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.

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.

Identification of isomorphic malaria vectors using a DNA probe.

About 7,000 recombinant clones, derived from chromosomally-identified families of wild-caught females of Anopheles dirus species D, were screened. The most promising clone was totally specific to species D when tested against single F1 females of all four species of the complex. In fresh specimens the clone was positive for DNA levels 150 times less than the normal DNA content of single individuals. Fresh adult males and females, larvae, and dried specimens have been successfully identified. The clone was sequenced; it is 124 bp long and appears to be repeated in the genome about 1.8 x 10(4) times.

Genetics of esterases and 6-phosphogluconate dehydrogenase in the Anopheles maculatus complex.

Electromorphic variation for some esterases and 6-phosphogluconate dehydrogenase enzymes in the Anopheles maculatus complex is controlled by four loci which are unlinked to sex. Esterase loci are linked to each other: Est-1-36%-Est-4-16.5%-Est-3; but unlinked to Pgd-2. Linkage data were obtained by selfing the F1 generation from selected parents and analysing genotypes in the F2; the classical dihybrid-cross. The analysis consists of testing observed data for goodness of fit to a) ratios expected from Mendelian ratios without linkage and b) if they do not fit then computing a likely degree of linkage and computing expected ratios with such linkage for further tests. Confidence limits are given for the most likely levels of linkage. This method can provide useful information for population-genetic studies on anopheline mosquitoes, whose laboratory rearing is generally difficult. Through indirect evidence, the enzyme loci are correlated to polytene chromosomes. The esterases probably lie on chromosome three (polytene arms 3 and 4) and Pgd-2 on the second chromosome (arms 2 and 5).

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.

Scanning electron microscopic observations and differentiation of eggs of the Anopheles dirus complex.

Microscopic observations have revealed differences among the eggs of species A, B, C and D of the Anopheles dirus complex. The eggs of species A and C are similar in size and shape. They are intermediate in size between the eggs of species B, which is the largest, and that of species D, which is the smallest. The pattern of outer chorionic cells between the frill and the float is species specific. The pattern consists of rows of irregularly shaped cells in species D and different numbers of rows of regularly shaped cells in species A, B and C. Scanning electron microscopy revealed that the deck tubercles are arranged in aggregates which are more widely spaced in species A than in species B. The aggregates are large in species C, of moderate size in species A and B, and small in species D. The egg characters may be useful in separating species A, B, C and D of the An. dirus complex.

Metaphase karyotypes of Anopheles of Thailand and Southeast Asia: I. The Hyrcanus Group.

Metaphase karyotypes of 6 species of the Hyrcanus Species Group of the subgenus Anopheles show constitutive heterochromatin variation in X and Y chromosomes. Anopheles peditaeniatus exhibits the most extensive variation in the size and shape of heterochromatic sex chromosomes, with 3 types of X and 5 types of Y chromosomes. Anopheles nitidus shows the least variation, with only 2 types of X chromosomes. Anopheles sinensis and An. crawfordi each have 2 forms of metaphase karyotype in the heterochromatin of the Y chromosome. It is not known whether the 2 forms of metaphase karyotype in these 2 species represent inter- or intraspecific differences. The 2 forms of heterochromatic sex chromosomes observed in An. argyropus and An. nigerrimus may suggest the existence of sibling species complexes within each of these species.

Monandry (monogamy) in natural populations of anopheline mosquitoes.

Polymorphic Y chromosomes within two species of anopheline mosquitoes provide markers for testing if females are inseminated by one or more males in nature. Wild-caught females of Anopheles dirus (sp. A) and An. maculatus s.s. produced 291 and 55 families, respectively, which showed a single type of Y chromosome. One family of the former species showed two types of Y chromosomes. These field data support the idea, established from laboratory studies, that female mosquitoes are largely monandrous (monogamous). Such information is important in interpretation of population biological data and, practically, in attempts to control insect pests by use of genetically designed males.

