The loss of Halloween gene function seriously affects the development and reproduction of Diaphorina citri (Hemiptera: Liviidae) and increases its susceptibility to pesticides.

The citrus industry has suffered severe losses as a result of Huanglongbing spread by Diaphorina citri. Controlling the population of D. citri is the key to preventing and controlling the spread of Huanglongbing. Ecdysteroids are key hormones that regulate insect development and reproduction. Therefore, the Halloween gene family involved in the ecdysone synthesis of D. citri is an ideal target for controlling the population growth of this insect. In this study, we successfully cloned four Halloween genes expressed during D. citri development. Silencing of one of the four genes resulted in a significant decrease in 20E titers in nymphs and significant decreases in the developmental, survival and emergence rates. Inhibiting Halloween gene expression in adults impeded the growth of the female ovary, diminished yolk formation, lowered vitellogenin transcription levels, and hence impaired female fecundity. This showed that Halloween genes were required for D. citri development and reproduction. DcCYP315A1 and DcCYP314A1 were highly expressed when D. citri was exposed to thiamethoxam and cypermethrin, and silencing these two genes made D. citri more sensitive to these two pesticides. Inhibition of DcCYP315A1 and DcCYP314A1 expression not only significantly delayed the development and reproduction of D. citri but also increased its susceptibility to pesticides. Therefore, these two genes are more suitable as potential target genes for controlling D. citri.

SPARC plays an important role in the oviposition and nymphal development in Nilaparvata lugens Stål.

BACKGROUND: The brown planthopper (Nilaparvata lugens Stål)is a notorious rice pest in many areas of Asia. Study on the molecular mechanisms underlying its development and reproduction will provide scientific basis for effective control. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is one of structural component of the extracellular matrix, which influences a diverse array of biological functions. In this study, the gene for SPARC was identified and functionally analysed from N.lugens. RESULTS: The result showed that the NlSPARC mRNA was highly expressed in fat body, hemolymph and early embryo. The mortality increased significantly when NlSPARC was downregulated after RNA interference (RNAi) in 3 ~ 4th instar nymphs. Downregulation of NlSPARC in adults significantly reduced the number of eggs and offspring, as well as the transcription level of NlSPARC in newly hatched nymphs and survival rate in progeny. The observation with microanatomy on individuals after NlSPARC RNAi showed smaller and less abundant fat body than that in control. No obvious morphological abnormalities in the nymphal development and no differences in development of internal reproductive organ were observed when compared with control. CONCLUSION: NlSPARC is required for oviposition and nymphal development mainly through regulating the tissue of fat body in N.lugens. NlSPARC could be a new candidate target for controlling the rapid propagation of N.lugens population. Our results also demonstrated that the effect of NlSPARC RNAi can transfer to the next generation in N.lugens.

V-ATPase subunit a is required for survival and midgut development of Locusta migratoria.

The vacuolar-type H+ -ATPase (V-ATPase) is an ATP-dependent proton pump, which regulates various cellular processes. To date, most functional studies on V-ATPases of insects have focused on subunits of the V1 complex, and there is little information on the VO genes. In this study, two cDNA sequences of LmV-ATPase a were identified in Locusta migratoria. RT-qPCR analysis revealed that LmV-ATPase a1 and LmV-ATPase a2 are differentially expressed in various tissues and developmental stages. Injection of dsRNA for the common region of LmV-ATPase a1 and LmV-ATPase a2 into third-instar nymphs resulted in a significant suppression of LmV-ATPase a. The injected nymphs ceased feeding, lost body weight and finally died at a mortality of 98.6%. Furthermore, aberrations of midgut epithelial cells, the accumulation of electron-lucent vesicles in the cytoplasm, and a partially damaged brush border were observed in dsLmV-ATPase a-injected nymphs using transmission electron microscopy. Especially, the mRNA level of wingles, and notch genes were dramatically down-regulated in the dsLmV-ATPase a-injected nymphs. Taken together, our results suggest that LmV-ATPase a is required for survival and midgut development of L. migratoria. Hence, this gene could be a good target for RNAi-based control against locusts.

The RNA helicase DDX3 is required for ovarian development and oocyte maturation in Locusta migratoria.

