Heredrtas 113: 277-2x9 (19901
Chromosomal localization and copy number of 18s + 28s ribosomal RNA genes in evolutionarily diverse mosquitoes (Diptera, Culicidae) A. KUMAR and KARAMJIT S. RAI Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556,U.S.A.
KUMAR,A. and RAI, K. S. 1990. Chromosomal localization and copy number of 18s + 28s ribosomal RNA genes in evolutionarily diverse mosquitoes (Diptera, Culicidae). - Hereditus 113: 277-289. Lund, Sweden. ISSN 0018-0661. Received June 25, 1990. Accepted October 17, 1990 In situ hybridization using 3H-labeled 18s and 28s ribosomal DNA @DNA) probes from Aedes albopictus was performed on the mitotic chromosomes of 20 species of mosquitoes belonging to 8 genera of subfamilies Culicinae and Anophelinae. In all but one species examined, the rDNA family was localized to a single chromosome per haploid genome. Aedes triseriatus was the only exception, with the rDNA cistrons present on chromosome 1 and on chromosome 3. The ribosomal RNA genes were located on chromosome I in Ae. albopictus, Ae. aegypti, Ae. fzavopictus, Ae. seatoi, A. polynesiensis, Ae. alcasidi, Ae. annandalei. Ae. mascarensis, Ae. hendersoni, Ae. atropalpus, Ae. epactius, Culex pipiens quinquefasciatus. Wyeomyia smithii, and Sabethes cyaneus; chromosome 2 in Ae. mediovittatus and Haemugogus equinus; chromosome 3 in Armigeres subalbatus and Tripteroides bambusa; and the heteromorphic X and Y chromosomes in Anopheles quadrimaculatus. The variation in the location of ribosomal RNA genes on the different chromosomes and at different positions on the chromosome arm among the mosquito species examined is suggestive of considerable chromosome repatterning through translocations and inversions in the karyotypic evolution of mosquitoes. Dot-blot hybridization was used to estimate copy number of rRNA genes; the copy number per haploid genome ranged from 39 3.27 in So. cyaneus to 1023 f 68.14 in Ae. flavopictus. A . Kumar, Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.
The ribosomal RNA gene family is composed of Hsu et al. 1975; SCHAFERand SCHAFER1980; multiple tandemly repeated units. Each unit con- VITELLIet al. 1982). The mosquito family Culicidae is divided into tains an intergenic ‘nontranscribed’ spacer (IGS, formerly NTS) and a transcribed portion consisting three subfamilies: Toxorhynchitinae, Culicinae and of an external transcribed spacer (ETS), 18s rRNA Anophelinae. Based on their cytological studies on gene, internal transcribed spacer (ITS) containing four nematocerous families, RAOand RAI(1987a) the 5.8s rRNA gene, and 28s rRNA gene (BECKING- proposed that the Mochlonyx-like ancestor of the family Chaoboridae with 2 n = 8 may be the anHAM 1982). The copy number of these units ranges from 45 per haploid genome in Sciara coprophila cestral stock from which Culicidae evolved. They also proposed that two main evolutionary lines (GEnBI and CnousE 1976) to over 3000 in the grasshopper, Locusta migratoria (OISHIet al. 1985). RI- arose from this stock-one that led to Anophelinae TOSSA and SPIEGELMAN (1965) were the first to es- through Chagasia-like ancestor with 2n = 8, and the tablish a simple relationship between the number of other splitting into two independent lines leading to nucleolus organizer regions (NORs) (secondary Toxorhynchitinae and Culicinae. Little is known on constrictions) and the number of sites of ribosomal the phylogenetic relationships between different gene cluster coding for 18s and 28s ribosomal genera or taxonomic groupings of these subfamilies and STONE1977; RAO1985). These three RNA molecules in Drosophila melanogaster. In (KNIGHT general, this relationship has been found to be con- subfamilies together include 34 genera and approxisistent throughout eukaryotic genomes (PARDUE et mately 3100 species. The karyotypes of about 200 et al. 1972, species are known to date (WHITE1980). The dipal. 1970; PARDUE1974; HENDERSON 1974a, b; EVANS et al. 1974; PARDUE and Hsu 1975; loid number of chromosomes of all but one species
7uh/e i Taxonom) and source of genera and species examined .___.
_ _ _ I
Subfamil?
Trik
Subsenus
Genus
Species
Strain (year of collection), source
G'
__.~._____.______
Culicinae
Aedini
Sabethini
Culicini Aedini Anophelinae *
Rockefeller Inst.. USA (1959). D. W. Jenkins: Memphis. USA (1986). S . Jones alhoprcrirs Mauritius (1974). C. Courtois; Oahu. Hawaii (1971). L. Rosen; Malaysia (1973). B. Knudson frai opicrus Nagasaki. Japan (1988). Y. Eshita sea I111 Bangkok, Thailand (1972), D. Gould pfyircsreiisis Suva. Fiji (1982). D. Gubler ulcusrdi Taiwan (1981). J. Lien atiiiairdulei Taiwan (1963, G. A. H. McClelland Mauritius (1965). R. Marnet mu.wurensr .r Pr-oroniar~lecijurr-iser-iarits St. Joseph Co., USA (19691, R. Beach; Potato Creek, St. Joseph Co., USA (1990). D. Wesson Iiender-soilI St. Joseph Co., USA (1981). S. Paulson; Eddy St., St. Joseph Co.. USA (1990). S. Hanson Ochler~~rari~sarropa1pu.r Coosa. USA (1972), R. Brust epar'trus Sansal, El Salvador (1968). S. Breeland Gymnonietopu medioi.irtatus Puerto Rico (1981) R. Novak Tripteroides Tripteroides hambusa Kyushu Island, Japan (1987), W. Hawley Sahethes Sahethes cxanrus Balboa heights, Panama (1983), J. L. Peterson Wjeomyia Wyeompia smithii UNDERC* (1989) Culer Culer pipiens quinquefasciarus Gainesville, USA (l969), R. Lowe At-nii,qere> Arnrigeres suhaibarrrs Japan (1971). M. Sasa Huema,qo,qrrs Hacmagogits eqitinus Panama (1979). J. L. Peterson Anripheler Anoplirles qiradrimacrrlariis Savannah, USA (1971), H. Schoof
A edc
Sr~qomrin
urg\prr
74
9 46 40 45 5 43 19
21 60 60
50 2 21 2
43 53 21 36 52
0 125 1 I4 26 40
Approximate generations in colon)
* L'nivervty of Notre Dame Environmental
Re\earch Center at Land O'Lakes. Wirconsin
is 6 (2n = 6). The only exception, Chagasia bathana niques, the mosquito karyotypes do not demonstrate of the subfamily Anophelinae, possesses 4 pairs of any linear differentiation for NORs along the length chromosomes (KREUTZER1978). In general, species of chromosomes. A different approach, using silver of the subfamilies Toxorhynchitinae and Culicinae staining technique of GOODPASTURE and BLOOM demonstrate a considerable uniformity with respect (1975), has also failed to locate ribosomal RNA to their karyotypes. A typical karyotype comprises genes on the mitotic mosquito chromosomes (RAO 3 pairs of homomorphic chromosomes: a pair of and RAI 1987a). small chromosomes (chromosome I), a pair of large In situ hybridization has been extensively used chromosomes (chromosome 2) and a pair of inter- in the localization of ribosomal RNA gene cluster mediate-sized chromosomes (chromosome 3) (RAI on the chromosomes of both animals and plants 1963, 1966; WHITE1980: RAOand RAI 1987a). In (PARDUE et al. 1970; HENDERSON et al. 1972, 1974a, a karyotype, all three pairs of chromosomes may b; EVANSet al. 1974; PARDUE and Hsu 1975: Hsu be metacentric, or one or two chromosome pairs et al. 1975; SCHAFER and SCHAWR 1980; VITELLI et et al. 1988; VISSERet al. may be slightly submetacentric (RAI 1963, 1966: al. 1982: DELUCCHINI WW1T.E 1980: MUNSTERMANN and M A R C H I 1986: 1988). In insects such as Drosophila and the grassRAOand RAI 1987a). The lengths of all three pairs hoppers, in situ hybridization has been used to locof chromosomes often vary in different species (RAI ate the rRNA genes on their chromosomes (SPEAR 1963. 1966; BAKER and ASLAMKHAN 1969: R ~ ~ a n 1974; d HENNIG et al. 1975; SCHAFER and SCHAFER RAI 1987a). In contrast, the species of the subfami- 1980: SINIBALDI and CUMMINGS 1981; WHITEet al. lies Anophelinae possess 2 pairs of metacentric 1982). However, in situ hybridization has not so far chromosomes of unequal size and one pair of het- been used to locate the ribosomal RNA genes on eromorphic sex chromosomes, X and Y (KITZ- the chromosomes of mosquitoes. In this paper we MILLF;R 1967). Using standard chromosome techreport the successful in situ localization of ribo-
Hrieditus 113 llY90)
soma1 RNA genes on the mitotic chromosomes of 20 species of mosquitoes belonging to 8 genera of subfamilies Culicinae and Anophelinae. In addition, the copy number of rRNA genes has been determined in 13 species, using dot-blot hybridization.
18s+
zxs
RIBOSOMAL R Y A G E ~ E SI N MOSQUIIOES
279
at 35°C. The slides were dehydrated in 70 % ethanol (2x10 min), 95 % ethanol (2x5 min) and air-dried. The slides were coated with Kodak NTB-2 emulsion diluted 1:l with distilled water at 43°C and stored at 4°C for 11-25 days. After exposure, the slides were developed, fixed and stained in S 5% Giemsa (30 min) as described in PARDUE (1985).
