Pharmacological characterization of a β‐adrenergic‐like octopamine receptor in Plutella xylostella

The β‐adrenergic‐like octopamine receptor (OA2B2) belongs to the class of G‐protein coupled receptors. It regulates important physiological functions in insects, thus is potentially a good target for insecticides. In this study, the putative open reading frame sequence of the Pxoa2b2 gene in Plutella xylostella was cloned. Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. PxOA2B2 was transiently expressed in HEK‐293 cells. It was found that PxOA2B2 could be activated by both octopamine and tyramine, resulting in increased intracellular cyclic AMP (cAMP) levels, whereas dopamine and serotonin were not effective in eliciting cAMP production. Further studies with series of PxOA2B2 agonists and antagonists showed that all four tested agonists (e.g., naphazoline, clonidine, 2‐phenylethylamine, and amitraz) could activate the PxOA2B2 receptor, and two of tested antagonists (e.g., phentolamine and mianserin) had significant antagonistic effects. However, antagonist of yohimbine had no effects. Quantitative real‐time polymerase chain reaction analysis showed that Pxoa2b2 gene was expressed in all developmental stages of P. xylostella and that the highest expression occurred in male adults. Further analysis with fourth‐instar P. xylostella larvae showed that the Pxoa2b2 gene was mainly expressed in Malpighian tubule, epidermal, and head tissues. This study provides both a pharmacological characterization and the gene expression patterns of the OA2B2 in P. xylostella, facilitating further research for insecticides using PxOA2B2 as a target.     

Identification and localization of two sensory neuron membrane proteins from Spodoptera litura (Lepidoptera: Noctuidae)

Sensory neuron membrane proteins (SNMPs), which are located on the dendritic membrane of olfactory sensory neurons (OSNs), are proposed to be associated with odor reception in insects. Recent studies have demonstrated that SNMP1 is essential for electrophysiological responses of OSNs to the sex pheromone, cis‐vaccenyl acetate (cVA) in Drosophila melanogaster. To investigate the function of Lepidoptera SNMPs, we cloned two SNMP genes, SlituSNMP1 and SltiuSNMP2, from Spodoptera litura (Lepidoptera: Noctuidae). Sequence alignment and phylogenetic analysis showed that both genes bear the general characteristics of SNMPs, including six conserved cysteine residues and two transmembrane domains. Further expression profile experiments showed that SlituSNMP1 is mainly expressed in the antenna, while SlituSNMP2 is broadly expressed in various tissues. By in situ hybridization experiments, it was found that SlituSNMP1 expressing cells are surrounded by the SlituSNMP2 expressing cells in the pheromone sensitive sensilla, suggesting different functions of the two SNMPs in insect olfaction.     

EXPRESSION OF SNMP1 AND SNMP2 GENES IN ANTENNAL SENSILLA OF Spodoptera exigua (HÜBNER)

Sensory neuron membrane proteins (SNMPs) are olfactory‐specific, two‐transmembrane proteins. Previous publications reported that SNMP1 is expressed on the dendrite membrane of pheromone‐sensitive neurons in Heliothis virescens and is an essential cofactor for pheromone detection in Drosophila. In this study, we cloned two SNMP genes (GenBank accession nos. JX469106 and JX469107) from the antenna of the beet armyworm moth Spodoptera exigua (Lepidoptera: Hübner). These SNMP genes are classified into two highly conserved subclades, indicating their importance in physiological activity of lepidopteran insects. SexiSNMP1 is antenna‐specific in male and female adults, while SexiSNMP2 is antenna‐abundant but also expressed in other chemosensory tissues, particularly proboscises and maxillary palps of adults both sexes. In situ hybridization revealed that both SNMPs are broadly expressed in long and short trichoid and basiconic sensilla. We infer that SNMP1 and SNMP2 act in the detection of the sex pheromone and general odorants.     

