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Henosepilachna vigintioctopunctata is one of the most serious insect pests to a large number of nightshades and cucurbits. RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) offers a reduced risk approach to control the beetle. Identification of amenable target genes and determination of appropriate life stage for dsRNA treatment are two critical steps in order to improve RNAi efficiency. In the present paper, we identified three vATPase genes, namely HvvATPaseC, HvvATPaseE and HvvATPaseH. We found that the three transcripts were widely expressed in the eggs, first- to fourth-instar larvae, prepupae, pupae and adults. They were abundantly transcribed in the hindgut and Malpighian tubules, in contrast to the epidermis and fat body. Three days' ingestion of dsvATPaseC, dsvATPaseE and dsvATPaseH by the fourth-instar larvae significantly decreased corresponding transcript level by 90.1, 88.9 and 97.2%, greatly reduced larval fresh weight by 28.0, 29.9 and 28.0%, and caused 66.7, 100 and 78.7% larval lethality respectively. Comparably, 3 days' exposure of the third-instar larvae to dsvATPaseC significantly reduced HvvATPaseC mRNA level by 89.5%, decreased approximately 80% of the larval fresh weight, and killed 100% of the treated larvae. Therefore, the three vATPase genes, especially HvvATPaseE, are potential amenable target genes and young larvae are more susceptible to dsRNA. Our findings will enable the development of the dsRNA-based pesticide to control H. vigintioctopunctata. [ABSTRACT FROM AUTHOR]

Henosepilachna vigintioctopunctata is a serious insect pest which attacks a large number of nightshades and cucurbits in Asian countries, Brazil and Australia. Prolonged application of traditional pesticides has caused environmental pollution and exerted deleterious effects on human health. Finding new approaches with high target specificity and low environmental contamination has become an urgent task. RNA interference (RNAi) induced by double‐stranded RNA (dsRNA) is expected to be applicable to managing this pest. Here we evaluated the effects of Escherichia coli‐expressed dsRNAs targeting ecdysone receptor (EcR) gene via dietary delivery in laboratory and foliar spraying in a greenhouse. The target transcript was successfully knocked down when the 4th‐instar larvae had fed on potato foliage dipped with dsEcR in a laboratory bioassay. Around 85% of the HvEcR RNAi larvae remained as prepupae or became abnormal pupae, and failed to emerge into adults. Ingestion of dsEcR‐immersed foliage by the 3rd‐instar larvae effectuated a comparable RNAi response and brought about more severe defects: all the resultant larvae arrested development, remained as prepupae and finally died. For assay in the greenhouse, a dsEcR‐contained E. coli suspension was directly sprayed to the foliage of greenhouse‐growing potato plants and the 3rd‐ and 4th‐instar larvae were transferred to the leaves. High RNAi efficacy was obtained and identical RNAi phenotypes were observed in treated larvae. In addition, spraying dsEcR reduced leaf damage. Our results indicate a possibility of practical application of dsEcR as an environmentally friendly RNA pesticide to control H. vigintioctopunctata larvae. [ABSTRACT FROM AUTHOR]

Chemoresistance conferred by leukemia propagating cells (LPCs) in a therapy‐induced niche (TI‐niche) within the bone marrow is one of the main obstacles in leukemia treatment. Effective approaches to circumvent the TI‐niche protection and to eliminate the resident LPCs remain to be exploited. Here, developed is a niche‐targeted nanosystem using leukemic cell membrane‐coated mesoporous silica nanoparticles (DAazo@CMSN) for co‐delivering daunorubicin for leukemia cell chemotherapy and a TGFβRII neutralizing antibody (aTGFβRII) to block niche signaling. DAazo@CMSN effectively targets the TI‐niche. Through an azobenzene‐based hypoxia‐responsive linker, sequential delivery of the two active molecules overcomes niche‐mediated chemoresistance, attenuates systemic burden, and prolongs survival in a mouse model of leukemia. This work demonstrates a proof‐of‐principle for biomimetic and microenvironment‐activated multiplexed nanoparticulate drug delivery strategies for overcoming therapy‐induced chemoresistance in leukemia. [ABSTRACT FROM AUTHOR]

