Coxiella-like bacteria in Haemaphysalis wellingtoni ticks associated with Great Hornbill, Buceros bicornis.

Birds are known to be the most mobile hosts and are therefore considered to be hosts with potential to contribute to the long-distance spread and transmission of tick-borne pathogens. In the present study, ticks were collected from a hornbill nest at Chaiyaphum Province, Thailand. They were screened for the presence of Coxiella bacteria using conventional PCR. The evolutionary relationships of positive Coxiella-like bacteria (CLB) were analysed based on the gene sequences of 16S rRNA, groEL and rpoB. Among all 22 tested ticks, CLB infections were found in 2 Haemaphysalis wellingtoni individuals. In a phylogenetic analysis, the Coxiella 16S rRNA gene detected in this study formed a separate clade from sequences found in ticks of the same genus. In contrast, the phylogenetic relationships based on groEL and rpoB revealed that these two genes from H. wellingtoni ticks grouped with CLB from the same tick genus (Haemaphysalis). This study is the first to report the presence of CLB in H. wellingtoni ticks associated with the Great Hornbill, Buceros bicornis in Thailand. Three genes of CLB studied herein were grouped separately with Coxiella burnetii (pathogenic strain). The effects of CLB in the ticks and Buceros bicornis require further investigation.

Coxiella-like bacteria in Haemaphysalis wellingtoni ticks associated with Great Hornbill, Buceros bicornis

@#Birds are known to be the most mobile hosts and are therefore considered to be hosts with potential to contribute to the long-distance spread and transmission of tick-borne pathogens. In the present study, ticks were collected from a hornbill nest at Chaiyaphum Province, Thailand. They were screened for the presence of Coxiella bacteria using conventional PCR. The evolutionary relationships of positive Coxiella-like bacteria (CLB) were analysed based on the gene sequences of 16S rRNA, groEL and rpoB. Among all 22 tested ticks, CLB infections were found in 2 Haemaphysalis wellingtoni individuals. In a phylogenetic analysis, the Coxiella 16S rRNA gene detected in this study formed a separate clade from sequences found in ticks of the same genus. In contrast, the phylogenetic relationships based on groEL and rpoB revealed that these two genes from H. wellingtoni ticks grouped with CLB from the same tick genus (Haemaphysalis). This study is the first to report the presence of CLB in H. wellingtoni ticks associated with the Great Hornbill, Buceros bicornis in Thailand. Three genes of CLB studied herein were grouped separately with Coxiella burnetii (pathogenic strain). The effects of CLB in the ticks and Buceros bicornis require further investigation.

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.

Cytogenetic, crossing and molecular evidence of two cytological forms of Anopheles argyropus and three cytological forms of Anopheles pursati (Diptera: Culicidae) in Thailand.

Nine and 11 isolines of Anopheles argyropus and Anopheles pursati, respectively, were established from individual females collected from cow-baited traps, and the characteristics of metaphase chromosomes were investigated in their F1-progenies. As determined by the different amounts of extra heterochromatin on sex chromosomes, 2 types of X (X1, X2) and Y (Y1,Y2), and 2 types of X (X1, X2) and 3 types of Y (Y1, Y2, Y3) chromosomes were obtained from An. argyropus and An. pursati, respectively. These types of sex chromosomes comprised 2 [Forms A (X1, Y1) and B (X1, X2, Y2)] and 3 [Forms A (X1, X2, Y1), B (X1, X2, Y2) and C (X2, Y3)] karyotypic forms of An. argyropus and An. pursati, respectively. All karyotypic forms acquired from An. pursati are new one that were discovered in this study, of which Forms A, B and C were found generally in Chiang Mai Province, while only 1 isoline of Form B was obtained in Ratchaburi Province. Form A was recovered from An. argyropus only in Ubon Ratchathani Province, whereas Form B from that species was found commonly in both Ubon Rathchathani and Nakhon Si Thammarat Provinces. Crossing experiments among the 2 and 3 isolines representing 2 and 3 karyotypic forms of An. argyropus and An. pursati, respectively, indicated genetic compatibility in yielding viable progenies and synaptic salivary gland polytene chromosomes through F2-generations. The conspecific natures of these karyotypic forms in both species were further supported by very low intraspecific sequence variations (average genetic distance: An. argyropus = 0.003-0.007, An. pursati = 0-0.005) of ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII).

