THE OVARIAN POLYTENE CHROMOSOME OF THE COMPLEX SPECIES barbirostris VAN DER WULP.

Supratman ~ukowati',Herri ~ndris',Shinta '

KROMOSOM POLITEN OVARlUM DARI SPESIES KOMPLEKS Anopheles barbirostris VAN DER WULP

Abstrak. Malaria masih merupakan masalah kesehatan masyarakat di Indonesia. Salah satu upaya pemberantasan penyakit tersebut adalah dengan cara pengendalian vektornya. Sebagai dasar untuk menentukan strategi pengendalian vektor secara tepat guna adalah dengan pemahaman tentang spesies dan bioekologi serta habitatnya secara rinci. Untuk mengetahui adanya spesies sibling Anopheles barbirostris di Indonesia, telah dilakukan penelitian spesies sibling dengan teknik kromosom mitotik. Penelitian dilakukan di 5 daerah yaitu: (I) Ambarawa, Jawa Tengah, (2) Tara-Tara, Sulawesi Utara, (3) Boru-Boru, (4) Konga dan (5) Singaraja, tiga yang terakhir di Flores, Nusa Tenggara Timur. Analisis kromosom ovarium An. barbirostris Van der Wulp dilakukan dari ovarium nurse cell yang ditangkap dari 5 populasi alam yang berbeda daerah geografinya di Indonesia. Foto peta baku kromosom politen An. barbirostris telah dihasilkan dan didiskripsi dalam makalah. Spesimen-spesimen populasi tersebut menunjukan kariotipe kromosom mitotik yang serupa (2~6)yang tersusun dari dua pasang autosom dan satu kromosom kelamin (X). Foto peta kromosom politen An. barbirostris yang berasal ovarian nurse cell telah dapat dihasilkan dan diidentzj?kusi sehingga dapat digunakan sebagai acuan untuk mempelajari adanya polimo@sme dan atau spesies komplek takson tersebut. Analisis pola penggelangan kromosom politen An. barbirostris dari 5 populasi alam dengan daerah geografi berbeda, tidak ditemukan perbedaan pola kromosom, semua sampel menunjukkan pola penggelangan yang homosekuensial. Foto peta kromosom politen baku An. barbirostris dapat digunakan sebagai acuan untuk mempelajari adanya spesies sibling takson tersebut.

Kata kunci: Anopheles barbirostris, malaria, kromosom politen, spesies sibling

INTRODUCTION ment of efficient control measures against Malaria continues to be a public malaria have been subjects of greatly in- health problem of high priority in the creased interest in recent years. majority of malaria endemic countries The mosquito species Anopheles throughout the Southeast Asia region, such barbirostris is widely distributed in Indo- as Indonesia. Vector control remains the nesia, occurring in Sumatra, Java, Kali- most effective measure to prevent malaria mantan, Sulawesi, Timor, Flores and Irian transmission. The study of mosquito com- Jaya (I). This species is the principal vector plexes and the understanding of their bio- for human malaria in Sulawesi and Flores logy, evolution, epidemiology of malaria (293), and the filariasis vector in Sulawesi, transmission, and the possibly of develop- Timor and Flores (4p5),even though in some

' Health Ecology Research & Development Center,NIHRD Bul. Penel. Kesehatan, Vol. 33, No. 2,2005: 89-98