DDX3 represents a well-defined subfamily of DEAD-box RNA helicase and exerts multiple functions in RNA metabolism, cell cycle, tumorigenesis, signal pathway, and fertility. Our previous study has shown that LmDDX3, the ortholog of DDX3 in Locusta migratoria, is ubiquitously expressed, and with a high abundance in testis and ovary. Knockdown of LmDDX3 results in a lethal phenotype in nymph, but it still remains unclear for its role in reproductive process. In this study, we therefore characterized LmDDX3 expression in female adult locust and analyzed its function in oocyte development. LmDDX3 was expressed in all tissues examined with significant more transcripts in ovary and hindgut. In ovary, a strong expression level was detected at the day just after adult eclosion, and a dramatic reduction then occurred during the oocyte development. LmDDX3 RNAi led to a reduced vitellogenin (Vg) expression in fat body via partially at least, the JH signaling pathway, and caused an upregulation of vitellogenin receptor (VgR) in ovary, and thus blocked the ovarian development and oocyte maturation. Sequence and phylogenetic analysis indicated that LmDDX3 was closely related to termite DDX3. Taken together, these data reveal a critical role for LmDDX3 in regulating the transcription of Vg and VgR, two major factors in vitellogenesis that is a key process required for ovary development and oocyte maturation in locust, and contribute thereof a new putative target for locust biological control.

Assessing Cotton Genotypes for Resistance to Aphis gossypii (Hemiptera: Aphididae).

Aphis gossypii Glover (Hemiptera: Aphididae) is a polyphagous species frequently associated with the presence of sooty mold and viruses lethal to plants. The purpose of this work was to characterize possible resistance categories of cotton genotypes against A. gossypii. Initially, a preliminary test was carried out with 78 genotypes, 15 of which were selected for infestation ability assays and the determination of the cumulative aphid-day rates. Posteriorly, these genotypes were also evaluated through antixenosis and antibiosis assays. The genotypes FM 910, FM 966 LL, Mocó, Gossypium hirsutum var. punctatum L. (Malvaceae), Variedade Reba = BTK-12, Deltapine, Hi-Bred, Acala 4-42, IAC PV010-1664, IAC 21, Reba B-50 PR and FMT 709 inhibited the aphid colonization. In the infestation ability assay, G. hirsutum punctatum, IAC PV010-1664 and Acala 4-42 were the least infested. In a multiple-choice assay, Deltapine Smooth Leaf and Variedade Reba = BTK-12 were significantly less infested, suggesting antixenosis. In the antibiosis assay, Gossypium arboreum L. (Malvaceae) 1 showed the lowest number of nymphs, number of nymphs per adult per day and, number of nymphs at 10 d after the birth of the first nymph in addition to reducing the reproductive period, nymphal survival, adult longevity and, developmental time. In the FM 910, the number of nymphs produced per day and, at 10 d after the birth of the first nymph decreased, which also indicated resistance. The results obtained here are unprecedented and can be explored in breeding programs to develop insect-resistant cotton cultivars.

Under the skin: Ixodes ticks in the subcutaneous tissue of red foxes (Vulpes vulpes) from Germany.

BACKGROUND: Ixodes spp. are vectors of zoonotic pathogens. All three active life stages (larvae, nymphs, adults) need to feed on a host in order to develop. Usually ticks parasitize attached to the external surface of their hosts' skin. Interestingly, in some cases ticks can also be found in the subcutaneous tissue in a variety of hosts, such as red foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and dogs. METHODS: The visceral side of 126 red fox-furs from Germany was examined visually searching for ticks. The localization of ticks was recorded and assigned to ten specific body parts. Morphological identification of ticks was performed according to standardized taxonomic protocols. Ticks which could not be further identified were examined genetically via conventional PCR targeting the 16S rRNA and cox1 gene. Hematoxylin and eosin (H&E) staining was used for histopathological examination. RESULTS: In 111 out of 126 (88.1%) examined coats, at least one tick was found in the subcutaneous tissue. A total of 1203 ticks were removed from the subcutaneous tissue. Well-preserved ticks could be identified based on morphological criteria, but most ticks were in a progressed state of decomposition. Here, morphological species identification was not successful. Also, PCR methods did not lead to a successful species identification. The following species and development stages were found by morphological identification: Ixodes ricinus (female, n = 289; male, n = 8; nymph, n = 1), I. hexagonus (female, n = 2), I. canisuga (female, n = 1). Male I. ricinus were found individually or copulating in pairs with females. Subcutaneous ticks were localized at three predominant affected body parts: ears, axillar and inguinal region. Histological examination of subcutaneous ticks revealed a granulomatous panniculitis. CONCLUSIONS: To the authors' knowledge, this is the first finding of highly prevalent subcutaneous ticks in red foxes from Germany. Subcutaneous location of ticks seems to be very common in red foxes and the rule rather than the exception. Deep embedment of longirostra and long feeding times of females seem to put the subcutaneous location in favor. Most foxes were infested in the inguinal area, where the skin is thin and less hairy.

