Sperm ultrastructure in Chironomoidea (Insecta, Diptera).

The fine structure of spermatozoa from several species of chironomids, of Culicoides sp. (Ceratopogonidae) and of Odagmia pontina (Simulidae) was studied. A synapomorphic feature, consisting of nine kidney-shaped structures forming the centriole adjunct, was found in the chironomid species. All members of Chironomoidea share a mono-layered acrosome and a flagellar axoneme, provided with accessory tubules with 15 protofilaments in their tubular wall. The axoneme has a 9+9+2 pattern, but in an unidentified species of chironomid, a 9+9+0 model was observed where the central complex and the spokes are missing. Sperm motility is, however, maintained in all the examined species. The spermatozoa of this taxon have the tendency to complete maturation during their progression along the deferent ducts. Thus, in the proximal region of these ducts, they often show remnants of the spermatid cytoplasm.

Sergeia podlipaevi gen. nov., sp. nov. (Trypanosomatidae, Kinetoplastida), a parasite of biting midges (Ceratopogonidae, Diptera).

Three strains of a trypanosomatid protozoan were isolated from the midguts of two naturally infected species of biting midges [Culicoides (Oecacta) festivipennis and Culicoides (Oecacta) truncorum] and characterized by light and electron microscopy and by molecular techniques. Morphological characteristics and sequences of the 18S rRNA, 5S rRNA, spliced leader RNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase genes indicate that the studied flagellates represent a novel phylogenetic lineage within the Trypanosomatidae. Based on phylogenetic analyses, the novel endosymbiont-free, monoxenous trypanosomatid was classified as Sergeia podlipaevi gen. nov., sp. nov. Interestingly, it is closely related to another trypanosomatid species that parasitizes the sand fly Lutzomyia evansi, a blood-sucking dipteran from South America. The type strain of S. podlipaevi sp. nov., ICUL/CZ/2000/CER3, was obtained from Malpighian tubes. Of 2518 females of seven species of biting midges trapped in the Czech Republic, more than 1.5 % were infected by trypanosomatid parasites. An unrelated insect species, Culicoides (Monoculicoides) nubeculosus, was experimentally infected with S. podlipaevi, demonstrating that its host range extends to different subgenera of biting midges.

Nematode (Nematoda: Mermithidae) and hairworm (Nematomorpha: Chordodidae) parasites in Early Cretaceous amber.

The present report describes a mermithid nematode (Nematoda: Mermithidae) and a gordiid hairworm (Nematomorpha: Chordodidae) from Early Cretaceous Burmese amber dated at 100-110 million years. The mermithid, Cretacimermis protus sp. n., is emerging from a biting midge (Diptera: Ceratopogonidae) while the hairworm, Cretachordodes burmitis, gen. n., sp. n. had already emerged from its host. These rare specimens represent the first fossil mermithid parasite of a ceratopogonid midge and second oldest described nematode and the earliest known and only Mesozoic fossil of the phylum Nematomorpha. A list of previously described fossil mermithids is included.

Characterization of cell lines developed from field populations of Culicoides sonorensis (Diptera: Ceratopogonidae).

Two cell lines, ABADRL-Cs-W3 (W3) and ABADRL-Cs-W8A (W8), were developed from a field population of Culicoides sonorensis Wirth & Jones. The cell lines were characterized by isozyme phenotyping and the ability to support the replication of bluetongue virus (BLU) and epizootic hemorrhagic disease virus (EHDV) (Orbivirus, Reoviridae). Comparison of isozymes found in the cell lines with those found in adult C. sonorensis colony insects confirmed that the cell lines were of C. sonorensis origin. There was, however, sufficient isozyme variation present in the cell lines to construct a unique isozyme profile for each cell line. Although both cell lines supported BLU and EHDV replication to the same level, one-step growth curves for BLU indicated that virus replication was faster and attained a peak titer earlier in the W3 cell line than in the W8 cell line. Viral proteins and RNA were detected earlier in the W3 cell line as well. The accelerated virus growth kinetics observed in the W3 cell line and the adherent nature of the cells makes it more suitable for certain Orbivirus studies.

RGD tripeptide of bluetongue virus VP7 protein is responsible for core attachment to Culicoides cells.

Bluetongue virus (BTV) is an arthropod-borne virus transmitted by Culicoides species to vertebrate hosts. The double-capsid virion is infectious for Culicoides vector and mammalian cells, while the inner core is infectious for only Culicoides-derived cells. The recently determined crystal structure of the BTV core has revealed an accessible RGD motif between amino acids 168 to 170 of the outer core protein VP7, whose structure and position would be consistent with a role in cell entry. To delineate the biological role of the RGD sequence within VP7, we have introduced point mutations in the RGD tripeptide and generated three recombinant baculoviruses, each expressing a mutant derivative of VP7 (VP7-AGD, VP7-ADL, and VP7-AGQ). Each expressed mutant protein was purified, and the oligomeric nature and secondary structure of each was compared with those of the wild-type (wt) VP7 molecule. Each mutant VP7 protein was used to generate empty core-like particles (CLPs) and were shown to be biochemically and morphologically identical to those of wt CLPs. However, when mutant CLPs were used in an in vitro cell binding assay, each showed reduced binding to Culicoides cells compared to wt CLPs. Twelve monoclonal antibodies (MAbs) was generated using purified VP7 or CLPs as a source of antigen and were utilized for epitope mapping with available chimeric VP7 molecules and the RGD mutants. Several MAbs bound to the RGD motif on the core, as shown by immunogold labeling and cryoelectron microscopy. RGD-specific MAb H1.5, but not those directed to other regions of the core, inhibited the binding activity of CLPs to the Culicoides cell surface. Together, these data indicate that the RGD motif present on BTV VP7 is responsible for Culicoides cell binding activity.

Metaphase chromosomes of Culicoides variipennis Diptera:Ceratopogonidae).

The cytogenetics of Culicoides variipennis (Coquillett) tissue derived from a continuous cell line is presented. The karyotype consisted of 68.5% diploid (2n = 6), 30% tetraploid (4n = 12), and 1.5% octaploid (8n = 24) for the metaphase spreads examined. Distinguishing cytological features were not seen, and the 3 pairs of homomorphic chromosomes were not distinguished from each other.