Cytogenetical analysis of the Simulium damnosum complex (Diptera: Simuliidae) in Guinea Bissau

Abstract. In a 3-year study during 1990–92, larval collections of the Simulium damnosum complex from the River Corubal system in Guinea Bissau revealed that the only sibling species present were S. sirbanum and the Konkouré form of S. konkourense , but not S. damnosum s.s. which had been found at some of the localities (Saltinho and Cusselinta) in a previous survey by Quillévéré et at. (1981). Their differential distributions were seasonally consistent between years. S. sirbanum was concentrated in the upper reaches of the Corubal, moving downstream at the end of the dry season to exploit new breeding grounds, whereas S. konkourense seemed to be concentrated in the lower reaches and moved upstream in the middle of the dry season.
Since the previous survey, it appears that S. konkourense has largely replaced S. sirbanum and S. damnosum s.s. in the lower reaches of the Corubal. S. sirbanum was consistently associated with man-biting behaviour and, although infection studies were not performed, it is likely that S. sirbanum is the main vector of onchocerciasis in Guinea Bissau as S. konkourense does not seen to be anthropophilic.
Among five polymorphic inversions in S. sirbanum , the frequency of inversion IL-B increased during the dry season; IL-2 and IIL-7 showed homozygous disadvantage whereas IIL-3 and IIIL-6 did not; IS-2 tended to be fixed in all populations. Simulium konkourense populations in the River Corubal differ from those found upstream in its tributary the River Koumba, in the Fouta Djallon of Guinea, by their sex chromosomes (having no sex-linked inversions) and biting preferences, indicating that the populations are not freely interbreeding. Inversion IIIL-X was found for the first time in populations of S. konkourense outside the Fouta Djallon area and a new inversion IIL-Y was identified in samples from the Rivers Corubal and Féfiné.     

DNA probes for the identification of members of the Simulium damnosum complex (Diptera: Simuliidae)

Genomic libraries in plasmid have been constructed from various sibling species of blackflies of the Simulium damnosum complex from West Africa. Three cloned repetitive sequences, which show variation in copy number between sibling species, have been isolated. These clones can be used as probes for the dot-blot identification of larvae, pupae or adults into the three main West African subcomplexes, i.e. damnosum, squamosum and sanctipauli subcomplexes. The sequences also show some intraspecific variation in copy number.     

尼日利亚西南部奥孙河沿线黑蝇Simulium damnosum Theobald complex的形态分类研究(双翅目: 蚋科)（英文）

黑蝇Simulium damnosum sensu lato是由多个姊妹种组成的复合体,这些种在生态学和盘尾丝虫病的传播方面各不相同。本文对奥孙河沿线的尼日利亚西南部森林区域的黑蝇S. damnosum s.l.复合体的组成以及成虫的形态学特征进行了研究。本研究所用的黑蝇S. damnosum s.l.成虫标本来源于奥孙河沿线的3个区,分别是Osun Eleja, Osun Ogbere和Osun Budepo。标本采集通过人体诱捕的方式,采集时间从2008年2月至2009年6月上午7:00到下午6:00,每两周采集1次。通过观察成虫的翅毛簇（wing tufts）和其他形态特征,对其进行分类研究。结果表明：存在同域分布的森林种和稀树草原种。在奥孙河沿线3个区内,森林种为优势种,占总捕获量的99.18%,而稀树草原种仅占0.82%;森林种和稀树草原种在多度上存在显著差异(P<0.05)。所捕获的所有稀树草原种的翅毛簇均为灰白色,而捕获的森林种的翅毛簇颜色存在显著差异(P<0.05)。为了更清楚地揭示该地区黑蝇S. damnosum s.l.的物种组成,建议进行更深入的研究。     

尼日利亚西南部奥孙河沿线黑蝇Simulium damnosum Theobald complex的形态分类研究(双翅目:蚋科)

黑蝇Simulium damnosum sensu lato是由多个姊妹种组成的复合体,这些种在生态学和盘尾丝虫病的传播方面各不相同.本文对奥孙河沿线的尼日利亚西南部森林区域的黑蝇S.damnosum s.l.复合体的组成以及成虫的形态学特征进行了研究.本研究所用的黑蝇S.damnosum s.l.成虫标本来源于奥孙河沿线的3个区,分别是Osun Eleja,Osun Ogbere和Osun Budepo.标本采集通过人体诱捕的方式,采集时间从2008年2月至2009年6月上午7:00到下午6:00,每两周采集1次.通过观察成虫的翅毛簇(wing tufts)和其他形态特征,对其进行分类研究.结果表明:存在同域分布的森林种和稀树草原种.在奥孙河沿线3个区内,森林种为优势种,占总捕获量的99.18%,而稀树草原种仅占0.82%;森林种和稀树草原种在多度上存在显著差异(P<0.05).所捕获的所有稀树草原种的翅毛簇均为灰白色,而捕获的森林种的翅毛簇颜色存在显著差异(P<0.05).为了更清楚地揭示该地区黑蝇S.damnosum s.l.的物种组成,建议进行更深入的研究.     

