Diagnosis by Random Amplified Polymorphic DNA Polymerase Chain Reaction of Four Cryptic Species Related to Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) from Paraguay, Argentina, and Brazil

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Diagnosis by Random Amplified Polymorphic DNA Polymerase Chain Reaction of Four Cryptic Species Related to Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) from Paraguay, Argentina, and Brazil RICHARD THOMAS Department

C. WILKERSON, V. GAFFIGAN,

THOMAS EDUARDO

J. PARSON&l BERG0,2

AND

TERRY JOSE

A. KLEIN,

CONSOLIM3

of Entomology, Walter Reed Army Institute of Research, Washington, DC 20307-5100

J. Med. Entomol. 32(5): 697-704 (1995) ABSTRACT Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was applied to samples from widespread populations of the poorly characterized Anopheles (Nyssorhynchus) alhitarsis Lynch-Arribtizaga species complex, and 4 genetically differentiated species were distinguished. A screen of 65 random decamer oligonucleotide primers identified 12 primers, which produced 19 reproducible species-specific genetic markers and 4 markers common to 2 or more species. These markers were correlated in nearly all individuals of each species throughout the ranges sampled, including populations as far apart as 2,500 km. Each individual analyzed was from a different isofemale progeny brood, with associated morphological specimens. These specimens will facilitate studies to relate these species to previously reported chromosomal and enzymatic variation as well as to their feeding behavior and potential as malaria vectors. We hypothesize that 3 of the species have recognized valid names: An. (Nys.) alhitarsis Lynch-Arribdzaga, An. (Nys.) mxwajoara Galvgo and Damasceno, and An. (Nys.) deaneormm Rosa-Freitas, whereas the 4th is undescribed.

KEY WORDS Anopheks (Nyssorhynchus) alhitarsis Species Complex, random amplified polymorphic DNA, malaria vectors

MANY LINES OF EVIDENCE indicate that Anopheles (Nyssorhynchus) dbitarsis Lynch-Arrib5lzaga constitutes a complex of cryptic species. Anopheles albitarsis sensu Zuto (=Albitarsis Complex) has been documented or suggested to be differentiated at the epidemiological, behavioral, morphological, cytological, and biochemical levels (Galv50 and Damasceno 1944, Kreutzer et al. 1976, Steiner et al. 1982, Linthicum 1988, Rosa-Freitas 1989, Narang et al. 1993). It is considered an important vector of malaria parasites in certain localities, whereas in others its role is secondary or unimportant (reviewed by Rosa-Freitas et al. 1990). Because of a lack of voucher specimens for comparative study, there is little identifiable correspondence between the differentiated forms that have been described in the various studies. Random amplified polymorphic DNA (RAPD) analysis is a polymerase chain reaction (PCR)based technique (Welsh and McClelland 1990, Williams et al. 1990), which provides a quick and effective means of identifying genetic markers to distinguish closely related species. RAPDs have

I Department of Plant Pathology,Universityof Nebraska,406 Plant Sciences Hall, Lincoln, NE 68583-0722. 2 Superentendencia do Controle de Endemicas, Rua Palo Souza, #166, Bairro da Luz, 01027, S%oPaula, Brazil. 3 Itaipu Binacional (Ecomuseu), 85890 Foz do Iguacu, Paranli, Brazil.

been used as a tool for genetic mapping, strain, species and population identification, and systematics on a wide variety of organisms (for example, Black et al. 1992, Chapco et al. 1992, Hadrys et al. 1992, Kambhampati et al. 1992, Bowditch et al. 1993, Gawel and Bartlett 1993, Perring et al. 1993, Williams et al. 1993). Multiple fixed RAPD markers easily were obtained to distinguish laboratory populations of the morphologically indistinguishable malaria vector An. (CeZZiu,)guwzbiue Giles and An. (Gel.) urubiensis Patton (Wilkerson et al. 1993). Using morphology to distinguish members of an Anopheles species complex is complicated greatly by a lack of genetically characterized, pure samples. Because the component species may not be distributed allopatrically, the potential for mixed samples causes doubt in whether variations, which are often slight between cryptic species, actually represent species differences or simply polymorphic characters within a species. Because of the ease with which fixed RAPD markers were obtained for mosquito species maintained in laborawe investigated whether RAPDs tory colonies, would be equally useful for analyzing natural populations of the poorly characterized Albitarsis Complex. Our goal in this study was to use RAPDs to determine how many species were represented in collections from southern Brazil, Paraguay, and

0022-2585/95/0697-0704$02.00/O

0 1995 Entomological Society of America

698

Table Map reference

JOURNALOF

1.