Molecular characterization of insulin-like peptides in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

Insulin-like peptides (ILPs) including insulin, insulin-like growth factor (IGF) and relaxin are evolutionarily conserved hormones in metazoans, and they are involved in diverse physiological processes. The migratory brown planthopper (BPH), Nilaparvata lugens, encodes four ILP genes (Nlilp1, Nlilp2, Nlilp3 and Nlilp4) but their physiological roles are largely unknown. Sequence analysis showed that NlILP1 contained a relaxin-specific G protein-coupled receptor-binding motif and a variant motif of cysteine residues, and NlILP2 and NlILP4 resembled vertebrate IGFs. RNA interference (RNAi)-mediated gene silencing showed that depletion of each of Nlilp1, 2 and 3 significantly delayed the developmental duration of nymphs, and this effect could be exacerbated by double or triple gene depletion. Depletion of Nlilp1, Nlilp2 or Nlilp3 induces the accumulation of glucose, trehalose and glycogen, which is contradictory to depletion of the insulin receptor (NlInR1) in the BPH. Depletion of Nlilp1 significantly enhanced starvation resistance in both females and males although its extent was smaller than NlInR1 depletion. A parental RNAi assay showed that depletion of each of Nlilp1-4 dramatically impaired female fecundity. These findings indicate that NlILP1-4 have redundant and distinct roles in physiological processes in the BPH, thereby enhancing our understanding of the contribution of each NlILP to the ecological success of this species in natural habitats.

Using de novo transcriptome assembly and analysis to study RNAi in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae).

Phenacoccus solenopsis is one of the major polyphagous crop pests in India. Inadequate genomic or transcriptomic resources have limited the molecular studies in this insect despite its huge economic importance. The existing molecular sequence resources of this insect were supplemented through RNA sequencing, de novo transcriptome assembly and analysis, which generated 12, 925 CDS from 23,643 contigs with an average size of 1077.5 bp per CDS and 85.1% positive BLAST hits with NCBI Non redundant (nr) database. Twenty three genes involved in RNAi machinery identified through BLASTx search against NCBI nr database suggested the existence of robust RNAi in mealybug. RNAi in P. solenopsis was demonstrated through knockdown of IAP (Inhibitor of Apoptosis), AQP (Aquaporin), CAL (Calcitonin), VATPase (V-type proton ATPase subunit F 1), bursicon, chitin synthase, SNF7 and α-amylase by injecting sequence specific dsRNA of respective genes in adult female. Additionally, feeding RNAi has been demonstrated in 2nd instar nymph through dsRNA uptake in plant. The knockdown of core RNAi machinery genes such as Dicer, Argonaute and Staufen significantly hampered RNAi efficiency in this insect. However, downregulation of dsRNases improved RNAi efficiency. Sequential studies for understanding RNAi in P. solenopsis using transcriptome sequences have also been reported. The present study provides a base for future research on developing RNAi as strategy for management of this pest.

Insecticidal effect of aconitine on the rice brown planthoppers.

The brown planthopper, Nilaparvata lugens (Stål), severely damages rice production and develops high level resistance to several classes of insecticides. To find potential insecticidal resources is always important. As an environmentally friendly compound, aconitine exhibits potential pesticide features. In the present study, the pesticide and knockdown effects of aconitine were first tested on the brown planthopper. The results showed that the knockdown rates for an aconitine concentration of 200 ppm was 83.6%. The insecticidal LD50 was 22.68 ng/pest (95% CI, 17.75-28.99). The molecular mechanisms responding to aconitine application were analyzed through transcriptional sequencing. Compared to that of the knockdown nymphs of the brown planthoppers, the enzymes CYP3A4, UDP-glucuronosyltransferase (UGT), GST, carboxylesterase (EC3.1.1.1), and GABAergic synapse were up-regulated. We inferred that aconitine might be neurotoxic to the brown planthoppers, and the conscious nymphs resist the drug neurotoxicity through the upregulation of CYP3A4, UGT, and GABA receptor mutation. Although aconitine is not safe for mammals, it may be a leading compound to develop novel insecticides.

