Recent studies on the epidemiology and control of Guatemalan onchocerciasis, chiefly made by the Guatemala-Japan Cooperative Project on Onchocerciasis Research and Control, are reviewed. Epidemiological features of Guatemalan onchocerciasis are summarized as to characteristic altitudinal distribution of endemic areas, disease manifestation, vector taxonomy, biology and transmission dynamic of the disease. Extensive insecticide studies in the field and laboratory demonstrate that the characteristic situations of Guatemalan streams where Simulium ochraceum, the main vector of onchocerciasis, breeds require ingenious methods of larviciding. Finally, the feasibility of an area vector control is indicated by the successful control operation in the San Vicente Pacaya Pilot Area, in which a new fixed-dose larviciding method was applied.
Recent studies on the biology and ecology of adult blackflies in relation to the transmission of human onchocerciasis in Guatemala are reviewed. First, earlier studies on the transmission of the disease since its discovery by Dr. R. Robles in 1915 are outlined. Second, eleven blackfly species caught on humans are evaluated for vector status on the basis of their natural and experimental infections with third-stage larvae of Onchocerca volvulus, and Simulium ochraceum,* S. metallicum* and S. callidum are confirmed as natural vectors of the disease in Guatemala in descending order of importance, whereas S. gonzalezi, S. haematopotum, S. veracruzanum and S. horacioi are potential vectors. Third, the migration and fate of O. volvulus microfilariae ingested by female blackflies are highlighted on the basis of the findings of the cibarial armature of S. ochraceum and physiological incompatibility of S. metallicum as main barriers against microfilariae, both of which greatly decrease the number of ingested O. volvulus microfilariae developing to the third-stage larvae per female. Fourth, among many ecological factors of female blackfly populations, geographical and altitudinal distributions, habits of blood feeding, host preferences for blood feeding, preference for human body parts, parous rates, daily and seasonal fluctuations of biting activities, in particular, of parous females, gonotrophic cycle, longevity, flight range, and annual transmission potential are reviewed, and their influences on the transmission dynamics of the disease agents are considered. Fifth, effects of air temperatures on the O. volvulus-S. ochraceum complex are examined, with a special reference to the characteristic altitudinal distributions of the disease. The importance of reliable identification of both the vector blackfly species and filarial larvae found in female blackflies is emphasized to understand the transmission of the disease. [*It is now known that these two species are actually species complexes.].
The male, pupa and mature larva of Simulium (Asiosimulium) wanchaii Takaoka & Choochote, one of the four species of the small Oriental black fly subgenus Asiosimulium, are described for the first time based on samples collected from Thailand. The male S. (A.) wanchaii is characterized based on the enlarged hind basitarsus and the ventral plate which is much wider than long. The pupa and larva are characterized by the gill with 19 filaments and the deep postgenal cleft, respectively. Keys are provided to identify all the four species of the subgenus Asiosimulium for females, males, pupae and mature larvae.
A total of 29 female black flies were captured by a hand net as they swarmed around humans in Tam Dao National Park, Vinh Phuc Province, Vietnam. They included one species of the subgenus Gomphostilbia (Simulium (Gomphostilbia) asakoae Takaoka & Davies) and five species of the subgenus Simulium, of which one species is described as Simulium (Simulium) vietnamense sp. nov. and the other four species (S. (S.) chungi Takaoka & Huang, S. (S.) grossifilum Takaoka & Davies, S. (S.) maenoi Takaoka & Choochote, and S. (S.) rufibasis Brunetti) are newly recorded from Vietnam.
Simulium (Nevermannia) chomthongense sp. nov. is described from female, male, pupal and larval specimens collected from Doi Inthanon National Park and Doi Phahompok National Park, Chiang Mai, Thailand. This new species, first reported as S. (Eusimulium) sp. A, and later regarded as S. (N.) caudisclerum Takaoka & Davies, described from peninsular Malaysia, is distinguished from S. (N.) caudisclerum in the male by the number of enlarged upper-eye facets and the relative size of the hind basitarsus against the hind tibia and femur, and in the pupa by the relative length of the stalks of paired filaments against the common basal stalk and the color of the dorsal surface of abdominal segments 1- 3 (or 4). Taxonomic and molecular notes are provided to separate this new species from four other known species of the vernum species-group, which share an accessory sclerite on the larval abdomen, a rare characteristic in this species-group.
Simulium (Gomphostilbia) sofiani sp. nov. is described on the basis of reared adult female, male, pupal and larval specimens collected from Cameron Highlands, Pahang state, Malaysia. This new species is placed in the ceylonicum species-group within the subgenus Gomphostilbia and is easily distinguished from all the related known species by the combination of the following characteristics: an elongate sensory vesicle and yellow hair tuft on the stem vein of the wing in the female, the greater number of large upper-eye facets (15 or 16 vertical columns and 15 or 16 horizontal rows) and almost entirely darkened hind basitarsus in the male, and the gill bearing a long common basal stalk and 8 filaments arranged as [(1+2)+(1+2)] +2 filaments from dorsal to ventral in the pupa.
