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

Abstract

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.

Keywords: apoptosis; brown planthopper; ceramide; neutral ceramidase; reproduction.

FIGURE 1

Sphingolipid pathway in insects. SPT, serine palmitoyl transferase; KDS, 3-dehydrosphinganine reductase; CerS, ceramide synthases; CER, ceramidase; SPHK, sphingosine kinase; CerK, ceramide kinase; SPL, sphingosine-1-phosphate lyase; SPPase, sphingosine-1-phosphate phosphatase; SMS, sphingomyelin synthase and/or ceramide choline phosphotransferases; SMase, sphingomylinases; CGT, ceramide glucotransference; and GBA, glucosyl ceramidase.

FIGURE 2

Levels of ceramides and dihydroceramides vary in BPH at different developmental stages. BPH at indicated developmental stages were subjected to measurements of the concentrations of ceramides (A) and dihydroceramides (B). The data were expressed as mean of two replications. (C) The total ceramide concentrations (Cer Total) and total dihydroceramide concentrations (dCer Total) were caculated. (D) The correlation analysis between NCER transcript levels (blue line) and total ceramide concentrations (orange line) in BPH at different developmental stages. The data of NCER transcription were expressed as mean ± SE (n = 3). 1st, the first instar nymph; 2nd, the second instar nymph; 3rd, the third instar nymph; 4th, the fourth instar nymph; 5th, the fifth instar nymph; F, Female; M, Male.

FIGURE 3

NCER knockdown reduces female BPH fecundity. The 5th instar nymphs were injected with dsRNA of NCER or GFP. CERi, dsRNA-NCER injected; GFPi, dsRNA-GFP injected. The BPH females that emerged in 24 h (A,C,E,G) or 48 h (B,D,F,H) after dsRNA injection were utilized for the bioassay experiments. (A,B) The RNAi effenciency after dsRNA injection in BPH females. (C,D) The number of eggs laid by each female in seven days. (E,F) The hatching rate of eggs laid by BPH female. (G,H) The survival rates of offspring produced by BPH female. (I) The developmental status of BPH ovaries. BPH ovaries were dissected from 1, 3, 5 days-old females for the photography. The data was expressed as mean ± SE (N = 3). Significance is shown by “*” at the P < 0.05 and “**” at the P < 0.01 level.

FIGURE 4

NCER knockdown induces apoptosis in BPH oocytes. Ovaries were collected from GFPi or CERi females at 1, 3, or 5 days post injection with dsRNA and subjected to transmission electron microscopic (TEM) analysis. The difference of cellular structure was pointed by black arrow.

FIGURE 5

NCER knockdown increases the levels of (dihydro)ceramides without affecting the levels of sphingoid bases. Ovaries were collected from GFPi or CERi females at 3rd day post injection with dsRNA injection and subjected to LC-MS/MS for the levels of ceramides in BPH ovaries (A) or BPH bodies (B); the levels of dihydroceramides in BPH ovaries (C) or in BPH bodies (D); the levels of sphingosine and dihydrosphingosine in BPH ovaries (E) or in BPH bodies (F). CERi, BPH bodies with dsRNA-NCER injected. GFPi, BPH bodies with dsRNA-GFP injected. CEROi, BPH ovaries with dsRNA-NCER injected. GFPOi, BPH ovaries dsRNA-GFP injected. The data was expressed as mean ± SE (N = 3). Significance is shown by “*” at the P < 0.05 and “**” at the P < 0.01 level.