Molecular phytogeny and typing of blackflies (Diptera: Simuliidae) that serve as vectors of human or bovine onchocerciasis

Abstract. A subregion of the mitochondrial large subunit (16s) rRNA gene was amplified by polymerase chain reaction (PCR) from nine species of blackflies (Diptera: Simuliidae) which serve as natural or experimental vectors of human or bovine Onchocerca parasites. PCR products from each species of blackfly were tested by directed heteroduplex analysis (DHDA), and their genotypes established according to diagnostic banding patterns of the heteroduplex products. Three alleles of mitochondrial 16s rRNA were found to exist in members of the Simulium (Ewardsellum) damnosum sensu lato complex from West Africa, and two alleles were found in the Neotropical Simulium (Psilopelmia) ochraceum Walker complex and the Simulium (Simulium) metallicum Bellardi complex. Different single alleles were detected in Austrosimulium bancrofti , in English S.(S.)noelleri and in two North American laboratory vectors: Simulium (Psilozia) vittatum Zetterstedt and S.(S.)decorum Walker. Phylogenetic analysis of 16s sequences indicated that blackflies from West Africa and the Americas formed distinct clades. Neotropical onchocerciasis vectors were found to be more closely related to Nearctic and Palaearctic non-vector Simulium species than to the African vectors of onchocerciasis.     

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

Abstract. 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 pM 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 pM 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 pM 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S.(Ps.)ochraceum (24.8 pM 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 pM 0.6mU) than in Simulium (Notolepria) gonzalezi (5.9pM1.9mU), 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.     

Cytological approaches to simuliid biosystematics in relation to the epidemiology and control of human onchocerciasis

This paper presents an overview of advances in cytological research on the biosystematics of vector simuliid complexes in the areas of identification, age grading, and the evolution of resistance in relation to the epidemiology and control of human onchocerciasis. Systematic theory is discussed and relevant examples are given to show its application in predicting and resolving current problems in species identification for New World and Old World vector complexes. These complexes include Simulium damnosum s.l. and S. neavei s.l. from Africa and S. exiguum s.l., S. metallicum s.l., S. ochraceum s.l., and S. oyapockense s.l. from Latin America. The evolution of resistance in S. damnosum s.l. and the need for future molecular research as part of resistance management strategies are discussed.     

An epizootic of patent iridescent virus disease in multiple species of blackflies in Chiapas, Mexico

Simulium blackfly larvae (Diptera: Simuliidae) were collected from rivers and streams at 500-1500 m a.s.l. in Chiapas State of southern Mexico. Among 45 sites surveyed over an area of 2300 km2 (around 15 degrees 15'N 92 degrees 20'W), some Simulium larvae from three sites were opalescent violet-blue, interpreted as patent infection with invertebrate iridescent virus (IIV). Dissection confirmed the presence of putative Iridovirus particles, 130nm diameter, but no IIV isolates were obtained from homogenates injected into Galleria mellonella (L) larvae (Lepidoptera: Pyralidae). All Simulium with patent IIV infection died before metamorphosis, whereas approximately 60% of asymptomatic Simulium survived to adulthood in the laboratory. During 1997, standard monthly samples from two parallel rivers 42-50 km north-west of Tapachula comprised the following species proportions (and rates of patent IIV infection): 41.8% (47%) Simulium mexicanum Bellardi complex, 31.3% (31.4%) S. rubicundum Knab, 10.1% (13.1%) S. paynei, 6.5% (2.9%) S. callidum (Dyar & Shannon), 6.3% (5.1%) S. ochraceum Walker complex, 3.1% (0.7%) S. downsi Vargas et al., 0.7% S. samboni Jennings and 0.2% S. metallicum Bellardi complex, showing a strong correlation between blackfly abundance and the prevalence of patent infection. An epizootic of IIV in January and February (infection rates 41-100%) was followed by absence of larvae (March-August) until the end of the rainy season, when numbers collected on nylon strings rose to approximately 1/cm with patent IIV infection rates of 0-12.5% during September-December. Further investigations are underway to isolate this IIV and assess its potential usefulness for biological control of Simulium pests and vectors of onchocerciasis.     