DNA extraction and dot-blot hybridization. DNAs were extracted from honey-fed adult female Mosquito species and chromosome preparation. mosquitoes according to BLINand STAFFORD ( 1976) -The details of genera and species of mosquitoes with minor modifications as described in BRADemployed during the present study are given in FIELD and WYATT(1983). A typical yield from 10 Table 1. All mosquito species used were from la- adults was 8-15 pg DNA, depending on the size boratory stocks, except Wyeomyia smithii. The lar- of mosquito species used. vae of Wy. smithii were collected from the pitcher Since the number of 18s rRNA genes equals the plants (Sarracenia purpurea) at the University of number of 28s genes or the ribosomal repeat units, Notre Dame Environmental Research Center at 18s DNA was used as a standard and the nitrocelLand O'Lakes, Wisconsin. Mitotic chromosome lulose membrane was hybridized with '*P-labeled preparations were obtained from colchicine-treated pE2 DNA. To isolate the 18s rDNA insert, plasmid brain ganglia of fourth instar larvae, following the pE2 (20 pg) was digested with EcoRI to excise the complete 1.8 kb 18s DNA under the conditions method of RAOand RAI(1987a). recommended by the supplier. The digest was run DNA probes and nick-translation. - Three recomin 0.8 % agarose, and the fragments were purified binant plasmids: pP11, pP1 and PE2 were used as from the agarose, using a Geneclean kit (Bio 101 DNA probes (BLACKet al. 1989). Plasmid p P l l Inc. USA). contains the 28Sp gene plus about 800 bp of inStandard DNA and species DNA were blotted tergeriic spacer (IGS). Plasmid pP1 contains the onto a nitrocellulose membrane, using a Schleicher complete 28Sa gene and some internal transcribed and Schuell manifold. The nitrocellulose membrane spacer (ITS) of the ribosomal cistron. Plasmid pE2 was vacuum dried for 2 h at 80"C, and prehybridcontains the complete 1.8 kb 18s gene. These re- ized in 50 % deionized formamide, SxSSC, 0.5 R combinant plasmids are pUC 118 subclones derived SDS, 5 % dextran sulphate, 5 x Denhardt's solution from a lambda EMBL3 rDNA clone from a ge- and 100 pg/ml denatured calf-thymus DNA at 42°C nomic library of Ae. albopictus (BLACKet al. 1989). overnight. 32P-labeledpE2 DNA was directly added The subcloned DNAs were purified according to to the prehybridization mixture and the hybridizaMANIATIS et al. (1982). Each subcloned DNA was tion was carried out for 16-24 h at 42°C (SAMBROOK nick-translation with 3H-dCTP (18 Ci/mmole) and et al. 1989). Following hybridization, the membrane ?H-dGTP (10 Ci/mmole) using a BRL (Bethesda was washed twice in 1 x wash buffer (10 x wash Research Laboratory USA) nick-translation kit ac- buffer = 3M NaCl, 0.6 M Tris-HCI, pH 8.0, 0.02 cording to a standard protocol of MANIATIS et d. M EDTA, pH 8.0) for 10 rnin each at room tem(1982). For dot-blot hybridization, pE2 DNA was perature, twice in 1 x wash buffer and 1 % SDS for nick-translated with "P-dCTP (3000 Ci/mmole) as 30 min each at 60°C, and finally twice in 0.01 x described above. wash buffer for 30 min each at room temperature. In situ hybridization. - Pretreatment of the slides The membrane was dried at room temperature, and in situ hybridization were carried out according wrapped in the Saran wrapTMand exposed to Kodak to PARDUE (1985). In situ hybridization was con- X-OMAT X-ray film with a Dupont intensifying ducted overnight at 40°C using 1 6 ~ 1 0 ~ 4 0 ~ screen 1 0 ~ at -70°C for a range of time. The copy cpm per slide in 50 p1 hybridization buffer ( ~ x S S C , number of rRNA repeat units was determined by SO % formamide, 5 % dextran sulphate, 1 % Den- comparing the dot intensity of the species DNA hardt's solution, 0.037 pg probe DNAs, 7 pg calf- with the appropriate standard DNA of the autorathymus DNA). The three rDNA probes (pP1, pP11 diograms that were in linear range of the film reet al. 1989) using a EC- densiand pE2) were mixed for each in situ hybridization sponse (ZIMMERMAN reaction. The non-specific bound probes were re- tometer and a Zeineh software 1-D scanner (Fisher moved by incubating slides in 2xSSC (3x10 min) Scientific Inc. USA).
Materials and methods
Fig. l a d . In situ hybridization of "-labeled rDNA probes to mitotic chromosomes from the genus Aede.5, subgenus Stegoniyia: (a) Ae. oexyptr, (b) Ae.,fluwpicri(.s,( c ) A e searoi. (d)Ae. alhopictus. The arrows point to sites of hybridization. Bar = 10 pn.
Chr~onzosomeider~ificatioriiri czrliciires. - To definitively identify chromosome pair(s) involved in in situ hybridization. chromosome measurements were made on the photomicrographs of 8-1 0 meta-
phase per species in culicines, using a digitizer. The data on chromosome measurements are not presented here but may be obtained on request.
IXS
+ ?XS
RIBOSOMAL RNA GENES IN MOSQUITOES
28 1
Fig. 2a-f. In situ hybridization of 3H-labeled rDNA probes to mitotic chromosomes from the genus Aedes, subgenera Stegomyia and Protomacleaya: (a)Ae. polynesiensis, ( b )Ae. alcasidi, (c)Ae. annandalei, ( d ) Ae. mascarensis, (e)Ae. triseriatus, and (f) Ae. hendersoni. Bar = 10 pm.
Fig. 3a-e. In situ hybridization of 3H-labeled rDNA pro-
Results Hybridization of rDNA probes to mitotic chromosomes. - In situ hybridization of mitotic chromosomes with 3H-labeled rDNA probes showed that a single site per haploid karyotype was hybridized in all species except in Aedes triseriatus. In Ae. triseriatus, rDNA probes hybridized to two sites per haploid karyotype. The results for various species were as follows: rDNA probes hybridized on one arm of chromosome 1 in all species of Aedes examined (Fig. 1, 2 and 3a-b) except in Ae. triseriatus and Ae. mediovittatus. In Ae. annandalei, which possesses submetacentric chromosome 1, the site of hybridization was located on the long arm (Fig. 2c).
bes to mitotic chromosomes from the genera Aedes, (subgenera Ochlerotatus and Gymnometopa) Wyeomyia and Tripteroides: (a) Ae. atropalpus, ( b )Ae. epactius, ( c ) Ae. mediovittatus, (d) W y . smithii, and (e) Tp. bambusa. Bar = 10 p m .
Probes hybridized to one site each on chromosome 1 and chromosome 3 in Ae. triseriatus (Fig. 2e) and to a site on chromosome 2 in Ae. mediovittatus (Fig. 3c). In the species of other culicine genera, rDNA probes hybridized on chromosome 1 in Wyeomyia smithii (Fig. 3d), Culex pipiens quinquefasciatus (Fig. 4b) and Sabetkes cyaneus (Fig. 4c); chromo-
Her-rdtrus 113 (1990)
Fig. 4a-d. In situ hybridization of 3H-labeled rDNA probes to mitotic chromosomes from the genera Haemagogus. Culex, Sabethes, and Armigeres: (a) Hq. equinus, (b) C.r. pipiens quinquefasciutus, (c) Su. ryatieus, and ( d ) Ar. subalbatus. Bar = 10 +m.
some 2 in Haemagogus equinus (Fig. 4a); and chromosome 3 in Tripteroides bamhusa (Fig. 3e) and Armigeres slcbalbatus (Fig. 4d). The location of hybridization sites on the specific chromosome varied in different species (Fig. 6). For example, the hy-
bridization sites were located close to the telomere in Ae. atropalpus, Ae. epactius and Tp. bambusa; in an intercalary position in Hg. equinus and Ae. triseriatus; and near the centromere in the rest of the culicine species examined.
Heredifas 113 (19901
18s + 2RS RIBOSOMAL RNA GENES IN MOSQUITOES
283
Discussion
Fig. 5. In situ hybridization of 3H-labeled rDNA probes to mitotic chromosomes of Anopheles quadrimaculatus. Bar = 10 pm.
In Anopheles quadrimaculatus, probes hybridized strongly on the X and Y chromosomes (Fig. 5). Autoradiographic silver grains completely covered the heterochromatic arms of both sex chromosomes. Hybridization of rDNA probes to interphase nuclei (Fig. 7). -Except in Ae. triseriatus, a single cluster of silver grains was observed over interphase nuclei of all species. Fig. 7a shows interphase nuclei with a single cluster of silver grains in Ae. albopictus. In most of the cases, silver grains were clustered at the periphery of interphase nuclei. Two clusters of silver grains were detected over the interphase nuclei of Ae. triseriatus (Fig. 7b). However, some interphase nuclei with a single cluster of silver grains were also noted, suggesting that both chromosome pairs may organize a common nucleolus as well as separate nucleoli. Alternatively, these nuclei may simply represent polar views of the nuclei. Estimation of rRNA gene copy number. - Fig. 8 shows an autoradiograph of dot-blot hybridization. The copy number was determined in 13 species for which the 1C-DNA content is known (Table 2). The copy number per haploid genome varied nearly 26fold among mosquito species examined the minimum copy number was 39f3.27 in Sa. cyaneus, and the maximum was 1023k68.14 in Ae.flavopictus. No correlation was observed between the 1CDNA content and the copy number of rRNA genes (r = 0.28, df = 12, p>0.05).
The present study has shown that the rRNA gene cluster is confined to a single chromosome per haploid genome among all mosquito species, except Ae. triseriatus, belonging to 8 genera of Culicidae. Further, the ribosomal genes are conserved on chromosome 1 in all the species of Aedes except in Ae. mediovittatus and Ae. triseriatus. The ribosomal RNA genes are also conserved on chromosome 1 in Cx. p . quinquefasciatus, Sa. cyaneus and Wy. smithii. In contrast, they are located on chromosome 2 in Ae. mediovittatus and H g . equinus and chromosome 3 in Ar. subalbatus and Tp. bambusa. The only species examined from the subfamily Anophelinae, An. quadrimaculatus, possesses ribosomal genes on the sex chromosomes. In the species of Aedes and Culex where linkage group-chromosome correlations have been made, chromosome 1 pair has been shown to be involved in sex-determination ( M A C D O N A LRAI D ~ 1970; ~ BAKER^^ al. 1971a, b; DENNHOFER 1972; BHALLA et al. 1974). The sex is determined by a single pair of alleles or a segment of chromosomes for which the males are heterozygous and the females are homozygous (GILCHRIST and HALDANE 1947). Even in those cases where no correlations have been made, it has been assumed that this is true for them as well (MOTARA and RAI 1978). This indicates that the ribosomal genes are conserved on the sex chromosomes in a majority of mosquito species. In other insect species, the NORs have been localized on the X and Y chromosomes in Drosophila melanogaster, D. simuhns and D . hydei (SPEAR1974; H E N N I Cal.~ 1975), ~ the X chromosome only in D . virilis (ENDOW and GALL1975), the Y and microchromosomes (autosomes) in D . nasuta nasuta and D . n. albomicans (HAGELE and RANGANATH 1983), the X and microchromosomes in the ‘mulleri’ complex of Drosophila (BICUDO 1981) and Zaprionus indianus (KUMAR and GUITA 1987), the microchromosomes only in D. tumiditarsus (SINIBALDI and CUMMINCS 1981), and the X and autosomes in the grasshoppers (WHITEet al. 1982). In situ localization of ribosomal genes on chromosome 1 in Ae. aegypti is in contrast to the finding of MACDONALD and RAI(1970). Based on the presence of a slightly unstained region on chromosome 3 in some preparations in the above species, they suggested that the same may represent NORs. However, such situations are not uncommon. Hsu et al. (1975) demonstrated that the weak constrictions noted in the karyotypes of some mammalian species are not always the sites of ribosomal genes. In Sa. cyaneus, MUNSTERMANN and MARCHI(1986) ob-
CHROMOSOME 1
CHROMOSOME 2
CHROMOSOME 3
SPECIES
Aedes aegypti, Ae. albopictus, Ae. flavo pictus. Ae. seatoi, Ae. polynesiensis, Ae. alcasidi. Ae. mascarensis. Ae. hendersoni, Sabethes cyaneus, Wyeomyia smithii, Culex pipiens quinquefasciatus
Aedes annandalei Aedes triseriatus
Aedes atropalpus, Ae. epactius
IXI
I X I
Aedes mediovittatus
I x 1
Tripteroides bambusa
I x i
Arrn/geres subalbatus Haemagogus equinus
I X I
IXI
Anopheles quadrimawlatus
Fig. 6. Diagrammatic repre5entation of mitotic chromosomes of mosquito species in situ hybridized with 3H-labeled
rDN.4 probes.
