CYTOGENETIC STUDIES ON FEW CULICINE MOSQUITOES SPECIES
DISSERTATION SUBMITTED IN PARTIAL rULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF
Maettv of ^fjilosiciptip in Zoologp
x:
BY Poonam Varshncy
DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2004 ^r l>S'-34'^^. \ \ '^" \ i\. V X^i^^-; •-'
5 APR 2005
DS3438 cUjedicated
to mt p.amn t6 ^^chnou/ledgmentf
All the thanks are due to Almighty God, who bestowed upon mc the capabihties necessary to achieve this target.
It is matter of pride and pleasure, for me to accord my most sincere thanks to my supervisor Dr. Anjum Ara^ senior lecturer (Women's College) Aligarh Muslim University, Aligarh for suggesting the problem and skillful supervision.
I am also thankful to Prof. Mohammad Hayat^ chairman, Department of Zoology and Prof. S.M. Hadi^ Dean Faculty of life sciences, Aligarh Muslim University, Aligarh for providing various facilities.
My gratitude also goes to Dr. Waseem Ahmad Faridi^ Deptt. of Zoology, AMU, Aligarh and Dr. Niamat Alt, P.G. Department of Zoology Sher-e-Kashmir University, Shrinagar, Kashmir, for their kind help during my dissertation work.
Finally my special thanks to Babu Bhai (Lab Attendant) and my colleagues Mrs. Rafat Siddiqui and Mr. Mehdi, for altruistic cooperation.
Lastly, I am also thankful to Azad & Brothers, Classic Computer for typing and graphic work with patience.
Poonam Varsheny Dr. (Ms.) Anjum Ara /^^^ DEPARTMENT OF ZOOLOGY Senior Lecturer Cl ^H ALIGARH MUSLIM UNIVERSITY M sc M Phil Ph D (Aiig) V\ k^ ± .^1*11 AUGARH-202002 (INDIA)
Oa\e&. .2>.:..^.:..o^.
Certificate
I certify that "Cytogenetic Studies on Few Mosquitoes
Species", is the original work of Miss Poonam Varsfiney and is
suitable for the partial fulfilment of the degree of Master of Philosophy
in Zoology of Aligarh Muslim University, Aligarh.
This work has done under my supervision.
Dr. (Ms.) Anjum Ara Women's College AMU, Aligarh. CONTENTS
Page No.
Introduction 1 -6
Historical Review 7-10
Material & Methods 11-16
Observations and Results 14-21
Discussion 22-25
Conclusion 26
Summary 27-28
References 29-35
INTRODUCTION
In the past several decades, considerable progress has been made in the field of mosquito cytogenetics, because mosquito population posed a problem in pest control programme by developing resistance against some insecticides. This stimulated interest in genetic control of this dipteran. Because of their importance as pests as well as vectors of many important and distressing human diseases, they have been studied in almost all parts of the world, extensively from the standpoints of bionomics, physiology, systematics, disease transmission, insecticidal resistance, chemical and biological control etc.
Although the cytogenetic work especially with Anopheline species started around 1940s, knowledge in this field is still very scanty, particularly regarding the tropical mosquitoes and among them the oriental species.
A valuable summary of previous work and synopsis of workable techniques have been made by Breland and his group.
Breland (1959, 1960, a,b 1961, 1963) Breland and Gassner (1961,
1962). Breland (1961) described mitotic chromosome complement of twenty four species of mosquitoes belonging to nine genera.
Among them were seven species of Culex, seven species of Aedes, two of Orthopodomiya three of Psorophora and one each of
Anopheles, Haemagogus, Culiseta, Taxorhynchites and
Uranotaenia. Breland 1959 was the first to extend the study of mosquito chromosomes by the use of squash technique. This method has provided a great stimulus and has been instrumental in accumulation of considerable information. French et. al. (1962) have indicated advantages of colchicine pretreatment for these studies. Certain improved procedures for fixation and storage of cytological material have been emphasized by Rai (1963a).
Amirkhanian (1968) devised a simple air drying technique which involved the hydrolysis of tissues in acid alcohol and staining the chromosome material in crystal violet solution. This technique was also applicable for staining the salivary gland chromosomes and other tissues of larvae and adult mosquitoes. Cell culture techniques to maintain mosquito tissues in vitro have been developed by establishing cell line. Grace (1966) established a cell line from the well developed larvae (about to pupate) of Aedes aegypti, by employing a culture medium containing haemolymph of the moth Antheracea eucalypti. Singh (1967) established three lines of Aedes albopictus, and two of Aedes aegypti using a culture medium without insect haemolymph. Schneider (1969) established three diploid cell lines of Anopheles Stephens! using the early larval tissues. An insect chromosomal isolation techniques for the metaphase chromosome harvest from cultured cells of the mosquito Aedes albopictus was developed by Mukherjee and De Giorgio (1981).
Breland and Gassner (1961), confirmed six as the diploid chromosome number in brain cells of fourth instar larvae of Aedes aegypti and added detailed information in the mitotic karyotype of this species. Some observations on both mitotic and meiotic chromosomes in the same species and other mosquitoes were made by Akstein (1962).
Rai and Craig (1961) also reported mitotic metaphase from five species of Aedes and one each of Corethra, Anopheles, and Culex. The usual
diploid picture 2n=6 except (Corethra 2n=8) was found. They agreed with Breland in finding one short chromosomal pair and two relatively larger ones in Aedes aegypti. All the three pairs show intimate somatic pairing during prophase as is the characteristic of other mosquito
species. Mukherjee et. al., (1966) presented the karyotypes of nineteen species of mosquitoes belonging to four genera. In another paper of
(1970) they described again the comparative karyotypes of eleven species of mosquitoes belonging to four different genera. The species studied by these cytologist included Aedes implicatus, Aedes sierremis,
Aedes veripalpus, Aedes fitchii, Aedes impigens, Aedes sinerens,
Anopheles franciscanus, Anopheles ear lei, Culex apical is and
Psorophora signipennis.
