite DNA has been detected in queens in relation to workers, because the amount of satellite DNA in queens is about 25% of the amount found in workers. Restriction analysis of the total DNA with methylation-sensitive enzymes suggests that this DNA is not methylated. Analysis of the electrophoretic mobility of satellite DNA on non-denaturing polyacrylamide showed that this satellite DNA is only very lightly curved. Their possible transcription was analyzed using reverse transcription and polymerase chain reaction (RT-PCR). The satellite DNA is transcribed on the two DNA strands at the same level in worker and queen pupae, as well as in worker adults.

Sequences homologous to the hobo transposable element in E strains of Drosophila melanogaster.

Hobo is one of the three Drosophila melanogaster transposable elements, together with the P and I elements, that seem to have recently invaded the genome of this species. Surveys of the presence of hobo in strains from different geographical and temporal origins have shown that recently collected strains contain complete and deleted elements with high sequence similarity (H strains), but old strains lack hobo elements (E strains). Besides the canonical hobo sequences, both H and E strains show other poorly known hobo-related sequences. In the present work, we analyze the presence, cytogenetic location, and structure of some of these sequences in E strains of D. melanogaster. By in situ hybridization, we found that euchromatic hobo-related sequences were in fixed positions in all six E strains analyzed: 38C in the 2L arm; 42B and 55A in the 2R arm; 79E and 80B in the 3L arm; and 82C, 84C, and 84D in the 3R arm. Sequence comparison shows that some of the hobo-related sequences from Oregon-R and iso-1 strains are similar to the canonical hobo element, but their analysis reveals that they are substantially diverged and rearranged and cannot code for a functional transposase. Our results suggest that these ubiquitous hobo-homologous sequences are immobile and are distantly related to the modern hobo elements from D. melanogaster.

The ITR binding domain of the Mariner Mos-1 transposase.

Mariner-like elements are widespread eukaryotic transposons, but Mos-1 is the only natural element that is known to be active. Little is known about the biochemistry of mariner transposition. The first step in the process is the binding of the transposase to the 5' and 3' inverted terminal repeats (ITRs) of the element. Using the 3' ITR of the element, we have determined the binding properties of a recombinant Mos-1 transposase produced in bacteria, and we have used deletion derivatives to localize the minimal ITR binding domain between amino acids 1 and 141. Its features and structure indicate that it differs from the ITR binding domain of the transposase encoded by Tc1-related elements.

Dynamics of the hobo transposable element in transgenic lines of Drosophila melanogaster.

The impact of the hobo transposable element in global reorganization of the Drosophila melanogaster genome has been investigated in transgenic lines generated by injection of hobo elements into the Hikone strain, which lacked them. In the present extensive survey, the chromosomal distribution of hobo insertion sites in the line 28 was found to be homogeneous and similar for all chromosomal arms, except 3L, when compared with other transgenic lines. However, some original features were observed in this line at the genetic and chromosomal levels. Several hotspots of insertion sites were observed on the X, second and third chromosomes. Five sites with a high frequency of hobo insertions were present on the 3L arm in most individuals tested, suggesting the action of selection for hobo element in some sites. The presence of doublets or triplet was also observed, implying that hobo inserts can show local jumps or insertions in preferred regions. This local transposition occurred independently in 11 specific genomic regions in many individuals and generations. The dynamics of this phenomenon were analysed across generations. These results support the use of the hobo system as an important tool in fundamental and applied Drosophila genetics.

Phylogenetic analysis of the functional domains of mariner-like element (MLE) transposases.

We have analyzed the sequences of mariner-like element (MLE) transposases, in order to obtain a clearer picture of their phylogenetic relationships. In particular, we have considered their two known structural domains, as well as the nucleic acid sequences of the MLE inverted terminal repeats (ITR). The most consistent tree was obtained using sequences of the catalytic domain of the transposase. The trees obtained with the amino acid sequences of the ITR-binding domain and the ITR sequences themselves were similar to that obtained with the catalytic domain. However, a major difference indicated that the cecropia sub-family is divided into two sub-groups. These new trees were used to examine the evolutionary divergence of mariner-like transposable elements, with particular reference to the possibility that recombination events or gene conversions created mosaic elements during the evolution of transposons.

Drosophila resistance genes to parasitoids: chromosomal location and linkage analysis.

Insect hosts can survive infection by parasitoids using the encapsulation phenomenon. In Drosophila melanogaster the abilities to encapsulate the wasp species Leptopilina boulardi and Asobara tabida each involve one major gene. Both resistance genes have been precisely localized on the second chromosome, 35 centimorgans apart. This result clearly demonstrates the involvement of at least two separate genetic systems in Drosophila resistance to parasitoid wasps. The resistance genes to L. boulardi and A. tabida are not clustered as opposed to many plant resistance genes to pathogens cloned to date.

