Male‐specific alleles in the Ryukyu drywood termite Neotermes sugioi

Abstract Sex‐determination systems often show remarkable diversity in upstream signals, although downstream genes are broadly conserved. Therefore, the downstream genes have been investigated in various taxa, but the most upstream signals determining sex in insects have been well‐described mainly in model organisms, including fruit flies and honey bees, and not in hemimetabolous insects such as termites. Identification of sex‐linked genetic markers in termites is important to the survey of primary sex‐determination signals. Here, we report male‐specific alleles at the microsatellite locus NK12‐1 in the Ryukyu drywood termite Neotermes sugioi (Kalotermitidae). This study provides the third example of a genetic marker linked with sexual phenotype in termites, which is a small but important step to elucidate the evolutionary process of the sex‐determination system in termites.


| INTRODUC TI ON
Sex determination is one of the fundamental differentiation processes in the embryonic development of most animals. Although some of the major genes (doublesex and transformer) acting at the bottom of the sex-determining cascade are broadly conserved, the most upstream signals in the sex-determination cascade are remarkable diverse (Price, Egizi, & Fonseca, 2015;Suzuki, 2010;Verhulst, van de Zande, & Beukeboom, 2010). This diversity poses a major barrier to the study of the evolutionary process of sex-determination systems, as it prevents the identification of the most upstream signals in phylogenetically distant species and makes interspecific comparisons difficult. To the best of our knowledge, the most upstream signals of sex determination in hemimetabolous insects have not been revealed, unlike in model organisms, such as fruit flies and honey bees. Thus, how the sex-determination process evolved in Hemimetabola remains unclear.
At the other six loci, there were no significant differences in the frequency of heterozygosity between males and females in both Okinawa and Ishigaki populations (Fisher's exact test, p > .05; Figure   S1, Tables S3 and S4). Ten out of 144 females were all homozygous at all the nine loci we genotyped. Even when these potentially parthenogenetically produced females were excluded from the analysis, the results were almost identical: males tended to be heterozygous compared with females at the three loci (p < .0001 for NK12-1 and NK14-5 in both populations, p = .0065 for NK8-2 in Okinawa population, p > .05 for others; Table S4).

| D ISCUSS I ON
Our microsatellite analysis revealed male-specific alleles in the locus NK12-1 of N. sugioi (Figure 1), suggesting that this locus may be located closer to the most upstream sex-determination signals.
Moreover, the number of male-specific alleles was less than that of alleles shared between sexes, which might be arising from difference between the Y and X chromosomes. The Y chromosome is transmitted from male to male offspring only, while the X chromosome is transmitted from female to male and female offspring and from male to female offspring. Therefore, compared to Y chromosome, the mutation rate per generation will be high on the X chromosome and the variation is likely to be maintained in the population.
Contrary to the clear link of NK12-1, those of NK14-5 and NK8-2 are obscure. This incomplete linkage can be explained by the distance from sex-determining gene. These loci might be located on the sex chromosome but relatively far from the sex-determining gene compared with NK12-1, and recombination broke the linkage between these two loci and the most upstream signal of sex determination in some populations. Interestingly, in a colony AG20200611c4 at NK8-2, all males were homozygous (148/148) and all females were heterozygous (148/154). This pattern can be explained by a heterozygous father (X linked 154 bp and Y linked 148 bp) and a homozygous mother (X linked 148 bp). The heterozygous males (148/154) were actually found in a colony AG20200611c2, where the allele 148 bp was found only in males (Table S3). Thus, this result is also consistent with the hypothesis that this locus is linked to the sex-determination gene but the link is easily broken by recombination due to its long distance from the sex-determination gene.
Another potential mechanism explaining weak linkage of NK14-5 and NK8-2 is multi-chromosome translocation complexes in male F I G U R E 1 Sexual differences in the microsatellite genotype frequencies (NK12-1) of the Ryukyu drywood termite Neotermes sugioi (Kalotermitidae). In each panel, filled and open bars indicate the data of male and female N. sugioi termites, respectively. All males have the male-specific alleles (163 and 169 bp)