Showing papers in "Insect Science in 2016"

CRISPR/Cas9-mediated targeted gene mutagenesis in Spodoptera litura.

Abstract: Custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricultural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A (Slabd-A) gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of insects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA (sgRNA) into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura.

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth, Plutella xylostella.

Abstract: Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field-evolved indoxacarb resistance has been reported in several lepidopteran pests, including the diamondback moth Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong Province of China in 2012, was 750-fold more resistant to indoxacarb and 70-fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of complementary DNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the sixth segment of domain IV of the PxNav protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among 10 field-collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34- to 870-fold) and metaflumizone (1- to 70-fold). The two mutations were never found to co-exist in the same allele of PxNav , suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species.

Invasive bark beetle-associated microbes degrade a host defensive monoterpene

Abstract: Conifers respond to herbivore attack with defensive chemicals, which are toxic to both insects and their associated microorganisms. Microorganisms associated with insects have been widely reported to metabolize toxic chemicals, which may help both microorganisms and host insects overcome host conifer defense. Dendroctonus valens LeConte, an introduced exotic pest from North America to China, has killed millions of healthy pines. Alpha-pinene is the most abundant defensive monoterpene in Chinese Pinus tabuliformis. Although microorganisms associated with D. valens have already been investigated, little is known about their bioactivities when encountering host defensive monoterpenes. In this study, we evaluated the influences of different concentrations of α-pinene to D. valens and the three most frequently isolated yeasts and bacteria of D. valens, and further assayed microorganisms' capabilities to degrade α-pinene. Results showed that the gallery lengths and body weight changes of bark beetles were significantly affected by 6 mg/mL and 12 mg/mL of α-pinene applied in media compared to controls. The tolerance of experimental microorganisms to α-pinene varied depending on the microbial species. Two out of three yeast strains and all three bacterial strains degraded 20%-50% of α-pinene compared to controls in 24 h in vitro. The microorganisms capable of α-pinene degradation in vitro and their tolerance to high levels of α-pinene suggested that D. valens-associated microorganisms may help both microorganisms and the bark beetle overcome host α-pinene defense.

Satyrization and satyrization-resistance in competitive displacements of invasive mosquito species.

Abstract: Competitive displacements or reductions of resident populations of insects, often effected by a related species, may be caused by a variety of mechanisms. Satyrization is a form of mating interference in which males of one species mate with females of another species, significantly decreasing their fitness and not generating hybrids. Satyrization has been established to be the probable cause of competitive displacements of resident mosquitoes by invasive species, especially of Aedes aegypti by Aedes albopictus, two important vectors of dengue and chikungunya viruses. Mathematical models predict that even low levels of asymmetric mating interference are capable of producing competitive displacements or reductions. Couplings of virgin Ae. aegypti females with Ae. albopictus males effectively sterilize these females through the monogamizing actions of male accessory gland products, but the converse interspecific mating does not impact the future reproduction of Ae. albopictus females. Populations of Ae. aegypti exposed to satyrization quickly evolve resistance to interspecific mating, which is believed to ameliorate reproductive interference from, and promote co-existence with, Ae. albopictus. The evolution of satyrization resistance among Ae. aegypti in laboratory cages is accompanied by fitness costs, such as reduced fecundity and slower receptivity to conspecific males. Cage experiments and field observations indicate that Ae. albopictus males are capable of satyrizing females of other species of the Stegomyia subgenus, potentially leading to competitive displacements, and possible extinctions, especially of endemic species on islands. Examination of other examples of reproductive interference in insects reveals few parallels to the mechanism and outcomes of satyrization by Ae. albopictus. We conclude by posing the hypothesis that satyrization may favor the ecological success of Ae. albopictus, and suggest many lines for future research on this phenomenon.

Identification of heat shock cognate protein 70 gene (Alhsc70) of Apolygus lucorum and its expression in response to different temperature and pesticide stresses

