MARCH1993
CyrocnNtrrcs
oF AN. cuLrcrFAcrEs
27
ANOPHELES CULICIFACIES COMPLEX: CYTOGENETIC
CHARACTERIZATIONOF RAMESHWARAM ISLAND POPULATIONS
S. K. SUBBARAO,I NUTAN NANDA,T R. K. CHANDRAHAS, nNo V. P. SHARMAT
ABSTRACT. A nophel.es
culicifaciessensulato collectedfrom Rameshwaramisland, Tamil Nadu state.
India was identified as speciesB based on the diagnostic inversion karyotype Xab 2grtn' as observedin
polytene chromosomes.Among male mitotic karyotypes made from larval neurogonial cells, two tlpes
were observed: one with an acrocentric Y-chromosome and the other with a sub-metacentric Ychromosome,both had sub-metacentricX and metacentric autosomes.The Rameshwarampopulation is
identical to speciesB in its genetic relationship with speciesA and C as determined by experimental
hybridizations (sterile and fertile male hybrids, respectively).
INTRODUCTION
Cytotaxonomic studies have so far identified
4 reproductively isolated populations in the
taxon AnophelesculicifaciesGiles. These populations have been given the speciesstatus and
are provisionally designatedas speciesA and B
(Greenand Miles 1980),speciesC (Subbaraoet
al. 1983)and speciesD (Subbaraoet al. 1988a,
Vasanthaet al. 1991).The 4 sibling specieswere
identified on the basis of positive assortative
mating observedamong populations differing in
paracentricinversions.Sibling speciesare also
distinguishableby structural variation in the Ychromosomes(Vasantha et al. 1982, 1983; Suguna et al. 1989).
Extensive surveys were carried out in the
country to establish the distribution of sibling
species (Subbarao et al. 1988a). Longitudinal
and spot surveyswere also conducted to establish biologicaldifferencesamongsibling species
(Subbaraoet al. 1987,Joshi et al. 1988).Incrimination studies using a two-site immuno-radiometric assayestablishedspeciesA, C and D as
vectorsofmalaria (Subbaraoet al. 1988c,1992).
Further, it was shown that speciesB may play
a legligible role, if at all, in malaria transmis-
cuLicifaciessibling speciesidentified by hybrid
sterility/fertility in genetic crosses.Results are
reported in this paper.
MATERIALS
AND METHODS
Anopheles culicifacies adults were collected
from: 1) Manali, about 40 km from Madras city,
2) Kolamanjanur, Rayandapuram and Vepur
chekadi villages near the Sathanur dam area in
North Arcot district, and 3) Karaiyur, Rajakoil,
Natarajapuram and Tharvaithoppu villages on
Rameshwaram island in the district Ramanathapuram in Tamil Nadu state (Fig. 1). Three
collectionswere madein December1990and in
May and June 1991from Tharvaithoppu village
on Rameshwaramisland.
For sibling speciesidentification, ovarieswere
removed from half-gravid females collected in
the field and were fixed in 1:3 glacial acetic acid
and methanol. Polytene chromosomeswere prepared according to the method of Green and
Hunt (1980) and sibling specieswere identified
following the diagnostic inversion karyotypes
given in Subbaraoet al. (1988a).
Adult An. culicifaciescollected from Rameshwaram island were brought to Delhi for estabIishing a laboratory colony. Mosquitoes were
On Rameshwaramisland only speciesB was
reared in the insectary maintained at 28 + 1"C
found (Subbarao 1984), but An. culicifacicss.l.
and70-80% RH with simulated dawn and dusk
had been incriminated and is consideredas the
conditions.