Cytogenetic, crossing and molecular evidence of two cytological forms of Anopheles argyropus and three cytological forms of Anopheles pursati (Diptera: Culicidae) in Thailand

Nine and 11 isolines of Anopheles argyropus and Anopheles pursati, respectively, were established from individual females collected from cow-baited traps, and the characteristics of metaphase chromosomes were investigated in their F1-progenies. As determined by the different amounts of extra heterochromatin on sex chromosomes, 2 types of X (X1, X2) and Y (Y1,Y2), and 2 types of X (X1, X2) and 3 types of Y (Y1, Y2, Y3) chromosomes were obtained from An. argyropus and An. pursati, respectively. These types of sex chromosomes comprised 2 [Forms A (X1, Y1) and B (X1, X2, Y2)] and 3 [Forms A (X1, X2, Y1), B (X1, X2, Y2) and C (X2, Y3)] karyotypic forms of An. argyropus and An. pursati, respectively. All karyotypic forms acquired from An. pursati are new one that were discovered in this study, of which Forms A, B and C were found generally in Chiang Mai Province, while only 1 isoline of Form B was obtained in Ratchaburi Province. Form A was recovered from An. argyropus only in Ubon Ratchathani Province, whereas Form B from that species was found commonly in both Ubon Rathchathani and Nakhon Si Thammarat Provinces. Crossing experiments among the 2 and 3 isolines representing 2 and 3 karyotypic forms of An. argyropus and An. pursati, respectively, indicated genetic compatibility in yielding viable progenies and synaptic salivary gland polytene chromosomes through F2-generations. The conspecific natures of these karyotypic forms in both species were further supported by very low intraspecific sequence variations (average genetic distance: An. argyropus = 0.003-0.007, An. pursati = 0-0.005) of ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII).

Metaphase karyotypes of Anopheles paraliae (Diptera:Culicidae) in Thailand and evidence to support five cytological races.

Sixteen isoline colonies of Anopheles paraliae were established from wild-caught females collected from cow-baited traps at 4 locations in Thailand. They showed 3 types of X (X1, X2, X3) and 5 types of Y (Y1, Y2, Y3, Y4, Y5) chromosomes based on the number and amount of major block(s) of heterochromatin present in the heterochromatic arm, and were designated as Forms A (X3, Y1), B (X1, X2, X3, Y2), C (X3, Y3), D (X1, X2, X3, Y4) and E (X3, Y5). Form A was found in Songkhla Province, Form B was obtained in Ratchaburi, Nakhon Si Thammarat and Songkhla Provinces, Form C was acquired in Chanthaburi Province, Form D was recovered in Ratchaburi and Songkhla Provinces, and Form E was encountered in Ratchaburi Province. Hybridization experiments among the 7 isoline colonies, which represented the 5 karyotypic forms of An. paraliae, revealed genetic compatibility in providing viable progenies and synaptic salivary gland polytene chromosomes through F2-generations, and thus suggest the conspecific nature of these karyotypic forms. These results were supported by the very low intraspecific sequence divergence (mean genetic distance = 0.000-0.002) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII) of the 5 forms.

Geographic distribution and genetic compatibility among six karyotypic forms of Anopheles peditaeniatus (Diptera: Culicidae) in Thailand.

Fifty-three isolines of Anopheles peditaeniatus were established from individual wild-caught females collected from cow-baited traps in 17 provinces of Thailand. Three types of X (X1, X2, X3) and 6 types of Y (Y1,Y2, Y3, Y4, Y5, Y6) chromosomes were determined based on different amounts of major block(s) of heterochromatin. These sex chromosomes comprised 6 karyotypic forms designated as Forms A (X3, Y1), B (X1, X2, X3, Y2), C (X3, Y3), D (X1, X2, X3, Y4), E (X1, X2, X3,Y5) and F (X2, X3, Y6). Form F is a new metaphase karyotype discovered in this study and is commonly found in all regions. Form A was found only in Lampang province, whereas Form E is widespread throughout the country. Forms B, C and D were obtained from the northern, northeastern, western and southern regions. Crossing experiments among the 11 isoline colonies representing the 6 karyotypic forms of An. peditaeniatus indicated genetic compatibility yielding viable progenies and complete synapsis of salivary gland polytene chromosomes through to the F2-generations. The results suggested the conspecific nature of these karyotypic forms which were further supported by very low intraspecific variation (genetic distance = 0.000-0.003) of nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII).

Cytotaxonomy, morphology and ecology of the Simulium nobile species group (Diptera: Simuliidae) in Thailand.

