Cuticular Hydrocarbon Plasticity in Three Rice Planthopper Species.

Insect cuticular hydrocarbons (CHCs) are organic compounds of the surface lipid layer, which function as a barrier against water loss and xenobiotic penetration, while also serving as chemical signals. Plasticity of CHC profiles can vary depending upon numerous biological and environmental factors. Here, we investigated potential sources of variation in CHC profiles of Nilaparvata lugens, Laodelphax striatellus and Sogatella furcifera, which are considered to be the most important rice pests in Asia. CHC profiles were quantified by GC/MS, and factors associated with variations were explored by conducting principal component analysis (PCA). Transcriptomes were further compared under different environmental conditions. The results demonstrated that CHC profiles differ among three species and change with different developmental stages, sexes, temperature, humidity and host plants. Genes involved in cuticular lipid biosynthesis pathways are modulated, which might explain why CHC profiles vary among species under different environments. Our study illustrates some biological and ecological variations in modifying CHC profiles, and the underlying molecular regulation mechanisms of the planthoppers in coping with changes of environmental conditions, which is of great importance for identifying potential vulnerabilities relating to pest ecology and developing novel pest management strategies.

Three-dimensional architecture of a mechanoreceptor in the brown planthopper, Nilaparvata lugens, revealed by FIB-SEM.

Trichoid sensilla are the most common mechanoreceptors in insects; depending on their distribution, they can act as either exteroceptors or proprioceptors. In this study, the internal structure of the trichoid sensillum from Nilaparvata lugens was studied, using focused ion beam scanning electron microscopy (FIB-SEM). We reconstructed a three-dimensional (3D) model derived from the FIB-SEM data set. The model displayed characteristic mechanosensory sensilla components, including a hair inserted in the socket, a dendrite going through the laminated cuticle, and an electron-dense tubular body at the dendrite terminal. The detailed 3D model showed the relationship between the microtubules within the tubular body and those outside of the tubular body. We also found an autocellular junction in the tormogen cell, indicating that the tormogen cell grows around the dendrite sheath to form a hollow column shape during sensilla morphogenesis.

EndophilinA2 protects against angiotensin II-induced cardiac hypertrophy by inhibiting angiotensin II type 1 receptor trafficking in neonatal rat cardiomyocytes.

Cardiac hypertrophy is one of the major risk factors for chronic heart failure. The role of endophilinA2 (EndoA2) in clathrin-mediated endocytosis and clathrin-independent endocytosis is well documented. In the present study, we tested the hypothesis that EndoA2 protects against angiotensin II (Ang II)-induced cardiac hypertrophy by mediating intracellular angiotensin II type 1 receptor (AT1-R) trafficking in neonatal rat cardiomyocytes (NRCMs). Cardiac hypertrophy was evaluated by using cell surface area and quantitative RT-PCR (qPCR) analyses. For the first time, we found that EndoA2 attenuated cardiac hypertrophy and fibrosis induced by Ang II. Moreover, EndoA2 inhibited apoptosis induced by excessive endoplasmic reticulum stress (ERS), which accounted for the beneficial effects of EndoA2 on cardiac hypertrophy. We further revealed that there was an interaction between EndoA2 and AT1-R.The expression levels of EndoA2, which inhibits AT1-R transport from the cytoplasm to the membrane, and the interaction between EndoA2 and AT1-R were obviously decreased after Ang II treatment. Furthermore, Ang II inhibited the co-localization of AT1-R with GRP-78, which was reversed by EndoA2 overexpression. In conclusion, our results suggested that EndoA2 plays a role in protecting against cardiac hypertrophy induced by Ang II, possibly by inhibiting AT1-R transport from the cytoplasm to the membrane to suppress signal transduction.

Autophagy is involved in the protective effect of endophilin A2 on H2O2-induced apoptosis in H9C2 cardiomyocytes.

