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. 2015 Mar 18:5:9245.
doi: 10.1038/srep09245.

Si-CSP9 regulates the integument and moulting process of larvae in the red imported fire ant, Solenopsis invicta

Daifeng Cheng  1 , Yongyue Lu  1 , Ling Zeng  1 , Guangwen Liang  1 , Xiaofang He  1
Affiliations

Si-CSP9 regulates the integument and moulting process of larvae in the red imported fire ant, Solenopsis invicta

Daifeng Cheng et al. Sci Rep. .

Abstract

Chemosensory proteins (CSPs) have been predicted to be involved in development; however, direct evidence for their involvement is lacking, and genetic basis is largely unknown. To determine the function of the chemosensory protein 9 (Si-CSP9) gene in Solenopsis invicta, we used RNA interference to silence Si-CSP9 in 3rd-instar larvae. The 3rd-instar larvae failed to shed their cuticle after being fed Si-CSP9-directed siRNA, and expression profiling of RNAi-treated and untreated control larvae showed that 375 genes were differentially expressed. Pathway enrichment analysis revealed that 4 pathways associated with larval development were significantly enriched. Blast analysis revealed that one fatty acid amide hydrolase (FAAH) gene was up-regulated and 4 fatty acid synthase (FAT) genes and one protein kinase DC2 gene (PKA) were down-regulated in the enriched pathways. Significantly higher expression of these genes was found in 4th-instar larvae, and Pearson correlation analysis of the expression patterns revealed significant relationships among Si-CSP9, PKA, FAAH, and FAT1-4. Moreover, we confirmed that expression levels of Si-CSP9, FAAH, and FAT1-4 were significantly reduced and that the development of 3rd-instar larvae was halted with PKA silencing. These results suggest that Si-CSP9 and PKA may be involved in the network that contributes to development of 3rd-instar larvae.

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Figures

Figure 1
Figure 1. Expression of Si-CSP9 in developmental stages, castes and tissues.
(A): expression of Si-CSP9 in eggs, 1st-instar larvae, 2nd-instar larvae, 3rd-instar larvae, 4th-instar larvae, pupae and adults; (B): expression of Si-CSP9 in worker, alate females and alate males; (C): expression of Si-CSP9 in the antenna, head, thorax, leg and abdomen of workers. Means ± SE that are labelled with the same letter within each treatment are not significantly different.
Figure 2
Figure 2. Expression of Si-CSP9 during developmental process between L3 and L4.
0-L3: newly emerged 3rd-instar larvae; 24-L3: 24-h-old 3rd-instar larvae; 48-L3: 48-h-old 3rd-instar larvae; 72-L3: 72-h-old 3rd-instar larvae; 96-L3: 96-h-old 3rd-instar larvae; 0-L4: 4th-instar larvae. Means ± SE that are labelled with the same letter within each treatment are not significantly different.
Figure 3
Figure 3. Expression of Si-CSP9 after silencing.
(A): Expression of Si-CSP9 after being silenced for 24 h. (B): Expression of Si-CSP9 after being silenced for 48 h. (C): Expression of Si-CSP9 after being silenced for 72 h. SiRNA: small interference RNA; disSiRNA: small interference RNA with a disordered sequence; CK: sugar-water. In all groups, for treatment compared with CK, “**” denotes P < 0.01; “ns” denotes not significant.
Figure 4
Figure 4. Mortality and phenotype of larvae after silencing of Si-CSP9.
(A): Number of dead larvae after silencing of Si-CSP9. (B): Phenotype of larvae after silencing of Si-CSP9. In all groups, for treatment compared with CK, “**”denotes P < 0.01; “ns” denotes not significant.
Figure 5
Figure 5. Diversity of ontological categories of differentially expressed genes.
(A): Ontological categories in biological process. (B): Ontological categories in cell component. (C): Ontological categories in molecular function.
Figure 6
Figure 6. Neighbour-joining trees of PKA, FAAH, and FAT1-4 created using DNAMAN software.
The tree is collapsed to nodes with 50% or greater bootstrap support (n = 1000 replicates), and the bootstrap values are listed at each node. (A): Neighbour-joining tree of PKA. (B): Neighbour-joining tree of FAAH. C: Neighbour-joining tree of FAT1-4. Information on the genes is given in Supplementary Table S1.
Figure 7
Figure 7. Development-specific expression of PKA.
Means ± SE that are labelled with the same letter within each treatment are not significantly different.
Figure 8
Figure 8. Development-specific expression of FAAH.
Means ± SE that are labelled with the same letter within each treatment are not significantly different.
Figure 9
Figure 9. Development-specific expressions of FAT1-4.
Means ± SE that are labelled with the same letter within each treatment are not significantly different.
Figure 10
Figure 10. Phenotype and mortality of larvae after PKA silencing.
(A): Phenotype of larvae with PKA silencing. (B): Phenotype of larvae in CK. (C): Number of dead compared within the silenced PKA samples and normally expressed PKA samples. “**” denotes P < 0.01.
Figure 11
Figure 11. Effects on expression of Si-CSP9 after silencing of PKA.
Tr: samples with PKA silenced; CK: samples with PKA normally expressed.
Figure 12
Figure 12. Effects on expression of FAAH after silencing of PKA.
Tr: samples with PKA silenced; CK: samples with PKA normally expressed.
Figure 13
Figure 13. Effects on expression of FAT1-4 after silencing of PKA.
(A): expression of FAT1; (B): expression of FAT2; (C): expression of FAT3; (D): expression of FAT4. Tr: samples with PKA silenced; CK: samples with PKA normally expressed.
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