served recurrent association of the nucleolus with chromosome 1 in polytene nuclei. They suggested that the NORs are probably located on chromosome 1 in this species. Our result of in situ hybridization confirms their finding. In addition to chromosome 1. ribosomal genes are also located on chromosome 3 in Ae. rriseriurus. Sometimes i t was difficult to determine whether the large pair of chromosomes with hybridization is chroinosome 2 or chromosome 3. The difference between the lengths of these chromosomes is only 0.17 Kni (data not shown). It appeared that in some inetaphase plates hybridization sites are located on chromosome 2 and in others on chromosome 3 from the \ame individual. Since in majority of the cases, 4gnal is located o n chromosome 3. we concluded
that the second site of ribosomal gene is on chromosome 3. Recently, DELUCCHINI et al. (1988) reported extra ribosomal sites in the genome of Triturus vulguris meridiotiulis. These sites contain only IGS and are devoid of 18s and 28s genes. Unlike the Triturirs situation, both chromosome 1 and chromosome 3 of Ae. [riser-iatus still hybridize to rDNA probes that lack pP11, and therefore are devoid of IGS sequences (data not shown). Thus the second site of hybridization in Ae. tt.iser-iatus appears to be a bona fide rDNA site. and not just transposed IGS sequences. Ae. triseriufltsand Ae. henrlersoni are sibling species and crosses between them are compatible biand CRAIG1968). Presence directionally (TRLIMAN of an additional site of ribosomal gene cluster in
Hereditus 113 (1990)
IRS+ 28s RIBOSOMAL R N A GENES IN MOSQUITOES
285
Fig. 8. Autoradiograph of dot-blot hybridization. The 18s DNA standards were blotted in column 1 and 2: Al, 0.7 ng; B1, 1.4 ng; CI, 2.8 ng; D1, 5.6 ng; El, 7.0 ng; F1, 1.4 ng; GI, 100 ng lambda DNA as a negative control; A2G2, each with 1.4 ng 18s DNA. Each standard 18s DNA sample was supplemented with 100 ng lambda DNA as a carrier. The species DNAs were arranged as follows: A3&A4, Aedes aegypti; B3&B4, Ae. albopictus; C3&C4, Ae. flavopictus; D3&D4, Ae. seatoi; E3&E4, Ae. polynesiensis; F3&F4, Ae. alcasidi; A5&A6, Ae. annandalei; Fig. 7. In situ hybridization of 3H-labeled rDNA probes B5&B6, Ae. mascarensis; C5&C6, Ae. triseriatus; to interphase nuclei: (a) Aedes albopictus and (b) Ae. D5&D6, Ae. hendersoni; E5&E6, Ae. atropalpus; F5&F6, triseriatus. Bar = 10 pm. Ae. epactius; G5&G6, Ae. mediovittatus; A7&A8, Tripteroides bambusa; B7&B8, Sabethes cyaneus; C7&C8, Ae. triseriatus and its absence in Ae. hendersoni and Wyeomyia smithii; D7&D8, Culex pipiens quinquefasciatus; E7&E8, Armigeres subalbatus; F7&F8, Haemagogus other Aedes species indicate that it may be of recent equinus; and, G7&G8, Anopheles quadrimaculatus. Each origin. To account for this observation, a transloca- sample dot except G7&G8 contains 1 p g DNA, while tion is postulated, where a substantial amount of G7&G8 contain 0.5 pg DNA. The hybridization and the ribosomal gene cluster has been translocated from post-hybridization washing were carried out as described chromosome 1 to chromosome 3, possibly via extra- in the Materials and methods section. The intensity of chromosomal circular rDNA intermediates (Ro- species sample dots was compared with 1.4 ng (0.76~10’ copies of 18s gene; 1 picogram DNA = 0 . 9 8 ~ 1 0kb, ~ CHAIX et al. 1974). The significance of two sites of 1985a) standard DNA. To test if the copy the rRNA genes in Ae. triseriatus is not clear. Pres- CAVALIER-SMITH ence of two sites of the rRNA genes in Ae. number estimate varies as a function of stringency, the whole experiment was repeated at low stringency conditriseriatus resembles the analogous situation found tions (25 % formamide and the hybridization at 35°C with in D . hydei (HENNIGet a]. 1975), grasshoppers subsequent washing at 48°C). The copy number estimate (WHITEet al. 1982), mammals (Hsu et al. 1975), of the rRNA genes at low stringency conditions was in and urodele amphibians (MACGREGOR and SHER- agreement with that at high stringency conditions (data WOOD 1979; DELUCCHINI et al. 1988; WILEYet al. not shown).
1989), where multiple sites of the rRNA genes are known to occur. In addition to the physical mapping of the rRNA genes on the chromosomes of mosquitoes, another reason to undertake this study was to examine the extent to which chromosome repatteming has taken place during speciation of mosquitoes. The general uniformity in the karyotypes of Culicinae has led to speculate that the speciation of Culicinae has
more relied on point mutation than chromosome repatteming (see MOTARA and RAI1978). However, the latter studies by MOTARA and RAI(1978), MUNSTERMANN (1981) and RAOand RAI (1987a) have given clear indications of chromosome repatteming through translocations and inversions. The chromosome complements of different Aedes species can-
286
Her-editus 113 (IYYUJ
A K L M A R FT 4L
Table 2 The copy number of rRXA gene5 among mosquito species
IC-DNA content (P€)"
No. of rRNA genesilkg DNA~
0.81
o.s2x10y
423k3 1.9
1.24
0.67~10'
82 1545.23
Ae. flaropictus Ae. seator Ae. polvnesiensis Ae. ulcmidi Ae. annandaler Ae mascarensis Ae. triwr-iarus (St. Joseph Co., 1990) Ae. hendersoni ( S . Joseph Co., 1990)
1.33 0.97 0.73 0.97
0.77~10~ 0 . 1 8 10' ~ 0.55~10' 0.79~10' 0.71x109 0.5 1x10' 0 . 3 410' ~
-
o.19x109
-
Ae atrnpalpus
-
0.34~10' 0.2Sx1OY 0.22~10~ 0.22x1OY
-
Ae. epuctius Ae. medrovirrarus Tp bamhusu So. cymeus W?. smirhii Cx. p yuinquejasciatus Ar. .suhalhatus Hg.equinus An. quadrrmaculatus
Species (Strain)
No. of rRNA
geneshaploid genome ( ~ s E M ) ~
~
Ae. aegypri (Rockefeller) Ae. albopicrus (Oahu)
-
I .52
~
0.79 0.86 0.54 1.24 1.12 0.2s
O.OSx10'
0.91~10' 0 . 1 6 1~0' 0. 15x10' 0.04~10~
0.96~10~
1023568.14 1785l3.M) 398119.73 762k48.73 -
512k36.12
-
39f 3.27 783+S 1.8 87f 3.18 189k47.46 45C 6.35 47W36.60
not be distinguished from each other. However, of mosquito species supports this assumption. The they can be distinguished from each other in a few physical location of the rRNA gene cluster on the cases based on their C-banding patterns (MOTARA chromosome arm close to the telomere in Ae. atroand RAI 1978; RAOand R.41 1987a). The chromo- palpus, Ae. epactius and Tp. bambusa, at an intersome complement of Ae. mediovittatus is the only calary position in Hg. equinus and Ae. triseriatus, exception that can be distinguished from the chro- and close to the centromere in the rest of the culimosome complements of other Aedes species. The cines can be explained by paracentric inversions chromosome complement of Ae. mediotittatus con- (see Fig. 6). Further, no correlation exists between tains a pair of much smaller chromosome 1 , a pair the taxonomic grouping of the species and the chroof much larger chromosome 2 and a pair of interme- mosome location of the rRNA genes. For example, diate-sized chromosome 3 (see Fig. 3c). This sug- the rRNA genes are located on chromosome 1 in gests that a considerable chromosome repatterning Sa. cyaneus and Wy. smithii and on chromosome 3 has occurred in the karyotypic evolution of this spe- in Tp. bamhusa even though these species are incies. In situ hybridization to chromosome 2 of this cluded in the same tribe Sabethini. Similarly, the species supports this assumption. The metaphase species of Aedes, H g . equinus and Ar. suhalbatus complement of H g . equinus with the rRNA gene are classified within the tribe Aedini, but the rRNA cluster on chromosome 2 can be derived from a genes are located on chromosome 1 in the species metaphase complement containing the rRNA gene of Aedes except Ae. mediovittatus, chromosome 2 cluster on chromosome 1 by a translocation be- in H g . equinus, and chromosome 3 in Ar. subaltween chromosome I and chromosome 2 , possibly hatus. 1976) of a small chrovia the 'shift' (STRICKBERGER In a cell line of Ae. albopipictus that was derived mosome segment containing the rRNA gene cluster. from minced neonate larvae (SINGH1967), SPRADA similar mechanism can place the rRNA gene LING et al. (1974) estimated 430 copies of rRNA cluster from chromosome 1 to chromosome 3 in Tp. genes per haploid genome using Cot-curve analysis. hurnhusa and Ar. subalhatus. The location of the Recently, PARKand FALLON (1990) estimated 30C rRNA gene cluster on chromosome 1 in a majority 400 rRNA genes in the same cell line, and 35C.500
Hereditas 113 (1990)
18s+ 28s RIBOSOMAL R N A GENES I N MOSQUITOES
287
