Knickkopf (LmKnk) is required for chitin organization in the foregut of Locusta migratoria.

The foregut, located at the front of the digestive tract, serves a vital role in insects by storing and grinding food into small particles. The innermost layer of the foregut known as the chitinous intima, comes into direct contact with the food and acts as a protective barrier against abrasive particles. Knickkopf (Knk) is required for chitin organization in the chitinous exoskeleton, tracheae and wings. Despite its significance, little is known about the biological function of Knk in the foregut. In this study, we found that LmKnk was stably expressed in the foregut, and highly expressed before molting in Locusta migratoria. To ascertain the biological function of LmKnk in the foregut, we synthesized specific double-stranded LmKnk (dsLmKnk) and injected it into locusts. Our findings showed a significant decrease in the foregut size, along with reduced food intake and accumulation of residues in the foregut after dsLmKnk injection. Morphological observations revealed that newly formed intima became thinner and lacked chitin lamella. Furthermore, fluorescence immunohistochemistry revealed that LmKnk was located in the apical region of new intima and epithelial cells. Taken together, this study provides insights into the biological function of LmKnk in the foregut, and identifies the potential target gene for exploring biological pest management strategies.

CYP6FD5, an antenna-specific P450 gene, is potentially involved in the host plant recognition in Locusta migratoria.

Cytochrome P450 monooxygenases (P450s) are a large superfamily of heme-thiolate proteins and play a vital role in the biosynthesis and inactivation of endogenous substances as well as the detoxification of exogenous substances. They also function as odor-degrading enzymes (ODEs) in insect olfactory sensory systems. In the present study, a P450 gene was obtained from the antennae of Locusta migratoria and named as CYP6FD5. Multiple alignment of P450 proteins revealed that LmCYP6FD5 contained five conserved motifs, including the helix C motif, an oxygen-binding site, helix K motif, a meander region, and the haem-binding motif. The expression of LmCYP6FD5 in various tissues and antennal development stages was determined by using RT-qPCR. Our results showed that LmCYP6FD5 was antenna-specific and highly expressed throughout the antennal developmental stages of female and male locusts. Furthermore, the role of LmCYP6FD5 in the perception of host plant volatiles was assessed using RNAi in combination with electroantennogram (EAG) and behavioral responses. Our findings showed that after silencing LmCYP6FD5, the EAG responses of female and male locusts to the main volatiles of gramineous plants, including trans-2-Hexen-1-al, cis-3-Hexenyl acetate, and decanal, were significantly diminished. Moreover, a significant decrease in EAG response of male antennae to benzaldehyde was also observed. In addition, behavioral assay showed that the locust response to single volatile from host plant or wheat remained unchanged after the silencing of LmCYP6FD5. Antenna-specific expression and EAG responses of locusts to host plant volatiles still suggested that LmCYP6FD5 was potentially involved in host plant recognition, although no behavioral changes were observed.

Both LmDicer-1 and two LmDicer-2s participate in siRNA-mediated RNAi pathway and contribute to high gene silencing efficiency in Locusta migratoria.

Dicers belong to a class of large RNase III multidomain ribonucleases and are central components of the RNA interference (RNAi) pathways. In insects, Dicer-2 has been known to cleave long double-stranded RNA (dsRNA) in small interfering RNA (siRNA)-mediated-RNAi pathway. However, Dicer-1 is responsible for cleaving precursor microRNAs (pre28 miRNAs) in miRNA-mediated RNAi pathway. In this study, we identified one LmDicer-1 and two LmDicer-2 (LmDicer-2a and LmDicer-2b) genes in Locusta migratoria. The RNAi of RNAi assay showed that knockdown of each of the Dicer genes reduced RNAi efficiency against a target gene (Lmß-Tubulin), suggesting that all these genes participated in the siRNA-mediated RNAi pathway. Sequence analyses of the siRNAs generated from dsLmß-Tubulin after silencing each LmDicer gene showed no significant difference in the pattern of siRNAs mapped to dsLmß-Tubulin. This result indicated that all the three LmDicers are capable of generating siRNAs from the dsRNA. We then generated recombinant proteins consisting of different domains using Escherichia coli expression system and incubated each recombinant protein with dsLmß-Tubulin. We found that the recombinant Dicer proteins successfully cleaved dsLmß-Tubulin. However, LmDicer-2a-R lacking dsRBD domain lost activity, suggesting that dsRBD domain is critical for Dicer function. Furthermore, overexpression of these proteins in Drosophila S2 cells improved RNAi efficiency. Our siRNA affinity chromatography and LC-MS/MS analysis identified LmDicer-2a, LmDicer-2b, LmR2D2, LmAgo2a, LmAgo1, LmStaufen and LmTARBP2 as constituents of RNA-induced silencing complex. Taken together, these data show that both LmDicer-1 and two LmDicer-2s all participate in siRNA-mediated RNAi pathway and likely contribute to high RNAi efficiency in L. migratoria.