The polytene chromosomes of 512 larvae of the Simulium nobile species group collected from 16 stream sites in northern, central and southern Thailand were examined. Band-by- band comparisons relative to the established standard chromosome map for the subgenus Simulium distinguished the 2 species of this group, S. nobile and S. nodosum. The differences are mainly due to the position of the nucleolar organizer, pseudochromocenter, and fixed and polymorphic inversions. S. nobile is a monomorphic species, while S. nodosum is a polymorphic species. The morphology of these taxa is described based on the gill filaments and the color of the antennae and female genitalia. S. nobile is distributed in southern Thailand at low altitudes, whereas the distribution of S. nodosum is in the north at higher altitudes.

Wolbachia and bacteriophage WO-B density of Wolbachia A-infected Aedes albopictus mosquito.

Wolbachia are maternally inherited symbiotic bacteria capable of inducing an extensive range of reproductive abnormalities in their hosts, including cytoplasmic incompatibility (CI). Its density (concentration) is likely to influence the penetrance of CI in incompatible crosses. The variations of Wolbachia density could also be linked with phage WO density. We determined the relative density (relative concentration) of prophage WO orf7 and Wolbachia (phage-to-bacteria ratio) during early developmental and adult stages of singly infected Aedes albopictus mosquito (Wolbachia A-infected) by using real-time quantitative PCR. Phage WO and Wolbachia did not develop at the same rate. Relative Wolbachia density (bacteria-to-host ratio) was high later in development (adult stages) whilst relative prophage WO density (phage-to-bacteria ratio) was low in the adult stages. Furthermore, 12-d-old adults of singly infected female mosquito had the highest Wolbachia density. In contrast, the larval stage 4 (L4) contained the highest prophage WO-B orf7 density. The association of hosts-Wolbachia-phage among diverse species is different. Thus, if phage and Wolbachia are involved in CI mechanism, the information of this association should be acquired for each specific type of organism for future use of population replacement or gene drive system.

Bacteriophage WO-B and Wolbachia in natural mosquito hosts: infection incidence, transmission mode and relative density.

Bacteriophages of Wolbachia bacteria have been proposed as a potential transformation tool for genetically modifying mosquito vectors. In this study, we report the presence of the WO-B class of Wolbachia-associated phages among natural populations of several mosquito hosts. Eighty-eight percent (22/25) of Wolbachia-infected mosquito species surveyed were found to contain WO-B phages. WO-B phage orf7 sequence analysis suggested that a single strain of WO-B phage was found in most singly (23/24) or doubly (1/1) Wolbachia-infected mosquitoes. However, the single Wolbachia strain infecting Aedes perplexus was found to harbour at least two different WO-B phages. Phylogenetic analysis suggested that horizontal transmission of WO-B phages has occurred on an evolutionary scale between the Wolbachia residing in mosquitoes. On an ecological scale, a low trend of co-transmission occurred among specific WO-B phages within Wolbachia of each mosquito species. Assessment of the density of WO-B phage by real-time quantitative polymerase chain reaction (RTQ-PCR) revealed an average relative density of 7.76 x 10(5)+/- 1.61 x 10(5) orf7 copies per individual mosquito for a single Wolbachia strain infecting mosquitoes, but a threefold higher density in the doubly Wolbachia-infected Aedes albopictus. However, the average combined density of WO-B phage(s) did not correlate with that of their Wolbachia hosts, which varied in different mosquito species. We also confirmed the presence of WO-B-like virus particles in the laboratory colony of Ae. albopictus (KLPP) morphologically, by transmission electron microscopy (TEM). The viral-like particles were detected after purification and filtration of Ae. albopictus ovary extract, suggesting that at least one WO-B-like phage is active (temperate) within the Wolbachia of this mosquito vector. Nevertheless, the idea of utilizing these bacteriophages as transformation vectors still needs more investigation and is likely to be unfeasible.

Closely related Wolbachia strains within the pumpkin arthropod community and the potential for horizontal transmission via the plant.

