Stable introduction of Wolbachia wPip into invasive Anopheles stephensi for potential malaria control

Abstract

The spread and invasion of the urban malaria vector Anopheles stephensi has emerged as a significant threat to ongoing malaria control and elimination efforts, particularly in Africa. The successful use of the maternally inherited endosymbiotic bacterium Wolbachia for arbovirus control has inspired the exploration of similar strategies for managing malaria vectors, necessitating the establishment of a stable Wolbachia-Anopheles symbiosis. In this study, we successfully transferred Wolbachia wPip into An. stephensi, resulting in the establishment of a stable transinfected HP1 line with 100% maternal transmission efficiency. We demonstrate that wPip in the HP1 line induces nearly complete unidirectional cytoplasmic incompatibility (CI) and maintains high densities in both somatic and germline tissues. Despite a modest reduction in lifespan and female reproductive capacity, our results suggest the Wolbachia infection in the HP1 line has little impact on life history traits, body size, and male mating competitiveness, as well as the ability of its larvae to tolerate rearing temperatures up to 38°C, although wPip densities moderately decrease when larvae are exposed to a constant 33°C and diurnal cyclic temperatures of 27-36°C and 27-38°C. These findings highlight the potential of the HP1 line as a robust candidate for further development in malaria control.

Fig 1. Generation of a stable wPip transinfection in An. stephensi through intensive PCR-screenings over multiple generations.

In each generation, 8 to 16 female parents from each isofemale line were individually assayed by PCR to detect wPip infection after producing offspring. Offsprings from positive mothers were pooled to establish subsequent generations for further screening. Generation 0 consists of individuals that survived embryonic microinjection.

Fig 2. CI crosses and Wolbachia densities in the whole bodies and different tissues of HP1 mosquitoes.

(A) Results of reciprocal crosses between the HP1 line and wild non-infected Hor line. (B) An. stephensi embryos in the compatible crosses with normal development. (C) An. stephensi embryos in the CI crosses with the early death during development. (D) Relative densities of wPip in HP1 females or males at 7 days old. (E) Relative density of wPip in ovaries, midguts, fat bodies, salivary glands of 7-day-old non-blood-fed HP1 females. wsp and rps6 genes were used as target and host reference genes, respectively. Error bars represent the standard errors based on 3 biological replicates for A and 5 biological replicates for D and E. Different lowercase letters above each column indicate significant differences, P < 0.05, Student’s t test for D, ANOVA analysis, followed by Tukey post-hoc tests for A and E.

Fig 3. The impacts of wPip on the longevity of HP1 line.

(A) The survival curves of unmated males, provided with sugar water only. (B) The survival curves of unmated females, provided with sugar only. (C) The survival curves of mated females, provided with sugar only. (D) The survival curves of mated females, privided with both sugar and blood meal. The dead mosquitoes were removed with an aspirator and recorded daily after the first blood meal. The curves represent the mean percentage of mosquitoes surviving from three biological replicates each day. Log-rank test was used to analyze significant differences in the longevity of adult mosquitoes between HP1 line and Hor line. P < 0.05 for A to D.

Fig 4. The impact of wPip on the reproduction of HP1 line.

(A) The fecundity of HP1 line and Hor line. (B) The eggs hatch rates of HP1 line and Hor line. (C) Egg hatch rates in laboratory cage populations with different Hor female: HP1 male: Hor male ratios. The red line illustrates the observed egg hatching rates, while the black line illustrates the expected egg hatching rates, assuming equal mating competitiveness of HP1 and Hor males and random mating. (D) HP1 male mating competitiveness index. Error bars represent the standard errors based on the biological replicates (n = 31 for A and B, n = 3 to 5 for C, and n = 5 for D). Different lowercase letters above each column indicate significant differences in values, P < 0.05, Student’s t test for A and B, One sample t test for C and D.

Fig 5. The tolerance of HP1 line to high temperatures.

Wolbachia densities of male (A) and female (B) in HP1 adults after larvae exposure to either constant temperatures or different diurnal cyclic temperatures. The constant temperature of 27 ± 1°C is used as the control group. wsp and rps6 genes are used as target gene and host reference gene, respectively. Error bars represent the standard errors based on eight biological replicates. Different lowercase letters above each column indicate significant differences in values, P < 0.05 (ANOVA analysis, followed by Tukey post-hoc tests).