Abstract: Heat shock cognate protein 70 (Hsc70) is a very important stress-resistance protein of insects against environmental stresses. We employed fluorescent real-time quantitative polymerase chain reaction and Western-blot techniques to analyze the transcriptional and translational expression profiles of AlHSC70 under extreme temperature (4°C and 40°C) or 4 pesticide stresses in Apolygus lucorum. The results showed that the expression of AlHSC70 were significantly induced by cyhalothrin or extremely high temperature (40°C) in both transcriptional and translational levels (P < 0.05), while the transcriptional and translational level of AlHSC70 decreased significantly in treatments of chlorpyrifos or extreme cold temperature (4°C) (P < 0.05). Moreover, after Apolygus lucorum treated by imidacloprid or emamectin benzoate, the expression of AlHSC70 was only up-regulated significantly at the transcriptional level (P < 0.05), although obviously up-regulated at the translational level of AlHSC70. Therefore, this study confirmed that the Alhsc70 gene played important roles in response to both temperature and pesticide stresses, especially for cyhalothrin or extremely high temperature (40°C). In addition, the significant polynomial regression correlations between temperature and the Alhsc70 expression level were shown in all the nymph and adult stages (P < 0.01), indicating temperature was an important factor to affect the relative expression of Alhsc70.

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis (Lepidoptera: Crambidae)

Abstract: Two transgenic rice lines (T2A-1 and T1C-19b) expressing cry2A and cry1C genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis (Walker) (Lepidoptera: Crambidae). The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a cry1Ab/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A-1 > Huahui 1 > T1C-19b among rice lines, and leaf > stem > root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar: higher level at early stages than at later stages with an exception that high Cry1C level in T1C-19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the jointing stage with an exception for T1C-19b providing an excellent performance at the maturing stage. By comparison, T1C-19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A-1, being close to Huahui 1. The results suggest cry1C is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C-19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.

Expression profiles of glutathione S-transferase superfamily in Spodoptera litura tolerated to sublethal doses of chlorpyrifos.

Abstract: Chlorpyrifos (CPF) is a broad-spectrum organophosphate insecticide. Glutathione S-transferases (GSTs) in insects are a family of detoxification enzymes and they play critical roles in CPF detoxification. Spodoptera litura is one of the most destructive agricultural pests in tropical and subtropical areas in the world. In this study, 37 Slgsts from 46 unique transcripts of gsts in S. litura transcriptome data, including eight previously reported GSTs, were identified and their expression patterns in susceptible and 12-generation-CPF-treated strains were analyzed to understand the roles of these Slgsts in sublethal doses of CPF tolerance. The results indicate that the members of the S. litura GST superfamily could be distinguished into three major groups: one group, including six cytosolic Slgsts (SlGSTe1, SlGSTe3, SlGSTe10, SlGSTe15, SlGSTo2 and SlGSTs5) and two microsomal Slgsts (SlMGST1-2 and SlMGST1-3), was directly responsible for CPF induction in both 12-generation-treated and susceptible strains; the second group, including three cytosolic Slgsts (SlGSTe13, SlGSTt1 and SlGSTz1) and one microsomal Slgst (SlMGST1-1), was induced only in the 12-generation-treated strain; the third group, including eight cytosolic Slgsts (two epsilon, three delta, one omega, one zeta and one unclassified Slgst), was expressed 1.52-5.15-fold higher in the 12-generation-treated strain than in the susceptible strain.

Insight into the possible mechanism of the summer diapause of Delia antiqua (Diptera: Anthomyiidae) through digital gene expression analysis.

Abstract: The onion fly, Delia antiqua, is a major underground agricultural pest that can enter pupal diapause in the summer and winter seasons. However, little is known about its molecular regulation due to the lack of genomic resources. To gain insight into the possible mechanism of summer diapause (SD), high-throughput RNA-Seq data were generated from non-diapause (ND) and SD (initial, maintenance and quiescence phase) pupae. Three pair-wise comparisons were performed and identified, 1380, 1471 and 435, and were significantly regulated transcripts. Further analysis revealed that the enrichment of several functional terms related to juvenile hormone regulation, cell cycle, carbon hydrate and lipid metabolism, innate immune and stress responses, various signalling transductions, ubiquitin-dependent proteosome, and variation in cuticular and cytoskeleton components were found between ND and SD and between different phases of SD. Global characterization of transcriptome profiling between SD and ND contributes to the in-depth elucidation of the molecular mechanism of SD. Our results also offer insights into the evolution of insect diapause and support the importance of using the onion fly as a model to compare the molecular regulation events of summer and winter diapauses.

Landscape distribution of food and nesting sites affect larval diet and nest size, but not abundance of Osmia bicornis.