Among 2960 species of mosquitoes (Knight and Stone 1977), karyotypes have been described for less than two hundred species only
Kitzmiller (1976). A common features of these species is that they possess three pairs of chromosomes, with often only minor morphological difference in their over all length and centromeric positions, particularly in Culicines Rai (1966), Kitzmiller (1976).
After the development of banding patterns techniques around
1968, it became a general practise to "band" the chromosome before
analysis, these techniques of differential staining of metaphase
chromosomes are now in use for the study of mosquito metaphase
chromosomes. Since the individual chromosomes of many species are
similar in size and morphology, the differential banding pattern
facilitates the detection of each chromosome, these banding techniques are therefore in common practise in taxonomy. They are of much importance for the correct and exact identification and classification of different species of insects as well as other animals, these patterns are generally consistent for a taxon except for minor variations. These techniques also made the identification of individual chromosome pairs possible, even to the extent of sister chromatid exchange.
The four mosquito species used for the karyotype studies in the present investigation belong to two genera i.e. Aedes and Culex. These four species are as follows:
Aedes togoi (Finlaya)- Aedes togoi occurs in eastern Siberia,
Korea, Japan, China, Hongkong and Taiwan. It has also been reported from the east cofet of Thailand (Gould et al. 1968), West Malaysia
(Ramalingam 1969) and British Columbia.
Culex sinensis (Theobald). This mosquito is not very common in
India, though widely distributed throughout the oriental region. In India, it has been recorded from Rajpur district, Bihar, Keirpur, Katihar,
Pumea district, Orissa, Bengal, Assam Khasi hills district and Dibrugarh etc.
Culex vishnui (Theobald). One of the commonest of Indian mosquitoes, this species is present from north-west frontier to Assam and Burma, and through peninsular India to Ceylon. It is less common at high elevations and in the western himalayas at altitudes of over 5,000' ft. Its range extends to China and Japan, and throughout the oriental region as far south east as new Guinea.
Culex pipiens fatigans (Widemann) This species is also very common in India. Found in all parts of the Indian region, it occurs up to
5,000'ft. or more in the hills. It is common in the tropics and subtropics of both new and old worlds.
REVIEW OF LITERATURE- A HISTORICAL
ACCOUNT
Stevens (1910) who observed for the first time the meiotic chromosomes of Culex pipiens is generally credited with initiating chromosomal studies on mosquitoes and first report of polytene
chromosomes in mosquitoes was made by Bogojawlensky (1934)
from the salivary glands; malpighian tubules; midgut and the
anterior portion of the hind gut of Anopheline larvae. Polytene
chromosomes oi Culex pipiens were reported by Sutton in (1942).
The significance of cytogenetic studies of mosquitoes was
realised only after the timely publication of a review of literature
by Kitzmiller (1953) and a survey of the chromosomal
complements in several species of mosquitoes by Kitzmiller and
Frizzi(1954).
Rozeboom and Kitzmiller (1958) have emphasized the
genetic aspect in their review of hybridization and speciation in
mosquitoes. A synopsis of workable techniques and the
contribution of considerable new findings have been made by Breland and his collaborators Breland (1961, 1963), Breland and
Gassner (1961, 1962), Breland and Riemann, (1961), Long (1961).
Breland (1961) described in detail, the mitotic and meiotic chromosomes of twenty four species of mosquitoes. Among them were, seven species of Culex, seven species of Aedes, two of
Orthopodomyia three of Psorophora and one each of Anopheles,
Haemagogus, Culiseta, Taxorhynchites and Uranotaenia. Rai and
Craig (1961) reported mitotic metaphases from five species of
Aedes and one each from Corethra, Anopheles and Culex. The morphology of mitotic chromosome from brain tissue of fourth instar larvae has been studied by Rai (1963) in twelve species of mosquitoes.
Other review by Davidson and Mason (1963) and Kitzmiller
(1963,1967) are also quite significant. Kitzmiller and Mason
(1967) have dealt in detail with the formal genetics of
Anophelines. "Genetics of insect vectors of disease" edited by
Wright and Pal (1967) is a very important publication and serves as a land mark in the field of insect genetics.
Mukherjee et. al., (1966) presented the karyotypes of nineteen mosquito species belonging to four genera. In another paper (1970) they described the karyotypes of eleven species belonging to four genera. The species studied by these workers included, Aedes implicatus, Aedes pullatus, Aedes si err ens is.
Aedes veripalpus, Aedes sinerens, Aedes fitchii, Aedes impigens.
Anopheles franciscanus, Anopheles ear lei, Culex apical is and psorophora signipennis.
Chowdaiah et. al., (1971) have made a brief review of the cytogenetic studies in oriental mosquitoes. The other important publication include, the "Genetic control of insect pests" by
Davidson (1974); and the "Use of genetics in insect control" edited by pal and Whitten (1974). Another review by Kitzmiller
(1976) has appeared in volume 18 of "Advances in Genetics", which contains a brief but quite accurate synopsis of genetics, cytogenetics and evolution of mosquitoes.
Chowdaiah (1980) made a detailed reviews of the recent advances in the genetics of Culicine mosquitoes. Motara (1982) has reported that the red eye allele (re) on chromosome 1 is the most important in the production of abnormal progeny. He analyzed the sex locus of these mosquitoes and provided experimental data supporting the hypothesis of the sex locus in Culicine mosquitoes being a segment or block of genes on chromosome I.
Hartberg et. al. (1985) have described the mitotic chromosome studies of Aedes mediovittatus. The cytogenetic studies and iQ/s^yme profile of Sabethes cyaneus (Culicine) mosquito were studied by Munstermann et. al., (1986). Pattnaik et. al., (1989) described the mitotic and meiotic chromosomes of
Culex quinquefasciatus.
