A Mucin2-Like Gene, NlMuc2, is Required for Early Embryonic Development in Nilaparvata lugens

doi:10.1016/j.rsci.2025.04.004

Abstract

Abstract:

In Nilaparvata lugens, a serious and widespread rice pest, high fecundity is one of the important reasons for large populations and outbreaks. As embryogenesis is a critical process associated with insect fecundity, this study investigated the function of the Mucin2-like gene (NlMuc2) in the embryogenesis of N. lugens. The results showed that NlMuc2 was highly expressed in female reproductive organs and in 1-day-old eggs. Knockdown of NlMuc2 via RNA interference (RNAi) in 3rd instar nymphs resulted in increased mortality. In 5th instar nymphs, NlMuc2 knockdown led to a dramatic reduction in egg hatchability from 86.0% to 24.0%, offspring numbers from 330.4 to 81.5, egg count from 382.4 to 217.0, and resulted in 86.0% of eggs exhibiting inverted embryos. In newly emerged females, NlMuc2 knockdown retarded ovarian development and decreased the number of mature eggs, with 51.0% of eggs containing inverted embryos. Transcriptome sequencing analysis of eggs revealed that numerous genes were downregulated after NlMuc2 knockdown, with 16 and 15 downregulated genes enriched in the Wnt and MAPK pathways, respectively. Temporal and spatial expression profiling of selected differentially expressed genes, including Axin, δ-Catenin, Glypican-4-like, and Ror-like in the Wnt pathway, and MKK4 in the MAPK pathway, showed expression patterns similar to NlMuc2. Knockdown of Ror-like, Glypican-4-like, or MKK4 reduced the total number of eggs. Knockdown of Axin or MKK4 reduced the egg hatchability. A similar phenotype of eggs with inverted embryos was also observed in eggs laid by dsMKK4-and dsAxin-treated females. Thus, NlMuc2 is involved in embryonic development mainly by regulating the Wnt and MAPK signaling pathways. These findings may provide new targets for pesticide design and RNAi-based control of N. lugens, and will also provide new insights into insect embryonic development and the function of insect mucins.

Key words: mucins, Nilaparvata lugens, embryogenesis, katatrepsis

Wang Weixia, Zhu Tingheng, Wei Qi, Wan Pinjun, He Jiachun, Lai Fengxiang, Fu Qiang. A Mucin2-Like Gene, NlMuc2, is Required for Early Embryonic Development in Nilaparvata lugens[J]. Rice Science, 2025, 32(4): 549-560.

Figures/Tables 7

Fig. 1. Motif consensus and locations in NlMuc2. The top three most highly enriched motifs of NlMuc2 in Nilaparvata lugens.

Fig. 2. Spatial and temporal expression patterns of Nilaparvata lugens mucin-2 (NlMuc2). A, Expression patterns of NlMuc2 in different tissues of N. lugens. Total RNA was extracted from salivary glands (SG), digestive tracts (DT), female internal reproductive organs (FIRO), male internal reproductive organs (MIRO), fat bodies (FB), legs (LG), wings (WG), and cuticles (CT). Tissues were dissected from adults 24‒72 h after emergence. Different lowercase letters above error bars indicate significant differences determined by one-way analysis of variance followed by Tukey’s honestly significance difference (HSD) tests (P < 0.05). B, Expression patterns of NlMuc2 across developmental stages of N. lugens. Samples were extracted from whole insects collected every 24 or 48 h from the start of each stage. E1d‒E6d, Eggs from 1 to 6 d after laying; N0d‒N11d, Nymphs from 0 to 11 d after hatching; F0d, F2d, and F4d, Newly emerged, 2-day-old, and 4-day-old females, respectively; M0d, M2d, and M4d, Newly emerged, 2-day-old, and 4-day-old males, respectively. 18S rRNA and ribosomal protein S11 were used as internal controls for qRT-PCR. Data show relative NlMuc2 mRNA levels (mean ± SE with three independent experiments). C, Dynamic expression patterns of NlMuc2 in eggs throughout embryonic development at the transcript level by in situ hybridization. Blue signals were detected using NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) and alkaline phosphatase-conjugated anti-DIG (digoxigenin) antibodies after hybridization with DIG-labeled antisense RNA probes. Scale bars, 300 μm. D, Dynamic expression patterns of NlMuc2 in eggs at the protein level by immunohistochemistry. Brown signals were visualized using DAB (3,3′-diaminobenzidine) and HRP (horseradish peroxidase)-conjugated goat anti-rabbit IgG. Eggs were collected every 24 h from rice leaves. Scale bars, 300 μm. E, Analysis of NlMuc2 protein levels during egg development stages with Western blotting. E1d‒E6d, Eggs from 1 to 6 d after laying. Immunoblotting used anti-NlMuc2 antibodies. Red arrow indicates NlMuc2 (166.9 kDa). For electrophoresis, 30 μg of protein was load into each well of a 3%-8% NuPAGE gel.

