Despite decades of control success and a competent network of country-wide health infrastructure, malaria remains an important health threat in rural Thailand. All 4 known human malaria parasites have been reported present, with Plasmodium falciparum and Plasmodium vivax predominant. The expansion and intensity of multi-drug resistant Plasmodium falciparum is the most serious development to occur the last several decades. Members of 3 anopheline species complexes, Anopheles dirus, Anopheles minimus, and Anopheles manculatus, are considered to be primary malaria vectors in the country. Representatives within all 3 taxa are difficult or impossible to separate morphologically from one another, and insufficient information exists about population genetics between sibling species and vector status. Vector control in Thailand has been the primary means of malaria control, mainly by the use of routine residual insecticide spray inside houses. The use of DDT in vector control has resulted in measurable successes to interrupt malaria transmission in many parts of the country. Since 1949, DDT has been the predominant compound used: however, its public health use has continued to decline as a result of perceived operational difficulties, political issues and environmental concerns. The increased use of pyrethroids to impregnate bednets and for intradomiciliary spraying are generally more accepted by rural populations and are rapidly replacing the use of DDT. Organized malaria control activities have reduced malaria morbidity from 286/1,000 population in 1947 to 1.5/1,000 population by 1996. Despite encouraging trends in dramatically reducing malaria, the rates of disease may be re-emerging in the country as evidence from an increased annual parasite index from 1.78/1,000 in 1997 to 2.21 in 1998. The possible reasons for the apparent increase in incidence are discussed in terms of the technical, operational and social obstacles in malaria control in Thailand.

Status of malaria in Thailand.

Among Oriental anopheline mosquitoes (Diptera: Culicidae), several major vectors of forest malaria belong to the group of Anopheles (Cellia) leucosphyrus Donitz. We have morphologically examined representative material (> 8000 specimens from seven countries) for taxonomic revision of the Leucosphyrus Group. Six new species are here described from adult, pupal and larval stages (with illustrations of immature stages) and formally named as follows: An. latens n. sp. (= An. leucosphyrus species A of Baimai et al., 1988b), An. cracens n. sp., An. scanloni n. sp., An. baimaii n. sp. (formerly An. dirus species B, C, D, respectively), An. mirans n. sp. and An. recens n. sp. Additionally, An. elegans (James) is redescribed and placed in the complex of An. dirus Peyton & Harrison (comprising An. baimaii, An. cracens, An. dirus, An. elegans, An. nemophilous Peyton and Ramalingam, An. scanloni and An. takasagoensis Morishita) of the Leucosphyrus Subgroup, together with An. baisasi Colless and the An. leucosphyrus complex (comprising An. balabacensis Baisas, An. introlatus Baisas, An. latens and An. leucosphyrus). Hence, the former Elegans Subgroup is renamed the Hackeri Subgroup (comprising An. hackeri Edwards, An. pujutensis Colless, An. recens and An. sulawesi Waktoedi). Distribution data and bionomics of the newly defined species are given, based on new material and published records, with discussion of morphological characters for species distinction and implications for ecology and vector roles of such species. Now these and other members of the Leucosphyrus Group are identifiable, it should be possible to clarify the medical importance and distribution of each species. Those already regarded as vectors of human malaria are: An. baimaii[Bangladesh, China (Yunnan), India (Andamans, Assam, Meghalaya, West Bengal), Myanmar, Thailand]; An. latens[Borneo (where it also transmits Bancroftian filariasis), peninsular Malaysia, Thailand]; probably An. cracens (Sumatra, peninsular Malaysia, Thailand); presumably An. scanloni (Thailand); perhaps An. elegans (the Western Ghat form of An. dirus, restricted to peninsular India); but apparently not An. recens (Sumatra) nor An. mirans[Sri Lanka and south-west India (Karnataka, Kerala, Tamil Nadu)], which is a natural vector of simian malarias. Together with typical An. balabacensis, An. dirus and An. leucosphyrus, therefore, the Leucosphyrus Group includes about seven important vectors of forest malaria, plus at least a dozen species of no known medical importance, with differential specific distributions collectively spanning > 5000 km from India to the Philippines.

