Articles by John J. Shepard in JoVE
Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay Philip M. Armstrong1, Theodore G. Andreadis1, Shannon L. Finan1, John J. Shepard1, Michael C. Thomas1 1Center for Vector Biology and Zoonotic Diseases, Department of Environmental Sciences, The Connecticut Agricultural Experiment Station We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.
Other articles by John J. Shepard on PubMed
Molecular Phylogeny and Evolutionary Relationships Among Mosquitoes (Diptera: Culicidae) from the Northeastern United States Based on Small Subunit Ribosomal DNA (18S RDNA) Sequences Journal of Medical Entomology. May, 2006 | Pubmed ID: 16739399 The phylogenetic relationships of Culicidae native to the northeastern United States were investigated by analyzing small subunit ribosomal DNA (18S rDNA) sequences obtained from 39 species representing nine genera. Molecular phylogenies were consistent with traditional classifications based on morphological characters except for the placements of Psorophora Robineau-Desvoidy and Uranotaenia Lynch Arribalzaga. In our analyses, 1) Anopheles Meigen was strongly supported as the sister taxon to the remaining Culicidae; 2) Toxorhynchites Theobald was represented as a distinct monophyletic sister group to the Culicinae; 3) Psorophora formed a basal clade to Culiseta Felt, Coquillettidia Dyar, and Culex L. but also was shown as a sister taxon to Aedes Meigen and Ochlerotatus Lynch Arribalzaga; 4) Coquillettidia perturbans (Walker) seems to be a sister group to Culiseta; 5) placement of Uranotaenia was inconclusive and seemed to be either a sister group to the Aedes and Ochlerotatus or a basal taxon to all other culicines; and 6) Aedes and Ochlerotatus formed two separate and distinct clades, providing phylogenetic data consistent with the recent elevation of Ochlerotatus to the generic level as proposed by Reinert (2000). The utility of 18S rDNA for evaluating phylogenetic and evolutionary relationships among mosquito taxa was demonstrated at the genus and species levels. To our knowledge, this study represents the first molecular-based phylogenetic study of mosquito species occurring within this geographic region of North America and contains the largest number of species that have been examined among the genera Aedes and Ochlerotatus.
Ultrastructural Characterization and Comparative Phylogenetic Analysis of New Microsporidia from Siberian Mosquitoes: Evidence for Coevolution and Host Switching Journal of Invertebrate Pathology. Jan, 2012 | Pubmed ID: 22001630 A survey of mosquito larvae infected with microsporidia was conducted from 2005 to 2008 in the Tomsk, Kemerovo and Novosibirsk regions of western Siberia, Russia. Twenty-one morphologically and genetically unique species of microsporidia were isolated from nine species of Anopheles, Aedes, Culex and Ochlerotatus mosquitoes including: (1) 14 proposed new species of Amblyospora (A. bakcharia, A. baritia, A. bogashovia, A. chulymia, A. hristinia, A. jurginia, A. kazankia, A. mavlukevia, A. mocrushinia, A. modestium, A. salairia, A. severinia, A. shegaria, and A. timirasia); (2) a newly proposed genus and species, Novothelohaniaovalae and; (3) six species of Amblyospora (A. flavescens, A. kolarovi, A. rugosa), Parathelohania (P. divulgata and P. tomski) and Trichoctosporea (T. pygopellita) from which gene sequences had not been previously obtained. Detailed ultrastructure of meiospores revealed unique cytological features associated with the length, arrangement and ratio of broad to narrow coils of the polar filament, comparative thickness of the exospore and endospore, and overall size of each species reaffirming their value in distinguishing taxonomic relationships. SSU rDNA sequences obtained from each species of microsporidia were unique when compared with GenBank entries. Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and Neighbor Joining analyses yielded similar topologies with a high degree of congruence between parasite and host at the generic level. Species that parasitize Aedes/Ochlerotatus and Culex mosquitoes segregate into distinct monophyletic groupings mirroring their host phylogeny, while species from Anopheles mosquitoes group as a sister clade basal to the entire group of mosquito-parasitic microsporidia as their Anopheles hosts cluster as a sister clade to the entire group of culicine mosquitoes. This provides strong evidence for host-parasite coevolution by descent at the generic level and limited host lineage switching between unrelated taxa. Among parasites of Aedes/Ochlerotatus and Anopheles mosquitoes, we found several instances where a single mosquito species serves as a host for two or more related species of microsporidia, an observation consistent with host switching and independent parasite speciation. Among the microsporidian parasites of Culex mosquitoes, we found only one parasite per host indicating a higher degree of host specificity and less host switching among parasites of this genus. Findings suggest a degree of host-parasite co-speciation with host switching occurring occasionally when the "normal" host is unavailable in the aquatic ecosystem. Frequency of host switching seems to be occurring in proportion to host relatedness and does not cross generic boundaries in this system.