in larvae, and about 1200 copies in the pupae and length of 9.0 kb in Ae. aegypti (GALEand CRAMPadults of Ae. aegypti, using dot-blot hybridization. TON 1989) and 15.6 kb in Ae. albopictus (PARKand However, the interpretation of their data was based FALLON1990), the proportion of haploid genome on the 1C-DNA content of 1.45 pg for Ae. albopic- represented by the rDNA in these species is 0.96 tus cell line (SPRADLING et al. 1974) and 1.30 pg for and 2.10 % respectively. In conclusion, the present study has shown that Ae. aegypti (DITTMANN et al. 1990). During the present study, we estimated 423k31.9 and 821k45.23 the rRNA gene cluster is located on a single chrocopies of rRNA genes per haploid genome in the mosome per haploid genome in all species of mosadults of Ae. aegypti and Ae. albopictus respec- quitoes examined except in Ae. triseriatus. Howtively. However, our interpretation was based on ever, the species differed nearly 26-fold in the the 1C-DNA content of 0.81 pg in Ae. aegypti and abundance of rRNA genes. The presence of the 1.24 pg in Ae. albopictus (RAOand RAI 1987b). The ribosomal RNA gene cluster on different chromohaploid nuclear DNA content has been shown to somes and at different positions on the chromosome vary nearly 3-fold among world strains of Ae. albo- arm among mosquito species indicates that a conpictus (KUMARand RAI 1990). Our preliminary siderable chromosome repatterning through translostudy has shown a 5-fold variation in rRNA gene cations and inversions has occurred in the karyocopy number between two strains of Ae. albopictus typic evolution of the members of Culicinae and that differed 3-fold in their 1C-DNA contents; the corroborates earlier findings of RAIet al. (1982) and (1981). Savannah strain from the USA with 1.65 pg 1C- MUNSTERMANN DNA content has 1083 copies of rRNA genes per haploid genome and the Koh Samui strain from Acknowledgements. -We thank Drs S. Kambhampati and L. E. for critically reading the manuscript; Mr Brian Thailand with 0.62 pg 1C-DNA content has Munstermann Turco, Mrs Peggy Hodges and Miss Dawn Verleye for rearing the 218f12.5 copies (KUMAR and RAI,unpublished). In mosquitoes in the insectary; and Mrs Tracy Frost for help in typthree species examined from the Ae. albopictus sub- ing. This work was supported by NIH research grant 5R01 A1 group (viz.: Ae. albopictus, Ae. flavopictus and Ae. 21443 to KSR. We also thank the reviewers for suggesting changes seatoi), 1.4-fold difference in the haploid DNA con- in the earlier draft of this paper. tent is accompanied by over 5-fold difference in the copy number of rRNA genes. Among other species of mosquitoes examined, the copy number spanned References M. 1969. Karyotypes of some with a minimum value of 39k3.27 in Sa. cyaneus BAKER,R. H. and ASLAMKHAN, Asian mosquitoes of the subfamily Culicinae (Diptera Culici. to a maximum value of 783k51.8 in Wy. smithii. It dae). - J . Med. Entomol. 6: 44-52 has been suggested that there is a positive correla- BAKER, R. H., SAKAI,R. K. and MIAN,A. 1971a. Linkage grouption between the copy number of rRNA genes and chromosome correlation in a mosquito: inversions in Culex 1C-DNA content (BIRNSTIEL et al. 1971; CAVALIER- tritaeniorhynchus. - J . Hered. 62: 31-36 SMITH1985b). In contrast, no such correlation was BAKER,R. H., SAKAI,R. K. and MIAN,A. 1971b. Linkage groupchromosome correlation in Culex tritaeniorhynchus. - Science observed during the present study similar to the 171: 585-587 observation made in the flax genotrophs by CULLIS BECKINGHAM, B. 1982. Insect rDNA. -In: The Cell Nucleus (eds H. BUSCHand L. ROTHBLUM), Academic Press, New York, (1976). As with the chromosome localization of the p. 205-263 rRNA genes, no correlation is observed between the S. C., CAIAIBA,A. C. I., CARVALHO, W. M. P. and SANBHALLA, taxonomic position of the species and the copy TOS,J. M. 1974. Translocations, inversions and correlations of number of the rRNA genes in mosquitoes examlinkage groups to chromosomes in the mosquito Culex pipiens fatigans. - Can. J . Genet. Cytol. 16: 337-850 ined. For example, the difference in the copy number of the rRNA genes between Wy. smithii and BICUDO,H. E. M. C. 1981. Nucleolar organizer activity and its regulatory mechanisms in Drosophila species of the ‘mulleri’ Sa. cyaneus with nearly the same 1C-DNA content complex and their hybrids. - Caryologia 34: 231-253 is as much as 20-fold, although both species are BIRNSTIEL,M. L., CHIPCHASE, M. and SPIERS,J. 1971. The ribosomal RNA cistrons. - Progr. Nucleic Acid Res. Mol. Biol. included in the tribe Sabethini. At present, the fac11: 351-389 tors controlling the copy number of the rRNA genes BLACK,W. C. IV, MCLAIN,D. K. and &I, K. S. 1989. Patterns among different species of mosquitoes are unknown of variation in the rDNA cistron within and among world popuand remain to be investigated. lations of a mosquito, Aedes albopictus (Skuse). - Genetics 121: 539-550 On an average rDNA constitutes only about 1 %, or at maximum only 3.7 % of total genome for eu- BLIN,N. and STAFFORD,D. W. 1976. A general method for isolation of high molecular weight DNA from eukaryotes. - Nukaryotes (see CAVALIER-SMITH 1985b) with more cleic Acids Res. 3: 2303-2308 values ranging around 1 % (SCHAFERand KUNZ BRADFIELD, J. Y. and WYATT,G. R. 1983. X-linkage of a vitellogenin gene in Locusta migratoria. - Chromosoma 88: 19&193 1987). Based on the average ribosomal repeat unit
olbopicri~s(Skuse). - Theor. Appl. Genet. 79: 748-752 M,ACDOSALD, P. T. and RAI, K. S. 1970. Correlation of linkage groups with chromo\omes in the mosquito Aedes aegypfi L. - Gerretics 66: 4 7 5 4 8 5 S. 1979. The nucleolus orgaM ~ C G R E C ~H.O C. R , and SHERWOOD, nirers of Pletkodon and Aiieides located by in situ nucleic acid hybridization with Xenopits 'H-ribosomal RNA. - Chromo.sonlo 72: 271-280 T., FRnwti. E. F. and SAMBROOK, J. 1982. Molecular M~\I.ATIS. Cloning: A Laboratory Manual. - Cold Spyin,? Hurhor Laboriltol~\.. .vt'\+'Y#rk M O T A R M. ~ , A. and R.u. K. S . 1978. Giernsa C-handing patterns iii A d e \ (Stegoniyiu) mosquitoes. - Chroniosoma 70: 51-58 h l L \ S T E R > 1 4 U \ . L. E. 1981. Enzyme linkage maps for tracing chromosomal evolution in Ardes mosquitoes. -In: Application of Generic..s 011 Cito/o,q? i n Insec~t.Yj.sremoric.s und Evolution ( e d M. W. SIOCK).U n w Oflduho. Moscon8.p. 129-140 ML\STERMAY\. L. E. and MAR('ii1. A. 1986. Cytogenetic and isot \ i x ~ a .S A. and G \ i i ~J . G. 1975. Ditferential replication of \atellite DNA iii polyploid ti\we o f D r ~ i . ~ o p / i ii./iur i / r s . Clrr~iryme profile of Soherhe.\ cymeus: a mosquito of the neotropical nin\ornir 5iJ: 175-195 J . tiered 77: 241-248 canop). F\ q \ \ , H J.. B%c k i A \ I L R A . dnd P4mi I.. 51. L. 1974. Location OISHI. M..LWKF.J. and W v ~ r r G. . R. 1985. The ribosomal DNA ot gcnw coding for I X S and l X S ribo\omal R N A in the human gene\ of Locu.\tu migraror-ia: copy number and evidence for riiniowmiu 48: 4 0 5 4 2 6 underreplication i n a polyploid tissue. - Cun. J . Bioc.lieni. Cell. + \ i P i o \ . J. IOXY. The riho\omal gene\ of the Biol. hi: 106&1070 .\ ' l l ~ q \ / l f l Elll. .I Rrrx h n 185: 3 I 1-3 17 P \ R D IE. 51. L. 1974. Localization of repeated DNA sequences in G I K i i i . S. A . and C w i 5 5 . H. V. 1976. Further studie> on the Xenopiis chromosomes. - Cold Spring Harbor Synip. Quunr. rihwamal R h A cistroii\ ot Sc iura ~ o p i n i p h i l u [Diptsra). G c ~ n t , f i r .83: r 8 1-90 . In situ hybridization. - In: N d e i c . Acid G i i t I I H ~ C I ,H. M .and Hxi.i>.&\i. J . B. S. 1347. Se\-lmkaee and H>hi-idirurion:A Pi-ucr i d Approach (eds B. D. HAMES (2nd S . \ex detemiinatioii i n a mosquito. Ci1le.1 niole.srrrr Herc,rlirii.\ J. I+IGGI\S!. I.K.L. Press. Ovjord, p. 179-203 .?.i: !75-190 P 4 ~ i ) i . t . 41. L.. GERBI. S. A,. ECKHARDT, R. A. and GALL.J. G. ( h ) i w , % \ i ~ K k .c. and Bi o o v . s. E. 1975. Vi\ualr7ation (it nu1970. Cytological localimion of DNA complementary to ribosomal RNA in polytene chromosomes of Diptera. - Chromocleolu\ organirer in mammalian chromoromer wing \il\er mining. - C / ~ r ~ ~ n i o S.+ \ ~ ~37-50 nr~i uiniu 29: 268-290 H \ c ~ ~ . i . iK . . and R,\v-.\\*rti. H . A. 1983. The chromoionie\ of P ~ K I1..Xhl. L. and H ~ v T. , C. 1975. Localization of 18s and 28s t h o 1 h o w p t i i l i i race\: I l r ~ ~ w p / r nru/ (~i i ~ r t i i i u \ i t r ~ i and D n ribowrnal genes i i i the chromosome$ of the lndian Muntjac. -u/hi)niicum 111. Localintion of nucleolar organiier region\. ./. ( E l / B l I J l 64: 25 1-254 ~ ; e n ~ t ohi): u 123-1?X P ~ R KY.\\G-JA . and F.xi.i.cn, A. M. 1990. Mosquito ribosomal t { l \ i l F v b ~ ) \ ,A . s . E I ~E. ~51..~y L~.IT. ~ 'r. . a l l d ~ ~ i \ \ ( j ~Kl t.~c. . RNA genes: characterization of gene structure and evidence for IWk. Thc chromororne location of ribomnal DN.4 i n the change, in copy numher during development. - Insect Biomou\r. - C'hroirioriniit 4Y: 155-16C) 1 licni. 3011): 1-1 I . 4. s.. WARHI R I O \ . D and . A l \ % < X ) i > .K . c. 1Y77. R4i. K . S. 1963. A comparative \tudy of mosquito karyotypes. I-ocation of ribowma1 DNA i n thc. human chromosome comple.Arm Entomol. Sor Anr 56: 160-170 ment. P ~ o c.Vuri. . ,-kud. Sl:I LrSA 69: 339.1-3398 R\i. K. S. 1966. Funher observations on the somatic chromosome t I f \ t ~ ~ ~ iA i ~ . S.. \ . W . \ R H L R I O D. \ . and ;\r\\cjou. K. C. 1971b. cyto!og} of some mosquitoes (Diptera: Culicidae). - A n n . EnLocaliution of rDNA i n the chimpanzee (Puri rro,y/odvre\) ronrol Soc . A m . 59: 242-246 chromosome complenient. - C / i r ~ ~ n i ~ ~ . s46: o n i4i 3 ~534 I R.M. K. S . , PASHLEY, D. P. and ML,NSTERMANN, L. E. 1982. Genett-iEv\ic.. W., LnK. B. and L ~ o \ c l \ i ,0. 1975. The location of ics of speciation in aedine mosquitoes. -- In: Rec,ent DerelopnucleoluE organizer regions 111 D~o\r:plriluInder Chroniofirenis i n Genetics if Insect Disease Vectors (ed,s W. M. M. wmo S l : 57-63 S T E l \ E K . W. J. T ~ B A C H N IK. C Ks.. RAIond s. NAKANF), Stipes, H v . T C.. SPIRI.I.(I. S . E. and PARDL 1. M. L. 1975. Distribution Ckunipoign. 111. p. 83-1 29 of I X S c 2 8 S riboromal genes rn mammalian genomes. - C / I ~ ( I . RAO. P. N. 1985. Nuclear DNA and chromosomal evolution in niouJmo 53: 25-36 mosquitoes. - Ph.D Thesis. Unii3.of Notre Dame. US4 KITZMIL.LEH. J. H. 1967. Mosquito c)togenetic\. - In: Gerirrrc I R m . P. N. and R4i. K.S. 1987a. Comparative karyotypes and I J ~Inrrc.tr Irc tors of Ilrseose ( e d s J. W WRIGHT m l f R. P.\LI. chrommomal evolution in some genera of nematocerous (DipE/tci'/
Chromosomal localization and copy number of 18s + 28s ribosomal RNA genes in evolutionarily diverse mosquitoes (Diptera, Culicidae) A. KUMAR and KARAMJIT S. RAI Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556,U.S.A.
