The brown planthopper, Nilaparvata lugens, is a pest that threatens rice (Oryza sativa) production worldwide. While feeding on rice plants, planthoppers secrete saliva, which plays crucial roles in nutrient ingestion and modulating plant defense responses, although the specific functions of salivary proteins remain largely unknown. We identified an N. lugens-secreted mucin-like protein (NlMLP) by transcriptome and proteome analyses and characterized its function, both in brown planthopper and in plants. NlMLP is highly expressed in salivary glands and is secreted into rice during feeding. Inhibition of NlMLP expression in planthoppers disturbs the formation of salivary sheaths, thereby reducing their performance. In plants, NlMLP induces cell death, the expression of defense-related genes, and callose deposition. These defense responses are related to Ca²⁺ mobilization and the MEK2 MAP kinase and jasmonic acid signaling pathways. The active region of NlMLP that elicits plant responses is located in its carboxyl terminus. Our work provides a detailed characterization of a salivary protein from a piercing-sucking insect other than aphids. Our finding that the protein functions in plant immune responses offers new insights into the mechanism underlying interactions between plants and herbivorous insects.

Plants deploy receptor-like kinases and nucleotide-binding leucine-rich repeat receptors to confer host plant resistance (HPR) to herbivores¹. These gene-for-gene interactions between insects and their hosts have been proposed for more than 50 years². However, the molecular and cellular mechanisms that underlie HPR have been elusive, as the identity and sensing mechanisms of insect avirulence effectors have remained unknown. Here we identify an insect salivary protein perceived by a plant immune receptor. The BPH14-interacting salivary protein (BISP) from the brown planthopper (Nilaparvata lugens Stål) is secreted into rice (Oryza sativa) during feeding. In susceptible plants, BISP targets O. satvia RLCK185 (OsRLCK185; hereafter Os is used to denote O. satvia-related proteins or genes) to suppress basal defences. In resistant plants, the nucleotide-binding leucine-rich repeat receptor BPH14 directly binds BISP to activate HPR. Constitutive activation of Bph14-mediated immunity is detrimental to plant growth and productivity. The fine-tuning of Bph14-mediated HPR is achieved through direct binding of BISP and BPH14 to the selective autophagy cargo receptor OsNBR1, which delivers BISP to OsATG8 for degradation. Autophagy therefore controls BISP levels. In Bph14 plants, autophagy restores cellular homeostasis by downregulating HPR when feeding by brown planthoppers ceases. We identify an insect saliva protein sensed by a plant immune receptor and discover a three-way interaction system that offers opportunities for developing high-yield, insect-resistant crops.

Heat shock protein 70 genes participate in obligatory pupal diapause in Pieris melete to survive unfavorable conditions. In this study, three full-length cDNAs of PmHsc70, PmHsp70a and PmHsp70b were identified, and their expression patterns in response to diapause and short-term temperature stresses were investigated. Summer and winter diapause were induced in the pupae and non-diapause individuals were used as a control. The pupae from each diapause group were subjected to either hot or cold conditions and the expression levels of the HSP genes were measured. Our results showed that up-regulation of PmHsc70 and PmHsp70b were detected both in summer and winter diapause, but not for PmHsp70a. Under cold stress, PmHsp70a and PmHsp70b were upregulated in summer and winter diapause, while heat shock significantly induced upregulation of all three genes. In non-diapause pupae, none of the genes responded to cold or heat stress. Furthermore, we found that incubation at 39 ${}^{\circ }$C for 30 min was the most sensitive heat stress condition for PmHsc70 expression in summer diapause. On the other hand, the same temperature was effective for PmHsc70, PmHsp70a, and PmHsp70b expression in winter diapause. During summer diapause, expression of all three genes was upregulated in response to high-temperature acclimation at 31 ${}^{\circ }$C, but only PmHsp70a and PmHsp70b were upregulated when acclimated to a low temperature of 4 ${}^{\circ }$C in winter diapause. These results suggest that the PmHsc70, PmHsp70a, and PmHsp70b respond differently to pupal diapause and temperature stress, and that PmHsc70 is more sensitive to heat shock than to cold stress.

Clostridium butyricum, recognized as a probiotic, is widely distributed in the intestines of various animals. In this study, the C. butyricum strain YF1 was isolated from the intestine of the ricefield eel (Monopterus albus) using an anaerobic culture method and was identified through morphological, physiological, biochemical, and 16S rRNA sequence analyses. Notably, C. butyricum YF1 exhibited a rapid growth rate and was found to produce ten types of short-chain fatty acids, particularly high-yield acetic acid and butyric acid. Additionally, YF1 demonstrated a high tolerance to elevated temperatures (70 °C), bile salts (0.1% to 0.5%), artificial intestinal fluid, and artificial gastric fluid, while being sensitive to most antibiotics. Further whole-genome sequencing revealed that C. butyricum YF1 has a total genome size of 4,314,266 bp and contains 3853 coding genes. Specifically, 82 tRNAs, 21 rRNAs, 288 repeat sequences, 13 prophages, and two gene islands were detected. Moreover, gene function analysis indicated that the highest number of genes were annotated to metabolic processes, and the butyric acid metabolism pathway was found to be complete. Meanwhile, 598 virulence genes and 186 resistance genes were predicted. In conclusion, the findings from this study contribute to probiotic development and provide innovative approaches for the sustainable and healthy cultivation of ricefield eels.