Collection

localities

Country, state/ province

for Albitarsis

Locality

MEDICAL

Complex

ENTOMOLOGY

species determined

Coordinates

Vol. 32, no. 5

by RAPD-PCR

Date

Collection no.

Species(n)

Brazil 10 11 4 2 6 5

sso Paul0 %o Paul0 szo Paul0 Pali Pari ParaG Parand

Ilha Comprida 6 km SW Registro Ponte Melo Peixoto Capanema Ilha de Maraj6 Santa Helena Near Guaird

24” 42.75’ S, 47” 31.6’ W 24” 36.8’ S, 47” 53.1’ W 22” 39.0s’ s, 53” 01’ W 1” 24’ S, 47” 11’ W 1” 00’ s, 49” 30’ w 24” 56’ S, 54” 23’ W 24” 04’ S, 54” 15’ W

9 Feb. 1989 26 Jan. 1992 10 Feb. 1992 Aug. 1993 Aug. 1991 30 Jan. 1992 1 Feb. 1992

3

Espirito Santo

Aguia Brdnca

18” S9’ s, 40” 44’ w

Rio Acaray

20 Jan. 1992

BR 73 BR .500, SO1 BR 508 BR 403 BFUR 001 BR 503 BR 504, 510, 511 BFURio 002

B(1)

25” 29’ S, 54” 42’ W

4 Feb. 1992

PA 1

A(9)

Hernandarias

2Y 22’ s. 54” 45’ w

6 Feb. 1992

PA 2

A(8), B(6)

Near National Airport

Not known

8 Feb. 1992

PA 3

B(9)

Posadas 90 km W Posadas Corrientes 11 km W Mercedes Santa Torn6 Baradero

27” 23’ S, 55” 53’ W Not known 27” 28’ s, 59” 50’ w 29” 12’ s, 58” OS’ w 28” 33’ S, S6” 03’ W 33” 48’ s, 59” 30’ w

30 Jan. 1992 31 Jan. 1992 31 Jan. 1992 1 Feb. 1992 2 Feb. 1992 6 Feb. 1992

AR AR AR AR AR AR

NW

Paraguay

8

9

Alto Paran Alto Pa-an& Alto Paran& Argentina

12 13 14 15 16 17

Misiones Corrientes Corrientes Corrientes Corrientes Buenos Aires

northern Argentina. We also sought to assign isofemale progeny broods reared from wild-caught females, with preserved voucher specimens of all stages, to their respective, genetically defined species. This will help in the recognition of morphological characters to distinguish the various species. This, in turn, may facilitate studies to relate the species to previously reported chromosomal and enzymatic variation as well as to their host preferences and potential as malaria vectors.

Materials

and Methods

Identification. A recent review by Peyton et al. (1992) is the b asis for the definition of the subgeAll species in the Albitarsis nus Nyssorhynchus. Complex were identified in the adult by: hindtarsomere 5 without a basal dark band and vein 1A mostly pale-scaled (Argyritarsis Section); sternum I with a distinct row of white scales on each side; and posterolateral scale tufts beginning on segments III or IV [on the very similar An. braxiliensis (Chagas) the tufts begin on segment II, see Linthicum (1988) f or additional immature characters]. Collection Methods. In 1992 we (R.C.W., T.A.K., E.B., and J.C.) collected females belonging to the Albitarsis Complex in southern Brazil, Paraguay, and northern Argentina. Additional specimens from Par& Brazil, were collected and reared by personnel at the U.S. Army Medical Research Unit in Rio de Janeiro (USAMRU-B). Brazil collections BR 73 (Ilha Comprida, Iguape), BR 501 (Registro), BR 403 (Capanema), and BWR 001 (Ilha de Marajo) were made from human-bait cap-

