Male-killer symbiont screening reveals novel associations in ladybirds.

While male-killing bacteria are known to infect across arthropods, ladybird beetles represent a hotspot for these symbioses. In some host species, there are multiple different symbionts that vary in presence and frequency between populations. To further our understanding of spatial and frequency variation, we tested for the presence of three male-killing bacteria: , and , in two ladybird species from a previously unexplored UK population. The two-spot ladybird, , is known to harbour all three male-killers, and we identified infection in the Merseyside population for the first time. However, in contrast to previous studies on two-spot ladybirds from continental Europe, evidence from egg-hatch rates indicates the strain present in the Merseyside population does not cause embryonic male-killing. In the related ten-spot ladybird, , there is only one previous record of a male-killing symbiont, a , which we did not detect in the Merseyside sample. However, PCR assays indicated the presence of a in a single specimen. Marker sequence indicated that this was divergent from that found in sympatric . Genome sequencing of the -infected additionally revealed the presence of cobionts in the form of a parasitoid wasp and the parasitic fungi . Further study of from this population is needed to resolve whether it is the ladybird or wasp cobiont that harbours , and to establish the phenotype of this strain. These data indicate first that microbial symbiont phenotype should not be assumed from past studies conducted in different locations, and second that cobiont presence may confound screening studies aimed to detect the frequency of a symbiont in field collected material from a focal host species.