PIWI proteins and Piwi‐interacting RNAs (piRNAs) are crucial for gametogenesis, embryogenesis, and stem cell maintenance in animals. The Piwil1 ( Piwi-like RNA-mediated gene silencing 1 ) gene has rapidly evolved in the protogynous hermaphrodite ricefield eel ( Monopterus albus ) compared to other teleosts. Notably, there is a significant reduction in DNA methylation levels and an increase in the expression levels of the Piwil1 gene in testes compared to those in ovaries. However, the role of Piwil1 in the gonadogenesis of ricefield eel remains unclear. This study elucidated the histomorphological characteristics of ricefield eel gonads at different developmental stages and analyzed the expression and localization of Piwil1 using qRT‐PCR, western blot, and immunofluorescence. Histological analysis showed that the female gonad contained oocytes at different developmental stages, the intersex gonad had both ovarian and testicular tissues, and the male gonad included spermatogenic cells and testicular lobules. Quantitative real‐time PCR and western blot indicated significantly higher Piwil1 expression in the ovary, intersexual gonad, and testis compared to other tissues, with levels increasing during gonadal differentiation and peaking in the testis. Immunofluorescence analysis revealed that the PIWIL1 protein was abundantly localized in the cytoplasm of immature oocytes, granulosa cells surrounding the oocytes, spermatogonia, and primary spermatocytes. Importantly, in both the ovary and testis, the expression of Piwil1 initially increased but then decreased at the mRNA and protein levels. These results suggest that Piwil1 might play a crucial role in gonadal development and gametogenesis in ricefield eels.

KDF (potassium diformate) has been recognized as a highly promising additive due to its beneficial effects on growth and immunity improvement. In order to expand the application of KDF in the aquatic farming practice, the effects of KDF on ricefield eels were thoroughly evaluated. In the present study, the growth performance, blood biochemistry, hepatic health and disease resistance were assessed in the juvenile ricefield eels fed diets supplemented with 0 (D0), 3 (D1), 6 (D2), 9 (D3), 12 (D4) and 15 (D5) g/kg KDF. After 8 weeks feeding, fish weight gain rate (WGR) was improved by KDF addition, and the estimated optimum dietary KDF was determined at 8.91 g/kg according to the regression analysis between dietary KDF level and WGR. Fish condition factor (CF) was increased by dietary KDF supplementation compared to the control (D0). The VSI of fish decreased significantly and then kept stable when the dietary KDF level was above 9 g/kg (p < 0.05). The supplementation of KDF in diet had no significant effect on the moisture, crude ash, and crude protein contents of the whole fish (p > 0.05), while the crude lipid content in the D4 group was significantly lower than that in the control (p < 0.05). Compared to the control group, the inclusion of 9 g/kg KDF (D3) in the diet significantly decreased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (p < 0.05). Liver catalase (CAT) activity increased significantly with increasing the level of KDF in diet, but it remained stable at KDF levels at and above 9 g/kg (D3) (p < 0.05). The significant improvement of glutathione peroxidase (GSH-Px) activity and total antioxidant capacity (T-AOC) were found in the D1 and D4 groups, respectively (p < 0.05). Liver malondialdehyde (MDA) content showed a non-monotonic pattern across KDF levels, and the addition of KDF in diet did not significantly alter MDA content compared to the control (p > 0.05). Fish in the D4 group showed the lowest liver malondialdehyde (MDA) content. Serum ALP activity was markedly elevated in the D3 group compared with the control (p < 0.05). Supplementation of KDF in diets enhanced hepatic ALP and ACP activities, which differed significantly from the control in the D3 and D4 groups, respectively (p < 0.05). The histological results indicated that while no significant alteration of liver cell structure was observed, fish liver showed a lower number and size of lipid droplets with the KDF level increasing, and the hepatic sinusoids appeared more prominent in the high KDF group (D5). The Aeromonas hydrophila challenge test demonstrated that the addition of 9 g/kg KDF (D3) in fish diet significantly increased the survival rate of juvenile ricefield eel (p < 0.05). In summary, dietary supplementation with KDF significantly enhanced growth performance, improved several biomarkers associated hepatic health, and strengthened pathogen resistance in juvenile ricefield eels.