1 2 3 4 S 7

A(l), DC-3 A(g), D(2) MS) A(S) NW

tures. Collection AR 2 (90 km W Posadas, Argentina) were blood-fed females resting indoors. All others were collected in a Shannon trap, with the collectors and a propane lantern as attractants. Human and trap collections were conducted from dusk to 1 or 2 h after dusk. Specimen Sources and Treatment. Forty-eight to 72 h after a blood meal, the mosquitoes were separated and induced to oviposit on water by removal of a wing. Each F1 individual for morphological study consisted of a pinned adult and associated larval and pupal exuviae in 80% ethanol. The pinned adult and associated exuviae were given a unique code number so they could be associated with each other and other progeny from the same female. Other adults and larvae from reared individual females were given a unique family code, frozen in liquid nitrogen for DNA analysis, and are maintained at -70°C in our laboratory at the Smithsonian Institution. Specimens for morphological study will be deposited in the National Museum of Natural History, Smithsonian Institution, Washington, DC; U.S. Army Medical Research Unit, Rio de Janeiro; Insituto Oswald0 Cruz, Rio de Janeiro; and the Nlicleo de Pesquisa Taxonomica e Sistematica em Entomologia Medica (NUPTEM) at the Universidade de Sao Paulo. Collection localities and locality codes are listed in Table 1. A letter code followed by a number is the country code and collection number (for example, AR 7 is collection 7 from Argentina); each brood from the same female is indicated by a number in parenthesis [for example, AR 7(2) would be assigned to all progeny from the 2nd female from

September

1995

WILKEHS~N

ET AL.: DIAGNOSIS

collection 71 (Table 1; Fig. 1). Each individual and its exuviae from a given brood was then assigned a unique number [for example, AR 7(2)-l, is the 1st specimen to come from female AR 7(2)]. Isolation of DNA. DNA was isolated by phenolchloroform extraction from individual larvae or adults as described in Wilkerson et al. ( 1993). Amplification of RAPD-PCR. Procedures essentially followed Bowditch et al. (1993). Briefly, total reaction vohunes of 25 ~1 were used with the following final concentrations: 11 mM Tris-HC1 (pH 8.3); 50 mM KCl; 1.9 mA4 MgCl,; 0.1 mg/ml BSA; 0.1 mM each of dATP, dCTP, dGTP, and TTP; 0.24 pmol//_d (6 pmol) primer; 0.24.0 ng/pl template DNA; 0.02-0.06 U/p1 (OS-l.5 units) Tb9 DNA polymerase. A Perkin-Elmer Cetus model 480 thermocycler was used for all reactions with the following parameters: 1 min denaturation at 94°C followed by 45 cycles of 1 min at 94’C, annealing 1 min at 35°C and elongation 2 min at 72’C, all with minimum ramp times. Agarose Gel Electrophoresis. Using standard methods (Sambrook et al. 1989), amplification products were analyzed using the USA Scientific Extra Wide mini horizontal system (catalog no. 3488-0000), Ocala, FL) which uses 100 ml 1.5% agarose minigels with 0.8 pg/ml ethidium bromide run at SO \‘/25 mA for ~3 h in TBE (89 mh! Tris base, 89 mA4 boric acid, and 2 mM EDTA, pH 8.3). Amplification products were observed and photographed losing short wave (312 nm) UV. Molecular weight standards were provided by lambda DNA digested with Hind III, and phiX174 DNA digested with Hne III (Sigma, St. Louis, MO). The approximate molecular weight of amplification products was calculated using a program written for Lotus l-2-3 by A. F. Cockburn (United States Department of Agriculture, Gainesville, FL). Oligonucleotide Primers. Primers were purchased from Operon (Alameda, CA) and were 10 bases in length. Operon primer kits A, B, C (20 primers per kit), and DOl-DO5 were screened. Primers discussed in the text have the following sequences (5’-3’): B16, TTTGCCCGGA; B05, TGCGCCCTTC; DOl, ACCGCGAAGG; C07, GTCCCGACGA; B02, TGATCCCTGG; AOl, CAGGCCCTTC; C19, GTTGCCAGCC; C16, CACACTCCAG; C15, GACGGATCAG; Bll, GTAGACCCGT; A12, TCGGCGATAG; and A08, GTGACGTAGG.