The MTase15 regulates reproduction in the wing-dimorphic planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

S-Adenosyl-l-methionine-dependent methyltransferases (SAMMTases) modulate important cellular and metabolic activities in both prokaryotes and eukaryotes. Here, we functionally characterized an SAMMTase gene (MTase15) in the migratory brown planthopper (BPH), Nilaparvata lugens, which is the most notorious rice pest in Asia. The cDNA sequence of MTase15 is 2764 nt in length with an open reading frame of 1218 nt encoding 405 amino acid residues. Quantitative real-time PCR analysis showed that MTase15 was readily detected from egg to adult stages and extensively distributed in various body parts of adult females and males, with slightly high levels in ovary and testis, respectively. In addition, MTase15 was transcriptionally regulated by the insulin signalling pathway in BPH. RNA-interference-mediated knockdown of MTase15 (dsMtase15) resulted in deficiencies in vitellogenin synthesis and oogenesis, and female infertility. Males with Mtase15 knockdown retained the capability of producing sperms with normal viability, but less sperm was transferred to wild-type (wt) females during copulation, and eggs laid by these wt females arrested embryogenesis. These findings not only assign a functional role to MTase15, but also provide a link between the insulin signalling pathway and epigenetic regulation in BPH reproduction.

Silencing cathepsin L expression reduces Myzus persicae protein content and the nutritional value as prey for Coccinella septempunctata.

Gut-expressed aphid genes, which may be more easily inhibited by RNA interference (RNAi) constructs, are attractive targets for pest control efforts involving transgenic plants. Here we show that expression of cathepsin L, which encodes a cysteine protease that functions in aphid guts, can be reduced by expression of an RNAi construct in transgenic tobacco. The effectiveness of this approach is demonstrated by up to 80% adult mortality, reduced fecundity, and delayed nymph production of Myzus persicae (green peach aphids) when cathepsin L expression was reduced by plant-mediated RNAi. Consistent with the function of cathepsin L as a gut protease, M. persicae fed on the RNAi plants had a lower protein content in their bodies and excreted more protein and/or free amino acids in their honeydew. Larvae of Coccinella septempunctata (seven-spotted ladybugs) grew more slowly on aphids having reduced cathepsin L expression, suggesting that prey insect nutritive value, and not just direct negative effects of the RNAi construct, needs to be considered when producing transgenic plants for RNAi-mediated pest control.

The functional difference of eight chitinase genes between male and female of the cotton mealybug, Phenacoccus solenopsis.

The cotton mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is a polyphagous insect that attacks tens of plant and causes substantial economic loss. Insect chitinases are required to remove the old cuticle to allow for continued growth and development. Though insect chitinases have been well studied in tens of insects, their functions in mealybug are still not addressed. Here, we sequenced the transcriptomes of adult males and females, from which eight chitinase genes were identified. We then used the method of rapid amplification of cDNA ends to amplify their full length. Phylogenetic analysis indicated that these genes clustered into five subgroups. Among which, group II PsCht2 had the longest transcript and was highly expressed at second instar nymph. PsCht10, PsCht3-3 and PsIDGF were highly expressed in the adult females, whereas PsCht4 and PsCht4-1 were significantly expressed at the male pupa and adult male. Next, we knocked down all eight chitinase genes by feeding the double-stranded RNA. Knockdown of PsCht4 or PsCht4-1 led to the failure of moult and, silencing PsCht5 resulted in pupation defect, while silencing PsCht10 led to small body size, suggesting these genes have essential roles in development and can be used as a potential target for pest control.

Evaluation of potential RNA-interference-target genes to control cotton mealybug, Phenacoccus solenopsis (Hemiptera: Pseudococcuidae).

