DOI: 10.5061/dryad.j3tx95xnn
title: Sexually transmitted mutualist nematodes promote host growth across dung beetle species

description: 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.
description: <strong>Dung beetle husbandry</strong>:
 <em>Onthophagus taurus </em>(originally collected in North
 Carolina, United States)<em>, Onthophagus binodis
 </em>(Western Australia, Australia)<em>, </em>and
 <em>Digitonthophagus gazella</em> (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 <em>ad libitum</em> cow dung. The <em>O.
 taurus</em> and <em>O. binodis</em> colonies were held
 at 24ºC while the <em>D. gazella</em> colonies were held at 29
 ºC. <strong>Identification and maintenance of
 nematode cultures</strong>: <em>Diplogastrellus
 </em>nematodes were collected from the genitalia of multiple
 <em>Onthophagus taurus</em> 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. <strong>Experimental rearing of
 dung beetle larvae with and without symbiotic
 nematodes</strong><em>: </em>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
 <em>Diplogastrellus </em>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.
description: # Sexually Transmitted Mutualist Nematodes Promote Host Growth Across Dung
 Beetle Species
 [https://doi.org/10.5061/dryad.j3tx95xnn](https://doi.org/10.5061/dryad.j3tx95xnn) 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. ## Description of the data and file structure The Excel file contains details on the species (*O. binodis*, *O. taurus*, or *D. gazella*), the rearing treatment (with or without nematodes), sex (male or female), thorax width (body size in millimeters), eclosure time (i.e., egg-to-adult development time in days), survival (binary variable), and experimental block (first or second). Individuals that were part of the experiment but died before sex, size, or development time could be measured have missing data (indicated as not available 'NA').