Poor genetic differentiation but clear cytoform divergence among cryptic species in Simulium damnosum complex (Diptera: Simuliidae)

A complex cryptic species taxonomy has been developed for the flies that vector Onchocerca volvulus L euckart in A frica. This taxonomy names cytoforms of Simulium damnosum T heobald s.l. based on chromosomal inversion polymorphisms. Researchers have linked variation in cytoforms to variation in geographical distributions and biological traits, but have failed to demonstrate that many of these cytoforms are differentiated genetically or morphologically. This may be explained by recent divergence and incomplete lineage sorting of ancestral polymorphisms. Alternatively, it may be the outcome of ongoing gene flow, with inversions maintaining sets of co‐adapted alleles and selection driving inversion frequency differences across habitats. Here, our goals were to test species delimitations in the S. damnosum complex, and determine if the poor differentiation between these species is better explained by the ancestral polymorphisms or gene‐flow hypotheses. Geometric morphometric analysis and more intensive genetic sampling of populations in N igeria only reinforced the lack of clear divergence among S. damnosum complex cytoforms. By contrast, the first statistical estimates of evolutionary relationships based on chromosomal inversion polymorphism data revealed clear signs of divergence, which lends support to the ancestral polymorphisms hypothesis. But the latter analyses may be biased by non‐neutral evolution of inversion polymorphisms; more rigorous tests of cytoform‐based species hypothesis will require deeper sampling across populations and neutral genetic markers along with explicit modelling of genetic isolation and migration.     

Identification of Anopheles (Nyssorhynchus) albitarsis complex species (Diptera: Culicidae) using rDNA internal transcribed spacer 2-based polymerase chain reaction primes

Anopheles (Nyssorhynchus) marajoara is a proven primary vector of malaria parasites in Northeast Brazil, and An. deaneorum is a suspected vector in Western Brazil. Both are members of the morphologically similar Albitarsis Complex, which also includes An. albitarsis and an undescribed species, An. albitarsis "B". These four species were recognized and can be identified using random amplified polymorphic DNA (RAPD) markers, but various other methodologies also point to multiple species under the name An. albitarsis. We describe here a technique for identification of these species employing polymerase chain reaction (PCR) primers based on ribosomal DNA internal transcribed spacer 2 (rDNA ITS2) sequence. Since this method is based on known sequence it is simpler than the sometimes problematical RAPD-PCR. Primers were tested on samples previously identified using RAPD markers with complete correlation.     

The cytotaxonomy of Simulium sanctipauli and Simulium soubrense (Diptera: Simuliidae)

It is noted that the chromosomal inversion 2L-7, which has been used in the past to separate S. Sanetipauli from S. soubrense, occurs as an intraspecific polymorphism and hence cannot be considered diagnostic, although in some populations 2L-7 can still strogly indicate the presence of two species. However, two newly recognised inversions, 1L-A and 2L-A, can be used in combination to identify S. sanctipauli, S. soubrense and a new species S. soubrense B. The absence of the relevant heterozygotes for these two new inversion confirms the separate specific status of S. sanctipauli from S. soubrense from S. soubrense B as well as providing a reliable means of larval identification. The misuse of 2L-7 as a species diagnostic inversion has undoubtedly led to past misidentifications of S. sanctipauli and S. soubrense, and it is possible, for example, that only S. sanctipauli is resistant to organophosphate insecticides in Ivory Coast and not S. soubrense. Beffa form appears to be a distinctive geographic race of S. soubrense, but forme konkouré remains as yet unassigned. A cytotaxonomic key for the identification of members of the S. sanctipauli sub-complex is presented.     

Polytene chromosomes of Simulium craigi (Diptera: Simuliidae)

Simulium craigi Adler and Currie is a polymorphic species based on polytene chromosome banding patterns in the long arm of chromosome III (IIIL). Three cytotypes are described based on the predominant IIIL sequences. These correspond to three broad geographic areas: cytotype CC from Pennsylvania; cytotype AF from Ontario and Manitoba; and cytotype ACF/BCF from New Hampshire. In the absence of sympatric populations, these cytotype differences are best explained by clinal variation within a single species. The relationship of S. craigi to other described members of the S. vernum group is discussed.     