Simulium chayamaritae Takaoka and Srisuka from Thailand belongs to the Simulium darjeelingense species-group of Simulium (Simulium) (Diptera: Simuliidae). The female of this species is described for the first time based on a female reared from a pupa collected from Chiang Mai, Thailand. It is characterized by the sensory vesicle elongate and the inner margins of the arms of the genital fork divergent, then convergent apically. It is similar to the female of S. eshimai Takaoka and Adler of the same speciesgroup from Vietnam. Taxonomic notes are given to separate it from two other species of the S. darjeelingense species-group from India and Malaysia, and 28 of 31 other species of the subgenus Gomphostilbia recorded from Thailand.
Simulium (Gomphostilbia) dhangi sp. nov., S. (G.) sumbaense sp. nov. and S. (Nevermannia) wayani sp. nov. are described from the Lesser Sunda Archipelago, Indonesia. Simulium (G.) sumbaense sp. nov. is placed in the S. varicorne species-group and is characterized by the pupal gill with eight filaments arranged as (1+1+1+1+2)+2 from dorsal to ventral, while S. (G.) dhangi sp. nov., unplaced to group, is characterized by the pupal gill composed of two inflated trunks and four slender filaments all arising basally, and the short larval antenna as long as the stem of the labral fan. Simulium (N.) wayani sp. nov. belongs to the S. ruficorne species-group and is characterized by the female spermatheca with an unsclerotized neck, and pupal gill with four inflated filaments. The number of species of black flies from the archipelago increases from 19 to 22.
Simulium (Simulium) contractum Takaoka from Sulawesi, Indonesia was known only as the pupa. Its female, male and mature larva are described for the first time. The tentative assignment of this species in the Simulium dumogaense species-group is confirmed by the adult characters including the female and male genitalia. The female and male of this species are similar to those of Simulium (Simulium) tumpaense Takaoka and Roberts but are distinguished by the yellowish femora.
A rare non-sex mosaic abnormality represented by genitalia-like appendages on the ventral surface of abdominal segment 8 of a male black fly collected in Hokkaido, Japan, is reported. The appendages consist of a pair of style-like projections each arising from a coxite-like base, inverted-Y shaped ventral plate-like structure, and isolated round structure. This male was morphologically and molecularly identified as an abnormal form of S. (S.) iwatense (Shiraki), the only species in the Simulium (Simulium) ornatum species-group in Japan, although certain morphological characteristics of this male including the reduced number of uppereye (large) facets and elongate cerci are different from those of S. (S.) iwatense.
Simulium ( Simulium) rufibasis Brunetti originally described from India was once considered a geographic generalist widely distributed in the Oriental Region. In this study, the species previously regarded as S. (S.) rufibasis in Taiwan was morphologically re-evaluated and found to be distinguished in the male and pupa from true S. (S.) rufibasis from India. This new species is described as S. ( S.) hehuanense sp. nov. based on a female, a male and their pupal exuviae in Taiwan. This new species is placed in the S. rufibasis subgroup of the S. tuberosum species-group, and is similar to S. (S.) yamatoense Takaoka, Adler & Fukuda from Japan and Korea, but it is barely distinguished by the slenderer forebasitarsi of the female and male. Another new, related species, S. (S.) xiulinense sp. nov., is described based on a male and its pupal exuviae. This new species is distinguished from S. (S.) hehuanense sp. nov. by the number of male upper-eye (large) facets in 20 or 21 vertical columns and 22 horizontal rows (19 vertical columns and 19 horizontal rows in the latter species), and presence of a pair of dorsolateral shiny spots on male abdominal segment 5 (absence in the latter species). Both new species inhabit streams at high elevations (ca, 2,600 m). They differ from S. (S.) sp. (probably S. (S.) arisanum Shiraki) from Taiwan, of the same subgroup, which breeds in streams at low elevations (433-685 m), by the pupal head and thorax covered with tubercles (bare in the latter species).
Simulium (Gomphostilbia) okinawense Takaoka and S. (G.) tokarense Takaoka, both from the Nansei Islands, Japan, were morphologically reexamined and genetically analysed by using the COI gene sequences. The female, male, pupa and mature larva of the two species are redescribed. Morphological reexamination shows that both species are more similar to species in the S. asakoae species-group than to those in the S. ceylonicum species-group, by having a medium-long female sensory vesicle, yellow tuft hairs (S. (G.) okinawense) or yellow tuft hairs mixed with a few to several dark hairs (S. (G.) tokarense) at the base of the radial vein in the female and male, and medium-long larval postgenal cleft. However, the body of the male ventral plate (viewed ventrally) is parallel-sided (S. (G.) okinawense) or parallelsided or slightly narrowed (S. (G.) tokarense) and not emarginated basally, differing from those of most species in the S. asakoae species-group. Our genetic analysis shows that S. (G.) tokarense is in the S. asakoae species-group, and S. (G.) okinawense formed a separate sister clade with other members of the S. asakoae species-group with high bootstrap support. From the results of morphological and genetic analysis combined, S. (G.) okinawense and S. (G.) tokarense are transferred from the S. ceylonicum species-group to the S. asakoae species-group.
