MARCH 1993 Merepnesr K.rRyotyprs oF ANopHELEs METAPHASE KARYOTYPES OF ANOPHELES OF THAILAND AND SOUTHEASTASIA: I. THE HYRCANUSGROUP VISUT BAIMAI,' RAMPA RATTANARITHIKUL, eNo UDOM KIJCHALAO' . AB.STRACT.Metaphase karyotypes of 6 speciesof the_HyrcanusSpecies Group of the subgenus Anophelesshow constitutive heterochromatin variation in X and y cfrro'-osomes. A'nophelespeditaei- iotus exhibits the most extensivevariation in the size and shape of h;;t;;1;;-atic sex ch.o-oso-es, with 3 types -ofX and 5 tlpes of Y chromosomes.Anophel.es n;i;a"s strowsihe -tt"u" l"u.t variation, with only 2 types of X chromosomes.Anoph.ebs sinensis and'An. crawfordi "r"li 2 forms "f ;"t"p;;;; karyotype in the heterochromatin of the Y chromosome. It is not k"";; whether the 2 forms oi metaphase karyotype in these 2 speciesrepresent inter- or intraspecifrc diif"."rr""*. The 2 forms of heterochromatic sex chromosomesobserved in An. argyropn ^nA ii.- iijirrt^* -"y ."gg*i1h" existenceof siblingspecies complexes within eachof theie species. INTRODUCTION the different amount and distribution of consti- The genusAnopheles is one of the most im- tutive heterochromatincan be a usefulcvtotax- portant groupsof mosquitoesin terms of disease onomictool in separatingclosely related species, particularly transmissionin the world. This genusconsists homosequentialspecies as exempli- ofmore than 400described species (Colless 19b6. fied in the LeucosphyrusGroup (Baimai et al. Reid 1968,Harrison and Scanlon1925, Harrison 1981,1988a, 1988b; Baimai 1988). 1980, Harrison et al. lggl). The subgenera We report here on the metaphasekaryotlpe of 6 species, Anopheles and Cellia are the 2 larsest of the 6 including various forms, of tle Hyrcanus subgenerabelonging to the genus.Harrison et Group of the subgenus Anopheles, which occur al. (1991) recently updated the checklist of in Thailand. Morphologicalidenti- fication of Anophelesfound in Thailand, and included 35 speciesbelonging to the Hyrcanus Group has been a major problem 1nd 37 speciesin the subgeneraAnopheles and for mosquito Cellia, respectively. Although considerable taxonomists of this region (Reid 1g68,Harrison 1972, knowledgeon the morphology, systematicsand Hanison and Scanlon192b, Kanda et al. 1e81). geographicdistribution of the known speciesof Anophelesin the SoutheastAsian Region has beendocumented since the publication of Har- MATERIALS AND METHODS rison and Scanlon(19Tb), relatively few species The are known cytologically.Of those speciesthat 6 speciesof the Hyrcanus Group studied are known chromosomally,mitotic chromosome arc An. sinensis Wiedemann, An. nigerrirnus information is notably sparse(Kitzmiller 1967. Giles, An. crawfordi Reid, An. nitidus Harrison, Kanda 1968,Vasantha et al. 1982,Avirachan et Scanlon and Reid, An. argyropus (Swellengre- al.1969). bel) andAn. peditaeniattts(Leicester) (Table tt. During our studieson the populationcytoge- Adult femalesof these specieswere collected netics of human malaria vectors in Th;ila;d from animal and/or human bait at different localities and_Southeast Asia, we had an opportunity to during our researchprogram, which study metaphasekaryotypes in 40 anopheiine was initiated in 1980.Fully blood-fed females were speciescollected from natural populations.Our identifiedmorphologically to speciesas far p-ossible. aim in this seriesof publicationi is to provide as The healthyspecimens were brought data on metaphasekaryotypes of the Anopheles back and reared individually in our laborator"ies at Mahidol of Thailand and neighboring countries for a University and at the Armed Forces better understanding of chromosomalevolution ResearchInstitute of Medical Sciencesto obtain single (isofemale in this areaof anophelinediversity. Even more families lines).The brain gan- glia importantly, karyotypic differentiation due to of fourth-instar larvaewere usedfor meta- phase_chromosome preparations employing a modifiedmethod as describedby Baimai (lgi7). The air-dried slides were stainid with Giemsa r.Department and mounted in Permount (Fisher) . of Biology, Faculty of Science, Mah- for perma- rclolUniversiry, Rama 6 Road, Bangkok 10400,Thai_ nent preparations.The best prophase and/or land. metaphasespreads 2 of mitotic karyotypeswere Department of Medical Entomology, U.S. Army photographedwith Kodak Technical Film using Medical Component, Armed Forces Research Institute an Olympus Photomicroscopewith a green ofMedical filtei Sciences,Bangkok 10400, Thailand. and an oil immersion (x620) lens. 60 JouRner,oF THEAMERTcAN Mosqutro CotttRot-Assocllrlon VoL. 9, No. I Table 1. The number of females (isolines) of 6 species(including karyotypic forms) within the Hyrcanus Group of the subgenusArnph.ebs collected and examined cytologically from different wild populations in - Thailand, Taiwan and Indonesia. No. of isolines Date of Species/Form Locality examined collection stnenslS Form A Mae Sariang,Mae Hong Son Province 3 September1985 Chiayi County,Taiwan z August 1986 Form B Mae Sariang,Mae Hong Son Province 4 September1985 Chiayi County,Taiwan 3 August 1986 nigerrimus Form A Jambi Province,Sumatra, Indonesia I April 1986 Nachaluai,Ubon RatchathaniProvince 1 January1987 Form B Bangpa-in,Ayutthaya Province I February 1983 crawfordi Form A Makarm, ChanthaburiProvince July 1982 Form B Tung Ka Ngok, PhangngaProvince June 1982 argyropus Form A Maetang,Chiangmai Province 3 November1983 Rongkwang,Phrae Province 2 February 1984 Form B Maetang,Chiangmai Province 8 November1983 Makarm, ChanthaburiProvince a November 1983 nitidus Tung Ka Ngok, PhangngaProvince I June 1982 Sadao,Songkhla Province 1 January 1986 peditaeniatus Makarm, ChanthaburiProvince z November1983 Maetang,Chiangmai Province A November 1983 Makarm, ChanthaburiProvince November1983 Maetang,Chiangmai Province I November1983 Makarm, ChanthaburiProvince 1 November 1983 Rongkwang,Phrae Province J February 1984 Makarm, ChanthaburiProvince z November 1983 Maetang.Chiangmai Province z November1983 RESULTS Taiwan were examined cytologically. The X chromosomeis submetacentric,comprising the The chromosomalanalysis of the Hyrcanus euchromaticshort arm with a considerableblock Group has revealed that the metaphase kary- of pericentric heterochromatin and the totally otpe (2n : 6) usually consistsof one pair of heterochromatic long arm with a conspicuous metacentricand one pair of submetacentricau- secondaryconstriction present in some prepa- tosomesand onepair of heteromorphicsex chro- rations (Figs. 2, 4, 5). The 2 types of Y chro- mosomes(XX in femalesand XY in males).The mosomeshave been observedin the same pop- X chromosomeis generallyeither metacentric ulations at both localities (Table 1). The Yr or submetacentric.One arm clearly consistsof chromosome is subtelocentric or acrocentric euchromatin while the opposite arm is totally with only a small portion of the short arm pres- heterochromatic.Secondary constriction(s) may ent (Fig. 1). The long arm is approximatelythc sometimesbe observedin the heterochromatic same length as that of the X chromosome.It arm. Variation of the X chromosomedue to occasionallyshows a secondary constriction different amounts of major block(s) of consti- similar to the X. ChromosomeY2 is clearly a tutive heterochromatinhas also been observed submetacentric with the short arm approxi- in 5 species.The Y chromosome,on the other matelyone-half the lengthof the long arm (Figs. hand, is almost entirely heterochromatic.The Y 3, 5). The Y2 appears to have arisen from the may be telocentric,subtelocentric (acrocentric), presumedancestral Yr simply by meansof the submetacentric, or metacentric of different additiott of an extra block of heterochromatin sizes.Specific differences ofthe metaphasechro- onto the short arm, transforming it to a sub- mosomeso{'the 6 speciesof the HyrcanusGroup metacentric configuration. Based on the differ- are briefly describedbelow. ent types of Y chromosomes,two forms of met- Anopheles sinensis:Twelve families of this aphasekaryotypes have been recognizedin Al. speciesfrom Mae Sariang,Mae Hong Son Prov- sinensis,viz., form A (X, Yl) and form B (X, ince in northern Thailand and Chiayi County in Yz). These 2 forms of metaphasekaryotype have 62 JounNar, oF THE ArrteRrceNMosqurto CoNtnol AssocrATroN VoL. 9, No. I tected in a male larva of one family of form B matin (Figs.11, 13). The Yr is a small telocen- from Taiwan (Fig.5). This is a rarephenomenon tric, whereasthe Yz chromosomeis a largesub- occurring adventitiously in anopheline mosqui- telocentric that is about twice the Iength of the toes. Y1 (Figs. 10, 12). Thus, the Yz could have been AnopheLesnigeruimus: There are 2 distinct derived from the presumedancestral Y, chro- forms of metaphasekaryotype with respect to mosomethrough the acquisitionof a largeblock the sexchromosomes. In form A (Figs.6, 7), the of distal heterochromatinin the long arm. Forms Xr chromosomeis submetacentric.The short A and B have been discoveredin allopatric pop- arm is euchromatic with only a very small por- ulations at Phangnga and Chanthaburi Prov- tion of pericentricheterochromatin, whereas the inces.However, it is not possibleto determine Iong arm is totally heterochromatic.The Yr whether these 2 forms of metaphasekaryotype chromosomeis subtelocentricor acrocentric. merelyrepresent intraspecific variation or inter- Form A apparentlycorresponds to the mitotic specific differences. chromosomesof An. nigerrimus from Balloki, Anopheles argyropus: Seventeen isofemale Pakistanas describedby Aslamkhanand Baker lines of An. argyrop.tsfrom Chanthaburi, Phrae