Nuclear DNA Content in Aedes Mosquitoes

Heredity 59 (1987) 253—258 The Genetical Society of Great Britain Received 16 November 1986

Inter and intraspecific variation in nuclear DNA content in Aedes mosquitoes

P. Nagesh Rao and Department of Biological Sciences, University of K. S. Rai Notre Dame, Notre Dame, IN 46556, U.S.A. Haploid nuclear DNA of 23 species of Aedes, as determined by Feulgen cytophotometry, was found to vary 3-fold. This was accompanied by a 2-fold variation in total chromosomal length. There was a significant correlation (r = 9765,P< 0.001) between these two parameters. Genome size varied from 0-87 pg to 1-3 pg among 10 strains of Aedes albopictus, from wide geographic regions. Large scale differences in chromosomal DNA amounts have accompanied speclation and evolution in aedine mosquitoes.

INTRODUCTION sympatric distribution over a wide range stretching from Madagascar in the west through the Indo- Amountsof nuclear DNA vary greatly among and Malayan and the Oriental regions, China, Japan, within taxa (Bachman et al., 1972; Sparrow et a!., the Pacific islands and extending as far east as 1972; Rees and Jones, 1972; Hinegardner, 1976; Hawaii, and is recently reported to be in southern Sherwood and Patton, 1982). For example, the United States (Knight and Stone, 1977; Rai, 1987). genome sizes of several Drosophila species show Both these subgroups have been the object of a 25-fold variation in C-values (Laird, 1973). extensive genetic studies with particular emphasis Similarly Rees et a!. (1978) observed a 3-fold vari- on the genetics of speciation (Rai, 1983; Rai, 1987). ation in nuclear DNA content among species of A striking cytological feature of Aedes is the acridid grasshoppers with uniform karyotype. Bier constancy in the chromosome number (2n =6) and and Muller (1969) measured the genome sizes of the lack of pronounced variation in chromosome a variety of insects and found that primitive groups morphology among various species (Rai et aL, have larger genomes than more recently evolved 1982; Rao, 1985). Most species so far studied are insect groups. In mosquitoes, very little work has characterised by a small pair of metacentric been done in this field. Jost and Mameli (1972) chromosome and two pairs of larger metacentric and Spradling et al. (1974) have provided some or submetacentric chromosomes. However, careful data on relative and absolute amounts of DNA in measurements of chromosomal arms, giemsa C- two species of Aedes and six species of three other banding studies, meiotic analyses of species genera. hybrids and linkage map comparisons have shown The genus Aedes (Culicidae) is subdivided into the existence of individual differences among the 38 subgenera (Knight and Stone, 1977) and karyotypes of various species (Rai, 1980; Dev and includes more than 1000 species (White, 1980). Rai, 1984; Sherron and Rai, 1984; Rao and Rai, One of the largest subgenera, Stegomyia, contains 1987). The uniformity in the chromosome number about 110 described species divided into seven and yet the worldwide distribution and habitat groups including the scutellaris group which is diversity of various species, make Aedes an inter- subdivided into the scutellaris and albopictus sub- esting genus to study the extent of changes in groups. Ae. scutellaris subgroup is widely dis- nuclear DNA amount and its possible evolutionary tributed in Southeast Asia and the South Pacific role. (Marks, 1954; Huang and Hitchcock, 1980), and Feulgen cytophotometry is an important inves- has a predominantly allopatric distribution; on the tigative technique to determine quantitative other hand the albopictus subgroup has largely genomic changes across and within species. This 254 P. N. RAO AND K. S. RAI paper presents data on nuclear DNA in (a) 23 cent relative humidity (Craig and Vande Hey, species of Aedes, belonging to five subgenera with 1963). different distribution and habitat preferences, with Testes from 12-24 h old male pupae, were dis- particular emphasis on the Aedes scutellaris group, sected in insect saline, fixed in formalin—acetic and (b) 10 geographic strains of the widespread acid-ethanol (6:1:14) (Sharma and Sharma, species Ae. albopictus. Other objectives of this 1980), and squashed in 45 per cent acetic acid. study were to correlate total chromosomal length Each slide also contained on one side a fine smear to nuclear DNA amounts in order to ascertain the of chicken red blood cells (Dhillon et aL, 1977). evolution of chromosomal DNA between and For Feulgen staining, the tissue was hydrolysed within species and to provide information on in 5 N HC1 for 30 mm at room temperature and which to base future analyses of genome organi- washed throughly in cold running water. Staining sation. was done in SchifT's reagent for I h and excess stain was washed oil with three rinses in aq. 10 per cent potassium metabisuiphite solution MATERIALS AND METHODS (McLeish and Sunderland, 1961). Relative feulgen units were calculated accord- Table I provides the names of the 23 species with ing to Patau (1952), using the two-wavelength the sites and dates of their original collection. Field method (505 m and 555 nm, determined through collected species were raised at 21°C in the labora- absorbance versus wavelength curve) (Berlyn and tory. The rest of the species were reared in the Mikshe, 1976), with a Zeiss Microscope Photo- insectary maintained at 25 2°C and 80 10 per meter 01. Thirty to fifty primary spermatocytes at