First report of a trichomycete fungus (Zygomycota: Trichomycetes) inhabiting larvae of Simulium ochraceum sensu lato Walker (Diptera: Simuliidae) from the Galapagos Islands

A collection of the larval black fly Simulium ochraceum sensu lato Walker was made at six lotic habitats on San Cristobal Island, Galapagos Islands, Ecuador, during May 2003. Sixty-eight larvae were assayed for the presence of gut fungi (Zygomycota: Trichomycetes). A trichomycete, designated as Smittium sp. was found inhabiting the hindgut of 17.6% of S. ochraceum s.l. larvae. Implications of this finding with respect to both host and trichomycete biogeography are discussed.     

Larval description of Simulium (Notolepria) cuasiexiguum and Simulium (Chirostilbia) obesum and new records of black fly species (Diptera: Simuliidae) in the states of São Paulo and Minas Gerais, Brazil

We describe the larval stage of Simulium cuasiexiguum Shelley, Luna Dias, Maia-Herzog & Lowry and Simulium obesum Vulcano and provide new distribution records of four black fly species in the State of S?o Paulo (Simulium cuasiexiguum, Simulium metallicum s.l. Bellardi, Simulium stellatum Gil Azevedo, Figuer6 & Maia-Herzog and Simulium oyapockense s.l. Floch & Abonnenc) and three species in the State of Minas Gerais (Simulium duodenicornium Pepinelli, Hamada & Trivinho-Strixino, Simulium obesum and Lutzsimulium pernigrum Lutz).     

Structure and function of the cibarial armature in Simuliidae

Abstract. Cibarial armature morphology in adult female blackflies (Diptera: Simuliidae) is described using scanning electron microscopy. Three distinct types of armature are recognized, comprising those with teeth, e.g. Simulium ochraceum, S. ornatum, S. veracruzanum and S. vorax ; those with spicules, e.g. Austrosimulium bancrofti, S.damnosum, S.exiguum, S.metallicum and S.neavei; and those lacking these projections, e.g. Prosimulium rufipes and S.lineatum.
Whereas the armature is poorly developed in vectors of human onchocerciasis such as S. damnosum, S. exiguum, S. metallicum and S. neavei , the well-developed armature in S. ochraceum, S. veracruzanum and S. vorax does not prevent these species becoming infected with Onchocerca spp. (Nematoda: Onchocercidae). Hence the armature is not primarily a mechanism to counteract microfilaria superinfection.
Since cibarial armatures are more developed in the haematophagous females than in the males of certain Families of flies, e.g. Ceratopogonidae, Culicidae, Phlebotominae and Simuliidae in the sub-order Nematocera, evidently the armature has evolved in response to the blood-feeding habit. As the suction of imbibed blood by the cibarial pump may require a valve mechanism to prevent back-flow, it is suggested that the armature is primarily for this purpose. Secondarily, the cibarial armature presents a damaging barrier against ingested microfilariae.     

A cytogenetic analysis of the Simulium ochraceum species complex (Diptera: Simuliidae) in Central America.

A cytobiotaxonomic study of the medically important insect vector Simulium ochraceum s.l. revealed two sibling species and one cytotype from various endemic and nonendemic zones of human onchocerciasis in Guatemala and Mexico. Polytene chromosome maps and idiograms as well as notes on the biology of the three taxa designated S. ochraceum A, S. ochraceum B, and S. ochraceum C within the subgenus Psilopelmia are presented. All three taxa exhibit distinct sex chromosomes and taxon-specific suites of autosomal inversion polymorphisms. Simulium ochraceum C differs from both S. ochraceum A and S. ochraceum B by five interspecific inversions designated IIS-7,8 and IIIL-12, 13 + 14, 15. The three taxa exhibit niche and biting preferences, with S. ochraceum A being highly anthropophilic. Analysis of autosomal inversion polymorphism profiles indicates S. ochraceum A has long-range dispersal capability. Our results are consistent with the general findings that in the Simuliidae, sibling speciation may be suspected wherever a morphospecies occupies different niches in a stream continuum. We find for the first time an apparent partitioning of taxa by altitude. Simulium ochraceum A may be a primary vector of human onchocerciasis in Guatemala, as its vertical distribution is coincident with that of the highest areas of nodule prevalence in the human population. A genotypic component of variation in vectorial capacity of S. ochraceum A populations seems to occur, since the Y4 chromosome and its X chromosome counterpart are associated with hyperendemic areas of human onchocerciasis. Our observation that a supernumerary band, 37B1Hb, is associated with sex determination in two of the taxa may be of significance for the elucidation of the molecular basis of sex determination and possible resolution of issues pertinent to the general model of sympatric speciation in the Simuliidae.     