RNA interference (RNAi) of vital insect genes is a potential tool for targeted pest control. However, selection of the right target genes is a challenge because the RNAi efficacy is known to vary among insect species. Cotton mealybug, Phenacoccus solenopsis, is a phloem-feeding economically important crop pest. We evaluated the RNAi of 2 vital genes, Bursicon (PsBur) and V-ATPase (PsV-ATPase) as potential targets in P. solenopsis for its control. PCR fragments of PsBur and PsV-ATPase were amplified using cDNA synthesized from the total RNA. The PCR amplicons were cloned into Potato virus X (PVX) to develop recombinant PVX for the inoculation of Nicotiana tabacum plants for bioassays with healthy P. solenopsis. Reverse-transcription-polymerase chain reaction (RT-PCR) was used to validate the expression of transgenes in the recombinant-PVX-inoculated plants (treated), and suppression of the target genes in the mealybugs exposed to them. The RT-PCR confirmed the expression of transgenes in the treated plants. Mealybug individuals on treated plants either died or showed physical deformities. Further, the population of mealybug was significantly reduced by feeding on N. tabacum expressing RNAi triggers against PsBur and PsV-ATPase. The results conclude that RNAi is activated in P. solenopsis by feeding on N. tabacum expressing RNAi triggering elements of PsBur and PsV-ATPase genes through recombinant PVX vector. Further, V-ATPase and Bursicon genes are potential targets for RNAi-mediated control of P. solenopsis.

Environmental and hormonal control of body color polyphenism in late-instar desert locust nymphs: Role of the yellow protein.

Locusts show body color polyphenism that is considered to be an adaptation to various biotic and abiotic environmental changes. In Schistocerca gregaria, wild-type late-instar nymphs growing under crowded conditions (gregarious form) develop yellow and black body coloration, whereas they assume various body colors under isolated conditions (solitarious form). Black and green body colorations are induced by the neuropeptide corazonin (Crz) and juvenile hormone (JH), respectively. To characterize the molecular mechanisms controlling body color polyphenism, we investigated factors influencing body coloration in S. gregaria. We report here that yellow body coloration in the last nymphal instar is caused by the yellow protein of the takeout family (YPT) in this locust. YPT transcription was enhanced under high-temperature conditions during which the nymphs turned bright yellow and had little black patterning. RNAi-mediated YPT knockdown suppressed the appearance of yellow individuals and yellow staining in the exuviae. In albino nymphs, injection of JH induced yellow and green coloration and enhanced the YPT expression levels in both yellow and green individuals. YPT knockdown also suppressed yellow staining in the exuviae but did not prevent the appearance of yellow individuals. Therefore, another factor or pigment may contribute to the observed yellow body color. Injection of Crz into wild-type nymphs caused darkening and suppressed yellowing and YPT expression at high temperatures. Thus, Crz signaling could inhibit yellowing by suppressing YPT expression. Rearing cup substrate color significantly influenced YPT expression in albino nymphs both under isolated and crowded conditions. In contrast, substrate color affected YPT expression in wild-type nymphs only under isolated conditions. From these results, we conclude that YPT is an important factor in the control of body color polyphenism in S. gregaria, and its expression is influenced by temperature, JH, Crz, and substrate color of the growing environment.

The specific host plant DNA detection suggests a potential migration of Apolygus lucorum from cotton to mungbean fields.

The polyphagous mirid bug Apolygus lucorum (Heteroptera: Miridae) has more than 200 species of host plants and is an insect pest of important agricultural crops, including cotton (Gossypium hirsutum) and mungbean (Vigna radiata). Previous field trials have shown that A. lucorum adults prefer mungbean to cotton plants, indicating the considerable potential of mungbean as a trap crop in cotton fields. However, direct evidence supporting the migration of A. lucorum adults from cotton to mungbean is lacking. We developed a DNA-based polymerase chain reaction (PCR) approach to reveal the movement of A. lucorum between neighboring mungbean and cotton fields. Two pairs of PCR primers specific to cotton or mungbean were designed to target the trnL-trnF region of chloroplast DNA. Significant differences in the detectability half-life (DS50) were observed between these two host plants, and the mean for cotton (8.26 h) was approximately two times longer than that of mungbean (4.38 h), requiring weighted mean calculations to compare the detectability of plant DNA in the guts of field-collected bugs. In field trials, cotton DNA was detected in the guts of the adult A. lucorum individuals collected in mungbean plots, and the cotton DNA detection rate decreased successively from 5 to 15 m away from the mungbean-cotton midline. In addition to the specific detection of cotton- and mungbean-fed bugs, both cotton and mungbean DNA were simultaneously detected within the guts of single individuals caught from mungbean fields. This study successfully established a tool for molecular gut-content analyses and clearly demonstrated the movement of A. lucorum adults from cotton to neighboring mungbean fields, providing new insights into understanding the feeding characteristics and landscape-level ecology of A. lucorum under natural conditions.

