Population dynamics of male-killing and non-male-killing spiroplasmas in Drosophila melanogaster

Abstract

The endosymbiotic bacteria Spiroplasma spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 x 10(8) dnaA copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 x 10(9) dnaA copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 x 10(-5) for NSRO and 3 x 10(-4) for NSRO-A.

FIG. 1.

Infection dynamics of male-killing and non-male-killing spiroplasmas throughout the early developmental stages of D. melanogaster, expressed in terms of the number of dnaA copies per individual insect (A) or per host ef1α gene copy (B). Five replicated measurements for the egg and first-instar stages and 10 replicated measurements for the later stages were obtained. Note that for the tiny eggs and first-instar larvae five samples, each containing 100 individuals, were used for DNA extraction and quantitative PCR analysis. The error bars indicate standard errors. An asterisk indicates that there is a significant difference between NSRO-infected females and NSRO-A-infected females (asterisks above the lines) or between NSRO-A-infected females and males (asterisks below the lines) (P < 0.05, as determined by the Mann-Whitney U test).

FIG. 2.

Infection dynamics for male-killing and non-male-killing spiroplasmas during aging of D. melanogaster adults, expressed in terms of the number of dnaA copies per insect (A) or per host ef1α gene copy (B). Ten replicated measurements were obtained. The error bars indicate standard errors. An asterisk indicates that there is a significant difference between NSRO-infected females and NSRO-A-infected females (asterisks above the lines) or between NSRO-A-infected females and males (asterisks below the lines) (P < 0.05, as determined by the Mann-Whitney U test).

FIG. 3.

Relationship between the age of female adults of D. melanogaster infected with male-killing spiroplasma strain NSRO and the sex ratio of their offspring. Five replicated measurements were obtained. The error bars indicate standard deviations.

FIG. 4.

Threshold density hypothesis for the expression of male killing caused by spiroplasmas.