To investigate the breeding habitat preference of black flies, a comprehensive black fly survey was conducted for the first time in Peninsular Malaysia. Preimaginal black flies (pupae and larvae) were collected manually from 180 stream points encompassing northern, southern, central and east coast of the Peninsular Malaysia. A total of 47 black fly species were recorded in this study. The predominant species were Simulium trangense (36.7%) and Simulium angulistylum (33.3%). Relatively common species were Simulium cheongi (29.4%), Simulium tani (25.6%), Simulium nobile (16.2%), Simulium sheilae (14.5%) and Simulium bishopi (10.6%). Principal Component Analysis (PCA) of all stream variables revealed four PCs that accounted for 69.3% of the total intersite variance. Regression analysis revealed that high species richness is associated with larger, deeper, faster and higher discharge streams with larger streambed particles, more riparian vegetation and low pH (F=22.7, d.f.=1, 173; P<0.001). Relationship between species occurrence of seven common species (present in >10% of the sampling sites) was assessed. Forward logistic regression analysis indicated that four species were significantly related to the stream variables. S. nobile and S. tani prefer large, fast flowing streams with higher pH, large streambed particles and riparian trees. S. bishopi was commonly found at high elevation with cooler stream, low conductivity, higher conductivity and more riparian trees. In contrast, S. sheilae was negatively correlated with PC-2, thus, this species commonly found at low elevation, warmer stream with low conductivity and less riparian trees. The results of this study are consistent with previous studies from other geographic regions, which indicated that both physical and chemical stream conditions are the key factors for black fly ecology.
A new black fly species, Simulium (Gomphostilbia) pangsidaense, is described on the basis of adult female, male, pupal exuviae and mature larvae from Pang Sida National Park, Sa Kaew Province, Eastern Thailand. This new species is placed in the Simulium ceylonicum species-group. It is distinguished from three Thai members of the S. ceylonicum species-group by the following characteristics: from S. (G.) curtatum Jitklang et al. and S. (G.) sheilae Takaoka & Davies by the wide pupal terminal hooks (triangular terminal hooks in the latter two species), and from S. (G.) sheilae and S. (G.) trangense Jitklang et al. by the number of male upper-eye facets in 13 vertical columns and 14 or 15 horizontal rows (10 or 11 vertical columns and 12 or 13 horizontal rows in the latter two species). This is the fourth member of the S. ceylonicum species-group recorded from Thailand.
Although the microbiome of blood-feeding insects serves an integral role in host physiology, both beneficial and pathogenic, little is known of the microbial community of black flies. An investigation, therefore, was undertaken to identify culturable bacteria from one of Malaysia's most common black flies, Simulium tani Takaoka and Davies, using 16S rDNA sequencing, and then evaluate the isolates for antibiotic resistance and virulence genes. A total of 20 isolates representing 11 bacterial species in four genera were found. Five isolates showed β-hemolysis on Columbia agar, and virulence genes were found in three of these isolates. Some degree of resistance to six of the 12 tested antibiotics was found among the isolates. The baseline data from this study suggest rich opportunities for comparative studies exploring the diversity and roles of the microbiome of S. tani and other Southeast Asian black flies.
Most female black flies in the genus Simulium are blood-sucking flies and they can cause various parasitic diseases in human and animal. A total of 94 species of black flies have been reported in Malaysia, however, their biting behavior and role as vector of infectious agents remain understudied. To fill in this knowledge gap, we attempted to survey adult black flies from field populations in Peninsular Malaysia. In a survey carried out in 2017 at Tasik Kenyir, Terengganu, three females were caught while attracted and landed on human skin. Further morphological and molecular analyses showed that the specimens were identical to Simulium (Gomphostilbia) aziruni Takaoka, Hashim & Chen of the Simulium gombakense species-group. This is the first report on a black fly species attracted to human in Malaysia which serves as a steppingstone towards in-depth studies for black flies in this region.
Simulium (Simulium) hackeri Edwards, 1928 of the Simulium variegatum species-group from Malaysia was described initially based on the female specimen from Cameron Highlands, Pahang. In the present study, the pupa and larva of this species are described for the first time. Their morphological characters resemble those of the Simulium variegatum species-group by having six gill filaments per side, abdomen with dorsal spine-combs at least on segments 7 and 8, cocoon with wall-pocket shaped and with or without an anterodorsal projection. Postgenal cleft of the larva medium-sized, rarely small, ventral papillae small or absent. The DNA barcode of this species is also reported herein.
