MARCH 1993 CyrocnNtrrcs oF AN. cuLrcrFAcrEs

ANOPHELES CULICIFACIES COMPLEX: CYTOGENETICCHARACTERIZATION OF RAMESHWARAM ISLAND POPULATIONS

S. K. SUBBARAO,I NUTAN NANDA,T R. K. CHANDRAHAS, nNo V. P. SHARMAT

ABSTRACT. A nophel.es culicifacies sensu lato collected from Rameshwaram island, Tamil Nadu state.India was identified as species B based on the diagnostic inversion karyotype Xab 2grtn' as observed inpolytene chromosomes. Among male mitotic karyotypes made from larval neurogonial cells, two tlpeswere observed: one with an acrocentric Y-chromosome and the other with a sub-metacentric Y-chromosome, both had sub-metacentric X and metacentric autosomes. The Rameshwaram population isidentical to species B in its genetic relationship with species A and C as determined by experimentalhybridizations (sterile and fertile male hybrids, respectively).

27

INTRODUCTION

Cytotaxonomic studies have so far identified4 reproductively isolated populations in thetaxon Anopheles culicifacies Giles. These popu-lations have been given the species status andare provisionally designated as species A and B(Green and Miles 1980), species C (Subbarao etal. 1983) and species D (Subbarao et al. 1988a,Vasantha et al. 1991). The 4 sibling species wereidentified on the basis of positive assortativemating observed among populations differing inparacentric inversions. Sibling species are alsodistinguishable by structural variation in the Y-chromosomes (Vasantha et al. 1982, 1983; Su-guna et al. 1989).

Extensive surveys were carried out in thecountry to establish the distribution of siblingspecies (Subbarao et al. 1988a). Longitudinaland spot surveys were also conducted to estab-lish biological differences among sibling species(Subbarao et al. 1987, Joshi et al. 1988). Incrim-ination studies using a two-site immuno-radio-metric assay established species A, C and D asvectors ofmalaria (Subbarao et al. 1988c, 1992).Further, it was shown that species B may playa legligible role, if at all, in malaria transmis-

On Rameshwaram island only species B wasfound (Subbarao 1984), but An. culicifacics s.l.had been incriminated and is considered as themajor vector of malaria on the island (Sabesanet al. 1984). A similar situation exists in SriLanka (Subbarao 1988, Wickramasinghe andSamarasinghe 1991). To resolve this paradox, acytogenetical study of An. culicifacies s.l. popu-Iations from Rameshwaram island and a fewareas in Tamil Nadu state was undertaken: 1)to establish sibling species composition by po-lytene chromosome and mitotic chromosome ex-amination, and 2) to analyze genetic relation-ships of the island population with other An.

'Malaria Research Centre (ICMR), 22-Sham NathMarg, Delhi - l l0 054, India.

'� MRC, Field Station. Madras. Tamil Nadu. India.

cuLicifacies sibling species identified by hybridsterility/fertility in genetic crosses. Results arereported in this paper.

MATERIALS AND METHODS

Anopheles culicifacies adults were collectedfrom: 1) Manali, about 40 km from Madras city,2) Kolamanjanur, Rayandapuram and Vepurchekadi villages near the Sathanur dam area inNorth Arcot district, and 3) Karaiyur, Rajakoil,Natarajapuram and Tharvaithoppu villages onRameshwaram island in the district Ramana-thapuram in Tamil Nadu state (Fig. 1). Threecollections were made in December 1990 and inMay and June 1991 from Tharvaithoppu villageon Rameshwaram island.

For sibling species identification, ovaries wereremoved from half-gravid females collected inthe field and were fixed in 1:3 glacial acetic acidand methanol. Polytene chromosomes were pre-pared according to the method of Green andHunt (1980) and sibling species were identifiedfollowing the diagnostic inversion karyotypesgiven in Subbarao et al. (1988a).