The chromosomal studies in two Brazilian populations of
Aedes aegypti from Sao Jose do Rio preto and Marilia (Sao Paulo
State) were made by Lima catelani et. al. (1994), they also described the karyotypic studies of Aedes fJuviat His in^995)^ The variation in Y chromosome in Aedes aegypti described by Owusu-
Daakuet. al., (1998).
10 ^ MATERIAL AND METHODS
Source of Material:
The species of mosquitoes used in present investigation are classified in tj*€ two important genera i.e. Culex and Aedes of the family Culicidae.
Most of the mosquitoes used in the present work were reared in our laboratory, however some of them were field collected, gravid females were collected from the field and houses etCj^ were transferred to the insectary and then allowed to deposit eggs in the individual pans.
The hatched larvae of these individual females were also kept in separate pans. The eggs of Aedes togoi were imported from the mosquito cytogenetic laboratory of Dr. Takeo Tedano, Department of
Medical Zoology St. Marianna University Kawasaki, Japan.
Laboratory Rearing and Routine Maintenance:
For rearing, the adult mosquitoes, were kept in wooden cages
(18" X 18" X 18") with wire netting in the insectary at a temperature of
24°C ± rc, and Relative humidity (RH) 80% ±10%. A cotton pad
soaked with 5% sucrose solution in a small petridish was provided together with a beaker filled with water to be renewed alternately, in each cage. For blood feeding, females (about 5 days old) starved for 24 hours were provided with an albino rat wrapped in wire mesh, for 2-3 hours during the evening each week. A plastic cup (6cm diameter and 3
11 cm deepV, containing tap water and lined with a small strip of filter paper, was kept for oviposition in each cage, oviposition occurred on
th th the moist filter paper on 4 and 5 day after blood meal, one or two days after oviposition, water drained out from the oviposition cup without removing the egg paper for Aedes species to allow embryonation and conditioning of the eggs.
For hatching of aedine eggs, the paper was immersed in tap water about 2.5 cm deep in plastic rearing pans (36.5 x 27x9cm) for several days. Hatching was usually completed within two days after immersion. Crushed feed or yeast tablets were given to the larvae.
Scum if formed was removed from the surface of water with a strip of
filter paper on alternate days.
In case of Culex species egg rafts were immediately transferred to the white enamel pans containing tap water for hatching. At 24+l"C
pupation started 9-10 days after hatching, though few larvae took a
much longer time (up to 20 days) to pupate in condition of
overcrowding. Since at this temperature the pupal stages lasted almost
3 days, pupae were picked on alternate days and transferred to the
wooden cages.
Preparation of Slides:
The larval brain and gonadal tissues were used for the
preparation of chromosome slides. For securing the sufficient number
12 of metaphases from the brain cells, the larvae were pretreated with
0.1% colchicine solution for a duration of 2-5 hours. To obtain an
adequate number of metaphase plates 20-30 larvae were dissected at a
time. The chromosomal preparations were made from dissected tissue
of the larvae by using a modified air drying technique standardized in
this laboratory.
In order to assess the results of the preparative techniques,
hypotonic solutions of the following salts were tried on the dissected
tissues. The best preparations were obtained with salt solutions of the
following molarities.
• 0.15M potassium chloride solution in case of early meiotic
stages.
• 0.06M to 0.08M sodium chloride solution in case of
mitotic and later meiotic stages.
• 0.015M sodium citrate solution in case of mitotic
metaphase.
Molarity was determined by the following formulae.
Weight in grams =^o^^^^^^^ ^^- ^ Molarity requiredx Volume required 1000
_ MwXMxV ^~ Tooo
13 Sexing was done at the larval and pupal stages. In female,thoracic region is much wider than in the males, and the female pupae are much bigger in size than the male pupae.
The dissected material in the hypotonic solution was thoroughly minced with the help of a pair of small scissors with a curved tip until the fine suspension was obtained. This suspension was made fine using
a syringe and a broad needle of no. 18. After mincing the cell
suspension was transferred by a pasteur pipette into a 15 mU centrifuge
tub&5, and the cells were sedimented at 800 rpm for 4 minutes. The
supernatant was removed and replaced with hypotonic solution to allow
resuspension for some time followed by after centrifugation. The
hypotonic solution was then removed and replaced with 3-5 ml of
freshly prepared fixative (Methanol and glacial acetic acid in a ratio of 3:1).
The pellet of cells was then dispersed into fixative by gentle agitation
with the help of pasteur pipetteJ^ and the volume was slowly increased
by addition of more fixative. The tube containing material was kept in
the refrigerator for fixation at least for two hours. After this duration,
the cell suspension was further centrifuged and resuspended with tvv'o
changes in the fresh fixadve. After the last centrifugation the
supernatant was discarded and a small volume of fixative was again
added to the residue to obtain a turbid cell suspension. This cell
suspension was dropped on previously chilled slides with the help of
14 pasteur pipette. These glass slides were dipped in chromic acid previously for 3 hours, washed with water and kept under running water overnight. These slides were then immersed in absolute ethyl alcohol and refrigerated for 24 hrs. Two coplin jars each containing distilled water were also refrigerated for sometime^ prior to slide making. The chilled slides from the refrigerated alcohol jar were transferred to one of the coplin jars having distilled water and were vigorously shaken until the surface of the slide appeared smooth. The slides were then transferred to the other coplin jar having distilled water and were again shaken well. A pasteur pipette was used to drop 3 or 4 drop; of cell suspension over the wet slide, it was then shaken vigorously to remove excess of liquid accumulated over the surface and air dried.