A black fly species of the Simulium feuerborni species-group of Simulium (Nevermannia) from Cameron Highland, Peninsular Malaysia, previously regarded as S. feuerborni Edwards, originally described from East Java, is described as Simulium pairoti sp. nov. based on complete life stages. High intraspecific variations in the arrangement of the six pupal gill filaments, length of the stalk of the ventral paired filaments, and length of the anterodorsal projection of the cocoon, are noted in this species. This new species is readily distinguished from its congeners by having the characters of male genitalia with simple lamellate ventral plate, short inwardly-twisted styles, several parameral hooks, and a simple narrow median sclerite. Morphological data reported herein plus the chromosomal and molecular data presented elsewhere support S. pairoti as a novel pseudocryptic species.
In recent decades, the numbers of cryptic taxa have increased significantly with current progress in DNA barcoding, yet, most of these cryptic taxa have not been formally named and recognized as valid species. To address this issue, we provide a guide for applying the procedure of describing new cryptic species in the family Simuliidae. Simulium (Simulium) vanluni from Pahang, Peninsular Malaysia, previously treated as S. nobile De Meijere, is described as a new species by using an integrated morpho-taxonomical and genetic approach. This new species is morphologically identical to S. nobile from Java and S. kiuliense Smart & Clifford from Borneo, but their distinctiveness is supported by an expanded multigene phylogeny analysis.
Surveys of pupae and larvae of black flies were carried out in Bali, Lombok, Sumbawa and Flores in the Lesser Sunda Archipelago, Indonesia, where 10 species were known. A total of 14 simuliid species including four new species and five new records of the genus Simulium were collected, bringing the number of species from the Lesser Sunda Archipelago to 19. They are classified into four subgenera: two in Nevermannia, nine in Gomphostilbia, seven in Simulium and one in Wallacellum. One of four new species, Simulium (Simulium) baliense, is described based on females, males, pupae and larvae from Bali and Lombok. This new species, which is placed in the Simulium striatum species-group of the subgenus Simulium, is closely related to S. (S.) argyrocinctum De Meijere from Java and Sumatra, but it is distinguished from the latter species by the smaller number of the male enlarged upper-eye facets and larval abdomen lacking dorsal pairs of conical protuberances. The distribution record of S. (S.) upikae Takaoka & Davies from Flores is corrected as that of S. (S.) eximium De Meijere. Some aberrant characters of the pupal gill filaments of S. (G.) atratum De Meijere, S. (G.) floresense Takaoka, Hadi & Sigit and S. (G.) sunapii Takaoka, Sofian-Azirun & Suana are illustrated. Characteristics of the fauna of black flies in this archipelago are briefly noted. Keys to all 19 species are provided for females, males, pupae and larvae.
Blackflies (Diptera: Simuliidae) are ecologically and medically important insects but they have been understudied in Malaysia. Accordingly, a study on the temporal variation in diversity and community structure of preimaginal blackflies was conducted for the first time in Malaysia. A total of 865 preimaginal blackflies were collected in 120 samplings from five streams across three monsoon seasons from February 2018 until January 2019. Ten species were recorded and most frequently collected species were Simulium cheongi, Simulium vanluni and Simulium jeffreyi. Relatively common species were Simulium roslihashimi, Simulium tani complex and Simulium trangense. No significant changes of rainfall was observed between three monsoon seasons as well as the seasons with species and physiochemical parameters except acidity (pH) (P 20%) indicated that S. vanluni and S. jeffreyi were commonly associated with wider, deeper and fast-flowing streams with low conductivity and larger streambed particle. In contrast, S. cheongi was associated with smaller, slower and small streambed particle. This first extensive bimonthly study has uncovered the species community structure as well as the changes of stream physicochemical parameters over time although they were not greatly and significantly influenced by the monsoon seasons. Species distribution, richness and abundance, however, were highly determined by the stream width, depth and velocity, therefore, were vital in shaping diversity and community structure of preimaginal blackflies.
The population genetic structures of S. vanluni, S. cheongi and S. jeffreyi were determined from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) across different states in Peninsular Malaysia. High levels of genetic diversity and genetic differentiation were observed among three species. All three species revealed an intermediate level of gene flow among the populations. Negative values of Fu's Fs and low values of Raggedness index supported the hypothesis of population expansion in S. vanluni, S. cheongi and S. jeffreyi.
Simulium (Gomphostilbia) aziruni Takaoka, Hashim & Chen was described initially based only on a pupa and a mature larva collected from Peninsular Malaysia. Herein, we describe the morphological characters of the female of S. aziruni for the first time. It resembles those of the other members of the Simulium gombakense species-group by the genital fork with a distinct projection directed medioposteriorly from each arm and claw with a large basal tooth. Cytochrome c oxidase I (COI) barcoding analysis indicates that S. aziruni is the sister species of S. maleewongae, but both are distantly separated by a genetic distance of 4.9%.