places of its distribution, e.g. in Sumatra major malaria vector in India, An. culicifa- and Java, does not play an important role cies, has been identified as a complex of (6) three sibling species ('O1"). The other vec- tors species which have been identified as The An. barbirostris population species complexes are An. balabacensis (6 differs in their tendency to feed on both species) (I6),An. maculatus (3 species) (I7), human and blood; this characterris- An. subpictus (2 species) (I2); and An. tic has obvious relevance to the role of the farauti (3 species) From these exam- species in the transmission of disease. In ples, it may be concluded that there is a Java, the population of this species is pri- need for new research studies using new marily zoophilic, though in Sulawesi, Ti- technologies such as cytogenetics, electro- mor and Flores populations, it does appear phoresis and DNA in the field of malaria to be more anthropophagic (7). These ob- vectors in Indonesia, aimed at a better un- servations seem to suggest that An. barbi- derstanding of malaria epidemiology and rostris could be a cluster of closely related control strategies. species, each of which may play a vital role in malaria or filariasis transmission in its The application of cytogenetics in the areas of distribution. study of sibling species and evolution of the malaria vectors in Indonesia is In recent years, new technologies somewhat limited, despite serious pro- have become available for the identificati- blems with regards to controlling malaria on of malaria vector species complexes, vectors. We present a photomap of the which promise a much better under- ovarian polytene chromosomes of An. bar- standing of malaria vectors. Polytene chro- birostris that we have designated as mosome differences in Anopheles, as "standard" for that taxon. We also report shown by a comparative study of closely the results of survey on polytene chromo- related species, involve changes in banding some arrangements relative to "standard" sequence due to paracentric inversion of arrangement in a natural population of An. fixed inversions and differences in unique barbirostris. arrays of chromosomal polymorphism which have led to findings of sibling spe- cies of malaria vectors, e.g., An. gambiae MATERIALS AND METHODS (839),An. culicifacies complex cOmP1ex Natural populations of An. barbiros- ('Op" ,An. subpictus complex (I2), An. dirus tris Van der Wulp were sampled from 5 complex (I3). Many taxa of medical im- geographically isolated populations in In- portance are known to be complexes of donesia: (1) Ambarawa, Central Java, (2) morphologically-cryptic species. Anophe- Tara-Tara, North Sulawesi, (3) Boru-Boru, les gambiae, the most important malaria (4) Konga and (5) Singaraja. Numbers 3-5 vector in Africa, has been shown to be a are all located in Flores, East Nusa Teng- complex of at least six sibling species, with gara. marked differences in behavior and specific polytene chromosome patterns. The mosquitoes were kept alive and Practical and reliable cytotaxonomic cha- fed until fully engorged with guinea pig racters were established for identification blood, and maintained in a cool, humid of members of the An. gambiae com lex environment. The fully blood-fed females by White and Davidson & Hunt (14.14 A were maintained at about 20' C with re- The Ovarium Polytene Chromosome...... ( Sukowati at.al)

lative humidity of about 80%. About 24-36 tene chromosome complements of An. bar- hours after feeding (development time for birostris comprise three synapse elements: production of half a gravid female), ova- a short pair of sex chromosomes (chro- ries were dissected, put on labeled filter mosome 1/X chromosome) and two longer paper and placed in Carnoy's fluid and pairs of autosomes 2 and 3 that are labeled where they were kept at low temperature as chromosome 2R/2,3U3,3W4 and 3U5 (4'~)for at least 24 hours before making a (Figure 1). The right and left arms are polytene chromosome preparation. somewhat unequal in autosomes 2 and 3, The polytene chromosomes were the right being the longer element. The spread and processed according to the me- ovarian polytene chromosome complement thods of Green and Hunt and Sukowati & of An. barbirostris has been arbitrarily divided into 50 zones, starting with the free Baimai (201 21). The chromosome photo- graphs were made using a phase contrast end of an X-chromosome running through microscope and on Kodak Technical Pan the free ends of arms 2W2 and 3R/4 and Film 24 15. The standard chromosome map finishing at the free end of arm 3U5 as was made using methods described by follows; X-chromosome covering zones 1- 5, 2W2 zones 6-16, 2U3 zones 17-25, Hunt, and Sukowati and Baimai (22,2'). A series of photographs were selected to 3W4 zones 26-39, and 3L/5 zones 40-50 as produce the map. Accumulated prints of described below. There were no inversion each chromosome showing several of polymorphisms detected in this study. All stretching were made for comparative pur- the wild samples examined showed similar poses. The chromosome maps were, then chromosomal arrangements to the standard assembled by joining the photographs in photomap. such a way that the curvature of alternate X segments compensated for that of the pre- The Chromosome ceding segment. As in the general rule for the genus Anopheles, the X chromosome of An. bar- The standard photographic map of (2 1,25,26,27) 1, An. barbirostris was constructed from a birostris is the shortest one series of photographs taken from wild most preparation observed in this study, caught populations. Arbitrary divisions the X-chromosome has very distinct bands along the photomap basically followed the with a flared and large centromeric region. methods of Baimai et al., Baimai et a1 and Zone 1 marks the free end of chromosome X. The occurrence of two largely diffuse Sukowati & Baimai (23924121). Determinati- on of banding sequences for polytene chro- and light dotted bands with the same cur- mosomes of wild specimens was accom- vature is a consistent feature at the tip, plished by comparison with the standard followed by two dark bands and a short photomap directly through a camera Luci- dark band at the constriction area. Fol- da attached to the microscope, or by com- lowing the constriction area is a light broad paring photograph. dotted band, two light and two dark bands, and continues with four dotted bands RESULTS respectively. Zone 2 begins with two dark bands followed by three dotted bands, a A standard photomap based on the double dark band at the constriction, and ovarian nurse cell polytene chromosomes three dotted bands. Zone 3 is marked by a for the taxon An. barbirostris Van der closely spaced set of four bands (one dark, Wulp collected from Jambu, Ambarawa, two dotted and one dark band, respective- Central Java is presented in Figure 1. Poly- Bul. Penel. Kesehatan, Vol. 33, No. 2,2005: 89-98

