Preservation of larvae retrieved from cadavers is important in ensuring the quality and integrity of entomological specimens used for the estimation of post-mortem interval (PMI). The process of killing and preserving larvae could distort the larvae leading to inaccurate estimation of PMI. In this study, the effects of killing Chrysomya megacephala larvae with hot water at different temperatures and subsequent maintenance in various preservatives were determined. Larvae not killed by hot water but preserved directly were used as control. The types of preservative used were 10% formalin, 70% ethanol and Kahle's solution. The morphological features examined were length, turgidity, curvature and coloration of larvae. Larvae killed in 80ºC hot water have shorter mean length (12.47 ± 2.86 mm) compared to those in 60ºC hot water (12.95 ± 2.69 mm). Increasing the duration of preservation in all types of preservative caused elongations of larvae treated or untreated with hot water. There were no significant changes in larval turgidity preserved in Kahle's solution compared to other two preservatives and were unaffected by the duration of storage. Larvae preserved in Kahle's solution experienced the least changes in coloration and shape compared to other preserved larvae in 70% ethanol or 10% formalin. Larvae directly immersed alive in 70% ethanol experienced the most changes in curvature, coloration and turgidity. This study suggested that killing larvae with hot water at 80ºC and preservation in Kahle's solution is the optimum method resulting in least changes in morphological features of Ch. megacephala larvae.
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.
Seven populations of Simulium parahiyangum Takaoka & Sigit (Diptera: Simuliidae), a geographically widespread nominal species of black fly in Southeast Asia, were morphologically and molecularly studied. Three morphoforms based on male and pupal morphological features, and two primary lineages based on the COI gene sequence analysis were recognized. Morphoform 1 (lineage 1) from Sarawak, Malaysia, is identified as S. parahiyangum sensu stricto and morphoform 2 (lineage 2) from Thailand and Vietnam, and morphoform 3 (lineage 1) from Peninsular Malaysia are each regarded as distinct species, although morphoform 3 is partially homosequential for the COI gene with morphoform 1. Morphoforms 2 and 3 are described as Simulium ngaoense sp. nov. and Simulium sazalyi sp. nov., respectively. Overall, S. parahiyangum is not a single geographic generalist but a composite of multiple species.
Simulium undecimum sp. nov. is described from Thailand. This new species is assigned to the Simulium multistriatum species-group, one of the 20 species-groups of the subgenus Simulium in the Oriental Region. It is characterized by the female cibarium with minute processes, male ventral plate with a narrow body having two vertical rows of distinct teeth on the posterior surface and without setae on the anterior and lateral surfaces, pupal gill with eight short filaments decreasing in length from dorsal to ventral, and divergent at an angle of around 90 degrees when viewed laterally, spine-combs only on abdominal segments 7 and 8, and cocoon wall-pocket shaped with anterolateral windows. Taxonomic notes to separate this new species from related species in Thailand and other countries are given. This new species is the 11th nominal member of this species-group recorded in Thailand. An analysis of the COI gene sequences shows that it is most closely related with S. malayense Takaoka & Davies (cytoform A) from Thailand but they are distantly separated by 3.01-8.87%.
Two lineages of stream toads in the genus Ansonia from Malaysian Borneo have long been suspected to be specifically distinct on the basis of molecular data. We assessed the taxonomic status of these lineages using morphological and additional genetic data. In mtDNA phylogeny, each lineage-one from Bario, Kelabit Highlands of Sarawak, the other from Mt. Mulu of Sarawak and the Crocker Range of Sabah-is separated from other congeners by large genetic distances, comparable with those observed between heterospecific species in the genus. These lineages are also morphologically distinguishable from other species, and are considered to represent valid, independently evolving species. We therefore describe them as A. kelabitensis sp. nov. and A. kanak sp. nov.