Phylogenetic studies have implicated frequent horizontal transmission of Wolbachia among arthropod host lineages. However, the ecological routes for such lateral transfer are poorly known. We surveyed the species of two arthropod communities, one on pumpkin and the other on loofah plants, for Wolbachia, constructed wsp gene phylogenies of those Wolbachia strains found to infect community members, and established ecological links among infected members. Four taxonomically diverse insects in the pumpkin arthropod community contained very closely related Wolbachia wsp sequences (<1.5% divergence by Kimura-2-parameter distances). These insects, namely, the whitefly Bemisia tabaci, the planthopper Nisia nervosa, the flea beetle Phyllotreta sp., and the fleahopper Halticus minutus, were all collected from pumpkin leaves. They were ecologically linked through feeding on the same leaf substrate. Unlike other infected leaf insects, the whitefly population appeared to have a permanent breeding relationship with pumpkin plants, and high and stable, but not fixed, monthly Wolbachia infection rates. Our findings suggest potential roles for the plant in Wolbachia transmission and for whiteflies in being an infection source for other pumpkin leaf-feeding insects.

SCAR markers and multiplex PCR-based identification of isomorphic species in the Anopheles dirus complex in Southeast Asia.

The Anopheles dirus Peyton & Harrison complex of mosquitoes (Diptera: Culicidae) comprises seven known species, including important malaria vectors in Southeast Asia. Specific identification of each species of the complex, which cannot be distinguished using morphological characters, is crucial for understanding vector ecology and implementing effective control measures. Derived from individual random amplified polymorphic DNA (RAPD) markers, sequence characterized amplified regions (SCAR) were analysed for the design of specific paired-primers. Combination of six SCAR primers resulted in the development of a simple, robust, single multiplex PCR able to identify three important malaria vectors among the four most common species (A, B, C, D) of the complex: species A from several Southeast Asian countries, species B from Perlis, Malaysia, and species C and D from Thailand.

Host age effect and expression of cytoplasmic incompatibility in field populations of Wolbachia-superinfected Aedes albopictus.

The Asian tiger mosquito, Aedes albopictus (Skuse), is a known vector of dengue in South America and Southeast Asia. It is naturally superinfected with two strains of Wolbachia endosymbiont that are able to induce cytoplasmic incompatibility (CI). In this paper, we report the strength of CI expression in crosses involving field-caught males. CI expression was found to be very strong in all crosses between field males and laboratory-reared uninfected or wAlbA infected young females. In addition, crossing experiments with laboratory colonies showed that aged super-infected males could express strong CI when mated with young uninfected or wAlbA infected females. These results provide additional evidence that the CI properties of Wolbachia infecting Aedes albopictus are well suited for applied strategies that seek to utilise Wolbachia for host population modification.

Genetic population structure and introgression in Anopheles dirus mosquitoes in South-east Asia.

Genetic structure and species relationships were studied in three closely related mosquito species, Anopheles dirus A, C and D in Thailand using 11 microsatellite loci and compared with previous mitochondrial DNA (mtDNA) data on the same populations. All three species were well differentiated from each other at the microsatellite loci. Given the almost complete absence of mtDNA differentiation between An. dirus A and D, this endorses the previous suggestion of mtDNA introgression between these species. The high degree of differentiation between the northern and southern population of An. dirus C (RST = 0.401), in agreement with mtDNA data, is suggestive of incipient species. The lack of genetic structure indicated by microsatellites in four populations of An. dirus A across northern Thailand also concurs with mtDNA data. However, in An. dirus D a limited but significant level of structure was detected by microsatellites over ~400 km in northern Thailand, whereas the mtDNA detected no population differentiation over a much larger area (>1200 km). There is prior evidence for population expansion in the mtDNA. If this is due to a selective sweep originating in An. dirus D, the microsatellite data may indicate greater barriers to gene flow within An. dirus D than in species A. Alternatively, there may have been historical introgression of mtDNA and subsequent demographic expansion which occurred first in An. dirus D so enabling it to accumulate some population differentiation. In the latter case the lack of migration-drift equilibrium precludes the inference of absolute or relative values of gene flow in An. dirus A and D.

A multiplex PCR-based method derived from random amplified polymorphic DNA (RAPD) markers for the identification of species of the Anopheles minimus group in Southeast Asia.

Effective control of Anopheles minimus s.l., an important malaria vector in Southeast Asia, is based on the accurate identification of species within An. minimus complex, which cannot be distinguished using morphological characters. Derived from individual random amplified polymorphic DNA markers, sequence characterized amplified regions were analysed for the design of species-specific paired-primers. Combination of these primers resulted in the development of a simple, robust multiplex PCR able to identify both species An. minimus A and C belonging to the complex, hybrids AC, and three sympatric and closely related species, An. aconitus, An. pampanai and An. varuna. Hybrids AC do not possess alleles of both parents but exhibit novel adaptive potentials resulting from recombination among parental genes leading to hybrizyme.