KUMAR,A. and RAI, K. S. 1990. Chromosomal localization and copy number of 18s + 28s ribosomal RNA genes in evolutionarily diverse mosquitoes (Diptera, Culicidae). - Hereditus 113: 277-289. Lund, Sweden. ISSN 0018-0661. Received June 25, 1990. Accepted October 17, 1990 In situ hybridization using 3H-labeled 18s and 28s ribosomal DNA @DNA) probes from Aedes albopictus was performed on the mitotic chromosomes of 20 species of mosquitoes belonging to 8 genera of subfamilies Culicinae and Anophelinae. In all but one species examined, the rDNA family was localized to a single chromosome per haploid genome. Aedes triseriatus was the only exception, with the rDNA cistrons present on chromosome 1 and on chromosome 3. The ribosomal RNA genes were located on chromosome I in Ae. albopictus, Ae. aegypti, Ae. fzavopictus, Ae. seatoi, A. polynesiensis, Ae. alcasidi, Ae. annandalei. Ae. mascarensis, Ae. hendersoni, Ae. atropalpus, Ae. epactius, Culex pipiens quinquefasciatus. Wyeomyia smithii, and Sabethes cyaneus; chromosome 2 in Ae. mediovittatus and Haemugogus equinus; chromosome 3 in Armigeres subalbatus and Tripteroides bambusa; and the heteromorphic X and Y chromosomes in Anopheles quadrimaculatus. The variation in the location of ribosomal RNA genes on the different chromosomes and at different positions on the chromosome arm among the mosquito species examined is suggestive of considerable chromosome repatterning through translocations and inversions in the karyotypic evolution of mosquitoes. Dot-blot hybridization was used to estimate copy number of rRNA genes; the copy number per haploid genome ranged from 39 3.27 in So. cyaneus to 1023 f 68.14 in Ae. flavopictus. A . Kumar, Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.
The ribosomal RNA gene family is composed of Hsu et al. 1975; SCHAFERand SCHAFER1980; multiple tandemly repeated units. Each unit con- VITELLIet al. 1982). The mosquito family Culicidae is divided into tains an intergenic ‘nontranscribed’ spacer (IGS, formerly NTS) and a transcribed portion consisting three subfamilies: Toxorhynchitinae, Culicinae and of an external transcribed spacer (ETS), 18s rRNA Anophelinae. Based on their cytological studies on gene, internal transcribed spacer (ITS) containing four nematocerous families, RAOand RAI(1987a) the 5.8s rRNA gene, and 28s rRNA gene (BECKING- proposed that the Mochlonyx-like ancestor of the family Chaoboridae with 2 n = 8 may be the anHAM 1982). The copy number of these units ranges from 45 per haploid genome in Sciara coprophila cestral stock from which Culicidae evolved. They also proposed that two main evolutionary lines (GEnBI and CnousE 1976) to over 3000 in the grasshopper, Locusta migratoria (OISHIet al. 1985). RI- arose from this stock-one that led to Anophelinae TOSSA and SPIEGELMAN (1965) were the first to es- through Chagasia-like ancestor with 2n = 8, and the tablish a simple relationship between the number of other splitting into two independent lines leading to nucleolus organizer regions (NORs) (secondary Toxorhynchitinae and Culicinae. Little is known on constrictions) and the number of sites of ribosomal the phylogenetic relationships between different gene cluster coding for 18s and 28s ribosomal genera or taxonomic groupings of these subfamilies and STONE1977; RAO1985). These three RNA molecules in Drosophila melanogaster. In (KNIGHT general, this relationship has been found to be con- subfamilies together include 34 genera and approxisistent throughout eukaryotic genomes (PARDUE et mately 3100 species. The karyotypes of about 200 et al. 1972, species are known to date (WHITE1980). The dipal. 1970; PARDUE1974; HENDERSON 1974a, b; EVANS et al. 1974; PARDUE and Hsu 1975; loid number of chromosomes of all but one species
7uh/e i Taxonom) and source of genera and species examined .___.
_ _ _ I
Subfamil?
Trik
Subsenus
Genus
Species
Strain (year of collection), source
G'
__.~._____.______
Culicinae
Aedini
Sabethini
Culicini Aedini Anophelinae *
Rockefeller Inst.. USA (1959). D. W. Jenkins: Memphis. USA (1986). S . Jones alhoprcrirs Mauritius (1974). C. Courtois; Oahu. Hawaii (1971). L. Rosen; Malaysia (1973). B. Knudson frai opicrus Nagasaki. Japan (1988). Y. Eshita sea I111 Bangkok, Thailand (1972), D. Gould pfyircsreiisis Suva. Fiji (1982). D. Gubler ulcusrdi Taiwan (1981). J. Lien atiiiairdulei Taiwan (1963, G. A. H. McClelland Mauritius (1965). R. Marnet mu.wurensr .r Pr-oroniar~lecijurr-iser-iarits St. Joseph Co., USA (19691, R. Beach; Potato Creek, St. Joseph Co., USA (1990). D. Wesson Iiender-soilI St. Joseph Co., USA (1981). S. Paulson; Eddy St., St. Joseph Co.. USA (1990). S. Hanson Ochler~~rari~sarropa1pu.r Coosa. USA (1972), R. Brust epar'trus Sansal, El Salvador (1968). S. Breeland Gymnonietopu medioi.irtatus Puerto Rico (1981) R. Novak Tripteroides Tripteroides hambusa Kyushu Island, Japan (1987), W. Hawley Sahethes Sahethes cxanrus Balboa heights, Panama (1983), J. L. Peterson Wjeomyia Wyeompia smithii UNDERC* (1989) Culer Culer pipiens quinquefasciarus Gainesville, USA (l969), R. Lowe At-nii,qere> Arnrigeres suhaibarrrs Japan (1971). M. Sasa Huema,qo,qrrs Hacmagogits eqitinus Panama (1979). J. L. Peterson Anripheler Anoplirles qiradrimacrrlariis Savannah, USA (1971), H. Schoof
A edc
Sr~qomrin
urg\prr
74
9 46 40 45 5 43 19
21 60 60
50 2 21 2
43 53 21 36 52
0 125 1 I4 26 40
Approximate generations in colon)
* L'nivervty of Notre Dame Environmental
Re\earch Center at Land O'Lakes. Wirconsin
is 6 (2n = 6). The only exception, Chagasia bathana niques, the mosquito karyotypes do not demonstrate of the subfamily Anophelinae, possesses 4 pairs of any linear differentiation for NORs along the length chromosomes (KREUTZER1978). In general, species of chromosomes. A different approach, using silver of the subfamilies Toxorhynchitinae and Culicinae staining technique of GOODPASTURE and BLOOM demonstrate a considerable uniformity with respect (1975), has also failed to locate ribosomal RNA to their karyotypes. A typical karyotype comprises genes on the mitotic mosquito chromosomes (RAO 3 pairs of homomorphic chromosomes: a pair of and RAI 1987a). small chromosomes (chromosome I), a pair of large In situ hybridization has been extensively used chromosomes (chromosome 2) and a pair of inter- in the localization of ribosomal RNA gene cluster mediate-sized chromosomes (chromosome 3) (RAI on the chromosomes of both animals and plants 1963, 1966; WHITE1980: RAOand RAI 1987a). In (PARDUE et al. 1970; HENDERSON et al. 1972, 1974a, a karyotype, all three pairs of chromosomes may b; EVANSet al. 1974; PARDUE and Hsu 1975: Hsu be metacentric, or one or two chromosome pairs et al. 1975; SCHAFER and SCHAWR 1980; VITELLI et et al. 1988; VISSERet al. may be slightly submetacentric (RAI 1963, 1966: al. 1982: DELUCCHINI WW1T.E 1980: MUNSTERMANN and M A R C H I 1986: 1988). In insects such as Drosophila and the grassRAOand RAI 1987a). The lengths of all three pairs hoppers, in situ hybridization has been used to locof chromosomes often vary in different species (RAI ate the rRNA genes on their chromosomes (SPEAR 1963. 1966; BAKER and ASLAMKHAN 1969: R ~ ~ a n 1974; d HENNIG et al. 1975; SCHAFER and SCHAFER RAI 1987a). In contrast, the species of the subfami- 1980: SINIBALDI and CUMMINGS 1981; WHITEet al. lies Anophelinae possess 2 pairs of metacentric 1982). However, in situ hybridization has not so far chromosomes of unequal size and one pair of het- been used to locate the ribosomal RNA genes on eromorphic sex chromosomes, X and Y (KITZ- the chromosomes of mosquitoes. In this paper we MILLF;R 1967). Using standard chromosome techreport the successful in situ localization of ribo-
Hrieditus 113 llY90)
soma1 RNA genes on the mitotic chromosomes of 20 species of mosquitoes belonging to 8 genera of subfamilies Culicinae and Anophelinae. In addition, the copy number of rRNA genes has been determined in 13 species, using dot-blot hybridization.