Molecular characterization and tissue localization of sensory neuron membrane protein from Chinese honey bee, Apis cerana cerana (Hymenoptera: Apidae)

Sensory neuron membrane protein (SNMP) is an olfactory receptor with photoaffinity analogs, capable of binding the pheromone membrane protein receptor deduced from receptor membrane protein with the pheromone–pheromone binding protein complex. However, this hypothesis has not yet been experimentally verified. In this experiment, the cDNA sequence encoding an open reading frame (ORF) of the SNMP gene AccSNMP1 (GenBank, KC012595) was cloned from Chinese honey bee, Apis cerana cerana Fabricius. Results from sequence analysis showed that this gene is 1,563 bp long, and that the ORF encodes 520 amino acids with a predicted molecular weight of 58.02 kDa, and has a theoretical isoelectric point of 5.83. Furthermore, there are two putative transmembrane domains. Multiple sequence alignment indicated that the AccSNMP1 gene from A. cerana cerana had different degrees of identity with the corresponding genes in nineteen other insects at the amino acid level. Phylogenetic analysis of the aligned sequences showed that A. cerana cerana is closely related to Apis mellifera Linnaeus and Bombus impatiens Cresson. Its distribution in tissues, as quantified using real-time RT-PCR, indicated that AccSNMP1 is highly expressed in the antennae and legs of A. cerana cerana, and there was a significant difference (p < 0.05) in gene expression between those tissues and tissues in the thorax, abdomen, snout, and head (not including antennae). Western blotting also confirmed the existence in the antennae of AccSNMP1 with an M _W of 58.0 kDa, which is the same as the expected value of 58.02 kDa. An immunohistochemistry study showed that AccSNMP1 is expressed in the trichoid sensilla of A. cerana cerana antenna. Therefore, the results of this study provide the basis for further studies of the function of SNMP from A. cerana cerana.     

Effect of Bt broccoli and resistant genotype of <Emphasis Type="Italic">Plutella xylostella</Emphasis> (Lepidoptera: Plutellidae) on development and host acceptance of the parasitoid <Emphasis Type="Italic">Diadegma insulare</Emphasis> (Hymenoptera: Ichneumonidae)

The ecological implications on biological control of insecticidal transgenic plants, which produce crystal (Cry) proteins from the soil bacterium Bacillus thuringiensis (Bt), remain a contentious issue and affect risk assessment decisions. In this study, we used a unique system of resistant insects, Bt plants and a parasitoid to critically evaluate this issue. The effects of broccoli type (normal or expressing Cry1Ac protein) and insect genotype (susceptible or Cry1Ac-resistant) of Plutella xylostella L. (Lepidoptera: Plutellidae) were examined for their effects on the development and host foraging behavior of the parasitoid, Diadegma insulare (Cresson) (Hymenoptera: Ichneumonidae) over two generations. Parasitism rate and development of D. insulare were not significantly different when different genotypes (Bt-resistant or susceptible) of insect host larvae fed on non-Bt broccoli plants. D. insulare could not discriminate between resistant and susceptible genotypes of P. xylostella, nor between Bt and normal broccoli plants with different genotypes of P. xylostella feeding on them. No D. insulare could emerge from Bt broccoli-fed susceptible and heterozygous P. xylostella larvae because these larvae were unable to survive on Bt broccoli. The parasitism rate, developmental period, pupal and adult weights of D. insulare that had developed on Bt broccoli-fed Cry1Ac-resistant P. xylostella larvae were not significantly different from those that developed on non-Bt broccoli-fed larvae. Female D. insulare emerged from Cry1Ac-resistant P. xylostella that fed on Bt plants could successfully parasitize P. xylostella larvae. The life parameters of the subsequent generation of D. insulare from P. xylostella reared on Bt broccoli were not significantly different from those from non-Bt broccoli. The Cry1Ac protein was detected in P. xylostella and in D. insulare when hosts fed on Bt broccoli. These results are the first to indicate that Cry1Ac did not harm the development or host acceptance of an important endoparasitoid after two generations of exposure. We suggest that using other Bt crops and resistant insect species would likely lead to similar conclusions about the safety of the presently used Bt proteins on parasitoids.     

