The Development of Adultoid Reproductives and Brachypterous Neotenic Reproductives From the Last Instar Nymphs in Reticulitermes labralis (Isoptera: Rhinotermitidae): A Comparative Study

Lower termites are characterized by a unique flexibility in development (Korb et al. 2009). Caste development in the genus Reticulitermes within Rhinotermitidae, as lower termites, is highly plastic, so that environmental stimuli can direct the developmental pathway (Elliott and Stay 2007, Korb and Hartfelder 2008). In Reticulitermes, newly hatched individuals (larvae) develop into workers or nymphs, which are distinguished by the absence or presence of wing pads, respectively. The primary reproductives develop via the last instar nymphs molting into winged sexuals (alate adults) that fly off at the same time in great swarms, mate, and create new colonies. In the absence of the primary reproductives, some of the workers and nymphs develop into secondary reproductives to continue colony growth. The workers become apterous neotenics, and the nymphs become brachypterous neotenics (Thorne et al. 1999, Korb and Hartfelder 2008). Roisin and Pasteels (1991) and Sieber (1985) reported that in Kalotermitidae and Termitidae, adultoid reproductives (AR) occurred as alate individuals who shed their wings and reproduce within the natal colony to become secondary reproductives. Neoh et al. (2010) suggested that the AR in Macrotermes gilvus (Termitidae) developed from alate adults with torn wings and retained at the original nest as secondary reproductives. However, in Reticulitermes urbis and R. labralis, the AR with floppy wings develop from nymphs and have the secondary reproductive function (Ghesini and Marini 2009, Xing et al. 2015). Field and laboratory colonies of Reticulitermes contain a large number of secondary reproductives derived from nymphs, in which the secondary reproductives play important roles in colony growth (Huang et al. 2013, Wu et al. 2013). Therefore, the study of differentiation of secondary reproductives from nymphs has important significance for understanding the expansion of the colony. All the individuals in a termite colony share similar genetic backgrounds. The maintenance and transformation of the caste structure are controlled by environmental factors and pheromones that induce gene expression based on their endocrine signature (Korb and Hartfelder 2008; Korb et al. 2009, 2012). Juvenile hormone (JH) is a central regulator of termite postembryonic development and life history traits (Korb 2015). Nijhout and Wheeler (1982) proposed a model for caste differentiation of termites, in which continuous low JH titers would induce alate adult differentiation, whereas high JH titers followed by low titers would induce neotenic reproductive differentiation (Cornette et al. 2008). In Reticulitermes flavipes, the development of a worker into an apterous neotenic reproductive requires somewhat elevated JH synthesis immediately prior to the molt (Elliott and Stay 2008). The increase in JH titers can predict the differentiation of secondary reproductives in Kalotermes flavicollis (Nijhout and Wheeler 1982). The reproductive maturity of female individuals is signified by the emergence of vitellogenic oocytes in insects. Vitellogenin has been considered a sex-specific protein that is exclusively required for oocyte vitellogenesis (Korb and Hartfelder 2008). During vitellogenesis, vitellogenin is synthesized in the fat body, secreted into the hemolymph and taken up by the developing oocyte. JH in hemolymph stimulates vitellogenin gene expression and promotes vitellogenesis of oocytes (Borst et al. 2000, Parthasarathy et al. 2010). For Reticulitermes speratus queens, the increased JH titers are positively correlated with high levels of vitellogenin gene expression (Maekawa et al. 2010). However, at different stages of oogenesis in R. flavipes, JH does not continuously stimulate vitellogenesis of the reproductives. For example, when the oocytes were at the early stage of vitellogenesis, the rate of JH synthesis was high; when the oocytes were at the stages before and after vitellogenesis, the rate of JH synthesis was low (Elliott and Stay 2007). In Zootermopsis angusticollis, an elevated JH titer prevents precocious ovarian activity in immature alates or stimulates oogenesis in mature queens. Therefore, JH plays a dual role in the reproductives depending on their developmental stage (Brent et al. 2005, Maekawa et al. 2010). Although both AR and brachypterous neotenic reproductives (BN) from the last instar nymphs are observed in field and laboratory colonies of Reticulitermes, the developmental differences between them are still unclear. In particular, the development of AR has been rarely studied. To better understand the regulation mechanisms of the development of AR and BN and to elucidate the biological characteristics and reproductive functions of AR, in this study, the dynamic changes in JH levels, vitellogenin gene expression and oogenesis during the development of AR and BN of R. labralis from the last instar nymphs are investigated and compared for the first time.