The Insect Ecdysone Receptor is a Good Potential Target for RNAi-based Pest Control

RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests.     

Pathways of Amino Acid Degradation in Nilaparvata lugens (St?l) with Special Reference to Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase (LKR/SDH)

Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens.     

Sublethal effects of abamectin on the development,fecundity, and wing morphs of the brown planthopper Nilaparvata lugens

The brown planthopper Nilaparvata lugens negatively affects rice yield by sucking nutrients from the rice stem and spreading viruses. In this study, the effects of sublethal concentrations of abamectin on development, fecundity, and wing morphs of N. lugens were investigated. Treatment with sublethal concentrations of abamectin showed no significant effects on the developmental period of F₀ nymphs. Although LC₁₀ and LC₂₅ treatments resulted in 10% and 25% mortality, the LC₂₅ treatment significantly prolonged the lifespan of macropterous and brachypterous females. Abamectin-treated brachypterous females showed significantly lower fecundity than control females. Furthermore, abamectin treatment showed no significant effects on the developmental period of F₁ nymphs. However, LC₂₅ treatment significantly inhibited the fecundity of brachypterous F₁ females and significantly reduced the proportion of macropterous F₁ females and brachypterous F₁ males. LC₂₅ treatment showed no significant effects on the fecundity of macropterous F₁ females. Considering the proportion of macropterous and brachypterous F₁ females, the relative fitness of the macropterous F₁ nymphs in the control, LC₁₀, and LC₂₅ treatment groups was 1, 1.02, and 0.84, respectively, and that of brachypterous F₁ nymphs was 1, 0.79, and 0.93, respectively. Sublethal concentrations of abamectin inhibited N. lugens emergence. The present findings indicate the potential of abamectin for N. lugens field control.     

A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust

Application of RNA interference (RNAi) for insect pest management is limited by variable efficiency of RNAi in different insect species. In Locusta migratoria, RNAi is highly efficient through injection of dsRNA, but oral delivery of dsRNA is much less effective. Efforts to understand this phenomenon have shown that dsRNA is more rapidly degraded in midgut fluid than in hemolymph due to nuclease enzyme activity. In the present study, we identified and characterized two full-length cDNAs of double-stranded RNA degrading enzymes (dsRNase) from midgut of L. migratoria, which were named LmdsRNase2 and LmdsRNase3. Gene expression analysis revealed that LmdsRNase2 and LmdsRNase3 were predominantly expressed in the midgut, relatively lower expression in gastric caeca, and trace expression in other tested tissues. Incubation of dsRNA in midgut fluid from LmdsRNase3-suppressed larvae or control larvae injected with dsGFP resulted in high levels of degradation; however, dsRNA incubated in midgut fluid from LmdsRNase2-suppressed larvae was more stable, indicating LmdsRNase2 is responsible for dsRNA degradation in the midgut. To verify the biological function of LmdsRNase2 in vivo, nymphs were injected with dsGFP, dsLmdsRNase2 or dsLmdsRNase3 and chitinase 10 (LmCht10) or chitin synthase 1 (LmCHS1) dsRNA were orally delivered. Mortality associated with reporter gene knockdown was observed only in locusts injected with dsLmdsRNase2 (48% and 22%, for dsLmCht10 and dsLmCHS1, respectively), implicating LmdsRNase2 in reducing RNAi efficiency. Furthermore, recombinantly expressed LmdsRNase2 fusion proteins degraded dsRNA rapidly, whereas LmdsRNase3 did not. These results suggest that rapid degradation of dsRNA by dsRNase2 in the midgut is an important factor causing low RNAi efficiency when dsRNA is orally delivered in the locust.     

Effects of elevated CO2 on the nutrient compositions and enzymes activities of Nilaparvata lugens nymphs fed on rice plants

Elevated CO 2 may reduce the tolerance of Nilaparvata lugen (N. lugens) to adverse environmental factors through the biological and physiological degeneration of N. lugens. In an artificial climate box, under 375 and 750 μL L 1 CO 2 levels, the rice stems nutrient content, the nutrient content and enzyme activities of N. lugens nymph fed on rice seedlings exposed to ambient and elevated CO 2 were studied. The results showed that rice stems had significantly higher protein and total amino acid levels under ambient than elevated CO 2 levels. Nymphs had significantly higher protein levels in the ambient CO 2 treatment, while their glucose levels were significantly lower under ambient CO 2 conditions. Significantly higher trypsin activity was observed in nymphs grown in elevated CO 2 . Significantly lower activities of the protective enzymes total superoxide dismutase and catalase were observed in the nymphs under ambient CO 2 . Meanwhile, the activity of the detoxification enzyme glutathione S-transferase was significantly higher in the ambient CO 2 treatment. Measuring how energy and resources were allocated to enzymes in N. lugens nymphs under elevated CO 2 conditions can provide a more meaningful evaluation of their metabolic tolerances to adverse climatic conditions.     

