Hybridization between Aedes simpsoni and Aedes woodi with observations on the genetic basis of morphological differences*

Bull. Org. mond. Sant 1972 46, 345-352Bull. Wld Hith Org. j192463532 Hybridization between Aedes simpsoni and Aedes woodi with observations on the genetic basis of morphological differences* W. K. HARTBERG ' The public health importance of the genus Aedes makes it imperative that a better understanding of the genetic differences and isolating barriers between the species be developed. Aedes simpsoni and Aedes woodi from near Dar es Salaam, Tanzania, were used in this investigation. Analysis of F1, F2, and backcross progeny obtainedfrom forced matings indicates that single factorsform the genetic basis ofsome ofthe differences between the two species. There is evidence that Ae. simpsoni and Ae. woodi are separated by both premating and postmating reproductive isolating mechanisms. The genus Aedes of the family Culicidae is the largest and one of the most important of the mos- quito genera. The genus is divided into 24 sub- genera, including Stegomyia with about 98 species. Aedes simpsoni (Theobald) and Aedes woodi Edwards are members of Group A of Edwards (1932) of the subgenus Stegomyia. Group A consists ofmore than 30 species that are largely confined to the Ethiopian region. Aedes simpsoni is widely distributed in Africa south of the Sahara Desert and is the dominant species in some localities. Aedes woodi is restricted to a few localities within the range of Aedes simpsoni where it is seldom, if ever, found in large numbers. McClelland (1962), using forced mating, was able to cross these two species and obtain fertile hybrids. In the F2 generation and in backcrosses to Ae. woodi he noted in some progeny that characters of Ae. woodi assorted independently. Some infertility or breakdown in viability was also noted in the back- cross progeny. No backcrosses to Ae. simpsoni were made. The production of fertile hybrids, F2S, and back- cross progeny by artificial mating between these two closely related species makes it possible to deter- mine the genetic basis for the differences between * This research was supported by Public Health Service research grant CC 00261 from the Center for Disease Control, Atlanta, Ga., USA. 1 Entomologist/Geneticist, World Health Organization, East Africa Aedes Research Unit, Dar es Salaam, Tanzania. Present Address: Department of Biology, Georgia Southern College, Statesboro, Ga., USA. them. In addition, genetic isolating barriers other than behavioural characteristics, such as F1 sterility or hybrid breakdown, may be revealed. The public health importance of the subgenus Stegomyia, which contains several species that serve as vectors of arboviruses, makes it imperative that a better understanding of the genetics and speciation within the subgenus Stegomyia be developed. The greater our knowledge in these areas, the greater are the chances of discovering and successfully employ- ing biological or autocidal control measures. More knowledge of the genetic basis of the differences between Ae. simpsoni and Ae. woodi and of the isolating barriers between them will contribute to an understanding of the comparative genetics of Stego- myia mosquitos, as well as a better understanding of evolution within the subgenus. MATERIALS AND METHODS The adult Aedes simpsoni and Aedes woodi used in the present study were reared in the laboratory from eggs laid by field-collected females. Females of the two species taken in biting catches or net catches from the Mbagala area near Dar es Salaam, Tan- zania, were brought to the laboratory and isolated in individual shell vials (2 x 7 cm) with moist cotton- wool in the bottom. To obtain eggs, they were given a blood meal from a human arm and allowed to oviposit on the moist cotton-wool in the bottom of the vial. Forced-mated females from the experi- mental crosses were handled in the same way. 2810 - 345 - 346 W. K. HARTBERG Eggs were conditioned by slow drying of the moist cotton-wool for 3-6 days under insectary conditions. Hatching was accomplished by immersion of the eggs