Metaphase karyotypes of Anopheles of Thailand and Southeast Asia: V. The Myzomyia Series, subgenus Cellia (Diptera:Culicidae).

Metaphase karyotypes of 9 species of the Myzomyia Series show intra- and interspecific differences based on quantitative variation and distribution of constitutive heterochromatin in the sex chromosomes or the centromeric regions of the autosome pairs or both. Anopheles jeyporiensis and Anopheles aconitus each exhibit 4 forms of mitotic karyotypes, which may reflect interspecific differences within each taxon. The well-defined genetic species within the Anopheles minimus and the Anopheles culcifacies complexes clearly exhibit distinctive metaphase karyotypes that can be used as diagnostic characters for separating these sibling species, which are difficult to identify by morphological criteria alone. Our analysis on metaphase karyotypes of Anopheles pampanai, Anopheles varuna, and Anopheles flavirostris also confirms their morphological identification based on heterochromatin differences in the sex chromosomes and autosome 2.

Metaphase karyotypes of Anopheles of Thailand and Southeast Asia. VI. The Pyretophorus and the Neomyzomyia series, subgenus Cellia (Diptera:Culicidae).

A total of 6 species of the Pyretophorus (4 species) and Neomyzomyia (2 species) series of the subgenus Cellia of Anopheles were used for metaphase karyotype analysis. Anopheles subpictus and An. vagus exhibit 4 and 2 forms of mitotic karyotypes, respectively, which are attributable to different types of Y chromosomes. Such distinctive mitotic chromosomes may reflect interspecies differences within each of these 2 taxa. Two distinct species, An. indefinitus and An. sundaicus, show similar metaphase karyotypes, particularly with regard to the size and shape of the sex chromosomes. Likewise, An. tessellatus and An. kochi, which are distinct species of the Neomyzomyia Series, also have metaphase karyotypes that resemble each other. They exhibit a typical feature of telocentric sex chromosomes resembling those of the An. dirus complex and the other species of the Leucosphyrus Group. Like the other cases of the Oriental Anopheles, heterochromatin has played a significant role in chromosome evolution of the 6 species.

Cytogenetic evidence for a fifth species within the taxon Anopheles dirus in Thailand.

Crossbreeding and chromosomal evidence are presented for the existence of a fifth sibling species within the taxon of Anopheles dirus in Thailand. The new species is morphologically identifiable as Anopheles balabacensis "Fraser's Hill form." Structural differences in mitotic chromosomes and extensive asynapsis in hybrid polytene chromosomes indicate that significant genetic divergence exists between this species and its closest relatives, An. dirus species A, B, C and D and An. balabacensis.

Cytogenetic evidence for two species within the current concept of the malaria vector Anopheles leucosphyrus in Southeast Asia.

Karyotypes and crossing relationships were investigated for three allopatric populations of Anopheles leucosphyrus in Southeast Asia: South Kalimantan, Sumatra and Thailand. The mitotic karyotypes of these populations were similar to those previously observed in other species of the An. leucosphyrus group. Populations from Thailand and South Kalimantan exhibited telocentric and subtelocentric sex chromosomes, respectively, with a distinctive band of intercalary heterochromatin in the X chromosome. Strikingly different submetacentric X and Y chromosomes were observed in the population from Sumatra, and it seems likely that the evolution of these chromosomes occurred through the acquisition of constitutive heterochromatin. Sterile F1 males were observed in crosses between the Sumatra population and the populations from South Kalimantan and Thailand. No genetic incompatibility was observed in crosses between the latter two populations. We believe that the present concept of An. leucosphyrus includes two allopatric species, one inhabiting Borneo, West Malaysia and southern Thailand and one confined to Sumatra.

Anopheles dirus species E: chromosomal and crossing evidence for another member of the dirus complex.