Chitin synthase (CHS) plays a critical role in chitin synthesis and excretion. In most insects, CHSs have been segregated into 1 and 2 classes. CHS1 is responsible for chitin production in the ectodermally-derived epidermal cells. CHS2 is dedicated to chitin biosynthesis in the midgut peritrophic matrix (PM). Henosepilachna vigintioctopunctata is a serious pest of Solanaceae and Cucurbitaceae plants. In this study, we identified HvCHS1 and HvCHS2. We found that HvCHS1 was abundantly transcribed in the larval tracheae and epidermis, whereas HvCHS2 was mainly expressed in the guts. Escherichia coli HT115 expressed double stranded RNAs targeting HvCHS1 and HvCHS2 (ds CHS1 and ds CHS2) were used to immerse potato foliage and the treated leaves were provided to the newly-molted fourth- and third-instar larvae. Ingestion of ds CHS1 by the fourth-instar larvae significantly diminished the target mRNA level and had slight influence on the expression of HvCHS2. In contrast, consumption of ds CHS2 significantly lowered the target mRNA level but triggered the transcription of HvCHS1. Knockdown of HvCHS1 , rather than HvCHS2 , arrested larval development and impaired larva-pupa-adult transition. A large proportion of HvCHS1 hypomorphs became stunting prepupae, deformed pupae or misshapen adults. Moreover, knockdown of HvCHS1 damaged gut integrity, decreased cuticle thickness, and delayed the formation of newly-generated cuticle layer during ecdysis. Furthermore, depletion of HvCHS1 inhibited the development of trachea system and thinned tracheal taenidia. Ingestion of ds CHS1 at the third-instar stage caused similar but severe negative effects. Our results demonstrated that Hv CHS1 is responsible for chitin biosynthesis during ecdysis. Moreover, HvCHS1 is a potential amenable target gene and young larvae are more susceptible to dsRNA. [Display omitted] • HvCHS1 and HvCHS2 are identified in H. vigintioctopunctata. • Knockdown of HvCHS1 impairs larva-pupa-adult transition. • Silencing HvCHS1 influences gut structure and cuticle formation. • Depletion of HvCHS1 inhibits trachea generation and thins tracheal taenidia. • Ingestion of dsCHS1 by young larvae causes severe negative effects. [ABSTRACT FROM AUTHOR]

An exploration of novel control strategies for Leptinotarsa decemlineata is becoming more pressing given rapid evolution of insecticide resistance and rise of production loss of potato. Dietary delivery of bacterially expressed double-stranded RNA (dsRNA) is a promising alternative for management. An important first step is to uncover possible RNA-interference (RNAi)-target genes effective against both young and old larvae. Taiman (Tai) is a basic-helix-loop-helix/Per-Arnt-Sim transcription factor that is involved in the mediation of both juvenile hormone (JH) and 20-hydroxyecdysone (20E) signaling. In the present paper, we found that continuous ingestion of ds Tai for three days by third (penultimate)-instar larvae caused approximately 20% larval mortality and 80% pupation failure. The larval lethality resulted from failed cuticle and tracheae shedding, which subsequently reduced foliage consumption and nutrient absorption, and depleted lipid stores. In contrast, pupation failure derived from disturbed JH and 20E signals, and disordered nutrient homeostasis including, among others, inhibition of trehalose metabolism and reduction of chitin content. Knockdown of LdTai caused similar larval lethality and pupation impairment in second and fourth (final) larval instars. Therefore, LdTai is among the most attractive candidate genes for RNAi to control L. decemlineata larvae. Unlabelled Image • DsRNA of LdTaiman (ds Tai) inhibits pupation in 3rd instar larvae. • Ds Tai leads to similar pupation impairment in 2nd and 4th larval instars. • Defects by ds Tai result from disturbed metabolism, and JH and 20E signals. • LdTaiman is a potential target for RNAi to control L. decemlineata larvae. [ABSTRACT FROM AUTHOR]

Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata . In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAP s, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2 , whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAP s responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval–larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that Ld UAP1 and Ld UAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata . [ABSTRACT FROM AUTHOR]