Lightweight males of Podisus nigrispinus (Heteroptera: Pentatomidae) neglect lightweight females due low reproductive fitness / Machos leves de Podisus nigrispinus (Heteroptera: Pentatomidae) negligenciam fêmeas leves devido ao baixo desempenho reprodutivo

Abstract Sexual choice by male stink bugs is important because females that experience food shortages lay fewer eggs with lower viability compared with well-fed females. In this study, we investigated whether Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) males fed with a low-quality diet during its nymphal stage show selectivity for sexual partners resulting in high-quality progeny. Lightweight males and females were obtained from nymphs fed weekly with Tenebrio molitor L. (Coleoptera: Tenebrionidae) pupae. By contrast, heavyweight males and females were fed three times a week and received an extra nutritional source: cotton leaves, Gossypium hirsutum L. (Malvaceae). Lightweight males preferred to mate with heavy females (77.78 ± 14.69%), whereas heavyweight males did not discriminated between light or heavyweight females. Females mated with lightweight males showed similar levels of reproduction to those mated with heavyweight males. The results provide an indication of the importance of male and female body weight for sexual selection in Asopinae stink bugs.

Resumo A seleção sexual por machos de percevejos é importante porque fêmeas que passaram por escassez alimentar poem poucos ovos com baixa viabilidade em comparação com fêmeas bem alimentadas. Nesse estudo, investigamos se machos de Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) alimentados com dieta de baixa qualidade durante seu estágio ninfal apresenta seletividade por parceiras sexuais resultando em progênie de alta qualidade. Machos e fêmeas leves foram obtidos de ninfas alimentadas semanalmente com pupas de Tenebrio molitor L. (Coleoptera: Tenebrionidae). Em contraste, machos e fêmeas pesados foram alimentados três vezes por semana e receberam uma fonte nutricional extra: folhas de algodão, Gossypium hirsutum L. (Malvaceae). Machos leves preferiram acasalar com fêmeas pesadas (77,78 ± 14,69%), enquanto machos pesados não distinguiram fêmeas leves ou pesadas para acasalamento. Fêmeas que acasalaram com machos leves apresentaram níveis de reprodução semelhantes em comparação com aquelas acasaladas com machos pesados. Os resultados fornecem uma indicação da importância do peso corpóreo de machos e fêmeas para a seleção sexual em percevejos Asopinae.

DIETARY SILVER NANOPARTICLES REDUCE FITNESS IN A BENEFICIAL, BUT NOT PEST, INSECT SPECIES.

Silver nanoparticles (AgNPs) have antimicrobial and insecticidal properties and they have been considered for their potential use as insecticides. While they do, indeed, kill some insects, two broader issues have not been considered in a critical way. First, reports of insect-lethal AgNPs are often based on simplistic methods that yield nanoparticles of nonuniform shapes and sizes, leaving questions about the precise treatments test insects experienced. Second, we do not know how AgNPs influence beneficial insects. This work addresses these issues. We assessed the influence of AgNPs on life history parameters of two agricultural pest insect species, Heliothis virescens (tobacco budworm) and Trichoplusia ni (cabbage looper) and a beneficial predatory insect species, Podisus maculiventris (spined soldier bug), all of which act in agroecosystems. Rearing the two pest species on standard media amended with AgNPs led to negligible influence on developmental times, pupal weights, and adult emergence, however, they led to retarded development, reductions in adult weight and fecundity, and increased mortality in the predator. These negative effects on the beneficial species, if also true for other beneficial insect species, would have substantial negative implications for continued development of AgNPs for insect pest management programs.

MOLECULAR IDENTIFICATION OF CYSTEINE AND TRYPSIN PROTEASE, EFFECT OF DIFFERENT HOSTS ON PROTEASE EXPRESSION, AND RNAI MEDIATED SILENCING OF CYSTEINE PROTEASE GENE IN THE SUNN PEST.