Abstract: Habitat fragmentation is a major threat for beneficial organisms and the ecosystem services they provide. Multiple-habitat users such as wild bees depend on both nesting and foraging habitat. Thus, they may be affected by the fragmentation of at least two habitat types. We investigated the effects of landscape-scale amount of and patch isolation from both nesting habitat (woody plants) and foraging habitat (specific pollen sources) on the abundance and diet of Osmia bicornis L. Trap-nests of O. bicornis were studied in 30 agricultural landscapes of the Swiss Plateau. Nesting and foraging habitats were mapped in a radius of 500 m around the sites. Pollen composition of larval diet changed as isolation to the main pollen source, Ranunculus, increased, suggesting that O. bicornis adapted its foraging strategy in function of the nest proximity to main pollen sources. Abundance of O. bicornis was neither related to isolation or amount of nesting habitat nor to isolation or abundance of food plants. Surprisingly, nests of O. bicornis contained fewer larvae in sites at forest edge compared to isolated sites, possibly due to higher parasitism risk. This study indicates that O. bicornis can nest in a variety of situations by compensating scarcity of its main larval food by exploiting alternative food sources.

RFID monitoring indicates honeybees work harder before a rainy day

Abstract: Storms are usually accompanied by a drop in temperature, and an increase in wind and barometric pressure and rainfall, which have negative impacts on most activities, survival and reproduction in insects (Gillot, 2005). The majority of studies have mainly focused on how the flight activity of various flying insects such as honeybees, bumble bees, horse flies and leafminers were directly influenced by intraday weather changes (Burnett & Hays, 1974; Lundberg, 1980; Casas, 1989; Vicens & Bosch, 2000). However, accumulating evidences showed that animals can make behavioral changes before storms, which is enormously important for their survival in severe weather conditions. Before upcoming storms birds unusually chirp and bathe with sand; native frogs croak and hide their egg masses; spiders spin shorter and produce thicker webs and wasps hide their comb before rains (Galacgac & Balisacan, 2009; Acharya, 2011). In early 1893, honeybees were reported as more active before storms (Inwards, 1893). In this study, we compared the working habits of foragers on days that were followed by a sunny day and those that were followed by a rainy day using the Radio Frequency Identification (RFID), which was developed and manufactured by the Honeybee Research Institute of Jiangxi Agricultural University in collaboration with the Guangzhou Invengo Information Technology Co., Ltd., and we showed that honeybees worked harder before a rainy day. Three honeybee (Apis mellifera) colonies were maintained at the Honeybee Research Institute, Jiangxi Agricultural University, Nanchang, China. Each colony had four full frames, with approximately 6000 workers and a

Fitness components of Drosophila melanogaster developed on a standard laboratory diet or a typical natural food source

Abstract: Drosophila melanogaster is often used as a model organism in evolutionary biology and ecophysiology to study evolutionary processes and their physiological mechanisms. Diets used to feed Drosophila cultures differ between laboratories and are often nutritious and distinct from food sources in the natural habitat. Here we rear D. melanogaster on a standard diet used in our laboratory and a field diet composed of decomposing apples collected in the field. Flies developed on these two diet compositions are tested for heat, cold, desiccation, and starvation resistance as well as developmental time, dry body mass and fat percentage. The nutritional compositions of the standard and field diets were analyzed, and discussed in relation to the phenotypic observations. Results showed marked differences in phenotype of flies from the two types of diets. Flies reared on the field diet are more starvation resistant and they are smaller, leaner, and have lower heat resistance compared to flies reared on the standard diet. Sex specific effects of diet type are observed for several of the investigated traits and the strong sexual dimorphism usually observed in desiccation resistance in D. melanogaster disappeared when rearing the flies on the field diet. Based on our results we conclude that care should be taken in extrapolating results from one type of diet to another and especially from laboratory to field diets.

Identification of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism.

Abstract: The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography-tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and α-amylase) or other reactions (β-1,3-glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a heterogenous group consisting of polycalin, REPATs, serpin, C-Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase-2 (McCHS-2), chitinase (McCHI), and β-N-acetylglucosaminidase (McNAG) enzymes, involved in PM chitin metabolism, were also identified. McCHS-2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS-2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.

Further insight into reproductive incompatibility between putative cryptic species of the Bemisia tabaci whitefly complex.