Two new black fly species, Simulium (Gomphostilbia) kaohsiungense and S. (G.) shoufengense, are described from females, males and pupae in Taiwan. These new species are placed in the S. asakoae species-group. Simulium (G.) kaohsiungense sp. nov. is characterized by a combination of the elongate female sensory vesicle, male upper-eye (large) facets in 15 vertical columns and 15 or 16 horizontal rows, male hind basitarsus 1.02-1.15 times the width of the hind femur, and widened pupal terminal hooks. These characteristics distinguish this new species from most species of the S. asakoae species-group. Simulium (G.) shoufengense sp. nov. is similar in the female and male to S. (G.) asakoae Takaoka & Davies originally described from Peninsular Malaysia but is distinguished from the latter species in the pupa by the combined length of the primary and secondary stalks of the dorsal triplet of gill filaments as long as or longer than the common basal stalk of the gill, and dorsum of abdominal segments 1 and 2 not darkened. The species previously regarded in Taiwan as S. (G.) metatarsale Brunetti, originally described from India, is morphologically and molecularly identified as S. (G.) asakoae.
The species status of two cytoforms of two species complexes in the Simulium (Simulium) tuberosum species-group, which are almost indistinguishable in the pupal stage, was morphologically and genetically evaluated. Cytoform 'L' of the S. (S.) tani Takaoka & Davies complex, previously recognized as S. (S.) suzukii Rubtsov in Taiwan, is described as a new species, S. (S.) jianshiense. It is morphologically distinguishable as adults from two members of the S. (S.) suzukii complex: cytoform 'C' from Hokkaido, selected to represent the type of S. (S.) suzukii sensu stricto, and cytoform 'D' from Okinawa Island and, based on our chromosomal analysis, also from Amami Island. This new species is genetically separated from both cytoforms of the S. (S.) suzukii complex with a genetic distance of 5.31-6.67%. Cytoform 'D' is distinguished from cytoform 'C' by the color of the male forecoxa and relative length of the female sensory vesicle to the third maxillary palpomere. Accordingly, the name S. (S.) ryukyuense Ogata, once regarded as a synonym of S. (S.) suzukii, is revalidated and applied to cytoform 'D'. The genetic distance between S. (S.) ryukyuense and S. (S.) suzukii sensu stricto is 1.24-1.60%.
Simulium (Gomphostilbia) omutaense Ogata & Sasa, 1954 is the only named species in the Simulium batoense species-group of the subgenus Gomphostilbia Enderlein recorded from Honshu and Kyushu, Japan. It represents the northernmost distribution of this species-group, of which most members are distributed in the Oriental region. This species, the only member of the Simulium omutaense subgroup, is unique among the seven subgroups of the S. batoense species-group by having the pupal gill with one long filament and seven short filaments, similar to the arrangement of the pupal gill filaments in the S. zonatum subgroup of the S. epistum species-group in the same subgenus. This species is fully redescribed based on adults, pupal exuviae and mature larvae, and is most similar to species of the S. decuplum subgroup, based on adult morphological characteristics, although the pupal gill of the latter subgroup is markedly different by having 10 or 12 short filaments. Its close relationship to the S. decuplum subgroup is supported by a DNA analysis using COI gene sequences, with genetic distances of 9.30-11.02%. On the other hand, genetic distances between S. (G.) omutaense and species of the S. zonatum subgroup were 16.32-16.93%. Our study shows that a similar arrangement of the pupal gills in two different species-groups, which is rarely seen, has evolved independently and its occurrence does not necessarily reflect phylogenetic relationships.
Species in the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) in Taiwan and Japan are morphologically reevaluated. Simulium (S.) yanpingense sp. nov. is described as the third member in this species-group from Taiwan and a revised description of S. (S.) quinquestriatum (Shiraki) is provided. The species previously regarded as S. (S.) quinquestriatum in Japan is described as a new species, S. (S.) tanegashimaense. Simulium (S.) yanpingense sp. nov. is similar to S. (S.) pingtungense Huang & Takaoka from Taiwan, in many characteristics including the haired basal portion of the female radius, dark leg color, and male scutum with brassy hairs, but it is distinguished from the latter species by the number of male upper-eye (large) facets, which are in 17 (rarely 18) vertical columns and 17 or 18 horizontal rows in this new species but in 19 vertical columns and 20 horizontal rows in S. (S.) pingtungense. Simulium (S.) tanegashimaense sp. nov. is similar to S. (S.) quinquestriatum originally described from Taiwan by having the male scutum with yellow fine hairs, but is distinguished from the latter species by the number of male upper-eye (large) facets, which are in 17 (rarely 16) vertical columns and 17 horizontal rows in this new species but in 19 or 20 vertical columns and 19 or 20 horizontal rows in S. (S.) quinquestriatum. The morphological differences between these two new species and their related species from India, Malaysia, Thailand and Vietnam are noted.
