Phlebotomine sand flies (Diptera, Psychodidae) from Pha Tong cave, Northern Thailand with a description of two new species and taxonomical thoughts about Phlebotomus stantoni.

BACKGROUND: In South-East Asia, Thailand is the country with the highest number of human autochthonous cases of leishmaniases mostly due to Leishmania martiniquensis. Their transmission remains unresolved to date even though sand flies are known vectors of leishmaniases. As such, we focused a study on the sand fly fauna of a cave in Thailand to explore the biodiversity of potential Leishmania vectors. MAIN RESULTS: We carried out an inventory in Pha Tong cave. We caught and identified 570 Phlebotomine sand flies (452 females and 118 males) and identified 14 species belonging to the genera Phlebotomus, Idiophlebotomus, Chinius, Sergentomyia and Grassomyia. Among these 14 species, two could not be related to known sand fly species. Herein, we propose the description of two new sand fly species, previously unknown to science. The first new species, Phlebotomus shadenae n. sp. is a sand fly of the subgenus Anaphlebotomus. It is morphologically close to Ph. stantoni, a species widely distributed throughout Southeast Asia. However, it differs by the length of the genital filaments in males or by the length of the ducts of the spermathecae in females as well as the high divergence of cytochrome b sequences. Additionally, we revised the systematics of the subgenus Anaphlebotomus and reinstated, by examination of its holotype, the validity of Ph. maynei, an Indian wrongly considered as a synonym of Ph. stantoni in the past. The second new species, Sergentomyia maiae n. sp., differs from a species in the same group, Se. barraudi, by an original cibarial double row of vertical teeth as well as by molecular data. CONCLUSIONS: We propose the description of two new sand fly species for Science with morphological and molecular evidence. Ph. shadenae n. sp. was also found to be distributed in the south of Thailand and in Laos. Future studies need to determine whether these two species can play a role as vectors of Leishmania parasites, Trypanosomatids or Phlebovirus. Most of the species caught in the present study are strictly cavernicolous except Grassomyia sp. and a few Sergentomyia.

Field evaluation of four commercial light traps, trap placement, and effect of carbon dioxide on phlebotomine sand fly collection in northern Thailand.

Several light trap devices have been invented and developed to assess the abundance of sand flies. Traps available in the market have different designs and attractant combinations to catch sand fly vectors. We evaluated the efficacy of four commercial light traps and determined the effect of trap placement and carbon dioxide (CO2) on sand fly collection in northern Thailand. Trap evaluations were conducted at two natural caves located in Chiang Rai province, Thailand. In the first part of the study, the efficacies of four trap types including the Centers for Disease Control miniature light trap (CDC LT), Encephalitis Vector Survey trap (EVS), CDC Updraft Blacklight trap (CDC UB), and Laika trap (LK) were evaluated and compared using a Latin square experimental design. The second half of the study evaluated the influence of trap placement and CO2 on sand fly collection. Additionally, CDC LT were placed inside, outside, and at the entrance of caves to compare the number of sand flies collected. For the trap efficacy experiment, a total of 11,876 phlebotomine sand flies were collected over 32 trap-nights. Results demonstrated that CDC LT, CDC UB, and LK collected significantly more sand flies than EVS (P > 0.05). However, there were no significant differences between the numbers of sand flies collected by CDC LT, CDC UB, and LK. A total of 6,698 sand flies were collected from the trap placement and CO2 experiment over 72 trap-nights. Results showed that CO2 did not influence the numbers of sand flies captured (P < 0.05), whereas trap placement at the entrance of the caves resulted in collection of significantly more sand flies than traps placed inside and outside of the caves. We found the CDC LT, CDC UB, and LK without CO2 captured the greatest amount of sand flies. This was particularly observed when traps were placed at the entrance of a cave, perhaps because of the greater passage of stimuli caused by wind flow at the entrance of the cave. The light traps in this study can be used effectively to collect sand fly vectors in northern Thailand.

The Prevalence of Bartonella Bacteria in Cattle Lice Collected from Three Provinces of Thailand.

Cattle lice are obligatory blood-sucking parasites, which is the cause of animal health problems worldwide. Recently, several studies have revealed that pathogenic bacteria could be found in cattle lice, and it can act as a potential vector for transmitting louse-borne diseases. However, the cattle lice and their pathogenic bacteria in Thailand have never been evaluated. In the present study, we aim to determine the presence of bacterial pathogens in cattle lice collected from three localities of Thailand. Total genomic DNA was extracted from 109 cattle louse samples and the Polymerase Chain Reaction (PCR) of 18S rRNA was developed to identify the cattle louse. Moreover, PCR was used for screening Bartonella spp., Acinetobacter spp., and Rickettsia spp. in cattle louse samples. The positive PCR products were cloned and sequenced. The phylogenetic tree based on the partial 18S rRNA sequences demonstrated that cattle lice species in this study are classified into two groups according to reference sequences; Haematopinus quadripertusus and Haematopinus spp. closely related to H. tuberculatus. The pathogen detection revealed that Bartonella spp. DNA of gltA and rpoB were detected in 25 of 109 samples (22.93%) both egg and adult stages, whereas Acinetobacter spp. and Rickettsia spp. were not detected in all cattle lice DNA samples. The gltA and rpoB sequences showed that the Bartonella spp. DNA was found in both H. quadripertusus and Haematopinus spp. closely related to H. tuberculatus. This study is the first report of the Bartonella spp. detected in cattle lice from Thailand. The finding obtained from this study could be used to determine whether the cattle lice can serve as a potential vector to transmit these pathogenic bacteria among cattle and may affect animal to human health.

