Discussion
The remarkable sensitivity of insect olfactory system has intrigued scientists for decades. The behavioral responses of moths to odor molecules reveal that insects have a highly sensitive olfactory system (Kaissling, 1987; Ishida and Leal, 2005). ODEs acting as rapid odorant inactivators play the major roles in insect behavior (Leal, 2013). However, the molecular mechanism of ODEs involved in the odorant reception of insects is not fully understood. Here, we identified theG. molesta CXEs and demonstrated functions of several target GmolCXEs in regulating the adult and larva behavior by metabolizing sex pheromone components or food ester volatiles in G. molesta .
In the present study, we identified 23 candidate GmolCXE genes by the larval heads’ transcriptome analysis. The number of theGmolCXEs identified in G. molesta was similar to the identified species in Lepidoptera from C. suppressalis (19 CXEs) (Liu et al., 2015), S. inferens (20 CXEs) (Zhang et al., 2014),S. littoralis (20 CXEs) (Durand et al., 2010b), S. litura (24 CXEs) (Zhang et al., 2016). OurGmolCXE1 and GmolCXE5 were widely distributed in the olfaction associated organs of adults and highly expressed in antennae, which was similar to the reported finding in S. litura (Zhang et al., 2016) and Ectropis obliqua (Sun et al., 2017). Besides expression in adults, seven genes (GmolCXE7 , 10 , 13 , 14and 20 -22 ) were highly expressed in larval heads. However,GmolCXE1 , 5 , 13 , 14 and 21 expression ofG. molesta were significantly up-regulated after fresh fruits (fuji apples and crown pears) and female adult stimulation. No publications reported the screening of CXEs involved in behaviors (foraging, mating, etc.) via by odor-stimulation experiments. GmolCXE1 and GmolCXE13 in the evolutionary tree were along with ApolODE/ PDE that degraded the pheromone of A. Polyphemus in vitro (Ishida & Leal, 2005). Consequently, we inferred theGmolCXE1 , 5 and GmolCXE13 , 14 , 21might be involved in the process of odor molecule reception during mating and foraging of G. molesta respectively.
Previous studies demonstrated that male moths of Lepidoptera insect showed 0.5-1.0 sec behavioral responses to sudden changes of pheromone concentration in the environment during this pheromone-oriented flight (Kramer, 1975; Vogt et al., 1985). The purified ApolCXE degraded the A. polyphemuspheromone with a half-life of 15 msec (Vogt et al., 1985). In this study, the male moths of G. molesta displayed a weak responses to ester sex pheromones of female moths when GmolCXE1 andGmolCXE5 were knocked down by RNAi. Similarly, foraging of the larvae were weaken and the responses of larvae to ethyl butanoate and ethyl hexanoate were decreased after the GmolCXE14 andGmolCXE21 silencing. CXEs acting as ODEs can rapidly inactivate ester odor molecules (sex pheromones, odorant volatiles) in order to restore the sensitivity of the sensory neuron (Vogt et al., 1985; Leal, 2013). Once the CXEs were knocked down, odorant molecules would continuously stimulate the olfactory receptor neurons so that restore the sensitivity of the sensory neuron could not cannot be restored and reset. That’s why G. molesta spent much time looking for food or mates after GmolCXEs silencing. No studies were reported the behavioral experiments of CXEs knocked down by RNAi. Nevertheless, other molecules (OBPs, ORs, etc.) associated with odor recognition were widely reported that male moths reduced the responses to sex pheromone components and plant volatiles after these genes silencing (Dong et al., 2017; Zhang et al., 2017a). The above mentioned results verified the GmolCXE1 andGmolCXE5 involved in regulating the courtship and mating behavior of male adults, and the GmolCXE14 and GmolCXE21 involved in regulating the foraging behavior of larvae in G. molesta . Therefore, make clear that these GmolCXEs degrades the corresponding ester odor molecules in the process of odor recognition is the question we’re going to solve.
The calculated molecular masses of GmolCXE1, 5 14, 21 (59.30~64.79 kDa) were similar to that of purified GmolCXE1, 5, 14, 21 (59 kDa < MW< 65 kDa) measured by gel filtration (Figure S2). (Z )-8-dodecenyl acetate and (E )-8-dodecenyl acetate are major ester sex pheromone components of G. molesta (Cardé et al., 1979). Ethyl butanoate and ethyl hexanoate were verified to be major ester odorant volatiles of ripe crown pears in this paper. Odorant volatiles of host plants and sex pheromones, plays critical roles in the foraging, courtship and mating of insect (Leal, 2013; Li and Liberles, 2015). Previous studies had demonstrated ApolCXE could degrade sex pheromone component Z11-16: Ac of A. Polyphemus by electrophysiological test (Ishida and Leal, 2005), and SexiCXE10 could degrade ester sex pheromones (Z11-14:Ac, Z9-16:Ac and Z11-16:Ac) and plant volatiles ((Z)-3-hexenyl acetate) by GC (He et al., 2015). Here, by EAG, GC-MS and fluorescence binding assays, we verified that the GmolCXE1 and GmolCXE5 degraded the (Z/E )-8-dodecenyl acetate to produce (Z/E )-8-dodecenol respectively. Study has indicated that PjapPDE degrade sex pheromones of Popillia japonica , and the degradation products are inactive into the sensillar lymph of pheromone-detecting sensilla on the P. japonica antennae (Ishida and Leal, 2008). Meanwhile, we also demonstrated ethyl butanoate and ethyl hexanoate were degraded by GmolCXE14 and GmolCXE21 respectively. Our results supported the report that the ApolCXE rapidly degraded the sex pheromones of A. polyphemusunder conditions where there were no competing pheromone-protein interactions (Vogt et al., 1985).
There is a clear sense that CXEs are an important part in the process of odor signal (Leal, 2013). Many studies researched on that adult CXEs could degrade ester sex pheromone components (Ishida and Leal, 2008; Chertemps et al., 2015; He et al., 2015), few studies showed adult CXEs could degrade ester plant volatiles (Durand et al., 2010a; He et al., 2015). Here, not only did we show that adult’ GmolCXE1 and GmolCXE5 degraded ester sex pheromones helping male adults to find female ones for mating, but we also showed that larval GmolCXE14 and GmolCXE21 degraded ester plant volatiles helping them forage host plants (crown pear).