Simulium takahasii (Rubtsov), which was originally described from Japan, and recorded from Korea and China, is the first among the 19 species of the subgenus Wilhelmia Enderlein recorded from East Asia. It is striking in mating, blood-feeding and ovipositing in captivity and in experimentally transmitting Dirofilaria immitis (Leidy) and Brugia pahangi (Buckley & Edeson), and it is a severe biter of cattle and horses, rarely of humans. Nevertheless, updated information about its morphological characteristics was lacking, making comparisons with related species described from China difficult, since species of the subgenus Wilhelmia are almost indistinguishable from one another, in particular, in their female terminalia, male genitalia and most of larval features. In this study, as many morphological characteristics as possible of S. takahasii based on specimens from Japan are redescribed. New information about many features of this species including the length of the female sensory vesicle against the third palpal segment, number of male upper-eye (large) facets, arrangement of the eight pupal gill filaments, presence or absence of tiny dark setae on the dorsum of the larval abdomen and the number of rows and hooklets of the larval posterior circlet will be useful in evaluating the species status of several Wilhelmia species in China including the species regarded as S. takahasii.
Perspicuous assessments of taxonomic boundaries and discovery of cryptic taxa are of paramount importance in interpreting ecological and evolutionary phenomena among black flies (Simuliidae) and combating associated vector-borne diseases. Simulium tani Takaoka & Davies is the largest and perhaps the most taxonomically challenging species complex of black flies in the Oriental Region. We use a DNA sequence-based method to delineate currently recognized chromosomal and morphological taxa in the S. tani complex on the Southeast Asian mainland and Taiwan, while elucidating their phylogenetic relationships. A molecular approach using multiple genes, coupled with morphological and chromosomal data, supported recognition of cytoform K and morphoform 'b' as valid species; indicated that S. xuandei, cytoform L, and morphoform 'a' contain possible cryptic species; and suggested that cytoform B is in the early stages of reproductive isolation whereas lineage sorting is incomplete in cytoforms A, C, and G.
The population genetic structure of Simulium tani was inferred from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) and II (COII) along an elevational gradient in Cameron Highlands, Malaysia. A statistical parsimony network of 71 individuals revealed 71 haplotypes in the COI gene and 43 haplotypes in the COII gene; the concatenated sequences of the COI and COII genes revealed 71 haplotypes. High levels of genetic diversity but low levels of genetic differentiation were observed among populations of S. tani at five elevations. The degree of genetic diversity, however, was not in accordance with an altitudinal gradient, and a Mantel test indicated that elevation did not have a limiting effect on gene flow. No ancestral haplotype of S. tani was found among the populations. Pupae with unique structural characters at the highest elevation showed a tendency to form their own haplotype cluster, as revealed by the COII gene. Tajima's D, Fu's Fs, and mismatch distribution tests revealed population expansion of S. tani in Cameron Highlands. A strong correlation was found between nucleotide diversity and the levels of dissolved oxygen in the streams where S. tani was collected.
To determine the geographic origin of the black fly Simulium suzukii on Okinawa Island, Japan, macrogenomic profiles derived from its polytene chromosomes were compared with those of mainland and other insular populations of S. suzukii and of the isomorphic Simulium tani species complex. The Okinawan population is a chromosomally unique cytoform, designated 'D,' which is essentially monomorphic and differs by about 27 fixed rearrangements from the chromosomal standard sequence for the subgenus Simulium and by two fixed differences from its nearest known relative, representing the type of S. suzukii, on the main islands of Japan. Chromosomal band sequences revealed two additional, sympatric cytoforms of S. suzukii, designated 'A' and 'B,' each with species status, in Korea, and a third cytoform, designated 'C,' on Hokkaido, Japan. A new cytoform, 'K,' of S. tani from Malaysia, representing the type of S. tani, is more closely related to cytoforms in Thailand, as are populations from Taiwan previously treated as S. suzukii but more closely aligned with S. tani and newly recognized as cytoform 'L' of the latter nominal species. Rooting of chromosomal band sequences by outgroup comparisons allowed directionality of chromosomal rearrangements to be established, enabling phylogenetic inference of cytoforms. Of 41 macrogenomic rearrangements discovered in the five new cytoforms, four provide evidence for a stepwise origin of the Okinawan population from populations characteristic of the main islands of Japan. The macrogenomic approach applied to black flies on Okinawa Island illustrates its potential utility in defining source areas for other species of flies including those that might pose medical and veterinary risks.
