Sexually anomalous individuals of the black fly Simulium trangense (Diptera: Simuliidae) infected with mermithid parasites (Nematoda: Mermithidae).

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.

A novel molecular and chromosomal lineage of the anthropophilic Simulium (Simulium) rufibasis subgroup (Diptera: Simuliidae) in Taiwan.

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.

Simulium (Gomphostilbia) merapiense sp. nov. (Diptera: Simuliidae) from Java, Indonesia.

Simulium (Gomphostilbia) merapiense sp. nov. is described based on females, males, pupae, and mature larvae from Yagyakarta, Java, Indonesia. This new species is placed in the Simulium epistum species-group, and is characterized by the pupal gill with eight short filaments all arising at the same level from a short stalk, somewhat enlarged basal fenestra, entirely bare pupal head and thoracic integument, and small and short larval postgenal cleft. These characters rarely are found in the subgenus. Taxonomic notes are given to separate this new species from related species of the S. epistum species-group.

Two new Species of Simulium (Simulium) (Diptera: Simuliidae) From Mount Murud, Sarawak, Malaysia.

Two new species of black flies, Simulium (Simulium) murudense and Simulium (Simulium) cheedhangi, are described on the basis of females, males, pupae, and larvae collected in Mount Murud, Sarawak, Malaysia. Both species belong to the Simulium melanopus Edwards species group. S. (S.) murudense sp. nov. is distinguished from most known species by a combination of the haired basal portion of the radial vein and the darkened fore coxae, and S. (S.) cheedhangi sp. nov. is characterized in the female by having a medium-sized claw tooth and in the pupa by six somewhat inflated gill filaments. Notes are given on the S. melanopus species-group in Sarawak and Sabah.

Cytogenetic and Molecular Evidence of Additional Cryptic Diversity in High Elevation Black fly Simulium feuerborni (Diptera: Simuliidae) Populations in Southeast Asia.

Simulium feuerborni Edwards is geographically widespread in Southeast Asia. Previous cytogenetic study in Thailand revealed that this species is a species complex composed of two cytoforms (A and B). In this study, we cytologically examined specimens obtained from the Cameron Highlands, Malaysia, and Puncak, Java, Indonesia. The results revealed two additional cytoforms (C and D) of S. feuerborni. Specimens from Malaysia represent cytoform C, differentiated from other cytoforms by a fixed chromosome inversion on the long arm of chromosome III (IIIL-5). High frequencies of the B chromosome (33-83%) were also observed in this cytoform. Specimens from Indonesia represent the cytoform D. This cytoform is differentiated from others by a fixed chromosome inversion difference on the long arm of chromosome II (IIL-4). Mitochondrial DNA sequences support genetic differentiation among cytoforms A, B, and C. The pairwise F(ST) values among these cytoforms were highly significantly consistent with the divergent lineages of the cytoforms in a median-joining haplotype network. However, a lack of the sympatric populations prevented us from testing the species status of the cytoforms.

Revision of the Simulium (Simulium) melanopus species-group (Diptera: Simuliidae) in Sabah, Malaysia.

Species of the Simulium (Simulium) melanopus species-group in Sabah are taxonomically revised by examining type specimens of S. (S.) crassimanum S. (S.) laterale, and S. (S.) nigripilosum, all described from females by Edwards in 1933, and newly collected samples from the vicinity of Mt. Kinabalu. The females of these three species are redescribed, and their males and pupae are described for the first time based on adults reared from pupae. Simulium (S.) liewi Takaoka, 2007 and S. (S.) kinabaluense Smart & Clifford, 1969 are synonymized with S. (S.) crassimanum and S. (S.) laterale, respectively. Simulium (S.) cheedhangi Takaoka, Sofian-Azirun & Ya'cob, 2015 is newly recorded from Sabah. Two new related species, S. (S.) lardizabalae and S. (S.) timpohonense, are described from males reared from pupae. Keys to identify eight species of the S. melanopus species-group in Sabah are provided for females, males, pupae and mature larvae.

Two new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from peninsular Malaysia, with keys to 10 peninsular Malaysian species of the Simulium batoense species group.

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.