Mitotic chromosome preparations
major vector of malaria on the island (Sabesan
were made from neurogonial cells of late III or
et al. 1984). A similar situation exists in Sri
early IV instar larvae by the squashtechnique
Lanka (Subbarao 1988, Wickramasinghe and
of Breland (1961)with slight modifications.The
Samarasinghe1991).To resolvethis paradox,a
brain was dissectedfrom male and female larvae
cytogeneticalstudy of An. culicifaciess.l. popu- (sexed
by examiningthe gonads)and placedin
Iations from Rameshwaram island and a few
0.1%colchicinefor 1-1.5 h, stainedin2Tolacto.
areas in Tamil Nadu state was undertaken: 1)
aceto orcein for 10 min and squashed.Isofemale
to establishsibling speciescompositionby po- lines
having different Y-chromosome karylytenechromosomeand mitotic chromosomeexotJ,peswere isolated by examining 5-10 progeny
amination, and 2) to analyze genetic relation- of single females.
ships of the island population with other An.
Stocks were used in crosseswith An. culicifacies from Rameshwaram island as in Table 1.
Reciprocalcrossesamong stocks were carried
'Malaria Research
out in 30 x 30 x cm cloth cages.Only Fr adults
Centre (ICMR), 22-Sham Nath
Marg, Delhi-l l0 054, India.
of the Rameshwarampopulation were used in
' MRC, Field
Station. Madras. Tamil Nadu. India.
all the crosses.Mosquitoes were offered fresh
VoL. 9, No. 1
JouRual oF THE Avnmcex MoSQUIToCoNtRol, AssocrlrtoN
28
t
i
ARUTiACHAL
PRAOESH
PUNJAB
HARYANA
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,r*r
TIEcHALA/.L.lg
,
KKIII,I
\.L..
..,
(.
'l
RAJASTHAN
UTTAR
PRADESH
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I|IADHYA
PRADISH
T RI P U R A
MI ZORAIt.I
MAHARASHTR
O RI S S A
GOA
KARNATAKA
Madras city
llorth Arcot
R a m e hs w a r a m
island
xrnnLn
$
0
.j
S RI
LANKA
0
Fig. 1. Map of India showing areas surveyed.
Table 1. Stocks used in crosseswith Anophelesculicifaci.esfrom Rameshwaramisland.
Species
Inversiongenotype observable
in polytene
chromosomes
MetaphaseYchromosome
X+. +b 2+"' +h'
Sub-metacentric
B
Xab 2gr +h'
Acrocentric
C
xab 2+gr h1
Sub-metacentric
R
Xab 2gt +h'
RAC
RSM
Xab 2g1 +h'
Xab 2gt +h'
Acrocentric *
sub-metacentric
Acrocentric
Sub-metacentric
Collection areasfor
establishingthe
stocks
District Ghaziabad,
Uttar Pradesh
(UP) in July 1990.
District Shahjahanpur, UP in August
1990.
District Allahabad,
UP in March 1988.
Rameshwaramisland
Rameshwaramisland
Rameshwaramisland
MARCH 1993
CvrocpNn'rrcs or Ary. cuLrcIFAcrEs
29
Table 2. Y-chromosometypes observedin the Rameshwaramcolony of Anophelcs culbifaciesestablished
from
material collected in May and June 1991.
Periodexamined
June, 1st week
June, 3rd week
July 1991
Sept.1991
Jan. 1992
No. examined
47
8
2l
10
t4
Y-chromosome
Acrocentric
10
10
0
I
water-soakedraisins and.I% glucosewater on
cotton pads. Femaleswere offered rabbits as a
bloodmealsource.In all crosses,eggswere collected in a pool and counted after 2 days to
determine the hatch rate. Larvae were reared
and sexedat the pupal stage.Reproductiveorgansof hybrid malesand femaleswere dissected
out and examinedunder the light microscope.
Sub-metacentric
Unidentified
q
28
D
t)
10
9
0
4
Resultsfrom crossesbetweenspeciesA and B
(crosses1 and 2) were as reportedearlier (Subbarao et al. 1988d). i.e.. the hatch rate was
normal in A female and B male cross and 0%
hatch in the reciprocal cross. Hybrid females
were fertile and hybrid males had extremely
underdevelopedor rudimentary testeslobes and
vasa deferens and fully developed ejaculatory
duct and accessoryglands,i.e.,sterile.