18s+
zxs
RIBOSOMAL R Y A G E ~ E SI N MOSQUIIOES
279
at 35°C. The slides were dehydrated in 70 % ethanol (2x10 min), 95 % ethanol (2x5 min) and air-dried. The slides were coated with Kodak NTB-2 emulsion diluted 1:l with distilled water at 43°C and stored at 4°C for 11-25 days. After exposure, the slides were developed, fixed and stained in S 5% Giemsa (30 min) as described in PARDUE (1985).
DNA extraction and dot-blot hybridization. DNAs were extracted from honey-fed adult female Mosquito species and chromosome preparation. mosquitoes according to BLINand STAFFORD ( 1976) -The details of genera and species of mosquitoes with minor modifications as described in BRADemployed during the present study are given in FIELD and WYATT(1983). A typical yield from 10 Table 1. All mosquito species used were from la- adults was 8-15 pg DNA, depending on the size boratory stocks, except Wyeomyia smithii. The lar- of mosquito species used. vae of Wy. smithii were collected from the pitcher Since the number of 18s rRNA genes equals the plants (Sarracenia purpurea) at the University of number of 28s genes or the ribosomal repeat units, Notre Dame Environmental Research Center at 18s DNA was used as a standard and the nitrocelLand O'Lakes, Wisconsin. Mitotic chromosome lulose membrane was hybridized with '*P-labeled preparations were obtained from colchicine-treated pE2 DNA. To isolate the 18s rDNA insert, plasmid brain ganglia of fourth instar larvae, following the pE2 (20 pg) was digested with EcoRI to excise the complete 1.8 kb 18s DNA under the conditions method of RAOand RAI(1987a). recommended by the supplier. The digest was run DNA probes and nick-translation. - Three recomin 0.8 % agarose, and the fragments were purified binant plasmids: pP11, pP1 and PE2 were used as from the agarose, using a Geneclean kit (Bio 101 DNA probes (BLACKet al. 1989). Plasmid p P l l Inc. USA). contains the 28Sp gene plus about 800 bp of inStandard DNA and species DNA were blotted tergeriic spacer (IGS). Plasmid pP1 contains the onto a nitrocellulose membrane, using a Schleicher complete 28Sa gene and some internal transcribed and Schuell manifold. The nitrocellulose membrane spacer (ITS) of the ribosomal cistron. Plasmid pE2 was vacuum dried for 2 h at 80"C, and prehybridcontains the complete 1.8 kb 18s gene. These re- ized in 50 % deionized formamide, SxSSC, 0.5 R combinant plasmids are pUC 118 subclones derived SDS, 5 % dextran sulphate, 5 x Denhardt's solution from a lambda EMBL3 rDNA clone from a ge- and 100 pg/ml denatured calf-thymus DNA at 42°C nomic library of Ae. albopictus (BLACKet al. 1989). overnight. 32P-labeledpE2 DNA was directly added The subcloned DNAs were purified according to to the prehybridization mixture and the hybridizaMANIATIS et al. (1982). Each subcloned DNA was tion was carried out for 16-24 h at 42°C (SAMBROOK nick-translation with 3H-dCTP (18 Ci/mmole) and et al. 1989). Following hybridization, the membrane ?H-dGTP (10 Ci/mmole) using a BRL (Bethesda was washed twice in 1 x wash buffer (10 x wash Research Laboratory USA) nick-translation kit ac- buffer = 3M NaCl, 0.6 M Tris-HCI, pH 8.0, 0.02 cording to a standard protocol of MANIATIS et d. M EDTA, pH 8.0) for 10 rnin each at room tem(1982). For dot-blot hybridization, pE2 DNA was perature, twice in 1 x wash buffer and 1 % SDS for nick-translated with "P-dCTP (3000 Ci/mmole) as 30 min each at 60°C, and finally twice in 0.01 x described above. wash buffer for 30 min each at room temperature. In situ hybridization. - Pretreatment of the slides The membrane was dried at room temperature, and in situ hybridization were carried out according wrapped in the Saran wrapTMand exposed to Kodak to PARDUE (1985). In situ hybridization was con- X-OMAT X-ray film with a Dupont intensifying ducted overnight at 40°C using 1 6 ~ 1 0 ~ 4 0 ~ screen 1 0 ~ at -70°C for a range of time. The copy cpm per slide in 50 p1 hybridization buffer ( ~ x S S C , number of rRNA repeat units was determined by SO % formamide, 5 % dextran sulphate, 1 % Den- comparing the dot intensity of the species DNA hardt's solution, 0.037 pg probe DNAs, 7 pg calf- with the appropriate standard DNA of the autorathymus DNA). The three rDNA probes (pP1, pP11 diograms that were in linear range of the film reet al. 1989) using a EC- densiand pE2) were mixed for each in situ hybridization sponse (ZIMMERMAN reaction. The non-specific bound probes were re- tometer and a Zeineh software 1-D scanner (Fisher moved by incubating slides in 2xSSC (3x10 min) Scientific Inc. USA).
Materials and methods
Fig. l a d . In situ hybridization of "-labeled rDNA probes to mitotic chromosomes from the genus Aede.5, subgenus Stegoniyia: (a) Ae. oexyptr, (b) Ae.,fluwpicri(.s,( c ) A e searoi. (d)Ae. alhopictus. The arrows point to sites of hybridization. Bar = 10 pn.
Chr~onzosomeider~ificatioriiri czrliciires. - To definitively identify chromosome pair(s) involved in in situ hybridization. chromosome measurements were made on the photomicrographs of 8-1 0 meta-
phase per species in culicines, using a digitizer. The data on chromosome measurements are not presented here but may be obtained on request.
IXS
+ ?XS
RIBOSOMAL RNA GENES IN MOSQUITOES
28 1
Fig. 2a-f. In situ hybridization of 3H-labeled rDNA probes to mitotic chromosomes from the genus Aedes, subgenera Stegomyia and Protomacleaya: (a)Ae. polynesiensis, ( b )Ae. alcasidi, (c)Ae. annandalei, ( d ) Ae. mascarensis, (e)Ae. triseriatus, and (f) Ae. hendersoni. Bar = 10 pm.
Fig. 3a-e. In situ hybridization of 3H-labeled rDNA pro-
Results Hybridization of rDNA probes to mitotic chromosomes. - In situ hybridization of mitotic chromosomes with 3H-labeled rDNA probes showed that a single site per haploid karyotype was hybridized in all species except in Aedes triseriatus. In Ae. triseriatus, rDNA probes hybridized to two sites per haploid karyotype. The results for various species were as follows: rDNA probes hybridized on one arm of chromosome 1 in all species of Aedes examined (Fig. 1, 2 and 3a-b) except in Ae. triseriatus and Ae. mediovittatus. In Ae. annandalei, which possesses submetacentric chromosome 1, the site of hybridization was located on the long arm (Fig. 2c).
bes to mitotic chromosomes from the genera Aedes, (subgenera Ochlerotatus and Gymnometopa) Wyeomyia and Tripteroides: (a) Ae. atropalpus, ( b )Ae. epactius, ( c ) Ae. mediovittatus, (d) W y . smithii, and (e) Tp. bambusa. Bar = 10 p m .
Probes hybridized to one site each on chromosome 1 and chromosome 3 in Ae. triseriatus (Fig. 2e) and to a site on chromosome 2 in Ae. mediovittatus (Fig. 3c). In the species of other culicine genera, rDNA probes hybridized on chromosome 1 in Wyeomyia smithii (Fig. 3d), Culex pipiens quinquefasciatus (Fig. 4b) and Sabetkes cyaneus (Fig. 4c); chromo-
Her-rdtrus 113 (1990)
Fig. 4a-d. In situ hybridization of 3H-labeled rDNA probes to mitotic chromosomes from the genera Haemagogus. Culex, Sabethes, and Armigeres: (a) Hq. equinus, (b) C.r. pipiens quinquefasciutus, (c) Su. ryatieus, and ( d ) Ar. subalbatus. Bar = 10 +m.
some 2 in Haemagogus equinus (Fig. 4a); and chromosome 3 in Tripteroides bamhusa (Fig. 3e) and Armigeres slcbalbatus (Fig. 4d). The location of hybridization sites on the specific chromosome varied in different species (Fig. 6). For example, the hy-
bridization sites were located close to the telomere in Ae. atropalpus, Ae. epactius and Tp. bambusa; in an intercalary position in Hg. equinus and Ae. triseriatus; and near the centromere in the rest of the culicine species examined.
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283
Discussion
Fig. 5. In situ hybridization of 3H-labeled rDNA probes to mitotic chromosomes of Anopheles quadrimaculatus. Bar = 10 pm.
In Anopheles quadrimaculatus, probes hybridized strongly on the X and Y chromosomes (Fig. 5). Autoradiographic silver grains completely covered the heterochromatic arms of both sex chromosomes. Hybridization of rDNA probes to interphase nuclei (Fig. 7). -Except in Ae. triseriatus, a single cluster of silver grains was observed over interphase nuclei of all species. Fig. 7a shows interphase nuclei with a single cluster of silver grains in Ae. albopictus. In most of the cases, silver grains were clustered at the periphery of interphase nuclei. Two clusters of silver grains were detected over the interphase nuclei of Ae. triseriatus (Fig. 7b). However, some interphase nuclei with a single cluster of silver grains were also noted, suggesting that both chromosome pairs may organize a common nucleolus as well as separate nucleoli. Alternatively, these nuclei may simply represent polar views of the nuclei. Estimation of rRNA gene copy number. - Fig. 8 shows an autoradiograph of dot-blot hybridization. The copy number was determined in 13 species for which the 1C-DNA content is known (Table 2). The copy number per haploid genome varied nearly 26fold among mosquito species examined the minimum copy number was 39f3.27 in Sa. cyaneus, and the maximum was 1023k68.14 in Ae.flavopictus. No correlation was observed between the 1CDNA content and the copy number of rRNA genes (r = 0.28, df = 12, p>0.05).