A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Malaysia, with keys to 32 species of the Simulium ceylonicum species-group.

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 species of the Simulium batoense species-group of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Peninsular Malaysia.

Two new black fly species, Simulium (Gomphostilbia) tekamense and Simulium (Gomphostilbia) jerantutense, are described based on adult females emerged from pupae in Peninsular Malaysia, and assigned to the binuanense subgroup of the batoense species-group in the subgenus Gomphostilbia. Simulium (G.) tekamense sp. nov. is characterized in the female by the subcosta with 0-2 hairs, and presence of a  deep notch on the apex of the mediolongitudinal ridge of the cibarium, and in the pupa by one of two paired gill filaments of the middle triplet much thicker than the counter filament. Simulium (G.) jerantutense sp. nov. is characterized in the female by the short claw tooth 0.46 times the length of the claw, and in the pupa by the gill filaments arranged as [2+1+(1+2)]+2 filaments from dorsal to ventral. Taxonomic notes are given to distinguish these new species from related species.

Diversity, Distribution and Host Blood Meal Analysis of Adult Black Flies (Diptera: Simuliidae) from Thailand.

Understanding the factors associated with the species diversity and distribution of insect vectors is critically important for disease epidemiology. Black flies (Diptera: Simuliidae) are significant hematophagous insects, as many species are pests and vectors that transmit pathogens to humans and other animals. Ecological factors associated with black fly species distribution have been extensively examined for the immature stages but are far less well explored for the adult stage. In this study, we collected a total of 7706 adult black fly specimens from various locations in forests, villages and animal shelters in Thailand. The integration of morphology and DNA barcoding revealed 16 black fly taxa, including Simulium yvonneae, a species first found in Vietnam, which is a new record for Thailand. The most abundant species was the Simulium asakoae complex (n = 5739, 74%), followed by S. chumpornense Takaoka and Kuvangkadilok (n = 1232, 16%). The Simulium asakoae complex was dominant in forest (3786 of 4456; 85%) and village (1774 of 2077; 85%) habitats, while S. chumpornense predominated (857 of 1175; 73%) in animal shelter areas. The Simulium asakoae complex and S. nigrogilvum Summers, which are significant pests and vectors in Thailand, occurred at a wide range of elevations, although the latter species was found mainly in high (>1000 m) mountain areas. Simulium chumpornense, S. nodosum Puri and the S. siamense Takaoka and Suzuki complex occurred predominately in low (<800 m)-elevation areas. Simulium furvum Takaoka and Srisuka; S. phurueaense Tangkawanit, Wongpakam and Pramual; and S. nr. phurueaense were only found in high (>1000 m) mountain areas. A host blood meal analysis revealed that the S. asakoae; S. chamlongi Takaoka and Suzuki; S. nigrogilvum; S. chumpornense; and the S. striatum species group were biting humans. This is the first report of the latter two species biting humans. We also found that S. chumpornense was biting turkeys, and S. chamlongi was biting chickens, which are new host blood sources recorded for these species. In addition, we found that the S. feuerborni Edwards complex was biting water buffalo, which is the first report on the biting habits of this species.

Co-occurrence of dual lineages within Simulium (Gomphostilbia) atratum De Meijere in the Indonesian Archipelago along Wallace's Line.

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.

Multi-Locus Sequence Analysis Indicates Potential Cryptic Speciation in the Chigger Mite Neoschoengastia gallinarum (Hatori, 1920) Parasitising Birds in Asia.

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.

Occurrence of five distinct clades of mermithid nematodes (Nematoda: Mermithidae) infecting black fly larvae (Diptera: Simuliidae) in tropical streams in Malaysia.

Mermithids are the most common parasites of black flies and are associated with host feminization and sterilization in infected hosts. However, information on the species / lineage of black fly mermithids in Southeast Asia, including Malaysia requires further elucidation. In this study, mermithids were obtained from black fly larvae collected from 138 freshwater stream sites across East and West Malaysia. A molecular approach based on nuclear-encoded 18S ribosomal RNA (18S rRNA) gene was used to identify the species identity / lineage of 77 nematodes successfully extracted and sequenced from the specimens collected. Maximum likelihood and neighbor-joining phylogenetic analyses demonstrated five distinct mermithid lineages. Four species delimitation analyses: automated simultaneous analysis phylogenetics (ASAP), maximum likelihood Poisson tree processes with Bayesian inferences (bPTP_ML), generalized mixed yule coalescent (GMYC) and single rate Poisson tree processes (PTP) were applied to delimit the species boundaries of mermithid lineages in this data set along with genetic distance analysis. Data analysis supports five distinct lineages or operational taxonomic units for mermithids in the present study, with two requiring further investigation as they may represent intraspecific variation or closely related taxa. One mermithid lineage was similar to that previously observed in Simulium nigrogilvum from Thailand. Co-infection with two mermithids of different lineages was observed in one larva of Simulium trangense. This study represents an important first step towards exploring other aspects of host - parasite interactions in black fly mermithids.

From bites to barcodes: uncovering the hidden diversity of black flies (Diptera: Simuliidae) in Vietnam.

BACKGROUND: Prompt and precise identification of black flies (Simuliidae) is crucial, given their biting behaviour and significant impact on human and animal health. To address the challenges presented by morphology and chromosomes in black fly taxonomy, along with the limited availability of molecular data pertaining to the black fly fauna in Vietnam, this study employed DNA-based approaches. Specifically, we used mitochondrial and nuclear-encoded genes to distinguish nominal species of black flies in Vietnam. METHODS: In this study, 135 mitochondrial cytochrome c oxidase subunit I (COI) sequences were established for 45 species in the genus Simulium in Vietnam, encompassing three subgenera (Gomphostilbia, Nevermannia, and Simulium), with 64 paratypes of 27 species and 16 topotypes of six species. Of these COI sequences, 71, representing 27 species, are reported for the first time. RESULTS: Combined with GenBank sequences of specimens from Malaysia, Myanmar, Thailand, and Vietnam, a total of 234 DNA barcodes of 53 nominal species resulted in a 71% success rate for species identification. Species from the non-monophyletic Simulium asakoae, S. feuerborni, S. multistriatum, S. striatum, S. tuberosum, and S. variegatum species groups were associated with ambiguous or incorrect identifications. Pairwise distances, phylogenetics, and species delimitation analyses revealed a high level of cryptic diversity, with discovery of 15 cryptic taxa. The current study also revealed the limited utility of a fast-evolving nuclear gene, big zinc finger (BZF), in discriminating closely related, morphologically similar nominal species of the S. asakoae species group. CONCLUSION: This study represents the first comprehensive molecular genetic analysis of the black fly fauna in Vietnam to our knowledge, providing a foundation for future research. DNA barcoding exhibits varying levels of differentiating efficiency across species groups but is valuable in the discovery of cryptic diversity.

DNA barcoding of black flies (Diptera: Simuliidae) in Indonesia.

BACKGROUND: DNA barcoding is a valuable taxonomic tool for rapid and accurate species identification and cryptic species discovery in black flies. Indonesia has 143 nominal species of black flies, but information on their biological aspects, including vectorial capacity and biting habits, remains underreported, in part because of identification problems. The current study represents the first comprehensive DNA barcoding of Indonesian black flies using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. METHODS: Genomic DNA of Indonesian black fly samples were extracted and sequenced, producing 86 COI sequences in total. Two hundred four COI sequences, including 118 GenBank sequences, were analysed. Maximum likelihood (ML) and Bayesian inference (BI) trees were constructed and species delimitation analyses, including ASAP, GMYC and single PTP, were performed to determine whether the species of Indonesian black flies could be delineated. Intra- and interspecific genetic distances were also calculated and the efficacy of COI sequences for species identification was tested. RESULTS: The DNA barcodes successfully distinguished most morphologically distinct species (> 80% of sampled taxa). Nonetheless, high maximum intraspecific distances (3.32-13.94%) in 11 species suggested cryptic diversity. Notably, populations of the common taxa Simulium (Gomphostilbia) cheongi, S. (Gomphostilbia) sheilae, S. (Nevermannia) feuerborni and S. (Simulium) tani in the islands of Indonesia were genetically distinct from those on the Southeast Asian mainland (Malaysia and Thailand). Integrated morphological, cytogenetic and nuclear DNA studies are warranted to clarify the taxonomic status of these more complex taxa. CONCLUSIONS: The findings showed that COI barcoding is a promising taxonomic tool for Indonesian black flies. The DNA barcodes will aid in correct identification and genetic study of Indonesian black flies, which will be helpful in the control and management of potential vector species.