Identification and RNAi-based functional analysis of chitinase family genes in Agrotis ipsilon.

BACKGROUND: Chitin is a major component in the extracellular matrix of insects, and its metabolism largely affects insect development and molting. As essential degradative enzymes, chitinases are encoded by multiple genes that differ in size, expression pattern and function in insects. However, our limited knowledge on the functions of different chitinases in Agrotis ipsilon has prevented our application of new technologies to target these genes as new pest management strategies. RESULTS: We revealed 11 full-length complementary DNA sequences of chitinase genes (AiChts) from A. ipsilon transcriptome. Although the domain architecture of these chitinases varied greatly, they all contained at least one chitinase catalytic domain. Developmental stage- and tissue-dependent expression profiles showed that most AiChts had the highest expression in the pupal stage. Furthermore, AiCht2, AiCht6, AiCht7 and AiCht10 were mainly expressed in the integument, whereas AiCht8 and AiCht-h had the highest expression in the midgut. The RNA interference (RNAi) experiment revealed that knockdown of AiCht10 or the imaginal disc growth factor gene (AiIDGF) induced high larval mortality. Larvae failed to shed the old cuticle during molting after the injection of double-stranded RNA targeting AiCht10 (dsAiCht10), whereas the larval bodies shrunk and blackened after the injection of dsRNA targeting AiIDGF (dsAiIDGF). CONCLUSION: Our results revealed for the first time the important functions of AiCht10 and AiIDGF in A. ipsilon. These genes are essential for larval development, and can potentially serve as new targets for RNAi-based pest management. © 2022 Society of Chemical Industry.

Increased RNAi Efficiency by dsEGFP-Induced Up-Regulation of Two Core RNAi Pathway Genes (OfDicer2 and OfAgo2) in the Asian Corn Borer (Ostrinia furnacalis).

RNA interference (RNAi) is a sequence-specific gene silencing mechanism that holds great promise for effective management of agricultural pests. Previous studies have shown that the efficacy of RNAi varies among different insect species, which limits its wide spread application in the field of crop protection. In this study, we identified and characterized six core RNAi pathway genes including OfDicer1, OfDicer2, OfR2D2, OfAgo1, OfAgo2, and OfAgo3 from the transcriptomic database of the Asian corn borer (Ostrinia furnacalis). Domain analysis showed that the six deduced proteins contained the necessary functional domains. Insect developmental stage- and tissue-specific expression analysis showed that five genes were expressed in all the stages and tissues examined except OfAgo3, which showed low expression in larvae, and high expression in pupae and adults and in the midgut. RT-qPCR was performed to examine the response of these six genes to exogenous double-stranded RNA (dsRNA). Interestingly, the transcript levels of OfDicer2 and OfAgo2 were significantly enhanced after the injection of dsEGFP at different time points and tissues investigated. Consequently, the RNAi efficiency in targeting the insect endogenous genes can be greatly enhanced in the hemolymph or midgut. Taken together, our investigations suggest that RNAi efficiency can be enhanced by pre-injection of dsRNA to induce the RNAi core machinery genes, which could be a useful strategy to improving RNAi efficiency for studying gene functions under laboratory conditions.

The stability and sequence cleavage preference of dsRNA are key factors differentiating RNAi efficiency between migratory locust and Asian corn borer.