Sunn pest, Eurygaster integriceps, is a serious pest of cereals in the wide area of the globe from Near and Middle East to East and South Europe and North Africa. This study described for the first time, identification of E. integriceps trypsin serine protease and cathepsin-L cysteine, transcripts involved in digestion, which might serve as targets for pest control management. A total of 478 and 500 base pair long putative trypsin and cysteine gene sequences were characterized and named Tryp and Cys, respectively. In addition, the tissue-specific relative gene expression levels of these genes as well as gluten hydrolase (Gl) were determined under different host kernels feeding conditions. Result showed that mRNA expression of Cys, Tryp, and Gl was significantly affected after feeding on various host plant species. Transcript levels of these genes were most abundant in the wheat-fed E. integriceps larvae compared to other hosts. The Cys transcript was detected exclusively in the gut, whereas the Gl and Tryp transcripts were detectable in both salivary glands and gut. Also possibility of Sunn pest gene silencing was studied by topical application of cysteine double-stranded RNA (dsRNA). The results indicated that topically applied dsRNA on fifth nymphal stage can penetrate the cuticle of the insect and induce RNA interference. The Cys gene mRNA transcript in the gut was reduced to 83.8% 2 days posttreatment. Also, it was found that dsRNA of Cys gene affected fifth nymphal stage development suggesting the involvement of this protease in the insect growth, development, and molting.

Molecular Characterization of Six Small Heat Shock Proteins and Their Responses Under Cadmium Stress in Oxya chinensis (Orthoptera: Acridoidea).

Small heat shock proteins (sHSPs) have been implicated in many physiological processes and play important roles in the response to various stresses. In this study, the full-length sequences of six sHSPs: OcHSP19.1, 19.8, 20.4, 20.7, 21.1, and 23.8 were obtained from the rice grasshopper Oxya chinensis transcriptome database. The deduced amino acid sequences of the six OcsHSPs contain a typical α-crystallin domain, which consists of approximately 100 amino acid residues and five ß-strands. The phylogenetic analysis suggested that OcHSP23.8 was orthologous to the sHSPs of other species and that OcHSP19.1, 20.4, 20.7, and 21.1 were species specific, whereas OcHSP19.8 did not cluster closely to Orthoptera but was placed on the basal end of the cluster. Developmental stage-dependent and tissue-specific expression patterns were evaluated using quantitative real-time polymerase chain reaction. The six genes were expressed in all developmental stages and showed clear tissue specificity. The cadmium acute experiment indicates that Cd(2+) can induce the six genes. However, various response patterns were observed among these genes under Cd(2+) stress conditions. OcHSP19.1, 19.8, 20.4, and 20.7 were highly induced by 2.61 mM Cd(2+) at 24 h. OcHSP23.8 was significantly upregulated by 2.61 mM Cd(2+) at 6 h. For OcHSP21.1, the highest expression levels were found after treatment with 0.87 mM Cd(2+) for 24 h, 1.74 mM Cd(2+) for 36 h, and 2.61 mM Cd(2+) for 12 h. These differential characteristics will facilitate future investigations into the physiological functions of sHSPs.

Molecular characterisation of the vitellogenin gene (AlVg) and its expression after Apolygus lucorum had fed on different hosts.

BACKGROUND: The polyphagous pest Apolygus lucorum is now the dominant pest of Bacillus thuringiensis (Bt) cotton in China. In this study, the transcriptional and translational profiles of AlVg influenced by different hosts were identified, and then the correlations between AlVg gene or AlVg protein expression and key population proliferation parameters of A. lucorum were further clarified. RESULTS: AlVg or AlVg expression can be significantly regulated by different host nutrients (P < 0.05). AlVg or AlVg expression was significantly higher in A. lucorum reared on Bt and conventional cotton than in A. lucorum reared on garland chrysanthemum and broad bean (P < 0.05), but there was no significant difference between AlVg or AlVg expression in A. lucorum reared on Bt and conventional cotton (P > 0.05). In addition, there were significant linear regression correlations between AlVg or AlVg expression and total mortality rate of nymphs, female lifespan, per female fecundity and egg hatching rates (P < 0.05). CONCLUSION: Our results confirm that AlVg or AlVg is the key parameter affecting female fertility of A. lucorum. AlVg and AlVg expression can be influenced by different host nutrients except for Bt toxin. © 2015 Society of Chemical Industry.