Bacteria abundance and diversity of different life stages of Plutella xylostella (Lepidoptera: Plutellidae), revealed by bacteria culture‐dependent and PCR‐DGGE methods

Microbial abundance and diversity of different life stages (fourth instar larvae, pupae and adults) of the diamondback moth, Plutella xylostella L., collected from field and reared in laboratory, were investigated using bacteria culture‐dependent method and PCR‐DGGE analysis based on the sequence of bacteria 16S rRNA V3 region gene. A large quantity of bacteria was found in all life stages of P. xylostella. Field population had higher quantity of bacteria than laboratory population, and larval gut had higher quantity than pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Twenty‐five different bacterial strains were identified in total, among them 20 strains were presented in larval gut, only 8 strains in pupae and 14 strains in adults were detected. Firmicutes bacteria, Bacillus sp., were the most dominant species in every life stage. 15 distinct bands were obtained from DGGE electrophoresis gel. The sequences blasted in GenBank database showed these bacteria belonged to six different genera. Phylogenetic analysis showed the sequences of the bacteria belonged to the Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was the most abundant species in larval gut. In pupae, unculturable bacteria were the most dominant species, and unculturable bacteria and Serratia sp. were the most dominant species in adults. Our study suggested that a combination of molecular and traditional culturing methods can be effectively used to analyze and to determine the diversity of gut microflora. These known bacteria may play important roles in development of P. xylostella.     

Downregulation of ubiquitin gene expression in the deltamethrin‐resistant diamondback moth (Plutella xylostella L.)

Plutella xylostella (L.) has long been regarded as a very important universal pest of cruciferous crops. It is therefore important to understand its mechanisms of insecticide resistance. In our previous study, we obtained a differently expressed sequence with relatively high homology to the ubiquitin (UBI) gene as assessed via cDNA representational difference analysis (RDA). To further study this product, we cloned UBI open reading frame (ORF) sequences identified from both sexes of the deltamethrin‐resistant strain (DRF and DRM) and the deltamethrin‐sensitive strain (SF and SM) of P. xylostella. The deduced protein sequence of DRM was different from the other samples. The expression level was measured using real‐time quantitative polymerase chain reaction (PCR) and Western blot. The results of both analyses revealed the expression levels, in order from highest to lowest, to be in SF, SM, DRF and DRM. This indicates that the long‐term use of deltamethrin on P. xylostella results in decreased expression of the UBI gene.     

Remarkable susceptibility of the diamondback moth (Plutella xylostella) to ingestion of Pir toxins from Photorhabdus luminescens

Genes encoding Pir toxins were cloned and sequenced from Photorhabdus luminescens (Enterobacteriaceae) strain Hm. Cultures of Escherichia coli expressing the Pir A and B proteins were highly toxic when fed to larvae of Plutella xylostella L. (Lepidoptera: Plutellidae), as had been reported previously. Histological examination of P. xylostella larvae fed with recombinant E. coli revealed gross abnormalities of the midgut epithelium, with profound swelling and shedding of the apical membranes. However, the recombinant E. coli had no effect on the growth or mortality of larval Heliothis virescens F. (Lepidoptera: Noctuidae), Manduca sexta L. (Lepidoptera: Sphingidae), Lymantria dispar L. (Lepidoptera: Lymantriidae), or Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Based on these results, P. xylostella is at least 300‐fold more susceptible to Pir toxins than other insect species tested, suggesting that they may not be broadly useful as insecticidal proteins. Because Pir B has sequence similarities with N‐terminal portions of Cry proteins from Bacillus thuringiensis, we also tested the recombinant E. coli against a strain of P. xylostella that is resistant to the Cry 1A toxin, but found no difference in mortality between resistant and susceptible strains.     

Effects of mixed cropping on population densities and parasitism rates of the diamondback moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (Lepidoptera: Plutellidae)

Effects of mixed cropping and barrier crops on the population density and parasitism of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), were evaluated in field plots of cabbage grown in Bali, Indonesia. The densities of P. xylostella at larval and pupal stages, as well as the overall density at larval plus pupal stages, were significantly lower in cabbage/coriander mixed cropping subplots than in cabbage monoculture subplots. Parasitism of P. xylostella by the larval parasitoid Diadegma semiclausum (Hellen) (Hymenoptera: Ichneumonidae) was not significantly different between the mixed and monocultural cropping systems. These results do not support the so-called enemies hypothesis, but suggest that disruption of the host searching behavior of female moths by neighboring non-host plants is the mechanism behind the associational resistance observed in the coriander mixed cropping system. The inclusion of a Napier grass barrier between mixed crop and monoculture subplots did not affect the influence of mixed cropping on larval and pupal densities. Therefore, Napier grass, which is used locally as a fence for preventing livestock invasion of fields, would not obstruct the pest-reducing effect of coriander/cabbage mixed cropping.     