Phosphoglucomutase and trehalase isoenzymes of Venezuelan simulium vectors of Onchocerca volvulus

Phosphoglucomutase (PGM) and trehalase (Tre) isoenzymes of five species of Simulium blackflies (Diptera: Simuliidae), vectors of onchocerciasis in Venezuela, were investigated by means of a portable electrophoresis field kit. Tre differed between S. incrustatum and S. oyapockense s.l. Electrophoretic variation of Tre in other members of the S. amazonicum and S. incrustatum groups merit further investigation. PGM appears to be more useful for separating populations within species complexes. Multiple populations and/or seasonal changes in population structure of S. guianense s.l., S. exiguum s.l. and S. metallicum s.l. were inferred from elecrophoretic variation of PGM.     

广东蚋属二新种记述 (双翅目:蚋科)

广东蚋属二新种记述（双翅目：蚋科）安继尧郝宝善＊严格（军事医学科学院微生物流行病研究所北京１０００７１）＊广州军区防疫队１９９５－０５－０５收稿,１９９６－０８－２２收修改稿１９９０年４月,在广东省上川岛采蚋时,发现黄足真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉ...     

Spatial and temporal abundance,substrate partitioning and species co-occurrence in a guild of Neotropical blackflies (Diptera: Simuliidae)

Blackfly larvae were sampled monthly from two smallNeotropical mountain streams 500 m apart from each other in northernVenezuela during a 15-month period, and ten habitat variables weremeasured at two altitudes (800 and 900 m) to determine local spatialand temporal variations in abundance, stream variables, speciesco-occurrence, and substrate preference within the blackflyguild. The blackfly species composition of each stream was the same,with five species (in order of abundance): Simulium ochraceumWalker s.l., S. paynei Vargas, S. metallicum Bellardicytospecies E, Simulium sp., and S. horacioi Okazawa &Onishi. There were clear differences in community structurebetween streams (not among altitudes), although S. ochraceums.l. was the most abundant species in both habitats. Principal component andcorrelation analyses revealed that structural (great depth, width,flow, and discharge) and chemical (low conductivity) factors werethe stream variables best associated to the spatial distribution ofmost blackfly species. Blackfly abundance was greater in the largerstream. Larvae were most abundant on submerged rocks and fallenleaves, although the patterns of substrate use within the guildrevealed substrate partitioning among most blackfly species.There was one general annual peak of abundance at the end of therainy season. We suggest that blackfly community structure in theseNeotropical headwaters streams, over the small spatial scaleexamined here, is largely influenced by interactions among streamsize and velocity-related factors (width, streamflow, discharge andsubstrate availability-stability) and the rainfall regime.     

滋生于海南岛五指山瀑布岩壁上蚋属二新种(双翅目,蚋科)

首次记述我国大陆孳生于海南岛五指山瀑布岩壁上蚋属2新种,即瀑布蚋Simulium ( Simulium ) waterfallum sp. nov.和副瀑布蚋S. ( S. ) parawaterfallum sp. nov.二者均隶属于蚋亚属Simulium的多条蚋组multistriatum-group以其孳生环境命名.文中对其两性成虫、蛹和幼虫进行描述并分别与其近缘种进行分类讨论.模式标本存放于贵阳医学院生物学教研室.     

