Lin-Quan Ge

Molecular basis for insecticide-enhanced thermotolerance in the brown planthopper Nilaparvata lugens Stål (Hemiptera:Delphacidae).

Climate change is likely to have marked ecological effects on terrestrial ecosystems, including the activities of insect pests. Most attention has focused on the increasing geographical ranges of pests; however, if extrinsic factors enhance their thermotolerance, populations may express increased voltinism and longer daily and annual activity periods. These changes in pest populations have the potential for severe consequences, including increased crop losses and decreased food security at the global level. The brown planthopper (BPH) Nilaparvata lugens Stål (Hemiptera: Delphacidae) is a serious pest of rice crops in temperate and tropical regions of Asia. It is often present in rice microclimates at temperatures close to its maximum thermotolerance. Recent BPH outbreaks in tropical Asia are considered to be associated with excess use of pesticides and increasing temperature. This study tested whether exposure to sublethal concentrations of triazophos (tzp), an insecticide widely used in Asian rice production, enhances thermotolerance of BPH. Tzp exposure (40 ppm at 40 °C) significantly decreased mortality (from 94% in controls to 50% at 48 h post‐treatment) and increased lethal mean time (LT50) of adults by 17.2 h. To investigate the underlying molecular mechanism of this tzp‐enhanced thermotolerance, we selected Hsp70 and Arginine kinase (Argk) for detailed study. Transcripts encoding both proteins in third‐instar nymphs and brachypterous adult females were up‐regulated, compared with controls, after exposure to tzp. RNAi silencing of both genes demonstrated that Hsp70 and Argk are essential for survival and tzp‐increased thermotolerance. We propose that tzp induces thermotolerance in BPHs by increasing the expression of genes that act in cell protection mechanisms. The significance of our proposal relates to the importance of understanding the influence of sublethal concentrations of insecticides on pest biology. In addition to its influence on thermotolerance, tzp also enhances BPH reproduction. We infer that exposure to a pesticide stressor can produce cross‐tolerance, that is, increased tolerance to one stressor also increases tolerance to other stressors, including temperature. Aside from needing a better understanding of these effects in nature and in other pest/cropping systems, we suggest that pest management programmes can be improved with better understanding of the influences of stressors, including increased environmental temperatures and sublethal concentrations of insecticides, on pest biology.

Proteomic analysis of insecticide triazophos-induced mating-responsive proteins of Nilaparvata lugens Stål (Hemiptera: Delphacidae).

The brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a classic example of a resurgent pest induced by insecticides. It has been demonstrated that triazophos treatment causes an increase in the content of male accessory gland proteins (Acps) that can be transferred to females via mating, influencing female reproduction. However, the mechanism of this type of insecticide-induced Acps in males and the subsequent stimulation of reproduction in females are not well understood. To identify changes in the types of Acps and reproductive proteins in mated females, we conducted a comparative proteomic analysis. Six samples were categorized into four different groups: (1) untreated unmated males compared to treated unmated males (UUM vs TUM); (2) treated unmated males compared to treated mated males (TUM vs TMM); (3) untreated unmated females compared to treated unmated females (UUF vs TUF); (4) treated unmated females compared to treated mated females (TUF vs TMF). Protein expression changes among the four different groups were examined by two-dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Of the 500-600 reproducibly detected protein spots on each gel, 107 protein spots were differentially expressed between the four different groups. Of the 63 proteins identified by LC-MS/MS analysis, 38 were up-regulated and 25 were down-regulated in the four different groups. Some novel proteins related to fecundity were observed including spermatogenesis-associated protein 5, testis development protein NYD-SP6, arginine kinase, actin-5C, vitellogenin, and ovarian serine protease nudel. The elevated expression of novel fecundity proteins in six samples of N. lugens females and males due to exposure to triazophos was confirmed by quantitative real-time PCR (qRT-PCR). The results suggest that these proteins may participate in the reproductive process of N. lugens adult females and males. Our findings fill a gap in understanding the relationship between insecticide-treated males and the stimulated reproduction of N. lugens females.

