4. Discussion
GOBPs are a member of the OBPs family play important roles as the first barrier for insects to perceive odorants. Therefore, understanding the role of GOBPs in an insect’s function requires inseparable from multisensory ecological interaction with the host (Stöckl and Kelber, 2019). In this study, we successfully addressed these behaviors by generating a homozygous strain with CRISPR-Cas9. Applied this genome editing system to produce a large deletion already be useful in Helicoverpa armigera and other insects in which the CRISPR-Cas9 system is effective (Wang et al., 2018; Wang et al., 2020b).
The homozygous GOBP1, GOBP2 and GOBP1/2 strain after knockout do not affect the insect’s viability under our rearing conditions, but it results in weakening the larva and adults’ sensitivity to food odorants. GOBPs are mainly localized in biconical sensilla and postulated to be involved in recognizing general odorants from host plants and the environment (Vogt et al., 1991; Wang et al., 2003; Steinbrecht et al., 1992; Laue et al., 1994). In this study, the larvae after the gene knockout were fed for five consecutive generations, there was no difference compared with the WT strain, and they could successfully emerge without showing a decrease in food sensitivity, mainly because there was more artificial diet around the larvae and this time its touch, taste or other olfactory genes play an important role. When the larvae were placed in a petri dish to keep them away from food and allowed to make choices, we found that they were significantly different from the WT strain. Its sensitivity to food odorants presents difficulties. This result indicates that GOBPs are indeed involved in identifying host odorants. However, not all larvae cannot find the location of the food, and it may be caused by the following reasons: a) some odorants in the diet may be recognized and operated to the receptor by other OBPs or CSPs, which as well as play crucial roles in insect chemoreception, such as recognizing, capturing and transporting hydrophobic chemicals from the environment to olfactory receptors (Pelosi et al., 2006; Pelosi et al., 2005; Liu et al., 2012); b) vision may play a role in identifying food; c) due to limited space, it could happen to find food after crawling aimlessly.
EAG only represents an overall activity of all the sensilla on the antenna and, therefore, even compassionate specialist olfactory receptor neurons may not show up if their total number is low (Deng et al., 2012). When the gene was knocked out, most of the moth’s antennae showed a weakened response or even no response to odorants (Figure 3a), indicating that GOBPs are closely related to the recognition of these odorants. Males and females have different responses to the same odorants, and compared to males, females respond more obviously to the tested odorants, which means that females can perceive more odorants than males. The same result can also show in the wind tunnel test. This may be related to their final task. In fact, many moths have shown plasticity of olfactory-guided behavior, dependent not only on the nature of the chemical but also on the physiological status (e.g., age, growth conditions, hormone or mating status) of the individual (Anton et al., 2007; Kubli, 2003; Jing et al., 2020). Our previous studies have also proved this result by stimulating host odorants, ovarian development, and changes in GOBPs expression before and after oviposition (Jing et al., 2019; Jing et al., 2020). For males and looking for hosts to maintain normal physiological activities after emergence, it is more important to find females to complete mating. Therefore, male adults may pay more attention to recognize sex pheromones. For females, looking for food and attracting males to complete mating, it is more important to find suitable oviposition sites, which determines that females need to be able to recognize more odorants. The wind tunnel test experiment in this study also strongly proved sex pheromones are more attractive to males, while host odorants are more attractive to females.