Figure 1. Standard Photomap of The Ovarium Nurse Cell Polytene Chromosomes of Anopheles barbirostris from Indonesia. Centromeric end of Each Chromosome Arm is Indicated by an Arrow The Ovarium Polytene Chromosome...... ( Sukowati at.al)

ly). Broad light areas containing four prises one dotted band, four thin dotted diffuse bands follow this region. The re- bands, one curvature small dark band, two maining part of zone 3 is composed of two dotted light bands, three pairs of dotted dark bands and one dotted light band in bands, two dark bands, one thin dark band, between. Zone 4 has a prominent landmark and a pair of dotted band res-pectively. of three dark bands; these are followed by Zone 12 has one long puffing area with a one dark dotted band, a diffuse dotted band series of two small black dotted bands, two and ends with a doubled dark band at the light dotted bands, one thick black band on constriction. The centromeric region is co- the constriction, two pairs of light dotted vered by zone 5 that has one prominent bands, two black dotted bands at the dark band, followed by a large puff that middle of puffing, a pair of black bands, consists of 5 diffuse dotted bands and four two light dotted bands, two pairs of dotted dark dotted bands at the contromer. bands and ends with a pair of prominent black bands. Zone 13 starts with one pair Chromosome arm 2R/2 of dotted bands, followed by two pairs of The free end, including zone 6, has diffused dotted bands, one curved black four dotted bands at the tip followed by band, one dotted band, two thick promi- doubled dark bands, two dotted light nent dark bands, three pairs of light dotted bands, three dark bands, two light dark bands, and one doubled small dark band bands and ends with doubled dotted dark respectively. Zone 14 is characterized by bands. Zone 7 begins with three light dot- two prominent dark bands with space in ted bands, followed by three closely dotted between which consist of two pairs of dark bands, two pairs of light dotted bands, dotted light bands, followed by several and one dark band, three light dotted light dotted bands, two pairs of curvature bands, one prominent dark band, one dot- black bands with space in between, fol- ted dark band, and one dark band respec- lowed by three light dotted bands, four tively. Zone 8 begin with one light dotted black dotted bands and four dotted light band, followed by a doubled closed dotted bands respectively. Zone 9 represents ano- dark band, three light dotted bands, two ther characteristic, consisting of several dark bands, two light dotted bands, one dark bands, starting with one thick dark dark band, two dotted bands, and two light band, followed by three dotted bands with bands respectively. Zone 9 is marked by curvature, three thin black bands, a pair of one thick bands, three dotted bands, and dotted bands, one doubled dark band at the light area, following the constriction area constriction, and five dotted bands with are three short thick dark bands, three thin curvature respectively. Zone 16, covering dark bands, two wide diffuse bands, one the centromeric region, is characterized by dotted dark band and one thick curvature five prominent dark bands. Zone 16, be- dark band. Zones 10 and 11 consist of gins with a doubled dark band, one light several dark bands. Zone 10 start with band, five prominent dark band, several three medium dotted bands, continued by wavy bands, a pair of prominent dark two dotted bands, a pair of curvature dark bands with one dotted band in between, bands, two light bands, one small thick several diffuse light bands, and a pair of band, a light area, two dotted bands, three small dark bands at the centromeric region. conspicuous dotted dark bands and ends with two light dotted bands. Zone 11 com- Bul. Penel. Kesehatan, Vol. 33, No. 2,2005: 89-98