Results As an initial screen for suitable RAPD markers, 65 primers were run on 10 individuals from 4 localities. Many primers produced potentially diagnostic bands, but only those that produced bright or consistent, or both, bands were tested on larger samples (see Wilkerson et al. 1993 for primer selection criteria). To identify individuals belonging to different species, we looked for multiple bands from one or more primers that always occurred

OF CRYPTIC Anopheles

BY RAPD-PCR

699

together in particular individuals; that is, sets of bands in apparent linkage disequilibrium. If RAPD-PCR regularly produces bands that are fixed and unique to a particular species, members of a species can be identified because they always share unique multiple bands. We selected the 12 best primers: these produced 19 unique and 4 shared bands, which enabled the identification of 4 genetically distinct taxa in the Albitarsis Complex from Brazil, Paraguay, and Argentina (Figs. 14; Tables 1 and 2). To facilitate discussion, these 4 taxa will be referred to by letter designations A, B, C, and D. Species A was identified by 3 unique marker bands produced by 3 primers, B16, AOl, and C15; species B by 3 unique markers produced by 3 primers, C07, B02, and C15; species C by 7 unique markers produced by 4 primers, DOl, AOl, C19, and C16; and, species D by 6 unique markers produced by 5 primers, DOl, Clri, Bll, A12, and A08. The marker bands referred to above generally were present in all members of their respective putative species, with occasional exceptions (Table 2). For example, the primer A01 1.41 kilo base pairs (kbp) marker band diagnostic for species C was absent in 3% (2/61) of individuals classified as species C. However, all of the individuals lacking the 1.41-kbp band showed the other 6 marker bands for species C. In this study there was only a single individual that lacked more than one of its expected marker bands. This was an individual that had 5 marker bands of species C, but lacked both markers with primer AOl. the 1.22- and 0.85kbp For a band to be considered a marker for a particular species, it had to have been scored as absent in all individuals ascribed to any of the other species. In addition to the marker bands, all primers produced other bands of varying intensity that were polymorphic within and among the putative species. Additionally, some bands were fixed in more than 1 species (Table 2). For example, a 0.80kbp band produced with primer DO1 was seen in most individuals of species C and D, but not in species A and B. Species A was present at 12 localities sampled in the southern part of the range of the complex, but was not collected in northern Brazil at Ilha de Maraj6 and Capanema (Fig. 1; Table 1). Species A was sympatric with species B at 3 sites (Fig. 1; Table l), and species A, B, and D were collected together near Guaira, Brazil. Species B was found at 6 sites with populations as far apart as 2,500 km (Registro-Capanema). Species B and C were sympatric at Capanema, Brazil. Species C was the only species collected at the Ilha Comprida and also was found near the type locality of An. nuzrajonru on Ilha de Maraj6; these 2 populations are also =2,FiOO km apart. Discussion Visual analysis of banding patterns produced by RAPD-PCR reactions on mosquitoes in the Albi-

700

JOURNAL OF MEDICAL

ENTOMOLOGY

Vol. 32, no. 5

0” -

Brazil

; /

13(A&D). 14 (A & D) 3

x



%-r-N/_,’

Argentina

Fig. 1. Collection localities and distribution of 4 species belonging to the Albitarsis Complex, determined by RAPD-PCR. (See Table 1) 1, Ilha de Marajo; 2, Capanema; 3, Aguia Branca; 4, Ponte Melo Peixoto; 5, near Guatra; 6, Santa Helena; 7, Hernandarias; 8, Rio Acaray; 9, near National Airport; 10, Ilha Comprida; 11, 6 km SW Registro; 12, Posadas; 13, 90 km W Posadas; 14, Corrientes; 15, 11 km W Mercedes; 16, Santa Tome; 17, Baradero.

