JUNE 1987 Hygntolzmton Bennren tr.l :{gpps scvtELLARrs Corupr.px

INCREASEDFEMALE MORTALITY AS A BARRIER TO HYBRIDIZATION BETWEEN MEMBERS OF THE COMPLEX OF MOSQUTTOES

R. E. DUHRKOPF

Department of Bialagy, Ba.ylor Uniuersity, Waro, TX 76798

ABSTRACT. Interspecific crosses between the mosquitoeg Aedes polyncsiensis and Aedes mdnyensis have shown a unidirectiona_l pattern of compatibility. Aedes plynesicnsis females inseminated by Ae. nwlayercb males fail to produce viable offspring while the reciprocal cross is viable. In both crosses, rates of insemination are comparable to control rates. The Ae. polynesbnsis females fail to lay eggs.One apparent reason for thig is that the Ae. plynesiensis females have a high rate of mortality aftcr inseminatioiAy Le. malayensis males. Such mortality is an effective barrier to hybridization in that-croes, and is a new cliss of isolating mechanism.

INTRODUCTION unidirectional pattern of compatibility (Yen and Barr 1973, Wright and Barr 1980, Wright anc The Aedes scutellaris complex of mosquitoes Wang 1980,Trpis et al. 1981b). is comprised ofabout 30 speciesdistributed from During attempts at these crosses it was ob- Andaman the Islands in the west to the Mar- served that the Aedespolynesi.ensis females in- quesas and Tuamoto Archipelago in the east, seminated by Ae. m.ahyenslsmales apparently and reaching as far north as Okinawa. Several had a higher rate of mortality than colony fe- members have been implicated as important males. This was true in a variety of attempts vectors of filariasis in the South Paciftc. Aedes involving several different laboratory strains. polyncsiensis Marks has been shown to be highly Even in attempted crossesof very large numbers susceptible to both Brueia Whnnqi and,Bngia (>500 females) mortality was so great that few, malayi (Duhrkopf and Trpis 1980).It is distrib- if any, eggswere laid. Such mortality could prove uted throughout the eastern South Paoific, to be a substantial isolating mechanism, pre- reaching as west far as the Ellice Islands and as venting interspecific hybridization in one direc- far east as the Marquesas, Tuamotos and Pit- tion through the post-fertilization death of the cairn Island (Macdonald 1976).Aed.es malayen- female prior to oviposition. This study is an srs Colless is refractory to filarial infection investigation of the extent of that post-fertiliz- (Macdonald 1976 and Trpis et al. 1981). It is ation mortality. distributed throughout the western South Pa- cific and Southeast Asia. The limits of its dis- MATERIALS AND METIIODS tribution are the Malaysian Peninsula on the east, the Andaman Islands on the west, and All mosquitoeswere from coloniesmaintained and on the north. in the Laboratories of Medical Entomology at The factors involved in frlarial susceptibility The Johns Hopkins University School of Hy- have been shown to follow a nonMendelian pat- giene and Public Health. Aedes malayensis tern of inheritance (Trpis et al. 1981a). It is BANG strain was originally obtained from the possiblethat a rickettsial symbiont is associated SEATO Medical ResearchLaboratory in Bang- with filarial susceptibility (Duhrkopf and Trpis kok in 1969. Aedes polynesiensis APIA strain 1981).During the analysis ofthe genetic system was collectedby Barry Engber in Apia, Western involved in susceptibility, several crosseswere Samoain t977. All mosquitoeswere reared in a attempted between members of the complex. controlled environment of 26'C and 80% RH on Most of these crossesshowed a unidirectional a 16:8 light:dark regimen. pattern of compatibility. Of importance to this Larvae were reared in 29 x 18 cm rectangular paper is the seriesof reciprocal crossesbetween pans at a density of 100 per liter. Larvae were Aedes polyncsiensrs and.Ae. malayensis. When fed on diluted liver powder suspension. Upon Aedes rnal,ayensisfemales are inseminated by pupation, the pupae were removed and sexed. Ae. polynesicnsdsmales, viable hybrids result. The sexes were separated and held for emer- When Aedes polyrwsiznsis females are insemi- gence.Newly emerged adults were temporarily nated by Ae. malayensrrsmales, no viable off- held in cylindrical paper containers, 18 cm high spring are produced. Similar patterns have pre- x 18 cm diameter. All crosseswere set up with viously been reported in the Aedes scutellaris adults which were no more than 12 hours old. complex (Tesfa-Yohannesand Rozeboom1974, The initial experiment was to compare the Macdonald 1976),and inthe Culcx pipicns com- rates of insemination in control versus experi- plex (Laven 1951). In both cases,a rickettsial mental populations. Four groups of 20 cages symbiont has been reported as the causeofthe were set up. They were small, cylindrical paper JounNel oF THE AupRrclN Moseurro CoNrnol AssocnnoN VoL. B, No.2 cages,9 cm high x g cm in diameter. A vial of Table1. Percentagesofinsemination in controland water was inserted through a hole in the bottom, expedmentatfumal* piece and a of cotton soaked with l0% sucrose Aedespolywsi.ensis Aedesm.alnyensis was kept at the top. Five males and five females were placed in each cage.In one set of 20 cages, Experi- Experi- all males and femaleswere Ae. polynesi.ensis.In Day Control mental Control mental the secondset of 20 cages,all malesand females 1 0000 wereAe. malayensis.In the third set of 20 cages, 2 0000 the males wereAe. polynesi.ensisand the females 3 1001010 were Ae. m,alo.yensis,and in the fourth set of 4 40 20 50 40 cages,the males were Ae. malayenslsand the o 80 70 90 90 6 100 100 100 100 females wete Ae. polyncsiensis.Every day, 10 n 100 100 100 100 females were randomly removed from various cagesand dissectedto inspect the spermathecae for the presenceof sperm. experimentalsand 78 andTt% in the controls). A similar procedure was used to investigate No such difference is seen in the data for the the rates of survival in the different populations. Aedes mal,ayensrsfemales (90 and 88% in the Eight groups of 20 small cageswere set up as experiments and 92 and96% in the controls). above. In the first two groups, all males ani In Figs. 1 and 2, the survival curyesare shown females werc Aedespolynesiensis. In the second. over the 20 day period. Fig. 1 presents the sur- two groups,all males and females wete Ae. tna- vival curves for the four populations of Aedes layensis. In the third two groups the males were polynesicnsis females, and Fig. 2 presents the Ae. polyncsiensrsand the females were Ae. ma- survival curves for the four populations of Ae. layensis,and in the final two groups, the males nwla.yensisfemales. The curves demonstrate the wete Ae. malayensisand the females were Ae. differences between the populations which were polynesiensis.For eachday over a 20 day period, presented in Table 2. It can be seen that the the number of surviving females in each cage curves for the two Ae. polyncsiazsrs experimen- was recorded.In this way, there were two repli- tal lines are different from those of the two cates for each group-Ae. polyrwsiensiscontrol control lines, while all four curves for the Ae. females (inseminated by Ae. polyrwsicnsis m,alayensisfemdes are similar. males),Ae. malayenslscontrol females (insemi- The Log-rank Survival Analysis results in a nated by Ae. malayensismales), Ae. malnryensis statistic which is distributed as a Chi-square. experimental females (inseminated by Ae. poly- Differences between the Aedes malayensis nesiensismales), and Ae. polynesicnsisexperi- groups were not significant (x2 : 3.93, 3 d.f.). mental females (inseminated by Ae. malayensis There were significant differences between the males).Analysis of the data was done using Log- Aed.espolynzslensis groups (x2 : 47.L6,3 d.f., P rank Survival Analysis (Anderson et al. 1980). < 0.001). Further comparisons of the Aedes polyrrcsiensisgroups showed no significant dif- (12 = RESULTS ference between the two control groups 0.78, 1 d.f.) and no significant difference be- The results of the insemination tests are tween the two experimentalgroups (x2 :3.I7, shown in Table 1. The purpose of the insemi. 1 d.f.). Thus, the experimental groups had a nation test was to determine whether femalesin significantly reducedsurvival when comparedto interspecific crosseswere being successfullyfer- the control groups. tilized. The results of the insemination tests show that, by the sixth day, all of the females DISCUSSION had sperm in their spermathecae.Thus, the Aedesrnalayensrs males were successfullyinsem- Aedes polyncsierxis has been the subject of inating the Ae. polynesiensisfemales, and Ae. intense study for the past 30 years becauseof polynesiensismales were successfullyinseminat- its susceptibility to filarial parasites. As previ- ing Ae. rnalayensisfemales. ously mentioned, crossesbetween Ae. polync- The results of the mortality experiments are sicnsh and Ae. mala.yensis have demonstrated shown in Table 2 and Figs. 1 and 2. In Table 2, an unidirectional pattern of compatibility the daily survival rates are presented for the (Tesfa-Yohannesand Rozeboom1974, Macdon- eight groups. From day 8 through day 20, the ald 1976, Trpis et al. 1981a). That pattern is number of females in the Aedes polyncsiensis believedto be due to the presenceofa rickettsial experimental populations was less than the symbiont (Wright and Wang 1980, Wright and number in the control populations. The finai Barr 1980, Trpis et al. 1981b). Unidirectional results were many fewer females in the experi- pattern of compatibility in membersof the Aedcs mentals than the controls (46 and 36% in the scutellaris complex are well known. Woodhill JUNE 1987 HvsnrptzltloN BARRIERrN AEDEs scurELLARrs Coupr,px 183

Table 2. The percentageof femalessurviving on each day. Aed,espolyncsiensis Aedes mala.yercis

Dav cIr cIIr E12 EII' cI8 cII3 EI' EII4 1 97 100 99 99 100 100 100 99 2 97 100 99 99 100 100 100 99 o 95 99 98 97 100 100 100 99 4 94 99 98 90 100 100 100 98 5 92 99 97 90 100 100 100 97 6 92 97 96 85 99 99 100 97 7 91 95 93 7L 97 99 100 96 8 91 S2 87 69 96 99 100 96 I 91 89 84 67 95 99 100 96 10 89 88 77 67 95 99 97 96 11 88 85 74 51 95 99 97 95 t2 86 83 7r 51 95 99 97 95 13 86 81 69 51 95 99 97 95 t4 83 79 66 49 95 99 95 93 15 81 77 54 46 93 97 93 93 16 80 76 51 44 93 96 93 93 t7 78 73 51 44 93 96 93 90 18 ?8 72 46 36 93 96 93 88 19 78 72 46 36 92 96 93 88 20 78 7t 46 36 92 96 90 88 1Ae. polynesicnsrs females X Ae. polyncsicinsismales. 2Ae. polynzsiensrc females X Ae. malayensrsmales. 3Ae. malayensis females X Ae. malayer*ls males. aAe. malayensu females X Ae. polyrwsicnsis mabs.

r00

,l e7o z360 X50

1 2 3 4 5 6 7 8 9 l0 ll 12 13 14 15 16 r7 18 19 20 ]. DAY Fig. 1. The percent survival of Aedeppolyncsrpnsrs females- lD Aedespolyrwsrznsis females X Ae. polynesiensis males (CI), O Ae. polyncsiensisfemales X Ae. polynesiarsrsmales (CII), f Ae. polyncsiensisfemales X Ae. malayensismales (EI), E Ae. polynesiensrsfemaleg X Ae. malaryensrsmales (EII).

(1949,1950, 1954) and Smith-White and Wood- Gubler (1970a,1970b, 1971) showedthat once hill (1954)documented a seriesof unidirectional Ae. polynesiensls females are inseminated by crossesbetween membersof the complex. Aedesalbopittus (Skuse) males, they are essen; In a series of papers directed towards using tially sterilized, and that presenting them with such findings in control of Aedespolynesicnsis, Ae. polyncsiznsls males after insemination by 184 JounNer.or rHE ArrrpnrceNMosgurto CoNtnor. Assocnmor.r VoL.3,No.2

100

90

I z 3 4 5 6 7 8 9 l0 11 12 13 t4 15 16 l7 18 19 20

DAY Fig. 2. The percent survival of.Aedes nnlayensis females-O Ae. malayensis females X Ae. malayensrsmales (CI), O Ae. nnlayensis females X Ae. malayensis males (CII), I Ae. mnlnyensis females X Ae. polynesicnsis males (EI), E Ae. malayensirfemales X Ae. polynesiensrsmales (EII).

Ae. albopictusmales does not result in returned inated by Ae. malayenslsmales, and that only fertility. In addition, he showedthat in a series 8.6% of the Ae. malayensisfemales were insem- of mixed populations, a population of Ae. ahop- inated by Ae. polyncsiensls males. H6wever, ictus will competitively displace a population of their dissections were on the second and third Ae. polyrwsiensls. Finally, this was related to days, and it seems likely that more would be oviposition behavior. He showed that the two inseminated at a later time. Macdonald (1976) specieswould most likely oviposit in the same reported results similar to those of Tesfa-Yo- places, resulting in larval competition, and be- hannes and Rozeboom. However, the more cause of the unidirectional nature of interspe- crowded conditions in the present study could cific matings, in locations inhabited by both have promoted higher levels of insemination. ,Ae. albopictus could competitively dis- Of greater importance is the documentation place Ae. polyncsiensis.However, none of these that when Aedes polynesiensisfemales are in- studiesmention any increasein the mortality of seminated by Ae. malayenslsmales, there is an the femalesof one specieswhen inseminated by increase in the mortality of the females. It males of the other. shouldbe noted that this study was done without The results of this study show that bothAedes providing the femaleswith a blood meal. In the polynesiensisand Ae. malayensisfemales can be original crossesdescribed in Trpis et al. (1981a), successfullyinseminated by males of the other the mortality amongstthe femaleswas such that species.No attempt has been made here to judge very large populations had to be used. In many competitivenessof the two speciesin inseminat- cases,in excessof 500 females were necessary. ing females of the opposite species.It is likely Even with large numbers, few survived long that males of the opposite specieswould be at a enoughto oviposit. Bloodfeedingseemed to be a competitive disadvantage when compared to confounding variable with referenceto the pres- males of the same speciesas the female. How- ent study, because,in the previous study (Trpis ever. under the situations described in this et al. 1981) it appeared that few of the Aed.es study, Ae. polynesierxismales can successfully polyncsicnsisfemales inseminated by Aedesma- inseminate Ae. moloryensisfemales, andAe. ma- Layensismales successfullytook a blood ineal. layensis males can successfullyinseminate Ae. So, it was felt that bloodfeedingwas a variable polynesierwis females. These results differ which was not necessaryfor showing the reduc- slightly from those of both Tesfa-Yohannesand tion in the numbers of females following inter- Rozeboom(1974) and Macdonald (f976). Tesfa- specific insemination. Yohannes and Rozeboom reported that only The results of the experiment carry with them 60%of the Ae. polyncsiensisfemales were insem- at least two possible implications. The first is JUNE 1987 HvsnrutzlttoN BARRIERrN AEDEy scurELLAnIs Couplnx 185 that Aedes malayensis males might prove to be ACKNOWLEDGMENTS another means of possible control of Ae. poly- I would like to thank Dr. Milan Trpis for nesiensis.Since Ae. malayensisis not a vector of discussions during the initial planning filariasis, use of males would not significantly helpful period of this experiment. It would also like to add to the vector population. However, it should Dr. W. Keith Hartberg for a critical re- again be emphasized that no study has been thank view of the manuscript. done of the competitiveness of Ae. malayensis males compared to Ae. polyrrcsiensismales. It seems likely that such a scheme of biological REFERENCES CITED the Ae. control would have little effect upon Anderson, S., A. Anquier, W. W. Hauk, D. Oakes,W. polynesiensispopulation. Vandaeleand H. I. Weisberg.1980. Statistical meth- Secondly, this paper presents an apparently ods for comparative studies. John Wiley and Sons, new post-copulatory isolating mechanism in New York. mosquitoes. Classical post-copulatory isolating Dobzhansky,T. 1951.Genetics and the origin of spe- mechanismsinvolve such things as gametic and. cies. Columbia Univ, Press,New York. zygotic mortality and hybrid inviability or ste- Dobzhansky,T., F. J. Ayala, G. L. Stebbins and J' W. H. Freemanand Co., rility. However, no record is known of the death Valentine. 1977.Evolution. W. Francisco. a result of interspecific insemi- San of the female as Duhrkopf, R. E. and M. Trpis. 