Abstract: The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), with its global distribution and extensive genetic diversity, is now known to be a complex of over 35 cryptic species. However, a satisfactory resolution of the systematics of this species complex is yet to be achieved. Here, we designed experiments to examine reproductive compatibility among species with different levels of mitochondrial cytochrome oxidase I (mtCOI) divergence. The data show that putative species with mtCOI divergence of >8% between them consistently exhibited complete reproductive isolation. However, two of the putative species, Asia II 9 and Asia II 3, with mtCOI divergence of 4.47% between them, exhibited near complete reproductive compatibility in one direction of their cross, and partial reproductive compatibility in the other direction. Together with some recent reports on this topic from the literature, our data indicates that, while divergence in the mtCOI sequences provides a valid molecular marker for species delimitation in most clades, more genetic markers and more sophisticated molecular phylogeny will be required to achieve adequate delimitation of all species in this whitefly complex. While many attempts have been made to examine the reproductive compatibility among genetic groups of the B. tabaci complex, our study represents the first effort to conduct crossing experiments with putative species that were chosen with considerations of their genetic divergence. In light of the new data, we discuss the best strategy and protocols to conduct further molecular phylogenetic analysis and crossing trials, in order to reveal the overall pattern of reproductive incompatibility among species of this whitefly complex.

Temporal changes of symbiont density and host fitness after rifampicin treatment in a whitefly of the Bemisia tabaci species complex

Abstract: Microbial symbionts are essential or important partners to phloem-feeding insects. Antibiotics have been used to selectively eliminate symbionts from their host insects and establish host lines with or without certain symbionts for investigating functions of the symbionts. In this study, using the antibiotic rifampicin we attempted to selectively eliminate certain symbionts from a population of the Middle East-Asia Minor 1 whitefly of the Bemisia tabaci species complex, which harbors the primary symbiont "Candidatus Portiera aleyrodidarum" and two secondary symbionts "Candidatus Hamiltonella defensa" and Rickettsia. Neither the primary nor the secondary symbionts were completely depleted in the adults (F0) that fed for 48 h on a diet treated with rifampicin at concentrations of 1-100 μg/mL. However, both the primary and secondary symbionts were nearly completely depleted in the offspring (F1) of the rifampicin-treated adults. Although the F1 adults produced some eggs (F2), most of the eggs failed to hatch and none of them reached the second instar, and consequently the rifampicin-treated whitefly colony vanished at the F2 generation. Interestingly, quantitative polymerase chain reaction assays showed that in the rifampicin-treated whiteflies, the density of the primary symbiont was reduced at an obviously slower pace than the secondary symbionts. Mating experiments between rifampicin-treated and untreated adults demonstrated that the negative effects of rifampicin on host fitness were expressed when the females were treated by the antibiotic, and whether males were treated or not by the antibiotic had little contribution to the negative effects. These observations indicate that with this whitefly population it is not feasible to selectively eliminate the secondary symbionts using rifampicin without affecting the primary symbiont and establish host lines for experimental studies. However, the extinction of the whitefly colony at the second generation after rifampicin treatment indicates the potential of the antibiotic as a control agent of the whitefly pest.

Cuticular protein LmTwdl1 is involved in molt development of the migratory locust

Abstract: The cuticle, an essential structure for insects, is produced from cuticular proteins and chitin via a series of biochemical reactions. Tweedle genes are important members of the cuticular protein family and have four conserved motifs binding to chitin. Tweedle family genes have been found to play a profound effect on cuticle development. Here, we report that the cuticular protein gene LmTwdl1 of Locusta migratoria belongs to the Tweedle family. In situ hybridization showed that LmTwdl1 is localized to epidermal cells of the cuticle. The expression patterns of LmTwdl1 showed low expression in the cuticle during the early and middle stages of the fifth-instar nymphs; in contrast, its expression rapidly increased in the late stages of fifth-instar nymphs. We performed RNA interference to examine the function of LmTwdl1 in locusts. Silencing of LmTwdl1 resulted in high mortality during the molting process before the next stage. Also, the epicuticle of nymphs failed to molt, tended to be thinner and the arrangement of chitin in the procuticle appeared to be disordered compare to the control group. These results demonstrate that LmTwdl1 plays a critical role in molting, which contributes to a better understanding of the distinct functions of the Tweedle family in locusts.