The increasing attention on Vietnam as a biodiversity hotspot prompted an investigation of the potential for cryptic diversity in black flies, a group well known elsewhere for its high frequency of isomorphic species. We analyzed the banding structure of the larval polytene chromosomes in the Simulium tuberosum species group to probe for diversity beyond the morphological level. Among 272 larvae, 88 different chromosomal rearrangements, primarily paracentric inversions, were discovered in addition to 25 already known in the basic sequences of the group in Asia. Chromosomal diversity in Vietnam far exceeds that known for the group in Thailand, with only about 5% of the rearrangements shared between the two countries. Fifteen cytoforms and nine morphoforms were revealed among six nominal species in Vietnam. Chromosomal evidence, combined with available molecular and morphological evidence, conservatively suggests that at least five of the cytoforms are valid species, two of which require formal names. The total chromosomal rearrangements and species (15) now known from the group in Vietnam far exceed those of any other area of comparable size in the world, supporting the country's status as a biodiversity hotspot. Phylogenetic inference based on uniquely shared, derived chromosomal rearrangements supports the clustering of cytoforms into two primary lineages, the Simulium tani complex and the Southeast Asian Simulium tuberosum subgroup. Some of these taxa could be threatened by habitat destruction, given their restricted geographical distributions and the expanding human population of Vietnam.
The transmission of zoonotic filarial parasites by black flies has so far been reported in the Chiang Mai and Tak provinces, Thailand, and the bites of these infected black flies can cause a rare disease-human zoonotic onchocerciasis. However, species identification of the filarial parasites and their black fly vectors in the Chiang Mai province were previously only based on a morphotaxonomic analysis. In this study, a combined approach of morphotaxonomic and molecular analyses (mitochondrial cox1, 12S rRNA, and nuclear 18S rRNA (SSU HVR-I) genes) was used to clarify the natural filarial infections in female black flies collected by using human and swine baits from two study areas (Ban Lek and Ban Pang Dang) in the Chiang Mai province from March 2018 to January 2019. A total of 805 and 4597 adult females, belonging to seven and nine black fly taxa, were collected from Ban Lek and Ban Pang Dang, respectively. At Ban Lek, four of the 309 adult females of Simulium nigrogilvum were positive for Onchocerca species type I in the hot and rainy seasons. At Ban Pang Dang, five unknown filarial larvae (belonging to the same new species) were detected in Simulium sp. in the S. varicorne species-group and in three species in the S. asakoae species-group in all seasons, and three non-filarial larvae of three different taxa were also found in three females of the S. asakoae species-group. This study is the first to molecularly identify new filarial species and their vector black fly species in Thailand.
Sexually anomalous individuals, typically intersexes or gynandromorphs, bear a mixture of male and female traits. Twelve sexually anomalous individuals of the black fly Simulium (Gomphostilbia) trangense Jitklang, Kuvangkadilok, Baimai, Takaoka & Adler were discovered among 49 adults reared from pupae. All 12 sexually anomalous adults were parasitized by mermithid nematodes, although five additional parasitized adults had no overt external anomalies. Sequence analysis of the 18S rRNA gene revealed that the mermithids, possibly representing a new species, are related to Mesomermis spp., with genetic distances of 5.09-6.87%. All 12 anomalous individuals had female phenotypical traits on the head, thorax, forelegs, midlegs, and claws, but male features on the left and right hind basitarsi. One individual had mixed male and female genitalia. The findings are in accord with the trend that mermithid infections are associated with sexually anomalous adult black flies.
The Simulium rufibasis subgroup is one of three subgroups of the Simulium (Simulium) tuberosum species-group; it is characterized by a pair of clustered stout hairs on the ventral surface of female abdominal segment 7. A member of the S. rufibasis subgroup in Taiwan was investigated morphologically and genetically using the universal cytochrome c oxidase subunit I (COI) barcoding gene and polytene chromosomal banding pattern. The Taiwanese material is morphologically similar to S. rosliramlii Takaoka & Chen from Vietnam and represents the second species of the S. rufibasis subgroup known from Taiwan. It also represents a novel molecular lineage that is distinct from three other primary lineages identified as S. doipuiense, S. doipuiense/S. rufibasis, and S. weji previously reported from Thailand. The mitochondrial evidence for a distinct lineage in Taiwan is supported by chromosomal analysis, which revealed unique sex chromosomes. For nomenclatural stability, we associate the name S. arisanum Shiraki with the Taiwanese entity. Originally described from females from Taiwan, S. arisanum until now has remained an enigmatic species.