Population genetics of the biting midge Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) from Thailand and its genetic relationships with global populations.

Culicoides oxystoma Kieffer is a vector of viruses, filarial nematodes and protozoa of the genus Leishmania transmitted to humans and other animals. Understanding genetic diversity, genetic structure and genetic relationships among geographically widespread populations will provide important information related to disease epidemiology. In this study, genetic diversity, genetic structure and genetic relationships between Thai C. oxystoma and those reported from other countries were inferred based on mitochondrial cytochrome oxidase I (COI) and nuclear internal transcribed spacer 1 (ITS-1) sequences. A high level of genetic diversity was found in C. oxystoma from Thailand. The maximum K2P intraspecific genetic divergence for COI gene and ITS-1 sequences were 4.29% and 6.55%, respectively. Despite high genetic diversity, no significant genetic differentiation was found within the 13 Thai populations. This could be a result of unspecialized habitat requirement of the larval habitat, abundance and continuous distribution of host blood sources, potential for long distance movement with host via trading. Mitochondrial genealogy analysis of the global population of C. oxystoma revealed three (A, B and C) genetically divergent lineages. Specimens from Thailand were included in the main lineage (A) with those from all other countries except those from Senegal that formed lineage B and those of Lineage C that was exclusively found in Bangladesh. The nuclear (ITS-1) genetic markers genealogy indicated that Thai C. oxystoma belong to the same genet.

Common but not connected: high genetic structure and cryptic genetic diversity in the ubiquitous biting midge Culicoides peregrinus Kieffer.

The biting midge Culicoides peregrinus Kieffer is a significant pest and vector species, and knowledge of its genetic diversity and genetic structure is critically important for designing an effective control program. However, such information is limited to only small sample-size DNA barcoding studies. Therefore, in this study, we used mitochondrial cytochrome c oxidase I (COI) to examine genetic structure and diversity of C. peregrinus from northeastern Thailand. In addition, we also inferred genetic relationships between C. peregrinus from Thailand and those reported from other countries across the geographic range of the species. Maximum intraspecific genetic divergence (3.83%) within Thai specimens was relatively high compared to other Culicoides species. Genetic structure analysis revealed that 71% (32 from 45) of population comparisons were highly significantly different. A high level of genetic structure among populations, even between those in close geographic proximity (22 km geographic distance) suggested that there has been little or no movement between local populations. This is possibly due to the ability to exploit diverse types of breeding site and a generalist feeding habit which enables C. peregrinus to complete its life cycle within cattle pens. Genetic relationships between Thai C. peregrinus and those reported from other countries revealed three genetically divergent lineages (A, B and C) associated with geographic origins. Specimens from Thailand + China formed lineage A, those from Australia formed lineage B and India + Bangladesh belonged to lineage C. These genetically divergent lineages also agree with morphological variation of the wing pale marking spots. Further investigation using independent genetic loci from nuclear genes will be very useful to resolve taxonomic status of these divergent lineages.

Molecular detection of filarial nematode from Culicoides biting midges (Diptera: Ceratopogonidae) in northeastern Thailand.

Culicoides Latreille biting midges are important blood feeding insects. Many species are pests and vectors of the disease causing agents including viruses, protozoa and filarial nematodes which can be transmitted to humans and other animals. However, knowledge of the role of Culicoides as vectors of filarial nematodes is limited, particular in Thailand, where at least 100 species of the genus Culicoides have been reported. In this study, a molecular approach using the 12S rRNA gene sequence was used to detecting the filarial nematode in four common biting midge species, C. actoni Smith, C. oxystoma Kieffer, C. peregrinus Kieffer and C. mahasarakhamense Pramual, Jomkumsing, Piraonapicha & Jumpato in animal shelters from northeastern Thailand. A total of 1,721 specimens were used for molecular screening. An unidentified Onchocercidae sp. was detected in a specimen of C. mahasarakhamense collected from Maha Sarakham province. This filarial species shows 93% sequence similarity with an unidentified Onchocercidae sp. isolated from Culex mosquitoes. Phylogenetic analyses revealed that Onchocercidae sp. from C. mahasarakhamense formed a clade with strong bootstrap support (100%) with filarial species detected in birds. Thus, it is very likely that the Onchocercidae sp. found in this study employes birds as vertebrate hosts which agrees with feeding behavior of C. mahasarakhamense which is known to feed on chicken. Further study is requiring to examine whether this biting midge species is a competent vector of this Onchocercidae sp.