Enzymes-based resistant mechanism in pyrethroid resistant and susceptible Aedes aegypti strains from northern Thailand.

Previous studies have shown that permethrin resistance in our selected PMD-R strain of Aedes aegypti from Chiang Mai, Thailand, was associated with a homozygous mutation in the knockdown resistance (kdr) gene and other mechanisms. In this study, we investigated the metabolic mechanism of resistance of this strain compared to the PMD strain which is susceptible to permethrin. The permethrin susceptibility of larvae was determined by a dose-response bioassay. Two synergists, namely piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate (BNPP), were also added to determine if the resistance is conferred by oxidase or esterase enzymes, respectively. The LC(50) value for PMD-R (25.42 ppb) was ∼25-fold higher than for PMD (1.02 ppb). The LC(50) was reduced 3.03-fold in PMD-R and 2.27-fold in PMD when the oxidase inhibitor (PBO) was added, but little or no reduction was observed in the presence of BNPP, indicating that oxidative enzymes play an important role in resistance. However, the LC(50) previously observed in the heterozygous mutation form was reduced ∼eightfold, indicating that metabolic resistance is inferior to kdr. The levels of cytochrome P450 (P450) extracted from fourth instar larvae were similar in both strains and were about 2.3-fold greater in microsomal fractions than in crude supernatant and cytosol fractions. Microsome oxidase activities were determined by incubation with each of three substrates, i.e., permethrin, phenoxybenzyl alcohol (PBOH), and phenoxybenzaldehyde (PBCHO), in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NAD(+)), PBO, and BNPP. It is known that hydrolysis of permethrin produces PBOH which is further oxidized to PBCHO by alcohol dehydrogenase (ADH) and then to phenoxybenzoic acid (PBCOOH) by aldehyde dehydrogenase (ALDH). When incubated with permethrin, a small amount of PBCOOH was detected in both strains (about 1.1-1.2 nmol/min/mg protein), regardless of the addition of NADPH. The addition of PBO resulted in about 70% and 50% reduction of PBCOOH in PMD and PMD-R, respectively. The addition of BNPP reduced PBCOOH about 50% and 35% in PMD and PMD-R, respectively. Using PBOH as substrate increased PBCOOH ∼16-fold and ∼40-fold in PMD and PMD-R, respectively. Using PBCHO as substrate increased PBCOOH ∼26-fold and ∼50-fold in PMD and PMD-R, respectively. The addition of NADPH, and particularly NAD(+), increased the level of PBCOOH. Together, the results have indicated the presence of a metabolic metabolism involving P450, ADHs, and ALDHs in both PMD and PMD-R strains, with greater enzyme activity in the latter.

High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand.

OBJECTIVES: To develop rapid monitoring tools to detect the F1534C permethrin-resistance mutation in domain IIIS6 of the Aedes aegypti voltage-gated sodium channel gene and determine the frequency and distribution of this mutation in Thailand. METHODS: A TaqMan SNP genotyping and an allele specific PCR (AS-PCR) assay were developed and validated by comparison with DNA sequencing of homozygous susceptible and homozygous resistant laboratory strains, their reciprocal-cross progenies, and field-caught mosquitoes. To determine the resistance phenotype of wild-caught A. aegypti, mosquitoes were exposed to 0.75% permethrin paper. The AS-PCR assay was used to screen 619 individuals from 20 localities throughout Thailand. RESULTS: Overall, both assays gave results consistent with DNA sequencing for laboratory strains of known genotype and for wild-caught A. aegypti. The only slight discrepancy was for the AS-PCR method, which overestimated the mutant allele frequency by 1.8% in wild-caught samples. AS-PCR assays of permethrin-exposed samples show that the mutant C1534 allele is very closely associated with the resistant phenotype. However, 19 permethrin-resistant individuals were homozygous for the wild-type F1534 allele. DNA sequencing revealed all these individuals were homozygous for two other mutations in domain II, V1016G and S989P, which are known to confer resistance (Srisawat et al. 2010). The F1534C mutation is widespread in Thailand with mutant allele frequencies varying among populations from 0.20 to 1.00. CONCLUSIONS: These assays can be used for the rapid detection of the F1534C resistance mutation in A. aegypti populations. The F1534C, and other, mutations underlie an extremely high prevalence of pyrethroid resistance in Thailand.

The specific status of Anopheles minimus s.l. collected from Taiwan.

At least three members (species A, C, and E) of the Anopheles minimus complex have been described in the Orient. This study investigated the specific status of An. minimus collected in the southern part of Taiwan by crossing experiments with species A from Thailand and species E from Japan. Crosses between Taiwan An. minimus and species A revealed genetic compatibilities. Post-zygotic isolation was observed in crosses between Taiwan An. minimus and species E. Hybrid progeny were only obtained from Taiwan female X species E male. F2 hybrid progeny were not obtained, since the hybrid males were sterile or almost sterile, with atrophied testes or abnormal spermatozoa. The hybrid females backcrossed with either Taiwan F1 progeny and species E males, and laid eggs with lower fertility and viability. This study supports previous published data regarding the analysis of the D3 region of the 28S gene of ribosomal DNA that An. minimus species A is indigenous to Taiwan. Whether other members of the An. minimus complex exist in Taiwan is not conclusive and needs more study.