We compared the stability of double-stranded RNA (dsRNA) in each of two body fluids (hemolymph, midgut fluid) and in each of two tissues (integument, midgut), and the uptake of dsRNA in each of two cultured tissues (integument, midgut) between the migratory locust (Locusta migratoria) and the Asian corn borer (Ostrinia furnacalis). We further compared the abundance of putative small interfering RNAs (siRNAs) generated from each of two dsRNAs (dsß-actin, dsEf1α) and the preference of dsRNA cleavages between the two insect species. Our studies showed a rapid degradation of dsRNA in the midgut fluids of both insect species and in O. furnacalis hemolymph. However, dsRNA remained reasonably stable in L. migratoria hemolymph. When nuclease degradation of dsRNA in cultured tissues was inhibited, dsRNA uptake was not significantly different between the two species. We further showed that the silencing efficiency against target genes was consistent with the abundance of putative siRNAs processed from the dsRNA. In addition, O. furnacalis showed a strong preference in cleaving dsRNA when the nucleotide G was in the position of "1" at 5'-end whereas L. migratoria showed broad spectrum in cleavage sites to generate siRNA. Taken together, our study revealed that silencing efficiency of a target gene by RNAi was directly related to the dsRNA degradation by nucleases and the abundance of siRNAs generated from the dsRNA.

Destruxin A inhibits scavenger receptor B mediated melanization in Aphis citricola.

BACKGROUND: Destruxin A (DA) is a mycotoxin secreted by entomogenous fungi, such as Metarhizium anisopliae, which has broad-spectrum insecticidal activity. Insect innate immunity provides resistance against the invasion of entomopathogenic fungi. Previous studies have shown that DA could inhibit the immune response, however, the suppressive mechanism of DA on prophenoloxidase system is still unknown. RESULTS: Based on the transcriptome of Aphis citricola, we screened the scavenger receptor class B(AcSR-B)and identified that it significantly responds to DA. Spatio-temporal expression analysis showed that AcSR-B is highly expressed in adult stage and is mainly distributed in the abdominal region. We further revealed that both M. anisopliae and Escherichia coli could suppress the expression of AcSR-B at 24 h, and that the expressed recombinant protein rAcSR-B possessed agglutination activity to M. anisopliae and E. coli. DA could suppress the protein expression of AcSR-B. In addition, RNA interference of AcSR-B caused death of A. citricola in a dose-dependent manner, and RNA interference of AcSR-B increased mortality in A. citricola under the same lethal concentration of DA. The inhibiting effect of AcSR-B silencing was similar with the DA treatment upon phenol oxidase (PO) activity of A. citricola hemolymph. DA could not decrease PO activity further after AcSR-B silencing. CONCLUSION: Destruxin A inhibits melanization by suppressing AcSR-B in A. citricola. Our findings are helpful in understanding the underlying molecular mechanism of the DA suppressing immune system, and uncover a potential molecular target for double-stranded RNA (dsRNA) insecticides.

Identification of Rab family genes and functional analyses of LmRab5 and LmRab11A in the development and RNA interference of Locusta migratoria.

Rab proteins constitute the largest family of small GTPases, which play pivotal roles in intracellular membrane trafficking in all eukaryotes. A number of Rab genes have been identified in eukaryotes; however, very little information about these genes has been reported in insects. In the current study, for the first time we identified and characterized 27 Rab family genes from Locusta migratoria. Phylogenetic analysis and comparison of domain architecture indicated that Rab family genes are highly conserved among insect species. Tissue-dependent expression profiles indicated that expression of Rab genes was highest in the ovary, except for LmRab3, which was most highly expressed in hemolymph. The biological function of each Rab gene was investigated using RNA interference (RNAi). Double-stranded RNA targeting each Rab gene was injected into the hemocoel of nymphs and revealed that suppression of two Rab genes (LmRab5 and LmRab11A) caused 100% mortality. In addition, nymphs injected with dsLmRab5 exhibited severe phenotypic defects in the gastric caeca and midgut, while dsLmRab11A arrested the molting process. We then applied the RNAi of RNAi technique to test if silencing either of these two genes would affect the suppression of the lethal giant larvae (LmLgl) reporter gene and found that suppression of LmRab5 diminished the RNAi efficiency of LmLgl, whereas suppression of LmRab11A enhanced RNAi efficiency of LmLgl. These results indicate that Rab genes contribute differently to RNAi efficiency in different tissues. Our study provides a foundation for further functional investigations of Rab genes and their contributions to RNAi efficiency in L. migratoria.

