Neutral Ceramidase Is Required for the Reproduction of Brown Planthopper, Nilaparvata lugens (Stål).

Ceramides are bioactive sphingolipids that have been implicated in insect development; however, their role in insect reproduction remains poorly understood. Here, we report the pivotal role of neutral ceramidase (NCER) in the female reproduction of the brown planthopper (BPH), Nilaparvata lugens (Stål), a significant pest in rice cultivation in Asia. LC-MS/MS demonstrated that, among different developmental stages of BPH, the levels of ceramides were highest in 1st instar nymphs and lowest in adults. The transcription of NCER was negatively correlated with the levels of ceramides at different developmental stages of BPH, in that the transcript levels of NCER were the highest, whereas ceramides levels were the lowest in BPH adults. Knocking down NCER through RNA interference (RNAi) increased the levels of ceramides in BPH females and ovaries, which resulted in a delay in oocyte maturation, a reduction in oviposition and egg hatching rate, as well as the production of vulnerable offspring. Transmission electron microscopy (TEM) analysis and TdT-mediated dUTP Nick-End Labeling (TUNEL) assays showed mitochondrial deficiency and apoptosis in NCER-deficient oocytes. Taken together, these results suggest that NCER plays a crucial role in female reproduction in BPH, likely by regulating the levels of ceramides.

Identification and Expression Profiles of 14 Odorant-Binding Protein Genes From Pieris rapae (Lepidoptera: Pieridae).

The small white butterfly, Pieris rapae (L.), is an important insect pest of Brassica crops. This species utilize olfactory cues to find their hosts and mates. However, the molecular mechanism underlying the olfactory perception in this species remains unclear. Here, we identified 14 odorant-binding proteins (OBP) genes-essential for insect olfaction-in P. rapae by exploring a previously published transcriptome dataset. Proteins encoded by all of these genes contain N-terminal signal peptides and six positionally conserved cysteine residues, which are characteristic of insect OBPs. These OBPs displayed high amino acid identity with their respective orthologs in other lepidopterans, and several conserved motifs were identified within these OBPs. Phylogenetic analysis showed that these OBPs were well segregated from each other and clustered into different branches. PrapOBP1 and PrapOBP2 were clustered into the 'general odorant-binding protein' clade, and PrapOBP3 and PrapOBP4 fall into the 'pheromone-binding protein' clade. The 14 OBP genes were located on seven genomic scaffolds. Of these, PrapOBP1, 2, 3, and 4 were located on scaffold332, whereas PrapOBP5, 6, 7, 8, and 9 were located on scaffold116. Ten of the 14 genes had antenna-biased expression. Of these, PrapOBP1, 2, 4, and 13 were enriched in male antennae, whereas PrapOBP7 and PrapOBP10 were female-biased. Our findings suggest that these OBPs may be involved in olfactory communication. To the best of our knowledge, this is the first report on the identification and characterization of OBPs in P. rapae, and our findings provide a solid foundation for studying the functions of these genes.

A neutral ceramidase, NlnCDase, is involved in the stress responses of brown planthopper, Nilaparvata lugens (Stål).

Ceramidases (CDases) are vital enzymes involved in the biosynthesis of sphingolipids, which are essential components of eukaryotic membranes. The function of these enzymes in insects, however, is poorly understood. We identified a neutral ceramidase (NlnCDase) from the brown planthopper, Nilaparvata lugens, one of the most destructive hemipteran pests of rice. The C12-ceramide was the most preferred substrate for the NlnCDase enzyme. The activity of the NlnCDase enzyme was highest in the neutral-pH range (pH 6.0). It was inhibited by EGTA, Cs+ and Fe2+, while stimulated by EDTA and Ca2+. Moreover, the NlnCDase has higher transcript level and activity in adults than in eggs and nymphs, and in the reproductive organs (ovaries and spermaries) than in other tissues (i.e. heads, thorax, legs, midguts), which suggested that the NlnCDase might be elevated to mediate developmental process. In addition, transcripts and activity of the NlnCDase were up-regulated under abiotic stresses including starvation, abnormal temperature, and insecticides, and biotic stress of resistant rice varieties. Knocking down NlnCDase by RNA interference increased female survival under starvation and temperature stresses, suggesting that NlnCDase might be involved in the stress response in N. lugens.

Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae).

NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems.

[Pathological characteristics mixed epithelial and stromal tumor of kidney].

OBJECTIVE: To study the clinicopathological features of mixed epithelial and stromal tumor of the kidney(MESTK). METHODS: Clinical and pathological characteristics of one case of MESTK was studied. RESULTS: A case of MESTK which uncommonly occurred in a 16-year old adolescent male presented with dysuria and a large mass in the right renal region without a history of estrogen/progestogen treatment and/or obesity or urogenital surgery. Radiology revealed a large cystic/solid mass within the right kidney. Grossly, it was well demarcated and had a solid and cystic appearance on sectioning. Microscopically, the tumor was composed of a mixture of stromal and epithelial components. The epithelial component was composed of flat to columnar cells forming glands or tubules. The stromal components essentially consisted of bland, loosely packed spindle cells in an edematous and myxoid background. In some areas, there were smooth muscle cells forming bands and fascicles but no ovarian-type stroma was present. Immunohistochemical staining revealed that the epithelial components were positive for AE1/AE3 and focally positive for estrogen receptor (ER),progesterone receptor(PR), CD10 and Vimentin, whereas the stromal components were positive for ER, PR, Desmin and smooth muscle actin(SMA). Both epithelial and stromal components were negative for HMB-45, S-100, alpha-inhibin and WT-T. Five months after resection, the patient was well without evidence of recurrence. CONCLUSION; MESTK occurred in a pubertal male, as in the current case, supports the hypothesis that proliferation of remnants of the primitive mesenchyme in the kidney in situation of sex-steroid abnormity may play an important role in the pathogenesis of male MESTK.