The application of a next-generation of sequencing technology in insects has greatly
promoted the efficiency and quantity of gene annotation [
19]. Meantime, a lot of antennal transcriptomes olfactory-related genes were identified
[
21-24]. In this research, we identified 26 OBP genes, 19 CSP genes, 55 OR genes and 20
IR genes from the C. pinicolalis antennal transcriptome, and all the genes are reported for the first time C. pinicolalis is a sibling species of C. punctiferlis, and had ever been recognized as the same species [
24]. In C. punctiferlis, totally 25 OBPs, 15 CSPs, 62 ORs and 10 IRs were identified from antennae transcriptome
[
20], and the numbers of OBPs, CSPs and ORs are similar with C. pinicolalis, whereas more IRs were identified from the C. pinicolalis antennal transcriptome. This may depend on the depth of the sequencing. The olfactory
gene sequences similarity was also analyzed, as shown in the evolution tree (Fig.
3) and olfactory gene identities (Additional file 3: Table S1), OBP, CSP, OR and IR
genes have highly similarity with C. punctiferlis. Most of the identities are more than 90%, even some nucleotides sequences can reach
to 99%. From the evolution tree, most olfactory relative genes can cluster with C. punctiferlis at highly scores (Fig. 3). Others were matched with the O. furnacalis [
25
</a>]or other Crambidae family insect. This results also can conclude <em>C. punctiferlis and C. pinicolalis</em> are close relationship species.</p>
As in other insects [
26-28] OBPs and CSPs were highly detected in the antennae of both male and female (Additional
file 3: Table S1). Among these genes, many of them were sexual bias expression (Fig.
1). PBPs were widely thought as sex pheromone binding function, normally have 3~5
PBPs in each insect. Many research reported at least one of the PBPs can well bind
with sex pheromones [
29-31] . In our analysis, PBP2 showed significantly male bias expression, and PBP1, PBP3
and PBP4 showed significantly male bias expression. In male moth, the main assignment
is to trail the sex pheromones to find female moth for mating. We speculated the PBP2
may play a critical role in pheromone binding. For females, besides mating and oviposition,
are often selective when deciding to mate, and ensures that they find a high-quality
mate that satisfies their reproductive needs. GOBP1 and GOBP2 genes, as well as OBP6,
OBP7 and OBP9, were also highly expressed in female, this may play some important
roles and need for further study. GOBPs are proposed to detect host plants volatiles,
food and oviposition sites and PBPs play key role in detecting sex pheromones [
32-34]. However, some studies have demonstrated that GOBPs were also strongly bound with
sex pheromones and possibly responsible for conducting the function [
35]. Although the transcriptome of C. pinicolalis and C. punctiferlis possess higher similarity, the C. pinicolalis adult rely on fresh masson pine branches for laying eggs, which the case is very different
in C. punctiferlis adult, they have wide variety of host plants selection. Therefore, both GOBPs and
PBPs from C. pinicolalis and C. punctiferlis might have greater interest in future research.
CSPs were found in insect contact and olfactory sensilla, but members showed peculiar
functions. Binding activity of volatile compounds has been described in a similar
way as OBPs. In C. pinicolalis antennae transcriptome, we totally identified 19 putative CSPs, and found five CSPs
(CSP4, CSP5, CSP11, CSP14, and CSP17) were significantly expressed in female antennae
(Fig. 1B). MsepCSP8 of Mythimna separate was specially expressed in female antennae and showed less sensitive to plant volatiles
after RNAi [
36]. Also in Locusta migratoria, nearly 17 CSPs abundantly expressed in the female reproductive organs [37]. CSPs
may plays a particular role in female, especially the female bias gene expression.
OR or IR genes are responsible for receiving and detecting odor molecules sent by
OBP during the recognition process. In total of 55 OR genes, 22 OR genes from male
and female antennae showed significant difference in nucleotides. In this dataset,
4, 18 ORs were specially expressed in male and female antennae. In Lepidoptera, OR1
and OR3-8 were identified as pheromone receptors. Our result obviously showed OR1,
OR3 and OR6 were specially expressed in male antennae, this may suggest OR1, OR3 and
OR6 genes focus on sex pheromones recognition. PRs in other Lepidoptera were reported
to bind with sex pheromones [
38]. OR34 also performed biased expression in male antennae, but till now, the function
is unknown. More numbers of ORs were highly expressed in female antennae (Fig. 2),
this is also discovered in mosquitos [
39], female need more receptors for host seeking. In Bombyx mori, more female biased ORs suggested to have function of oviposition cues or male-produced
courtship pheromones [
40]. This indicated more ORs bias in female C. pinicolalis may provide more receptors to recognize hundreds of host plants, not as sex pheromones
which only have one or two more components.
We have identified 20 IRs in C. pinicolalis which are one-fold more than IRs reported in C. punctiferlis. In some studies reported that these IR genes were localized in different tissue
in some species [6]. Indeed, the expression of IR may have a certain specificity. For example, there
are some IRs were expressed exclusively in Spodoptera littoralis, Helicoverpa armigera [
41,
42]. Also, different IR genes were detected in gustatory organs in Drosophila melanogaster [
43]. However, in this study the IR gene family from transcriptome data analyzed only
from the C. pinicolalis antennae and compared with C. punctiferlis antennal data, this explains the tissue specific IR gene localization may not apply
for this result.
NormFinder and geNorm programs are commonly used to screen the internal reference
genes during qRT-PCR and optimized the number of reference genes [
44, 4
5]. At the same time, the difference between reference genes can be compared, but only
one optimal gene can be screened when using the NormFinder [
46]. In this research, we used both methods to screen the reference gene. The GeNorm
result showed Actin and GAPDH were more stable during different development stages
of the C. pinicolalis, and NormFinder showed the RP49 as stable reference gene. This variation may be due
to different algorithms coded in this software. Different software were used for calculating
the reference gene stability at different developmental stages in yellow peach moth,
RP49 and GAPDH was found to be more stable [
47]. Since the expression of reference gene differs for different developmental stage
and tissue, therefore the selection of two or more reference genes is useful to calibrate
the expression level of the gene of interest. Cardoso et al. [
48
</a>] reported three different reference genes (Actin, 60S ribosomal protein L3, RPL13;
and peptidylprolyl isomerase, PPI) for different developmental stages in <em>Aphidius gifuensis </em>[<a href="#_ENREF_58">
49</a>]. Also, Actin, GAPDH and RP49 reported to be the most stable reference gene in the
<em>Calliphoridae</em> family [<a href="#_ENREF_59">
50
</a>]. According to our results, it is recommended to use GAPDH or RP49 at different developmental
stages of the<em> C. pinicolalis</em>. On other hand, ribosomal proteins are involved in translation and protein synthesis,
this recommended us to use RP49 and RPL13 for different tissues in yellow peach moth
[<a href="#_ENREF_57">
47</a>]. Similarly, our findings indicate that both RP49 and RPL13 are the best reference
genes for the different body part of the adult. </p>
We mainly performed a comprehensive analysis of antennal transcriptome of C. pinicolalis and mined many sexual bias expression olfactory related genes. Meanwhile, genes sequences
in C. pinicolalis showed same with its sibling species C. punctiferalis. This study provides a starting point to understand the genetic difference at the
molecular level and further studies.