Molecular Characterizations and Functional Analyses of LmR2D2 in the Locusta migratoria siRNA Pathway.

Small interfering RNAs (siRNAs) are non-coding RNAs with a length of 21~23 nucleotides (nt) and present in almost all eukaryotes. The formation of siRNA is a highly conserved post-transcriptional gene-silencing mechanism mediated by key proteins, including Dicer2, Argonaute2 (Ago2) and R2D2. R2D2 has been identified as a double-stranded RNA (dsRNA)-binding protein and reported as an integral component of the siRNA pathway in Drosophila. However, the involvement of R2D2 in the siRNA pathway of Locusta migratoria is still unknown. In the present study, we identified an LmR2D2 gene from the transcriptome of L. migratoria. It consists of a 954-bp open reading frame that encodes a protein of 318 amino acid residues. Further sequence analysis revealed that LmR2D2 possesses two tandem dsRNA-binding domains (dsRBD) at the N-terminus. Analysis of the developmental expression profile of LmR2D2 indicated that its transcript level was stable in third-instar nymphs of L. migratoria, whereas the tissue-dependent expression profile exhibited high levels of expression of LmR2D2 in the testis and ovary. When LmR2D2 was silenced by RNAi, the RNAi efficiency against Lmß-tubulin as a marker gene was significantly diminished, as indicated by the 37.7% increased Lmß-tubulin transcript level. Additionally, the prokaryotic expression system was used to obtain the LmR2D2 supernatant protein. By incubating the LmR2D2 protein with biotin-dsRNA, we found that LmR2D2 can bind to dsRNA in vitro, which supports our conclusion that LmR2D2 plays an essential role in the siRNA pathway of L. migratoria.

Do microbial protein elicitors PeaT1 obtained from Alternaria tenuissima and PeBL1 from Brevibacillus laterosporus enhance defense response against tomato aphid (Myzus persicae)?

Tomato aphid (Myzus persicae) is a destructive insect pest of tomato responsible for huge losses in the production as well in the vegetable industry. In the present in vitro study two protein elicitors, PeaT1 and PeBL1 were considered to study their efficacies to exhibit defense response against tomato aphid. Three different concentrations of both protein elicitors were applied on the tomato seedlings. After the application of PeaT1 and PeBL1, population growth rates of tomato aphid were decreased as compared to the control treatment. In host preference assay, the tomato aphid showed a preference to build a colony on the control as compared to the treated tomato plant, because tomato leaves provided hazardous surface for aphid after the formation of wax and trichome. The concentrations of protein showed significant (p < 0.05) results in life-history traits of the aphid. Jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) showed significant accumulation in tomato seedlings treated with PeaT1 and PeBL1. Elicitors treated plants produced resistance against M. persicae. Our finding suggests that PeaT1 and PeBL1 have shown high potentials against the damage of M. persicae, and both elicitors could be used as novel biological tools against tomato aphid.

A dsRNA-degrading nuclease (dsRNase2) limits RNAi efficiency in the Asian corn borer (Ostrinia furnacalis).