September

1

2

199.5

3

4

5

WILKEKSON

6

7

8

9

10

ET AL.: DIAGNOSIS 11 12

13

OF CRYPTIC Anopheles BY RAPD-PCR

14 15 16 17 18

701

1

2

3

4

5

6

7

8

9

IO 11 12 13 14 15 16 17 18 1920

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 1920

-

Fig. 2. KAPD amplifications showing diagnostic markers for species C. Lanes 1 and 18, DNA size standard; lanes 2-5, primer AOl; lanes 69, primer C16; lanes lo13, primer C19; lanes 14-17 primer Dol.

tarsis Complex permitted the identification of 4 reproductively isolated taxa in a straightforward manner. Many correlated bands, derived from multiple primers, occurred exchisively in various subsets of individuals that readily could be classed as distinct. For our analysis, individuals were distinguished by “eye” in a manner similar to that described in Narang and Seawright (1988) as the “allelic cluster” method. In our RAPD analysis, groupings (taxa) were defined on the basis of correlated, bright marker bands as well as the similarity of their background patterns. Such background patterns usually consisted of faint or poorly resolved bands that would be difficult to score on an individual basis, but when taken together presented a visual profile

1

2

3

4

5

6

7

8

9

-

Fig. 4. Diagnostic markers for species B (lanes 2-7) compared with species A (lanes S-11), species C (lanes 12-15) and species D (lanes 1619) using primers B02 (top), and CO7 (bottom). Lanes 1 and 20, DNA size standard (see Fig. 1); 2-3, Registro, Brazil; 4-5, Hemandarias, Paraguay; 6, Guaira, Brazil; 7, Capanema, Brazil; S9, Baradero, Argentina; 10-11, Posadas, Argentina; 1213, Iguape, Brazil; 14-15, Ilha de Marajo, Brazil; 1617, Corrientes, Argentina; 18, Santa Helena, Brazil; 19, Guaira, Brazil.

1011

-

Fig. 3. RAPD amplifications showing diagnostic markers for species A, with primer B16. Arrow on right indicates fragment of -1.75 kbp. Lane 1, DNA size standard (see Fig. 1); 2-3, Baradero Argentina; 4, Registro, Brazil; 5, Santa Tome, Arg.; 6 Posadas, Arg.; 7, Conientes, Arg.; 8, Mercedes, Arg.; 9, near Santa Helena, Brazil; 10, near Guaira, Brazil; 11, Ponte Melo Peixoto, Brazil.

distinctive among individuals of the same group. Overall, as in the case of laboratory colonies of cryptic s ‘ (Wilkerson et al. 1%X3), the RA Iroved to be an effective means multiple bands that were unique nearly so) in each of the taxa. Because the taxa were distinguished by strictly correlated, fixed markers rather than allele frestatistical analyses were not quency differences, necessary to support the hypothesis of reproductive isolation. Because taxa recognized by RAPD-PCR were sympatric at many localities, we considered these to be reproductively isolated groups and therefore to represent distinct species. The fidelity of the RAPD patterns over widely separated localities (~2,500 km in both B and C) indicated that the genetic differences were not merely artifacts of isolation by distance; that is, they do not simply represent points along clines of genetic variation, in a single, genetically contiguous taxon. In addition, sample sizes from many of our localities are not sufficient to rule out the occurrence of additional sympatric species at these localities.

JOUHNAL OF MEDICAL ENTO~~OLOGY

702

Table species

Primrr 1316 BOS DO1

co7 B02 A01

Cl9

Cl6 Cli5

Bll Al2 A08

2. of

Summary Alhitarsis

Approximate kbp

I .73 1.74 2.2-2.:3 0.96 0.80 0.5.5 0.38 1.88 2.03 1.41 1.22 o.riij 1.30 1.17 0.88 0.99 1.24 1.oo 0.86 1.78 1.26 1.29 1.09 1.OY

of

RAPD

markers

used

to

identify

Complex

Species A *x1/82 1121/121 t 34/M o/:34 o/34 o/34 o/33 O/l 7 *118/11x w120 o/120 o/120 t 2Y2F; o/25 O/25 o/*51 O/15 o/15 *15/1.5 t Km5 O/l 5 O/lS O/l rj O/L5

