RNAi-mediated silencing of the neverland gene inhibits molting in the migratory locust, Locusta migratoria.

7-dehydrocholesterol (7-DHC) is a key intermediate product used for biosynthesis of molting hormone. This is achieved through a series of hydroxylation reactions catalyzed by the Halloween family of cytochrome P450s. Neverland is an enzyme catalyzes the first reaction of the ecdysteroidogenic pathway, which converts dietary cholesterol into 7-DHC. However, research on the physiological function of neverland in orthopteran insects is lacking. In this study, neverland from Locusta migratoria (LmNvd) was cloned and analyzed. LmNvd was mainly expressed in the prothoracic gland and highly expressed on days 6 and 7 of fifth instar nymphs. RNAi-mediated silencing of LmNvd resulted in serious molting delays and abnormal phenotypes, which could be rescued by 7-DHC and 20-hydroxyecdysone supplementation. Hematoxylin and eosin staining results showed that RNAi-mediated silencing of LmNvd disturbed the molting process by both promoting the synthesis of new cuticle and suppressing the degradation of the old cuticle. Quantitative real-time PCR results suggested that the mRNA expression of E75 early gene and chitinase 5 gene decreased and that of chitin synthase 1 gene was markedly upregulated after knockdown of LmNvd. Our results suggest that LmNvd participates in the biosynthesis process of molting hormone, which is involved in regulating chitin synthesis and degradation in molting cycles.

Transcription factors, cap 'n' collar isoform C regulates the expression of CYP450 genes involving in insecticides susceptibility in Locusta migratoria.

BACKGROUND: The cap 'n' collar (Cnc) belongs to the Basic Leucine Zipper (bZIP) transcription factor super family. Cap 'n' collar isoform C (CncC) is highly conserved in the animal kingdom. CncC contributes to the regulation of growth, development, and aging and takes part in the maintenance of homeostasis and the defense against endogenous and environmental stress. Insect CncC participates in the regulation of various kinds of stress-responsive genes and is involved in the development of insecticide resistance. RESULTS: In this study, one full-length CncC sequence of Locusta migratoria was identified and characterized. Upon RNAi silencing of LmCncC, insecticide bioassays showed that LmCncC played an essential role in deltamethrin and imidacloprid susceptibility. To fully investigate the downstream genes regulated by LmCncC and further identify the LmCncC-regulated genes involved in deltamethrin and imidacloprid susceptibility, a comparative transcriptome was constructed. Thirty-five up-regulated genes and 73 down-regulated genes were screened from dsLmCncC-knockdown individuals. We selected 22 LmCncC-regulated genes and verified their gene expression levels using RT-qPCR. Finally, six LmCYP450 genes belonging to the CYP6 family were selected as candidate detoxification genes, and LmCYP6FD1 and LmCYP6FE1 were further validated as detoxification genes of insecticides via RNAi, insecticide bioassays, and metabolite identification. CONCLUSIONS: Our data suggest that the locust CncC gene is associated with deltamethrin and imidacloprid susceptibility via the regulation of LmCYP6FD1 and LmCYP6FE1, respectively.

Silencing of transcription factor E93 inhibits adult morphogenesis and disrupts cuticle, wing and ovary development in Locusta migratoria.

Ecdysone-induced protein 93F (E93) plays important roles during the metamorphosis process in insects. In this study, a cDNA of the LmE93 gene was identified from the transcriptome of Locusta migratoria, which consists of the 3378-nucleotide open-reading frame (ORF) and encodes 1125 amino acids with helix-turn-helix (HTH) motifs. Reverse transcription quantitative polymerase chain reaction analysis revealed that LmE93 was highest expressed in ovary. The LmE93 expression level was markedly low from the 3rd to 4th instar nymphs, and greatly increased in 1-day-old 5th instar nymphs with a peak on middle nymphal days, then declined in the late nymphal days. Moreover, injected dsLmE93 into 4th and 5th instar nymphs greatly reduced LmE93 transcripts, respectively, and prevented the process of metamorphosis, causing supernumerary nymphal stages. Hematoxylin-eosin staining of the integument showed that the apolysis occurred in advance in 4th instar nymphs, and old cuticle degradation was decreased in dsLmE93-injected locusts of 5th instar nymphs. Smaller and no fully developed wings with reduced columns between the anterior and posterior regions were found in N6 and N7 supernumerary nymphs. In addition, the development of the ovary in dsLmE93-injected locusts was severely blocked, the yolk was almost not formed and there was no development of ovarioles. The results indicated that LmE93 play key roles in the metamorphosis, cuticle, wing and ovarian development of locusts.