Common but not connected: high genetic structure and cryptic genetic diversity in the ubiquitous biting midge Culicoides peregrinus Kieffer

@#The biting midge Culicoides peregrinus Kieffer is a significant pest and vector species, and knowledge of its genetic diversity and genetic structure is critically important for designing an effective control program. However, such information is limited to only small sample-size DNA barcoding studies. Therefore, in this study, we used mitochondrial cytochrome c oxidase I (COI) to examine genetic structure and diversity of C. peregrinus from northeastern Thailand. In addition, we also inferred genetic relationships between C. peregrinus from Thailand and those reported from other countries across the geographic range of the species. Maximum intraspecific genetic divergence (3.83%) within Thai specimens was relatively high compared to other Culicoides species. Genetic structure analysis revealed that 71% (32 from 45) of population comparisons were highly significantly different. A high level of genetic structure among populations, even between those in close geographic proximity (22 km geographic distance) suggested that there has been little or no movement between local populations. This is possibly due to the ability to exploit diverse types of breeding site and a generalist feeding habit which enables C. peregrinus to complete its life cycle within cattle pens. Genetic relationships between Thai C. peregrinus and those reported from other countries revealed three genetically divergent lineages (A, B and C) associated with geographic origins. Specimens from Thailand + China formed lineage A, those from Australia formed lineage B and India + Bangladesh belonged to lineage C. These genetically divergent lineages also agree with morphological variation of the wing pale marking spots. Further investigation using independent genetic loci from nuclear genes will be very useful to resolve taxonomic status of these divergent lineages.

Molecular detection of filarial nematode from Culicoides biting midges (Diptera: Ceratopogonidae) in northeastern Thailand

@#Culicoides Latreille biting midges are important blood feeding insects. Many species are pests and vectors of the disease causing agents including viruses, protozoa and filarial nematodes which can be transmitted to humans and other animals. However, knowledge of the role of Culicoides as vectors of filarial nematodes is limited, particular in Thailand, where at least 100 species of the genus Culicoides have been reported. In this study, a molecular approach using the 12S rRNA gene sequence was used to detecting the filarial nematode in four common biting midge species, C. actoni Smith, C. oxystoma Kieffer, C. peregrinus Kieffer and C. mahasarakhamense Pramual, Jomkumsing, Piraonapicha & Jumpato in animal shelters from northeastern Thailand. A total of 1,721 specimens were used for molecular screening. An unidentified Onchocercidae sp. was detected in a specimen of C. mahasarakhamense collected from Maha Sarakham province. This filarial species shows 93% sequence similarity with an unidentified Onchocercidae sp. isolated from Culex mosquitoes. Phylogenetic analyses revealed that Onchocercidae sp. from C. mahasarakhamense formed a clade with strong bootstrap support (100%) with filarial species detected in birds. Thus, it is very likely that the Onchocercidae sp. found in this study employes birds as vertebrate hosts which agrees with feeding behavior of C. mahasarakhamense which is known to feed on chicken. Further study is requiring to examine whether this biting midge species is a competent vector of this Onchocercidae sp.

A new species of black fly, formerly cytoform C of the Simulium angulistylum complex (Diptera: Simuliidae), from a high mountain in northeastern Thailand.

