MARCH 1992 Suscpptrsrlrry on,4iv. euADRIMAcuLeuusCoupr,nx 61

COMPARATIVESUSCEPTIBILITY OF SPECIESA, B AND C OF ANOPHELES QUADRIMACULA"US COMPLEX TO INFECTION WITH SUBPERIODICBRUGIA MALAYI AND PAHANGI (NEMATODA:FILARIOIDEA)1

J. K. NAYAR,, J. W. KNIGHT,' P. E. KAISER,3 J. A. SEAWRIGHT3 ETqNS. K. NARANG3I

ABSTRACT. Susceptibilities of natural populations of sibling speciesA, B and C of the Anopheles quadrimaculntysc9mpl91 and the colonized strain A to subperiodicBrugin rnalnyi and Brugia pihangi were compared.All 3 sibling speciesshowed varying degreesof susceptibility to both B. pahangi and,B. rnalnyi, and they were considerably more susceptibleto B. pahangi than to B. rnalnyl. The rate and intensity of infection to B. pahangi were highest for speciesA (66.2% and7.4L3/female, respectively) and lowest,for speciesB (21.3% and,1.7Ls/female).For B. mahyi these valueswere higher for-speciesA (29.7% and.1.84 L3lfemale) than for speciesB (t3.3% and 0.86 L3lfemale) and c Qr.6% and 0.7b h/ female).The colonizedstrain A of An. qundrimnrulntuswas significantly more susceptibleto both Brugia speciesthan the natural populations of sibling speciesA, B and C.

INTRODUCTION vertebrate host adaptation and mosquito nutri- tion affected the susceptibility of our colonized Until recently, Anopheles quadrirnaculatus An. quadrimaculatusstrain A to B. pahangi and Say was considereda single species(Darsie and B. malayi (Nayar et al. 1990,Nayar and Knight Ward 1981).However, genetic studies on natural 1991). populations from the eastern United States re- Most Anophelesoccur as complexesof sibling vealed that it is a complex of at least 4 sibling species(Narang and Seawright 1990).Natural species,A, B, C and D (Narang et al. 1989a, and laboratory populations of sibling speciesof 1989b).All 4 speciesoccur in Florida. Anoph,eles the Anophebs gambiae Giles complex from Af- qua.drimaculafusis a proven vector of malaria rica show variation in susceptibility to the hu- parasites(Boyd 1949),but it is not a natural man filarial parasite, W. bancrofti (Mnzava et vector of Brugia species.However, colonized al. 1989,Hunt and Gunders1990). Two strains strains of An. qua.drimoculatushave been found of Atwphel.esbarbirostris Van der Wulp, which to be susceptible to several speciesof belong to a speciescomplex in Southeast Asia, filariae,e.g., Brugia pahangi (Buckley and Ede- also show variations in susceptibility to subper- son) (Schacher1962), Brugia patei (BrckLey, iodic B. rnala.yi(Choochote et al. 1984). These Nelson and Heisch) (Nayar et al. 1984),Dirofi- studies suggestthat the natural populations of lariaimmitis Leidy (Nayar and Sauerman1975), sibling speciesof the An. quadrimaculatuscom- Dirofilaria tenuis Chandler (Pisty 1958), and plex might also show variations in susceptibility Dirofilaria striata (Molin) (Nayar et al., unpub- to filarial parasites.In this report, we compared Iished data), but not Brugia rnalnyi (Buckley) susceptibilities of speciesA, B and C of the An. (Orihel and Pacheco 1966) or Wuchereria ban- quadrimaculatiuscomplex to the filarial para- crofti (Cobbold) (Yangco et al. 1984). We re- sites,B. pahangi and subperiodicB. malnyi. Sus- cently reported that genetically selected,colo- ceptibility is usually used in the sense of the nized mutant strains of An. qua.drimaculntusare ability of pathogensto mature in the insect to susceptibleto subperiodicB. malayi adapted to the stage at which they can be transmitted to and maintained in nude mice, and B. pahangi the host. In this paper,to measuresusceptibility, and B. patei adapted to and maintained in jirds the rate of infection with third stagelarvae and (Nayar et al. 1989). We subsequently showed the intensity of infection with third stagelarvae that in addition to genetic factors which control per female are considered. susceptibility to B. malnyi, other factors like

MATERIALS AND METHODS I Institute of Food and Agricultural Sciences,Uni- Natural populations of An. qu.adrimaculntus versity of Florida Experiment Stations Journal Series were collected twice, at 2-wk intervals, from 4 No. R-01499. locations in north and central Florida from 2Florida Medical Entomology Laboratory, IFAS- March 1 to June 15, 1990.The collectionsites University of Florida, 200 9th Street S.E., Vero Beach, FL 32962. were: a) Lake Rousseau,Citrus Co.; b) Lake 3Medical and Veterinary Entomology Research Octahatchee, Hamilton Co.; c) Bear Bay Laboratory, USDA-ARS, Gainesville,FL 32608. Swamp, Dixie Co.; and d) Lake Panasoffkee, a Currently at: BioSciences Research Laboratory, Sumter Co. Members of the 3 sibling species(A, USDA-ARS,P. O. Box 5674,Fargo, ND 58105. B and C) are present at one or more of these JouRNer,oF THEAMERIcAN MosQUITo ColqtRol- AssocrarroN VoL.8,No. 1