The excision of polydnavirus sequences from the genome of the wasp Cotesia congregata (Braconidae, microgastrinae) is developmentally regulated but not strictly restricted to the ovaries in the adult.

Cotesia congregata polydnavirus (CcPDV) is essential for the successful parasitism of Manduca sexta larvae by the braconid wasp Cotesia congregata. In the absence of PDV, parasitoid eggs are encapsulated. Molecular analysis has demonstrated that polydnavirus sequences are integrated in the wasp chromosomes, and an ultrastructural analysis has shown that PDV replication occurs in the calyx region in the ovaries of the wasp. The bracovirus sequences appear to be excised from the wasp genome in the calyx cells where the virus replicates. Following excision of the virus sequences, the flanking sequences are rejoined. We analysed the production of two polydnavirus circles during wasp development and in different body parts of the adults of both sexes. Our study indicates that the excision of viral sequences is developmentally regulated, beginning in the pupal stage. In the adult wasp, excision occurs ubiquitously. However, regulation in the adult seems to occur only in diploid individuals, as no excision is detected in haploid males produced from virgin females.

Genetic localization of a Drosophila melanogaster resistance gene to a parasitoid wasp and physical mapping of the region.

Drosophila melanogaster larvae usually react against eggs of the parasitoid wasp Leptopilina boulardi by surrounding them with a multicellular melanotic capsule. The genetic determinism of this response has been studied previously using susceptible (non-capsule-forming) and resistant (capsule-forming) strains. The results suggest that differences in their encapsulation response involve a single gene, resistance to Leptopilina boulardi (Rlb), with two alleles, the resistant one being dominant. Rlb confers specific protection against Leptopilina boulardi and is thus probably involved in parasitoid recognition. Recent studies have localized this gene on the right arm of the second chromosome and our aim was to precisely determine its genetic and molecular location. Using strains bearing deletions, we demonstrated that resistance to Leptopilina boulardi is conferred by the 55C; 55F3 region and that the 55E2-E6; F3 region is particularly involved. A physical map of the 55C; 56A region was then constructed, based on a set of overlapping cosmid and P1 phage clones. Using single and double digests, cross hybridization of restriction fragments, and location of genetically mapped genes and STSs, a complete, five-enzyme restriction map of this 830-kb region was obtained.

Satellite DNA transcription in Diadromus pulchellus (Hymenoptera).

Previous studies have shown that the satellite DNAs in Hymenoptera account for 1-25% of the genome. They mainly correspond to a single family, or to several subfamilies having the same evolutionary origin. We have now showed that the satellite DNAs in the genomes of the hymenopterans Diadromus pulchellus, Diadromus collaris, Eupelmus vuilletti and Eupelmus orientalis are transcribed in both males and females. Satellite DNA transcripts could only be extracted with NP40/Urea, indicating that they are strongly associated with proteins. The satellite DNA in D. pulchellus was transcribed on the two DNA strands. The satellite DNA transcripts were single-stranded and not polyadenylated in vivo. The transcripts were found in embryos, larvae and imagos stages. The transcripts detected included one major transcript (1.9 kb) and several discrete smaller transcripts. The in vivo synthesis of these satellite DNA transcripts was explored by identifying their putative initiation sites.

Features of DNA fragments obtained by random amplified polymorphic DNA (RAPD) assays

Random amplified polymorphic DNA (RAPD) fragments were prepared from samples of Calonectris diomedea (Cory's shearwater, Aves) and Haemonchus contortus (Nematoda) DNA by polymerase chain reaction (PCR) using decamers containing two restriction enzyme sites as primers. Six of 19 studied RAPD fragments probably originated from traces of commensal microorganisms. Many rearranged fragments, absent in the original genomic DNA, were synthesized and amplified during the processing of all the DNA samples, indicating that interactions occur within and between strands during the annealing step of PCR. The model of interactions between molecular species during DNA amplification with a single arbitrary oligonucleotide primer was modified to include nested primer annealing and interactions within and between strands. The presence of these artefacts in the final RAPD have a major effect on the interpretation of polymorphism studies.

Free serosal cells originating from the embryo of the wasp Diadromus pulchellus in the pupal body of parasitized leek-moth, Acrolepiosis assectella. Are these cells teratocyte-like?