in a 0.01 % solution of ascorbic acid (Mulla & Chaudhury, 1968). The rearing methods used were generally similar to those described by Craig & VandeHey (1962) for genetic research with Aedes aegypti. Rearing was conducted in an insectary room at 26.5°C i 1C and 80% R.H. The larvae were reared in white plastic pans (20 x 36 x 7 cm). The larvae were fed on a mixture of 10 parts of Liver Powder NF,1 8 parts of wheat germ, and 2 parts of brewers' yeast. Pupation occurred 5-8 days after hatching. Pupae were segregated according to sex (females are larger than males) and placed in emer- gence containers. Adults emerged 2-3 days after pupation, and their sex was rechecked at this time to insure that virgin adult mosquitos were obtained. The adult mosquitos were fed on boiled raisins or sugar cubes. Females and males 4-6 days old were mated by the forced-copulation technique of McDaniel & Horsfall (1957). The males were anaesthetized with CO2 or immobilized with cold, decapitated, and impaled (through the dorsum of the thorax) on a pin in the end of an applicator stick. Females were anaesthe- tized with ether and placed ventral side uppermost on a glass microscope slide with the tip of the abdomen projecting over the edge of the slide. Con- tact between the male and female was accomplished manually under a dissecting stereo-microscope. The male and female were left in copula until they disengaged themselves. In a few cases they failed to disengage after a minute, they were pulled apart manually. After successful copulation, the females were isolated in individual vials. Forced-matings were made as follows (the female being listed first): Ae. woodi x Ae. simpsoni Ae. simpsoni x Ae. woodi F1 x F, (PI: Ae. woodi x Ae. simpsoni) F1 x F1 (Pl: Ae. simpsoni x Ae. woodi) F1 (Ae. woodi x Ae. simpsoni) x Ae. simpsoni Ae. simpsoni x F1 (Ae. woodi x Ae. simpsoni) F1 (Ae. simpsoni x Ae. woodi) x Ae. simpsoni Ae. simpsoni x F, (Ae. simpsoni x Ae. woodi) To check for insemination, females were dissected in Ae. aegypti saline (Hayes, 1953) and the sperma- thecae were examined under a compound micro- scope for the presence of sperm. I Nutritional Biochemicals Corp., Cleveland, Ohio, USA. RESULTS Ae. woodi x Ae. simpsoni; Ae. simpsoni x Ae. woodi The forced matings between Ae. woodi and Ae. simpsoni proved to be easy, copulation occurring readily in the reciprocal crosses. However, out of the 12 pairs of Ae. simpsoni female x Ae. woodi male mated, eggs were obtained from only 1 female (118 eggs in the first egg batch, of which 104 hat- ched). As no oviposition had occurred after several further blood meals, the females were dissected and checked for insemination. It was found only that the female that had laid eggs had been inseminated. Out of the 13 pairs of Ae. woodi female x Ae. simp- soni male mated, eggs were obtained from 6 of the pairs (first egg batches were: 83, 88, 70, 43, 94, and one was not counted). Eggs from only two of the females hatched. Dissection disclosed that these two were the only ones that had been inseminated. It would appear that even though copulation occurs easily during the forced matings, sperm are not readily transferred. The appearance of the reciprocal F1 hybrids obtained was intermediate between the parental types. The thorax scaling pattern was intermediate: the anterolateral spots were intermediate in size between the parental types; the submedian lines were as in Ae. woodi; the scutellum had all three lobes silver-scaled as in Ae. simpsoni with a band of black scales between the lobes. The shape of the F1 female abdomen appeared intermediate between the rather pointed and thin abdomen of Ae. woodi and the more rounded abdomen of Ae. simpsoni. F1 x F1 matings; backerosses to Ae. simpsoni The results of forced matings between F1 hybrids and backcrosses of the hybrids to Ae. simpsoni, are given in Tables 1-6. After eggs were laid, or if no eggs were laid after several blood meals, the females were dissected to determine whether they were inseminated. The F1 x F1 matings proved to be easy, and co- pulation occurred readily, 25% and 33.3 %, respect- ively, of these matings being successful (Tables 1 and 2). However, in the backcross matings, with one exception, the matings were difficult. The exception was the F1 (Ae. simpsoni x Ae. woodi) x Ae. simp- soni cross, in which copulation took place readily. In all the backcross matings, the semen was often deposited externally on the females. Though copul- ation did occur, sperm was not always transferred. Even in those crosses in which copulation was difficult a surprising number of the backcross HYBRIDIZATION BETWEEN AEDES SIMPSONI AND AEDES WOODI Table 1. Results of mating of Fi female x Fi male (Pi: Ae. woodi x Ae. simpsoni) No. of eggs laid Female Insemi- per egg batch Remarks no. nated 1 st 2nd 3rd 1 _ _ _ - died 2 yes no eggs laid few sperm present; gravid 3 no no eggs laid 4 no 34 26 - based on failure of eggs to hatch 5 no 7 33 - based on failure of eggs to hatch matings were successful (Tables 3, 4, and 5). In the Ae. simpsoni x F1 (Ae. simpsoni x Ae. woodi) cross (Table 6), however, none of the matings was suc- cessful. Many females that laid eggs died before they could be dissected, and in these cases the state of insemina- Table 2. Results of mating of Fl female x Fl male (Pi: Ae. simpsoni x Ae. woodi) No. of eggs laid Fenmoale nated per egg batch Remarks 1 st 2nd 3rd 1 no - - - gravid 2 no 21 9 31 based on no hatch 3 no - - - 4 no 122 - - based on no hatch 5 yes 132 102 - eggs hatched 6 no - - - gravid 7 yes 27 68 66 eggs hatched 8 yes 95 72 51 eggs hatched 9 yes - - - 10 no 103 - - 11 yes 23 11 - based on hatch 12 no 123 101 - based on no hatch 13 no 13 78 - bades on no hatch 14 no - - - gravid 15 no 26 17 111 based on no hatch 16 no 27 11 63 based on no hatch 17 no - - - gravid 18 yes 42 87 - based on hatch tion was inferred from whether or not the eggs hatched. It may well be that these females were, in fact, inseminated, but that the sperm were unable to fertilize the eggs successfully or, if the eggs were fertilized, genetic incompatibility prevented them from developing normally. The fact that these females laid essentially normal-sized egg batches would suggest that insemination did take place, since Leahy & Craig (1965) showed that accessory gland substance introduced by the male was a Table 3. Results of mating of Fi female (Pi: Ae. simpsoni x Ae. woodi) x Ae. simpsoni male No. of eggs laid Female Insemi- per egg batch Remarks no. natedI 1 st 2nd 3rd 1 yes 97 78 - eggs hatched 2 yes 98 37 43 eggs hatched 3 yes 94 12 35 eggs hatched 4 yes 87 70 40 eggs hatched 5 yes 50 74 - eggs hatched 6 ? 17 51 27 egg vials lost before females dissected 7 ? 67 - -J or eggs hatched 8 yes 113 41 - eggs hatched4 9 no 61 38 - based on no hatch 10 lost - - - 11 no 21 69 67 based on no hatch 12 no 33 26 - based on no hatch 13 no - - - gravid 14 no 62 28 - based on no hatch 15 lost - - - 347 W. K. HARTBERG Table 4. Results of mating of Ae. simpsoni female x Fi male (Pi: Ae. woodi x Ae. simpsoni) No. of eggs laidFemale Insemi- per egg batch Remarks 1 st 2nd 3rd 1 yes 41 32 - eggs hatched 2 yes - - - gravid 3 yes 16 - - died 4 no - - - gravid 5 yes - - - seemed to be some agglutination of sperm: some sperm actively swimming 6 no - - - 7 yes - - - gravid 8 no - - - gravid 9 no - - - gravid 10 yes 71 - 44 eggs hatched 11 no 14 38 54 based on no hatch 12 yes - - - gravid 13 yes - - - gravid: just a few sperm present stimulant for oviposition in Ae. aegypti and in Ae. albopictus. However, the true state of insemination of these females remains questionable, since Gillett (1955, 1956) has shown that strains of Ae. aegypti differ in their capacity to lay eggs without the stimulus of insemination and this capacity is under genetic control. McClelland (1962) points out that Gillett's results fit the hypothesis ofa single dominant gene permitting ovulation. The same type of gene could be present in Ae. simpsoni and Ae. woodi. It Table 5. Results of mating of Fi female (Pi: Ae. woodi x Ae. simpsoni) x Ae. simpsoni male No. of eggs laid Female Insemi- per egg batch Remarks no. natedf__ 1 st 2nd 3rd 1 yes 92 - - eggs hatched 2 no - - - gravid 3 yes - _ - gravid; sperm immobilized 4 no - _ - gravid Table 6. Results of mating of Ae. simpsoni female x Fi male (Pi: Ae. simpsoni x Ae. woodi) No. of eggs laid Female Insemi- per egg batch Remarks no. nated 1 st 2nd 3rd 1 no - _ - gravid 2 died 3 no - - - gravid 4 no 5 died 6 lost 7 lost 8 no 9 no - 10 no - _ - gravid 11 no - _ - gravid 12 no - _ - gravid 13 no - - - gravid 14 no 12 58 - based on no hatch should be noted that most of the females that failed to lay eggs were found on dissection not to have been inseminated. It is interesting to note that a number of females in the backcrosses that were inseminated did not lay eggs. The reason for this is not known. In the backcrosses of F1 (Ae. woodi x Ae. simp- soni) males and females to Ae. simpsoni (Tables 4 and 5), two females were dissected in which some agglutination and immobilization of the sperm was noted. No record was kept of the percentage hatch of the eggs laid by the females in the present study. There was a tendency for the eggs to hatch " prematurely " before the cotton-wool had dried thoroughly during the conditioning. The newly emerged larvae died as the cotton-wool dried. Apparently the F2 and backcross-generation eggs do not need a strong hatching stimulus. The eggs of Ae. woodi also had the tendency to hatch " prematurely ", whereas the Ae. simpsoni eggs seldom hatched while the eggs were being conditioned. This difference in hatching can probably be attributed to the difference in the breeding sites of Ae. woodi and Ae. simpsoni. Ae. woodi has only been found breeding in the axils of the swamp sedges Cyperus grandis (Harper, 1955) and 348 HYBRIDIZATION BETWEEN AEDES SIMPSONI AND AEDES WOODI Table 7. Segregation of anterolateral spots phenotypes in F2 (P: Ae. simpsoni x Ae. woodi) Anterolateral spots of F2 progeny x2Cross Sex I12:1 ratio woodi-like hybrid-like simpsoni-like Pt: Ae. simsoni x Ae. woodi female 8 8 0 male 1 9 5 Ft x Ft totals 9 17 5 0.79a 0.70-0.50 a Calculated using Yate's correction. Cyperus exaltatus (unpublished report, East Africa assorted independently. Three of these characters, Aedes Research Unit) and is therefore not directly shape of anterolateral spots, length of submedian dependent on rainfall for breeding. Ae. simpsoni, lines, and silver scaling on the scutellum, were however, breeds in plant axils, tree-holes, etc., that analysed. are filled by rain. Thus it is advantageous for Ae. The shape of the anterolateral spots on the meso- simpsoni to require a stronger hatching stimulus notum of the F1 hybrids was intermediate between than Ae. woodi. those of the parental types. In the F2 progeny examined (Table 7) the shape of the anterolateral Genetic observations spots could be classed as woodi-like, hybrid-like, or The " premature " hatch of the eggs from the F2 simpsoni-like. The three types were recovered in a and backcross generations sharply reduced the 1: 2: 1 ratio. The data collected are compatible number of adults available for study. Enough with the hypothesis that the shape of the antero- individuals were examined, however, to give some lateral spots is controlled by a single, semi-dominant idea of the genetic basis of some of the morpholo- factor, the Ae. simpsoni character being semi-domi- gical differences between Ae. woodi and Ae. simpsoni. nant over the Ae. woodi character. In both the F2 and backcrosses to Ae. simpsoni, Table 8 records the data for the segregation of many of the characters of Ae. simpsoni and Ae. woodi anterolateral-spots phenotypes in the backcrosses Table 8. Segregation of anterolateral spots phenotypes in backcrosses to Ae. simpsoni Anterolateral spots of back- Cross Sex cross progeny x P1 : 1 ratio simpsoni-like hybrid-like Ae. simpsoni x Ft (Ae. woodi x Ae. simpsoni) female 11 17 male 14 5 total 25 22 0.086 a 0.80-0.70 Ft (Ae. woodi x Ae. simpsoni) x Ae. simpsoni female 1 19 male 7 8 total 8 27 9.26 a 0.01-0.001 Ft (Ae. simpsoni x Ae. woodi) x Ae. simpsoni female 21 36 male 48 54 total 69 90 2.52 a 0.20-0.10 a Calculated using Yate's correction. 