贵州蚋科一新种 (双翅目:蚋科)(英文)

本文记述采自贵州雷公山的蚋属一新种 ,以产地命名为黔蚋Simulium (Simulium )qianensesp .nov。新种隶属于杂色蚋组S .variegatumspecies group ,与台湾蚋S .taiwanicum最近似 ,但可依其特殊的盾饰、足的颜色、雄尾中骨端裂和幼虫后颊裂深并在后腹部具附骨等特征与其他近缘种相区别。模式标本存放于贵阳医学院生物教研室。     

贵州蚋属一新种(双翅目：蚋科)

记述采自贵州雷公山的蚋属一新种, 显著蚋Simulium (Simulium) prominentum, 新种。根据其蛹和幼虫的特征显著而命名, 对其幼期形态进行描述并与其近缘种作分类讨论。     

西藏林芝真蚋亚属三新种（双翅目：蚋科）

本文记述西藏林芝真蚋亚属Ｅｕｓｉｍｕｌｉｕｍ三种：凸端真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉｍｕｌｉｕｍ）ｃｏｎｃａｖｕｓｔｙｌｕｍｓｐ．ｎｏｖ．、林芝真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉｍｕｌｉｕｍ）ｌｉｎｇｚｉｅｎｓｅｓｐ．ｎｏｖ．、裂缘真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉｍｕｌｉｕｍ）ｓｃｈｉｚｏｌｏｍｕｎｓｐ．ｎｏｖ 。     

陕西省蚋属一新种记述(双翅目:蚋科)

Simulium (Simulium) liubaense, sp. nov. is described based on the female specimens collected from Liuba, Shaanxi Province, China. This species is assigned to the variegotum-group of the subgenus Simulium, and is closely related to S. (S.) jingfui Cai and An, 2008, S. (S.) taiwanicum Takaoka, 1979, S. (S.) hackeri Edwards 1928, S. (S.) chamlongi Takaoka and Suzuki, 1984. However it is clearly differentiated from them by the structure of cibarium, genital fork, genital plate, paraproct and cercus of the female. All the specimens are kept in the Medical Entomology Collection Gallery, Academy of Military Medical Sciences, Beijing.     

贵州蚋属一新种 (双翅目：蚋科)

 记述采自贵州雷公山的蚋属一新种, 显著蚋Simulium (Simulium) prominentum, 新种 。根据其蛹和幼虫的特征显著而命名, 对其幼期形态进行描述并与其近缘种作分类讨论。     

Identification of members of the Simulium ochraceum species complex in the three onchocerciasis foci in Mexico

Larval collections of Simulium ochraceum Walker (Diptera: Simuliidae) were made in a variety of streams in the three onchocerciasis foci in Mexico and identified by cytotaxonomic criteria, based on the banding pattern of polytene chromosomes in larval salivary gland nucleii. S. ochraceum cytotype A was found in the Soconusco focus, cytotype B in the Oaxaca focus and cytotype C (not previously recorded in Mexico) in the Chamula focus. Attempts to analyse chromosomes of adult specimens were unsuccessful. A preliminary study of the cuticular hydrocarbons of adult specimens by gas liquid chromatography provided evidence that this technique may be suitable for separating the cytotypes.     

广东蚋属二新种记述(双翅目：蚋科)

Five speices of Simuliidae, Simulium (Eusimulium) aureohirtum Brunetti,1911, S. (Simulium ) quinquestriatum (Shiraki, 1935) S.(Gomphostilbia ) pingxiangense An & Hao, 1990, S.(S.) shangchuanense sp. Nov. , S.(S.) spoonatum sp. Nov. were collected from Shangchuan Island, Guangdong Province, China. Both adult and pupal stages of the new species are described in this paper. All the specimens are kept in the Medical Entomological Specimen Building, Institute of Microbiology and Epidemiology Academy of Military Medical Sciences, Beijing.     

贵州纺蚋亚属二新种(双翅目： 蚋科)

记述了采自贵州雷公山自然保护区的纺蚋二新种,清水纺蚋Simulium (Nevermannia) qingshuiense sp. Nov.和桥落纺蚋S. (N.) qiaolaoense sp. Nov.,二者均以其产地命名。文中对其成虫、蛹和幼虫形态进行了全面描述,并与其近缘种进行分类讨论。模式标本存放在贵阳医学院生物学教研室。