Exposure to opposing temperature extremes causes comparable effects on Cardinium density but contrasting effects on Cardinium-induced cytoplasmic incompatibility

Abstract

Terrestrial arthropods, including insects, commonly harbor maternally inherited intracellular symbionts that confer benefits to the host or manipulate host reproduction to favor infected female progeny. These symbionts may be especially vulnerable to thermal stress, potentially leading to destabilization of the symbiosis and imposing costs to the host. For example, increased temperatures can reduce the density of a common reproductive manipulator, Wolbachia, and the strength of its crossing incompatibility (cytoplasmic incompatibility, or CI) phenotype. Another manipulative symbiont, Cardinium hertigii, infects ~ 6-10% of Arthropods, and also can induce CI, but there is little homology between the molecular mechanisms of CI induced by Cardinium and Wolbachia. Here we investigated whether temperature disrupts the CI phenotype of Cardinium in a parasitic wasp host, Encarsia suzannae. We examined the effects of both warm (32°C day/ 29°C night) and cool (20°C day/ 17°C night) temperatures on Cardinium CI and found that both types of temperature stress modified aspects of this symbiosis. Warm temperatures reduced symbiont density, pupal developmental time, vertical transmission rate, and the strength of both CI modification and rescue. Cool temperatures also reduced symbiont density, however this resulted in stronger CI, likely due to cool temperatures prolonging the host pupal stage. The opposing effects of cool and warm-mediated reductions in symbiont density on the resulting CI phenotype indicates that CI strength may be independent of density in this system. Temperature stress also modified the CI phenotype only if it occurred during the pupal stage, highlighting the likely importance of this stage for CI induction in this symbiosis.

Fig 1. Experimental design for testing the effects of prolonged temperature exposure on Cardinium’s CI phenotype.

Color of bars denotes temperature range experienced at that life stage (no bar = 27°C, red = 32–29°C, blue = 20–17°C). This experimental design was modified slightly for temperature shock and vertical transmission assays. In shock assays, temperature shocks did not extend to multiple life stages. Only the control and larval treatments were performed in the vertical transmission experiment.

Fig 2. Proportion of arrested whiteflies (proxy for CI-mortality) generated from crosses involving male wasps exposed to different temperatures.

Color of bars denotes temperature range (grey = 27°C, red = 32–29°C, blue = 20–17°C). Treatments are also separated by the stage at which the temperature exposure began. R = rescue cross, C+ female x C+ male. CI = CI cross, C- female x C+ male. Asterisks denote significant differences compared to crosses of the same type performed at 27°C. Numbers above whisker plots denote replicate number. N = 5–12 males, with a mean offspring per male = 17.7.

Fig 3. Proportion of surviving offspring (developed pupae) generated from crosses involving female wasps exposed to different temperatures.

Color of bars denotes temperature range (grey = 27°C, red = 32–29°C, blue = 20–17°C). Treatments are also separated by the stage at which temperature exposure began. N = control cross: C+ female x C- male. R = rescue cross: C+ female x C+ male. Asterisks denote significant differences compared to respective crosses at 27C. § denotes significant differences to the N cross within that temperature treatment. N = 15 females for each treatment, with the mean number of offspring per female = 9.25.

Fig 4. Density of Cardinium infection in adult a) male E. suzannae and b) female E. suzannae.

Densities were measured by estimating the amount of single copy Cardinium gyrB genes relative to single copy host ef1-alpa genes with qPCR. N = 5–10 for males (denoted by numbers above whisker plots) and N = 5 for females. Each sample was run with three technical replicates. Color of bars denotes temperature range (grey = 27°C, red = 32–29°C, blue = 20–17°C). Treatments are also separated by the stage at which temperature exposure began. Asterisks denote significant differences compared to the symbiont density at 27°C.

Fig 5. Duration of male E. suzannae pupal stage (in days) at different temperatures.

Color of bars denotes temperature range (grey = 27°C, red = 32–29°C, blue = 20–17°C). Treatments are also separated by the stage at which temperature exposure began. Asterisks denote significant differences compared to the duration of the pupal stage at 27°C. Numbers above plots denotes replicates. N = 12–22 male wasps.

Fig 6. Correlation between CI strength (as denoted by the proportion of arrested whiteflies) and the duration of the pupal stage of male E. suzannae.

Points refer to development time and corresponding offspring mortality for individual males used in CI crosses. The color of points denotes temperature range (black = 27°C, red = 32–29°C, blue = 20–17°C). Treatments are also separated by the stage at which temperature exposure began (□ = Larva, Δ = Pupa, O = Adult).

Fig 7. Proportion of arrested whiteflies (proxy for CI-mortality) generated from crosses involving male wasps exposed to different temperature shocks.

Color of bars denotes temperature treatment (grey = 27°C, red = heat shock at 40°C, blue = cold shock at 4°C). Treatments are also separated by the stage at stage temperature exposure began. R = rescue cross, C(+) female x C(+) male. CI = CI cross, C(-) female x C(+) male. Asterisks denote significant differences compared to respective crosses at 27°C. Numbers represent replicates for each cross. N = 8–10, with the mean offspring per male = 15.3.