Cytoform C, one of three cytoforms in the Simulium angulistylum Takaoka & Davies complex from a high mountain in northeastern Thailand was morphologically and molecularly investigated. All known life stages (larva, pupa, adult male and female except egg) were morphologically similar to, but distinguishable from S. angulistylum s. str. and S. isanense Takaoka, Srisuka & Saeung in the adults by the relative length of the fore and hind basitarsi and relative length of the tooth to the claw. It is also morphologically distinct from other species of the S. epistum species-group. Here, it is formally described as a new species, S. prayooki. Molecular genetic data based on mitochondrial cytochrome c oxidase subunit I (COI) also supported the morphological similarity between the new species and the two related known species (S. angulistylum s. str. and S. isanense) as phylogenetic analysis retrieved them all from a single clade and with a low level of interspecific genetic divergence (1.74%). This might possibly have resulted from incomplete lineage sorting as they are likely to share a recent common ancestor. Despite limitation of molecular genetic differentiation, the new species was distinctly different from two other cytoforms of S. angulistylum complex based on polytene chromosome banding patterns and ecology of the immature stages. Thus, this study highlights the necessity of using an integrated approach for fully understanding black fly biodiversity.

A new record of Rickettsia japonica in ticks infesting a Burmese ferret-badger in Thailand.

Ticks are important vectors of arthropod-borne diseases and they can transmit a wide variety of zoonotic pathogens to humans, domestic and wild animals. Rickettsia japonica is a member of SFG rickettsiae causing Japanese spotted fever (JSF) and can transmit to humans via infected ticks. In this study, we report the first case of Rickettsia japonica in Haemaphysalis hystricis tick collected from a roadkill Burmese ferret-badger ( Melogale personata ) in Loei province, northeastern Thailand. According to the DNA sequences and phylogenetic analyses of the outer membrane protein A and B genes ( ompA and ompB), the detected R. japonica was identical to those found in JSF patients in Korea, Japan, and China, and closely related to Rickettsia detected by ompA in a tick from Thailand. Further study on the prevalence of R. japonica and diversity of mammalian reservoir hosts will be useful to gain a better understanding of JSF epidemiology.

Description of the male of Simulium triglobus Takaoka & Kuvangkadilok (Diptera: Simuliidae) from Thailand.

Simulium triglobus Takaoka & Kuvangkadilok from Thailand, in the Simulium (Simulium) multistriatum species-group, is unique among species in the family Simuliidae in having the female terminalia with three spermathecae (rather than one spermatheca). This species was described from Nan province, northern Thailand based on larvae, pupae and females but its male has remained unknown. In this study, the male of S. triglobus is described for the first time based on adult males reared from pupae collected from the type locality. The most distinctive characteristic of the male of S. triglobus is the shape of the ventral plate, which is hexagonal when viewed ventrally. No other members of S. multistriatum species group known thus far have such a unique ventral plate. In addition, the number of upper-eye (large) facets and color patterns of the legs can be used to differentiate this species in the male from other members of the S. multistriatum species-group. Mitochondrial cytochrome c oxidase I sequences enabled association of adult male specimens of S. triglobus with previously known life stages. Phylogenetic analysis based on these sequences revealed that specimens of S. triglobus formed a strongly supported monophyletic clade, being genetically distinct from other members of S. multistriatum species-group in Thailand.

A new species of black fly, formerly cytoform C of the Simulium angulistylum complex (Diptera: Simuliidae), from a high mountain in northeastern Thailand

@#Cytoform C, one of three cytoforms in the Simulium angulistylum Takaoka & Davies complex from a high mountain in northeastern Thailand was morphologically and molecularly investigated. All known life stages (larva, pupa, adult male and female except egg) were morphologically similar to, but distinguishable from S. angulistylum s. str. and S. isanense Takaoka, Srisuka & Saeung in the adults by the relative length of the fore and hind basitarsi and relative length of the tooth to the claw. It is also morphologically distinct from other species of the S. epistum species-group. Here, it is formally described as a new species, S. prayooki. Molecular genetic data based on mitochondrial cytochrome c oxidase subunit I (COI) also supported the morphological similarity between the new species and the two related known species (S. angulistylum s. str. and S. isanense) as phylogenetic analysis retrieved them all from a single clade and with a low level of interspecific genetic divergence (1.74%). This might possibly have resulted from incomplete lineage sorting as they are likely to share a recent common ancestor. Despite limitation of molecular genetic differentiation, the new species was distinctly different from two other cytoforms of S. angulistylum complex based on polytene chromosome banding patterns and ecology of the immature stages. Thus, this study highlights the necessity of using an integrated approach for fully understanding black fly biodiversity.