B

c

D

o/:33 o/20 t32L32 OK32 o/32 o/32 *32/32 *:35/35 OL3.5 O/35 o/35 o/:35 t 22/22 o/22 o/22 o/35 O/L5 *15/15 O/l 5 t 15/1*5 o/15 (MlS o/1.5 O/lS

w30 t 24/m t 42/42 o/42 t41/42 *42/42 O/42 O/l 7 o/f% *59/fi1 *.5x/61 *59/Fi1 O/58 *58/.5x *558/5x *63/63 o/1 5 O/L5 O/L5 t 1m.5 o/15 o/15 o/15 O/l 5

O/Y o/9 tu/9 *H/M tw o/9 O/h

(VY o/9 o/9 O/Y o/9 tw9 o/9 o/9 o/9 *9/9 o/9 o/I) o/I) *9/9 *WI *9/9 *Y/9

Nunlhers listed below species indicate nunher of individuals with or without a marker/total nudwr of’ individuals tested. *, Spw_ies-specificmu-kcrs; t, marker in co~n~mmto > 1 species.

As with other genetic and chromosomal studies, our detection of a minimum of 4 species in the Albitarsis Complex demonstrated greater complexity within the group that has been recognized by morphological and behavioral studies. Previous studies attempted to relate malaria transmission with behavior and the relative length of the dark portion of hindtarsomere 2 (reviewed by RosaFreitas and Deane 1989, Rosa-Freitas et al. 1990). These attempts were based on observations regarding malaria transmission and biting-resting behavior; most were ambiguous. Galvao and Damasceno (1944) regarded An. nlbitnmis nlbitnrsis as exophilic, not related to malaria transmission, and having 77-84% of hindtarsomere 2 dark. In comparison, they described a new subspecies, An. nlhitamis cZonre.~tic~l.~,as endophilic, related to malaria transmission, and having 36-SO% of hindtarsomere 2 dark. AR nlbitnr-sis rlomestiats later was synonymized under An. mnrajonra by Linthiculn (1988). We agree with this synonymy because the type locality for both species is the same, and apparently no type specimens exist for An. czZhitnrsis domesticus to refute the synonymy. Rios et al. (1984) also concluded that there was no morphological or behavioral justification for the 2 subspecies, based on a sample of 18 populations from throughout Brazil. Linthicum’s (1988) revision recognized 2 species, An. nlbitnrsis .sensu stricto and An. wxv-jonm; however, his redescription of An. numzjonm was based primarily on specimens from Panam& not the type locality of Ilha de Maraj6, Brazil. Linthi-

Vol. 32, no. 5

cmm’s An. nznr-ajonm inchlded all specimens from Costa Rica to southern Brazil, except An. nlbitnmis specimens that since have been .s..s., including shown to belong to a morphologically and biochemically distinguishable taxon, An. denneonlm (Rosa-Freitas 1989). The great variation Linthicum observed in An. nuzrajonm is explained by our results that demonstrate a minimum of 4 species in the Albitarsis Complex, leaving little doubt that Linthicum was dealing with a mixture of taxa. Previous cytological and allozyme studies have recognized at least 3 cryptic species in the Albitarsis Complex (Kreuzter et al. 1976, Steiner et al. 1982, Rosa-Freitas 1989, Rosa-Freitas et al. 1990, Narang et al. 1993). However, as Narang et al. (1993) pointed out, there have been no associations made among the forms distinguished by cytology and allozymes, nor are associated observations of morphological variation documented by detailed description or voucher specimens. An exception is that of An. denneorrlm from RondGnia and Acre States in Brazil (Rosa-Freitas 1989), which can be diagnosed by larval n~orpl~ology, allozymes, and mitochondrial DNA (mtDNA) (Narang et al. 1993). We note that the occurrence of sympatric cryptic species adds a level of complexity to systematic studies; not knowing if ~1particular population sample contains a mixture of species causes difficulties in determining whether observed variation (at either the morphological or molecular level) is caused by within-species polymorphism, or to between-species differences. Collections from a particular locality may be iL mixture of several species and cannot be analyzed as a single population for comparison to collections from other localities without somehow first assigning individuals to respective constituent taxa. For example, Rosa-Freitas et al. (1990), performed distance analysis of morphological, behavioral, and allozyme data from collections from 10 localities, that these populations could be an d concluded separated into is groups. This is probably an artificial classification, because our data indicated that these component populations very likely represent an Imcharacterized mixture of cryptic species. Based on collection locality and preliminq morpliological analysis, we may tentatively assign the species identified in this study to thosi of previolis studies. Species A is probably Am nlbitnmis .E.s., because species A was the only taxon collected at Baradero, Argentina, the type locality of An. crlbitcrr.si,y, and our samples match the redescription of that species (Rosa-Freitas and Deane 1989). Species C is likely to be An. mm-ajonm, because it was the only species collected at Ilha de Mar:ljcj, Brazil, the type locality (Galvao and Damasceno 1942). Species D exhibits morphological characters in the larval stage that match diagnostic characters for An. denneomm (Deane et al. 1948, Rosa-Freitas et al. 1987, Rosa-Freitas 1989); that is, the larval head seta 3-C is distinctly branched. Species B cannot be related to any other described species.