Conventional Giemsa Staining:
This procedure was adopted initially to observe the mitotic index in somatic tissues and meiotic stages from gonadal tissues immediately after slide preparation. It can also be used for straining the pachytene chromosomes and rapid 'C banding staining. The slides were immersed in diluted giemsa staining solution. This solution was prepared by adding 1 ml. Of giemsa stock solution and 1 ml. sorenson's phosphate buffer (pH 6.9) to 48ml. of distilled water. The
15 slides were stained for about 4 minutes and rinsed briefly in distilled water before air drying.
Preparation of Giemsa Stock Solution:
The stain was prepared by dissolving 1 gram giemsa powder in
66 ml. of glycerine which was incubated for two hours at 50"C and then 66 ml of methanol was added to the warm solution. It was kept in refrigerator for one week before use.
Phosphate Buffer:
0.422 gm of sodium phosphate diabasic (Molecular weight =
177.99) was dissolved in 250ml of distilled water and 0.390 gm of sodium phosphate monobasic was dissolved in 250 ml of distilled water separately. The O.OIM sorenson's buffer of pH. 7.0 was obtained by adding 10.5ml of monobasic solution to 250 ml of diabasic solution.
This buffer was used to prepare a buffered giemsa solution of pH=7.0
16 ^ OBSERVATIONS AND RESULTS
The metaphase stage is most suitable period for taking the observations of chromosomal preparations. During the metaphase stage chromatids become shorter- and thicker^ and very prominent, the number, size and morphology of chromosomes can be studied under light microscope after appropriate treatment of the cellsy and their appearance at this stage is characteristic for each species.
Colchicine treatment is given to arrest the metaphase stages.
The size of chromosome is dependent on the length of the arms, while its shape is dependent on the position of the centromere which is seen as the constriction. At metaphase,each arm consist of chromatids lying side by side. Depending on the length of the arm and position of the centromere they are named metacentric, submetacentric, acrocentric^ and telocentric. When the two arms are equal in length or almost so, the chromosome is "metacentric", when one arm is only one third to one half as long as the other, the chromosome is "submetacentric", when one arm is only one seventh to one third as long as the other, the chromosome is
"acrocentric", when the centromere is very close to the end the
17 chromosome is "subtelocentric" and when the centromere is at the end, chromosome is "telocentric".
Metaphase chromosomes can be shown or photographed and then arranged in homologous pairs in a systematic manner to form
"Karyotype". Normally the karyotype is constant from cell to cell with an individual and with the exception of sex chromosomes, from individual to individual, within the same species.
Karyotyping is done to study chromosomal changes, characterizing chromosomal aberration that can not be detected by microscopic examination alone. The morphological consideration of size and centromeric position are the critical parameters used in the identification of chromosomes.
The typical diploid chromosome number of 6 has been observed constantly in all the species of mosquitoes studied so far, except in Corethra (2n=8).
The Culicine karyotype in the present study also shows three pairs of chromosomes which are distinguished not only by slight differences in their total arm lengths, but also by means of their characteristic shapes. The sex chromosomal pair is not
18 heteromorphic in any of the species in the present work but appears similar to the autosomes.
In support of the observations of these results the photographs of each metaphase chromosome complement is presented.
KARYOTYPIC DESCRIPTION:
Culex pipiens fatigans (Widemann):
The diploid chromosome complement (2n=6), shows that it consists of 3 pairs of chromosomes, all the three pairs of chromosmes are metacentric as shown in the photograph
(Fig. A & B).
The percent relative length in male chromosomal complement comprises 19.12% for chromosome I, 36.17% for chromosome II and 44.70% for chromosome III aproximately of the total haploid set length (Table. I).
Culex sinensis (Theobald):
The diploid chromosome complements consists of three pairs of metacentric chromosomes which can be distinguished by their lengths. The smallest i.e. chromosome I is much smaller than
19 chromosome II and III in male chromosome complement.
Chromosome II and III show little difference of length as shown in the photograph (Fig. C & D).
The percent relative length in male chromosomal complement comprises 19.37% for chromosome I, 35.64% for chromosome II and 44.96% for chromosome III approximately of the total haploid set length (Table IV).
Culex vishnui (Theobald) The chromosome complement consists of three pairs of metacentric chromosomes as shown in photograph (Fig. E).
The percent relative length in male chromosomal complement comprises about 21.91% for chromosome I, 35.06% for chromosome II and nearly 42.95% for chromosome III of total haploid set length (Table VII).
Aedes togoi (Finlaya) The diploid chromosome number is six
(2n=6), this shows that the chromosome complement consists of 3 pairs of chromosomes, all the three pairs of chromosomes are metacentric, the smallest, that is chromosome I is much smaller than chromosome II and III as shown in photographs (Fig. F & G).
20 The present relative lengths in male chromosomal complement comprises 16,28% for chromosome I, 36.84% for chromosome II, and 46.78% for chromosome III approximately of the total haploid set length (Table IX).
21 FIGURE-A: Mitotic metaphases cliromosomes of brain from fourth instar cT larvae of Culex pipiens fatigans and the corresponding karyotype.