Molecular characterization, tissue and developmental expression profiles of cryptochrome genes in wing dimorphic brown planthoppers, Nilaparvata lugens

Abstract: Cryptochromes (CRYs) are blue and UV light photoreceptors, known to play key roles in circadian rhythms and in the light-dependent magnetosensitivity of insects. Two novel cryptochrome genes were cloned from the brown planthopper, and were given the designations of Nlcry1 and Nlcry2, with the accession numbers KM108578 and KM108579 in GenBank. The complementary DNA sequences of Nlcry1 and Nlcry2 are 1935 bp and 2463 bp in length, and they contain an open reading frame of 1629 bp and 1872 bp, encoding amino acids of 542 and 623, with a predicted molecular weight of 62.53 kDa and 70.60 kDa, respectively. Well-conserved motifs such as DNA-photolyase and FAD-binding-7 domains were observed in Nlcry1 and Nlcry2. Phylogenetic analysis demonstrated the proteins of Nlcry1 and Nlcry2 to be clustered into the insect's cryptochrome 1 and cryptochrome 2, respectively. Quantitative polymerase chain reaction showed that the daily oscillations of messenger RNA (mRNA) expression in the head of the brown planthopper were mild for Nlcry1, and modest for Nlcry2. Throughout all developmental stages, Nlcry1 and Nlcry2 exhibited extreme fluctuations and distinctive expression profiles. Cryptochrome mRNA expression peaked immediately after adult emergence and then decreased subsequently. The tissue expression profiles of newly emerged brown planthopper adults showed higher expression levels of CRYs in the head than in the thorax or abdomen, as well as significantly higher levels of CRYs in the heads of the macropterous strain than in the heads of the brachypterous strain. Taken together, the results of our study suggest that the two cryptochrome genes characterized in the brown planthopper might be associated with developmental physiology and migration.

Identification and characterization of microRNAs in the white-backed planthopper, Sogatella furcifera.

Abstract: MicroRNAs (miRNAs) are a novel class of small, non-coding endogenous RNAs that play critical regulatory roles in many metabolic activities in eukaryotes. Reports of the identification of miRNAs in Sogatella furcifera (white-backed planthopper), the insect that acts as the only confirmed vector of the southern rice black-streaked dwarf virus (SRBSDV), are limited. In this study, a total of 382 miRNAs were identified in S. furcifera, including 106 conserved and 276 novel miRNAs, using high-throughput sequencing based on two small RNA libraries from viruliferous and non-viruliferous S. furcifera, and these miRNAs belonged to 52 conserved miRNA families and 58 S. furcifera-specific families, respectively. Comparison with miRNAs from 26 insect species and five other species in miRBase showed that more than half of the conserved miRNA families are highly conserved in Hexapoda, while other miRNAs are only conserved in non-dipterans. Furthermore, 4 117 target genes predicted for the 382 identified miRNAs could be categorized into 45 functional groups annotated by Gene Ontology. Compared with non-viruliferous cells, eight up-regulated miRNAs and four down-regulated miRNAs were identified in cells inoculated with SRBSDV, among which miR-14 and miR-n98a may be involved in the immune response to SRBSDV infection. Analyses of the identified miRNAs will provide insights into the roles of these miRNAs in the regulation and expression of genes involved in the metabolism, development and viral infection of S. furcifera.

The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana.

Abstract: The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes (TH and DDC), in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.

Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune‐related genes during dengue virus serotype‐2 infection

Abstract: Mosquito microRNAs (miRNAs) are involved in host-virus interaction, and have been reported to be altered by dengue virus (DENV) infection in Aedes albopictus (Diptera: Culicidae). However, little is known about the molecular mechanisms of Aedes albopictus midgut-the first organ to interact with DENV-involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA (mRNA) expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut.

Next generation sequencing of Apis mellifera syriaca identifies genes for Varroa resistance and beneficial bee keeping traits.

Abstract: Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next-generation sequencing (NGS) technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses characteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in-depth functional annotation methods to identify ∼600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK-STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function-specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development (Cactus, Relish, dorsal, Ank2, baz), Varroa hygiene (NorpA2, Zasp, LanA, gasp, impl3) and Varroa resistance (Pug, pcmt, elk, elf3-s10, Dscam2, Dhc64C, gro, futsch) functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry.