Characteristics of Halloween genes and RNA interference-mediated functional analysis of LmCYP307a2 in Locusta migratoria.

Halloween genes are involved in the biosynthesis of the molting hormone, which plays a key role in insect ecdysis, development, metamorphosis, and reproduction. Our previous work identified five Halloween genes from Locusta migratoria, but their functions are currently unknown. In this study, the sequences of these five Halloween genes were analyzed and characterized. LmCYP307a2, LmCYP306a1, LmCYP302a1, and LmCYP315a1 were primarily expressed in the prothoracic glands, while LmCYP314a1 was universally expressed in peripheral tissues, especially in the ovaries and Malpighian tubules. All five Halloween genes were mainly expressed from the 5th to the 7th d in 5th-instar nymphs. RNA interference (RNAi) silencing of LmCYP307a2 resulted in severe molting delays and molting failure, which could be rescued by supplementary 20-hydroxyecdysone. A hematoxylin and eosin staining analysis suggested that the RNAi of LmCYP307a2 inhibited the ecdysis process by inhibiting the apolysis and degradation of the old cuticle, and by promoting the synthesis of a new cuticle. Quantitative reverse transcription polymerase chain reaction results showed that the expressions of LmE74, LmCht5, and LmCht10 were dramatically down-regulated, while that of LmChsI was substantially up-regulated, after knockdown of LmCYP307a2. The results suggest that LmCYP307a2 is related to the molt process via regulation of chitin synthesis and degradation.

Roles of LmCDA1 and LmCDA2 in cuticle formation in the foregut and hindgut of Locusta migratoria.

Chitin deacetylases (CDAs, including CDA1 and CDA2) are considered key enzymes for body cuticle formation and tracheal morphogenesis in various insect species. However, their functions in the formation of the cuticular intima of the foregut and hindgut are unclear. Here, we investigated the roles of their respective genes LmCDA1 and LmCDA2 in this process, in the hemimetabolous insect Locusta migratoria. Transcripts of LmCDA1 and LmCDA2 were highly expressed both before and after molting in the foregut. In the hindgut, their expression was high only before molting. In both the foregut and hindgut, LmCDA1 protein was localized in the basal half of the chitin matrix (procuticle), whereas LmCDA2 was detected in the upper half of the procuticle. Knockdown of LmCDA1 by RNA interference (RNAi) in 5th-instar nymphs caused no visible defects of the hindgut cuticle. By contrast, the chitinous lamellae of the cuticular intima in the foregut of knockdown animals were less compact than in control animals. RNAi against LmCDA2 led to thickening of both the foregut and hindgut cuticles, with a greater number of thinner laminae than in the respective control cuticles. Taken together, our results show that LmCDA1 and LmCDA2 have distinct, but overlapping, functions in chitin organization in the foregut cuticle. However, in the hindgut, this process seems independent of LmCDA1 activity but requires LmCDA2 function. Thus, the CDAs reflect tissue-specific differences in cuticular organization and function, which need further detailed molecular and histological analyses for full comprehension.

Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria.

Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix of midgut. In Locusta migratoria, two duplicated Cht5s (LmCht5-1 and LmCht5-2) have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate (4MU-(GlcNAc)3 ), and higher Km value for the longer substrate (CM-Chitin-RBV) compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain (CBD) tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5-1 and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-(GlcNAc)3 , whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBV. These findings are helpful for further research to clarify their different roles in insect growth and development.