349 W. K. HARTBERG to Ae. simpsoni. In these crosses two types, simpsoni- like and hybrid-like, were recovered. This is what would be expected if a single semi-dominant factor controlled the shape of the spots. The data from two of the three crosses examined statistically fit the hypothesis. It is reasonable to assume, in light of the F2 results, that the deviation from the 1: 1 ratio in the cross F1 (Ae. woodi x Ae. simpsoni) x Ae. simpsoni resulted from the use of small numbers of mosquitos. The submedian lines on the mesonotum of the F1 hybrids resembled those of the Ae. woodi parent. The progeny from three backcrosses were scored for this character. In all three crosses there appeared a 1: 1 ratio of woodi-like to simpsoni-like submedian lines (Table 9). The data from the crosses are com- patible with the hypothesis that the phenotype of the submedian lines is controlled by a single factor with the Ae. woodi condition dominant. The data from the third cross, F1(Ae. simpsoni x Ae. woodi) x Ae. simpsoni, do not fit the hypothesis statistically; however, the deviation can no doubt be attributed to the small numbers involved. In the F1 and backcross progeny, the lateral lobes of the scutellum were silver-scaled as in the Ae. simp- soni parent. A few individuals in the F2 progeny were recovered that had the dark-scaled lobes as in Ae. woodi. This would suggest that the dark-lobed Ae. woodi condition is recessive. McClelland (1962) backcrossed hybrids of Ae. woodi x Ae. simpsoni to Ae. woodi and recovered individuals with either silver-scaled lateral lobes or dark-scaled lateral lobes. These observations and the observations in the present study strongly suggest that the scaling on the lateral lobes of the scutellum is controlled by a single factor, with the Ae. woodi dark-lobed cha- racter recessive to the silver-scaled lobes of Ae. simp- soni. Attempted hybridization with Ae. aegypti Several attempts were made to forcibly mate males of Ae. simpsoni and Ae. woodi to females of Ae. aegypti. Copulation between Ae. simpsoni males and Ae. aegypti females was difficult to achieve with the forced-copulation technique. Six pairs success- fully copulated and the females laid no eggs even after several blood meals. Dissection and examina- tion of the spermathecae showed that the females had not been inseminated. No successful copulations were achieved between Ae. woodi males and Ae. aegypti females. McClelland (1962) was able to get some successful inseminations in the forced mating he conducted between Ae. aegypti, Ae. woodi, and Ae. simpsoni. He also observed that successful forced matings were possible between Ae. simpsoni and some but not all Table 9. Segregation of submedian lines phenotypes in backcrosses to Ae. simpsoni Cross Ae. simpsoni x Fi (Ae. woodi x Ae. simpsoni) Fl (Ae. woodi x Ae. simpsoni) x Ae. simpsoni Submedian lines of Sex backcross progeny female male total female male total female male total woodi-like 1 5 12 27 8 11 19 32 53 85 simpsoni-like 13 7 20 12 4 16 25 33 58 0.77 a 0.50-0.30 a Calculated using Yate's correction. x2 1 :1 ratio p Fl (Ae. simpsoni x Ae. woodi) x Ae. simpsoni 1.14 a 4.72 a 0.30-0.20 0.05-0.02 350 HYBRIDIZATION BETWEEN AEDES SIMPSONI AND AEDES WOOD3 the strains of Ae. aegypti he used. It may well be that different strains of these mosquitos can be forcibly mated more readily than others. DISCUSSION Aedes simpsoni and Ae. woodi are separated by both premating and postmating reproductive isolat- ing mechanisms. The primary isolating mechanism is no doubt behavioural. There is evidence for a difference in the sequence of biting and mating in the two species in nature (Hartberg, unpublished data). The difficulty in getting sperm transfer in the forced matings would indicate mechanical isolation also. The observation in the present study of sperm being immobilized and agglutinated in two females (Tables 4, 5) would suggest a degree of gametic isola- tion. The small numbers of backcross progeny obtained by McClelland (1962) indicate that another isolating mechanism between the two species is hybrid breakdown. The fact that fertile hybrids were obtained between two closely related species indicates a high