Insecticide-Induced Changes in Protein, RNA, and DNA Contents in Ovary and Fat Body of Female Nilaparvata lugens (Hemiptera: Delphacidae)

ABSTRACT The widespread use of insecticides is known to cause resurgence in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) populations, a serious insect pest of rice, Orzya sativa L., crops in Asia. The current study investigated insecticide-induced changes in protein, RNA, and DNA levels in the ovary and fat body of adult female N. lugens. Potted rice plants were sprayed with different concentrations of imidacloprid, triazophos, and detamethrin. The results showed that all three insecticides caused a significant increase in protein and RNA contents in the ovary and fat body of adult females developed from nymphs fed on treated plants compared with those from on untreated plants, although there was no significant effect on DNA content. In general, protein and RNA contents in both ovary and fat body increased linearly with insecticide concentrations or days after emergence (DAEs) when they were applied. There was a significant correlation between protein content and RNA content in both ovary and fat body. In addition, the relationship between ratios of RNA to protein in ovary and fat body of adult female N. lugens and insecticide concentrations showed a significant positive correlation for most DAEs of imidacloprid and several DAEs of triazophos and deltamethrin treatments. These findings suggest that RNA in both ovary and fat body was more sensitive to insecticides and that the insecticide-induced changes in RNA content in turn influence protein synthesis in ovary and fat body. Consistently, RNA-to-protein ratios in ovary and fat body were ≈0.42 in all treatments and controls.

Effects of two pesticides, TZP and JGM, on reproduction of three planthopper species, Nilaparvata lugens Stål, Sogatella furcifera Horvath, and Laodelphax striatella Fallén

Three planthopper species, the brown planthopper (BPH), Nilaparvata lugens Stål, the white-backed planthopper (WBPH), Sogatella furcifera Horvath, and the small brown planthopper (SBPH), Laodelphax striatella Fallén, often co-occur in rice grown regions of China. The present investigation examined effects of two pesticides, triazophos (TZP) and jinggangmycin (JGM) (a fungicide), on reproduction of BPH, WBPH, and SBPH. The results showed that TZP stimulated the fecundity of the three planthopper species. Interestingly, JGM stimulated the fecundity of BPH but suppressed the fecundity of WBPH. In addition, TZP and JGM had a significant effect on the preoviposition period (PVD), the oviposition period (OPD), and the longevity of adult females (LAF) of BPH and WBPH. Based on these findings, to avoid resurgence occurrence of planthoppers, we suggest that the application of TZP should be banned in rice fields, that JGM should be used to control rice sheath blight at the early growth stages of rice (with WBPH occurrence and without BPH occurrence).

Jinggangmycin increases fecundity of the brown planthopper, Nilaparvata lugens (Stål) via fatty acid synthase gene expression

The antibiotic jinggangmycin (JGM) is mainly used in controlling the rice sheath blight, Rhizoctonia solani, in China. JGM also enhances reproduction of the brown planthopper (BPH), Nilaparvata lugens (Stål). To date, however, molecular mechanisms of the enhancement are unclear. Our related report documented the influence of foliar JGM sprays on ovarian protein content. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) protocols to analyze ovarian proteins of BPH females following JGM spray (JGM-S) and topical application (JGM-T). We recorded changes in expression of 284 proteins (142↑ and 142↓) in JGM-S compared to the JGM-S control group (S-control); 267 proteins were differentially expressed (130↑ and 137↓) in JGM-T compared to the JGM-T control group (T-control), of which, 22 proteins were up-regulated in both groups. Comparing the JGM-S to the JGM-T group, 114 proteins were differentially expressed (62↑ and 52↓). Based on the biological significance of fatty acids, pathway annotation and enrichment analysis, we designed a dsRNA construct to silence a gene encoding fatty acid synthase (FAS). FAS was more highly expressed in JGM-S vs S-control and JGM-S vs JGM-T groups. The dsFAS treatment reduced fecundity by about 46% and reduced ovarian and fat body fatty acid concentrations in JGM-S-treated females relative to controls. We infer FAS provides critically needed fatty acids to support JGM-enhanced fecundity in BPH.

RNAi knockdown of acetyl-CoA carboxylase gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparvata lugens.

A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reproduction of insects within the planthopper guild, including the brown planthopper (BPH) Nilaparvata lugens and the white-backed planthopper (WBPH) Sogatella furcifera, both serious resurgence rice pests. JGM exposure significantly increased BPH fecundity and population growth, but suppressed both parameters in laboratory and field WBPH populations. We used digital gene expression and transcriptomic analyses to identify a panel of differentially expressed genes, including a set of up-regulated genes in JGM-treated BPH, which were down-regulated in JGM-treated WBPH. RNAi silencing of Acetyl Co-A carboxylase (ACC), highly expressed in JGM-treated BPH, reduced ACC expression (by > 60%) and eliminated JGM-induced fecundity increases in BPH. These findings support our hypothesis that differences in ACC expression separates intraguild species at the molecular level.