Apoptosis plays a critical role in normal embryonic development and tissue homeostasis regulation. EndophilinA2 (EndoA2) is widely reported to regulate endocytosis. Additionally, EndoA2 has been demonstrated to be involved in tumor metastasis, neuroregulation and vascular function. In this study, we used siRNA and Ad-EndoA2 transfection strategy to investigate whether EndoA2 provides a protective effect against apoptosis induced by H2O2 in H9C2 cardiomyocytes and the underlying mechanisms. We found that EndoA2 siRNA knockdown promoted H2O2-induced apoptosis in H9C2 cardiomyocytes, evidenced by decreased cell number, increased apoptotic cells, and activation of caspase-3. In contrast, EndoA2 overexpression showed the opposite effects and inhibited H2O2-induced apoptosis in H9C2 cardiomyocytes. Further studies revealed that EndoA2 overexpression strengthened autophagy, evidenced by the increased LC3 II/I ratio and P62 degradation, whereas EndoA2 siRNA knockdown produced the opposite effects. Furthermore, we revealed that there was an interaction between Bif-1 and Beclin-1. Upon H2O2 treatment, the association of Bif-1 and Beclin-1 remarkably increased. EndoA2 overexpression further promoted the binding of Bif-1 with Beclin-1, whereas EndoA2 siRNA knockdown reduced this association. These data strongly suggested that EndoA2 inhibited H2O2-induced apoptosis in H9C2 cardiomyocytes, possibly by promoting Bif-1 to form a complex with Beclin-1 and strengthening autophagy. This study provides a novel target for heart diseases.

Evaluation of the protective effect of pVAX-EtMIC3-recombined plasmid against E. tenella in chicken.

Coccidiosis caused by protozoan parasites of the genus Eimeria has a severe economic impact on commercial production worldwide. Micronemes of Eimeria play important roles in invading intestinal cell processes. In this study, the DNA vaccine expressing Eimeria tenella microneme protein 3 (EtMIC3) was constructed to evaluate its immune protective effect against E. tenella infection in chickens. The results demonstrated that chickens immunized with pVAX-EtMIC3 produced strong immune responses in the body, as shown by significant lymphocyte proliferation, cytokine production, and antibody responses. The average body weight gains of chickens in all the vaccinated groups were higher than those of non-vaccinated and challenged groups. In general, oocyst shedding was reduced, and bloody feces and gut lesion scores decreased. In addition, the survival rate of the immunized chickens increased compared to that of the unvaccinated and challenged control chickens. In summary, this study indicated that pVAX-EtMIC3 could induce protective immune effects against coccidiosis and that EtMIC3 is a potential vaccine candidate against coccidiosis.

PCR Identification and Phylogenetic Analysis of Trichomonas gallinae from Domestic Pigeons in Guangzhou, China.

Avian trichomoniasis caused by Trichomonas gallinae is a serious protozoan disease worldwide. The domestic pigeon (Columba livia domestica) is the main host for T. gallinae and plays an important role in the spread of the disease. Based on the internal transcribed spacers of nuclear ribosomal DNA of this parasite, a pair of primers (TgF2/TgR2) was designed and used to develop a PCR assay for the diagnosis of T. gallinae infection in domestic pigeons. This approach allowed the identification of T. gallinae, and no amplicons were produced when using DNA from other common avian pathogens. The minimum amount of DNA detectable by the specific PCR assay developed in this study was 15 pg. Clinical samples from Guangzhou, China, were examined using this PCR assay and a standard microscopy method, and their molecular characteristics were determined by phylogenetic analysis. All of the T. gallinae-positive samples detected by microscopic examination were also detected as positive by the PCR assay. Most of the samples identified as negative by microscopic examination were detected as T. gallinae positive by the PCR assay and were confirmed by sequencing. The positive samples of T. gallinae collected from Guangzhou, China, were identified as T. gallinae genotype B by sequencing and phylogenetic analyses, providing relevant data for studying the ecology and population genetic structures of trichomonads and for the prevention and control of the diseases they cause.

Three-dimensional reconstruction of insect chemosensory sensillum.

The olfactory system is involved in food and mate recognition in insects. However, 3D structures of chemosensory sensilla in insects are unexplored yet. Here, the internal structures of an olfactory sensillum on the antenna of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae), one of the most important rice pests, are examined and imaged using focused ion beam scanning electron microscopy. Based on these images, a 3D structure is reconstructed in this study. We find that the trichoid olfactory sensillum possesses a multiporous wall encircling a lumen with one sensory cell. Besides, there are three accessory cells (ACs) and a glia cell with different cell contents surrounding the sensory cell. The abundant tubular membrane structures in the tormogen cell suggest its role in secreting proteins like odorant binding proteins into the receptor lymph, while three auxiliary cells with simpler cellular content closely enfold the sensory cell, probably to prevent leaking of the receptor lymph into the surrounding epidermis. In the sensory cell, the microtubules and two tandem basal bodies at the base of the microtubules are also reconstructed. They are considered as a propulsive engine to ensure dendrite vibration or spinning in the receptor lymph, so that the proteins and odorant molecules move faster in the receptor lymph, which improves recognition of environmental odors and enables the insect to immediately respond to this information.

Three-dimensional reconstruction of a whole insect reveals its phloem sap-sucking mechanism at nano-resolution.