Adult An. culicifacies collected from Ramesh-waram island were brought to Delhi for estab-Iishing a laboratory colony. Mosquitoes werereared in the insectary maintained at 28 + 1"Cand70-80% RH with simulated dawn and duskconditions. Mitotic chromosome preparationswere made from neurogonial cells of late III orearly IV instar larvae by the squash techniqueof Breland (1961) with slight modifications. Thebrain was dissected from male and female larvae(sexed by examining the gonads) and placed in0.1% colchicine for 1-1.5 h, stained in2Tolacto.aceto orcein for 10 min and squashed. Isofemalelines having different Y-chromosome kary-otJ,pes were isolated by examining 5-10 progenyof single females.

Stocks were used in crosses with An. culicifa-cies from Rameshwaram island as in Table 1.Reciprocal crosses among stocks were carriedout in 30 x 30 x cm cloth cages. Only Fr adultsof the Rameshwaram population were used inall the crosses. Mosquitoes were offered fresh

28 JouRual oF THE Avnmcex MoSQUITo CoNtRol, AssocrlrtoN VoL. 9, No. 1

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H A R Y A N A

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G O A

K A R N A T A K A

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U T T A RP R A D E S H

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T R I P U R A

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l l o r t h A r c o t

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Table 1. Stocks used in crosses with Anopheles culicifaci.es from Rameshwaram island.

M A H A R A S H T R

Fig. 1. Map of India showing areas surveyed.

Species

Inversion geno-type observable

in polytenechromosomes

Metaphase Y-chromosome

Collection areas forestablishing the

stocks

B

C

R

RACRSM

X+. +b 2+"' +h'

Xab 2gr +h'

xab 2+gr h1

Xab 2gt +h'

Xab 2g1 +h'Xab 2gt +h'

Sub-metacentric

Acrocentric

Sub-metacentric

Acrocentric *sub-metacentric

AcrocentricSub-metacentric

District Ghaziabad,Uttar Pradesh(UP) in July 1990.

District Shahjahan-pur, UP in August1990.

District Allahabad,UP in March 1988.

Rameshwaram island

Rameshwaram islandRameshwaram island

MARCH 1993 CvrocpNn'rrcs or Ary. cuLrcIFAcrEs 29

Table 2. Y-chromosome types observed in the Rameshwaram colony of Anophelcs culbifacies established frommaterial collected in May and June 1991.

Y-chromosomePeriod exam-ined

No. ex-amined Acrocentric Sub-metacentric Unidentified

June, 1st weekJune, 3rd weekJuly 1991Sept.1991Jan. 1992

28

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q

D

109

10

100I

478

2 l10t4

water-soaked raisins and. I% glucose water oncotton pads. Females were offered rabbits as abloodmeal source. In all crosses, eggs were col-lected in a pool and counted after 2 days todetermine the hatch rate. Larvae were rearedand sexed at the pupal stage. Reproductive or-gans of hybrid males and females were dissectedout and examined under the light microscope.

RESULTS

AnopheLes culicifacies collected from Manali(n: l7), Sathanur dam area (n : 7\ and Ra-meshwaram island l. (n : 115) were of the Xab2gt*h' karyotype. Hence, the populations wereidentified as species B. A colony was establishedwith adults collected from Rameshwaram island.

Mitotic karyotype: Both male and female F1progeny of field collected adult females had 3pairs of chromosomes (2n : 6), females hadhomomorphic and males heteromorphic sex-chromosomes. Among 39 F1 male larval neuro-gonial preparations made, two different mitotickaryotlpes at the metaphase stage were ob-served: 23 with the Y-chromosomes having anarm-ratio ranging between 3.6 to 4.8 and 16 withY-chromosome having an arm-ratio of 1.5 to 2.1.In both karyotypes, the X-chromosome had anarm-ratio ranging between 1.4 and 2.3. Y-chro-mosomes having a higher arm-ratio would bereferred to as acrocentric and that with the loweras sub-metacentric and also the X-chromosomeas sub-metacentric (Fig. 2). In the F2 generation,of the 26 preparations examined, only 10 couldbe identified, of which 3 had acrocentric and 7sub-metacentric Y-chromosomes. The frequen-cies of the 2 types of Y-chromosomes observedin the colony established subsequently from themosquitoes collected in May and June 1991 aregiven in Table 2.