FIGURE-B: Meiotic metaphase chromosomes of gonads from fourth instar 9 larvae of Culex pipiens fatigans and the corresponding karyotype. w^
)X » « III II ^f
III II H-I: Histogram representing the normal karyotype of cT Culex pipiens fatigans. I-Chromosomal data from larval tissue of cT Culex pipiens fatigans (based on 5 replicates of each)
^
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lla lib lllb Chromosome number
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FIGURE-D: Mitotic metaphases chromosomes of brain from fourth instar $ larvae of Culex sinensis and the corresponding karyotype. v> l( tr II III II I ? / III II I H-III: Histogram representing the normal karyotype of cT Culex sinensis. -Chromosomal data from larval tissue of cT Culex sinensis (based on 5 replicates of each) •••• •••> •••• •••» •••• •••> •••• •••> •••• •••• •••# •••• •••# •••• 0) •••• •••• E o •••• •••* u) 4 ••4? •••J •••• •••• o •••• •••• E •••• •••• •••• •••> o •••• ••*• •••• ••<•; •••• •••• o ^ •••J •••• •••» •••5 ••<•• •••• •••I •••• ••<•> C •••• •••• ••*• •••• ••*• c 2 •••J •••• •••• •••• •••• •••I ••<•• •••• •••• •••^ •••• ::: :::t •••• •••* •••I •••• •••• •••J •••• •••• •••I •••• •••J ••4j •••I •••• •••; ••^^ 0 •"'•' la lb lla lib Ilia !llb Chromosome number (1 Unit= 1|.im H-IV: Histogram representing the normal karyotype of V Ciilex sinensis. iV-Chromosomal data from larval tissue of 9 Culex sinensis (based on 5 replicates of each) 0) E o (/) o E o o *-> U) c _aj c re 0} lb Ha lib lllb Chromosome number (1 Unit= 1^;m) 1 o o 'J o ^ c u o o ^ u 1 ^ •J ;^ a. i"^ ^ f* rt L- ij 'J o ^ ^ 2 2 2 y. 'J E "^ in c-, t^ 1 o .E f-, "* '.J ir. 1- -J .. 1 1 (u T- —; o O o ^ O 00 +1 +1 +1 oo n 5 5+1 q^-_ q —; tu ;; cc ^s Qv S c -i- -i- t o O •J 1 i - ^ +1 r o O r-^t 1^ u. r^l 5 -5 y ri r'~, — C r^ o c/j E q o o N^M /~S o o o MB S^ +1 +1 H X r; O p t/5 r-~ -t o+1 r-^t sO Qv r,"* C3 > ~ Zo r- cj w j; o' 'n -t^ 2 - -5 — f^i -r u: < o ^ _3 v-* i ^ U. "1 1 1 ^ 2« ( c^ E- +1 '•n < o _] Z M UJ X. s U < ;; o ', , +1 o z o p o o fl> o r-i o —! 1 1 1 +1 u. c:; '^, o X\ +1 1 «") —' •r ri CJ o r % C3 X .r:_- (U '-< Z ' < ^ I o u o o 'J o c <« o i o iJ o '_) o Ci. ;:i o CJ CJ '_) -_) c3 c: C3 Ki « cz Co ^ "^ o u 1J •D o ^ ^ u E ^ r-- 'b O o b. o +1 o o o o +1 o c +1 +1 K r—V oc ^^ .^M 00 U -f H r^-1 "1 Lu < > X +1 +1 o +1 +1 in c r3 C -; n '^ ~ < < -J ;::: Q^ oo ^ , ') (^1 (' - "i o r; c (/j 1 < .^ "3) o +1 + 1 +1 +1 o o c: E E n 7Z o f' 1 O ^ :/•; -J -E C/J c +1 +1 +1 :: ') c f- (^t oc C3 ^ n o 2 Of r> oo P (yj r-- u. O +1 o -•—t o c o~^^ +1 +1 +1 o 'r-, +1 o r I u o in o •J X ,. i/J u< o L- — 2 • — J u^ < > x. r/ r^'^ i-n < r- =«5 -J < v^ o O ii c +1 +1 +1 ^ +1 oc +1 +1 (<**« _J C3C n ^ c -: u. /v < o PIGURE-E: Mitotic metaphases chromosomes of brain IVom fourth instar cf larvae of Ciilex vishniii and the corresponding karyotype. irj^\ ,t f r)) )j III II H-V: Histogram representing the normal karyotype of cT Culex vishnui. V-Chromosomal data from larval tissue of cT Culex vishnui (based on 5 replicates of each) UJ oS (0 o s o a£ X o u. O X IlU- 2 < UJ lb lla Mb CHROMOSOME NUMBER (1 Unit=1^m) q. O > 3" DO p^ r^ __ o r "1 „_ o 2. ^ K) ^.^ > o -1 3- r* fj so ° O < P > 00 j/5 3 3- m 3 1+ 1+ 1+ o o o • o 3 3 ^ ?0 o 3 TO o o ivb i b^ CO o C- 2 m VO U) -i. 3^ 3 c« M 2: CO H ^ Z ^.^ ^^ o 2. -, K) =5" — W 2 o <-~^ ;-J o 3 O '^ n L/1 O 3 <• S O C/3 3 TO ;^ 3 ?0 1+ 1+ O 1+ o o mg 1 g „ 2: n o 3 ^ TO O in o Ub) »b^ Ln M^ ^ « S,3^§ ?0 z 1+ O ^ W H r 7S m -n n r *- U) NJ 3" &J 2 H s > 3 :^. fD H b VO § < » i^ o^ ^^ oo 3 n 3 < 1+ 1+ 1+ W 5" fr"n o PI o o O • 2 3 4 3 TO O r b-f^ b UbJ M ^— "3-3 1+ 0 ^ z n o a H 0 a: -p^ ui ^ vO S 2 o so 0 ^ o U) b ^ Tl o ^ 1+ 3 § 1+ H 1+ 1+ m -. fB o o o r 0 -1 a 0 w ^b— ub< -b^ U) t-f< so a. S n X X o O 0 00 2 2 2 3" 3- o ft) n CD r-^ r-*- ^-f- ^ S &3 &3 0 jz O O o 3 ni O M 'sO VO 4^ •^ 3 ^2 O O 3 3 3 = 1+ 1+ O 1+ 1+ 1+ O 1+ o o 1+ o "a 3- o o o b b 00 O =^ O o b so o b b m 3 ° ^ OS so 00 _. -» 3 3 B !!0 " 2 O =^ ^ '^ 4^ 2 oo 00 1+ 1+ -p 3 ^2 O 1+ 1+ 1+ 1+ o o o O 3 3 3 = O O b b 00 O 3- O O b b b m 3 o :i tn 2 b so so O > 00 3 3 O r tyzi >o H S-2 P=1 > £ o p •fl bo bo bo bo b b 3 3 = •n 00 vo K) o o r ?o 1+ 1+ 1+ 1+ 1+ 1+ 1+ ° «- o o o O o o o b b b b > FIGURE-G: Mitotic metaphases chromosomes of brain from fourth instar 9 larvae of Aedes togoi and the corresponding karyotype. t#^/ d) (I M III II I Af IJ It It III II H-VI: Histogram representing the normal karyotype of cf Aedes togoi. VI- Chromosomal data from larval tissue of cT Aedes togoi (based on 5 replicates of each) ^*i 6 ^•4 ¥^m ^•4 S ^^m ^•4 ^^M ••4 o ^•^^ 0) E o w o E p o ^ I I lb ila Mb Ilia lilb Chromosome number (1 Unit=lMm) 0) 0) o o o :S S 'LM C 'C •*-» ••-J •4-J C c c o o « u « o o •*-ca» •*-• •4-C<»J ^1 u u u 00 o :2 2 2 O tii X (U o — Q 6 >< 2 _ |— 1^ I x: ••^ O < 'b o o o o <^ E o o •*-» •4-J -4—> •f-t •*—» c c c C If, "5 u u u c o o o o ca E « « o J= O 00 ^ ••-» u -1—> S3 o 2 2 <^ la 2 o 00 o E ^ 00 m ON O q q q q o +1 o d O o d d d d +1 +1 +1 +1 +1 +1 tii « « o o