Simulium (Simulium) rasuli sp. nov. is described from two females collected by a Malaise trap in Genting Highlands, Peninsular Malaysia. This new species is placed in the Simulium christophersi species-group of the subgenus Simulium. The female of this new specie is characterized by the scutum with three longitudinal vittae, dark legs, claw with a small subbasal tooth, and ovipositor valve triangular with its inner margin nearly straight. This new species is distinguished in the female from all the six named species of the species-group by the entirely brownish-black femora and tibiae.
Mitochondrial cytochrome c oxidase subunit I (COI) sequences were utilized to infer the population genetic structure of Simulium (Gomphostilbia) atratum De Meijere, an endemic simulid species to Indonesia. Both median-joining haplotype network and maximum-likelihood tree revealed two genetic lineages (A and B) within the species, with an overlap distribution in Lombok, which is situated along Wallace's line. Genetic differentiation and gene flow with varying frequencies (FST = 0.02-0.967; Nm = 0.01-10.58) were observed between populations of S. (G.) atratum, of which population pairs of different lineages showed high genetic differentiation. Notably, the high genetic distance of up to 5.92 % observed within S. (G.) atratum in Lombok was attributed to the existence of two genetically distinct lineages. The co-occurrence of distinct lineages in Lombok indicated that Wallace's line did not act as faunistic border for S. (G.) atratum in the present study. Moreover, both lineages also exhibited unimodal distributions and negative values of neutrality tests, suggesting a pattern of population expansion. The expansion and divergence time estimation suggested that the two lineages of S. (G.) atratum diverged and expanded during the Pleistocene era in Indonesia.
Neoschoengastia gallinarum is widely distributed in Asia, preferentially parasitising birds, and heavy infestations have clinical impacts on domestic fowl. In common with other trombiculid mites, the genetic diversity and potential variation in host preferences or pathology induced by N. gallinarum are poorly understood. This study aimed to unravel the geographical variation and population structure of N. gallinarum collected from galliform birds in Peninsular Malaysia and Thailand by inference from concatenated mitochondrial-encoded cytochrome c oxidase subunit I (COI), and nuclear-encoded internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA gene sequences, including a comparison with previously published data from southeastern China. Our multi-locus sequence analysis revealed three monophyletic clades comprising (A) specimens from Peninsular Malaysia, (B) the samples from Thailand together with a minority of Chinese sequences, and (C) the majority of sequences from China. Similarly, most species delimitation approaches divided the specimens into three operational taxonomic units. Analysis of molecular variance revealed 96.41% genetic divergence between Malaysian and Thai populations, further supported by the absence of gene flow (Nm = 0.01). In conclusion, despite the two countries sharing a land border, populations of N. gallinarum from Peninsular Malaysia and Thailand appear to be genetically segregated and may represent distinct cryptic species.
Simulium (Gomphostilbia) leparense sp. nov. is described from females, males, and pupae collected from Peninsular Malaysia. This new species is assigned to the ceylonicum species-group of the subgenus Gomphostilbia, and is characterized by the female and male scuta covered with dark-brown short hairs, smaller number of male upper-eye facets, presence of shiny paired spots on the male abdominal segments 2-8, and absence of grapnel-shaped hooklets on the pupal abdominal segment 9. The male and pupa of S. capillatum Takaoka, which was originally described from larvae collected from Sarawak and Sabah, are described for the first time. Keys to identify all 32 species of the Simulium ceylonicum species-group including 27 species from other countries are provided for females, males, pupae, and mature larvae.
Two new blackfly species, Simulium (Gomphostilbia) azhari and Simulium (Gomphostilbia) johorense, are described based on adult females, males, pupae, and larvae collected from Peninsular Malaysia, and assigned to the parahiyangum subgroup and the duolongum subgroup of the batoense species group of the subgenus Comphostilbia, respectively. S. (G.) azhari sp. nov. is characterized in the female by the narrow frons, and in the male by the broad style and the ventral plate moderately produced ventrally. S. (G.) johorense sp. nov. is also remarkable in having the female subcosta lacking hairs or bearing a reduced number of hairs ranging from one to five. The pupae of both new species share a similar arrangement of the eight gill filaments (i.e., stalks of dorsal and middle triplets and ventral pair arising at the same level from the short common basal stalk), although relative lengths of filaments of the ventral pair to those of the dorsal and middle triplets are different between the two new species. Taxonomic notes are given to distinguish these new species from other related species. Keys to identify all 10 species of the batoense species group in Peninsular Malaysia are provided for adult females, males, pupae, and mature larvae.