Using serial block-face scanning electron microscopy, we report on the internal 3D structures of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) at nanometer resolution for the first time. Within the reconstructed organs and tissues, we found many novel and fascinating internal structures in the planthopper such as naturally occurring three four-way rings connecting adjacent spiracles to facilitate efficient gas exchange, and fungal endosymbionts in a single huge insect cell occupying 22% of the abdomen volume to enable the insect to live on plant sap. To understand the muscle and stylet movement during phloem sap-sucking, the cephalic skeleton and muscles were reconstructed in feeding nymphs. The results revealed an unexpected contraction of the protractors of the stylets and suggested a novel feeding model for the phloem sap-sucking.

Since the 19^th century, scientists have been investigating how the organs of insects are shaped and arranged. However, classic microscopy methods have struggled to image these small, delicate structures. Understanding how the organs of insects are configured could help to identify new methods for controlling pests, such as chemicals that target the mouthparts that some insects use to feed on plants. Most insects that feed on the sap of plants suck out the nutrient via their stylet bundle ­ a thin, straw-like structure surrounded by a sheath called the labium. As well as drying out the plant and damaging its tissues, the stylet bundle also allows the insect to transmit viruses that cause further harm. To investigate these mouthparts in more detail, Wang, Guo et al. used a method called SBF-SEM to determine the three-dimensional structure of one of the most destructive pests of rice crops, the brown planthopper. In this technique, a picture of the planthopper was taken every time a thin slice of its body was removed. This continuous slicing and re-imaging generated thousands of images that were compiled into a three-dimensional model of the brown planthopper's whole body and internal organs. Previously unknown features emerged from the reconstruction, including a huge cell in the planthopper's abdomen which is full of fungi that provide the nutrients absent in plants. Next, Wang, Guo et al. used this technique to see how the muscles in the labium and surrounding the stylet move by imaging planthoppers that were frozen at different stages of the feeding process. This revealed that when brown planthoppers bow their heads to eat, the labium compresses and pushes out the stylet, allowing it to pierce deeper into the plant. This is the first time that the body of such a small insect has been reconstructed three-dimensionally using SBF-SEM. Furthermore, these findings help explain how brown planthoppers and other sap-feeding insects insert their stylet and damage plants, potentially providing a stepping stone towards identifying new strategies to stop these pests from destroying millions of crops.

Ten fatty acyl-CoA reductase family genes were essential for the survival of the destructive rice pest, Nilaparvata lugens.

BACKGROUND: Fatty alcohols are the precursors of sex pheromone components, wax esters and hydrocarbons in insects. Fatty acyl-CoA reductases (FARs) are important enzymes required for the reduction of fatty alcohol and thereby contribute to the production of cuticular hydrocarbon (CHC). RESULTS: Based on bioinformatics analyses we identified 17 FAR genes in the brown planthopper, Nilaparvata lugens. RNA interference against these genes demonstrated that ten NlFAR genes were essential for the survival of N. lugens. For instance, knockdown of NlFAR5, 6, 11 or 15 was lethal and caused a slender body shape, while the old cuticles of the respective animals remained attached to the abdomen or failed to split open from the nota. Knockdown of NlFAR9 resulted in a phenotype, with a smooth body surface and a decrease in CHC amounts. Similarly, CHC deficiency in N. lugens resulted in increased adhesion of water droplets and secreted honeydew to the insect surface and the inability of N. lugens to survive in paddy fields with varying humidity. Knockdown of NlFAR1, 4, 5, 6, 8, 9, 11 and 13 additionally resulted in female adult infertility. CONCLUSION: The present study illustrates the structural and functional differences of FAR family genes and provides potential targets for RNA interference-based rice planthopper management. © 2020 Society of Chemical Industry.

FAR gene enables the brown planthopper to walk and jump on water in paddy field.

Many insects can live on water and survive being caught in the rain. Current research has shown that insect cuticular hydrocarbons (CHC) confer desiccation resistance to maintain water balance. In this study, we identified a fatty acyl-CoA reductase gene (NlFAR) of the rice brown planthopper, Nilaparvata lugens that is essential for the production of CHCs, and found that NlFAR is essential for N. lugens to walk and jump on water when moving from one rice plant to another in paddy fields. NlFAR was mainly expressed in the integument at the beginning of each molt. Cuticular surface analysis by scanning electron microscopy and characterization of CHC extracts indicated that N. lugens with knockdown of NlFAR using RNA inference (RNAi) had a neater epicuticle layer and a significant decrease in CHC contents. Knockdown of NlFAR did not influence the desiccation resistance of N. lugens, but the dsNlFAR-treated insects were easily adhered and moistened by water droplets or their own secreted honeydew and unable to walk or jump on water. These results suggested that NlFAR is a crucial enzyme for CHC biosynthesis and cuticle waterproofing, but not for water retention of N. lugens, which may provide a potential strategy for pest management.

