Mechanisms of sex determination and transmission ratio distortion in Aedes aegypti

Abstract

Background: More effective mosquito control strategies are urgently required due to the increasing prevalence of insecticide resistance. The sterile insect technique (SIT) and the release of insects carrying a dominant lethal allele (RIDL) are two proposed methods for environmentally-friendly, species-targeted population control. These methods may be more suitable for developing countries if producers reduce the cost of rearing insects. The cost of control programs could be reduced by producing all-male mosquito populations to circumvent the isolation of females before release without reducing male mating competitiveness caused by transgenes.

Results: An RNAi construct targeting the RNA recognition motif of the Aedes aegypti transformer-2 (tra-2) gene does not trigger female-to-male sex conversion as commonly observed among dipterous insects. Instead, homozygous insects show greater mortality among m-chromosome-bearing sperm and mm zygotes, yielding up to 100% males in the subsequent generations. The performance of transgenic males was not significantly different to wild-type males in narrow-cage competitive mating experiments.

Conclusion: Our data provide preliminary evidence that the knockdown of Ae. aegypti tra-2 gene expression causes segregation distortion acting at the level of gametic function, which is reinforced by sex-specific zygotic lethality. This finding could promote the development of new synthetic sex distorter systems for the production of genetic sexing mosquito strains.

Fig. 1

Phylogenetic tree derived from a maximum-likelihood analysis of TRA-2 RRM domain protein sequences. The phylogeny suggests that the duplication of AAEL004293 probably occurred in a common ancestor of mosquitoes after the split of this ancestor from other diptera. The sequences of RRM domains were sourced from Ae. aegypti (Aeg); Ae. albopictus (Alb); Ae. polynesiensis (Poly); Cx. quinquesfaciatus (Culex); An. gambiae (Gambiae); Drosophila melanogaster and Musca domestica. AAAEL009222/AGW27097, AAAEL006416 and AAAEL009224 are the three other Ae. aegyptitra-2 homologs. The protein IDs for the RRM domains are shown on the branches of the phylogenetic tree. Bootstrap values are indicated in each node. The scale bar shows the branch length, representing the number of substitutions per site. Protein sequences in the phylogenetic tree can be found in the VectorBase and NCBI database with the following IDs: [VectorBase: AAAEL009222/AGW27097, AAAEL006416, AAAEL009224 CPIJ016646, AGAP006798], [GenBan: KJ147314, KJ147315, KJ147316, KJ147317, KJ147318, KJ147319, KJ147320, KJ147321, NP476766.1 and XP005185276.1]

Fig. 2

Determination of the sex ratio in tra-2 RNAi transgenic mosquitoes. a Pupal sex ratio among the progeny of 10 single-pair crosses (Crosses 1–10) between homozygous males and females of line 6. b Pupal sex ratio among the progeny of 10 single-pair crosses (M1–10) between male (M) offspring from Cross-1 and Cross-2 and Rock females. Dissection of testes allowed males to be classified into those with very few sperms (V), lower sperm density (L) and normal sperm density (N) in comparison with Rock males. V = between 4 and 45 sperm; L = between 109 and 304 sperm; N = more than 400 sperm. c Pupal sex ratio among the progeny of 10 single-pair crosses (F1–10) between female (F) offspring of Cross-1 and Cross-2 and Rock males. The transgenic mosquitoes used for the crosses in a, b and c varied between 3 and 9 days old. Numbers at the top of the columns show the ratios of males and females. A significant deviation from the 1:1 sex ratio in a Chi-square test is indicated by *P < 0.05 or *** P < 0.001. The symbol ♦ indicates crosses in which dead sperms were found in the spermathecae of living Rock females 23 days after insemination by M1–10 males. All the crosses produced at least 80 eggs per cross with hatching rates >80 %, except for family F3 with a hatch rate of 71.9 %. The details of hatching rates and statistical tests are presented in the Additional file 5

Fig. 3

Segregation ratio of wild-type and heterozygous offspring in crosses between heterozygous male and wild-type mosquitoes at two different hatching times. Hatched eggs were produced from mass crosses between Rock females and heterozygous males (♂M1-pro, ♂M2-pro, ♂M9-pro and ♂M10-pro, the male progenies of M1, M2, M9 and M10). a One thousand eggs from each cross, produced after the first blood meal, showed no significant deviation from the anticipated 1:1:1:1 ratio in all the four mass crosses (P > 0.05; df = 3; all X ²  ≤ 5.73). The hatching rates were in the range of 82.2–97.2 %. b Two hundred eggs produced as described in A but hatched 1 month later, showed a hatching rate of 74.5–97 % and pooled values of four mass crosses were significantly different from the anticipated 1:1:1:1 ratio due to the mortality of heterozygous females (^*** P < 0.001; all X ²  ≥ 24.20; df = 3). After removing the heterozygous females from the counts, pooled values of heterozygous males, wild-type males and females in B did not deviate significantly from the anticipated 1:1:1 ratio (P > 0.05; X ²  = 0.00; df = 2). The numbers at the head of each column show the sex ratios of transgenic and wild-type mosquitoes from the same broods. The transgenic mosquitoes used for these crosses varied between 3 and 9 days old. The details of hatching rates and statistical tests are presented in Additional file 5

Fig. 4

Transgenic male ages in relation to sex ratio in the next generation. Transgenic homozygous males (C1, C2, C3, C4 and C5) from lines 2 and 10 and control Rock males (R1, R2, R3, R4 and R5) were divided into 3-day-old and 15-day-old age groups. The males in each group were crossed with Rock females. C1-5 and R1-5 are single-pair crosses. The stacked numbers in each column are values observed in each cross. The 3-day-old parental males produced no significant deviation from the anticipated 1:1 sex ratio. The red dashed line indicates 10 crosses showing significant deviation from the anticipated 1:1 sex ratio (P < 0.001; df = 1; all X ²  ≥ 15.07) when parental males were 15 days old. The details of hatching rates and statistical tests are presented in Additional file 5

Fig. 5

Dead sperm in the spermathecae of female mosquitoes. a Dead sperm without staining. Scale bar = 2.5 μm. b Dead sperm stained with Orcein. Scale bar = 1 μm. c Stained sperm from the age-matched control wild-type males. Scale bar = 1 μm. Images captured with a DP50 digital camera fitted to an Olympus BX51 microscope

Fig. 6

Female-specific lethality and sex determination. a Punnett square showing tra-2 RNAi segregation in a cross between a heterozygous male and a wild-type female. The knockdown effect driven by the minimal CMV promoter was not enough to cause an obvious effect on sperm activity but specific lethality in mm zygotes was still evident. b Punnett square showing tra-2 RNAi segregation in a homozygous line, resulting in dysfunctional m-chromosome-bearing sperm during spermatogenesis and no surviving female progeny after fertilization. c Hypothetical model of sex determination in Aedesa egypti in which paternal tra-2 mRNA (tra-2_pat) is necessary for the activity of m-chromosome-bearing sperms