MARCH 1993 Merepnesr K.rRyotyprs oF ANopHELEs

METAPHASE 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 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 .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;;;; in the heterochromatin of the Y . 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 while the opposite arm is totally with only a small portion of the short arm pres- heterochromatic.(s) may ent (Fig. 1). The long arm is approximatelythc sometimesbe observedin the heterochromatic same length as that of the .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 ,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 (1969).In form B (Figs. 8, 9), the X2 and Y2 and Chiangmaiprovinces were studied. Two dif- chromosomesare distinctively different from ferent forms (A and B) of metaphasekaryotype thoseof form A. The Xz chromosomeis appar- havebeen recognized (Figs. 14-19). Form A ex- ently a metacentric with one arm consisting of hibits 2 types of X chromosomes.The Xr has a approximately 7a euchromatic and 2/ehetero- small metacentric shape.One arm is euchro- "large" "extensive" chromaticportions of the arm length while the matic with a or block of oppositearm is totally heterochromatic(Fig. 9). pericentricheterochromatin, whereas the oppo- This type of X chromosomeconfiguration is sitearm is totally heterochromatic(Fig. 14).The quite peculiar within this group. The Yz chro- Xz is different from the X1 chromosomein hav- mosomeis also unique to this group. It may be ing an extra block of distal heterochromatin in regardedas submetacentricwith one arm being the heterochromaticarm, making it a long arm typical heterochromatinwhile the oppositearm of submetacentricconfiguration with a second- is a somewhatlighter-staining kind of hetero- ary constrictionat the middle of the arm (Fig. that is apparently not as sticky as the 16).Chromosome Yr has a metacentricshape of other arm. Thus the secondarm of the Y2always approximatelythe samesize as the X1 (Fig. 14), appearsas a satellitechromosome (Fig. 8). Such but it is smallerthan the Xz (Fig. 15). variation in the sex chromosomesstrongly sug- Form B alsoshows 2 typesof X chromosomes geststhat these 2 forms of metaphasekaryotype similar in appearanceto those of form A (Figs. of An. nigercimus could represent interspecific 17-19).Chromosome Yz hasa largesubmetacen- differences.Among the 3 families available for tric shapewhich is slightly shorter than the Xz. chromosomeanalysis in this study, form A has In certain preparations,the distal block of het- beenfound both in Ubon RatchathaniProvince, erochromatinin the long arm of the Y2 chro- Thailand and Sumatra,Indonesia, while form B mosomeis more lightly stained compared with was detectedin Ayutthaya Province,Thailand. the remainingportions (Fig. 19). This seemsto The 2 forms of An. nigerrimus found in Thai reflect a different nature of repetitive DNA in populations do not correspondto the metaphase the heterochromatic arm. Such differences in karyotype of An. hyrcanus nigerrimus reported metaphasechromosomes suggest a possiblespe- by Avirachanet al. (1969). cies complex within the taxon An. argyropus. Anophelescrawfordi: This speciesalso ex- Furthermore, our limited records indicate that hibits variation in the X and Y chromosomes form A hasbeen found only in the north whereas with respectto the amount of heterochromatin. form B is apparently more widespread,occurring Two types of X and 2 types of Y chromosomes both in northern and in easternThailand. have been observed,representing 2 forms of Anopheles nitidus: This species occurs in metaphasekaryotype in An. crawfordi, viz., form southern Thailand. Specimensfrom Songkhla A (Xr, X2 and Yr) and form B (X1, X, and Yz). and PhangngaProvinces show 2 tlpes of X and The Xr is a small metacentric. One arm of the one type of Y chromosome.The X1 is metacen- X1 is euchromaticwith a very small portion of tric while X2 appearsas a large submetacentric pericentric heterochromatinpresent, whereas (Fig. 21),similar to the Xr and Xz chromosomes the oppositearm is entirely heterochromaticof of An. crawfordimentioned above. The Y chro- approximatelyequal length (Fig. 10).The Xz is mosomeis apparentlysubtelocentric (Fig. 20). submetacentric with a euchromatic short arm Thus, the Y chromosomeof An. nitidus is also similar to that of the Xr. However,the hetero- similar in someways to the Yz of An. crawfordi. chromatic long arm is approximatelytwice the Hence, it is rather difficult to distinguishAn. length of that of the Xr, apparently due to the nitidus from An. crawfordi form B on the basis acquisitionof an extra portion of heterochro- of metaphasechromosomes even though they