Simulium (Gomphostilbia) maleewongae sp. nov. is described based on the adult males and females, their pupal exuviae, and larvae from Thailand. This new species is placed in the Simulium gombakense species-group of Simulium (Gomphostilbia). It is characterized by the female cibarium with a cup-like appendage, male ventral plate deeply depressed ventromedially, pupal gill composed of an inflated structure and eight slender filaments, cone-shaped pupal terminal hooks, and cocoon with an anterodorsal projection. Taxonomic notes are given to separate this new species from 10 other species of the same species-group known from China, India, Malaysia, Thailand, Nepal, and Vietnam. Keys to identify all 11 species of the S. gombakense species-group are provided for females, males, pupae, and larvae.
Two new species, Simulium (Gomphostilbia) sunapii and S. (G.) rangatense, are described based on adults, pupae, and mature larvae from Flores, in the eastern part of the Sunda Archipelago, Indonesia. Simulium (G.) sunapii sp. nov. is placed in the S. asakoae species-group, representing the easternmost geographical record for the group in this archipelago. It is characterized by a small number of male upper-eye large facets in eight or nine vertical columns and 12 horizontal rows. Simulium (G.) rangatense sp. nov. is placed in the S. ceylonicum species-group and is characterized by the pupal gill with six filaments. This new species, together with two related species of the S. ceylonicum species-group in Flores, suggests the species radiation of this species-group might have been accompanied by a reduction of the number of pupal gill filaments from eight to four through six. Taxonomic notes are provided to distinguish these two new species from related species.
Simulium (Asiosimulium) saeungae sp. nov. (Diptera: Simuliidae) is described based on females, males, pupae, and mature larvae collected from Nan Province, Northern Thailand. It is characterized by the medium-long cerci in the female, enlarged hind basitarsus, and broad ventral plate with its posterior margin not deeply concave in the male, arborescent pupal gill with 42-56 filaments in the pupa and smaller number of primary rays of the labral fan (30-33) in the larva. This is the fifth species of the subgenus Asiosimulium, the second smallest among 10 subgenera in the Oriental Region. Taxonomic notes are given to distinguish this new species from the three known species from Thailand and one from Nepal.
Human anisakiasis is a zoonosis acquired by eating raw or undercooked infected seafood. Herein, we report a case of acute dysentery caused by anisakiasis in a 64-year-old man in Malaysia. A colonoscopy was performed and a nematode larva was found penetrating the mucosa of the ascending colon. Bleeding was observed at the site of penetration. Y-shaped lateral epidermal cords were seen from the cross section of the worm, which is a prominent feature of Anisakis larva. Molecular analysis using polymerase chain reaction of cytochrome oxidase 2 (cox2) gene confirmed the specimen to be larva of Anisakis simplex.
Four new species of black flies are described, and three others are reported as newly recorded, based on adults reared from pupae, pupae and larvae collected in and near Tam Dao National Park, Vinh Phuc Province, Vietnam. New species include Simulium (Gomphostilbia) hongthaii sp. nov., S. (G.) tamdaoense sp. nov. (both species placed in the asakoae species-group), S. (Simulium) taythienense sp. nov. and S. (S.) xuandai sp. nov. (the two latter species placed in the striatum species-group). Newly recorded species are S. (G.) brinchangense Takaoka, Sofian-Azirun & Hashim, S. (Nevermannia) aureohirtum Brunetti and S. (S.) brevipar Takaoka & Davies. These discoveries increase the number of species of black flies known in Vietnam from 21 to 28.
The megophryid frogs Leptobrachella brevicrus, Leptolalax dringi and Megophrys dringi are species exclusively known from highly localised areas in isolated mountain ranges on Borneo. The tadpoles and adults in this study were collected at the shared type locality for the three species in Gunung Mulu National Park, Sarawak, Malaysia (Borneo). The species identities of larvae were determined via comparison to syntopic adults using DNA barcoding techniques based on partial 16S rRNA mitochondrial gene sequences. The genetic data supported the status of the three taxa as valid species. Descriptions of colouration in life and after preservation, external morphological features, morphometric measurements and ecological notes in comparison to congeneric species are supplied. The tadpoles of L. brevicrus and L. dringi show similar adaptations to a fossorial lifestyle. These include an elongated, vermiform body, a relatively long tail and small eyes. Both were found in the gravel beds of a small mountain stream. In contrast, the larvae of M. dringi are adapted to occupying and feeding at the surface of pools within the stream.