September L

1995

WILKERSON

ET AL.: DIAGNOSIS

In a study of allozymes and mtDNA, Narang et al. (1993) also distinguished An. deaneorum and 3 additional “forms” of An. aZbitarsis from Brazil. Two of the forms, from Ilha de Marajo and Ilha Compride, were analyzed by us and proved to be An. marajoara (species C). The results of this study indicated that RAPDPCR can be very useful for analyzing uncharacterized complexes of sibling species. R&search should now focus on (1) attempting to correlate the species we report with those described in other studies; (2) conducting more detailed surveys throughout the entire range of the complex; (3) identifying morphological characters, if any, that distinguish the various species; (4) analyzing the effectiveness of this technique for rapid field diagnosis using sincrossing experiments gle markers; (5) conducting to assess degree of reproductive isolation; and (6) sequencing of selected genes for phylogenetic analysis. Our RAPD study permitted us to assign progeny voucher specimens, including both adults and larvae, to unique species that can now be subjected to detailed m&-phological examination in the hopes of identifying diagnostic characters for routine identification. Additionally, the characterized voucher specimens can be used for other or mtDNA types of analyses, such as allozyme studies. Because RAPDs are PCR-based, analysis requires only minute amounts of sample material, with a single primer reaction using only -1/5OOth of the DNA that can be obtained from a single individual. Because of this, RAPDs can be used in conjunction with any other DNA study, and it is possible to perform RAPD and chromosomal or allozyme analysis on the same individual (T.J.P., unpublished data). RAPD-PCR analysis should faciliY_ate the taxonomic descrintion of this and other cryptic species complexes. &ce the basic taxono; my is established, studies of the role of the various species in malaria transmission may be meaningfully pursued. Acknowledgments

We thank in Brazil, 0. Forattini (NUPTEM, University of Sao Paulo); J. Carlos and R. M. Tubaki (SUCEM, Sao Paulo); J. B. L ima and E. Milstrey (U.S. Army Medical Research Unit, Rio de Janeiro); J. P. da Silva, J. L. da Silva, and P. B. Torres (Funda@ National de Saude, Foz do IguaCu); G. Tomich and A. M. de Mello (Fundaqao da Saude, Curitiba); N. Pelegrini (Itaipu Binacional [Ecomuseu], Foz do Iguaqu); A. T. Tang (Funda@ National da Saude, Brasilia); and in Paraguay, B. Montiel, J. Gabriaguez, and M. Dominguez (Zona IV, SENEPA, Alto Parana). A. Marques (Funda@ National da Saude, Brasilia) provided invaluable administrative support as did J. Arias (Pan American Health Organization, Brasilia). In the United States, S. Mitra provided excellent laboratory assistance. We thank G. Duncan and J. Glick for comments on the manuscript. References

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BY RAPD-PCR

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29 Novernher

1994; accepted

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