RNA interference to reveal roles of ß-N-acetylglucosaminidase gene during molting process in Locusta migratoria.

ß-N-acetylglucosaminidases are crucial enzymes involved in chitin degradation in insects. We identified a ß-N-acetylglucosaminidase gene (LmNAG1) from Locusta migratoria. The full-length complementary DNA (cDNA) of LmNAG1 consists of 2 667 nucleotides, including an open reading frame (ORF) of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5'- and 3'-ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized ß-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real-time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA (dsRNA). As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAG1-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process of L. migratoria.

Characterization of zinc-binding properties of a novel imidase from Pseudomonas putida YZ-26.

The imidase from Pseudomonas putida YZ-26 consisting of 293-amino acid residues is a novel imidase with four subunits as the holo-enzyme and low molecular weight which is significantly different from known mammalian imidase. This study measured the zinc-binding properties of the imidase using inductively coupled plasma-atomic emission spectrometry and competition assay combined with activity determinations. Results show that each subunit of the imidase binds the zinc ion by 1:1 stoichiometry with apparent binding constant of 9.5 x 10(8)M(-1). The activity of the apo-imidase (20 microM) was recovered with the addition of zinc in the lower concentration (0-20 microM), whereas the enzymatic activity is decreased in the presence of high concentration of zinc (above 100 microM). The site-directed mutagenesis of His(247), His(86) or Cys(7), Cys(108) in imidase resulted in loss of activity and zinc-binding abilities at different degrees, showing that these residues may critically affect both enzymatic activity and conformation.

The flexibility of the non-conservative region at the C terminus of D-hydantoinase from Pseudomonas putida YZ-26 is extremely limited.

We previously reported that a deletion mutant (P478) with a residue Arg deleted at the C terminus of D-hydantoinase (P479) from Pseudomonas putida YZ-26 was dissociated into the monomer from its dimeric state. Based on the above result, a series of mutants of the enzyme with the C-terminal residues either deleted or substituted were prepared. The size-exclusion chromatography and bioactivity assay show that a C-terminal-substituted enzyme (R479D) and several truncated mutants (P478, P477, P476, and P475) are dissociated into the monomeric state as well, but their activities are largely retained. In contrast, two other mutants (R474 and R479A) are expressed in the form of random aggregates without any activity. Our experiments demonstrate that only the last four amino acids (-PVQR) at the C terminus of the enzyme can be deleted without seriously affecting its activity, although the enzyme is dissociated from a dimer into a monomer. These mutants also reveal some unique properties such as the enzymatic activity in vivo or in vitro, the effect of divalent metal ions, and the thermostability etc. in comparison to wild-type enzyme (P479). In addition, the three-dimensional structural modeling shows that the intact structure of the enzyme is essential, and the flexibility of the non-conservative region at the C terminus of the enzyme is quite limited.

[Molecular form and stability of D-hydantoinase deleted at C-terminal residue Arg].

This report is about only deleting one C-terminal residue of D-hydantoinase to result in obvious changes on its molecular form and stability. A recombinant D-hydantoinase (P479) and its mutant enzyme deleted at C-terminal residue Arg (P478) were prepared by methods of gene cloning, expression and purification. Results show that the subunit molecular weight of P479 and P478 is the same (54kDa) as determined by SDS-PAGE, whilst the molecular form of native P479 and P478 is a dimer and a monomer respectively in the completely operative conditions. Compared with P479, the enzymatic activity of P478 for substrate hydantoin maintained about 40% and pH stability was obviously increased, at the alkaline side in particular, as well as the anti-SDS ability was also raised. However, the thermal stability for P478 was clearly lowed as compared to P479. It implies from above data that the C-terminal residue Arg of the D-hydantoinase is a crucial one for subunit dissociation, but non-essential for catalysis.