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.