degree of genetic and chromosomal homology. Three of the morphological differences between Ae. simpsoni and Ae. woodi have been shown in the present study to be controlled by single genes. McClelland (1962) and Hartberg (1965) have demonstrated that many of the morphological differences between Ae. aegypti and Ae. mascarensis (two Stegomyia mosquitos of Group A closely related to Ae. simpsoni and Ae. woodi) are also controlled by single genes. In addi- tion, Hartberg & Craig (in press) present genetic data that suggest there are chromosomal differences between Ae. aegypti and Ae. mascarensis. It is likely that further studies with Ae. woodi and Ae. simpsoni would also uncover chromosomal differences, as such differences are often the consequence of genetic isolation. Crossing experiments are one means of determin- ing relationships at the subgeneric level. In crosses where fertile offspring can be obtained, even if only in one direction, it may be possible to determine the nature of the genetic mechanisms controlling the differences between the species and which of these mechanisms have been altered during speciation. Such investigations may provide a pattern showing evolutionary relationships. UMt HYBRIDATION ENTRE AEDES SIMPSONI ET AEDES WOODI; OBSERVATIONS SUR LE FONDEMENT GENETIQUE DES DIFFERENCES MORPHOLOGIQUES La presente etude visait a determiner le fondement genetique des differences existant entre deux especes de Stegomyia, Aedes simpsoni et Ae. woodi et a mettre en evidence certains des facteurs responsables de l'isolement de ces deux especes sur le plan de la reproduction. On a utilise des moustiques adultes issus au labora- toire d'aeufs de femelles capturees dans la region de Dar es-Salaam (Tanzanie), et r6alis6 les accouplements par la technique de la copulation forcee. En contr6lant l'aptitude des cufs a l'6closion ou en examinant la sper- matheque, on a pu verifier s'il y avait eu insemination effective. On a pratique sans difficulte des accouplements entre Ae. woodi et Ae. simpsoni, mais on n'a observ6 d'inse- mination que dans un petit nombre de cas. La copulation forcee entre les hybrides F1 s'est faite ais6ment, alors que les r6trocroisements entre les hybrides F1 et Ae. simpsoni se sont reveles difficiles et, en cas de succes, n'ont pas toujours abouti a l'insemination. Les hybrides F1 offraient des caracteres intermediaires entre ceux des insectes parents: les dimensions des taches antero-lat6rales etaient intermediaires; les lignes sous- medianes 6taient analogues a celles d'Ae. woodi; le scu- tellum presentait sur ses trois lobes des ecailles argentees, comme chez Ae. simpsoni, avec une bande d'ecailles noires entre les lobes; chez les femelles F1, la forme de l'abdomen etait intermediaire entre la forme relativement mince et pointue d'Ae. woodi et la forme plus arrondie propre a Ae. simpsoni. Tant chez les descendants F2 que chez les insectes issus de retrocroisements, on retrouvait, repartis de facon independante, les caracteres d'Ae. woodi et d'Ae. simp- soni. L'analyse des donnees relatives a trois de ces carac- teres conduit 'a formuler les hypotheses suivantes: a) la forme des taches ant6ro-laterales est control&e par un facteur unique, le caractere Ae. simpsoni etant semi-domi- nant par rapport au caractere Ae. woodi; b) le phenotype des lignes sous-medianes est sous la dependance d'un facteur unique, le caractere Ae. woodi etant dominant; c) I'aspect des 6cailles sur les lobes lateraux du scutellum est sous la dependance d'un facteur unique, le caractere t lobes sombres * d'Ae. woodi etant recessif par rapport au caractere # lobes argentes * d'Ae. simpsoni. Ces observations - et celles d'autres auteurs - indi- quent que les mecanismes qui conditionnent l'isolement d'Ae. woodi et d'Ae. simpsoni sur le plan de la repro- duction agissent a la fois avant et apres l'accouplement. On a tente it plusieurs reprises, sans succes, de r6aliser des accouplements forces entre males d'Ae. simpsoni et d'Ae. woodi et femelles d'Ae. aegypti. 351 352 W. 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(1968) Mosquito News, 28, 217-221