Many symbionts are sexually transmitted and impact their host’s development, ecology, and evolution. While the significance of symbionts that cause sexually transmitted diseases (STDs) is relatively well understood, the prevalence and potential significance of the sexual transmission of mutualists remain elusive. Here, we study the effects of sexually transmitted mutualist nematodes on their dung beetle hosts. Symbiotic Diplogastrellus monhysteroides nematodes are present on the genitalia of male and female Onthophagus beetles and are horizontally transmitted during mating and vertically passed on to offspring during oviposition. A previous study indicates that the presence of nematodes benefits larval development and life history in a single host species, Onthophagus taurus. However, Diplogastrellus nematodes can be found in association with a variety of beetle species. Here, we replicate these previous experiments, assess whether the beneficial effects extend to other host species, and test whether nematode-mediated effects differ between male and female host beetles. Rearing three relatively distantly related dung beetle species with and without nematodes, we find that the presence of nematodes benefits body size, but not development time or survival across all three species. Likewise, we found no difference in the benefit of nematodes to male compared to female beetles. These findings highlight the role of sexually transmitted mutualists in the evolution and ecology of dung beetles.

Dung beetle husbandry: Onthophagus taurus (originally collected in North Carolina, United States), Onthophagus binodis (Western Australia, Australia), and Digitonthophagus gazella (Florida, United States; and Queensland, Australia) were housed in large plastic containers filled with a two parts sand and one part soil mixture. Colonies were fed twice a week with ad libitum cow dung. The O. taurus and O. binodis colonies were held at 24ºC while the D. gazella colonies were held at 29 ºC.

Identification and maintenance of nematode cultures: Diplogastrellus nematodes were collected from the genitalia of multiple Onthophagus taurus males, originally collected in North Carolina. Dauer larvae were collected from the male aedeagus and suspended in 1ml Dulbecco’s phosphate buffered saline (DPBS). 10µl of this solution was then added to 10 Falcon tubes that were each filled with ca. 20ml of previously frozen cow dung. Each Falcon tube was closed with a lid that contained a mesh for ventilation and incubated at room temperature. To propagate these laboratory cultures, nematodes were rinsed off the cow dung surface with ca. 1ml of DPBS and transferred onto new Falcon tubes every 10-14 days.

Experimental rearing of dung beetle larvae with and without symbiotic nematodes: To generate experimental animals for each of the three dung beetle species, 6 females and 3 males were randomly selected from each laboratory colony and placed into ovipositing containers. These rectangular containers (27cm × 17cm × 28cm) were filled with a standard sterile mixture of sand and topsoil and topped off with an excess of defrosted cow dung. After five days, all containers were deconstructed, and the brood balls were collected. Eggs were removed from their natal brood balls using sterilized forceps. The eggs were then surface sterilized with a 100µl rinse of a 1% bleach and 0.1% Triton-X 100 solution followed by two rinses with 1ml distilled water. Once sterilized, the eggs were transferred into artificial brood balls constructed in 12-well tissue culture plates.

Once beetle larvae were added to their artificial brood balls, half of the individuals were inoculated with nematodes. A live nematode solution was created by rinsing nematodes off the surface of three of the previously described Diplogastrellus cultures. Nematode cultures were rinsed with about 3ml of DPBS, and the resulting solution was collected in multiple separate 1.5ml Eppendorf tubes and spun down in a centrifuge at 2000rpm for two minutes. The pellets (containing nematodes), as well as a 1ml aliquot of the supernatant (which does not contain nematodes based on visual inspection under a microscope) were saved, and the rest was discarded. The pellets were carefully resuspended in about 200µl of DPBS and combined into one tube. To calculate the number of nematodes in the sample, we transferred three 20µl aliquots of this solution separately onto a glass slide and counted the number of live nematodes under a dissecting microscope. We then calculated the average number of nematodes per 20µl stock solution across the three aliquots and diluted the original stock solution to a concentration of 1 live nematode per 1µl. Half of the eggs were inoculated with 20µl of this nematode solution. As a control treatment, we inoculated the other half of all larvae with 20µl of the (nematode-free) supernatant set aside previously. 

Beetle larvae (with or without nematodes) were incubated at 27 ºC throughout their juvenile development. This experiment was done in two blocks of data collection. During the first block, individuals were monitored every day and the date each individual hatched and molted into an adult was recorded. During the second block, individuals were monitored once every two days. Throughout both blocks, any death prior to adulthood was recorded, and once adulthood was reached (as indicated by the eclosure from the pupal cuticle), individuals were euthanized and stored in 70% ethanol. To measure adult body size, we took calibrated pictures of each individual’s thorax in a randomized order using a Pixelink PL-D797CU-T camera attached to a Leica MZ-16 stereomicroscope. Pronotum width was quantified using ImageJ.