• Knockdown of LdHR38 inhibits ecdysteroidogenesis in third instar larvae. • Silencing LdHR38 intensify the expression of early ecdysone genes but represses LdFTZ-F1. • Depletion of LdHR38 impairs larval-pupal ecdysis. • Silencing LdHR38 at the fourth instar causes similar but less severe defects. In insects, nuclear receptors (NRs) including EcR (NR1H1), USP (NR2B4), E75 (NR1D3), HR3 (NR1F), HR4 (NR6) and FTZ-F1 (NR5A3) mediate the 20-hydroxyecdysone (20E) signaling cascade to play a critical role during larval metamorphosis. In this present paper, we focused on hormone receptor 38 (HR38) in Leptinotarsa decemlineata , the only insect homolog of the NR4A subclass. RNA interference (RNAi) of LdHR38 in the penultimate (third) instar larvae reduced the expression of an ecdysteroidogenesis gene and declined the titer of 20E. Knockdown of LdHR38 intensified the expression of LdUSP , LdE75 , LdE74 , LdE93 , LdBroad and LdHR3 , whereas repressed the transcription of LdFTZ-F1. Disruption of 20E signaling inhibited chitin biosynthesis in the larval cuticle. Approximately 25% of the LdHR38 RNAi larvae died, around 40% of the resultant larvae remained as prepupae or become deformed pupae. The body surface of the HR38 depleted abnormal prepupae and pupae looked wet, just like the cuticle being covered with a layer of liquid. Moreover, the increase of larval mortality, and the impairment of pupation and emergence exhibited dose-dependent manners. Furthermore, silencing LdHR38 at the final (fourth) instar caused similar but less severe impairment of pupation. Dietary supplement with 20E for the third instar larvae did not rescue the high larval death and only slightly alleviated the low pupation rate in the LdHR38 RNAi hypomorphs. Accordingly, we propose that HR38 is necessary for tune of ecdysteroidogenesis and for mediation of 20E signaling during metamorphosis in L. decemlineata. [ABSTRACT FROM AUTHOR]

MicroRNA 182 has been found to have a distinct contribution in the clonal expansion of activated- and functioning of specialized-helper T cells. In this study we knocked down microRNA 182 in vivo and induced experimental autoimmune encephalomyelitis (EAE) to determine the influences of microRNA 182 in the Treg cells functional specialization through Foxo1 dependent pathway in the peripheral lymphoid organs. Down-regulation of microRNA 182 significantly increased the proportions of Foxp3 + T cells in the peripheral lymph nodes and spleen. In vivo study verified a positive correlation between microRNA 182 levels and symptom severity of EAE, and a negative correlation between microRNA 182 and the transcriptional factor Foxp3. In vitro polarization study also confirmed the contribution of Foxo1 in microRNA 182 mediated down-regulation of Foxp3 + T cells. Together, our results provide evidence that during the development of EAE, microRNA 182 repressed Treg cells differentiation through the Foxo1 dependent pathway. [ABSTRACT FROM AUTHOR]

Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS ) or each of three degradation genes (soluble trehalases LdTRE1a , LdTRE1b or membrane-bound LdTRE2 ) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae. [ABSTRACT FROM AUTHOR]

Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier ( spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier ( Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor ( LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus. [ABSTRACT FROM AUTHOR]

Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate of L. decemlineata during flight and proline dehydrogenase (ProDH) catalyzes the first step in proline catabolism. Here we identified a putative LdProDH gene; it had three cDNA isoforms which shared the same 5′UTR and coding region, but differed in the lengths of 3′UTRs (515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively). LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDH was expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH-dsRNA by adults significantly decreased its messenger RNA (mRNA) level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals (AATAAA) were tandemly located in the 3′UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3′UTR to control proline catabolism for the season-dependent activity of L. decemlineata adults. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Spodoptera exigua is a highly polyphagous pest and causes extensive damage to many field and truck crops. Since the larvae are not or only sublethally affected by Cry1Ac, the fused Cry1Ac/Cry1Ab, or the stacked Cry1Ac+CpTI transgenic cottons that are widely planted in China, S. exigua has become a major economic pest of cotton across a wide distribution since the commercialization of Bt cottons in 1997 in China. Proteinase inhibitors are potential candidates for enhancing Bt toxicity against, and for expanding control spectrum for insect pests. In the present paper, we first found that S. exigua larval midgut fluids could remarkably degrade activated Cry1Ca, and slightly hydrolyze Cry1Ac and Cry1Ab. Subsequently, we investigated interactions between the 3 Cry toxins and 7 proteinase inhibitors, i.e., phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, tannic acid, N-α-tosyl-l-phenylalanine chloromethyl ketone, N-α-tosyl-l-lysine chloromethyl ketone, elastatinal and ethylenediaminetetraacetic acid (EDTA), by monitoring larval growth and mortality rate. A 6-day dietary exposure of the newly molted 2nd instars to either the inhibitors (3 test concentrations) or the toxins (Cry1Ac, 31.30ng/cm2; Cry1Ab, 3.2ng/cm2; Cry1Ca, 0.6ng/cm2) alone only slightly affected larval growth. In contrast, exposure to the mixtures containing an inhibitor and a toxin, with the exception of those containing EDTA, synergistically reduced larval weight. In general, the synergisms were more obvious at higher inhibitor concentrations. Regarding larval mortality, the inhibitors except EDTA at the highest test concentration showed significant synergism to both Cry1Ab (0.2μg/cm2) and Cry1Ca (0.04μg/cm2). As the inhibitor concentration reduced, however, the synergistic effects decreased. These results indicated that trypsin-, chymotrypsin-, and elastase-like proteinases in S. exigua larval midgut were involved in proteolytical hydrolyzation of the 3 activated Cry toxins, and protection of Bt Cry toxins from proteinase degradation in the midgut by inhibitors may greatly enhance toxicity against S. exigua larvae. [Copyright &y& Elsevier]