Table I Taxonomy and source of species and strains of Aedes examined

Subgenus Species Strains Source, site, year collected

Stegomyia aegypli Rock Rockfeller Inst., 1959 heischii Kenya, 1972 metallicus Kenya, 1972 albopictus Mauritius Mauritius, 1972 Tokyo Tokyo, 1979 Hongkong Hongkong, 1978 Hawaii Oahu, 1971 Tana Madagascar, 1978 Pontaniak Indonesia, 1978 Pune India, 1984 Delhi India, 1984 Kollar India, 1984 Calcutta India, 1973 flavopictus Japan, 1981 pseudalbopictus Taiwan, 1982 seatoi Thailand, 1972 unilineatus Senegal, 1983 katherinensis Austrialia, 1982 alcasidi Taiwan, 1972 malayensis Thailand, 1968 hehrideu,s Vanvatu Is., 1982 polynesiensis Samoa Is., 1979 pseudoscutellaris Fiji, 1980 cooki Tonga Is., 1973 Ochierotatus excrucians Mich., USA., 1984* stimulons md., USA., 1984° communis Mich., USA., 1984* canadensis Mich., USA., 1984* Aedes cinereus Mich., USA., 1984° Howardina bahamensis Bahamas, 1970 Protomacleaya triseriatus Ind., USA., 1969* zoosophus Texas, 1981 ' Fieldcollected NUCLEAR DNA CONTENT IN MOSQUITOES 255 metaphase I, were scored from a total of three to Total chromosomal length measurements at five pupae. In most cases anaphase I cells were somatic metaphases were obtained according to also measured from the same slide to confirm the the procedure described by Rao and Rai (1987). values obtained for the metaphases. The chick erythrocytes stained simultaneously not only ser- ved as an internal standard for the day to day RESULTS variations but the values were also used to convert relative mosquito DNA values to absolute amounts HaploidDNA amounts (1C) of the 23 Aedes in picograms (pg), using 25 pg per nucleus as the species, the total chromosomal length of 18 species, value for the diploid chicken genome (Mirsky and and the results of the Duncan's Test are shown in Ris, 1951; Leslie, 1955; Rasch et a!., 1978). All table 2. The ANOVA was significant at the P < DNAamountsreported are 1C values or values of 0.001 level. the unreplicated haploid complement. In the aegypti group, the rock strain of Ae. A One-Way ANOVA with absolute DNA aegypti possessed 0812±0031 pg of haploid values as the response variable and the different DNA. Among the seven species examined in the species and strains as the treatment was performed. scutellaris subgroup, Ae. katherinensis with 1277± Duncan's Multiple Range Test was used to com- O02 pg had the highest IC nuclear DNA per cell. pare mean DNA values of some species and to It was approximately double the amount found in establish different groupings. Ae. pseudoscutellaris and Ae. cooki which

