Incomplete removal of Wolbachia with tetracycline has two-edged reproductive effects in the thelytokous wasp Encarsia formosa (Hymenoptera: Aphelinidae)

Abstract

Wolbachia pipientis are intracellular endosymbionts that induce parthenogenesis in the parasitoid Encarsia formosa. Previous studies that focused on effects of Wolbachia on the wasp usually used tetracycline to remove Wolbachia without concern for the joint influences of tetracycline and Wolbachia. Here we treated the wasps (F0 lines) with tetracycline to produce offspring (F1 lines) which were not fed tetracycline to avoid antibiotic influence. The quantitative data and fluorescence in situ hybridization showed that Wolbachia titers were reduced but not totally removed. The Wolbachia that infected the male offspring were unpredictably detected. Low dose tetracycline enhanced the fertility of 2-day-old F0 wasps after 24 h of treatment; however, compared with controls, the oocyte load of 3- to 6-day-old tetracycline-treated wasps decreased day by day, and tetracycline reduced the longevity of the wasps. The fecundity of controls was significantly higher than that of the treated F1-10 and F1-20 generations. Gene expression of vitellogenin reflected the same trend as that of wasp fecundities in both F0 and F1 lines. Moreover, female offspring proportions of F0 and F1 lines were related to the titer of infected Wolbachia, demonstrating that Wolbachia titer affected the sex determination of E. formosa.

Figure 1. The number of offspring produced by E. formosa wasp in each generation.

G0 denotes the parental generation, while G1- G4 denote the filial generations. G0, G1, G2, and G3 were all treated with 10 mg/ml tetracycline solution. N: number of the total wasps; n: number of the female wasps.

Figure 2. Relative Wolbachia titers in E. formosa.

F0-0, F0-10, F0-20, and F0-50 were lines treated with 0, 10, 20, and 50 mg/ml tetracycline, respectively. Wolbachia titers were measured 1 d later. The F1-0, F1-10, F1-20, and F1-50 lines were female offspring of F0-0, F0-10, F0-20, and F0-50 wasps, respectively. M denotes males. The values are means + SD, means with different letters are significantly different (LSD test, P < 0.05).

Figure 3. FISH detection of Wolbachia in females and males of E. formosa.

(A,B,C) An F0-0 unfed female. (D,E,F) An F1-10 female offspring of F0-10 that was treated with 10 mg/ml tetracycline. (G,H,I) An F1-10 male offspring of F0-10 that was treated with 10 mg/ml tetracycline. M: mouthpart. FM: fight muscles. L: legs. ST: somatic tissues. OV: ovary. OP: ovipositor. T: testis. G: genitalia. Left panels (A,D,G) fluorescence signals; middle panels (B,E,H) bright field; right panels (C,F,I) combined bright field and fluorescence.

Figure 4. The reproductive effects of tetracycline on F0 wasps.

(A) Parasitism rate. (B) Oocyte load per ovariole. (C) Relative expression levels of the vitellogenin gene. F0-0, F0-10, F0-20, and F0-50 denote lines treated with 0, 10, 20, and 50 mg/ml tetracycline, respectively. The values are means + SD; means with different letters are significantly different (LSD test, P < 0.05).

Figure 5. The effects of tetracycline on the oocyte load of wasps.

The F0-0, F0-10, and F0-20 lines were treated continuously with 0, 10, and 20 mg/ml tetracycline, respectively. The values are means + SD; means with different letters are significantly different (LSD test for 2 day among F0-0, F0-10 and F0-20; t test for other ages between F0-0 and F0-10; P < 0.05). The uppercase letters indicate comparisons among different ages of the same line; the lowercase letters indicate the comparisons among different lines of the same age.

Figure 6. Longevity of F0 generation with continuous feeding on tetracycline.

The values are means + SD; means with different letters are significantly different (LSD test, P < 0.05).

Figure 7. Reproductive effects of the Wolbachia titer on F1 lines.

(A) Parasitism rate. (B) Oocyte load per ovariole. (C) Relative expression levels of vitellogenin gene. F1-0, F1-10, and F1-20 are the offspring of the females in the F0-0, F0-10, and F0-20 lines, respectively. The values are means + SD; means with different letters are significantly different (LSD test, P < 0.05).

Figure 8. The relationship between the relative Wolbachia titers of wasps and the sex ratios of their offspring.

F0-10, F0-20, and F0-50 are lines treated continuously with 10, 20, and 50 mg/ml tetracycline, respectively. F1-10 and F1-20 are the female offspring of the F0-10 and F0-20 lines, respectively. The values are means ± SD; means with different letters are significantly different (LSD test, P < 0.05).