Cellular and potential molecular mechanisms underlying transovarial transmission of the obligate symbiont Sulcia in cicadas

Vertical transmission of symbionts in insects is critical to persistence of symbioses across host generations. The key time point and related cellular/molecular mechanisms underlying the transmission in most insects remain unclear. Here, we reveal that in the bacteriome–endosymbiont system of the cicada Meimuna mongolica, the obligate symbiont Candidatus Sulcia muelleri (hereafter Sulcia) proliferates and migrates to the ovaries mainly after the adult emergence of cicadas. Sulcia cells swell to approximately twice their previous size with the outer membrane changed to be more irregular during this process. Almost all the Sulcia genes involved in biosynthesis of essential amino acids, heat shock protein, energy metabolism, DNA replication and repair and protein export were highly expressed in all life stages of cicadas. Among which, genes involved in DNA replication and synthesis of leucine and arginine were upregulated in the newly emerged adults relative to fifth-instar nymphs. Signal transduction is the pronounced function exhibited in both Sulcia and the cicada bacteriomes in newly emerged adults. The results suggest host sensing of arginine and leucine integrate Sulcia's output of host-EAAs into mTORC1 signalling. This study highlights the importance of signalling pathways in regulating the host/symbiont interaction and symbiont transmission in sap-feeding auchenorrhynchous insects.     

Knockdown of a putative Halloween gene Shade reveals its role in ecdysteroidogenesis in the small brown planthopper Laodelphax striatellus

Ecdysteroid hormone 20-hydroxyecdysone (20E) plays fundamental roles in insect development and reproduction, whereas the primary role of ecdysone (E) is the precursor for 20E. A cytochrome P450 monooxygenase (CYP), encoded by a Halloween gene Shade (Shd, cyp314a1), catalyzes the conversion of E into 20E in representative insect species in Diptera, Lepidoptera and Orthoptera. We describe here the cloning and characterization of LsShd in a hemipteran insect species, the small brown planthopper Laodelphax striatellus. LsSHD has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal expression pattern of LsShd was determined through the fourth-instar and the early fifth-instar stages by qPCR. LsShd showed two expression peaks in day 2 and day 5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Dietary introduction of double-stranded RNA (dsRNA) of LsShd into nymphs successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, and caused nymphal lethality and delayed development. Ingestion of 20E did not increase LsShd expression level, but almost completely rescued LsEcR mRNA level, and relieved the negative effects on the survival and development in LsShd-dsRNA-exposed nymphs. In contrast, dietary introduction of E had little rescue effects. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects, and LsSHD functions to regulate metamorphotic processes by converting E to 20E even in a hemipteran insect, L. striatellus.     

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

Can Tropical Insects Stand the Heat? A Case Study with the Brown Planthopper Nilaparvata lugens (St?l)

The brown planthopper Nilaparvata lugens (Stål) is the most serious pest of rice across the world, especially in tropical climates. N. lugens nymphs and adults were exposed to high temperatures to determine their critical thermal maximum (CT_max), heat coma temperature (HCT) and upper lethal temperature (ULT). Thermal tolerance values differed between developmental stages: nymphs were consistently less heat tolerant than adults. The mean (± SE) CT_max of nymphs and adult females and males were 34.9±0.3, 37.0±0.2 and 37.4±0.2°C respectively, and for the HCT were 37.7±0.3, 43.5±0.4 and 42.0±0.4°C. The ULT₅₀ values (± SE) for nymphs and adults were 41.8±0.1 and 42.5±0.1°C respectively. The results indicate that nymphs of N. lugens are currently living at temperatures close to their upper thermal limits. Climate warming in tropical regions and occasional extreme high temperature events are likely to become important limiting factors affecting the survival and distribution of N. lugens.     