Table 1. Rate of infection and intensity of infection of 3 speciesof Anophelesquadrirnaculatus complex, on the 11th day after feeding on jirds infected with Brugia pahangi and B. malayi. Intensity of infection No. of females Infection rate L"/9 Species dissected (%) i+SE

Brugia pahangi A 77 66.2b 7.4+1.2b B ot 21.3d 1.7+ 0.8c t, t22 39.3c 5.5+ 1.5b Colonizedstrain A r27 94.5a 19.6+ 1.3a

A OI 29.7 t 1.84+ 0.79f B 98 13.3g 0.86+ 0.38c U 247 12.6g 0.75+ 0.25c Colonizedstrain A 86 64.0e 4.79+ O.79e t Duncan's (1955) multiple range test. Means followed by the same letters are not significantly different (P < 0.05). locations (Seawrightet al., unpublisheddata). speciesof B. malayi and their development is Laboratory colonized strain A of An. quadri- very similar in their mosquito hosts (Beckett macuhtus, highly susceptible to B. malnyi and Macdonald 1971). Therefore, different (Nayarand Knight 1991),which wasmaintained stagesof larval developmentwere categorizedas on a IjVo sucrosesolution containing O.t% p- describedby Schacher(1962). Wild-caught fe- aminobenzoicacid (PABA), was usedas a con- malesand the laboratory colonizedstrain A were trol for comparison with the natural popula- identified by electrophoretic methods (Narang tions. Field-collected femalesdid not feed read- et al. 1989a),DNA probes(Johnson 1990)5, or ily on Brugia-infected jirds in the laboratory. mitochondrialDNA (Mitchell 1990)6. Therefore,they were maintainedon a 10% su- Data were statistically analyzed using an crose solution with 0.1% PABA for at least 4 analysisof varianceand Duncan's (1955)mul- days and starved overnight before they were tiple range test. allowedto blood-feedon jirds infected with B. pahangi or B. malayi. About 50 femalesfrom the RESULTS colonizedstrain A were fed during the period of an hour on the same jird as the field-collected Sibling speciesA, B and C were present in the females. Since smaller numbers of field-col- collectionsfrom the 4locations.The Lake Rous- lected females fed on the infected jirds at the seaucollection was composedof speciesA, Lake time of feeding, 10 colonized females were dis- Octahatcheespecies A and B, Bear Bay Swamp sectedindividually in a drop of Hanks'balanced speciesC, and Lake Panasoffkeespecies A and salt solution and the number of microfilariae C. Sibling speciesD was not present in these ingestedwere counted. The mosquitoesfed on a collections.Although similar numbers of B. pa- jird with a microfilaremiaof 250 + 10 mf/20 pI hangi and,B. malayi microfilariae were ingested of blood, and it resulted in a mean ingestion of by each of the sibling species, based on the 80 + 12 microfilariae per blood meal (range 50- number of microfilariae ingested by the colo- 120 mf), which was consideredacceptable for nized strain A, they showedvarying degreesof our purposes. Only the fully engorged females susceptibility to both B. pahangi and B. malayi wereseparated and maintained on a 10%sucrose (Table 1). AII 3 specieswere significantly more solution with 0.1% PABA at 24 + 1"C. No susceptible to B. pahangi than to B. rnalnyi. mortality was observedin blood-fedfemales dur- SpeciesA and C were not sigrrificantly different ing the next 10 days. Preliminary experiments with colonized females showedthat developing larvae reached the third stage in the thoracic u muscles,but did not start to migrate into the Johnson,D. W. 1990.Quick blots and nonradioac- hemocoelor the head until the 12th day at the tive detection systems:improvement on methods for DNA hybridizations using mosquitoes.Ph.D. disser- temperatureused. On the 11th day, development tation, University of Florida. 88 pp. of filariae was determinedby dissectingthe head 6Mitchell, S. E. 1990.Mitochondrial and ribosomal and thorax of each female;the abdomenof each DNA analysis for the identification of sibling species female was frozen at -20"C for later identifica- of the mosquitoArcpheles qwdrimaculatw Say.Ph.D. tion of the species.Brugia pahangi is a cogenic dissertation,University of Florida. 81 pp. Mmcu 1992 Suscsprrsrllrv or. -4rv. g uA D Rr M A c uLe rus Corrapr,sx OJ