Description of the male of Simulium triglobus Takaoka & Kuvangkadilok (Diptera: Simuliidae) from Thailand

@#Simulium triglobus Takaoka & Kuvangkadilok from Thailand, in the Simulium (Simulium) multistriatum species-group, is unique among species in the family Simuliidae in having the female terminalia with three spermathecae (rather than one spermatheca). This species was described from Nan province, northern Thailand based on larvae, pupae and females but its male has remained unknown. In this study, the male of S. triglobus is described for the first time based on adult males reared from pupae collected from the type locality. The most distinctive characteristic of the male of S. triglobus is the shape of the ventral plate, which is hexagonal when viewed ventrally. No other members of S. multistriatum species group known thus far have such a unique ventral plate. In addition, the number of upper-eye (large) facets and color patterns of the legs can be used to differentiate this species in the male from other members of the S. multistriatum species-group. Mitochondrial cytochrome c oxidase I sequences enabled association of adult male specimens of S. triglobus with previously known life stages. Phylogenetic analysis based on these sequences revealed that specimens of S. triglobus formed a strongly supported monophyletic clade, being genetically distinct from other members of S. multistriatum species-group in Thailand.

A new record of Rickettsia japonica in ticks infesting a Burmese ferret-badger in Thailand

@#Ticks are important vectors of arthropod-borne diseases and they can transmit a wide variety of zoonotic pathogens to humans, domestic and wild animals. Rickettsia japonica is a member of SFG rickettsiae causing Japanese spotted fever (JSF) and can transmit to humans via infected ticks. In this study, we report the first case of Rickettsia japonica in Haemaphysalis hystricis tick collected from a roadkill Burmese ferret-badger (Melogale personata) in Loei province, northeastern Thailand. According to the DNA sequences and phylogenetic analyses of the outer membrane protein A and B genes (ompA and ompB), the detected R. japonica was identical to those found in JSF patients in Korea, Japan, and China, and closely related to Rickettsia detected by ompA in a tick from Thailand. Further study on the prevalence of R. japonica and diversity of mammalian reservoir hosts will be useful to gain a better understanding of JSF epidemiology.

Genetic diversity and population structure of Culex modestus across Europe: does recent appearance in the United Kingdom reveal a tendency for geographical spread?

In mainland Europe, the mosquito species Culex modestus Ficalbi (1890) is a bridge vector for West Nile virus (WNV) from its natural bird-mosquito cycle to mammals. The present study assessed the genetic diversity of Cx. modestus, as well as related Culex species, using the mitochondrial COI DNA barcoding region and compared this with the population structure across Europe. A haplotype network was mapped to determine genealogical relationships among specimens. The intraspecific genetic diversity within individual Culex species was below 2%, whereas the interspecific genetic divergence varied from 2.99% to 13.74%. In total, 76 haplotypes were identified among 198 sequences. A median-joining network determined from 198 COI sequences identified two major lineages that were separated by at least four mutation steps. A high level of intraspecific genetic diversity was not detected in Cx. modestus in samples submitted from different European populations, which indicates that morphologically identified specimens represent a single species and not a species complex. Therefore, it is deduced that different populations of Cx. modestus will show a similar potential to transmit WNV, lending support to concerns that the population present in southeast England represents a risk of transmission to humans.

Genetic Analysis of Populations of the Peach Fruit Fly, Bactrocera zonata (Diptera: Tephritidae), in Iran.

Bactrocera zonata (Saunders) as one of the most devastating species of the genus Bactrocera is a notorious polyphagous fruit fly pest attacking lots of commercially important host plant species in many parts of the world including Iran. Despite economic importance, the population genetics of B. zonata have remained relatively unexplored in the country. In this study, the genetic structure, genetic diversity, and demographic history of this pest in Iran were investigated on the basis of cytochrome oxidase c subunit 1 (COI) sequences. A total of 21 haplotypes were identified from 66 individuals collected from seven locations. Genetic diversity indices (number of haplotypes, haplotype diversity, and nucleotide diversity) revealed high genetic diversity without any isolation by distance among the geographic areas. An overall low level of genetic differentiation between populations revealed by genetic structure analysis reinforces the hypothesis of free movement of flies throughout the geographic areas. Non-significant correlation between genetic distances and geographic distances was an indication of a high level of gene flow among the studied populations of the pest. In addition, the recent population expansion following a recent past bottleneck could be a factor that might be contributed to the overall low level of the genetic structure. Mismatch distribution analysis as well as Tajima's D and Fu's FS tests strengthens the likelihood of a recent expanded population following a population bottleneck.