Cytogenetic and crossing data provide strong evidence for the existence of another species, dirus E in southwestern India, within the Dirus Complex of Anopheles. These findings are in accord with unpublished morphological observations. Our data suggest a significant genetic divergence between species E and its close relatives, An. dirus A, B and C in Thailand. These data also suggest that dirus E is an incipient sibling species of its geographically nearest relative, dirus D, and that it seemingly co-evolved through the process of allopatric speciation.

Population cytogenetics of the malaria vector Anopheles leucosphyrus group.

Until recently, very little was known of Anopheles species complexes and their relationships to epidemiology and malaria transmission in Southeast Asia. During the past eight years, extensive studies on the genetics of natural populations of anopheline mosquitoes in this region, involving the interdisciplinary efforts of taxonomists, operational entomologists and biologists, have revealed groups of cryptic species of Anopheles vectors, particularly the An. leucos phyrus group. This species group comprise seventeen species and two subspecies widely distributed in the forested areas of Southeast Asia. Among these species. An. dirus Peyton and Harrison, has been shown by cytogenetic and morphological studies to be a complex of at least seven isomorphic species, provisionally designated species A, B, C, D, E, F and takasagoensis, on the Southeast Asian mainland. Cytological identification of these species is based on distinct banding patterns of salivary gland polytene chromosomes as well as heterochromatin differences in mitotic karyotypes. The five species found in Thailand (A-D, F) exhibit distinct geographic distributions. Species A is widespread throughout Thailand except in the south. Species B had been found in sympatry with species C in southern Thailand and both seem to show north-south clinal geographic variation. Species D is common on the west side of southern Thailand and along the Thai-Burmese border in sympatry with species A. Species F, An. nemophilous Peyton and Ramalingam, has been found in a population at the Thai-Malaysian border in this study although it was known to be common in southern and western Thailand and Peninsular Malaysia. Species E is known only from western India. The five species found in Thailand also exhibit seasonal variation in relative abundance and different nocturnal biting cycles. Chromosomal polymorphisms have been observed in mitotic and polytene chromosomes of An. dirus A and D. Species B and C also show heterochromatin variation in the sex chromosomes, but are monomorphic for the standard sequence in polytene chromosomes. These biological characteristics of the An. dirus complex may have implications for understanding the epidemiology of malaria in Southeast Asia. Recent cytogenetic studies of wild-caught samples of An. leucosphyrus from Sumatra, Kalimantan and southern Thailand have revealed the presence of two distinct species within this taxon. Species A is widely distributed in southern Thailand, East Malaysia and Kalimantan, while species B is confined to Sumatra. The two isomorphic species are vectors of human malaria within their range of distribution.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic response of Aedes aegypti to different environmental temperatures.

Three groups of Aedes aegypti maintained 8 generations in the laboratory at 20 degrees C, 27 +/- 1 degree C (normal control), and at 35 degrees C, respectively were analyzed for allozyme variability. Of the 8 larval loci examined Est alpha-1 and Lap-2 in generation 8 were still variable, while the other allozymes became uniform. The 35 degrees C population had mean level of heterozygosity of about 6% of individual per locus, less than those observed in the 20 degrees C and the control populations which were 14% and 13%, respectively. It is suggested that in the experimental populations, the rate of loss of alleles can be increased by high temperature which must not be higher than the optimum temperature of enzymes.

Some observations on sympatric populations of the malaria vectors Anopheles leucosphyrus and Anopheles balabacensis in a village-forest setting in South Kalimantan.

Human bait collections of biting anopheline mosquitoes were made on five consecutive nights during September 1986 in a remote village located in a heavily forested area of South Kalimantan, Indonesia. Anopheles leucosphyrus and An. balabacensis comprised 97.7% of the total number of specimens collected outside houses in the village. Anopheles balabacensis were slightly fewer in total numbers than leucosphyrus. Mosquitoes were collected simultaneously in the village and the forest on two nights. The numbers of leucosphyrus and balabacensis biting in the forest were small in comparison with the populations encountered in the village. Approximately 75% of the specimens were checked individually for sporozoite infections using ELISA for P. falciparum and P. vivax. Sporozoites of P. falciparum were detected in one specimen of leucosphyrus and one of balabacensis. The sporozoite infection rate was 1.0% for leucosphyrus and 1.3% for balabacensis.