Chromosome arm 2Ll3 with a light dotted band in between, and a Zone 25 marks the free end of arm pair of light dotted bands. Zone 17 is 2L showing a typical hnnel shape at the located at the centromeric end of arm 2L, fiee end with dotted and flared bands at the beginning with two constricted dark bands very tip. It is followed by one puff con- followed by two dotted bands, four light sisting of several diffuse dotted bands, and bands, two dotted bands, two close dark continued by a doubled dark band, two bands, and several difise light bands con- dotted light bands, and one dark band res- tributing lighter area toward the centro- pectively. Zone 24 comprises one dark meric region. band, two pairs of dotted bands, three light bands, a pair of black bands in the con- Chromosome arm 3R/4 striction, and three pairs of light dotted The free end in zone 26 has a diffuse bands. Zone 23 is characteristically marked character tip followed by a pair of dotted by two pairs of dark bands located at the bands and moderate swelling area with beginning of this zone and continued by a four dotted bands. Zone 27 starts with one wavy diffise band, and three pairs of dotted band, followed by two dark bands curvature dotted bands which form a mo- with a light area in between, a series se- derate swelling. Zone 22 begins with a pair quence of 10 dotted bands, two close dark of doubled dark bands, followed by one bands, and one dotted band. Zone 28 be- dark band in the constriction, a pair of gins with a pairs of dark bands, one dotted dotted bands, two dark bands, and three band, one dark band, one short dark band dotted bands. Zone 21 is composed of one in the constriction, and three prominent black band, followed by two black bands in dark bands. Zone 29 has a pair of light the constriction, six dotted bands, one thin bands, followed by one dark band, two black band, a pair of prominent dark bands diffuse bands, one dotted band, two dark in the constriction, one light band, one thin bands, one light band, one prominent dark light band and one dark band with a light band, two light bands, and a pair of dark area in between and three curvature dotted bands in the constriction. Having several bands. Zone 20 begins with one black pairs of prominent dark bands characteris- dotted band, followed by a light dotted tically marks zone 30 and zone 3 1. Zone band, one dark band, two thin dark bands 32 and zone 33 are lighter than zone 30 with one light dotted band in bet-ween, a and 3 1; most of the bands are composed of light area consisting of four thin bands, many dotted and light bands, and less dark five dark bands, diffuse band, one dark bands. Zone 34 consists of a series of dark band, diffise band and three dark bands bands and dotted dark bands, which starts respectively. Zone 19 is composed of two with two dark bands, followed by six light dotted bands, two close dark bands, dotted dark bands, two dotted bands, a pair one dark dotted band, 5 thin dark bands, a of dark bands, one light band, and two pair of prominent dark bands, two pairs of pairs of dark bands respectively. Zone 35 light bands and ends with three dotted starts with one light dotted band, three bands. Zone 18 contains one dotted band, close prominent dark bands, one dark two prominent dark bands, one curved dark dotted band, two pairs of dotted light band, two dotted bands, one dark band, a bands, one thin band, a pair of dotted pair of small dark bands, one light band, a bands, and ends with a pair of small band pair of close dark bands, two dark bands in the constriction. Zone 36 starts with a The Ovarium Polytene Chromosome...... ( Sukowati at.al)

pair of curvature dotted bands, continued pair of contracted black bands at the con- by three light dotted bands, a pair of black striction, followed by one curved black bands, three light bands, a pair of dotted band, three thin dark bands, three wavy bands, three thin bands, two black bands, bands, and one dotted band respectively. one black dotted band, and two light bands. Zone 44 consists of two black bands at the Zone 37 consists of several black bands, constriction, three black bands, one dotted starts with one prominent dark band, wavy band, one light dotted band, a pair of followed by 5 black bands, two close dotted bands, two dotted bands, one black prominent dark bands, a pair of dotted band, four dotted light bands, and a pair of bands and one light band. Zone 38 has one light bands. Zone 43 is characteristically small puffing area with a series of dark, marked by several dark bands, starting with dotted bands and diffuse bands, consisting one light band, and continued by three dark of one black band, three dotted bands, two bands, a pair of constricted dark bands, one thin bands, and several diffuse bands dark band, three pair of dotted dark bands, which form a light area in the puff, two and two pairs of dark bands. Zone 42 is prominent dark bands, and ends with four composed of a pair light dotted bands, one dotted bands. Zone 39 is located at the dark band, three pairs of wavy dotted centromeric end of arm 3Rl4 beginning bands, a pair of dotted bands, one black with two constricted prominent dark bands, band, several diffuse bands, a pair of black followed by four pairs of light dotted bands, one light band, and two dotted bands, one dotted band, and two pairs of bands. Zone 41 starts with two black light dotted bands contributing to a lighter constricted black bands, followed by three area toward the centromeric region. light dotted bands, one black band, two light bands, one light band at thc con- Chromosome arm 3L/5 striction, and two dotted bands. At the The telomere in zone 50 shows se- centromeric region is zone 40; this zone is veral light bands followed by one black darker in appearance, composed of one band, then a small swelling part comprised dark band, a pair of prominent dark bands, of two black bands, a pair of curved black three pairs of dotted dark bands, five bands and one prominent dark band at the dotted dark bands, and diffuses flare dark constriction. Zone 49 consists of four pairs bands at end. of dark bands. Zone 48 starts with a pair of short black bands at the constriction, fol- DISCUSSION lowed by one dotted band, a pair of thin As in other dipterans , one of black bands, two pairs of dotted black the essential requirements for cytogenetic bands, one black band, one dotted black investigations of anopheline mosquitoes is band, a pair of dotted black bands and a a standard polytene chromosome map; for pair of light dotted bands. Zone 47 starts example, in An. subpictus, An. vagus (26), with one dotted band, followed by two An. a1bimanu.s (28),An. nili (27),An. acorzi- dotted bands, eight black bands in series, tus (28),An. culicifacies (25),An. dirus (13,23) and a pair of dotted bands. Zone 46 is and An. surlduicus (21).The standard poly- composed of three pairs of dotted black tene photomap of An. barbirostris has been bands, one light band, three dark bands, prepared and is presented in this article for several diffuse bands, one dark band, and comparative investigations into differen- three dotted bands. Zone 45 starts with a Bul. Penel. Kesehatan, Vol. 33, No. 2,2005: 89-98