r<-) oo 00 q q a 00 00 00 d d u in in H > o OS in o H o q q q q q d d d d d d J o +1 +1 +1 +1 +1 +1 +1 Q z E .S o o ON ON m d Z ca ^ ca ^ « X> U o Of b. W O O NO >n in H :S ^ ui o O o q d d O q d U J o +1 +1 d +1 +1 d +1 o +1 o o +1 c E E- m >n in (N ft* CO O a O rn ON < Q O r^ m o o m o q d d O d O d o +1 +1 d +1 +1 +1 +1 NO NO NO O c S I E vq NO NO d CN (N o aQi: u cd (U O o (M m in o E O o o o q q J= (/j d o -4-* o d d d d d +1 +1 U1 bu +1 +1 +1 +1 +1 c o m NO NO c u E E NO (N ^ o2 =s- fN in in u L. d d o Z DISCUSSION The diploid number of all mosquito species examined is six with a single exception in Corethra 2n=8. However even though the basic number is uniform, conventional studies have revealed considerable variations among different species pertaining to chromosome size, centromeric position and chromosome polymorphism White, (1949), Kitzmiller, (1976), Mukherjee et. al., (1968), Tadei et. al (1984), Kaiser et. al; (1988). Although Rai (1963) indicated that karyotypes of different genera and occasionally of different species of mosquitoes may be distinctive, on the whole mitotic karyotypes, particularly of Culicines, are remarkably uniform, this fact indicates that gross changes in whole chromosomes or chromosome complements of Culicines may not have played any important role in speciation. Furthermore, it is generally believed that in most Culicines because of this uniformity, the somatic karyotypes are not especially promising for gleaming information about the evolution of these karyotypes/Kitzmilleri 3^1963). In contrast Anophelines possess numerous different karyotypes. Studies of their polytene chromosomes have revealed 22 a great deal of chromosome polymorphism, particularly in the presence of inversion Mathiopoulos et al., (1995). This is true not only for different species but also for different populations in the same species( Torre et. al.,|(1997). Holstein (1957) has shown that different strains of Anopheles gambiae Giles from different geographical areas show a high chromosomal variability, the same is true for Anopheles punctipennis and Anopheles freeborni Aitken populations in United Jtate? and for other Anophelines Kitzmiller (1963). Unfortunately, the polytene chromosomes of Culicine mosquitoes are unsuitable for detailed mapping purposes. It may be possible to bypass these difficulties and gain insights concerning the evolution of karyotypes of Culicines by undertaking a cytogenetic examination of the meiotic and somatic chromosomes of inter and intraspecific hybrids between different populations. In Culicines, the three pairs of chromosome complement, consist of two large and one short pair of chromosomes are individually recognizable and can be designated in ascending order. The shortest chromosomes designated as chromosome I is sex determining and the largest has chromosome III. The second and third chromosomes are larger than the first chromosome. The ratio of chromosome I to II and III is different 23 in different species and appear to be a generic characteristics, Rai (1963). Kitzmiller (1953), Mukherjee et al (1966) illustrated the karyotypes of Culex and Aedes with same figure. However the ratio of I to II and III is lower in Culex than in Aedes. The sex chromosome pair in Culicines can not be identified. Moreover, unlike Anophelines the sex chromosome pair is homomorphic and appear similar to autosomes Mukherjee et. al., (1966). The present study deals with few mosquito species, three of them belong to genus Culex and one of them to genus Aedes. In the genus Culex, Culex pipiens fatigans, Culex sinensis and Culex vishnui have three pairs of chromosomes, all are metacentric, with two large and one short pair of chromosome. These species shows more similarities and no remarkable differences in their karyotypes, the only difference being in the chromosome complement of Culex sinensis where the chromosome III shows a high ratio between the short and long arm inclined towards the submetacentric form than metacentric ones. In an Aedine species i.e. Aedes togoi, which also possess three pair of metacentric chromosomes with two larger and one shorter chromosome pair, the two chromosomes pairs II and III 24 show only minor differences in their lengths, while chromosome I is much smaller than chromosome II and III. The sex chromosome pair is homomorphic in all the species described above and appears similar to autosomes and there is no discrimination of sex chromosome pair in Aedine and Culicine species. Furthermore the size of three chromosome pairs shows only minor differences from species to species and consequently it is difficult to distinguish one species from another based on traditional chromosome studies. In view of remarkable uniformity of the karyotypes in Culicines mosquitoes, with those of Aedines and Anophelines, it is tempting to suggest that mechanism of speciation may be different in different genera of mosquitoes, Anophelines may have undergone much more chromosomal repatterning than Culicines, while non-Anopheline genera may have depended more on point or genie mutations, during the course of evolution. 