Resultsfrom reciprocalcrossesbetweenspeRESULTS
cies A and,An. culicifacies s.l. from RameshAnopheLesculicifaciescollected from Manali waram island (R), and between speciesA and
(n: l7), Sathanurdam area (n : 7\ and Ra- the R line with acrocentric Y-chromosome
meshwaramisland l. (n : 115)were of the Xab (RAC) were similar to those from speciesA and
2gt*h' karyotype.Hence, the populations were B. In the cross between female speciesA and
identifiedas speciesB. A colonywasestablished the male sub-metacentricIine (RSM) there was
with adultscollectedfrom Rameshwaramisland. no egg laying, and in the reciprocalcrossthere
Mitotic karyotype: Both male and female F1 was egg laying but the hatch rate was 0.7Vo.
progeny of field collected adult females had 3 Males from the Rameshwaram colony (at the
pairs of chromosomes(2n : 6), females had time when males were of predominantly subhomomorphic and males heteromorphic sex- metacentric type) when crossedwith A females
chromosomes.
Among 39 F1 male larval neuro- producedsterile hybrid progeny(Table 3).
In the reciprocal crossesbetween species B
gonial preparations made, two different mitotic
karyotlpes at the metaphase stage were ob- and the R strain and ir. thosebetweenspeciesC
served:23 with the Y-chromosomeshaving an and the R strain, fertility was>90% and hybrid
arm-ratiorangingbetween3.6to 4.8 and 16 with femalesand maleshad fully developedreproducY-chromosomehavingan arm-ratioof 1.5to 2.1. tive organs.Backcrossesof Fr malesand females
In both karyotypes,the X-chromosomehad an to both parental strains were fertile (data not
arm-ratio rangingbetween1.4 and 2.3. Y-chro- shownin the table).
mosomeshaving a higher arm-ratio would be
referredto as acrocentricand that with the lower
as sub-metacentricand also the X-chromosome
DISCUSSION
as sub-metacentric(Fig.2). In the F2generation,
of the 26 preparationsexamined,only 10 could
AnophelesculicifaciesspeciesB from districts
be identified, of which 3 had acrocentricand 7
Faridabad (Haryana), Ghaziabadand Nainital
sub-metacentricY-chromosomes.The frequen- (UP) and Aurangabad(Maharashtra)had acrocies of the 2 types of Y-chromosomesobserved centric Y-chromosomes(Vasanthaet al. 1982).
in the colony establishedsubsequentlyfrom the
Lines establishedrecentlyin 1990from Shahjamosquitoescollectedin May and June 1991are hanpur (UP) and Alwar (Rajasthan)also had
given in Table 2.
acrocentric Y-chromosomes. Suguna et al.
Isofemalelines having acrocentricand those (1983, 1989) reported the same from North Arhavingsub.-metacentric
Y-chromosomes
had the
cot district, Tamil Nadu state. In RameshXab 2g1*h'(speciesB) inversion genotype in
waram, two populationsthat are homosequenpolytenechromosomes.
tial for the polytene chromosomearrangements
Genetic crosses.' Results from reciprocal but different male mitotic karyotype were obcrossesbetweenspeciesA and B, and between served:population l-Sub-metacentric Y-chrostrains from Rameshwaramand sibling species mosomeand population 2-acrocentric Y-chroA, B and C are given in Table 3.
mosome.
MARCH 1993
CyroceNnncs
oF .4N. cllLrcrFAcrEs
arrangementof speciesC disappeared(Subbarao
et al. 1988d).In "choice-mating"experiments
(loc. cit.) speciesA and B were found to mate
with their own kind, although a few matings
between speciesA and B were observed.86tween speciesB and C, speciesC were found to
prefer to mate with speciesB and a low frelue11cyof matings was observedwithin species
C. Thus, male hybrid sterility observedbeiween
speciesA and B was not the reason for the
elimination of the species A arrangement. In
spite of bidirectional hybrid male fertility betweenB and C, speciesC waseliminated.Therefore, the possibility of speciesvarying in their
biological fitness was suggested(Subbaraoet al.
