Inter-clonal competition over queen succession imposes a cost of parthenogenesis on termite colonies

In social insect colonies, selfish behaviour due to intracolonial conflict among members can result in colony-level costs despite close relatedness. In certain termite species, queens use asexual reproduction for within-colony queen succession but rely on sexual reproduction for worker and alate production, resulting in multiple half-clones of a single primary queen competing for personal reproduction. Our study demonstrates that competition over asexual queen succession among different clone types leads to the overproduction of parthenogenetic offspring, resulting in the production of dysfunctional parthenogenetic alates. By genotyping the queens of 23 field colonies of Reticulitermes speratus, we found that clone variation in the queen population reduces as colonies develop. Field sampling of alates and primary reproductives of incipient colonies showed that overproduced parthenogenetic offspring develop into alates that have significantly smaller body sizes and much lower survivorship than sexually-produced alates. Our results indicate that while the production of earlier and more parthenogenetic eggs is advantageous for winning the competition for personal reproduction, it comes at a great cost to the colony. Thus, this study highlights the evolutionary interplay between individual-level and colony-level selection on parthenogenesis by queens.

Methods
(a) Clonal drive in queen population

We collected 175 colonies with kings and queens of R. speratus in pine or Japanese cedar forests in Kyoto, Shiga, Wakayama, Nagano, and Chiba, Japan from May to September 2017–2019. All termites were extracted from the nest within 10 days of collection, and the phenotypes of kings and queens (primary or secondary) and the number of each were recorded. The data were included in sheets “colony information” and “no. of royals”. Of the 175 total colonies, 28 were used for the genotyping analyses. Twenty secondary queens were randomly selected from each colony and stored at −80°C for genotyping. Primary queens were found in four of the colonies and were also stored for genotyping. The genotyping data were included in sheet “genotype of SQs”. Of the 28 colonies used for the genotyping analysis, 23 were used for taking queen weight measurements. Termites in the 23 colonies were extracted from the nest within 2 days of collection, and fresh weights of secondary queens were measured to the nearest 0.1 mg. Prior research has established that the total weight of queens, rather than the total number of queens, is a more accurate indicator of colony size. Therefore, the total weight of queens was used to analyse the correlation between the indicator of colony size and the degree of clonal drive. The data were included in sheet “no. of clones in SQ population”.
To investigate whether the frequency of certain alleles at specific loci increases in the secondary queen population from colonies dominated by a single clone type in queens, we conducted Fisher's exact test with Bonferroni correction. To compile the dataset for this analysis, we first defined a colony as a driven colony if over 90% of the genotyped secondary queens were composed of a single clone, which was then classified as the dominated clone type. Five colonies met this criterion (180626F, 180605I, 180721A, 180605L, 180609A). Colonies not meeting this criterion were categorized as non-driven colonies. Secondly, we listed the alleles found in each non-driven colony (sheet “alleles in non-driven colonies”), representing the alleles present in the primary queens of these colonies. Thirdly, to investigate the frequency of each allele present before the occurrence of clonal drive, we counted the number of each allele found in non-driven colonies for this analysis. If only one allele was detected at a locus, it means that the primary queens was homozygous for that allele, and therefore, that allele was double counted. Fourthly, we recorded the alleles of the dominated clone type in each driven colony (sheet "alleles in dominated clones"), and the count of each allele was used for the analysis.

(b) Parthenogenetically-produced alates in the field

(i) Sampling of dealates walking on the ground

We collected a pre-foundation population of R. speratus by traps. Each trap consisted of a plastic board (210 × 297 mm), a guiding wall (50 mm height), and four sticky traps (16 × 80 × 90 mm). The sticky trap, which has openings on all sides, was attached at each edge of the guiding wall. The guiding wall of this trap was set for leading dealates walking on the plastic board into the sticky traps. Fifty traps (a total of 200 sticky traps) were set haphazardly on the ground of an open forest of pine trees with grassy areas in Hieidaira, Kyoto, Japan, from 5 to 7 May 2015 during the swarming season of R. speratus. A massive synchronized flight was observed only on May 5th in this study site. On 7 May 2015, we brought all traps back to the laboratory and collected all the dealates from the sticky traps. The head width (the maximum distance across compound eyes) of each sample was measured under a stereoscope (Olympus, Tokyo, Japan) using a digital imaging system (FLVFS-LS; Flovel, Tokyo, Japan). Since the condition of the sticky trap samples varied among individuals, we measured head width as a stable body size indicator. The dealates were stored at −80°C for the following microsatellite analysis. Then, we analysed the genotypes of each individual for all eight microsatellite loci analysed. The data were included in sheet “genotype of alates”.

(ii) Sampling of dealates after colony foundation

We collected a post-foundation population of R. speratus from experimentally buried brown rotten pine wood pieces which were suitable for termite nesting material. The pine wood was cut into pieces of approximately 20W × 40D × 10H cm size, autoclaved, and half-buried in the ground at the same site on April 28, 2015. On May 14, 2015, which was 9 days after the mating flight, all the buried pine wood was excavated and transported to the laboratory. The pine wood was meticulously dissected to extract founding units. We determined the sex of each individual by examining the morphology of the terminal abdominal sterna under a microscope. The individuals were placed separately in a collection tube (1.5 mL) and stored at −25ºC. Then, we analysed the genotypes of each individual for the eight microsatellite loci. The data were included in sheet “genotype of alates”.

 (c) Survivorship of sexually- and parthenogenetically-produced alates

Three colonies (colonies A−C) containing alates were collected from pine or Japanese cedar forests in Kyoto, Japan, just before the swarming season in 2016. These colonies were maintained at 20°C to keep alates from swarming until the experiment started. Just before the experiment, each colony was transferred to a room at 28°C, and alates stimulated by high temperature emerged from their chambers in wood. Alates were then separated by sex and maintained in Petri dishes lined with a moist unwoven cloth, and used for the experiments within a day of the flight. We used 40 males and 40 females randomly chosen from each of the three colonies. After removing the wings from the alates, we measured their fresh weight and used them in the following experiment. The wings of each individual were preserved in a test tube containing 99.5% ethanol. Then, we analysed the genotypes of each individual using the wings for four microsatellite loci.

To compare viability and immunity levels between sexually-produced alates and parthenogenetically-produced alates, we investigated the survivorship of alates both with and without exposure to the entomopathogenic fungus Metarhizium anisopliae (source: National Institute of Technology Evaluation Biological Resource Centre, NBRC31961) that occurs with the termites in nature. Each termite was randomly assigned to one of the two treatments. See Text S1 for the methods of pathogen treatment. In the control treatment without the pathogen, the alates were individually exposed to a conidia-free 0.025% Tween 20 solution (without pathogen). After exposure to a conidia-free solution, termites were placed individually in a well of a 24-well plate (COSTAR®3526, Corning Inc. NY) lined with filter paper moistened with distilled water. The plates were maintained at 25ºC in darkness and checked every day for 30 days to investigate alate survival. The data were included in sheet “survivorship of alates”.