25 CONCLUSION 1. The diploid chromosome number of all mosquito species examined is %and each complement consist of three pairs of chromosomes two relatively large and one slightly short chromosome pair. 2. The sex chromosome pair (chromosome I) is homomorphic in Culicines i.e. Culicine. and Aedine species 3. The chromosomes have been designated as chromosome I, II, III in the ascending order of their relative lengths. 4. The mechanism of speciation may be different in different genera of mosquitoes. Culicines may have depended more on point or genie mutations for their evolutionary diversity during the evolutionary course. 26 SUMMARY In the present work karyotypes of four species of mosquitoes i.e. Culex pipiens fatigans, Culex sinensis, Culex vishnui and Aedes togoi have been analysed some of these were field collected, gravid females were usually collected from the field and houses© and eggs of Aedes togoi were imported from Japan. Sexing was done at the larval and pupal stages. In female thoracic region is much wider than in the males, and the female pupae are bigger in size than the male pupae. The larval tissues (brain and gonadal tissue) were used in the preparation of chromosomal slides. A modified air-drying technique standardized in this laboratory was adopted in the preparation of mosquito chromosomes this include a combination of hypotonic prefixation treatment and air drying, as wells methods used by Hungerford (1965,1971). The normal karyotype of the four species of mosquitoes is studied and their percent relative length and centromeric indices were calculated. All the four species show chromosome complements consists of 3 pairs of chromosome each. 27 Depending upon the length of the arms and the position of centromere, the chromosomes are named as "metacentric" "submetacentric" and "acrocentric" and "telocentric". Typically the Culicine karyotype shows three pairs of chromosome, a pair of shorter sex chromosome (homomorphic) and two pairs of longer autosomes, that can be distinguished by slight differences in their total arm lengths. In the species Culex pipiens fatigans, Culex sinensis, Culex vishnui and Aedes togoi. all chromosomes are metacentric. 28 REFERENCES Akstein, E. 1962. The chromosomes of Aedes aegypti and of some other species of mosquitoes, Bull. Res. Counc. Israel 9:146-155. Amirkhanian, J.D. 1968 A combined HCL- acetic alcohol fixation and hydrolysis followed by cresyl violet staining for mosquito chromosome spreads, Stain Technology 43: 167-170. Bogojawlensky, K.S. 1934. Studien uber zellergrosse and zellen wachtum XI. Zeit. Zeit. F. Zellforsch 22: 47-53. Breland, O.P. 1959 Preliminary observations on the use of the squash techniques for the study of the chromosomes of mosquitoes, Texas Journal of Science 11: 183. Breland, O.P. 1960a, Restoration of the name Aedes hendersoni Cockerell and its elevation to full specific rank (Diptera: Culicidae), Ann. Ent. Soc. Amer. 53. 600. Breland, O.P. 1960b. Studies on the chromosomes of mosi\\ji\ioQS, Anat. Rec, 137, 343. Breland, O.P. 1961, Studies on the chromosomes of mosquitoes, Ann. Ent. Soc. Amer. 54:360-375. Breland, O.P. 1963. Some events in the meiosis of mosquito Culiseta inornata (Williston), Bull. Ent. Soc. Amer., 9: 173. 29 Breland, O.P. and Gassner; III G., 1961 Notes on the chromosome complement of the mosquito Aedes aegypti (L.), Texas Journal of Science 13: 391. Breland, O.P. and Gassner, III G. 1962. Preliminary studies on the chromosomes in the mosquito Dienoceritus Mathewsoni, Balkin & Hogue. Amer. Zool. 2:508. Breland, O.P. and Riemann J.G. 1961. Preliminary studies of spermatogenesis in the mosquito Culiseta inornata, Amer. Zool. 1:345. Chowdaiah, B.N. 1980 Recent advances in the genetics of Culicine mosquitoes, XVI. International Congr. Entom, Kyoto. Japan Abs. St. 1.3: 465. Chowdaiah, B.N; Rajaselcharan, P.T. and Shetty, N.J. 1971 Cytogenetic studies in oriental mosquitoes, J. Sci. Ind. Res. 30(4): 187-189. Davidson, G. 1974 Genetic control of insect pests, Academic Press London. Davidson, G. and Mason, G.F. 1963. Genetics of mosquitoes, Ann. Rev. Ent. ^-All. French, W.L.; Baker, R.H. and Kitzmiller, J.B. 1962. Preparations of mosquito chromosomes. Mosquito News 22: 377-383. Gould, D.J.; Yuill, T.M.; Moussa, M.A; Simasathien, P. and Rutledge, L.C, 1968. An insular out break of dengue 30 haemorrhagic fever III. Identification of vectors and observations on vector ecology, Amer. J. Trop. Med. Hyg. 17: 609-618. Grace, T.D.C. 1966 Establishment of a line of mosquito Aedes aegypti cells grown in vitro, Nature 211(5047): 366-367. Hartberg, W.K., R. Gulp, M. Kerr, J. Morghan and C. Odom. 1985. Mitotic chromosomes of the mosquito Aedes mediovittatus, Mosqsyst 17(1): 44-48. Holstein, M. 1957. Gytogenetics of Anopheles gambiae. Bull. W. H.O. 16:456. Hungerford, D.A. 1965. Leucocytes from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypo tonic KGL, Stain Technology 40: 233-237. Hungerford, D.A. 1971. Ghromosome structure and function in man I pachytene mapping in the male, improved methods and general discussion of initial results, Cytogenetics 10(1): 23-32. Kaiser, P.E. Seawright, J.A. and Birky, B. K. 1988. Ghromosome polymorphism in natural population of Anopheles quadrimaculatus say species A & B, Genome 30(2): 138-146. Kitzmiller, J. B. 1953. Mosquito genetics and cytogenetics. Revista Brasileira De Malariologia E Doences, Tropicalis 5(4): 285-360. Kitzmiller, J.B. 1963 Mosquito cytogenetics, Bull. W.H.O. 29: 345. 