MARCH 1993 METApHASE KlRvotvpes oF ANopHELES OD

can be readily separatedby morphologicalcri- closelyrelated speciesof Anopheles(Baimai et teria. al. 1988a,1988b, Baimai 1988,Green et al. 1985). AnopheLespeditaeniatus.' Twenty-seven wild- The presentchromosomal evidence indicates caughtfemales morphologically identified asAn. that the 6 morphologicalspecies ofthe Hyrcanus peditaeniatuscollected at Chiangmai,Phrae and Group examinedgenerally exhibit a similar au- Chanthaburiprovinces show remarkablevaria- tosomal configuration.On the other hand, re- tion in the sex chromosomes.Three types of X markable differences have been encounteredin and 5 types of Y chromosomeshave been de- the X and Y chromosomes,primarily due to the tected in these families. ChromosomesXr, X: amount and distribution of constitutive hetero- and Xs differ from eachother in the number and chromatin. It is generallybelieved that an ac- amount of major block(s) of heterochromatin quisition of extra heterochromatinis a common present in the heterochromatic arm, making phenomenonin the chromosomalevolution of them appear as metacentric or small or large higherorganisms (White 1973,John and Miklos submetacentric(Figs. 29-33).The euchromatic 1979).Our findings supportthis. arm of eachX chromosometype exhibitsa large The position of the centromerein the X and block of pericentricheterochromatin. The vari- Y chromosomesvaries from telocentricto sub- ation in the X chromosomesin this caseis likely telocentric, or acrocentricto submetacentricand dueto the acquisitionof extra heterochromatin. metacentric,making the chromosomesvariable Likewise,the evolutionof Y chromosometypes in total length as extensivelydemonstrated in rn An. peditaeniatuscould have arisen via the An. peditaeniatusand, to a lesserextent, in An. process of gain, rather than loss, of major nitidus.It is not known whetherheterochroma- block(s) of heterochromatin.Thus, the very tin variation in the sex chromosomesof An. smalltelocentric Yr chromosomecould be taken crawfordiis an indication of inter- or intraspe- as an ancestor(Fig. 22). ChromosomeYz (Figs. cific difference.Heterochromatin variation ob- 23,24) could have been derived from the pre- servedin the sex chromosomesof An. sinensis. sumedancestral Y1 simply through the addition An. argyropusand An. nigerrimusis, however, of an extra block of heterochromatin.Chromo- Iikely to representinterspecific difference, sug- som€sY3, Yr and Y5 (Figs. 25-28) could also gestingthe presenceof siblingspecies complexes havearisen in a similar fashion.Since there are in eachof thesetaxa. This karyotypicapproach various combinations of X and Y chromosomes may prove an excellent tool to explore the chro- occurring within a population, e.g., X2 or X3 mosomal interrelationshipsof various closelv with Yr or Y2 or Y3, X2 or X3 with Ya, and X3 related speciesgroups within the subgenera with Y5, thesetypes of X and Y chromosomes Anophelesand, Cellia, or among the subgeneraof may merely represent intraspecific variation the genusAnopheles as a whole. Further chro- with respect to the amount and distribution of mosomal data on the metaphase karyotype of heterochromatin. other speciesgroups of the Anophelesin Thai- A diagrammaticrepresentation of mitotic kar- land will be publishedin separatepapers. yotypes of the 6 speciesand forms within the Gain of heterochromatinhas played an im- Hyrcanus Group is shown in Fig. 34. portant role in chromosome evolution in the oriental Anopheles(Yasantha et al. 1982,Baimai DISCUSSION 1988).In all casesobserved in our studies,the Previous studies of the metaphasekaryotype directionof chromosomalchange has been from of Anopheleshave shown that in most cases,if telocentric or acrocentric to submetacentric or not all, the typical karyotype (2n: 6) consists metacentricdepending on the location of the of 2 pairs of autosomesand one pair of sex acquiredblock(s) of heterochromatin.A good chromosomes,The autosomesare generally sub- exampleis the sex chromosomevariation seen metacentricsand/or metacentrics.The sexchro- in An. peditaeniatusdescribed here, and in the mosomes are obviously heteromorphic. Thus, An. dirus complex (Baimai et al. 1984,Baimai the X is usually subtelocentricor submetacen- and Traipakvasin 1987).The biologicalsignifi- tric with short and long arms. The Y chromo- canceof having one or more major blocks of some is usually subtelocentric. The X and Y heterochromatin in the genome,particularly in chromosomescan be distinguishedon the basis sexchromosomes, is an unsolvedproblem (Bai- of their sizeand shape.Nonetheless, little atten- mai 1988).Why is it so common inthe Anopheles tion has been paid to the constitutive hetero- of SoutheastAsia? Could it be very useful irt chromatinpresent in the sexchromosomes and, speciesdifferentiation in Anopheles and other to a lesserextent, in the centromeric region of dipteran insects as a whole? The phenomenon the .The heterochromatin differen- of acquisitionof heterochromatinis of interest tiation as revealed in metaphase karyotype is in its own right and deservesfurther investisa- sometimes useful as a diagnostic character of tion. JouRNnr,oF THEAnrnRrcnN Moseurro CoNrRor,Assocre'rror.r VoL. I, No. 1

crawfordi

iltiForm A liluForm B lfltr:Form A lIlh Form B

nigerrimus ars/ropus

[ilhForm A [[hForm B lIuForm A lflluForm B

peditaeniatus Uflnn l]lrnrl u[:ll[UIl!'t I Xr Xz X3 Y1 Y2 Y3 llY+ Ys II III II III Fig' 34. Diagrammatic representation and comparison of metaphasekaryotypes of the 6 species(including forms) of the Hyrcanus Group of- noph.eles (Anopheles). Only one set of auiosomesII and III is present. Black and shadedareas represent variable heterochromatic portions. The centromeresare indicated by constrictions of each chromosome.Chromosome lengths, arm ratios and heterochromaticportions are shownin proportion.

ACKNOWLEDGMENTS Forces Research Institute of Medical Sciences, Bangkok' we thank B. A. Harrison and D. strickman for their useful comments on the manuscript. We also thank K. Jayana and K. Veisanit for technical assistance and Prachong Panthusiri REFERENCES CITED for preparing the illustrations. This work has ^'.lilIH##::;1;#""*T p*1 rypn"'i"din partby the uNDp/worrd J:fl?f::fi',f:i:f Bank/WHO Special Program for Research and Asia.pak. J. Zool.!:t_7. Training in'llropical Diseasesand by the Armed Avirachan,T. T., P. L. Seetharamand B. N. Chow- MARCH 1993 Mrrlpneso Kenvotvpns oF AN1eHELES ot

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