Polyandry in the medfly - shifts in paternity mediated by sperm stratification and mixing

Abstract

Background: In the Mediterranean fruit fly (medfly), Ceratitis capitata, a highly invasive agricultural pest species, polyandry, associated with sperm precedence, is a recurrent behaviour in the wild. The absence of tools for the unambiguous discrimination between competing sperm from different males in the complex female reproductive tract has strongly limited the understanding of mechanisms controlling sperm dynamics and use.

Results: Here we use transgenic medfly lines expressing green or red fluorescent proteins in the spermatozoa, which can be easily observed and unambiguously differentiated within the female fertilization chamber. In twice-mated females, one day after the second mating, sperm from the first male appeared to be homogenously distributed all over the distal portion of each alveolus within the fertilization chamber, whereas sperm from the second male were clearly concentrated in the central portion of each alveolus. This distinct stratified sperm distribution was not maintained over time, as green and red sperm appeared homogeneously mixed seven days after the second mating. This dynamic sperm storage pattern is mirrored by the paternal contribution in the progeny of twice-mated females.

Conclusions: Polyandrous medfly females, unlike Drosophila, conserve sperm from two different mates to fertilize their eggs. From an evolutionary point of view, the storage of sperm in a stratified pattern by medfly females may initially favour the fresher ejaculate from the second male. However, as the second male's sperm gradually becomes depleted, the sperm from the first male becomes increasingly available for fertilization. The accumulation of sperm from different males will increase the overall genetic variability of the offspring and will ultimately affect the effective population size. From an applicative point of view, the dynamics of sperm storage and their temporal use by a polyandrous female may have an impact on the Sterile Insect Technique (SIT). Indeed, even if the female's last mate is sterile, an increasing proportion of sperm from a previous mating with a fertile male may contribute to sire viable progeny.

Figure 1

Changes in the proportion of offspring sired by the first (P1) and second (P2) male over time from twice-mated females. The mean proportion of progeny sired by 52 wild-type females mated first to tGFP1 males and then to DsRedEx1 males is shown on the left, whereas the mean proportion of progeny sired by 50 females mated according to the reciprocal male order is shown on the right. Green bars represent the progeny attributable to tGFP1, whereas red bars represent the progeny attributable to DsRedEx1 males. Vertical bars indicate standard errors.

Figure 2

Fertilization chambers of once-mated females. The fertilization chambers were dissected from wild-type females once-mated to wild-type (2A-B), tGFP1 (2C), and DsRedEx1 (2D) males, respectively. Picture 2A was captured using phase contrast, whereas Pictures 2B, 2C and 2D are the result of merging of phase contrast and epifluorescence microscopy captured with the Zeiss filters sets 13 and 20. Scale bar = 15 µm.

Figure 3

Confocal merged images of fertilization chambers of twice-mated females. Green sperm was transferred by the first male (tGFP1 line) and the red sperm (DsRedEx1 line) by the second male. In 3A, the fertilization chamber was dissected 24h after the remating and immediately observed. In 3B, the fertilization chamber was dissected seven days after the remating. The two squares at the bottom of each picture show, from the left, the single red and green unmerged images, respectively. Scale bar = 15 µm.