Hard ticks taken from the Japanese serow, Capricornis crispus, in Yamagata Prefecture, Honshu, harboured infective larvae of onchocercid filariae after incubation from the 22nd to the 158th day. Haemaphysalis flava and H. japonica contained one to eight filarial larvae; females, males and a nymph of the ticks were infected. The 44 infective larvae recovered were 612-1,370 μm long, and 11 of them, 930-1,340 μm long, were studied in detail. The larvae possessed the morphologic characteristics of the larvae of the genus Cercopithifilaria, namely an oesophagus with a posterior glandular part, no buccal capsule and a long tail with three terminal lappets. Five types (A to E) of infective larvae were identified based on the morphologic characteristics. While to date five species of Cercopithifilaria have been described from the Japanese serow, a specific identification of the larvae found in this study was generally not possible. Only type E larvae could be tentatively assigned to Cercopithifilaria tumidicervicata, as they had a cervical swelling similar to that of the adults of this species. A key for the identification of the five larval types is presented. The study presents circumstantial evidences indicating that H. flava and H. japonica may transmit Cercopithifilaria spp. to Japanese serows. It also suggests the possibility that such filarial larvae will be found in hard ticks anywhere, because Cercopithifilaria is distributed worldwide, though this genus generally goes unnoticed, as its microfilariae occur in the skin, not in the blood, of host animals.
The final instar larva of Orthetrum borneense Kimmins, 1936, is described and figured for the first time based on exuviae from three male and six female larvae collected in Sarawak, Borneo (East Malaysia). It is compared with an early instar larva, which was matched to the adult O. borneense by DNA barcoding, and the known larvae of other species of this genus that occur in the region.
The widespread nominal black fly Simulium (Simulium) rufibasis Brunetti was reexamined morphologically, chromosomally, and molecularly to determine the status of populations in Japan and Korea with respect to S. rufibasis from the type locality in India and to all other known species in the S. (S.) tuberosum species-group. Morphological comparisons established that the species previously known as S. rufibasis in Japan and Korea is distinct from all other species. Consequently, it was described and illustrated as a new species, Simulium (S.) yamatoense. Simulium yokotense Shiraki, formerly a synonym of S. rufibasis, was morphologically reevaluated and considered a species unplaced to species-group in the subgenus Simulium. Chromosomal analyses of S. yamatoense sp. nov. demonstrated that it is unique among all cytologically known species of the S. tuberosum group and is the sister species of the Taiwanese species tentatively known as S. (S.) arisanum Shiraki. Populations of S. yamatoense sp. nov. included two cytoforms, based on the sex chromosomes. Cytoform A, including topotypical representatives, was found in Kyushu, Japan, whereas cytoform B was found in Korea and Honshu, Japan. Molecular analysis based on the COI mitochondrial gene generally corroborated morphological and chromosomal data that S. yamatoense sp. nov. is a distinct species and, like the chromosomal data, indicate that it is most closely related to S. arisanum, with interspecific genetic distance of 2.92-4.63%.
The presence of the Afro-Australian genus Conochironomus Freeman, 1961 (Diptera: Chironomidae) in Asia has been recognised only informally. An unpublished thesis included Conochironomus from Singapore, and the genus has been keyed from Malaysia without named species. Here, the Sumatran Conochironomus tobaterdecimus (Kikuchi & Sasa, 1980) comb. n. is recorded from Singapore and Thailand. The species is transferred from Sumatendipes Kikuchi & Sasa, 1980, rendering the latter a junior synonym (syn. n.) of Conochironomus Freeman. Conochironomus nuengthai sp. n. and Conochironomus sawngthai sp. n. are described as new to science, based on adult males from Chiang Mai, Thailand. All species conform to existing generic diagnoses for all life stages, with features from male and female genitalia, pupal cephalic tubercles and posterolateral 'spurs' of tergite VIII providing evidence for species distinction. Some larvae are linked to C. tobaterdecimus through molecular barcoding. Variation in other larvae, which clearly belong to Conochironomus and are common throughout Thailand, means that they cannot be segregated to species. Larval habitats include pools in river beds, urban storage reservoirs, drains with moderately high nutrient loadings, and peat swamps. Endochironomus effusus Dutta, 1994 from north-eastern India may be a congener but may differ in adult morphology, thereby precluding formal new combination until discrepancies can be reconciled. Many problems with vouchering taxonomic and molecular material are identified that need to be rectified in the future.