A new species of Simulium (Simulium) (Diptera: Simuliidae) from Langkawi Island, Peninsular Malaysia.

Simulium (Simulium) kisapense sp. nov. is described on the basis of reared adult, pupal, and larval specimens collected from Langkawi Island, Peninsular Malaysia, and placed in the multi-striatum species-group in the subgenus Simulium. This new species is characterized by the bare basal section of the female radial vein, the male ventral plate with setae, the eight pupal gill filaments divergent at an angle of >90 degrees, and the shoe-shaped cocoon with small lateral window(s). Taxonomic notes are given to separate this new species from S. (S.) hirtinervis Edwards and S. (S.) malayense Takaoka and Davies, both from Peninsular Malaysia, and several other known species from Bhutan, India, Nepal, and Thailand.

Descriptions of two new species of the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) from Taiwan and Japan, and a revised description of S. (S.) quinquestriatum (Shiraki).

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.

Redescription of Simulium (Gomphostilbia) omutaense Ogata & Sasa (Diptera: Simuliidae) from Japan and its phylogenetic relationship with other members of the S. batoense species-group.

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.

Natural infections and distributions of parasitic mermithids (Nematoda: Mermithidae) infecting larval black flies (Diptera: Simuliidae) in tropical streams of Malaysia.

Mermithids are parasites of black flies that cause host mortality along with physical and behavioural changes in infected hosts. However, there is a lack of knowledge on the distribution of mermithids infecting black fly larvae and the factors that influence these distributions in Asia, including Malaysia. A total of 13,116 mid- to late-instar black fly larvae belonging to 42 species were collected from 138 streams across East and West Malaysia and screened for the presence of mermithid parasites. Overall, 121 mermithids were obtained from 107 (0.82%) larvae of nine (21.4%) black fly species. The average number of mermithids per black fly host was 1.10 ± 0.04 (SE), ranging from one to three mermithids per host. Mermithid infection was highest in Simulium trangense, with a frequency of occurrence of 6.5%, followed by S. cheongi (5.8%) and S. angulistylum complex (2.9%). Infection was lowest in S. brevipar and S. tahanense, with a frequency of occurrence of 0.7% each. Regression analysis indicated that mermithid infections in larval black flies were significantly associated with cooler and shallower streams with more canopy cover, dense riparian vegetation, high dissolved oxygen, and lower conductivity and complete pH. Forward logistic regression further indicated that infections in S. cheongi were associated with shaded, cooler, slightly acidic streams with higher conductivity and dissolved oxygen. These findings suggest that mermithid infections in larval black flies in Malaysia are not randomly distributed and are influenced by the breeding habitat of their hosts.

Mitochondrial Diversity of the Asian Tiger Mosquito Aedes albopictus (Diptera: Culicidae) in Peninsular Malaysia.

Aedes albopictus is one of the main mosquito vectors responsible for transmitting arboviruses to humans and animals. The ability of this mosquito to support virus transmission has been linked to vector competence, which is partly attributed to the genetic disparities in Ae. albopictus population. At present, little is known about the biologically important traits of Ae. albopictus in Malaysia. Thus, the study aims to determine the genetic variation of Ae. albopictus based on the mitochondria-encoded sequences of cytochrome oxidase subunit I (COI). A statistical parsimony network of 253 taxa aligned as 321 characters of the COI gene revealed 42 haplotypes (H1-H42), of which H1 was the most widespread haplotype in Peninsular Malaysia. Three highly divergent haplotypes (H21, H30, and H31) were detected from the northern population. Overall, haplotype and nucleotide diversities were 0.576 and 0.003, respectively, with low genetic differentiation (FST = 0.039) and high gene flow (Nm = 12.21) across all populations.