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.

Whole-Genome Analysis of Antimicrobial-Resistant Salmonella enterica Isolated from Duck Carcasses in Hanoi, Vietnam.

Salmonella enterica is one of the most dangerous foodborne pathogens listed by the World Health Organization. In this study, whole-duck samples were collected at wet markets in five districts in Hanoi, Vietnam, in October 2019 to assess their Salmonella infection rates and evaluate the susceptibility of the isolated strains to antibiotics currently used in the prophylaxis and treatment of Salmonella infection. Based on the antibiotic resistance profiles, eight multidrug resistance strains were whole-genome-sequenced, and their antibiotic resistance genes, genotypes, multi-locus sequence-based typing (MLST), virulence factors, and plasmids were analyzed. The results of the antibiotic susceptibility test indicate that phenotypic resistance to tetracycline and cefazolin was the most common (82.4%, 28/34 samples). However, all isolates were susceptible to cefoxitin and meropenem. Among the eight sequenced strains, we identified 43 genes associated with resistance to multiple classes of antibiotics such as aminoglycoside, beta-lactam, chloramphenicol, lincosamide, quinolone, and tetracycline. Notably, all strains carried the blaCTX-M-55 gene, which confers resistance to third-generation antibiotics including cefotaxime, cefoperazone, ceftizoxime, and ceftazidime, as well as resistance genes of other broad-spectrum antibiotics used in clinical treatment such as gentamicin, tetracycline, chloramphenicol, and ampicillin. Forty-three different antibiotic resistance genes were predicted to be present in the isolated Salmonella strains' genomes. In addition, three plasmids were predicted in two strains, 43_S11 and 60_S17. The sequenced genomes also indicated that all strains carried SPI-1, SPI-2, and SPI-3. These SPIs are composed of antimicrobial resistance gene clusters and thus represent a potential threat to public health management. Taken together, this study highlights the extent of multidrug-resistant Salmonella contamination in duck meat in Vietnam.

A new black fly species of Simulium (Nevermannia) (Diptera: Simuliidae) from Vietnam.

Simulium pumatense sp. nov. is described from Vietnam, and is placed in the Simulium feuerborni species-group of the subgenus Simulium (Nevermannia) Enderlein. Its morphological characteristics include the relatively smaller numbers of the following three numerical features: inner teeth of the female mandible (15-18), minute conical processes (16) on the female cibarium, and male upper-eye facets (in 15 vertical columns and 16 horizontal rows). Keys are constructed to distinguish this species from four species of the same group in Vietnam. Our molecular analysis of the DNA barcoding COI gene shows that this species is most closely related to cytoform A of the S. feuerborni complex from Thailand.

The black flies (Diptera: Simuliidae) of Vietnam.

The biodiversity of black flies (Diptera: Simuliidae), which are biting insects of medical and veterinary importance, is strikingly high in Southeast Asian countries, such as Indonesia, Malaysia, Philippines and Thailand. In 2013, we began to explore the fauna of black flies in Vietnam, which has so far been poorly studied. In this monograph, the wealth of the biodiversity of black flies in Vietnam is also confirmed on the basis of the results of our recent investigations, though limited to five provinces in the country.      Morphotaxonomic studies of black flies obtained from Sapa, Lao Cai Province, northern Vietnam, in 2014 and Nghe An Province, northern Vietnam, in 2015, and reexaminations of black flies collected from Tam Dao, Vinh Phuc Province, northern Vietnam, in 2013, Thua Thien Hue Province, central Vietnam, in 2014, and Lam Dong Province, southern Vietnam, in 2014, were conducted. A total of 22 species are described as new, including one in the newly recorded subgenus Montisimulium Rubtsov, and three species are recognized as new records from Vietnam. This investigation brings the number of species of black flies known in Vietnam to 70, all of which are assigned to the genus Simulium Latreille, and are placed in four subgenera (25 in Gomphostilbia Enderlein, one in Montisimulium, seven in Nevermannia Enderlein, and 37 in Simulium Latreille s. str.). The numbers of species-groups recognized include seven in Gomphostilbia, three in Nevermannia and nine in Simulium, indicating a high diversity of putative phylogenetic lineages. New species include S. (G.) sanchayense sp. nov. (= the species formerly regarded as S. (G.) brinchangense Takaoka, Sofian-Azirun & Hashim), S. (S.) lowi sp. nov. (= the species formerly regarded as S. (S.) brevipar Takaoka & Davies), S. (S.) fuscicoxae sp. nov. [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)], S. (S.) suoivangense sp. nov. [= morphoform 'b' of the S. (S.) tani Takaoka & Davies (complex)]. Newly recorded species are S. (G.) parahiyangum Takaoka & Sigit, S. (N.) maeaiense Takaoka & Srisuka, and S. (S.) doipuiense Takaoka & Choochote (complex) [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)]. The substitute name, S. (S.) huense, is given for the species that was described under the name of S. (S.) cavum from southern Vietnam. A redescription of the female, male, pupa and larva of S. (G.) asakoae Takaoka & Davies is presented, and the female and larva of S. (G.) hongthaii Takaoka, Sofian-Azirun & Ya'cob are described for the first time. Keys to 10 subgenera in the Oriental Region and all 70 species recorded from Vietnam are provided for females, males, pupae and mature larvae.      As investigations extend nationwide in all the provinces in Vietnam, more new species and records are expected to be discovered. It is hoped that this monograph will be useful as a baseline taxonomic reference for future studies of black flies in Vietnam and neighbouring countries.

