Laura B. Dickson; Maria V. Sharakhova; Vladimir A. Timoshevskiy; Karen L. Fleming; Alex Caspary; Massamba Sylla; William C. Black, IV

“ Aedes aegypti from Senegal, West Africa (SenAae) have a low oviposition rate; those that do oviposit have a low fecundity and poor egg-to-adult survival. Furthermore rearrangements were detected on all three chromosomes in SenAae. These observations are consistent with the presence of at least two cryptic subspecies of Ae. aegypti in Senegal arising from reproductive isolation due to chromosome rearrangements. Genetic control strategies are being considered for the suppression of Ae. aegypti populations worldwide. Barriers to gene flow in African Ae. ”

“ Male (“M”) alleles at the SDL are dominant so that males are “Mm” heterozygotes and females are homozygous recessive “mm.” Presgraves and Orr showed that for the most part Anopheline mosquitoes follow Haldane’s rule for sterility and inviability while Aedes mosquitoes only follow Haldane’s rule for sterility. The authors speculated that faster male evolution is driven by sexual selection which occurs irrespective of the sex determination mechanism. ”

“ The critical test for cryptic species would be to identify a priori two or more types of mosquitoes based on genetic markers, cytogenetics or phenotype and then show that reproductive incompatibilities exist between these types and not within them. ”

“ Aedes aegypti (Aaa) is light in color, has pale scales on the first abdominal tergite, oviposits in artificial containers, and preferentially feeds on humans. Aedes aegypti formosus (Aaf) , has a dark cuticle, is restricted to sub-Saharan Africa, has no pale scales on the first abdominal tergite and frequently oviposits in natural containers. Scale patterns correlate with cuticle color in East Africa but not in Senegal, West Africa where black cuticle mosquitoes display a continuum of scaling patterns and breed domestically indoors. ”

“ Even though individuals belonging to cryptic taxa can mate with one another, they produce few or no eggs (Fig 2) and if eggs are produced, they have lower survival to adults.
The suggestion that one or more new cryptic subspecies exist in Senegal in no way contradicts the phylogenetic patterns among worldwide Ae.aegypti s.l. populations that have been derived over the last 35 years using allozyme markers [78] – [18] , microsatellites [25] , [26] , [27] , nuclear SNP loci [15] , [28] , [22] , mitochondrial DNA [20] , [26] and most recently a SNP-Chip [28] . ”

“ Instead our results suggest that taxa in some of the clades resolved in these phylogenetic studies may be partially or wholly reproductively isolated from one another and furthermore that some clades identified in these earlier studies may contain reproductively isolated taxa. The results presented here therefore serve as a reminder of the importance of evaluating reproductive isolation among closely related taxa rather than assuming that they belong to a single reproductively continuous population. ”

“ If the results of the insemination experiment with Bignona (G) and Bignona (F) (Fig 5) can be extended to the other SenAae, then there is no evidence for assortative mating or sperm inviability. But strong inferences cannot be made concerning assortative mating under laboratory conditions.
Chromosomal rearrangements have frequently been associated with speciation in taxa including mosquitoes [74] . Pericentric inversions are only rarely detected within animal species but can be abundant among species [75] , [76] . ”

“ With the goal of performing Quantitative Trait Locus (QTL) mapping of gene regions associated with DENV2 susceptibility, Aaa P1 laboratory strains that are known to be highly susceptible to DENV2 infection were crossed with Senegal Ae. aegypti (SenAae) that are refractory to DENV2 infection [29] to breed F1 intercross families. During this process reproductive incompatibilities were observed between Aaa collected outside Africa and SenAae. Furthermore reproductive incompatibilities were observed within and among SenAae collections. ”

“ The first four contrasts in Table 3A indicate that offspring of crosses involving ROCK/PK10 hybrid F1 males had lower egg-pupal survival than offspring of ROCK families as predicted by Haldane’s rule if Aaa and SenAae are separate taxa. The next four contrasts show that offspring of crosses involving ROCK/PK10 hybrid F1 males had the same low egg-pupal survival as offspring of PK10 families. ”

“ To assess fecundity in F1 siblings, another series of F1 intercrosses were established with SenAae from Sedhiou and Ziguinchor from the Casamance area of southern Senegal (Fig 1) to determine whether the results seen in the first experiment were unique to PK10. These collections also had a large proportion of SenAae without scales on the first abdominal tergite. Ziguinchor (F) and Sedhiou (F) were crossed with one another and to D2S3. Egg to pupal survival in P1 and F1 eggs were recorded as was F2 pupal-to-adult survival. ”

“ Specifically, Haldane’s rule predicts that in fully or partially reproductively isolated species with heterogametic sex chromosomes, the heterogametic sex will be preferentially sterile and inviable in interspecific crosses. Dominance and rapid male evolution are often cited as the primary forces causing Haldane’s rule. ”

“ An earlier laboratory study did not indicate any pre- or postzygotic barriers to gene flow between Aaa and Aaf in East Africa. However, similar attempts to construct F1 intercross families between Aaa laboratory strains and Senegal Ae. aegypti (SenAae) failed due to poor F1 oviposition and low F2 egg-to-adult survival. Insemination and assortative mating experiments failed to identify prezygotic mating barriers. ”

“ Fig 3A shows the number of eggs produced by a) D2S3, b) Sedhiou (F) , and c) Ziguinchor (F) families, as well as d) D2S3 females crossed to Sedhiou (F) males and e) D2S3 females crossed to Ziguinchor (F) . All but the Sedhiou families produced similar numbers of eggs. There was wide variation in pupal survival (pupae/eggs) within all five crossing types but all were statistically homogeneous (Fig 3B) .
The number of eggs produced when intercrossing the F1 siblings to generate F2 families in each of the five crossing types was also recorded (Fig 4A) . ”

“ Fifty Higgs White Eye (HWE) females were located in a cage with 25 HWE males and 25 Richard Toll (F) males to test for assortative mating by Aaa females. Of 44 families generated, 24 were white eye, not significantly more than the 22 expected if there was no discrimination (χ2 [1 d.f] = 0.36. P = 0.55) . To test for assortative mating by Richard Toll (F) females, again 50 females were located in a cage with 25 HWE males and 25 Richard Toll (F) males. All F1 offspring were wild type and offspring adults were backcrossed to HWE adults to assess their genotypes. ”

“ However, most of the sterility in the present study arose from the absence of egg-laying, especially when females were crossed to hybrid F1 males. It was documented in 1958 that uninseminated Ae. aegypti females tend not to oviposit even when fully gravid [68] . In 1965, it was shown that male accessory gland substances, rather than sperm, acted as oviposition stimulants [69] in Ae. aegypti. Similarly in Drosophila melanogaster, the male accessory gland protein Acp26Aa stimulates oviposition [70] – [71] . ”

“ BAC clones are named by genetic markers they are carrying.The standard (A, B) and heterozygote (C, D) arrangement of the inversion on chromosome 2 in IB12 and Sedhiou strains respectively.BAC clones are named by genetic markers they are carrying. Prior studies document inversion polymorphisms on chromosome 1 in Aaa [52] , [53] . The linkage distance of the white-eye marker [45] and the sex determining locus (SDL) were determined to test for chromosome rearrangements on chromosome 1. These markers are at 20 cM and 34 cM on the standard genetic map of chromosome 1 in Ae. ”