## 4 Discussion
### Scg-Met Knockdown Severely Disrupts the Reproductive Development of Adult Male Locusts  
_Scg-Met_ mRNA levels were significantly reduced by injecting the locusts with _dsScg-Met_ indicating that the RNAi procedure was effective (Figure 1A). In addition, we also investigated the levels of _Scg-Krh1_ and _Scg-E93_, situated downstream of Met in the MEKRE93 pathway in juvenile insects. The significant reduction in _Scg-Krh1_ demonstrates that JH signaling was impaired upon knocking down _Scg-Met_ (Figure 2A). E93 is an ecdysone inducible transcription factor, of which the expression is inhibited by Kr-h1, and vice versa [23; 69; 70; 71; 72]. In line with this, an increase in the relative mRNA levels of _Scg-_E93 was observed in the CA (Figure 2A). Therefore, the contrasting expression profiles of _Scg-Krh1_ and Scg-_E93_ are very well in agreement with the sustained _Scg-Met_ knockdown that was obtained by the regular _dsScg-Met_ injections, suggesting that a mutually inhibiting cross-talk between _Scg-_Kr-h1 and _Scg-_E93_ also persists in adult male locusts. Knockdown of _Scg-Met_ resulted in very strong phenotypic effects, indicating a pivotal role of this gene in the life history of male desert locusts:  
1. An obvious visual effect of the _Scg-Met_ knockdown was the impaired cuiucular pigmentation of the adult males (Figure 3). _S. gregaria_ adults are weakly pigmented after their final molt. Hardening of the cuticle upon molting, results in steady background coloration. In adult gregarious males, a remarkable change to a bright yellowticular coloration occurs when they become sexually mature [73]. In our colony, this color change normally manifests itself at days 9-12 after the final molt. Research has highlighted the role of yellow protein (YP) in this remarkable phenotypic transformation. YP has a high binding capacity for carotenoids originating from the diet and will be allocated towards the epidermis and cuticle when gregarious males become sexually mature. High levels of JH during adult male development will induce increasing YP levels, eventually resulting in the deposition of the yellow pigment in the cuticle [74]. The strongly reduced _Scg-YP_ expression in _dsScg-Met_ injected males is fully in line with the observed absence of a bright yellow coloration in the cuticle, in stark contrast with the _dsGFP_ injected (control) males (Figure 3). 2. In comparison to _dsGFP_ injected (control) males, the _Scg-Met_ knockdown locusts produced much lower amounts of the pheromone PAN (Figure 4). Gregarious desert locusts live in very dense populations. Besides visual cues, pheromone communication plays a crucial role in mating behavior. One of the best-known pheromones produced by gregarious male desert locusts is phenylacetonitrile (PAN). This pheromone is abundantly released by the legs and wings of gregarious male locusts and plays a crucial role in identifying an appropriate sexual partner by preventing male-male interactions, as well as in the post-copulatory guarding of the inseminated female mate [38]. Knockdown of _Scg-Met_ very strongly affected PAN emission, which illustrates that _Scg-_Met plays a crucial role in the emission of this male-to-male anti-aphrodisiac pheromone. 3. The development of the male reproductive organs, testes and accessory glands, was significantly inhibited by knocking down _Scg-Met_ during the adult stage (Figure 5). Interestingly, a recent study demonstrated that treating male _S. gregaria_ nymphs (final nymphal stage) with a JH mimic resulted in underdeveloped accessory glands and seminal vesicles, and impaired molting to the adult stage [75]. This suggests a different function of JH in the development of reproductive organs in the desert locust during distinct life stages, again highlighting the complex role of JH throughout the life history of insects. The role of JH in protein accumulation in AG has been described in several insect species [76]. A more recent study on _D. melanogaster Met_[27] mutants first demonstrated the role of Met in the development of the male AG.