Phylogeography of Simulium siamense Takaoka and Suzuki complex (Diptera: Simuliidae) in Thailand

The phylogeography of Simulium siamense complex was inferred from mitochondrial DNA sequences. A 586‐bp fragment of the cytochrome oxidase I was sequenced for 92 individuals from 13 populations throughout Thailand, representing five cytoforms (A, B, C, F and G). The cytoforms are not genetically different at the molecular level except for cytoform B, which is genetically distinct from the others. This might indicate that cytoform B is a distinct species. Further morphological and molecular work using other genes is needed to clarify this. Our results also argue for the need of integrated approach, both cytological and molecular studies to understanding biodiversity of black flies. The star‐like shape of the mtDNA genealogy is consistent with the sudden population expansion of the mismatch distribution analysis and large negative values of Fu's F_s and Tajima's D‐tests, indicating a population demographic expansion. The expansion time is estimated to be in the late Pleistocene (about 120 000 years ago). Therefore, the overall low level of genetic structure could be due to sharing a recent history. The ancestral haplotype was found in the mountainous area in northeastern Thailand, suggesting that this area could have been the refugium of the species complex during the Pleistocene glaciations. Our results are consistent with previous findings about population expansion in response to the Pleistocene climatic change, thus revealing the importance of this historical event in shaping the genetic structure and diversity of Southeast Asian mainland species.     

Cladistic analysis of Simulium (Trichodagmia) and Simulium (Thyrsopelma) (Diptera: Simuliidae)

A cladistic analysis of Simulium ( Trichodagmia ) sensu Crosskey and Howard, using 34 morphological characters of larvae (6 characters), pupa (5) and adults (23), yields nine most parsimonious trees under equal weights (length 101 steps CI 0.49 RI 0.73). Successive weighting based on the maximal rescaled consistency index preferred one of the nine (31.37 steps CI 0.62 RI 0.87 total fit_con3= 235.8), which was also one of two trees found under implicit weights with concavity values of 3–6. The cladogram justifies the recognition of two subgenera. Simulium ( Trichodagmia ) sensu stricto (containing S. muiscorum, sumapazense, S. wygodzinskyorum, S. nigrimanum, S. chalcocoma, S. huairayacu and S. lahillei ) is supported by the branchial tip sclerotization and the presence of cibarial teeth, larval body tegument covered with lanceolate hairs, female with simple claw, and gonapophysis size. Simulium ( Thyrsopelma ) (containing S. scutistriatum, S. hirtipupa, S. orbitale, S. guianense, S. perplexum and S. itaunense ) is supported by the hypostomial teeth.     

Patterns of polytene-chromosome replication in Simulium ornatipes (Diptera: Simuliidae)

Double labelling of Simulium ornatipes polytene chromosomes with H³- and C¹⁴-thymidine shows that chromosome synthesis follows three distinct phases viz. a short phase of initiation in puffs and interbands spreading to more condensed regions; a long continuous labelling phase, then a discontinuously labelled end phase as bands complete their replication in temporal sequence. Analysis of H³ labelling patterns indicates that while heterochromatic bands replicate there is no clear correlation between heterochromatic or C-banding regions and band replication time. The major characteristic governing band replication time appears to be band size and density. However, in some bands this relationship is modified, perhaps it is suggested, by DNA organisation influencing the efficiency of replicons. The existence of great variability in homologous band replication times, even within a chromosome pair, indicates that the control of band replication is highly autonomous. It is suggested that polymorphisms at the molecular level determine this variation. Replication time of active nucleolar organisers is very long in contrast to the short replication of condensed inactive organisers. This may reflect differential polytenisation of ribosomal DNA as a result of a developmental polymorphism, or the amplification of ribosomal DNA by active nucleolar organisers.     