Simulium (Simulium) phraense sp. nov. (Diptera: Simuliidae) is described from females, males, pupae, and larvae from Thailand. This new species is placed in the Simulium striatum species group and is most similar to Simulium (Simulium) nakhonense Takaoka & Suzuki (Diptera: Simuliidae) from Thailand among species of the same species group but is barely distinguished from the latter species by lacking annular ridges on the surface of the pupal gill filaments. The fast-evolving nuclear big zinc finger (BZF) gene has successfully differentiated this new species from its allies, S. (S.) nakhonense and Simulium (Simulium) chiangmaiense Takaoka & Suzuki (Diptera: Simuliidae) of the S. striatum species group. The BZF gene sequences show that this new species is more closely related to S. (S.) nakhonense than to S. (S.) chiangmaiense, further supporting its morphological classification.
A new black fly species, Simulium haiduanense Takaoka, Low & Huang (Diptera: Simuliidae), is described on the basis of females, males, pupae, and mature larvae from Taiwan. This new species is placed in the Simulium argentipes species-group of the subgenus Simulium (Diptera: Simuliidae) and is characterized by the yellowish female legs, ovipositor valves rounded apically and with its inner margin concave, claw with a small subbasal tooth, male style without a basal protuberance, pupal gill with eight filaments, corbicular cocoon, and larval abdomen lacking paired protuberances. It represents the first record of the S. argentipes species-group from Taiwan. Taxonomic notes are given to separate this new species from all eight species in the same species-group. The phylogenetic relationships of this new species with four related species are presented.
There are serious concerns over the adverse impacts of microplastics (MPs) on living organisms. The main objective of this study was to test the effects of MPs on the total length, weight, condition factor (CF), transcriptional level of antioxidant, anti and pro-apoptotic, and neurotransmitter genes, and the histopathology of the gill, liver, brain, kidney, and intestine in the larvae of zebrafish (Danio rerio). Fish were exposed to one of three levels of pristine low-density polyethylene (LDPE) fragments (5, 50, or 500 μg/L) for 10 or 20 days. No significant changes were observed in any of the selected biomarkers across MP concentrations at days 10 or 20. The expression of casp9 (caspase 9, apoptosis-related cysteine protease), casp3a (caspase 3, apoptosis-related cysteine protease a) and cat (catalase), however, were significantly lower in the larvae sampled at day 20 than day 10. We provide evidence that virgin short-term exposure to LDPE fragments has minimal impact on biomarker responses in D. rerio larvae.
The occurrence of Setaria digitata in a horse is reported for the first time in Malaysia. An 8-year-old Thoroughbred cross mare was referred to the University Veterinary Clinic with the primary complaint of corneal opacity and excessive eye discharge. After initial treatment with Terramycin eye ointment, corneal opacity cleared partially to reveal a moving thread-like cylindrical worm in the anterior chamber of the eye. The parasite was successfully removed surgically, and examination under the light microscope revealed that the isolated worm (length = 45 mm) was a 5th stage larva of S. digitata based on morphological criteria. Confirmation of the species of the worm was through molecular methods. The 12S rRNA gene was PCR-amplified, and the purified amplicon was directly sequenced. Phylogenetic analyses revealed that the isolated roundworm showed 100% sequence similarity with that of S. digitata in NCBI GenBank database (Accession no.: KY284626.1). This report is the first confirmed case of equine ocular setariasis by S. digitata in Malaysia. The current study provides evidence that S. digitata is an etiological agent of ocular infection and its presence in Malaysia.