The present study has shown that the rRNA gene cluster is confined to a single chromosome per haploid genome among all mosquito species, except Ae. triseriatus, belonging to 8 genera of Culicidae. Further, the ribosomal genes are conserved on chromosome 1 in all the species of Aedes except in Ae. mediovittatus and Ae. triseriatus. The ribosomal RNA genes are also conserved on chromosome 1 in Cx. p . quinquefasciatus, Sa. cyaneus and Wy. smithii. In contrast, they are located on chromosome 2 in Ae. mediovittatus and H g . equinus and chromosome 3 in Ar. subalbatus and Tp. bambusa. The only species examined from the subfamily Anophelinae, An. quadrimaculatus, possesses ribosomal genes on the sex chromosomes. In the species of Aedes and Culex where linkage group-chromosome correlations have been made, chromosome 1 pair has been shown to be involved in sex-determination ( M A C D O N A LRAI D ~ 1970; ~ BAKER^^ al. 1971a, b; DENNHOFER 1972; BHALLA et al. 1974). The sex is determined by a single pair of alleles or a segment of chromosomes for which the males are heterozygous and the females are homozygous (GILCHRIST and HALDANE 1947). Even in those cases where no correlations have been made, it has been assumed that this is true for them as well (MOTARA and RAI 1978). This indicates that the ribosomal genes are conserved on the sex chromosomes in a majority of mosquito species. In other insect species, the NORs have been localized on the X and Y chromosomes in Drosophila melanogaster, D. simuhns and D . hydei (SPEAR1974; H E N N I Cal.~ 1975), ~ the X chromosome only in D . virilis (ENDOW and GALL1975), the Y and microchromosomes (autosomes) in D . nasuta nasuta and D . n. albomicans (HAGELE and RANGANATH 1983), the X and microchromosomes in the ‘mulleri’ complex of Drosophila (BICUDO 1981) and Zaprionus indianus (KUMAR and GUITA 1987), the microchromosomes only in D. tumiditarsus (SINIBALDI and CUMMINCS 1981), and the X and autosomes in the grasshoppers (WHITEet al. 1982). In situ localization of ribosomal genes on chromosome 1 in Ae. aegypti is in contrast to the finding of MACDONALD and RAI(1970). Based on the presence of a slightly unstained region on chromosome 3 in some preparations in the above species, they suggested that the same may represent NORs. However, such situations are not uncommon. Hsu et al. (1975) demonstrated that the weak constrictions noted in the karyotypes of some mammalian species are not always the sites of ribosomal genes. In Sa. cyaneus, MUNSTERMANN and MARCHI(1986) ob-
CHROMOSOME 1
CHROMOSOME 2
CHROMOSOME 3
SPECIES
Aedes aegypti, Ae. albopictus, Ae. flavo pictus. Ae. seatoi, Ae. polynesiensis, Ae. alcasidi. Ae. mascarensis. Ae. hendersoni, Sabethes cyaneus, Wyeomyia smithii, Culex pipiens quinquefasciatus
Aedes annandalei Aedes triseriatus
Aedes atropalpus, Ae. epactius
IXI
I X I
Aedes mediovittatus
I x 1
Tripteroides bambusa
I x i
Arrn/geres subalbatus Haemagogus equinus
I X I
IXI
Anopheles quadrimawlatus
Fig. 6. Diagrammatic repre5entation of mitotic chromosomes of mosquito species in situ hybridized with 3H-labeled
rDN.4 probes.
served recurrent association of the nucleolus with chromosome 1 in polytene nuclei. They suggested that the NORs are probably located on chromosome 1 in this species. Our result of in situ hybridization confirms their finding. In addition to chromosome 1. ribosomal genes are also located on chromosome 3 in Ae. rriseriurus. Sometimes i t was difficult to determine whether the large pair of chromosomes with hybridization is chroinosome 2 or chromosome 3. The difference between the lengths of these chromosomes is only 0.17 Kni (data not shown). It appeared that in some inetaphase plates hybridization sites are located on chromosome 2 and in others on chromosome 3 from the \ame individual. Since in majority of the cases, 4gnal is located o n chromosome 3. we concluded
that the second site of ribosomal gene is on chromosome 3. Recently, DELUCCHINI et al. (1988) reported extra ribosomal sites in the genome of Triturus vulguris meridiotiulis. These sites contain only IGS and are devoid of 18s and 28s genes. Unlike the Triturirs situation, both chromosome 1 and chromosome 3 of Ae. [riser-iatus still hybridize to rDNA probes that lack pP11, and therefore are devoid of IGS sequences (data not shown). Thus the second site of hybridization in Ae. tt.iser-iatus appears to be a bona fide rDNA site. and not just transposed IGS sequences. Ae. triseriufltsand Ae. henrlersoni are sibling species and crosses between them are compatible biand CRAIG1968). Presence directionally (TRLIMAN of an additional site of ribosomal gene cluster in
Hereditus 113 (1990)
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285
Fig. 8. Autoradiograph of dot-blot hybridization. The 18s DNA standards were blotted in column 1 and 2: Al, 0.7 ng; B1, 1.4 ng; CI, 2.8 ng; D1, 5.6 ng; El, 7.0 ng; F1, 1.4 ng; GI, 100 ng lambda DNA as a negative control; A2G2, each with 1.4 ng 18s DNA. Each standard 18s DNA sample was supplemented with 100 ng lambda DNA as a carrier. The species DNAs were arranged as follows: A3&A4, Aedes aegypti; B3&B4, Ae. albopictus; C3&C4, Ae. flavopictus; D3&D4, Ae. seatoi; E3&E4, Ae. polynesiensis; F3&F4, Ae. alcasidi; A5&A6, Ae. annandalei; Fig. 7. In situ hybridization of 3H-labeled rDNA probes B5&B6, Ae. mascarensis; C5&C6, Ae. triseriatus; to interphase nuclei: (a) Aedes albopictus and (b) Ae. D5&D6, Ae. hendersoni; E5&E6, Ae. atropalpus; F5&F6, triseriatus. Bar = 10 pm. Ae. epactius; G5&G6, Ae. mediovittatus; A7&A8, Tripteroides bambusa; B7&B8, Sabethes cyaneus; C7&C8, Ae. triseriatus and its absence in Ae. hendersoni and Wyeomyia smithii; D7&D8, Culex pipiens quinquefasciatus; E7&E8, Armigeres subalbatus; F7&F8, Haemagogus other Aedes species indicate that it may be of recent equinus; and, G7&G8, Anopheles quadrimaculatus. Each origin. To account for this observation, a transloca- sample dot except G7&G8 contains 1 p g DNA, while tion is postulated, where a substantial amount of G7&G8 contain 0.5 pg DNA. The hybridization and the ribosomal gene cluster has been translocated from post-hybridization washing were carried out as described chromosome 1 to chromosome 3, possibly via extra- in the Materials and methods section. The intensity of chromosomal circular rDNA intermediates (Ro- species sample dots was compared with 1.4 ng (0.76~10’ copies of 18s gene; 1 picogram DNA = 0 . 9 8 ~ 1 0kb, ~ CHAIX et al. 1974). The significance of two sites of 1985a) standard DNA. To test if the copy the rRNA genes in Ae. triseriatus is not clear. Pres- CAVALIER-SMITH ence of two sites of the rRNA genes in Ae. number estimate varies as a function of stringency, the whole experiment was repeated at low stringency conditriseriatus resembles the analogous situation found tions (25 % formamide and the hybridization at 35°C with in D . hydei (HENNIGet a]. 1975), grasshoppers subsequent washing at 48°C). The copy number estimate (WHITEet al. 1982), mammals (Hsu et al. 1975), of the rRNA genes at low stringency conditions was in and urodele amphibians (MACGREGOR and SHER- agreement with that at high stringency conditions (data WOOD 1979; DELUCCHINI et al. 1988; WILEYet al. not shown).
