Molecular systematics of the Simulium jenningsi species group (Diptera: Simuliidae), with three new fast-evolving nuclear genes for phylogenetic inference.

A molecular phylogeny was inferred for the 22 nominal species of black flies in the Simulium jenningsi species group, which includes major pests of humans and livestock in North America. Females are structurally monomorphic, presenting a problem for identification of the pests. For each species, we sequenced approximately two kilobases from the mitochondrial genome (ND2, Cox I, proximal one-half of Cox II) and about six kilobases from the nuclear genome (ca. 2 kilobases each from 3 rapidly evolving nuclear genes: big zinc finger [BZF], "5-intron gene" [5intG], and elongation complex protein 1 [ECP1]) and analyzed them phylogenetically using maximum likelihood and Bayesian methods. The three nuclear loci have not previously been used in phylogenetic studies. The mitochondrial region recovered 6 group members as monophyletic. BZF, 5intG, and ECP1 sequences each permitted identification of 13 species and recovered the S. fibrinflatum and S. taxodium subgroups. Simulium aranti Stone and Snoddy and S. luggeri Nicholson and Mickel were consistently recovered at the base of the group. Simulium ozarkense Moulton and Adler, S. dixiense Stone and Snoddy, S. krebsorum Moulton and Adler, and S. haysi Stone and Snoddy branched off before two well-supported sister groups of the remaining species. This remainder consisted of species occupying slow, sandy lowland streams-S. definitum Moulton and Adler, S. jonesi Stone and Snoddy, and the S. taxodium subgroup (S. taxodium Snoddy and Beshear, S. chlorum Moulton and Adler, S. confusum Moulton and Adler, and S. lakei Snoddy)-as sister to two clades of species inhabiting swift, rocky upland streams-the S. fibrinflatum subgroup (S. fibrinflatum Twinn, S. notiale Stone and Snoddy, and S. snowi Stone and Snoddy) and a clade comprised of S. anchistinum Moulton and Adler, S. jenningsi Malloch, and S. nyssa Stone and Snoddy, plus species having cocoons without anterolateral apertures (S. infenestrum Moulton and Adler, S. podostemi Snoddy, S. penobscotense Snoddy and Bauer, and S. remissum Moulton and Adler). Simulium snowi Stone and Snoddy is here considered a synonym of S. notiale Stone and Snoddy. Trees inferred from BZF and 5intG were largely concordant with those from ECP1, but slightly less resolved. Combining mitochondrial and nuclear data sets did not greatly improve the performance of the ECP1 data set alone. We, therefore, propose ECP1 as the gold standard for identification of members of the S. jenningsi group. Maximum likelihood analysis of combined sequences from all three nuclear genes, with three morphological constraints imposed, yielded a tree proposed as the best hypothesis of relationships among group members, based on all available data.

Genetic variability and phylogenetic analysis of hosta virus X.

Triple gene block 1 (TGB1) and coat protein (CP) sequences of 30 hosta virus X (HVX) isolates from Tennessee (TN), USA, were determined and compared with available sequences in GenBank. The CPs of all known HVX isolates, including those from TN, shared 98.3-100% and 98.2-100% nucleotide and amino acid sequence identity, respectively, whereas TGB1 shared 97.4-100% nucleotide and 97-100% amino acid sequence identity. TGB1 of TN isolates were all longer by one codon from that of a Korean isolate, which is the only sequence publicly available. Phylogenetic analysis of nucleotide and amino acid sequences of TGB1 and CP of all known HVX isolates, separately or combined, revealed a close relationship, suggesting that all of them are derived from a common ancestor. Phylogenetic analysis with the type member of each genus of the family Flexiviridae confirmed that HVX is a member of a distinct species of the genus Potexvirus.

Comparison of aflatoxigenic and nonaflatoxigenic isolates of Aspergillus flavus using DNA amplification fingerprinting techniques.

Aspergillus flavus is a filamentous fungus that produces mycotoxins in many food and feed crops, such as maize (Zea mays L.). Isolates were analyzed for toxin production by nucleic acid profiles in an attempt to differentiate aflatoxigenic from nonaflatoxigenic isolates. A total of 41 aflatoxigenic and 34 nonalfatoxigenic isolates were included in the study. The isolates were evaluated initially using DNA amplification fingerprinting (DAF) without clear resolution of the groups. A weak association of aflatoxigenic isolates was observed, as evidenced by their clustering in 18 of 81 trees recovered from maximum parsimony analysis of binary characters derived from arbitrary signatures from amplification profiles (ASAP) data; nonaflatoxigenic isolates exhibited a pattern of paraphyletic laddering. Up to five markers unambiguously supported the aflatoxigenic isolate grouping, but the presence of alternative conflicting topologies in equally parsimonious trees precluded the observation of meaningful statistical support. With additional markers for genome of A. flavus, this method could be used to resolve toxigenic from nontoxigenic strains. This additional work could resolve aflatoxigenic isolates of A. flavus present on maize plants using ASAP, which would reduce labor intense costs and potentially lead to faster determination of resistant cultivars in breeding efforts.

Salivary apyrase in New World blackflies (Diptera: Simuliidae) and its relationship to onchocerciasis vector status.

Salivary gland apyrase is believed to be critical to blood-feeding in arthropod vectors. This enzyme was measured in six New World blackflies representing three taxonomic pairs of non-vectors and vectors of Onchocerca volvulus. In Simulium (Psilopelmia) ochraceum, a highly anthropophilic vector in Mexico and Guatemala, apyrase exhibited maximum activity between pH 8.0 and 9.0, mean 39.8 +/- 4.7 milliUnits/pair of gland equivalents (mU), and was enhanced when ATP was used as a substrate. In the zoophilic non-vector Simulium (Psilopelmia) bivittatum maximum activity was significantly less (5.1 +/- 0.7 mU) under all conditions examined. Preference for ADP or ATP as substrate was a function of the pH of the reaction for this species. Apyrase activity in Simulium (Simulium) metallicum Bellardi (29.5 +/- 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S. (Ps.) ochraceum (24.8 +/- 13.7 mU at pH 8.5) with ADP as substrate, but showed reduced activity with ATP. Both these Central American vectors had higher apyrase activity than found in Simulium (Notolepria) exiguum, a vector of O. volvulus in Ecuador and Colombia. However, maximum apyrase activity, measured at pH 8.0 with ADP as substrate, was greater in S. (N.) exiguum (10.9 +/- 0.6 mU) than in Simulium (Notolepria) gonzalezi (5.9 +/- 1.9 mU), a non-vector species widespread in Central America. Therefore, for the consubgeneric species pairs examined, a positive association was detected between higher concentrations of apyrase activity and their vector status for O.volvulus.