Silencing a sugar transporter gene reduces growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

The brown planthopper (BPH), Nilaparvata lugens, sugar transporter gene 6 (Nlst6) is a facilitative glucose/fructose transporter (often called a passive carrier) expressed in midgut that mediates sugar transport from the midgut lumen to hemolymph. The influence of down regulating expression of sugar transporter genes on insect growth, development, and fecundity is unknown. Nonetheless, it is reasonable to suspect that transporter-mediated uptake of dietary sugar is essential to the biology of phloem-feeding insects. Based on this reasoning, we posed the hypothesis that silencing, or reducing expression, of a BPH sugar transporter gene would be deleterious to the insects. To test our hypothesis, we examined the effects of Nlst6 knockdown on BPH biology. Reducing expression of Nlst6 led to profound effects on BPHs. It significantly prolonged the pre-oviposition period, shortened the oviposition period, decreased the number of eggs deposited and reduced body weight, compared to controls. Nlst6 knockdown also significantly decreased fat body and ovarian (particularly vitellogenin) protein content as well as vitellogenin gene expression. Experimental BPHs accumulated less fat body glucose compared to controls. We infer that Nlst6 acts in BPH growth and fecundity, and has potential as a novel target gene for control of phloem-feeding pest insects.

Adipose triglyceride lipase (Atgl) mediates the antibiotic jinggangmycin-stimulated reproduction in the brown planthopper, Nilaparvata lugens Stål

The antibiotic jinggangmycin (JGM) is an agrochemical product widely used in China for controlling rice sheath blight, Rhizoctonia solani. Unexpectedly, it stimulates reproduction in the brown planthopper (BPH), Nilaparvata lugens (Stål). However, the underlying molecular mechanisms of the stimulation are unclear. The present investigation demonstrates that adipose triglyceride lipase (Atgl) is one of the enzymes involved in the JGM-stimulated reproduction in BPH. Silence of Atgl in JGM-treated (JGM + dsAtgl) females eliminated JGM-stimulated fecundity of BPH females. In addition, Atgl knockdown significantly reduced the protein and glycerin contents in the ovaries and fat bodies of JGM + dsAtgl females required for reproduction. We conclude that Atgl is one of the key enzymes responsible for JGM-stimulated reproduction in BPH.

Suppressing male spermatogenesis-associated protein 5-like gene expression reduces vitellogenin gene expression and fecundity in Nilaparvata lugens Stål

In our previous study with the brown planthopper (BPH), Nilaparvata lugens, triazophos (tzp) treatments led to substantial up-regulation of a male spermatogenesis-associated protein 5-like gene (NlSPATA5) compared to untreated controls. Mating with tzp-treated males significantly increased fecundity (as numbers of eggs laid), relative to females mated with untreated males. Because SPATA5 acts in mammalian sperm development and is expressed in testes, we posed the hypothesis that NlSPATA5 occurs in BPH seminal fluid and it operates in fecundity via mating. We tested the hypothesis by investigating the influence of suppressing NlSPATA5 expression in BPH males on fecundity. Reduced expression of NlSPATA5 led to decreased male accessory gland protein content and reproductive system development compared to controls. These changes in males led to prolonged pre-oviposition periods and decreased fecundity in females. For both genders, we recorded no difference in the body weight, oviposition periods, and longevity compared to controls. NlSPATA5 suppression in males also led to decreased fat body and ovarian protein content, yeast-like symbionts abundance and ovarian development as well as vitellogenin gene expression in their mating partners. We infer that increased NlSPATA5 expression may be one molecular mechanism of tzp-driven reproduction and population increases in BPH.

Driving Pest Insect Populations: Agricultural Chemicals Lead to an Adaptive Syndrome in Nilaparvata Lugens Stål (Hemiptera: Delphacidae)

The brown planthopper (BPH) is a devastating pest of rice throughout Asia. In this paper we document the BPH biogeographic range expansion in China over the 20-year period, 1992 to 2012. We posed the hypothesis that the range expansion is due to a syndrome of adaptations to the continuous presence of agricultural chemicals (insecticides and a fungicide) over the last 40 years. With respect to biogeography, BPH ranges have expanded by 13% from 1992 to 1997 and by another 3% from 1997 to 2012. In our view, such expansions may follow primarily from the enhancing effects of JGM, among other agricultural chemicals, and from global warming. JGM treatments led to increased thermotolerance, recorded as decreased mortality under heat stress at 40 ± 1 °C (down from 80% to 55%) and increased fecundity (by 49%) at 34 °C. At the molecular level, JGM treatments led to increased abundances of mRNA encoding Acetyl Co-A carboxylase (Acc) (up 25%) and Hsp70 (up 32%) in experimental BPH. RNAi silencing of Hsp70 and Acc eliminated the JGM effects on fecundity and silencing Hsp70 reduced JGM-induced thermotolerance. Integrated with global climate change scenarios, such syndromes in pest insect species have potential for regional- and global-scale agricultural disasters.