1989), where multiple sites of the rRNA genes are known to occur. In addition to the physical mapping of the rRNA genes on the chromosomes of mosquitoes, another reason to undertake this study was to examine the extent to which chromosome repatteming has taken place during speciation of mosquitoes. The general uniformity in the karyotypes of Culicinae has led to speculate that the speciation of Culicinae has
more relied on point mutation than chromosome repatteming (see MOTARA and RAI1978). However, the latter studies by MOTARA and RAI(1978), MUNSTERMANN (1981) and RAOand RAI (1987a) have given clear indications of chromosome repatteming through translocations and inversions. The chromosome complements of different Aedes species can-
286
Her-editus 113 (IYYUJ
A K L M A R FT 4L
Table 2 The copy number of rRXA gene5 among mosquito species
IC-DNA content (P€)"
No. of rRNA genesilkg DNA~
0.81
o.s2x10y
423k3 1.9
1.24
0.67~10'
82 1545.23
Ae. flaropictus Ae. seator Ae. polvnesiensis Ae. ulcmidi Ae. annandaler Ae mascarensis Ae. triwr-iarus (St. Joseph Co., 1990) Ae. hendersoni ( S . Joseph Co., 1990)
1.33 0.97 0.73 0.97
0.77~10~ 0 . 1 8 10' ~ 0.55~10' 0.79~10' 0.71x109 0.5 1x10' 0 . 3 410' ~
-
o.19x109
-
Ae atrnpalpus
-
0.34~10' 0.2Sx1OY 0.22~10~ 0.22x1OY
-
Ae. epuctius Ae. medrovirrarus Tp bamhusu So. cymeus W?. smirhii Cx. p yuinquejasciatus Ar. .suhalhatus Hg.equinus An. quadrrmaculatus
Species (Strain)
No. of rRNA
geneshaploid genome ( ~ s E M ) ~
~
Ae. aegypri (Rockefeller) Ae. albopicrus (Oahu)
-
I .52
~
0.79 0.86 0.54 1.24 1.12 0.2s
O.OSx10'
0.91~10' 0 . 1 6 1~0' 0. 15x10' 0.04~10~
0.96~10~
1023568.14 1785l3.M) 398119.73 762k48.73 -
512k36.12
-
39f 3.27 783+S 1.8 87f 3.18 189k47.46 45C 6.35 47W36.60
not be distinguished from each other. However, of mosquito species supports this assumption. The they can be distinguished from each other in a few physical location of the rRNA gene cluster on the cases based on their C-banding patterns (MOTARA chromosome arm close to the telomere in Ae. atroand RAI 1978; RAOand R.41 1987a). The chromo- palpus, Ae. epactius and Tp. bambusa, at an intersome complement of Ae. mediovittatus is the only calary position in Hg. equinus and Ae. triseriatus, exception that can be distinguished from the chro- and close to the centromere in the rest of the culimosome complements of other Aedes species. The cines can be explained by paracentric inversions chromosome complement of Ae. mediotittatus con- (see Fig. 6). Further, no correlation exists between tains a pair of much smaller chromosome 1 , a pair the taxonomic grouping of the species and the chroof much larger chromosome 2 and a pair of interme- mosome location of the rRNA genes. For example, diate-sized chromosome 3 (see Fig. 3c). This sug- the rRNA genes are located on chromosome 1 in gests that a considerable chromosome repatterning Sa. cyaneus and Wy. smithii and on chromosome 3 has occurred in the karyotypic evolution of this spe- in Tp. bamhusa even though these species are incies. In situ hybridization to chromosome 2 of this cluded in the same tribe Sabethini. Similarly, the species supports this assumption. The metaphase species of Aedes, H g . equinus and Ar. suhalbatus complement of H g . equinus with the rRNA gene are classified within the tribe Aedini, but the rRNA cluster on chromosome 2 can be derived from a genes are located on chromosome 1 in the species metaphase complement containing the rRNA gene of Aedes except Ae. mediovittatus, chromosome 2 cluster on chromosome 1 by a translocation be- in H g . equinus, and chromosome 3 in Ar. subaltween chromosome I and chromosome 2 , possibly hatus. 1976) of a small chrovia the 'shift' (STRICKBERGER In a cell line of Ae. albopipictus that was derived mosome segment containing the rRNA gene cluster. from minced neonate larvae (SINGH1967), SPRADA similar mechanism can place the rRNA gene LING et al. (1974) estimated 430 copies of rRNA cluster from chromosome 1 to chromosome 3 in Tp. genes per haploid genome using Cot-curve analysis. hurnhusa and Ar. subalhatus. The location of the Recently, PARKand FALLON (1990) estimated 30C rRNA gene cluster on chromosome 1 in a majority 400 rRNA genes in the same cell line, and 35C.500
Hereditas 113 (1990)
18s+ 28s RIBOSOMAL R N A GENES I N MOSQUITOES
287
in larvae, and about 1200 copies in the pupae and length of 9.0 kb in Ae. aegypti (GALEand CRAMPadults of Ae. aegypti, using dot-blot hybridization. TON 1989) and 15.6 kb in Ae. albopictus (PARKand However, the interpretation of their data was based FALLON1990), the proportion of haploid genome on the 1C-DNA content of 1.45 pg for Ae. albopic- represented by the rDNA in these species is 0.96 tus cell line (SPRADLING et al. 1974) and 1.30 pg for and 2.10 % respectively. In conclusion, the present study has shown that Ae. aegypti (DITTMANN et al. 1990). During the present study, we estimated 423k31.9 and 821k45.23 the rRNA gene cluster is located on a single chrocopies of rRNA genes per haploid genome in the mosome per haploid genome in all species of mosadults of Ae. aegypti and Ae. albopictus respec- quitoes examined except in Ae. triseriatus. Howtively. However, our interpretation was based on ever, the species differed nearly 26-fold in the the 1C-DNA content of 0.81 pg in Ae. aegypti and abundance of rRNA genes. The presence of the 1.24 pg in Ae. albopictus (RAOand RAI 1987b). The ribosomal RNA gene cluster on different chromohaploid nuclear DNA content has been shown to somes and at different positions on the chromosome vary nearly 3-fold among world strains of Ae. albo- arm among mosquito species indicates that a conpictus (KUMARand RAI 1990). Our preliminary siderable chromosome repatterning through translostudy has shown a 5-fold variation in rRNA gene cations and inversions has occurred in the karyocopy number between two strains of Ae. albopictus typic evolution of the members of Culicinae and that differed 3-fold in their 1C-DNA contents; the corroborates earlier findings of RAIet al. (1982) and (1981). Savannah strain from the USA with 1.65 pg 1C- MUNSTERMANN DNA content has 1083 copies of rRNA genes per haploid genome and the Koh Samui strain from Acknowledgements. -We thank Drs S. Kambhampati and L. E. for critically reading the manuscript; Mr Brian Thailand with 0.62 pg 1C-DNA content has Munstermann Turco, Mrs Peggy Hodges and Miss Dawn Verleye for rearing the 218f12.5 copies (KUMAR and RAI,unpublished). In mosquitoes in the insectary; and Mrs Tracy Frost for help in typthree species examined from the Ae. albopictus sub- ing. This work was supported by NIH research grant 5R01 A1 group (viz.: Ae. albopictus, Ae. flavopictus and Ae. 21443 to KSR. We also thank the reviewers for suggesting changes seatoi), 1.4-fold difference in the haploid DNA con- in the earlier draft of this paper. tent is accompanied by over 5-fold difference in the copy number of rRNA genes. Among other species of mosquitoes examined, the copy number spanned References M. 1969. Karyotypes of some with a minimum value of 39k3.27 in Sa. cyaneus BAKER,R. H. and ASLAMKHAN, Asian mosquitoes of the subfamily Culicinae (Diptera Culici. to a maximum value of 783k51.8 in Wy. smithii. It dae). - J . Med. Entomol. 6: 44-52 has been suggested that there is a positive correla- BAKER, R. H., SAKAI,R. K. and MIAN,A. 1971a. Linkage grouption between the copy number of rRNA genes and chromosome correlation in a mosquito: inversions in Culex 1C-DNA content (BIRNSTIEL et al. 1971; CAVALIER- tritaeniorhynchus. - J . Hered. 62: 31-36 SMITH1985b). In contrast, no such correlation was BAKER,R. H., SAKAI,R. K. and MIAN,A. 1971b. Linkage groupchromosome correlation in Culex tritaeniorhynchus. - Science observed during the present study similar to the 171: 585-587 observation made in the flax genotrophs by CULLIS BECKINGHAM, B. 1982. Insect rDNA. -In: The Cell Nucleus (eds H. BUSCHand L. ROTHBLUM), Academic Press, New York, (1976). As with the chromosome localization of the p. 205-263 rRNA genes, no correlation is observed between the S. C., CAIAIBA,A. C. I., CARVALHO, W. M. P. and SANBHALLA, taxonomic position of the species and the copy TOS,J. M. 1974. Translocations, inversions and correlations of number of the rRNA genes in mosquitoes examlinkage groups to chromosomes in the mosquito Culex pipiens fatigans. - Can. J . Genet. Cytol. 16: 337-850 ined. For example, the difference in the copy number of the rRNA genes between Wy. smithii and BICUDO,H. E. M. C. 1981. Nucleolar organizer activity and its regulatory mechanisms in Drosophila species of the ‘mulleri’ Sa. cyaneus with nearly the same 1C-DNA content complex and their hybrids. - Caryologia 34: 231-253 is as much as 20-fold, although both species are BIRNSTIEL,M. L., CHIPCHASE, M. and SPIERS,J. 1971. The ribosomal RNA cistrons. - Progr. Nucleic Acid Res. Mol. Biol. included in the tribe Sabethini. At present, the fac11: 351-389 tors controlling the copy number of the rRNA genes BLACK,W. C. IV, MCLAIN,D. K. and &I, K. S. 1989. Patterns among different species of mosquitoes are unknown of variation in the rDNA cistron within and among world popuand remain to be investigated. lations of a mosquito, Aedes albopictus (Skuse). - Genetics 121: 539-550 On an average rDNA constitutes only about 1 %, or at maximum only 3.7 % of total genome for eu- BLIN,N. and STAFFORD,D. W. 1976. A general method for isolation of high molecular weight DNA from eukaryotes. - Nukaryotes (see CAVALIER-SMITH 1985b) with more cleic Acids Res. 3: 2303-2308 values ranging around 1 % (SCHAFERand KUNZ BRADFIELD, J. Y. and WYATT,G. R. 1983. X-linkage of a vitellogenin gene in Locusta migratoria. - Chromosoma 88: 19&193 1987). Based on the average ribosomal repeat unit
olbopicri~s(Skuse). - Theor. Appl. Genet. 79: 748-752 M,ACDOSALD, P. T. and RAI, K. S. 1970. Correlation of linkage groups with chromo\omes in the mosquito Aedes aegypfi L. - Gerretics 66: 4 7 5 4 8 5 S. 1979. The nucleolus orgaM ~ C G R E C ~H.O C. R , and SHERWOOD, nirers of Pletkodon and Aiieides located by in situ nucleic acid hybridization with Xenopits 'H-ribosomal RNA. - Chromo.sonlo 72: 271-280 T., FRnwti. E. F. and SAMBROOK, J. 1982. Molecular M~\I.ATIS. Cloning: A Laboratory Manual. - Cold Spyin,? Hurhor Laboriltol~\.. .vt'\+'Y#rk M O T A R M. ~ , A. and R.u. K. S . 1978. Giernsa C-handing patterns iii A d e \ (Stegoniyiu) mosquitoes. - Chroniosoma 70: 51-58 h l L \ S T E R > 1 4 U \ . L. E. 1981. Enzyme linkage maps for tracing chromosomal evolution in Ardes mosquitoes. -In: Application of Generic..s 011 Cito/o,q? i n Insec~t.Yj.sremoric.s und Evolution ( e d M. W. SIOCK).U n w Oflduho. Moscon8.p. 129-140 ML\STERMAY\. L. E. and MAR('ii1. A. 1986. Cytogenetic and isot \ i x ~ a .S A. and G \ i i ~J . G. 1975. Ditferential replication of \atellite DNA iii polyploid ti\we o f D r ~ i . ~ o p / i ii./iur i / r s . Clrr~iryme profile of Soherhe.\ cymeus: a mosquito of the neotropical nin\ornir 5iJ: 175-195 J . tiered 77: 241-248 canop). F\ q \ \ , H J.. B%c k i A \ I L R A . dnd P4mi I.. 51. L. 1974. Location OISHI. M..LWKF.J. and W v ~ r r G. . R. 1985. The ribosomal DNA ot gcnw coding for I X S and l X S ribo\omal R N A in the human gene\ of Locu.\tu migraror-ia: copy number and evidence for riiniowmiu 48: 4 0 5 4 2 6 underreplication i n a polyploid tissue. - Cun. J . Bioc.lieni. Cell. + \ i P i o \ . J. IOXY. The riho\omal gene\ of the Biol. hi: 106&1070 .\ ' l l ~ q \ / l f l Elll. .I Rrrx h n 185: 3 I 1-3 17 P \ R D IE. 51. L. 1974. Localization of repeated DNA sequences in G I K i i i . S. A . and C w i 5 5 . H. V. 1976. Further studie> on the Xenopiis chromosomes. - Cold Spring Harbor Synip. 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