Cryptic biodiversity in the cytogenome of bird-biting blackflies in North Africa.

Bird-biting blackflies in the Simulium (Eusimulium) aureum group (Diptera: Simuliidae) are widespread vectors of Leucocytozoon and Trypanosoma parasites. The polytene chromosomes of 619 larvae of the three nominal members of the S. aureum group in North Africa were evaluated cytogenetically for cryptic biodiversity. Seven chromosomal segregates were discovered among 29 populations in Algeria and Morocco. This diversity was based primarily on two chromosomal inversions, which have assumed unique roles in different lineages, including sex linkage, fixation, loss and autosomal polymorphism. Reproductive isolation was demonstrated for six of the seven segregates, doubling the number of species known in the area. Four species were linked with existing names: (a) Simulium mellah Giudicelli & Bouzidi, which is known only from North African high-salinity habitats; (b) Simulium petricolum (Rivosecchi), which is tentatively conspecific with continental European populations; (c) Simulium rubzovianum (Sherban) and its synonym Simulium latinum (Rubtsov), which is widely distributed from North Africa across Europe into Western Asia, and (d) Simulium velutinum (Santos Abreu) and its new synonym Simulium tenerificum Crosskey, which is restricted to North Africa and the Canary Islands. Of the remaining entities, two are new species precinctive to North Africa and one, known only from Morocco, is of undetermined taxonomic status.

A multi-locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages.

A multi-locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria-encoded cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA genes, and the nuclear-encoded 18S rRNA and 28S rRNA genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi-locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups.

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.

Spatial stratification of host-seeking Diptera in boreal forests of northern Europe.

The stratification of haematophagous Diptera was assessed in two boreal forests in northern Sweden by placing traps baited with carbon dioxide at 1.5 m, 5.0 m and 10.0 m above the ground. More than 40 000 specimens were captured, including 617 biting midges (Ceratopogonidae), 4029 mosquitoes (Culicidae) and 36 092 black flies (Simuliidae). Catches at the various trap heights reflected the general vertical distribution of the preferred hosts, with mammalophilic flies predominating (68.6%) in catches at 1.5 m and ornithophilic flies (42.4%) in catches at 10.0 m; however, most flies that use host birds at ground level were caught in the lowest traps (e.g. 85.1% of Simulium annulus were collected at 1.5 m). Within-species variation in vertical patterns between forests suggests plasticity in responses to environmental factors such as vegetative structure.

Blood-feeding ecology of mosquitoes in zoos.

To determine if the unique host assemblages in zoos influence blood-feeding by mosquitoes (Diptera: Culicidae), a sampling programme was conducted in Greenville and Riverbanks Zoos, South Carolina, U.S.A., from April 2009 to October 2010. A total of 4355 female mosquitoes of 14 species were collected, of which 106 individuals of nine species were blood-fed. The most common taxa were Aedes albopictus (Skuse), Aedes triseriatus (Say), Anopheles punctipennis (Say), Culex erraticus (Dyar & Knab), Culex pipiens complex (L.) and Culex restuans (Theobald). Molecular analyses (cytochrome b) of bloodmeals revealed that mosquitoes fed on captive animals, humans and wildlife, and took mixed bloodmeals. Host species included one amphibian, 16 birds, 10 mammals (including humans) and two reptiles. Minimum dispersal distances after feeding on captive hosts ranged from 15.5 m to 327.0 m. Mosquito-host associations generally conformed to previous accounts, indicating that mosquito behaviour inside zoos reflects that outside zoos. However, novel variation in host use, including new, exotic host records, warrants further investigation. Zoos, thus, can be used as experiment environments in which to study mosquito behaviour, and the findings extrapolated to non-zoo areas, while providing medical and veterinary benefits to zoo animals, employees and patrons.

Vertical distribution of haematophagous Diptera in temperate forests of the southeastern U.S.A.