The fatty acid elongase gene family in the brown planthopper, Nilaparvata lugens.

The cuticular hydrocarbon (CHC) biosynthetic pathways branches off from the synthesis of fatty acids. Fatty acid elongases (ELOs) are enzymes catalyzing the synthesis of long-chain fatty acids and thereby contribute to the diversification of CHCs. Based on bioinformatics analyses we identified 20 ELO genes in the brown planthopper, Nilaparvata lugens. RNA interference against these genes demonstrated that 9 NlELO genes were essential for the survival of N. lugens nymphs and adults. Indeed, knockdown of NlELOs 1, 3, 4, 7, 8, 9, 10, 12 and 18 caused lethal phenotypes with a thin and wizened body and reduced lipids in the fat body. Surface analysis by scanning electron microscopy and CHC quantification indicated that knockdown of NlELOs 2, 3, 8 and 16 additionally resulted in a smooth body surface and a decrease in CHC amounts. Therefore, we speculate that long-chain CHCs are needed for CHC attachment to the cuticle surface. CHC deficiency, in turn, resulted in increased adhesion of water droplets and secreted honeydew to the animal surface and the inability of N. lugens to survive in paddy fields with varying humidity. Our present study provides an initial comprehensive analysis of ELO gene functions in an insect, and may serve to better understand the biology of CHCs.

The flightin gene is necessary for the emission of vibrational signals in the rice brown planthopper (Nilaparvata lugens Stǻl).

In duet-based courtship, species- and sex-specific vibrational signals enable animals to identify the species and sex of the singer and also provide the necessary information with which to locate a partner. Substrate-borne communication has been described in a wide variety of insects. Here, we focus on the gene necessary for the emission of male vibrational signals and whether the male song fulfills such a functional role in the mating system of the brown planthopper (BPH, Nilaparvata lugens). We generated mute BPH adult males via RNA interference (RNAi) of the flightin gene, which encodes a myosin-binding protein expressed exclusively in the dorsal longitudinal muscle (DLM) in the basal two abdominal segments used for driving the vibration of the male-specific tymbal structure in short-winged (brachypterous) BPH adults. Transmission electron microscopy (TEM) observation showed that flightin knockdown disrupted the normal sarcomere structure of the abdominal DLM. No courtship song could be detected in the brachypterous males after RNAi treatment. Behavior and competition trials showed that the lack of male courtship songs prolonged copulation latency and even caused female rejection. Unexpectedly, the mute males exhibited greater competitiveness when competing against normal males.

Endophilin A2 attenuates cardiac hypertrophy induced by isoproterenol through the activation of autophagy.

Decreased autophagy has been reported to contribute to the progression of cardiac hypertrophy. Our previous research has demonstrated that endophilin A2 (EndoA2) attenuates H2O2-induced cardiomyocyte apoptosis by strengthening autophagy. However, the role of EndoA2 in the regulation of autophagy in cardiac hypertrophy is unknown. In this study, we tested the hypothesis that EndoA2 suppresses cardiac hypertrophy induced by isoproterenol (ISO) by activating autophagy. In vivo, we established a cardiac hypertrophy model by subcutaneous injection of ISO and used intramyocardial delivery of adenovirus vector harboring EndoA2 cDNA (Ad-EndoA2) to overexpress EndoA2. The cardiac hypertrophic response and autophagy level were measured. EndoA2 overexpression suppressed pathological cardiac hypertrophy and enhanced autophagy in rat hearts. In addition, the effects of EndoA2 on cardiac hypertrophy and autophagy were observed in cultured neonatal rat cardiomyocytes (NRCMs) with gain- and loss-of-function approaches to regulate EndoA2 expression. The results were consistent with those of the in vivo study. Furthermore, the involvement of EndoA2-mediated autophagy in the attenuation of ISO-induced cardiac hypertrophy was explored by pharmaceutical inhibition of autophagy. Pretreatment with 3-methyladenine (3-MA) clearly diminished the anti-hypertrophic effects of EndoA2 in ISO-treated NRCMs. The results presented here provide the first evidence that EndoA2 is involved in ISO-induced cardiac hypertrophy. The anti-hypertrophic effects of EndoA2 can be partially attributed to its regulation of autophagy.