Production,purification, and characterization of the cecropin from <Emphasis Type="Italic">Plutella xylostella</Emphasis>, pxCECA1, using an intein-induced self-cleavable system in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD–intein–pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.     

瓜实蝇嗅觉受体基因的克隆及表达谱分析

昆虫的嗅觉受体是一个高度变异的蛋白家族, 其中一类Or83b嗅觉受体在不同昆虫体内高度保守, 在昆虫的行为调控过程中起到十分重要的作用。为进一步探讨Or83b受体的功能, 本研究利用RT-PCR和RACE方法克隆获得瓜实蝇Bactrocera cucurbitae (Coquillett) Or83b-like受体的全长cDNA序列, 命名为BcucOr83b-like（GenBank登录号： HM745934）。测序结果表明, BcucOr83b-like开放阅读框全长1 422 bp,编码473个氨基酸残基。氨基酸序列比对表明, 此序列具有Or83b受体的典型特征, 序列中具有7个跨膜区和高度保守的C端区域。BcucOr83b-like与其他昆虫的Or83b具有较高的氨基酸序列一致性, 其中与桔小实蝇Bactrocera dorsalis（Hendel）Or83b的序列一致性高达99.6%。对该基因在瓜实蝇成虫不同组织和发育时期表达量的荧光定量PCR分析表明, BcucOr83b-like主要在瓜实蝇成虫触角中表达, 头部（去除触角）、 雌虫前足和翅中也有较高的表达; 瓜实蝇在各个发育时期的表达水平不同, 在刚羽化雌成虫中的表达量最高。本研究为深入研究瓜实蝇Or83b受体的功能提供了理论依据。     

Silicon‐mediated resistance against specialist insects in sap‐sucking and leaf‐chewing guilds in the Si non‐accumulator collard

Soil amendment with Silicon (Si) can increase plant resistance against insect herbivores, but the underlying mechanisms remain unclear. The mechanical resistance hypothesis (MRH) states that Si accumulated in epidermal cells directly and passively protects against herbivores by creating a mechanical barrier. The physiological resistance hypothesis (PRH) states that Si enhances resistance by activating plant biochemical and physiological processes. We tested both hypotheses by manipulating Si fertilization of the Si non‐accumulator collard, Brassica oleracea L. cv. acephala (Brassicaceae). Then, we assessed functional and ultrastructural plant responses and the developmental and reproductive performance of the leaf‐chewing larvae of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), and the sap‐sucking cabbage aphid, Brevicoryne brassicae L. (Hemiptera: Aphididae). There was a 20% increase in leaf Si content. Silicon deposition in epidermal cells was identified by confocal microscopy and directly coincided with lower performance of P. xylostella, but did not affect B. brassicae. On the other hand, we found no unequivocal evidence that Si‐mediated changes in primary and secondary metabolism improved plant resistance against the insects. Negative mechanical effects of Si on the insects may have masked beneficial effects of increased water, nitrogen, and mineral contents in Si‐treated collards. Silicon did not change leaf contents of hemicellulose, cellulose, and lignin. Although Si‐mediated increases in leaf glucosinolates (GLS) correlated with lower larval performance and higher oviposition preference of P. xylostella, both P. xylostella and B. brassicae are highly specialized in overcoming such secondary metabolites. Thus, mechanical resistance may have impaired P. xylostella, rather than the Si‐mediated increase in GLS. We suggest that the PRH may depend on the degree of insect feeding specialization, so that toxic Si‐mediated defenses may be more efficient against unadapted polyphagous herbivores. For them, a toxic barrier may be added to the mechanical resistance.     