Characterization of putative soluble and membrane-bound trehalases in a hemipteran insect, Nilaparvata lugens

Trehalose is the main blood sugar of insects, and the enzyme trehalase is involved in energy metabolism and controlling trehalose levels in cells. Two forms (soluble and membrane-bound) of trehalase and the corresponding genes (NlTre-1 and NlTre-2) were identified from the brown planthopper, Nilaparvata lugens. Both NlTre-1 and NlTre-2 contain trehalase signature motifs, and NlTre-2 contains a putative transmembrane domain. Comparison of trehalase activity and gene mRNA level at different developmental stages, or following application of 20-hydroxyecdysone (20E), suggests that NlTre-1 and NlTre-2 encode a soluble trehalase and a membrane-bound trehalase respectively. Soluble trehalase activity accounted for the majority of total trehalase activity in N. lugens. Only soluble trehalase activity and NlTre-1 mRNA level could be induced by 20E. Additionally, only soluble trehalase activity was significantly higher in macropterous individuals than in brachypterous morphs. These results indicate that only soluble trehalase is differentially expressed between macropterous and brachypterous individuals and is more responsive to hormone stimulus.     

Behavioral response of Nilaparvata lugens (Stål), Cyrtorhinus lividipennis Reuter and Paederus fuscipes Curtis to three synthetic volatile chemical compounds

Plant essential oils (EOs) and a wide range of chemicals affect insect pests in many ways, such as via stimulatory, deterrent, toxic and hormonal effects. Three different compounds ((E)-β-caryophyllene (E-β-C), D-limonene (D-lime) and trans-2-dodecenol (T-2-D)) were tested against Nilaparvata lugens, Cyrtorhinus lividipennis and Paederus fuscipes, and their behavioral response was assessed. The results showed that on average, more N. lugens nymphs were repelled by E-β-C and T-2-D than by D-lime. More C. lividipennis nymphs were attracted to T-2-D and D-lime than to E-β-C. However, P. fuscipes displayed no significant response to the three chemical compounds. The results also demonstrated that T-2-D has exerted significant repellency against N. lugens and a significant attraction for C. lividipennis, while E-β-C and D-lime have no significant effect on any tested insect. T-2-D was selected and tested in a greenhouse under semi-field conditions, where the observations confirmed the results of the laboratory experiments. From the results, it can be concluded that T-2-D at a concentration of 0.06 g/L is an effective synthetic volatile chemical compound and is the strongest repellent of N. lugens and the strongest attractant for C. lividipennis. This synthetic chemical compound can be used as a pest management tool in rice agroecosystems.     

Molecular and Functional Analysis of UDP-N-Acetylglucosamine Pyrophosphorylases from the Migratory Locust,Locusta migratoria

UDP-N-acetylglucosamine pyrophosphorylases (UAP) function in the formation of extracellular matrix by producing N-acetylglucosamine (GlcNAc) residues needed for chitin biosynthesis and protein glycosylation. Herein, we report two UAP cDNA’s derived from two different genes (LmUAP1 and LmUAP2) in the migratory locust Locusta migratoria. Both the cDNA and their deduced amino acid sequences showed about 70% identities between the two genes. Phylogenetic analysis suggests that LmUAP1 and LmUAP2 derive from a relatively recent gene duplication event. Both LmUAP1 and LmUAP2 were widely expressed in all the major tissues besides chitin-containing tissues. However, the two genes exhibited different developmental expression patterns. High expression of LmUAP1 was detected during early embryogenesis, then decreased greatly, and slowly increased before egg hatch. During nymphal development, the highest expression of LmUAP1 appeared just after molting but declined in each inter-molting period and then increased before molting to the next stage, whereas LmUAP2 was more consistently expressed throughout all these stages. When the early second- and fifth-instar nymphs (1-day-old) were injected with LmUAP1 double-stranded RNA (dsRNA), 100% mortality was observed 2 days after the injection. When the middle second- and fifth-instar nymphs (3- to 4-day-old) were injected with LmUAP1 dsRNA, 100% mortality was observed during their next molting process. In contrast, when the insects at the same stages were injected with LmUAP2 dsRNA, these insects were able to develop normally and molt to the next stage successfully. It is presumed that the lethality caused by RNAi of LmUAP1 is due to reduced chitin biosynthesis of the integument and midgut, whereas LmUAP2 is not essential for locust development at least in nymph stage. This study is expected to help better understand different functions of UAP1 and UAP2 in the locust and other insect species.