Vietnam, a Hotspot for Chromosomal Diversity and Cryptic Species in Black Flies (Diptera: Simuliidae).

The increasing attention on Vietnam as a biodiversity hotspot prompted an investigation of the potential for cryptic diversity in black flies, a group well known elsewhere for its high frequency of isomorphic species. We analyzed the banding structure of the larval polytene chromosomes in the Simulium tuberosum species group to probe for diversity beyond the morphological level. Among 272 larvae, 88 different chromosomal rearrangements, primarily paracentric inversions, were discovered in addition to 25 already known in the basic sequences of the group in Asia. Chromosomal diversity in Vietnam far exceeds that known for the group in Thailand, with only about 5% of the rearrangements shared between the two countries. Fifteen cytoforms and nine morphoforms were revealed among six nominal species in Vietnam. Chromosomal evidence, combined with available molecular and morphological evidence, conservatively suggests that at least five of the cytoforms are valid species, two of which require formal names. The total chromosomal rearrangements and species (15) now known from the group in Vietnam far exceed those of any other area of comparable size in the world, supporting the country's status as a biodiversity hotspot. Phylogenetic inference based on uniquely shared, derived chromosomal rearrangements supports the clustering of cytoforms into two primary lineages, the Simulium tani complex and the Southeast Asian Simulium tuberosum subgroup. Some of these taxa could be threatened by habitat destruction, given their restricted geographical distributions and the expanding human population of Vietnam.

The black flies (Diptera: Simuliidae) from Thua Thien Hue and Lam Dong Provinces, Vietnam.

Surveys of pupae and larvae of black flies (Diptera: Simuliidae) were carried out in Thua Thien Hue Province of central Vietnam, and Lam Dong Province of southern Vietnam in 2014. A total of 26 species belonging to the genus Simulium were collected, consisting of eight known species, one newly recorded species, and 17 new species (of which three species of the subgenus Nevermannia were described in 2014). The remaining 14 new species (nine of the subgenus Gomphostilbia and five of the subgenus Simulium) are described here based on females, males, pupae and mature larvae. The total number of species of black flies in Vietnam is now 46. Keys to identify all 26 species recorded from the two provinces of Vietnam are given for females, males, pupae and mature larvae.

Bifidobacterium species lower serum glucose, increase expressions of insulin signaling proteins, and improve adipokine profile in diabetic mice.

This study, using C57BL/6J mice with streptozotocin (STZ)-induced diabetes, aimed to determine whether Bifidobacterium species (spp.) both induces the expressions of proteins in the insulin signaling pathway and enhances the expressions of certain adipocytokines. The protein expressions of IκB kinase alpha (IKKα), IκB kinase beta (IKKß), nuclear factor-kappaB inhibitor alpha (IκBα), and the mitogen-activated protein kinase (MAPK) pathway were also investigated. Oral administration of Bifidobacterium spp. reduced blood glucose levels significantly and increased the protein expressions of insulin receptor beta, insulin receptor substrate 1, protein kinase B (Akt/PKB), IKKα, and IκBα. Extracellular-signal-regulated kinase 2 (ERK2) showed increased expression. Bifidobacterium spp. also induced the adiponectin expression and decreased both macrophage chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) expression. In addition, IKKß, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase expressions showed no significant changes in both groups. In conclusion, Bifidobacterium spp. may be the promising bacteria for treating diabetes.

