METHODS: Pupae and larvae of black flies were collected from available substrates in the stream from central Thailand. Pupae were individually separated in plastic tubes and maintained until adult flies emerged. The emerged adult flies associated with their pupal exuviae and cocoon as well as mature larvae preserved in 85% ethanol were used to describe the new species based on an integrated approach of morphological examination and molecular analysis of the COI gene.
RESULTS: The new species is characterized in the female by the medium-long sensory vesicle with a medium-sized opening apically, scutum with three faint longitudinal vittae, and the ellipsoidal spermatheca; in the male by the number of upper-eye (large) facets in 20 vertical columns and 21 horizontal rows, hind basitarsus slender, nearly parallel-sided, and median sclerite much wider and upturned apically; in the pupa by the head and thoracic integument densely covered with tiny tubercles, and the pupal gill of arborescent type with 28-30 filaments; and in the larva by the postgenal cleft deep, nearly reaching the posterior margin of the hypostoma, and dark pigmented sheath of the subesophageal ganglion. The DNA barcode successfully differentiated the new species from its congeners with an interspecific genetic divergence of 1.74-18.72%, confirming the morphological identification that the species is a new member of the subgenus Asiosimulium. Phylogenetic analyses also indicated that the new species is genetically closely related to Simulium phurueaense Tangkawanit, Wongpakam & Pramual, 2018, further supporting its morphological classification.
CONCLUSIONS: This is the ninth species assigned to the subgenus Asiosimulium within the genus Simulium Latreille, 1802. Taxonomic notes and identification keys are given to distinguish this new species from the eight known species members in its same subgenus. Additionally, a distribution map of all species members in this subgenus occurring in Thailand and other countries is provided.
METHODS: Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis.
RESULTS: Totals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo.
CONCLUSIONS: Previously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak.