Cross-breeding of Aedes (S.) polynesiensis Marks with an autogenous species of the A. scutellaris group*

Bull. Org. mond. Sante 1 1973, 49, 367-370Bull. Wld Hith Org. Cross-breeding of Aedes (S.) polynesiensis Marks with an autogenous species of the A. scutellaris group* JAMES C. HITCHCOCK1 & LLOYD E. ROZEBOOM 2 An autogenous species belonging to the Aedes (Stegomyia) scutellaris subgroup was found on the island of Tafahi, Kingdom of Tonga. A subcolony was established at Johns Hopkins University, Baltimore, Md., USA, where cross-breeding experiments were con- ducted with the Samoan strain of A. polynesiensis. The cross between Tafahi females and A. polynesiensis males produced viable hybrid progeny, which were inbred through five generations. The reciprocal cross was not successful. F1 Tafahi x A. polynesiensis hybrid males were backcrossed successfully to Tafahi females, but of the many eggs produced by backcrossing these F1TP males to A. polynesiensis females, only 0.7 % hatched. Of the eggs produced by the F2TP malex A. polynesiensis female backcross, about 7% were viable. The results indicate a close genetic relationship between the Tafahi species and A. polynesiensis. The one-directional compatibility observed suggests the existence of cytoplasmic factors for sterility, which might eventually be useful in the control of members of the A. scutellaris complex. The scutellaris subgroup of the genus Aedes com- prises some 30 species and subspecies. The combined range of these mosquitos extends from the Andaman Islands in the west to the Marquesas and Tuamotu Islands in the east; and from northern Australia in the south to Okinawa in the north. One of the most important species in the subgroup is Aedes polyne- siensis Marks. It is widely distributed throughout Polynesia, where it is the principal vector of aperiodic Wuchereria bancrofti, and it has been shown experi- mentally to be capable of transmitting dengue virus (1). There have been many studies on the taxonomy of the subgroup, but our knowledge of the genetic relationships between many of the species and sub- species is far from complete. Because these occur as isolated populations among the multitude of Pacific islands, they afford excellent opportunities to study * This work was supported in part by Research Grants Nos. 2 R22 AI 07770, 5 RO1 AI 00351, and 5 RO1 AI 07880 from the National Institutes of Health, US Public Health Service, Bethesda, Md., USA. 1 Scientist, Filariasis and Vector Mosquito Research Laboratory, World Health Organization, P.O. Box 446, Nuku'alofa, Kingdom of Tonga. 2 Professor of Medical Entomology, Department of Patho- biology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Md., USA. the process of speciation in mosquitos. The signifi- cance of such apparently esoteric research for dis- ease control has been emphasized by Spielman & Kitzmiller (2), who pointed out that many important vector species are polymorphic, and that one popu- lation may be of much greater importance as a disease vector than its subtly differing neighbour. Hence, critical discussion of a vector population requires that the population be precisely defined as a Mendelian entity. Examples supporting these re- marks are the Anopheles maculipennis group in Europe and the Anopheles gambiae group in Africa. One reason, therefore, to conduct experimental cross-breeding experiments between populations of the Aedes scutellaris subgroup is to establish genetic relationships, which in turn may reveal isolating bar- riers by which speciation occurred. Secondly, a study of the inheritance of morphological and biological characters depends upon the finding of populations showing measurable discontinuities that are capable of producing hybrids. Finally, the demonstration of genetic or cytoplasmic incompatibility may even- tually be exploited for control. Laven's (3) extensive cross-breeding experiments with populations of the Culex pipiens group led to the now famous field trial in Okpo, Burma, for control of a local population 3128 -367- J. C. HITCHCOCK & L. E. ROZEBOOM of C. pipiens fatigans through the release of incom- patible males (4). The WHO Scientific Group on the Genetics of Vectors and Insecticide Resistance (5) suggested that inter-specific or inter-strain sterility might be used to control species of the Aedes scutellaris subgroup. The first step in such a programme is to search for sterility barriers between the subgroup's members. By analogy from Culex, in the WHO Scientific Group's view, numerous crossing types are to be expected among the various so-called species and varieties in the scutellaris complex. Woodhill (6, 7) demonstrated nonreciprocal fer- tility between A. scutellaris and A. s. katherinensis. We report here the existence of nonreciprocal fertil- ity between Aedes polynesiensis and an as yet un- named population of the A. scutellaris subgroup from the island of Tafahi, Kingdom of Tonga. MATERIALS AND METHODS Experimental populations Because the taxonomic status of the species from Tafahi has not been settled, it will be referred to here as the " Tafahi species ". It is unlike other known members of the A. scutellaris subgroup in that the females are capable of producing the first batch of eggs without having taken a blood meal. A biting-landing collection of the Tafahi species was made at 10 h 30 on 30 June 1970 in a plantation on the northwestern slope of Tafahi, some 800 m uphill from the island's only village of Kolokakala and at an elevation of about 180 m. Tafahi is a high conical volcanic island about 3.2 km long, rising abruptly to 610 m. It is located at 15°51' S and 173°43' W, about 8 km from Niuatoputapu but over 160 km from any other island. The collected adult females were allowed to feed, and then taken back to Niuatoputapu to oviposit. A colony was established on Niuatoputapu. Auto- geny was first observed in this colony on 2 August 1970 among the second generation of adults. A sub- colony was established at the University of Cali- fornia, Los Angeles, Calif., USA, and a second was started in April 1971 at Johns Hopkins University, Baltimore. The latter has been maintained almost entirely without blood meals, and indeed the females show a reluctance to bite. The larvae are fed com- mercial guineapig food pellets. The strain of A. polynesiensis used was one from Samoa that has been maintained in the laboratories at Johns Hopkins University since 1958. Rearing and breeding In order to obtain virgin adults, pupae were taken from the colony rearing pans and, after preliminary sexing by size, transferred in groups of 3-5 to test tubes until the adults emerged. In most test tubes the adults were of one sex; mixed groups were dis- carded. Breeding cages consisted of 3.8-litre card- board cartons, covered at the top with bobbinet and with a cloth sleeve fastened on the side. Female and male mosquitos aged 1-2 days were placed in appropriate combinations in the cages, at a male/ female ratio of about 2: 1. A 10% sucrose solution was provided as a maintenance diet. The anauto- genous A. polynesiensis were allowed to engorge on blood, while no blood was offered to the autogenous Tafahi females. In all crosses involving hybrid females obtained from the Tafahi female x A. poly- nesiensis cross, blood was also withheld. Insemina- tion rates were determined by microscopic examina- tion of the spermathecae of samples of females taken from the breeding cages after egg laying had been completed. A small glass dish lined with paper towel- ling was placed in each cage as an oviposition trap. Eggs were counted with the aid of a dissecting micro- scope, and the average number of eggs per female was calculated. The papers containing the eggs were placed in bowls of water. Newly hatched larvae were counted and transferred to rearing pans, and the adults reared from these larvae were counted. In the discussion below, T stands for Tafahi spe- cies and P for A. polynesiensis, and the species of female is given first. Thus, TP signifies a hybrid produced by crossing a Tafahi female with an A. polynesiensis male. Three reciprocal crosses of the A. polynesien- sis x Tafahi species were made, and a fourth with only the Tafahi females and A. polynesiensis males. In one experiment, the cross between the Tafahi females and the A. polynesiensis males (TP x TP) was carried through the F5 generation. There was one backcross of F1TP hybrid females to A. polynesiensis males. This was not repeated, but a greater effort was made in backcrossing TP hybrid males to A. polynesiensis females. There were three such backcrosses with the F1 males, and two with the F2 males. There were two control Tafahi populations and one of A. polynesiensis. EXPERIMENTAL RESULTS The combined results are presented in Table 1. The cross-insemination rate of Tafahi females by 368 CROSS-BREEDING OF A. POLYNESIENSIS WITH A. SCUTELLARIS Table 1. Results of cross-breeding experiments with Aedes (S.) polynesiensis and Aedes sp. from Tafahi Cross a Parent females b Eggs c Adults d Generation % ~~~~~~Insemi- AverageGeneration No. nated No. per female % hatched No. % survived Fi T P 165 100 4 913 29.8 67.0 2 279 69.3 Fi P T 167 63.0 8549 51.2 0 - - F2 TP TP 102 97.7 1 635 16.0 83.2 916 67.4 F3 TP TP 102 - 1 412 13.8 53.8 390 51.3 F4 TP TP 114 - 808 7.1 41.1 225 67.8 Fes TP TP 89 - 1 518 17.1 32.8 324 65.1 backcross FiTP P 82 - 780 9.5 - 418 - backcross P FiTP 141 81.4 8 667 61.5 0.7 33 56.9 backcross P F2TP 62 100 8221 132.6 e 6.9 396 70.2 control T T 96 77.6 3866 40.3 83.9 1 908 58.8 control P P 40 100 3 315 82.9 81.1 2 588 96.3 a T = Tafahi species; P = A. polynesiensis. b Numbers of females placed in breeding cage from which eggs of indicated generation were obtained. Percentage of females insemi- nated was calculated from samples of 17-58 females taken from- breeding cages. c Average based on number of females placed in the breeding cage. d Percentage of adults surviving from first-stage larvae. 6 Two blood meals given to these females. A. polynesiensis males was 100%, while that of the A. polynesiensis females by Tafahi males was 63 %. The autogenous control Tafahi females produced an average of 40.3 eggs. The Tafahi females crossed with A. polynesiensis males produced an average of 29.8 eggs; the F1TP females an average of 16.0 eggs; and the F2, F3, and F4TP females averages of 13.8, 7.1, and 17.1 respectively. The low egg production was probably attributable to nonlaying females rather than fewer eggs per laying female. Hatching rates of the F1 and F2TP eggs were comparable to those of the Tafahi controls, although they were somewhat lower in subsequent generations. Survival rates of the hybrid adults were equal to that of the Tafahi con- trol population and sex ratios were essentially 1 : 1. A backcross of F1TP females to A. polynesiensis males produced relatively few eggs. Although the hatching rate was not recorded, a total of 418 adults were reared from 780 eggs; evidently the backcross larvae were as vigorous as those of the control Tafahi population. The anautogenous, blood-fed A. polynesiensis con- trol females produced an average of 82.9 eggs per female, of which 81 % hatched. Of 8 549 eggs pro- duced by 167 A. polynesiensis females that had been in contact with Tafahi males, none hatched, although 63% of the females had been inseminated. The first backcross of F1TP hybrid males to A. polynesiensis females resulted in a high (81.4%) insemination rate, but only 0.7% of 8 667 laid by these females hatched. In the second backcross of F2TP males to A. polynesiensis females, the insemi- nation rate was 100%, and 6.9% of 8221 eggs hatched. Larvae of both backcrosses showed sur- vival rates that were comparable to those of other crosses, although somewhat lower than the A. poly- nesiensis control population. DISCUSSION The experiments demonstrated that the auto- genous species from Tafahi is genetically compatible with A. polynesiensis from Samoa in one direction. A. polynesiensis males readily inseminated the Tafahi females; all females examined were positive for sperm. The cross-bred Tafahi females as well as those in the F1 and subsequent generations were autogenous. Although there was some indication of reduced hatchability of the TP hybrid eggs, the hybrid larvae were vigorous and there seems to be no reason to doubt that this hybrid population could maintain itself indefinitely. The reciprocal cross was not successful. Back- crosses of F1TP males to A. polynesiensis females produced few viable progeny. Backcrosses of F2TP 369 370 J. C. HITCHCOCK & L. E. ROZEBOOM males to A. polynesiensis females were more