Sequencing and comparison of the Rickettsia genomes from the whitefly Bemisia tabaci Middle East Asia Minor I

Abstract: The whitefly, Bemisia tabaci, harbors the primary symbiont 'Candidatus Portiera aleyrodidarum' and a variety of secondary symbionts. Among these secondary symbionts, Rickettsia is the only one that can be detected both inside and outside the bacteriomes. Infection with Rickettsia has been reported to influence several aspects of the whitefly biology, such as fitness, sex ratio, virus transmission and resistance to pesticides. However, mechanisms underlying these differences remain unclear, largely due to the lack of genomic information of Rickettsia. In this study, we sequenced the genome of two Rickettsia strains isolated from the Middle East Asia Minor 1 (MEAM1) species of the B. tabaci complex in China and Israel. Both Rickettsia genomes were of high coding density and AT-rich, containing more than 1000 coding sequences, much larger than that of the coexisted primary symbiont, Portiera. Moreover, the two Rickettsia strains isolated from China and Israel shared most of the genes with 100% identity and only nine genes showed sequence differences. The phylogenetic analysis using orthologs shared in the genus, inferred the proximity of Rickettsia in MEAM1 and Rickettsia bellii. Functional analysis revealed that Rickettsia was unable to synthesize amino acids required for complementing the whitefly nutrition. Besides, a type IV secretion system and a number of virulence-related genes were detected in the Rickettsia genome. The presence of virulence-related genes might benefit the symbiotic life of the bacteria, and hint on potential effects of Rickettsia on whiteflies. The genome sequences of Rickettsia provided a basis for further understanding the function of Rickettsia in whiteflies.

The expression of proteins involved in digestion and detoxification are regulated in Helicoverpa armigera to cope up with chlorpyrifos insecticide.

Abstract: Helicoverpa armigera is a key pest in many vital crops, which is mainly controlled by chemical strategies. To manage this pest is becoming challenging due to its ability and evolution of resistance against insecticides. Further, its subsequent spread on nonhost plant is remarkable in recent times. Hence, decoding resistance mechanism against phytochemicals and synthetic insecticides is a major challenge. The present work describes that the digestion, defense and immunity related enzymes are associated with chlorpyrifos resistance in H. armigera. Proteomic analysis of H. armigera gut tissue upon feeding on chlorpyrifos containing diet (CH) and artificial diet (AD) using nano-liquid chromatography-mass spectrometry identified upregulated 23-proteins in CH fed larvae. Database searches combined with gene ontology analysis revealed that the identified gut proteins engrossed in digestion, proteins crucial for immunity, adaptive responses to stress, and detoxification. Biochemical and quantitative real-time polymerase chain reaction analysis of candidate proteins indicated that insects were struggling to get nutrients and energy in presence of CH, while at the same time endeavoring to metabolize chlorpyrifos. Moreover, we proposed a potential processing pathway of chlorpyrifos in H. armigera gut by examining the metabolites using gas chromatography-mass spectrometry. H. armigera exhibit a range of intriguing behavioral, morphological adaptations and resistance to insecticides by regulating expression of proteins involved in digestion and detoxification mechanisms to cope up with chlorpyrifos. In these contexts, as gut is a rich repository of biological information; profound analysis of gut tissues can give clues of detoxification and resistance mechanism in insects.

Intercrop movement of convergent lady beetle, Hippodamia convergens (Coleoptera: Coccinellidae), between adjacent cotton and alfalfa

Abstract: A 2-year study was conducted to characterize the intercrop movement of convergent lady beetle, Hippodamia convergens Guerin-Meneville (Coleoptera: Coccinellidae) between adjacent cotton and alfalfa. A dual protein-marking method was used to assess the intercrop movement of the lady beetles in each crop. In turns field collected lady beetles in each crop were assayed by protein specific ELISA to quantify the movement of beetles between the crops. Results indicated that a high percentage of convergent lady beetles caught in cotton (46% in 2008; 56% in 2009) and alfalfa (46% in 2008; 71% in 2009) contained a protein mark, thus indicating that convergent lady beetle movement was largely bidirectional between the adjacent crops. Although at a much lower proportion, lady beetles also showed unidirectional movement from cotton to alfalfa (5% in 2008 and 6% in 2009) and from alfalfa to cotton (9% in 2008 and 14% in 2009). The season-long bidirectional movement exhibited by the beetles was significantly higher in alfalfa than cotton during both years of the study. The total influx of lady beetles (bidirectional and unidirectional combined) was significantly higher in alfalfa compared with that in cotton for both years. While convergent lady beetles moved between adjacent cotton and alfalfa, they were more attracted to alfalfa when cotton was not flowering and/or when alfalfa offered more opportunities for prey. This study offers much needed information on intercrop movement of the convergent lady beetle that should facilitate integrated pest management decisions in cotton utilizing conservation biological control.