Isofemale lines having acrocentric and thosehaving sub.-metacentric Y-chromosomes had theXab 2g1*h' (species B) inversion genotype inpolytene chromosomes.

Genetic crosses.' Results from reciprocalcrosses between species A and B, and betweenstrains from Rameshwaram and sibling speciesA, B and C are given in Table 3.

Results from crosses between species A and B(crosses 1 and 2) were as reported earlier (Sub-barao et al. 1988d). i.e.. the hatch rate wasnormal in A female and B male cross and 0%hatch in the reciprocal cross. Hybrid femaleswere fertile and hybrid males had extremelyunderdeveloped or rudimentary testes lobes andvasa deferens and fully developed ejaculatoryduct and accessory glands, i.e., sterile.

Results from reciprocal crosses between spe-cies A and, An. culicifacies s.l. from Ramesh-waram island (R), and between species A andthe R line with acrocentric Y-chromosome(RAC) were similar to those from species A andB. In the cross between female species A andthe male sub-metacentric Iine (RSM) there wasno egg laying, and in the reciprocal cross therewas egg laying but the hatch rate was 0.7Vo.Males from the Rameshwaram colony (at thetime when males were of predominantly sub-metacentric type) when crossed with A femalesproduced sterile hybrid progeny (Table 3).

In the reciprocal crosses between species Band the R strain and ir. those between species Cand the R strain, fertility was>90% and hybridfemales and males had fully developed reproduc-tive organs. Backcrosses of Fr males and femalesto both parental strains were fertile (data notshown in the table).

DISCUSSION

Anopheles culicifacies species B from districtsFaridabad (Haryana), Ghaziabad and Nainital(UP) and Aurangabad (Maharashtra) had acro-centric Y-chromosomes (Vasantha et al. 1982).Lines established recently in 1990 from Shahja-hanpur (UP) and Alwar (Rajasthan) also hadacrocentric Y-chromosomes. Suguna et al.(1983, 1989) reported the same from North Ar-cot district, Tamil Nadu state. In Ramesh-waram, two populations that are homosequen-tial for the polytene chromosome arrangementsbut different male mitotic karyotype were ob-served: population l-Sub-metacentric Y-chro-mosome and population 2-acrocentric Y-chro-mosome.

MARCH 1993 CyroceNnncs oF .4N. cllLrcrFAcrEs 31

arrangement of species C disappeared (Subbaraoet al. 1988d). In "choice-mating" experiments(loc. cit.) species A and B were found to matewith their own kind, although a few matingsbetween species A and B were observed. 86-tween species B and C, species C were found toprefer to mate with species B and a low fre-lue11cy of matings was observed within speciesC. Thus, male hybrid sterility observed beiweenspecies A and B was not the reason for theelimination of the species A arrangement. Inspite of bidirectional hybrid male fertility be-tween B and C, species C was eliminated. There-fore, the possibility of species varying in theirbiological fitness was suggested (Subbarao et al.1988d).

Species B populations examined from otherparts of India were of the acrocentric y-chro-mosome type, and epidemiological (Subbarao etal. 1988c) and incrimination data (Subbarao etal. 1988b) from several areas suggested thatspecies B is not a vector. Hence, the possibilityof the new population on Rameshwaram island,i.e., species B having sub-metacentric Y-chro-mosome being a vector should be examined. InSri Lanka where only species B was found andAn. culicifacies s.l. was incriminated (Wickra-masinghe and Samarasinghe 1991), a similarstudy is suggested.

ACKNOWLEDGMENTS

We thank the staff members of the Director-ate of Public Health and Preventive Medicine,Madras, Tamil Nadu, for extending the cooper-ation in carrying out the work. We also thankthe staff members of the Genetics Division,Delhi and Malaria Research Centre field stationat Madras, Tamil Nadu, for technical help.

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