success- ful, which is to be expected because some of the F2 hybrids should possess the A. polynesiensis genotype. Because of the one-directional compatibility be- tween these populations, and the reduced fertility in the F2 and later hybrid generations, it would appear reasonable to consider them as separate species. On the other hand, the TP hybrids appear to be capable of becoming established, and these can backcross to the Tafahi species and to a lesser extent to A. poly- nesiensis. If this occurred in nature the two popu- lations would blend, indicating subspecific status. Certainly the Tafahi species is closely related geneti- cally to A. polynesiensis. The nonreciprocal fertility suggests that this is another example of cytoplasmic factors leading to sterility, as described by Laven (8-10) for Culex pipiens and Smith-White & Woodhill (11) for A. scu- tellaris katherinensis and A. s. scutellaris. Our results differ somewhat from those of Smith-White & Woodhill in that whereas there was little or no reduc- tion in the sterility of successive backcrosses to A. s. katherinensis females, in our backcross of F2TP males to A. polynesiensis females, 6.9% of the eggs were viable. Our results lend encouragement to the suggestion that this phenomenon might eventually be useful in the control of A. polynesiensis and other members of the A. scutellaris complex. RtSUMt CROISEMENT D'AEDES (S.) POLYNESIENSIS MARKS AVEC UNE ESPECE AUTOGkNE DU GROUPE A. SCUTELLARIS On a maintenu en colonie une espece autogene appar- tenant au sous-groupe Aedes (Stegomyia) scutellaris origi- naire de l'ile de Tafahi (Royaume de Tonga) et procede a des essais de croisement avec une souche d'A. polyne- siensis, principal vecteur de la filariose de Bancroft aperiodique en Polynesie. Le taux d'ins6mination des femelles Tafahi (T) par les males A. polynesiensis (P) a atteint 100%. Le nombre d'aeufs produits par ces femelles (sans prise prealable d'un repas de sang) a e comparable a celui des femelles Tafahi temoins; 67% de ces eufs ont eclos et 69,3% des larves ont survecu jusqu'au stade adulte. Lors de croise- ments reciproques, 63% des femelles A. polynesiensis ont e inseminees par les males Tafahi mais aucune eclosion n'a e observee parmi les 8549 ceufs produits. Les hybrides issus du croisement entre femelles T et males P ont e maintenus en endogamie pendant 5 gene- rations, sans prise de repas de sang, avec des taux d'&clo- sion variant de 41 a 83 % et des taux de survie des larves variant de 51 a 69 %. Un seul retrocroisement entre males P et femelles F1 TP a pu etre obtenu. Lors de retro- croisements reciproques entre males F1 TP et femelles P, on n'a note que 0,7% d'eclosions sur 8667 ceufs produits. Les retrocroisements entre males F2 TP et femelles P ont fourni 8221 ceufs dont 6,9% ont eclos. La compatibilite genetique entre les femelles Tafahi et les males A. polynesiensis, et l'aptitude des hybrides TP a se maintenir sous forme de colonie ind6pendante mon- trent qu'il existe de fortes affinites g6n6tiques entre les deux especes. Le caractere non reciproque de la f6condite suggere l'existence d'une incompatibilit6 cytoplasmique que l'on pourrait envisager de mettre A profit dans la lutte contre les membres du complexe A. scutellaris. REFERENCES 1. ROSEN, L. ET AL. Amer. J. trop. Med. Hyg., 3: 878-882 (1954). 2. SPIELMAN, A. & KITZMILLER, J. B. (1967) In: Wright, J. W. & Pal, R., ed. Genetics of insect vectors of disease. Amsterdam, Elsevier, 1967, pp. 459-485. 3. LAVEN, H. Cold Spr. Harb. Symp. quant. Biol., 24: 166-173 (1959). 4. LAVEN, H. Nature, 216: 383-384 (1967). 5. WHO SCIENTIC GROUP ON THE GENETICS OF VECTORS AND INSECTICIDE RESISTANCE. Report. Wld Hlth Org. techn. Rep. Ser., No. 268, 1964, pp. 34-38. 6. WOODHILL, A. R. Proc. Linn. Soc. N.S.W., 74: 224- 226 (1949). 7. WOODHMLL, A. R. Proc. Linn. Soc. N.S.W., 75: 251- 253 (1950). 8. LAVEN, H. Evolution, 5: 370-375 (1951). 9. LAVEN, H. Z. indukt. Abstamm.- u. Vererb.-L., 85: 118-136 (1953). 10. LAVEN, H. In: Transactions of the IXth Inter- national Congress of Entomology, Amsterdam, August 17-24, 1951. Amsterdam, 1953, vol. II, pp. 293-296. 11. SMITH-WHITE, S. & WOODHILL, A. R. Proc. Linn. Soc. N.S.W., 79: 163-176 (1954).