Fig. 3. Knockdown of NlMuc2 causes lethality. A, NlMuc2 expression after dsNlMuc2 injection. B, Survival rates of 3rd-instar nymphs on rice after dsNlMuc2 injection. C, Survival rates of 3rd-instar nymphs on artificial diet after dsNlMuc2 injection. RNAi, RNA interference. dsRNA (70 ng per nymph; n = 30) was injected into 3rd-instar nymphs. dsGFP was used as control. Data are represented as mean ± SE from six independent experiments and analyzed by student’s t-test (**, P < 0.05).

Fig. 4. Effects on fecundity after dsNlMuc2 injection on 5th-instar nymphs of Nilaparvata lugens. A‒C, Number of laid eggs (A), hatched nymphs (B), and hatching rate (C) of 5th-instar nymphs of N. lugens after dsNlMuc2 injection. D, Morphology of unhatched eggs dissected from rice leaf sheaths. The red eyespots were marked by the red arrow. Yolk-like mass was marked by the yellow arrow. In dsGFP, unhatched eggs contained a normally developed embryo with a white-to-yellow yolk-like mass at the posterior or the red eyespot at the anterior or near the micropylar cap. In dsNlmuc2, unhatched eggs contained white-to-yellow yolk-like mass (yellow arrow) at the anterior, the red eyespot at the posterior. Scale bars, 500 μm Data are represented as mean ± SE from 15 independent experiments and analyzed by student’s t-test (**, P < 0.05).

Fig. 5. Effects of NlMuc2 knockdown on ovarian (A) and embryonic (B) development. Newly emerged females were injected with 70 ng dsNlMuc2, the control group was treated with dsGFP. Ovaries from at least 10 females for each group were dissected at 4, 6, and 8 d post-injection and photographed using a stereo microscopy. At 4 d post-injection, mature banana-shaped eggs appeared in the ovarioles of the dsGFP females, whereas in the dsNMuc2 females, the ovarioles were transparent and adhered together. At 6 d post-injection, mature eggs filled all ovarioles in the dsGFP group, whereas in the dsNlMuc2 group, less than 50% of the ovarioles filled mature eggs. At 8 d post-injection, the number of mature oocytes in the dsNlMuc2-injected group was lower than in the dsGFP group. The eggs laid within the rice leaf sheath were dissected and photographed at 5 d after oviposition. The red eyespot was marked by the red arrow. The yellow arrow indicated the micropylar cap of the egg. In the eggs laid by the dsGFP group, the red eyespot on the anterior side of the egg was observed at 4 or 5 d after laying. In the dsNlMuc2 eggs, 51.0% had the eyespot at the posterior. Scale bars, 500 μm.

Fig. 6. Transcriptome analysis of eggs from dsNlMuc2- and dsGFP-injected females. A, Top 10 enriched Gene Ontology (GO) terms for differentially expressed genes (DEGs). BP, Biological process; CC, Cellular component; MF, Molecular function. B, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of downregulated DEGs. The x-axis indicates the subcategories and the y-axis indicates -log10(Adjusted P-value). Asterisks (*) above the bars represent significant differences (adjusted P-value <  0.05).

Fig. 7. Effects of differentially expressed gene knockdown on female fecundity. A, RNA-seq validation by qRT-PCR analysis. Nilaparvata lugens 18S rRNA and ribosomal protein S11 were used as internal controls. B and C, Effects of differentially expressed gene knockdown on the total number of laid eggs (B) and hatching rate (C) after dsDEGs injection in 5th-instar nymphs of N. lugen. The results were calculated using dsGFP as control. Data were expressed as mean ± SE from three independent experiments and analyzed using a two-tailed unpaired t-test at the significant levels at P < 0.05 (**). ns, Not significant. Fecundity analysis is based on three biological replicates with each includes five pairs of females and males as a pool.

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