Cytosystematics of the Simulium tuberosum group (Diptera: Simuliidae) in Thailand

The polytene chromosomes of 3347 larvae of the Simulium tuberosum group in Asia were analysed, representing the largest ever cytogenetic study of black flies in the Oriental Region. Band‐by‐band comparisons, relative to the established standard chromosome map for the subgenus Simulium, revealed 17 cytogenetically distinct taxa in Thailand, plus an 18th in China. Six of these taxa correspond to morphologically described species (S. doipuiense, S. rufibasis, S. setsukoae, S. tani, S. yuphae and S. weji). Recognition of the 18 taxa is based largely on unique inversions, either fixed or sex linked, primarily in the long arm of chromosome III. The greatest cytological diversity was discovered in the S. tani lineage, with ten cytoforms. This marked chromosomal diversification within S. tani is based largely on two inversions that have assumed different roles over evolutionary time, variously functioning in different combinations as fixed inversions, sex‐linked inversions and autosomal polymorphisms. Shared unique chromosomal features, relative to the subgeneric standard chromosome map, allowed evolutionary relationships among the cytotaxa to be inferred. Fluctuations in climate during the Pleistocene might have promoted differentiation of the Southeast Asian S. tuberosum group in isolated refugia such as mountains. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 289–315.     

Intrastrain internal transcribed spacer heterogeneity in Ganoderma species

Intrastrain internal transcribed spacer (ITS) heterogeneity is first reported from Ganoderma, a fungal genus within Basidiomycetes. ITS amplification products from 4 strains, representing 4 Ganoderma species, were cloned and sequenced. Two to five different ITS types were found within a single strain. The clone sequences were analyzed along with other sequences from Ganoderma retrieved from GenBank. The results show that sequence variation within strains varies considerably with species and the heterogeneity may occur in the 3 parts (ITS1, ITS2, and 5.8S) of the ITS region.     

Assessment of rDNA IGS as a molecular marker in the Simulium damnosum complex

For five cytospecies of the Simulium damnosum Theobald complex of blackflies (Diptera: Simuliidae) from West Africa, both ends of the intergenic spacer region (IGS) of the rDNA have been sequenced with the aim of developing specific molecular markers. No specific differences in these two regions were detected between Simulium sanctipauli V. & D., Simulium sirbanum V. & D., Simulium soubrense V. & D., Simulium squamosum Enderlein and Simulium yahense V. & D., except in the number of A subrepeats at the 5' end of the IGS (two in S. squamosum and four or five in the others) and in position 310 of the 3' end (a C in S. squamosum and a G in the others). However, genetic distances within and between species overlapped. These DNA sequences had no strong phylogenetic signal, and the trees obtained were mostly unresolved. Although most sequences from S. squamosum clustered together, a few of them were more similar to those in other cytospecies. These results could be explained either by hybridization with genetic introgression or by ancestral polymorphism and recent speciation.     

The speciation continuum: ecological and chromosomal divergence in the Simulium arcticum complex (Diptera: Simuliidae)

Chromosome inversions may be involved in adaptation and speciation. We investigate ecological diversification among members of the Simulium arcticum species complex at different stages of chromosome divergence. Our analyses focus on two geographical scales. First, we assess ecological divergence of sibling species throughout North America using niche modelling methods. Then, using canonical correspondence analysis, we investigate habitat associations of sibling species and cytotypes in the northern Rocky Mountains ecoregion, where cytotypes tend to occur. Despite significant overlap in predicted distributions, all sibling species are ecologically unique. On the other hand, we discover various degrees of ecological divergence for cytotypes. Some cytotypes are ecologically distinct and perhaps are in their initial stages of incipient speciation. Other cytotypes are ecologically associated with one another or with particular sibling species. Thus, for members of the S. arcticum complex, ecological and chromosomal differences tend to develop early in lineage formation. Ecological distinctiveness of sibling species and cytotypes suggests that local adaptation may be involved in diversification of these chromosomal forms. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 13–27.     

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.     

Two new black fly species of Simulium (Simulium) (Diptera: Simuliidae) from South India

Two new black fly species, Simulium (Simulium) pothigaiense sp. n. and Simulium (Simulium) valparaiense sp. n., are described based on females, males, pupae and larvae. Simulium (Simulium) pothigaiense sp. n. is characterized by a claw simple without subbasal tooth and the base of radial vein with hair tuft in the females, maxillary palp with small sensory vesicle in the males, pupal gill with 10 short slender filaments and very short common basal stalk in the pupae and 5 hypostomal bristles per side lying parallel to lateral margin in the larvae. Simulium (Simulium) valparaiense sp. n. is characterized by hind basitarsus 6.6 times as long as its greatest width and claw with small distinct subbasal tooth in the females, gonostyle in medial view 2.3 times longer than coxite in the males, common basal stalk of pupal gill 0.31 times as long as interspiracular trunk, and larval thoracic and abdominal cuticle with pair of dorsolateral protuberances. Taxonomic notes are given to distinguish these two new species from closely related species. www.zoobank.org/urn:lsid:zoobank.org:pub:7285F6BC-607D-4F76-B6EE-9CAC67CDE651