The efficiency of RNA interference (RNAi) varies substantially among different insect species. Rapid degradation of double-stranded RNA (dsRNA) by dsRNA-degrading nucleases (dsRNases) has been implicated to cause low RNAi efficiency in several insect species. In this study, we identified four dsRNase genes (OfdsRNase1, OfdsRNase2, OfdsRNase3 and OfdsRNase4) from the Asian corn borer (Ostrinia furnacalis) transcriptome database. Bioinformatic analyses showed that each deduced protein sequence contained endonuclease NS domains and signal peptides. Gene expression analysis revealed that OfdsRNase2 was exclusively expressed in the midgut of larvae. RNAi efficiency was investigated in 2-d-old fifth-instar larvae (high expression of dsRNase2) and 2-d-old pupae (low expression of dsRNase2) by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein (OfLgl). Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae, but not in larvae, suggesting that OfdsRNase2 could contribute to lower RNAi efficiency in larval stages. This hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene, OfHex1, was significantly improved after knockdown of OfdsRNase2. When double luciferase assays were performed to evaluate the role of the four dsRNases in vitro, only OfdsRNase2 expressed in S2 cells significantly affected RNAi efficiency by degrading dsRNA. Taken together, our results suggested that the degradation of dsRNA by OfdsRNase2 in the midgut contributed to low RNAi efficiency in O. furnacalis larvae.

Multiple Argonaute family genes contribute to the siRNA-mediated RNAi pathway in Locusta migratoria.

Argonautes (Ago) are important core proteins in RNA interference (RNAi) pathways of eukaryotic cells. Generally, it is thought that Ago1, Ago2 and Ago3 are involved in the miRNA (microRNA), siRNA (small interfering RNA) and piRNA (Piwi-interacting RNA)-mediated RNAi pathways, respectively. As a main component of the RNA-induced silencing complex (RISC), Ago2 plays an indispensable role in using siRNA to recognize and cut target messenger RNAs resulting in suppression of transcript levels, but the contributions of Ago1 and Ago3 to the siRNA-mediated RNAi pathway remain to be explored in many insect species. In this study, we investigated the contributions of four Ago genes (named LmAgo1, LmAgo2a and LmAgo2b and LmAgo3) to RNAi efficiency in Locusta migratoria by using both in vivo and in vitro experiments. Our results showed that suppression of each of the Ago genes significantly impaired RNAi efficiency when targeting Lmß-tubulin transcripts, resulting in recovery of 48, 43.3, 61.4 or 26% of Lmß-tubulin transcripts following RNAi-mediated suppression of LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3, respectively. Furthermore, overexpression of LmAgo1, LmAgo2a, LmAgo2b, or LmAgo3 in a PAc5.1-V5/HisB vector and co-transfection with psicheck2 fluorescence vector in S2 cells reduced luciferase fluorescence by 38.3, 58.9, 53.3 or 55.6%, respectively. Taken together, our results showed that LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3 each make significant contributions to RNAi efficiency in L. migratoria and suggest that the involvement of all four enzymes could be one of the major factors supporting robust RNAi responses observed in this species.

20-Hydroxyecdysone regulates the prophenoloxidase cascade to immunize Metarhizium anisopliae in Locusta migratoria.

BACKGROUND: PPO (prophenoloxidase) cascade plays an important role in resisting invasion of entomogenous fungus. The 20-hydroxyecdysone (20E) exerts potent effect on the innate immunity in many insects. However, whether 20E controls the PPO cascade system against fungi and the regulatory mechanism in insects remains unclear. RESULTS: In this study, both the proteome and transcriptome of Locusta migratoria were determined followed by the induction of 20E. Pattern recognition receptor GNBP-2 (Gram-negative binding proteins) has been identified that responded to 20E at both messenger RNA (mRNA) and protein levels. The PPO gene expression in fat body and PO (phenoloxidase) activity in plasma was found significantly induced after 20E injection and during the high-20E developmental stage. However, when 20E signal was blocked by RNA interference (RNAi) of ecdysone receptor, the expression level of PPO and PO activity failed to be increased by 20E. Thus, 20E could not significantly induce the expression of PPO gene and PO activity after RNAi of GNBP-2. Furthermore, 20E treatment notably enhanced the resistance of L. migratoria against Metarhizium anisopliae. Followed by of GNBP-2 silencing, the mortality of nymphs was significantly increased under the stress of Metarhizium anisopliae, and 20E injection could not increase the resistance. CONCLUSION: The 20E regulates the PPO system to resist fungal invasion via regulating GNBP-2 in worldwide pest L. migratoria. Our results provide insight into the mechanism of how 20E enhances the antimicrobial immunity, and will be beneficial for modification of entomogenous fungi targeting on hormones and the immune system. © 2020 Society of Chemical Industry.