In braconid species, teratocytes are derived from a serosal cell membrane which envelops the developing parasitoid embryo. On hatching, this membrane dissociates into individual cells, the teratocytes, which then circulate in the haemolymph of the host. We describe herein such a membrane, surrounding the embryo in eggs of the ichneumonid parasitoid wasp, Diadromus pulchellus. This membrane consisted of a single sheet of tightly packed cells with large 12+/-1.4 &mgr;m nuclei. These cells were released after hatching in vitro and cells of the same size were detected in vivo, in the vicinity of the D. pulchellus embryo. The number of nuclei detected suggests that the serosal membrane consists of about 450+/-150 cells. These cells did not grow after hatching of the parasitoid egg in the parasitized host, Acrolepiosis assectella, during the development of the parasitoid wasp larva. Southern blot experiments, using D. pulchellus satellite DNA or the ribosomal genes as probes, showed that free-living floating cells of wasp origin were present in the body of the parasitized host. This is the first time that free-floating teratocyte-like cells have been described in species of the Ichneumonidae.

Hobo transposons causing chromosomal breakpoints.

Several laboratory surveys have shown that transposable elements (TEs) can cause chromosomal breaks and lead to inversions, as in dysgenic crosses involving P-elements. However, it is not presently clear what causes inversions in natural populations of Drosophila. The only direct molecular studies must be taken as evidence against the involvement of mobile elements. Here, in Drosophila lines transformed with the hobo transposable element, and followed for 100 generations, we show the appearance of five different inversions with hobo inserts at breakpoints. Almost all breakpoints occurred in hobo insertion sites detected in previous generations. Therefore, it can be assumed that such elements are responsible for restructuring genomes in natural populations.

Transmission pattern of hobo transposable element in transgenic lines of Drosophila melanogaster.

This study is an attempt to trace the fate of hobo elements in the genomes of E strains of Drosophila melanogaster that have been transfected with pHFL1, a plasmid containing an autonomous hobo. Such long-term population studies (over 105 generations) could be very useful for better understanding the population and genomic dynamics of transposable elements and their pattern of insertions. Molecular analyses of hobo elements in the transfected lines were performed using Southern blots of XhoI-digested genomic DNAs. The complete element was observed in all six injected lines. In two lines we observed, at generation 100, two deleted elements, which did not correspond to Th1 and Th2. The results obtained by the in situ method show that the number of hybridization sites increases in each line and prove that the hobo element may be amplified in an RM genome. The hobo activity does not seem to be systematically correlated with the number of hobo elements. After generation 85, the evolution of the hobo element's insertion site number depends on the injected line. In all lines, the total number of insertions remains quite small, between 0 and 11. Hobo elements are located on each of the chromosomal arms. We describe 'hotspots'-insertion sites present in all lines and in all generations. On the 3R arm, a short inversion appeared once at generation 85.

Excision of the polydnavirus chromosomal integrated EP1 sequence of the parasitoid wasp Cotesia congregata (Braconidae, Microgastinae) at potential recombinase binding sites.

Cotesia congregata polydnavirus (CcPDV) is essential for successful parasitism of Manduca sexta larvae by the braconid wasp Cotesia congregata. To determine the molecular mechanisms for the vertical transmission of CcPDV in the wasps, we analysed the different forms of the virus sequences containing the gene encoding the early parasitism-specific protein 1 (EP1). By a detailed molecular analysis, we demonstrated that the EP1 sequences are present in wasp DNA in two forms: a circular form as seen in the virus particles and a form integrated into the wasp genome. Moreover, we showed that the integrated form of the EP1 sequences is able to excise in the ovary cells. A fragment corresponding to an EP1 'empty locus' (without the viral sequence) was PCR-amplified from ovarian DNA. Comparison of the sequences isolated from the EP1 circle, the integrated form and the empty locus revealed that the extremities of the EP1 genomic sequences constitute a direct repeat. Strikingly, these sequences contain a potential binding site for a recombinase of the Hin family located in close vicinity to the position where the DNA strand exchange occurs. Thus, the data bear upon the possibility that the bracovirus circles are excised via a mechanism related to the Hin mediated Conservative Specific-Specific Recombination (CSSR) of prokaryotes.

Particle and genomic characteristics of a new member of the Ascoviridae: Diadromus pulchellus ascovirus.

A new member of the family Ascoviridae, Diadromus pulchellus ascovirus (DpAV), has been found in the lepidopteran nymphs of Acrolepiopsis assectella parasitized by the hymenopteran wasp Diadromus pulchellus. Virions have the standard features of the ascovirus group; each particle is about 220 nm long and 150 nm wide. They are multilayered, with two clear 7-nm-thick outer layers and one 15-nm-thick inner layer surrounding an electron-dense core (155 x 110 nm). However, the flattened rice-grain shape and fragility of the DpAV particles are unlike that of known ascoviruses infecting Noctuidae species. They form large vesicles containing virions in infected cells. The DpAV genome is about 116 kb long and has a circular and relaxed structure. It contains 6-8 repeated and interspersed sequences of 494 bp. The structural and genomic features of DpAV suggest that this virus belongs to an ascovirus sub-family different from that containing the ascoviruses previously found to infect species of Noctuidae (Federici et al., 1991).