1980. The degree of nation in mosquitoes.Many secondary sources susceptibility and levelsof infection in ten different alludeto this phenomenon.Dobzhansky (1951) straing of Aedespolyncsiensls Marks infected with briefly mentions experiments of Standfuss in subperiodic Brugia maWi and Brugin Pahangi. Am. 1896 with moths in which interspecific copula- J. Trop. Med. Hyg. 29:815-819. tion causeddamage to the female organs. Mayr Duhrkopf, R. E. and M. Trpis. 1981. The effect of (1963) mentions experiments by Sturtevant, tetracycline treatment on filarial susceptibility in Stalker and Spieth in which interspecific crosses members of the Aedes scutellnris complex. Mosq. in Drosophila lead to the death of the female. News41:729-732. Induced sterility in Aedes (Ste' papers in the Gubler. D. J. 19?0a. However, none of the referenced gorlyia) polynesicnsis Marks by cross-insemination bibliography discusssuch findings. Finally, Dob- with Aedes (Steeomyia) ahopictus Skuse. J. Med. zhansky et al. (1977) refer to the death of the Entomol. 7:63-70. female in crossesbetween Drosophilapseudoob- Gubler, D. J. 1970b. Competitive displacement of scura females and D. mehnoga.stermales as a Aedes (Stegomyin) polynesiensrs Marks by Aedes result of damage done to the female organs. (Stegomyin) ahopictus Skuse in laboratory popula- However, again, no primary referenceis given. tions. J. Med. Entomol. T:229-235. In some respects the importance of this is Gubler, D. J. 1971, Studies on the comparative ovi- (Stegomyiol albpicttts merely academic. Aedes polyncsiensis and Ae. position behavior of Aedes (Stegomyin) polynesicrtsisMarks' J. Med. not sympatric in their distribu- andAedes malayensisare Entomol. 8:675-682. tions, so the chancesof interspecific matings in Laven, H. 1951. Crossing experiments wilh Culex nature are very low. However, becauseof the strains. Evolution 5:370-375. $eat extent of unidirectional patterns of com- Macdonald, W. W, 1976. genetics in relation patibility in crosseswithin the Ae. scutellaris to filarial infections. /n: A. Taylor, E. R. MuIIer and complex, and between members of the Culex R. Muller (eds,). Genetic aepectsof host-parasite pipiens complex, if this pattern can be shown to relationships. Blackwell Scientific Publ., Oxford. hold in other crosses,it may very well be an Mayr, E. 1963. speciesand evolution. Belknap important natural barrier to hybridization be- Press.Carnbridge, Mass. Srnith-White, S., and A. R. Woodhill. 1954.The na- tween closely related species. ture and significance of nonreciprocal fertility in The exact nature of the cause of mortality the Aedes scutellarisand other mosquitoes. Proc. was not under investigation in this study. It Linn. Soc.N. S. W. 79:163-1?6. would be of interest to investigate pathological Tesfa-Yohannes.Tesfa-Michael and L. E. Rozeboom. changes in the ovaries of Aed.espolynesiensis 19?4.Experimental crossingsof Aedes (5.) polyne- femalesinseminated by Ae. ntalayensismales. It siensisMarks and A. scutellaris malaryensisColless. is possible that the well documented rickettsial J. Med. Entomol. 11:323-331. symbionts have something to do with this phe- Trpis, M., R. E. Duhrkopf and K. L. Parker. 1981a. nomenon. It might be possible that this is one Non-Mendelian inheritance of mosquito suscepti- to infection wilh BruSia malnyi and Brugia of the reasons for the persistence of the sym- bility pahangi. Science 2ll:l 435-1437. bionts. They may be a means by which the Trpis, M., J. B. Perrone, M. Reissigand K. L' Parker. species maintains its integrity by preventing 1981b.Control of cytoplasmicincompatibility in the interspecific hybridization, and they may also Aedesscutellans complex. J. Hered. 72:313-3t7. be a means of speciation in this complex of Woodhill, A. R, 1949.A note on experimental crossing mosquitoes. of Aed.es(Stegomyia) scutellaris Walker and'Aedes 186 JounNll oF THE AunnrclN Moseurro CoNrnol AssocrlrroN VoL.3,No.2

(Steeonryia)scutelhris hath,erensisWoodhill (Dip- Wright, J. D. and A. R. Barr. 1980.The ultrastructure tera: Culicidae). Proc. Linn. Soc. N. S. W. 74:224- and symbiotic relationships of Wohachia of mos- 226. quitoes of the Aedes scutellaris group. J. Ultrastruct. Woodhill, A. R. 1950.Further notes on experimental P'es,72:52-64, crossing within the Aedes scutellans group of spe- Wright, J. D. and B. Wang. 1980. Observations on cies. Proc. Linn. Soc. N. S. W. 75:25t-253. Wolbachiae in mosquitoes. J. Invertebr. Pathol. Woodhill, A. R. 1954.Experimental crossing of.Aedes 35:200-208. (Stegomyia') pseudoscutellarrs Theobald and Aed,es Yen, J. H. and A. R. Barr. 1973.The etiological agent (Stegomyia)polynesiensis Marks (Diptera: Culici- of cytoplasmic incompatibility in Culex pipicns. J. dae). Proc. Linn. Soc. N. S. W. 79:19-20. Invertebr. Pathol. 22:242-250.