Genetic diversity and DNA barcoding of the black fly (Diptera: Simuliidae) vectors of parasites causing human onchocerciasis in Guatemala.

Genetic variation based on mitochondrial cytochrome c oxidase I (COI) and II (COII) sequences was investigated for three black fly nominal species, Simulium metallicum Bellardi complex, S. callidum Dyar and Shannon, and S. ochraceum Walker complex, which are vectors of human onchocerciasis from Guatemala. High levels of genetic diversity were found in S. metallicum complex and S. ochraceum complex with maximum intraspecific genetic divergences of 11.39% and 4.25%, respectively. Levels of genetic diversity of these nominal species are consistent with species status for both of them as they are cytologically complexes of species. Phylogenetic analyses revealed that the S. metallicum complex from Guatemala divided into three distinct clades, two with members of this species from several Central and South American countries and another exclusively from Mexico. The Simulium ochraceum complex from Guatemala formed a clade with members of this species from Mexico and Costa Rica while those from Ecuador and Colombia formed another distinct clade. Very low diversity in S. callidum was found for both genes with maximum intraspecific genetic divergence of 0.68% for COI and 0.88% for COII. Low genetic diversity in S. callidum might be a consequence of the result being informative of only recent population history of the species.

Genetic diversity and DNA barcoding of the black fly (Diptera: Simuliidae) vectors of parasites causing human onchocerciasis in Guatemala

@#Genetic variation based on mitochondrial cytochrome c oxidase I (COI) and II (COII) sequences was investigated for three black fly nominal species, Simulium metallicum Bellardi complex, S. callidum Dyar & Shannon, and S. ochraceum Walker complex, which are vectors of human onchocerciasis from Guatemala. High levels of genetic diversity were found in S. metallicum complex and S. ochraceum complex with maximum intraspecific genetic divergences of 11.39% and 4.25%, respectively. Levels of genetic diversity of these nominal species are consistent with species status for both of them as they are cytologically complexes of species. Phylogenetic analyses revealed that the S. metallicum complex from Guatemala divided into three distinct clades, two with members of this species from several Central and South American countries and another exclusively from Mexico. The Simulium ochraceum complex from Guatemala formed a clade with members of this species from Mexico and Costa Rica while those from Ecuador and Colombia formed another distinct clade. Very low diversity in S. callidum was found for both genes with maximum intraspecific genetic divergence of 0.68% for COI and 0.88% for COII. Low genetic diversity in S. callidum might be a consequence of the result being informative of only recent population history of the species.

Population genetic structure and demographic history of the black fly vector, Simulium nodosum in Thailand.

An understanding of the genetic structure and diversity of vector species is crucial for effective control and management. In this study, mitochondrial DNA sequences were used to examine the genetic structure, diversity and demographic history of a black fly vector, Simulium nodosum Puri (Diptera: Simuliidae), in Thailand. A total of 145 sequences were obtained from 10 sampling locations collected across geographical ranges in the country. Low genetic diversity was found in populations of S. nodosum that could be explained by the recent population history of this species. Demographic history analysis revealed a signature of demographic expansion dating back to only 2600-5200 years ago. Recent population expansion in S. nodosum possibly followed an increase in agriculture that enabled its hosts', humans and domestic animals, densities to increase. Alternatively, the Thai populations could be a derivative of an older expansion event in the more northern populations. Mitochondrial DNA genealogy revealed no genetically divergent lineages, which agrees with the previous cytogenetic study. Genetic structure analyses found that only 27% of the pairwise comparisons were significantly different. The most likely explanation for the pattern of genetic structuring is the effect of genetic drift because of recent colonization.