Distribution of the transposable element mariner in anopheline mosquitoes.

We have surveyed the distribution of the transposable element mariner using PCR in 23 species of Anopheles mosquitoes, including all of the most important vectors of malaria in South-east Asia. Sequencing of the nine positive species revealed elements from the irritans, mauritiana and mellifera subfamilies. These are the first data showing the presence of three subfamilies of mariners in anophelines. The elements we encountered are likely to be inactive, based on the presence of multiple stop codons and/or frameshifts.

Population structure and population history of Anopheles dirus mosquitoes in Southeast Asia.

Separating the confounding effects of long-term population history from gene flow can be difficult. Here, we address the question of what inferences about gene flow can be made from mitochondrial sequence data in three closely related species of mosquitoes, Anopheles dirus species A, C, and D, from southeast Asia. A total of 84 sequences of 923 bp of the mitochondrial cytochrome oxidase I gene were obtained from 14 populations in Thailand, Myanmar, and Bangladesh. The genealogy of sequences obtained from two populations of AN: dirus C indicates no contemporary gene flow between them. The F(ST) value of 0.421 therefore probably represents a recent common history, perhaps involving colonization events. Anopheles dirus A and D are parapatric, yet no differentiation was seen either within or between species. The starlike genealogy of their haplotypes, smooth unimodal mismatch distributions, and excess of low frequency mutations indicate population expansion in An. dirus A and D. This, rather than widespread gene flow, explains their low within-species F(ST) values (0.018 and 0.022). The greater genetic diversity of An. dirus D suggests that expansion occurred first in species D and subsequently in species A. The current geographical separation and low hybrid fitness of these species also argue against ongoing interspecific gene flow. They suggest instead either historical introgression of mtDNA from An. dirus D into species A followed by independent range expansions, or a selective sweep of mtDNA that originated in An. dirus D. While not excluding contemporary gene flow, historical population processes are sufficient to explain the data in An. dirus A and D. The genealogical relationships between haplotypes could not be used to make inferences of gene flow because of extensive homoplasy due to hypervariable sites and possibly also recombination. However, it is concluded that this approach, rather than the use of fixation indices, is required in the future to understand contemporary gene flow in these mosquitoes. The implications of these results for understanding gene flow in another important and comparable group of malaria vector mosquitoes in Africa, the An. gambiae complex, are also discussed.

Distribution and diversity of Wolbachia infections in Southeast Asian mosquitoes (Diptera: Culicidae).

Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a variety of reproductive alterations in their hosts, the best known being cytoplasmic incompatability. The Wolbachia pipientis assemblage has been divided into two major groups (A and B) and 12 subgroups. We report herein the first systematic survey of Wolbachia in mosquitoes, and the first survey classifying Wolbachia infections by subgroup. Wolbachia were detected in 28.1% of 89 wild-caught mosquito species, based on a polymerase chain reaction assay using ftsZ and wsp gene primers. Infections were found in all major disease vector genera except Anopheles. Nine of the 12 Wolbachia subgroups were represented. Group B Wolbachia strains showed more phylogenetic concordance with their host taxa than group A strains. Of the 25 positive mosquito species, five were superinfected with group A bacteria strains (AA), eight were superinfected with A and B strains (AB), and one was superinfected with group B strains (BB). The widespread distribution of Wolbachia among mosquito species further supports their potential importance in the genetic control of disease vectors.

Isozyme evidence for three sibling species in the Anopheles sundaicus complex from Indonesia.

Electrophoretic studies of isoenzymes in three chromosomally distinct forms (A, B and C) of the mosquito Anopheles sundaicus Theobald (Diptera: Culiciae) were undertaken on wild samples collected from six geographically isolated populations in Indonesia. Analyses of 12 enzyme systems comprising 15 loci revealed significant allelic variations, genetic polymorphism, within and among the populations of the An. sundaicus complex. Phylogenetic dendrograms produced by analysis using the Biosys-1 program based on UPGMA methods show that all the populations of form A fall into one cluster, which is closely related to the form C cluster, whereas the populations of form B belong to a more distinct cluster. Allelic frequency and Wright's F statistics of Mpi (mannose phosphate isomerase) are sufficient to identify individuals of each cytological form. This isozyme data correlates with our previous cytological evidence for the existence of three isomorphic species within the taxon An. sundaicus in Indonesia. These three species of the An. sundaicus complex were found together sympatrically at one locality, Asahan in North Sumatra.