Enhancing the stability and ecological safety of mass‐reared transgenic strains for field release by redundant conditional lethality systems

Abstract: The genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically-based population control programs, most notably the Sterile Insect Technique (SIT), for both plant and animal insect pests. Tetracycline-suppressible (Tet-off) conditional lethal systems may function together so that transgenic strains will be viable and fertile on a tetracycline-containing diet, but female-lethal and male sterile in tetracycline-free conditions. This would allow their most efficacious use in a unified system for sterile male-only production for SIT. A critical consideration for the field release of such transgenic insect strains, however, is a determination of the frequency and genetic basis of lethality revertant survival. This will provide knowledge essential to evaluating the genetic stability of the lethality system, its environmental safety, and provide the basis for modifications ensuring optimal efficacy. For Tet-off lethal survival determinations, development of large-scale screening protocols should also allow the testing of these modifications, and test the ability of other conditional lethal systems to fully suppress propagation of rare Tet-off survivors. If a dominant temperature sensitive (DTS) pupal lethality system proves efficient for secondary lethality in Drosophila, it may provide the safeguard needed to support the release of sexing/sterility strains, and potentially, the release of unisex lethality strains as a form of genetic male sterility. Should the DTS Prosβ2(1) mutation prove effective for redundant lethality, its high level of structural and functional conservation should allow host-specific cognates to be created for a wide range of insect species.

The complete mitochondrial genome of Anopheles minimus (Diptera: Culicidae) and the phylogenetics of known Anopheles mitogenomes.

Abstract: Anopheles minimus is an important vector of human malaria in southern China and Southeast Asia. The phylogenetics of mosquitoes has not been well resolved, and the mitochondrial genome (mtgenome) has proven to be an important marker in the study of evolutionary biology. In this study, the complete mtgenome of An. minimus was sequenced for the first time. It is 15 395 bp long and encodes 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs) and a non-coding region. The gene organization is consistent with those of known Anopheles mtgenomes. The mtgenome performs a clear bias in nucleotide composition with a positive AT-skew and a negative GC-skew. All 13 PCGs prefer to use the codon UUA (Leu), ATN as initiation codon but cytochrome-oxidase subunit 1 (COI) and ND5, with TCG and GTG, and TAA as termination codon, but COI, COII, COIII and ND4, all with the incomplete T. tRNAs have the typical clover-leaf structure, but tRNA(Ser(AGN)) is consistent with known Anopheles mtgenomes. The control region includes a conserved T-stretch and a (TA)n stretch, and has the highest A+T content at 93.1%. The phylogenetics of An. minimus with 18 other Anopheles species was constructed by maximum likelihood and Bayesian inference, based on concatenated PCG sequences. The subgenera, Cellia and Anopheles, and Nyssorhynchus and Kerteszia have mutually close relationships, respectively. The Punctulatus group and Leucosphyrus group of Neomyzomyia Series, and the Albitarsis group of Albitarsis Series were suggested to be monophyletic. The monophyletic status of the subgenera, Cellia, Anopheles, Nyssorhynchus and Kerteszia need to be further elucidated.

Resistance status of the carmine spider mite, Tetranychus cinnabarinus and the twospotted spider mite, Tetranychus urticae to selected acaricides on strawberries.

Abstract: The carmine spider mite, Tetranychus cinnabarinus (Boisduval) and the twospotted spider mite, Tetranychus urticae Koch, are serious pests of strawberries and many other horticultural crops. Control of these pests has been heavily dependent upon chemical acaricides. Objectives of this study were to determine the resistance status of these two pest species to commonly used acaricides on strawberries in a year-round intensive horticultural production region. LC90 of abamectin for adult carmine spider mites was 4% whereas that for adult twospotted spider mites was 24% of the top label rate. LC90s of spiromesifen, etoxazole, hexythiazox and bifenazate were 0.5%, 0.5%, 1.4% and 83% of their respective highest label rates for carmine spider mite eggs, 0.7%, 2.7%, 12.1% and 347% of their respective highest label rates for the nymphs. LC90s of spiromesifen, etoxazole, hexythiazox and bifenazate were 4.6%, 11.1%, 310% and 62% of their respective highest label rates for twospotted spider mite eggs, 3%, 13%, 432,214% and 15% of their respective highest label rates for the nymphs. Our results suggest that T. cinnabarinus have developed resistance to bifenazate and that the T. urticae have developed resistance to hexythiazox. These results strongly emphasize the need to develop resistance management strategies in the region.

Drosophila melanogaster brain invasion: pathogenic Wolbachia in central nervous system of the fly.