31 Kitzmiller, J.B. 1967. Mosquitoes cytogenetics in genetics of insect vector of disease. (Eds. Wright, J.W. & Pal, R), Els. Pub. Co. Amsterdam. Kitzmiller, J.B. 1976. Genetics, cytogenetics and evolution of mosqmtOQS, Advances in Genetics 18: 316-383. Kitzmiller, J.B. and Frizzi G. 1954. A survey of the chromosomal complements in several species of mosquitoes (Diptera Culicidae), In Attl. Del. IX Congress Internazionale Di Genetica, Fluorece 1953, 2: Caryologia Suppl. 611. Kitzmiller, J.B. and Mason, G.F. 1967 Formal genetics of anophelines. In genetics of insect vectors of disease, (Eds. Wright, J.W, and Pal R.), Else. Pub. Co. Amsterdam. Knight, K.L. and Stone, A. 1977 A. catalogue of the mosquitoes of the world (Diptera Culicidae), 2'"' edit. Entomological Society of America. Lima catelani, A.R. De. A. and H.E.M. De campos Bicudo. 1994. Chromosome studies in two Brazilian populations of Aedes aegypti, Cytobios 79(319): 241-251. Lima- catelani. Alba Regina De Abreu and Hermione Elly Melara De campos Bicudo* 1995. Cytogentics of Aedes fluviatilis, Cytobios ^\{^16): 189-194. Long, J.D. 1961 Chromosome studies with first instar mosquito larvae. Part I technique, Proc. Calif. Mosq. Control. Ass. 29: 24. 'KO Mathiopoulos, Kostas, D. and Gregory C. and Lanzaro 1995. Distribution of genetic diversity in relation to chromosomal inversion in the malarial mosquito Anopheles gambiae, J. mole. Evol. 40(6): 578-584. Motara, M.A. 1982. Fine structure of the mosquito sex locus. Nucleus 2S{y)'. 113-116. Mukherjee, A.B. and De Giorgio 1981. Insect chromosome isolation; A method for metaphase chromosome harvest from cultural cells of the mosquito Aedes albopictus, Canadian Journal of Genetics and Cytology 23: 453-457. Mukherjee, A.B. Rees D.M. and Mukherjee, A.B. 1970 A. comparative study of the mosquito karyotypes, Cytologia 35: 57-62. Mukherjee, A.B.; Rees, D.M. Vickery, R. K. 1966 A comparative study of the karyotypes of four genera and nineteen species of mosquitoes present in Utah (Diptera: QxiXi^iCdiQ,), Mosquito News. 26: 150-155. Munstermann, Leonard E*. and Annalisa Marchi. 1986. Cytogenetic and isozyme profile of sabethes cyaneus, J. Hered 77 (4): 241-248. Owusu- Daaku, K. O; Wood, R.J. and Butter, R.D. J^97^ Variations in Y chromosome meiotic drive in Aedes aegypti (Diptera: Culicidae). A potential, Genetics approach to mosquito CQnX,Bull. Ent. Res. 87(6): 617-623. 33 Pal, R and Whitten, M. J. 1974. The use of genetics in insect control, Els. I North Holland. Pattnaik, Subasini, R.K. Verma, P.M, Achary, R. Prashad and C.C. Das. 1988. Mitotic and meiotic chromosomes and C- banding pattern in Culex quinquefasciatus, Proc Acad Sci. India Sect B. (Biolsci) 58(1): 1-6. Rai, K.S. 1963. A comparative study of mosquito karyotypes, Ann. Ent. Soc. America 46: 160-170. Rai, K.S. 1966 Further observations on the somatic chromosome cytology of some mosquitoes (Diptera: Culicidae), Ann. Entomol. Soc. America 59(2): 242-246. Rai, K.S. and Craig, G.B. 1961. A study of the karyotypes of some mosquitoes. Genetics 46: 891. Ramelingam, S. 1969 New record for Aedes (Finlaya) togoi (Theobald), in West Malaya, Med. J. Malaya 23: 288. Rozeboom, L.E. and Kitzmiller, J. B. 1958 Hybridization and speciation, Ann. Rev. of Entomology 3: 231. Schneider, I. 1969 Establishment of three diploid cell lines of Anopheles stephensi (Diptera: Culicidae), Journal of Cell Biology 42: 603-606. Singh, K.R.P. 1967 Cell cultures derived from larvae of Aedes aegypti (S.) Aedes aegypti (L.), Current Science 36: 506-508. Stevens, N. M. 1910. The chromosomes in the germ cells of Culex, The Journal of experimental, Zoology 8: 207-225. 34 Sutton, E. 1942 Salivary gland type chromosomes in mosquitoes, Proc. National Acad, of Sciences (Wash). 28: 268-272. Tadei, W.P., Joselita, M.M. Dos, S. and Rabbani G.M. 1984. Anophelines 5. Chromosomal polymorphism of Anopheles darlingi (Diptera: Culicidae), Acta Amazonica 12(2): 353-370. Torre, A; Delia, L. Merzagora, J. Powell, R. and Coluzzi, M. 1997. Selective introgression of Paracentric inversions between two sibling species of the Anopheles gambiae complex, Genetics 146(1): 239-244. White, M.J.D. 1949. Cytological evidence on the phylogeny and the classification of the Diptera, Evolution 3: 252- 261. Wright, J.W and Pal, R. (Editors) 1967 Genetics of Insect Vector of Disease, Elsevier Publishing Co. Amsterdam. -\ 7 ;. 03-3^^^ 35