Six new species of the Anopheles leucosphyrus group, reinterpretation of An. elegans and vector implications

Revision of the Leucosphyrus Group of Anopheles (Cellia) (Diptera, Culicidae). This is a comprehensive revision of 20 species of the Leucosphyrus Group of the Neomyzomyia Series of Anopheles (Cellia). Morphological description of the adults, male and female, male genitalia, pupa and fourth-instar larva are provided for each taxon in addition to bionomics, distribution data and systematic discussion for each species, including diagnostic characters. Identification keys for females and fourth-instar larvae are provided. When possible medical importance of each species is included. Illustrations of of the adults, fourth-instar larvae and pupae are provided. Distribution maps for each species are mainly based on the material examined; however, when possible published data were also used. Tables on adult character variations, fourth-instar larval and pupal setal branching are included as appendices. A neotype for An. takasagoensis Morishita and An. sulawesi Koesoemawinangoen, and a lectotype for An. balabacensis Baisas, are designated. The authorship of An. sulawesi previously cited as Waktoedi is corrected to Koesoemawinangoen.

Revision of the Leucosphyrus group of Anopheles (Cellia) (Diptera, Culicidae)

Anopheles species diversity and distribution of the malaria vectors of Thailand

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.

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

A polymerase chain reaction assay based on differences in the internal transcribed spacer regions of ribosomal DNA was developed for distinguishing 2 members of the Anopheles dirus sibling species complex. This assay distinguished An. dirus species A from species D by producing diagnostic bands, 374 base pairs (bp) in length for species A and 663 bp in length for species D. Both laboratory colonies and field collections from Hainan and Yunnan provinces of China were identified with 100% accuracy.

A diagnostic polymerase chain reaction assay for species A and D of the Anopheles Dirus (Diptera: Culicidae) species complex based on ribosomal DNA second internal transcribed spacer sequence.

Anopheles dirus complex is a major malaria vector in Southeast Asia and South China. Rut the role played by different member of the species complex in malaria transmission is not clearly known. Correct identification of the sibling species is a foundational requirement for working out a sound scheme in mosquito biosystematics. This paper reports the resuets of biosystematic studies on the chromosomal karyotype, egg microstructure, ribosomal DNA sequences of a second internal transcribed spacer region and polymerase chain reactions of the complex in China. Specimens of species A and D from Hainan and Yunnan Provinces were carefully analyzed and the importance of development aspect of the mosquito biosystematics in malaria control is discussed.

On the biosystematics of anopheles dirus complex (diptera:culicidae) in china*

OBJECTIVE To isolate and identify genes related to malaria parasite infection in vector mosquito, and to explore the mechanisms. METHODS Anopheles stephensi infected with Plasmodium yoelii was used as tester (T) group, while uninfected but normal blood fed as driver (D) one. Engorged female mosquitoes of two groups were collected separately at 24 hours after biting. An enriched subtractive cDNA pool was generated through the course of suppression subtractive hybridization (SSH) and selective PCR amplification. The subtracted library was screened by hybridization using T and D cDNA mixture as probes, respectively. The positive clones, which produced stronger signal when probed with T than with D, were sequenced and their sequence homologues in GenBank database were searched with BLAST by internet. RESULTS The analysis of subtraction efficiency showed that the differentially expressed genes in T comparing to in D were enriched significantly. In dot blot screening, 24 of 58 randomly selected clones (41.4%) were shown up-regulation in malaria infected mosquitoes. The BLAST search of 23 genes revealed that 12 were homologous to functionally known genes, 4 were homologous to functionally unknown entries, and 7 were novel without any relatives. Nine of the 23 genes (39.1%) also hit homologous sequences in the An. gambiae EST database generated from an immune competent cell line treated with lipopolysaccharide (LPS). CONCLUSION An enriched cDNA pool of the mosquito genes which up-regulated responsively at the early stage of malaria parasite infection was obtained. Expression screening against the pool indicated that various biochemical processes and mechanisms might be involved in the response of mosquito to parasite infection, especially those related with the innate immune system and energy metabolism.

Xiaochun Xu

Papers

A diagnostic polymerase chain reaction assay for species A and D of the Anopheles Dirus (Diptera: Culicidae) species complex based on ribosomal DNA second internal transcribed spacer sequence.

On the biosystematics of anopheles dirus complex (diptera:culicidae) in china*

[Enrichment and screening of up-regulated genes of the mosquito Anopheles stephensi in response to malaria parasite].