ces, if any, in different wild populations. AKNOWLEDGEMENT Differences in the banding patterns and the The authors wish to thank the presence of chromosomal polyrnorphisms National Institute of Research and Science within and between populations of An. (LIPI) and the Ministry of Research and barbirostris s.1. could be useful informa- Technology for supporting this study under tion as to the genetic differentiation and a special research programme called Riset possible speciation within this taxon. Such ,Unggulan Terpadu (RUT). We are espe- differences in polytene chromosome ban- cially indebted and grateful to the National ding patterns have been reported in the ho- Institute of Health Research and Deve- mosequential species of An. dirus complex lopment for its support and facilities for from Southeast Asia and of An. sun- this research. daicus complex from Indonesia and Thai- land (21). REFERENCES In our study, all the wild of An. bar- birostris s.1 specimens obtained from dif- 1. Koesoemowinangun WR. Anopheline in ferent geographical areas in Indonesia Indonesia. Lembaga Malaria, Kementrian appear to share most of the standard chro- Kesehatan, Republik Indonesia Jakarta; 1954. mosome arrangements in the polytene 2. Horsfall WR. Mosquitoes 'Their Bionomic complement. The absence of chromosomal and relation to disease' 1" reprint. Haher polymorphism in these five populations publishing company, New York; 1972 from difference geographical areas might 3. Lien JC, Koesman L, Partono F, Joesoef A, be due to no detectable differences in the Kosin E and Cross JH. A Brief Survey of banding patterns of polytenes (homosequ- Mosquitoes in North Sumatera, Indonesia, J ential) in this species. The homosequential Med Entomol. 1975; 12:233-239. banding patterns of polytene chromosomes 4. Partono F, Purnomo, Dennis DT, have been reported from some species: e.g. Atmosoedjono S, Sri Oemiyati and Cross JH. An. atropawus and (I7) An. labranchiae, sp. (Nematode: Filarioidea) An. finestus and An. aruni, and seven from Flores Island, Indonesia. J. Parasit. species from the An. hyrcanus group (30). 1977; 63: 540-546. Alternatively, this species might have in- 5. Reid JA, Harrison BA and Atmosoedjono S. version or polymorphism in the polytene Variation and vector status in Anopheles chromosome; however, this has not been barbirostris. Mosquito systematics. 1979; 1 1 found yet because only 5 populations in (3): 235-250. Indonesia are being studied. Therefore, 6. Ramachandra Rao T. The Anopheline of from the mitotic chromosomes studied 4 India. Indian council of Medical Research forms of the mitotic chromosome in An. New Delhi, Malaria Research Centre (ICMR), barbirostris complex in Indonesia were Delhi Press, New Delhi; 1981 identified3'. Nevertheless, this standard 7. Hoedoyo. Bionomics of Anopheles barbi- photomap of polytene chromosome pro- rostris Van der Wulp in several areas in vide a reference point for further studies, Indonesia. 18" Annual Scientific Seminar of where more analysis of natural populations the Malaysia Society of Parasitology and Tropical Medicine in Penang. Malaysia. 1982; would be camed out together with the 12 PP necessary taxonomic studies of chromo- somally identified materials. 8. Coluzzi M, Sabatini A, Petraca V and Di Deco MA. Chromosomal differentiation and The Ovarium Polytene Chromosome...... ( Sukowati at.aC)

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