Abstract: Hydrogen sulfide (H2S) has been shown to effect physiological alterations in several animals, frequently leading to an improvement in survival in otherwise lethal conditions. In the present paper, a volatility bioassay system was developed to evaluate the survivorship of Drosophila melanogaster adults exposed to H2S gas that emanated from a K2S donor. Using this bioassay system, we found that H2S exposure significantly increased the survival of flies under arid and food-free conditions, but not under humid and food-free conditions. This suggests that H2S plays a role in desiccation tolerance but not in nutritional stress alleviation. To further confirm the suggestion, the mRNA levels of two desiccation tolerance-related genes Frost and Desat2, and a starvation-related gene Smp-30, from the control and treated flies were measured by quantitative real-time PCR. These genes were up-regulated within 2h when the flies transferred to the arid and food-free bioassay system. Addition of H2S further increased Frost and Desat2 mRNA levels, in contrast to Smp-30. Thus, our molecular results were consistent with our bioassay findings. Because of the molecular and genetic tools available for Drosophila, the fly will be a useful system for determining how H2S regulates various physiological alterations. [ABSTRACT FROM AUTHOR]

Water is a critical resource for all terrestrial insects. The yellow-spined bamboo locust Ceracris kiangsu Tsai (Oedipodidae: Orthoptera), matures, copulates and reproduces in the summer and adults are observed mud-puddling in human urine-contaminated materials on hot days. Besides obtaining NaCl and NHHCO, the locust may also compensate for water deficiency. In a dual-choice test in the laboratory, adults prefer to remain on wet rather than dry filter paper at high temperature (36 °C). The females are more likely to move towards and stay on wet filter paper than males. The adults chew wet filter paper in the laboratory at higher temperature and the females consume more than the males. Moreover, foam plastic containing an aqueous solution of an insecticide, bisultap, results in greater mortality in females than a dry formulation, both in the laboratory and the field. The water content of the frass from males is significantly lower than that from females and may partially explain the stronger hygropreference behaviour in females. Other possible reasons for sexually different hygropreference are also discussed. The removal of the adult antennae substantially reduces discrimination between wet and dry filter paper and mortality resulting from wet bisultap formulations in the laboratory. Hence, antennae mediate, at least partially, hygropreference behaviour. [ABSTRACT FROM AUTHOR]

Abstract: Successive electroacupuncture (EA) stimulation on Zusanli ST36 acupoints of rats with experimental autoimmune encephalitis (EAE), which is an inflammatory disease mediated by autoreactive T cells, relieved disease severity, inhibited specific T cell proliferation and rebuilt the CD4+ T cell subset balance. In addition, EA-treated rats had significantly higher ACTH concentrations in vivo compared to untreated EAE rats. These results indicated that EA stimulation could relieve the severity of EAE by restoring balance to the Th1/Th2/Th17/Treg Th cell subset responses by stimulating the hypothalamus to increase ACTH secretion. [Copyright &y& Elsevier]

Abstract: Interlukin-17 (IL-17) is active in a variety of brain injuries, including ischemia. The objective of this study was to test the hypothesis that IL-17 potentiates neuronal injury after stroke. Increased expression of IL-17 and IL-17 receptor (IL-17R) in serum and cortex was evaluated by ELISA, RT–PCR and immunohistochemistry. In the in vitro model of oxygen–glucose deprivation (OGD), IL-17 showed a dose-dependent effect in promoting neuronal injury through IL-17-IL-17R combination which can be blocked by IL-17R/Fc chimera. Our results demonstrated the up-regulation of IL-17 and IL-17R following permanent middle cerebral artery occlusion and suggested that they contributed to stroke outcome. [Copyright &y& Elsevier]