Oral administration of Bifidobacterium spp. improves insulin resistance, induces adiponectin, and prevents inflammatory adipokine expressions.

The intestinal microbiome might be an important contributor to the development of type 2 diabetes. This study was designed to test the hypothesis that oral administration of Bifidobacterium species (spp.) (including B. longum, B. bifidum, B. infantis, and B. animalis) may both ameliorate insulin resistance and reduce the expressions of inflammatory adipocytokines. Male Swiss-Webster mice fed a high-fat diet with or without oral administration of Bifidobacterium spp. for 5 weeks were subjected to an insulin tolerance test and an oral glucose tolerance test. Plasma levels of glucose at 30, 60, 90 and 120 min after insulin injection or glucose administration were significantly lower in the Bifidobacterium spp. than in the control group (P < 0.05), showing the beneficial effect of oral administration on insulin resistance in obese Swiss mice. In addition, Bifidobacterium spp. increased the adiponectin mRNA level and decreased those of monocyte chemoattractant protein 1 and interleukin 6 in non-diabetic C57BL/6J mice fed a normal diet, indicating a molecular mechanism which may ameliorate the inflammatory state, thereby reducing insulin resistance. In conclusion, oral administration of Bifidobacterium spp. improves insulin resistance and glucose tolerance in obese mice by reducing inflammation, as it does in the lean state.

Three new species of Simulium (Nevermannia) (Diptera: Simuliidae) from Vietnam.

Three new species of black flies, Simulium (Nevermannia) langbiangense, S. (N.) phami, and S. (N.) bachmaense, are described on the basis of females, males, pupae and larvae collected in Vietnam. All three species are assigned to the Simulium feuerborni species-group of Simulium (Nevermannia). Simulium (N.) langbiangense sp. nov. is characterized by the female sensory vesicle with a large opening, and a short common basal stalk of the six pupal gill filaments, S. (N.) phami sp. nov. is most striking in having the pupal gill with five filaments, a character not reported in species of the S. feuerborni species-group, and S. (N.) bachmaense sp. nov. is characterized by the female genital fork with a triangular lobe-like projection pointed posteromedially on each arm, and the small larval postgenal cleft. This represents the first record of the S. feuerborni species-group from Vietnam.

Overall retrospective epidemiological study on tranmission mechanism of orientia tsutsugamushi in transmission disease vector

Orientia tsutsugamushi is causative agent of tsutsugamushi disease, they parasitize in mites and is transmited from mother mite to other generation through egg (vertical transmission). In addition, they is transmited from this mite strain to other mite strain (horizontal transmission), this enssure large distribution of Orientia tsutsugamushi in mite community. However, this phenomenon rarely occure in nature. Orientia tsutsugamushi transmit to human through the bite of mites due to some of mite strains

Orientia tsutsugamushi in unengorged chiggers of epidemiological area of tsutsugamushi by nested PCR technique

The study applied the Nested PCR technique with part of 56kilodalton type-specific antigen (TSA) gene DNAs of Orientia tsutsugamushi. Forty-eight pools (480 individuals) of unengorged larvae belonging to 4 trombiculid species: L. scutellare, L. pallidum, L. kistasatoi and L. fuji, collected from soil and ground surface of an endemic area for tsutsugamushi disease, were tested for 56kilodalton type-specific antigen (TSA) gene DNAs of Orientia tsutsugamushi, by nested PCR. Among them, 2 of 13 pools of L. scutellare were positive, and 2 serotypes of Kuroki and Gilliam were identified. Remaining 35 pools of 3 species were negative for Orientia tsutsugamushi. By using epidemiological' analysis, unengorged larvae of L. scutellare was suspected as the vector of tsutsu-gamushi disease in the studied area.

Study on trombiculid fauna and seasonal occurrence of several trombiculid species in an endemic area of tsutsugamushi disease

The study was carried out in an endemic area of tsutsugamushi disease, Taketa city, Japan, from January to December, 2000. Results: 1018 unengorged larvaes of 14 species were isolated from soil and ground surface by direct method and Suzuki method. Among them Helenicula miyagawai was isolated by only the Suzuki method. Leptotrombidium scutellare and l.pallidum suspected as vectors of tsutsugamushi disease in many areas of Japan were detected in this study. The seasonal occurrence of unengorged larvae of L.scutellare was from October to January with the probable peak in November. While L.pallidum occur almost throughout the year with two peak periods, one in May and the other in September.