in susceptibility to B. pahangi, but both were Table 2. The percentage of individuals of 3 species of significantly more susceptible than speciesB. An. quadrimaculatus complex and colonized strain A The rate of infection and intensity of infection recorded with various concentrations of infective were 66.2Voand.7.4 + I.2 Lslfemale for species third stage Brugia lawae. 4,39.3% and 5.5 + 1.5 La/femalefor speciesC, No. of L: Species Species Species Colonized and,2L.3%and 1.7 + 0.8 La/femalefor species larvae/9 A B C strain A B, respectively (Table 1). Females of all of the natural populations were significantly lower (P Brugia pahangi < 0.05) in susceptibility comparedwith the col- 0 33.8 78.7 60.7 5.5 onizedstrain A (Table 1). 1-5 29.9 14.8 t9.7 18.1 6-10 13.0 1.6 The 3 sibling speciesingested almost similar 6.6 t4.2 11-20 14.3 1.6 4.r 20.5 numbers of microfilariae of B. rnalnyi. SpeciesA 20-30 5.2 1.6 4.9 16.5 was more susceptible,with significantly higher >30 3.9 1.6 4.1 25.2 rate of infection (29.7%)and intensity of infec- Brugin mahyi tion (1.84+ 0.79 Ls/female)than both species 0 70.3 86.7 87.4 36.0 B and C (L3.3% and, 12.6% infective rate and + + 1-5 18.9 9.2 8.9 37.2 0.86 0.38 and 0.75 0.25 Ls/female,respec- 6-10 2.7 2.0 1.6 11.6 tively) (Table L). Species B and C were not 11-20 5.4 1.0 1.6 9.3 significantly different from each other in sus- 20-30 2.7 00 4.7 ceptibility to B. malayi. All 3 specieswere sig- >30 0 1.0 0.4 7.2 nificarrtly lower (P < 0.05) in susceptibility than the colonizedstrain A (Table 1). A summary of the susceptibility of the 3 sib- which are genetically very different from those Iing speciesand colonized strain A in terms of in nature. As pointed out by Hunt and Gunders numbers of La/female are shown in Table 2. (1990),only a very small portion of the species Although filaria larvae developedto the infective genepool is included when establishinga colony. stagein someof the mosquitoesof each species, Theoretically, this "bottleneck" effect may re- a lack of susceptibility to infection with both B. sult in genes affecting susceptibility being in- mala.yiand B. pahangi was prevalent among all cluded or excluded at an unnaturally high fre- 3 sibling speciesof the field-collected mosqui- quency. In general, speciesA from the natural toes. The variable numbers of Ls developedin populations were also more susceptiblethan the individual mosquitoesalso indicated considera- other 2 species,but more data should be col- ble genetic differenceswithin each species,with Iected from additional populations to ascertain a small percentageof very susceptiblemosqui- whether speciesA is genetically more suitable toes in each species. as a vector for the filaria. Vernick and Collins (1989)and Vernick et al. joint DISCUSSION (1989) suggestedthat the action of 2 un- linked esteraseloci (Est A and Est C) and Pif- Sibling speciesA, B, and C of the An. qtnd- B locus controls expression of the susceptible rimaculatus complex showed variation in sus- and refractory phenotypes of.Annphel.es gambiae ceptibility to both Brugia species.The physio- Ga strain to infection of the malarial parasite, logical reasonsfor variations in susceptibility of Plasmodium cynomolagi.Though we have not the sibling speciesto either B. pahangi or B. examinedthe geneticbasis of refractorinesstrait malnyi were not evaluated in this study. They or its linkage to other genesin 3 sibling species could be either due to damageto ingestedmicro- of the An. quadrimaculafuscomplex, there are filariae by cibarial and/or pharyngeal armatures significant esterase electromorph differences in the alimentary canal as shown by Bryan and amongthe3 species(Naranget al. 1989a,1989c). Southgate (1988) in sibling speciesof the An. We used B. pahangi and B. malayi to study gambi.aecomplex to W. bancroftl, or due to the susceptibilities of the sibling species of An. presence of other physiological factors which qua.drirnaculafuscomplex, because these fi lariae may inhibit developmentof ingestedmicrofilar- were readily available in animal models.Human iae in the thoracic musclesof these mosquitoes. infections with naturally occurring Brugia spe- The colonized strain A is more susceptibleto cies in North America, though rare, have been both species of Brugia than the field-caught reported during the last 30 years (Orihel and species A, B and C of An. quadrimaculatus, Beaver1989). These authors concluded that be- assuming that the females of these speciesin- causeBrugin specieshave been reported to infect gesteda similar number of microfilariae of each throughout the world and have a dem- Brugia species.This is probably due to the bio- onstrated ability to infect humans as well, it is logical characteristics of colony mosquitoes likely that zoonotic infections will be recognized 64 Jounner, oF THEAMERTCAN MosQUITo CoNtnoL Assocrarrou VoL.8,No. 1 with increasing frequency and will be shown to Narang,S. K., P. E. Kaiserand J. A. Seawright.1989b. have an evenwider geographicaldistribution. In Identification of speciesD, a new member of the this context, it is of interest that the sibling Anophelcsqtndrim.aculntas complex. A biochemical Assoc.5:317-324. species of the An. quadrirnaculatus complex key.J. Am. Mosq. Control Narang, S. K., S. R. Toniolo, J. A. Seawright and P. show variations in susceptibility to the Brugia E. 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