Table 2 Absolute IC nuclear DNA amounts and total chromosomal length

Duncan's Species/strains DNA (pg) SE.' groups" TCL (u)' aegypti 0812 0-03 1990d heischii 1121 003 21-62 metallicus I093 0-03 19-32 albopictus Mauritius 1-321 0-03 A 26-91 Tokyo 1286 003 A Hong Kong 1-259 003 A Oahu 1239 0-03 A B 21-23 lana 1148 002 B C 31-10 Pontaniak 1068 004 C Pune 1066 006 C 31-95 Delhi 1-021 001 CD 32.85 Kollar 0944 0-03 D 2407 Calcutta 0865 0.03 D 2399 flavopictus 1-330 0-02 3334 pseudalbopictus 1290 002 30-09 unilineatus 1064 004 2550 seatoi 0971 0-02 2711 katherinensis 1277 0-02 A 2951 alcasidi 0-974 0-02 B 21-44 malayensis 0943 0-03 B 1912 hebrideus 0-965 0-03 B 19-33 polynesiensis 0-725 0-02 C 2088 cooki 0-594 0-03 D pseudocutellaris 0591 001 D 1624 excrucians 1500 0-03 stimulans 1-439 0-04 2980d communis 1-013 005 canadensis 0-904 002 cinereus 1210 003 2180' bahamensis 1-375 0-03 triseriatus 1-520 0-06 35-88 zoosophus 1902 0-06 38-29 aSEstandard error;" P<000l; CICL=total chromsome length in microns; dRai(1963); 'Mukherjee eta!. (1970). 256 P. N. RAO AND K. S. RAI possessed the smallest genome of all Aedes variation was accompanied by a two-fold range in examined. Ae. alcasidi, Ae. malayensis and Ae. the total chromosomal lengths at somatic meta- hebrideus were grouped together and did not differ phases. This increase in chromosomal size was significantly in their DNA amounts. The 10 strains often observed in all the three pairs of chromo- of Ac. albopictus ranging from Hawaii to Mauritius soines, rather than one particular pair (Rai, 1963; had IC DNA amounts ranging from 1321± Dev and Rai, 1984; Sherron and Rai, 1984; Rao 0035 pg to 0865±003 pg. Duncan's Test estab- and Rai, 1987). There was a good correlation lished six groupings based on genome sizes with between the DNA amounts and the total chromosome overlapping. Both Ae. triseriatus and Ae. somal lengths (fig. 1). zoosophys of the subgenus Protomacleaya, with Based on biogeographical and morphological I 52 0•062 pg and I •902 0062 pg respectively considerations, Belkin (1962) had originally sug- have the highest DNA values of all aedine species gested that speciation in the Ae. scutellaris sub- studied here. group proceeded from east to west with the Linear regression analysis of the DNA amounts southeast Asian species being of more recent origin to the total chromosomal length of 18 species and Polynesian species more primitive. This has showed a significant correlation (r=0765; P< been confirmed by other lines of research (Rai, 0.001) (fig. 1). 1983). Our results show that island dwelling species in Polynesia possessed low DNA amounts whereas

40— the continental southeast Asian species had higher DNA values. This suggests that there has been a

38 gradual amplification of chromosomal DNA in the evolution of species in this subgroup. The mean nuclear DNA amount in the albopictus subgroup was higher than the scutellaris sub-

