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.
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 study on biodiversity of forensically important Diptera in the tropical rain forest in Malaysia is scarce. Thus, a preliminary survey was conducted at a jungle fringe near Kampung Bahagia Bukit Lagong, Sungai Buloh, Selangor. A rat carcass was offered to attract carrion flies and we collected an adult female calliphorid, Hypopygiopsis fumipennis (Walker, 1856) during the fresh stage of carcass decomposition. The female fly was allowed to oviposit on chicken liver in a container and the resulting larvae were reared to the adult stage. Along the developmental process, several individuals from each instar were collected and preserved in 70% ethanol and then processed on the slides. We recorded the duration of development for each instar and described its larval features for the first time. The third instar larvae of H. fumipennis showed accessory oral sclerite present, anterior spiracle with 13-15 papillae, intersegmental spines mostly unicuspid with pointed end, and posterior spiracles heavily sclerotized with inter-slit projections. Some larval differences between H. fumipennis and Hypopygiopsis violacea were noted.
Hypopygiopsis violacea, a species of fly of forensic importance, was recovered from a corpse and described for the first time. The morphological structures of the second and third instar larvae of four specimens were examined using light microscope. Observations were focused on three main morphological characters: cephalopharyngeal skeleton, anterior and posterior spiracles. Cephalopharyngeal skeleton of second instar larva is darkly pigmented and without accessory sclerite below the mouth hook. The anterior spiracles of second and third instar larvae have 8-9 papillae each, arranged in a single row. The posterior spiracle of second instar larva has two spiracular slits with no thickening of peritreme. This differentiates it from the third instar, whereby the latter has three slits for each posterior spiracle. Cephalopharyngeal skeleton of third instar larva is heavily pigmented. An accessory sclerite is found below the hook part of third instar larva but is absent in second instar. Peritreme of the posterior spiracle of third instar larva is thick almost complete encircling a button. The intersegmental spines of the cuticular surface are dome-shaped and unicuspid. Third instar larva of this species is large with size approximately 15 mm long. These findings provide important identification features of immature stages of Hy. violacea which could be useful in forensic entomology.
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.
Simulium (Simulium) lomkaoense sp. nov. is described from females, males, pupae, and larvae in Thailand. This new species is assigned to the Simulium malyschevi Dorogostaisky, Rubtsov & Vlasenko species-group of the subgenus Simulium, and appears to be closely related to Simulium baimaii Kuvangkadilok & Takaoka from Thailand in having a similar shape of the female and male genitalia, pupal gill with two inflated filaments, and simple wall-pocket-shaped cocoon. This new species is compared taxonomically with S. baimaii and other related species. It represents the third species of the S. malyschevi species-group known from Thailand.
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%.
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 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.
Larvae and adults reared from pupae of Simulium (Gomphostilbia) gombakense Takaoka & Davies from eight streams in five provinces of Thailand were genetically analyzed by using cytochrome c oxidase subunit 1 sequences. The material was composed of four lineages, of which lineages 2, 3 and 4 are genetically closely related to one another, but more distantly related to lineage 1, which is equivalent to typical S. (G.) gombakense from Peninsular Malaysia. Lineages 2, 3 and 4, which are morphologically indistinguishable, are designated as genoforms A, B and C within a species complex of S. (G.) paiense sp. nov., which is described based on specimens of lineage 2 (=genoform A). Simulium (G.) paiense sp. nov. is similar to S. (G.) gombakense from Peninsular Malaysia but appears to be barely distinguished by the relative length of the female fore basitarsus. Simulium (G.) thuathienense Takaoka & Sofian-Azirun is newly recorded from Thailand and its male is described for the first time. The female of S. (G.) gombakense is briefly described based on females reared from pupae collected from Peninsular Malaysia.
Two new species of black flies, Simulium (Simulium) srisukai and S. (S.) kiewmaepanense, are described from specimens collected in Thailand. Both species are assigned to the Simulium variegatum species-group of the subgenus Simulium (Simulium) Latreille. They are characterized by the darkened female femora and tibiae and six inflated pupal gill filaments, and the darkened female tibiae and six ordinary thread-like pupal gill filaments, respectively. Taxonomic notes are given to separate these new species from 10 related species among the group. These new species represent the third and fourth species of the S. variegatum species-group from Thailand.
Two new species of Simulium (Gomphostilbia), S. (G.) myanmarense and S. (G.) monglaense, are described from females, males, pupae and larvae from Myanmar. The two new species are placed in the S. asakoae species-group, and are similar to each other in the female and male but distinguished in the pupa by the presence or absence of an anterodorsal projection of the cocoon, and in the larva by a unique pattern of colored markings on the abdomen. Taxonomic notes are given to separate these species from related species. The COI gene sequences of both species are compared with those of eight species of the S. asakoae species-group and three species of the S. ceylonicum species-group. Both new species are most closely related to each other, further supporting their morphological classification in the S. asakoae species-group.
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%.
A new species of black fly, Simulium (Gomphostilbia) rampae, is described, based on adult male, its pupal exuviae and mature larvae collected from Doi Inthanon National Park, northern Thailand. This new species is placed in the Simulium asakoae species-group, and is characterized in the male by the high number of upper-eye facets in 17 vertical columns and 18 horizontal rows, in the pupa by the gill with a long common basal stalk, cone-shaped terminal hook, and cocoon with an anterodorsal projection, and in the larva by the medium-long postgenal cleft. A DNA analysis using COI gene supported its assignment to the S. asakoae species-group and showed its close relationship to S. (G.) udomi Takaoka & Choochote and S. (G.) chiangdaoense Takaoka & Srisuka. This is the fourth member of the S. asakoae species-group recorded from Thailand.
Simulium (Gomphostilbia) fukudae sp. nov. is described based on females, males, pupae, and larvae from Thailand. This new species is placed in the Simulium batoense species-group of Simulium (Gomphostilbia). It is characterized by the female frons almost bare, male paramere covered with minute setae, pupal gill composed of six slender filaments, pupal terminal hooks cone-shaped, and cocoon with an anterodorsal projection. Taxonomic notes are given to separate this new species from the most similar species, Simulium (G.) sextuplum Takaoka & Davies and Simulium (G.) paukatense Takaoka, both from Malaysia.
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.
A new species of black fly, Simulium (Gomphostilbia) isanense, is described based on females, males, pupae and mature larvae from Thailand. This new species is placed in the Simulium epistum species-group of the subgenus Gomphostilbia Enderlein. It is characterized by the pupal gill with eight filaments arranged as 3+3+2 from dorsal to ventral, of which an inner filament of the ventral pair is slightly longer than its counter filament. Taxonomic notes are provided to distinguish this new species from S. (G.) angulistylum Takaoka & Davies from Peninsular Malaysia, and three other related species. The difference between this new species and S. (G.) angulistylum is supported by genetic distances using the mitochondrial COI gene.