The effect of β-aminobutyric acid on the growth of herbivorous insects feeding on Brassicaceae

dl ‐β‐Aminobutyric acid (BABA) is a nonprotein amino acid that can enhance defences in a variety of plants against a wide range of pathogens. BABA can also reduce infestation by phytopathogenic nematodes and has recently been shown to suppress the growth of aphids feeding on legumes. This investigation examined the effect of applying BABA as a root drench to a range of Brassicaceae, including Arabidopsis thaliana, on the performance of two species of aphid (Myzus persicae and Brevicoryne brassicae) and the larvae of two species of Lepidoptera (Trichoplusia ni and Plutella xylostella). Application of BABA reduced the performance of all four insect species, and inhibition of insects occurred on all the plants tested. The results illustrate that BABA‐induced resistance (BABA‐IR) can affect generalist and specialist insect herbivores and inhibit insects feeding with mandibulate as well as sap‐feeding mouthparts. The BABA‐induced suppression of B. brassicae and P. xylostella feeding on A. thaliana provides a means to further examine the mechanisms of BABA‐IR to insects using this model plant.     

Expression and characterization of inhA gene from Bacillus thuringiensis 8010

InhA, a zinc metalloprotease secreted by Bacillus thuringiensis, specifically hydrolyzes antibacterial peptides produced by insect hosts. In this study, the inhA gene was cloned from B. thuringiensis 8010 using a pair of degenerate primers and the deduced 796 amino acid sequence showed a high degree of similarity with other InhA proteins in the Bacillus cereus group. The deduced amino acid sequence contained the zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family. Additionally, the inhA gene was expressed in Escherichia coli BL21 (DE3). The expressed InhA protein was shown to be toxic to the third larvae of Plutella xylostella, contrary to preliminary study concerning the effect of InhA on Bombyx mori. This study provided insights into the potential of InhA for the biological control of certain lepidopteran insects.     

Antennal SNMPs (sensory neuron membrane proteins) of Lepidoptera define a unique family of invertebrate CD36-like proteins

SNMP1-Apol is an antennal-specific protein of the wild silk moth Antheraea polyphemus; the protein is abundantly expressed and localized to the receptor membranes of sex-pheromone specific olfactory sensory neurons (OSNs). SNMP1-Apol is thought to function in odor detection based on its olfactory-specific expression, localization within OSNs, developmental time of expression, and apparent homology to the CD36 family of membrane-bound receptor proteins. In the current study, SNMP1-Apol homologues were identified from the moths Bombyx mori, Heliothis virescens, and Manduca sexta. These species posses antennal mRNAs encoding proteins with amino acid sequence identities ranging from 75-80%; these proteins are collectively designated SNMP1. A second M. sexta SNMP homologue, previously identified and partially sequenced [Robertson et al.: Insect Mol Biol 8:501-518, 1999] was fully sequenced and characterized. The encoded protein shares only 26-27% sequence identity with the SNMP1 proteins, and is thus designated SNMP2-Msex. The SNMP sequences were used to identify 14 and four possible homologues in Drosophila melanogaster and Caenorhabditis elegans genome databases, respectively; thus, greatly expanding CD36 family membership among the invertebrate lineages. Despite their sequence difference, SNMP1-Msex and SNMP2-Msex expression is localized to OSNs and occurs simultaneously with the onset of olfactory function. These findings suggest that SNMPs play a central role in odor detection in insects, and that the CD36 gene family is widely represented among animal phyla. The SNMPs are the only identified neuronal members of the CD36 family, and as such expand the activities of this gene family into roles influencing brain function and behavioral action.     

Resource concentration hypothesis: effect of host plant patch size on density of herbivorous insects

The resource concentration hypothesis (Root 1973) predicts that specialist herbivorous insects should be more abundant in large patches of host plants, because the insects are more likely to find and stay longer in those patches. Between August 1989 and January 1990 we experimentally tested Root's hypothesis by analyzing the numerical response of four species of herbivorous insects associated with patches of 4, 16, 64 and 225 cabbage plants, Brassica oleracea var. capitata. In addition, we studied the colonization of patches by adults of Plutella xylostella (L.) (Lepidoptera: Plutellidae), and the migration of their larvae in patches of different sizes. No herbivorous insect densities differed significantly with patch size. Adults of P. xylostella colonized all kind of patches equally. Larvae did not migrate between patches, and their disappearance rate did not differ between patches. The resource concentration hypothesis is organism-dependent, being a function of the adult and juvenile herbivore dispersal behavior in relation to the spatial scale of patchiness.