Abstract: The pathogenic Wolbachia strain wMelPop rapidly over-replicates in the brain, muscles, and retina of Drosophila melanogaster, causing severe tissue degeneration and premature death of the host. The unique features of this endosymbiont make it an excellent tool to be used for biological control of insects, pests, and vectors of human diseases. To follow the dynamics of bacterial morphology and titer in the nerve cells we used transmission electron microscopy of 3-d-old female brains. The neurons and glial cells from central brain of the fly had different Wolbachia titers ranging from single bacteria to large accumulations, tearing cell apart and invading extracellular space. The neuropile regions of the brain were free of wMelPop. Wolbachia tightly interacted with host cell organelles and underwent several morphological changes in nerve cells. Based on different morphological types of bacteria described we propose for the first time a scheme of wMelPop dynamics within the somatic tissue of the host.

Electroantennogram and behavioral responses of Cotesia plutellae to plant volatiles

Abstract: Plant volatiles have been demonstrated to play an important role in regulating the behavior of Cotesia plutellae, a major larval parasitoid of the diamondback moth (DBM), Plutella xylostella, but little is currently known about the function of each volatile and their mixtures. We selected 13 volatiles of the DBM host plant, a cruciferous vegetable, to study the electroantennogram (EAG) and behavioral responses of C. plutellae. EAG responses to each of the compounds generally increased with concentration. Strong EAG responses were to 100 μL/mL of trans-2-hexenal, benzaldehyde, nonanal and cis-3-hexenol, and 10 μL/mL of trans-2-hexenal and benzaldehyde with the strongest response provoked by trans-2-hexenal at 100 μL/mL. In the Y-tube olfactometer, C. plutellae, was significantly attracted by 1 μL/mL of trans-2-hexenal and benzaldehyde. β-caryophyllene, cis-3-hexenol or trans-2-hexenal significantly attracted C. plutellae at 10 μL/mL, while nonanal, benzyl alcohol, cis-3-hexenol or benzyl cyanide at 100 μL/mL significantly attracted C. plutellae. Trans-2-hexenal significantly repelled C. plutellae at 100 μL/mL. EAG of C. plutellae showed strong responses to all mixtures made of five various compounds with mixtures 3 (trans-2-hexenal, benzaldehyde, nonanal, cis-3-hexenol, benzyl cyanide, farnesene, eucalyptol) and 4 (trans-2-hexenal, benzaldehyde, benzyl alcohol, (R)-(+)-limonene, β-ionone, farnesene, eucalyptol) significantly attracting C. plutellae. These findings demonstrate that the behavior of C. plutellae can be affected either by individual compounds or mixtures of plant volatiles, suggesting a potential of using plant volatiles to improve the efficiency of this parasitoid for biocontrol of P. xylostella.

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests

Abstract: The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.

Sexual behavior and male volatile compounds in wild and mass-reared strains of the Mexican fruit fly Anastrepha ludens (Diptera: Tephritidae) held under different colony management regimes

Abstract: We compared the calling and mating behavior and volatile release of wild males Anastrepha ludens (Loew) with males from 4 mass-reared strains: (i) a standard mass-reared colony (control), (ii) a genetic sexing strain (Tap-7), (iii) a colony started from males selected on their survival and mating competitiveness abilities (selected), and (iv) a hybrid colony started by crossing wild males with control females. Selected and wild males were more competitive, achieving more matings under field cage conditions. Mass-reared strains showed higher percentages of pheromone calling males under field conditions except for Tap-7 males, which showed the highest percentages of pheromone calling males under laboratory cage conditions. For mature males of all strains, field-cage calling behavior increased during the last hour before sunset, with almost a 2 fold increase exhibited by wild males during the last half hour. The highest peak mating activity of the 4 mass-reared strains occurred 30 min earlier than for wild males. By means of solid phase microextraction (SPME) plus gas chromatography-mass spectrometry (GC-MS), the composition of volatiles released by males was analyzed and quantified. Wild males emitted significantly less amounts of (E,E)-α-farnesene but emitted significantly more amounts of (E,E)-suspensolide as they aged than mass-reared males. Within the 4 mass-reared strains, Tap-7 released significantly more amounts of (E,E)-α-farnesene and hybrid more of (E,E)-suspensolide. Differences in chemical composition could be explained by the intrinsic characteristics of the strains and the colony management regimes. Characterization of calling behavior and age changes of volatile composition between wild and mass-reared strains could explain the differences in mating competitiveness and may be useful for optimizing the sterile insect technique in A. ludens.