The vertical distribution of blood-feeding flies in two temperate forests in the southeastern U.S.A. was determined by placing 15 Centers for Disease Control and Prevention miniature light traps (12 CO(2)-baited, three unbaited controls), without lights, at three heights (1.5 m, 5.0 m, 10.0 m). More than 6550 haematophagous flies, representing 49 species in four families, were collected. Eighteen species were taken almost exclusively (90-100%) at 1.5 m or 10.0 m, and the mean number of flies per trap differed significantly with height for another six species. Five species exhibited shifts in vertical distribution between the two forests, indicating that forest structure could influence the height of host searching. Most (52.5%) mammalophilic flies were collected at 1.5 m, whereas most (56.4%) ornithophilic flies were taken at 10.0 m, suggesting that host associations influence vertical distributions. The significant differences in the composition of haematophagous fly populations among forest strata emphasize the importance of trap placement in vector surveillance and of understanding the ecological relationships of blood-feeding flies.

Seasonal colonization and decomposition of rat carrion in water and on land in an open field in South Carolina.

Decomposition and insect colonization of rat, Rattus rattus L., carrion on land and in water were compared during summer and winter in a plowed field in northwestern South Carolina. During winter, carcasses on land reached the dried-remains stage of decomposition, whereas carcasses in water reached the early-floating stage. During summer, carcasses in both habitats entered the final-remains stage of decomposition in 1-2 wk. Fewer than 30 species of carrion insects were recorded from the carcasses over the duration of the study, probably reflecting the small size of the carcasses and the depauperate fauna of the habitat. Three species of blow flies--Cynomyopsis cadaverina (Robineau-Desvoidy), Calliphora vicina Robineau-Desvoidy, and Lucilia illustris (Meigen)--colonized carrion on land during winter, but no insects colonized carrion in water during winter. Two species of blow flies, Cochliomyia macellaria (F.) and Phaenicia sericata (Meigen), and 1 species of flesh fly, Sarcophaga bullata Parker, colonized the carrion on land and in water during summer; the blow fly, Phormia regina (Meigen), colonized only the carrion on land. This study demonstrated seasonal variation in decomposition and colonization patterns of carrion in contrasting habitats, with important implications for forensic entomology.

Economic impact of black flies (Diptera:Simuliidae) in South Carolina and development of a localized suppression program.

The membership of a private golf club in South Carolina's Piedmont Physiographic Region was surveyed to determine perceptions regarding local black fly populations and the effects of these populations on golfing habits. The economic impact of black fly annoyance at this club was estimated to be $27,202. Adult monitoring indicated that the pest species were members of the Simulium jenningsi group; larval monitoring identified the Enoree River, Laurens County, as the primary source of the pest species. A localized suppression program was initiated during the late summer of 1994 and continued through the fall of 1995. Four larvicide applications were conducted in 1994 and 11 in 1995, using the biological insecticide Vectobac. Larval mortalities of 94.5 and 97% were produced during 1994 and 1995, respectively. Adult black fly populations were reduced 92 and 88% during 1994 and 1995, respectively, from pre-program populations. Improvements for future suppression programs are proposed.

Development of the first black fly (Diptera: Simuliidae) management program in Argentina and comparison with other programs.

In response to increasing pest populations of black flies, the government of Argentina initiated a pilot program to evaluate the effectiveness of larval control in the irrigation system of the Negro River Valley, an 18,240-km2 area in Patagonia. The extensive system of irrigation canals, drainage ditches, and natural waterways not only provides water for agriculture and general habitation but also affords habitat for immature black flies. Three species (Simulium bonaerense, Simulium wolffhuegeli, and Simulium nigristrigatum) are primary pests of humans and animals in this area. Trials were conducted using Vectobac AS in representative irrigation canals, a drainage ditch, and a medium-sized river. Most trials resulted in effective larval mortality and insecticide carry. Based on a comparison with 11 black fly suppression programs throughout the world, a suppression program for the Negro River Valley has a projected cost of approximately $1,623,360/year. This study represents the initial steps in development of the 1st areawide black fly suppression program in Argentina.

Comparative morphology of the pyloric armature of adult mosquitoes (Diptera: Culicidae).