Distribution and chromosomal polymorphism of the malaria vector Anopheles dirus species D.

Preliminary results indicate that species D of the Anopheles dirus complex is widespread west of the Thai-Burma border in Burma and Bangladesh. A chromosomal study of An. dirus species D in these areas has revealed that this malaria vector is highly polymorphic for chromosomal rearrangements in salivary gland polytene chromosomes. The data from the limited number of wild-caught samples suggest that different geographically isolated populations may occur with respect to the frequency of inversions 2La, 3Ra and 3La. The distribution of chromosomal polymorphisms may be associated with the geography and epidemiology of human malaria in this region.

Geographic distribution and biting behaviour of four species of the Anopheles dirus complex (Diptera: Culicidae) in Thailand.

A cytogenetic analysis of the species status of members of the Anopheles dirus group, from natural populations, over a six-year period has produced biogeographic and behavioural data which are presented herein. Species A, B, C and D have quite distinct geographic distributions in Thailand. Species A is the only species in most of the mainland but is absent from the southern half of the peninsula. Species B is dominant in the far south of the peninsula giving way to species C on the north-east side and rarely occurring on the west side of the peninsula; it is unknown from the northern half or the rest of the country. Species C is known from the middle eastern side of the peninsula and from a site far to the north along the Burmese border. Species D occurs down the mountains along the Burmese/Thai border and along the north half of the western peninsula. Each species appears to have distinct times of biting during the night. They also appear to have differential seasonal abundance. These data together with the differing geographic distributions suggest some implications for Plasmodium transmission and that care should be taken to identify these genetic species during malariometric studies.

Cytogenetic studies of some species complexes of Anopheles in Thailand and Southeast Asia.

Recent studies on cytogenetics, behavioral, geographical and distinct morphological characters on adult, pupal and larval stages have revealed that "balabacensis" is a species complex. Anopheles dirus the mainland species, is distributed widely in Thailand and is renowned for its role as primary vector of human malarial parasites. Further, evidence from cytogenetic and taxonomic studies suggests that "An. dirus" is a species complex comprising at least four distinct species provisionally designated: dirus A, B, C and D. These cryptic species are distinguishable only partially morphologically, but can be separated on the basis of metaphase chromosomes using the Giemsa and Hoechst 33258 staining techniques. Apparently, these siblings show distinct patterns of geographic distribution in Thailand and Peninsular Malaysia. The recognition of dirus as a complex of species in Thailand and Peninsular Malaysia requires a re-evaluation of the role that the individual members of this complex have in the transmission of malaria parasites in this region. Cytological analysis of gene rearrangements in ovarian polytene chromosomes has shown that An. maculatus is a sibling-species complex consisting of at least four species in Thailand provisionally designated: maculatus A, B, C and G. These siblings are sympatric in some populations. Furthermore, species B is so highly polymorphic for chromosome rearrangements that four geographic forms can be recognized. It is not known whether these four forms are subspecies or yet further species within the species B complex. These sibling-species must be differentiated in order to understand any differential capabilities in their transmission of human malaria parasites. Anopheles nivipes was elevated from synonymy under An. philippinensis to full species status by Reid, a decision recently confirmed by cross mating experiments. The Thailand Malaria Division does not differentiate these two species and only identifies An. philippinensis, yet, An. nivipes is by far the most common of the two species in Thailand. Furthermore, preliminary surveys of the ovarian polytene chromosomes of several widely separated populations of An. nivipes in Thailand have revealed at least two distinct chromosomal types of nivipes based on fixed inversions on the X chromosomes.

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).