34 group. Ae. flavopictus and Ae. pseudalbopictus had larger genomes and showed good pairing of chromosomes at metaphase 1 in their hybrids. In 32 contrast, crosses between Ae. sea toi, with a rela- .3- . 30. tively smaller genome and Ae. flavopictus showed O chromosomal size heteromorphism in two of the C C — 2v three pairs (Rai and Herman, 1985). The same was C - E observed between Ae. seatoi and Ae. unilineatus. 0 The actual patterns of quantitative change within the Ae. albopictus strains was interesting. The Indo-Malayan region, where Ae. albopictus is widely distributed, is presumably the center of its I :i origin. Strains from this region possess relatively lower DNA amounts. The expansion of the species to the various island regions is apparently associ- 20- ated with increase in nuclear DNA amounts. Similar correlation exists between nuclear genome iv sizes and historical migration of the species in the Ac. scutellaris subgroup. The nearly two-fold vari- v ation in DNA amount observed in strains of Ac. Haploid DNA values (pg} albopictus demonstrates the presence of extensive populational difference in cellular DNA. The role Figure 1Linearregressionof haploid DNA amounts to the it may play in its divergence is not well understood. total chromosomal length in 18 species of Aedes. Black and Rai (1987) showed that increase in DNA amounts in different strains was primarily due to DISCUSSION higher amounts of highly repeated elements, although they found that in general all three classes Athree-fold variation in haploid DNA genome of repeated DNA increased with genome sizes. sizes was found among the 23 species of Aedes. In Numerous studies have identified similar intras- general, there was no correlation between DNA pecific variation in genome size in other taxa (Sher- amounts and their systematic affinities. Also, this wood and Patton, 1982; Greenlee et a!., 1984). It NUCLEAR DNA CONTENT IN MOSQUITOES 257 is possible that these differences arise within significant correlation between the haploid DNA species and subsequently lead to or enhance diver- amounts and the developmental time (Ferrari and gence and speciation (Robertson, 1981). It is also Rai, unpublished). well known that intrachromosomal addition of Based on DNA amounts it was suggested that DNA within and between species have little effect organisms (Chironomus, 02 pg/ haploid genome on phenotypic or genotypic characters (Hut- [Wells et a!., 1976]; Apis mellifera, O35 pg/haploid chinson et a!., 1979, Sherwood and Patton, 1982). genome [Cramet a!., 1976b]), with low DNA The four species in the Ochieratus subgenus, amounts (less than O4 pg) would show the had larger genome sizes and exhibited a 66 per Drosophila-type of longterm interspersion of cent difference in DNA content. The total chromo- repeats while those with larger genomes (Musca, somal length of Ae. stimulans was the largest O•89 pg/haploid genome [Cram et a!., 1976b], will among the species reported by Rai (1963). Ae. exhibit quite typical short-term repeat pattern the caspius, a related species of the same subgenus, Xenopus-type. With Aedes showing such extensive found in saline waters, was reported to have an variation in DNA amounts it would be interesting haploid amount of 099 pg (Jost and Mameli, to study the pattern of repeated elements in this 1972). Ae. triseriatus and Ae. zoosophus are some- large genus. Black and Rai (1987) found that what specialised in their habitat preferences in that genomic organisation in Ae. albopictus and Ae. both are tree-hole mosquitoes. triseriatus was of the short interspersed type. There are numerous reports of large scale In conclusion, large scale differences in differences in the nuclear DNA content in related chromosomal DNA amounts have accompanied species (Hinegardner, 1976; Bennett and Smith, speciation and evolution in Aedes besides struc- 1976). Britten and Davidson (1971), suggested that tural changes brought about by chromosomal rear- DNA increases are important in the origin of rangements (Munstermann, 1981; Rai et aL, 1982). repetitive DNA which in turn may play a role in evolutionary diversification. In Dermestes, Fox Acknowledgment We thank Drs W. Black and J. Ferrari for (1969) reported significant differences in nuclear their help in numerous ways. This research received support DNA despite very similar karyotypes. The vari- from NIH grant 5R01 Al 21443 to K. S. Rai. ation in the total DNA content was mostly accoun- ted for by variation in the fast fraction, that is made up largely of highly repetitive DNA (Rees REFERENCES et a!., 1976). 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