The structure of the pyloric armature, hypothesized to aid in blood-meal digestion or parasite resistance, was compared quantitatively among the following 8 species in 5 genera of adult mosquitoes from the southeastern United States: Aedes albopictus, Aedes japonicus, Aedes triseriatus, Anopheles punctipennis, Culex pipiens s.l., Culex restuans, Orthopodomyia signifera, and Toxorhynchites rutilus. Females differed significantly among species in the structure of spines composing the armature, with Aedes spp. forming one general group, Culex spp. another, and An. punctipennis and Or. signifera a third. Relationships of species based on structural characters of the armature were consistent with recent culicid phylogenies. Although pyloric armature has been noted in mosquitoes and other insects, this is the first quantitative investigation of the mosquito pyloric armature.

Polytene chromosomes of Simulium arakawae, a pest species in the Simulium venustum group (Diptera: Simuliidae) from Japan.

The chromosomal relationships of Simulium arakawae, a black fly of medical-veterinary importance, are resolved relative to other members of the Simulium venustum species group and to the standard reference map for the subgenus Simulium. Simulium arakawae differs from the subgeneric standard by eight fixed inversions, apparently none of which are shared with any of the other 17 chromosomally studied nominal members of the group. The chromosomal features that indicate group membership might have been lost in S. arakawae, particularly if they were polymorphic in an ancestor.

Polytene chromosomes of Simulium arakawae, a pest species in the Simulium venustum group (Diptera: Simuliidae) from Japan

The chromosomal relationships of Simulium arakawae, a black fly of medicalveterinary importance, are resolved relative to other members of the Simulium venustum species group and to the standard reference map for the subgenus Simulium. Simulium arakawae differs from the subgeneric standard by eight fixed inversions, apparently none of which are shared with any of the other 17 chromosomally studied nominal members of the group. The chromosomal features that indicate group membership might have been lost in S. arakawae, particularly if they were polymorphic in an ancestor.

Molecular characterization and taxonomy of a new species of Caudosporidae (Microsporidia) from black flies (diptera: simuliidae) with host-derived relationships of the North American caudosporids.

A new species of microsporidium, Caudospora palustris (Microsporidia: Caudosporidae), is described from 3 species of black flies (Cnephia ornithophilia and diploid and triploid cytospecies of Stegopterna mutata), bringing to 7 the total species of caudosporids recorded from North America. This new species of caudosporid is recorded from swamp streams of the Coastal Plain from New Jersey to Georgia, with single records from the New Jersey mountains and the Upper Peninsula of Michigan. Densities of patently infected larvae (up to 10,600/m2) and spore production (nearly 8x10(11)/m2) are the greatest recorded for any microsporidium of black flies. The ultrastructure of this new species is presented, along with the first molecular characterization for a microsporidium of black flies. The phylogenetic position of black fly microsporidia within the phylum Microsporidia is presented; however, the analysis does not support the inclusion of C. palustris in any clade. Key features of all North American caudosporids are provided, and possible evolutionary trajectories are proposed based on optimization of caudosporid species on the phylogeny of their 22 known host species, including 16 that represent new host species records.

Molecular differentiation of two sibling species of the black fly Simulium vittatum (Diptera: Simuliidae) based on random amplified polymorphic DNA.

Larvae of the black fly morphospecies Simulium vittatum from Colorado, Montana, Nebraska, and New Hampshire were cytologically identified as either the IS-7 or the IIIL-1 cytospecies. DNA was PCR amplified from cytotyped larvae using eight 10-mer primers, labeled with 33P, and run on polyacrylamide gels. The entire data set of 96 amplicons produced incomplete separation of the two cytospecies when subjected to neighbor-joining and maximum parsimony analyses. However, when analyzed within geographical regions, separate species status was supported. Bootstrap support for distinctness of the two cytospecies was best in Colorado where they were collected in true sympatry. The IS-7 cytospecies was more polymorphic in the western states, where it differed most from IIIL-1, which was most variable in the eastern states. The frequencies of the 17 most common amplicons in the two cytospecies were inversely correlated. A model of speciation derived from the molecular evidence suggests that IS-7 evolved in the west and spread eastward, whereas IIIL-1 later originated in the east and spread westward.