Interestingly, the effect of GOBP2 gene knocked out seems to be greater than GOBP1, and there is no significant difference between the knockout of both GOBP1 and GOBP2 genes (Figure 3 and 4). Previous research has demonstrated that proteins in the GOBP2 class share high sequence similarity and can bind to a wide range of odorants with a broad specificity (Deng et al., 2012; Zhou et al., 2009; He et al., 2010; Gong et al., 2009) and can bind with the sex pheromones (Feng and Prestwich, 1997; He et al., 2010; Liu et al., 2010). Another experiment also showed that GOBPs from different families of lepidoptera. Lepidoptera seem to have different expression patterns among male and female moths, indicating the different physiological roles of GOBP1 and GOBP2 in the perception of semiochemicals (Yao et al., 2016). This difference may be due to the gender mentioned above, but more importantly, is the function of the protein itself. ITC and SPR are good methods used to thermodynamically and kinetically characterize the protein-ligand or protein-protein interactions’ binding mechanism, respectively (Jelesarov and Bosshard, 1999; Krishnamoorthy et al., 2020; Kim et al., 2020). Based on the SPR results, GOBP2 is better than GOBP1 in terms of the number of binding proteins and binding ability. From this result, we can hypothesize that when GOBP2 binds to a variety of other proteins, a complex is formed, which greatly broadens the binding range of the GOBP2 protein, especially with a strong binding ability CSP protein. Because although CSP is a distinct from OBPs, it shares no sequence similarity with OBPs (Lu et al., 2007; Gong et al., 2007). Whereas OBPs are primarily expressed in antennae, CSPs are expressed in various insect tissues, such as antennae, maxillary palps (Maleszka and Stange, 1997), proboscis, pheromone glands (Meillour et al., 2000), and the sub-cuticular epithelium (Wanner et al., 2004), etc. Furthermore, although a vast amount of structural evidence on OBPs and CSPs has been collected in the effort to elucidate their function, physiological studies have been sparse and have failed to offer compelling models for their behavior (Pelosi et al., 2006). The GOBPs can reaction with CSPs by SPR and ITC may provide a new idea for research or contribute to more in-depth research on both.
ITC can detect whether proteins are interacting and measures the heat released or absorbed during the protein-ligand interactions (Krishnamoorthy et al., 2020; Freyer and Lewis, 2008). The results of ITC are consistent tendency with the results of SPR. All reactions with ΔG < 0 are spontaneous reactions, ΔH<0 are exothermic reactions and ΔS<0 indicates reactions are process driven by enthalpy, and an unfavorable entropy compensation effect occurs. It also shows that non-covalent bonds such as hydrogen bonds and van der Waals forces may be formed in these reactions. ΔS<0 indicates that during the binding process, a specific protein is likely to undergo a conformational change, which will reduce the degree of freedom of the molecule, which is a factor that is not conducive to the binding of these proteins. The conformational change of this protein may have a great relationship with the pH value. It was demonstrated previously that the C-terminal dodecapeptide segment of Bmor PBP formed a regular helix α7 at pH 4.5, the compact structure allowing the ligand to enter the binding cavity would not be detectable once the complex was formed; and at pH 6.5 did not present the additional α7. The C-terminus of the protein folds into an α-helical domain and enters the bombykol binding site, thus assisting the release of the pheromone molecule from the cavity (Wojtasek and Leal, 1999; Horst et al., 2001). The pH-dependent conformational structural flexibility was also reported in other members of olfactory proteins, such as in Aedes aegypti(Leite et al., 2009), Antheraea polyphemus (Mohanty et al., 2004). In addition, the structures of PBP1 and PBP2 of Lymantria dispar had different changes at pH 5.5 and 7.3 by fluorescence binding assay, showing different affinity to chemicals (Yu et al., 2012). Therefore, we believe that the ΔS will also change under different pH values, especially at low pH, which may be a favorable response. Especially when GOBPs and CSPs are combined, it will be more interesting if their conformation is changed by pH after forming a complex. Therefore, more in-depth experiments need to continue.
In conclusion, we have successfully knockout GOBP1 and GOBP2 genes inC. punctiferalis with the CRISPR-Cas9 system. Through the tendency test of larvae to artificial diet, EAG, and the wind tunnel test of the adult, it was found that the knockout effect of the GOBP2 gene is better than GOBP1. The protein interaction test initially explained that this is due to the more substantial binding capacity and broader binding spectrum of GOBP2 than GOBP1. Also, this study provides an additional perspective on insect GOBP, OBP, and CSP genes and their functional contribution to the pest olfactory system.