1988d).
SpeciesB populations examined from other
parts of India were of the acrocentricy-chromosometype, and epidemiological(Subbaraoet
al. 1988c)and incrimination data (Subbaraoet
al. 1988b) from several areas suggestedthat
speciesB is not a vector. Hence,the possibility
of the new population on Rameshwaramisland,
i.e., speciesB having sub-metacentricY-chromosomebeing a vector should be examined.In
Sri Lanka where only speciesB was found and
An. culicifaciess.l. was incriminated (Wickramasinghe and Samarasinghe1991), a similar
study is suggested.
31
infection and vectorial capacityof AnopheLes
culic/ociesGilesin Rameshwaram
island(Tamil Nadu).
Indian J. Med. Res.80:43-46.
Subbarao,S. K. 1984.Biological speciesin malaria
vectorsof India, pp. 77-83.In: V. P. Sharma (ed.),
Proc. Indo-UK Workshopon Malaria. Malaria ResearchCentre (I.C.M.R.).Delhi.
Subbarao,S. K. 1988.'fhe Anophelesculicit'ocies
complex and control of malaria. ParasitologyToday
4:72-75.
Subbarao,S. K., K. Vasantha,T. Adak and V. P.
Sharma. 1983.Anophelesculicifaciescomplex. Evidencefor a new sibling speciesC. Ann. Entomol.
Soc.Am. 76:985-988.
Subbarao,S. K., K. Vasantha,T. Adak and V. P.
Sharma.1987.Seasonalprevalenceofsibling species
A and B of the taxon Anophelcsculicifaciesin villagesaroundDelhi. Indian J. Malariol. 24:9-16.
Subbarao,S. K., K. Vasantha and V. P. Sharma.
1988a.Cy'totaxonomyof malaria vectors in India,
pp. 25-37..In: M. W. Service(ed.),Biosystematics
of haematophagous
insects.Oxford Univ. Press.
Subbarao,S. K., K. Vasantha,H. Joshi, K. Raghavendra, C. Usha Devi, T. S. Sathyanarayan, A. H.
Cochrane, R. S. Nussenzweigand V. P. Sharma.
1992. Role of Arnphel.esculi.cifadcssibling species
in malaria transmission in Madhya Pradesh state,
India. Trans. R. Soc.Trop. Med. Hyg. 86:613-614.
Subbarao,S. K., K. Vasantha,K. Raghavendra,V. P.
Sharma and G. K. Sharma. 1988c.Anophel.esculicifacies;siblingspeciescompositionand its relationship to malaria incidence.J. Am. Mosq. Control
Assoc.4:29-33.
Subbarao,S. K., K. Vasantha and V. P. Sharma.
1988d.Studies on the crossesbetween sibling species of the Anophelesculicifaciescomplex. J. Hered.
ACKNOWLEDGMENTS
79:300-302.
Subbarao,S. K., K. Vasantha,H. Joshi, K. RaghavWe thank the staff members of the Directorendra, C. Usha Devi, T. S. Sathyanarayan, A. H.
ate of Public Health and Preventive Medicine,
Cochrane, R. S. Nussenzweigand V. P. Sharma.
Madras,Tamil Nadu, for extendingthe cooper1992. Role of Anophelesculicifaciessibling species
ation in carrying out the work. We also thank
in malaria transmission in Madhya Pradesh state.
India. Trans. R. Soc.Trop. Med. Hyg. 86:613-614.
the staff members of the Genetics Division,
Delhi and Malaria ResearchCentre field station Suguna,S. G., S. C. Tewari, T. R. Mani, J. Hiriyan
and R. Reuben. 1983.Anophelesculicifaciesspecies
at Madras, Tamil Nadu, for technical help.
complex in Thenpenniyar riverine tract, Tamil
Nadu. Indian J. Med. Res.77:445-449.
Suguna,S. G., S. C. Tiwari, T. R. Mani, J. Hariyan
and R. Reuben. 1989.A cytogeneticdescription ofa
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