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Wild bee populations are declining due to human activities, such as land use change, which strongly affect the composition and diversity of available plants and food sources. The chemical composition of food (i.e., nutrition) in turn determines the health, resilience, and fitness of bees. For pollinators, however, the term 'health' is recent and is subject to debate, as is the interaction between nutrition and wild bee health. We define bee health as a multidimensional concept in a novel integrative framework linking bee biological traits (physiology, stoichiometry, and disease) and environmental factors (floral diversity and nutritional landscapes). Linking information on tolerated nutritional niches and health in different bee species will allow us to better predict their distribution and responses to environmental change, and thus support wild pollinator conservation.

Long non-coding RNAs (lncRNAs) generally display tissue-specific distributions, and testis-specific lncRNAs form the highest proportion of lncRNAs in many species. Here, we presented a detailed analysis of testis-specific lncRNAs in the melon fly, Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. Most testis-specific lncRNAs were found to be long intergenic non-coding RNAs (lincRNA). The size distribution of these lncRNAs ranged between 600 and 1000 nucleotides. Testis-specific lncRNAs that harboured one isoform number and two exons were the most abundant. Compared to other male tissues, the testis had more highly expressed lncRNAs. The quantitative real-time polymerase chain reaction results of 10 randomly selected testis-specific lncRNAs showed expression patterns consistent with RNA-seq data. Further analysis of the most highly expressed testis-specific lncRNA, lnc94638, was undertaken. Fluorescent in situ hybridization assays localized lnc94638 to the apical region of the testis that contains mature spermatozoa. RNA interference-mediated knockdown of lnc94638 expression reduced spermatozoa numbers and impaired the fertility of Z. cucurbitae male. This study provides a catalogue of testis-specific lncRNAs, shows that the testis-specific lnc94638 is involved in spermatogenesis and has the potential to be used for treating male sterility.

This protocol was developed for the COST-Action “Super-B”, whose purpose was to coordinate research, outreach and policy towards sustainable pollination1-3. The protocol addresses the detection of parasites and pathogens across bee species, as one of several possible drivers of bee decline4,5. It consists of four major components:1. A sample collecting protocol, based around a dominant bee species driving pathogen distribution among other bee species2. A sample processing protocol, based on analyzing nucleic acids from individual bees3. A pathogen detection protocol, based on RT-qPCR with broad-range primers for several common pathogens4. A barcoding protocol, for accurate bee species identification The protocols have largely been adapted from existing knowledge and protocols but also include two key innovations: the use of passive reference nucleic acids and synthetic positive controls, that significantly improve the quality and robustness of the raw data, and thus the reliability of the analyses and conclusions.

none 23 si Safeguarding crop pollination services requires the identification of the pollinator species involved and the provision of their ecological requirements at multiple spatial scales. However, the potential for agroecological intensification of pollinator-dependent crops by harnessing pollinator diversity is limited by our capacity to characterise the community of pollinator species for each crop, and to determine how it is influenced by the different survey methods used, as well as by climatic variables at larger geographic scales. Here, we surveyed wild bees using a standardised protocol at an unprecedented scale including 62 commercial apple orchards in Western and Central Europe (i) to validate recent findings on pollinator community divergence as measured by common survey methods (netting and pan trapping) using conventional and alternative biodiversity metrics (phylogenetic and functional diversity), and (ii) to investigate the impact of climatic variation on the patterns observed. Our results confirm the significant divergence in pollinator communities measured using the two common methods at the larger, sub-continental scale, and we provide evidence for a significant influence of climate on the magnitude of pollinator community divergence (beta diversity and its turnover component) between survey methods, particularly when comparing colder to warmer sites and regions. We also found that warmer sites are more dissimilar than colder sites in terms of species composition, functional traits, or phylogenetic affinities. This result probably stems from the comparatively larger species pool in Southern Europe and because apple flowers are accessible to a wide spectrum of pollinator species; hence, two distant survey localities in Southern Europe are more likely to differ significantly in their pollinator community. Collectively, our results demonstrate the spatially-varying patterns of pollinator communities associated with common survey methods along a climate gradient and at the sub-continental scale in Europe. open Leclercq, N.; Marshall, L.; Weekers, T.; Anselmo, A.; Benda, D.; Bevk, D.; Bogusch, P.; Cejas, D.; Drepper, B.; Galloni, M.; Gérard, M.; Ghisbain, G.; Hutchinson, L.; Martinet, B.; Michez, D.; Molenberg, J.-M.; Nikolic, P.; Roberts, S.; Smagghe, G.; Straka, J.; Vandamme, P.; Wood, T.J.; Vereecken, N.J. Leclercq, N.; Marshall, L.; Weekers, T.; Anselmo, A.; Benda, D.; Bevk, D.; Bogusch, P.; Cejas, D.; Drepper, B.; Galloni, M.; Gérard, M.; Ghisbain, G.; Hutchinson, L.; Martinet, B.; Michez, D.; Molenberg, J.-M.; Nikolic, P.; Roberts, S.; Smagghe, G.; Straka, J.; Vandamme, P.; Wood, T.J.; Vereecken, N.J.

One of the main functions of the piwi-interacting RNA pathway is the post-transcriptional silencing of transposable elements in the germline of many species. In insects, proteins belonging to the Tudor superfamily proteins belonging to the Tudor superfamily play an important role in to play an important role in this mechanism. In this study, we identified the tudor gene in the oriental fruit fly, Bactrocera dorsalis, investigated the spatiotemporal expressional profile of the gene, and performed a functional analysis using RNA interference. We identified one transcript for a tudor homologue in the B. dorsalis transcriptome, which encodes a protein containing the typical 10 Tudor domains and an Adenosine triphosphate (ATP) synthase delta subunit signature. Phylogenetic analysis confirmed the identity of this transcript as a tudor homologue in this species. The expression profile indicated a much higher expression in the adult and pupal stages compared to the larval stages (up to a 60-fold increase), and that the gene was mostly expressed in the ovaries, Malpighian tubules and fat body. Finally, gene knockdown of tudor in B. dorsalis led to clearly underdeveloped ovaries in the female adult and reductions in copulation rate and amount of oviposition, indicating its important role in reproduction. The results of this study shed more light on the role of tudor in ovary development and reproduction.

Vitellogenin (Vg) and its receptor (VgR) play a key role in the reproductive process and development of insects. Aphids are a group of high-fecundity insect species with pseudoplacental viviparity, but the roles of their Vg and VgR genes have not been investigated yet. The brown citrus aphid, Aphis (Toxoptera) citricidus, is a major insect pest of citrus and the main vector of Citrus tristeza closterovirus. In this study, we identified and characterized these two genes, designated as AcVg and AcVgR, from the brown citrus aphid. We found that AcVg has lost the DUF1943 domain that is present in other insect Vgs. Silencing of AcVg and AcVgR led to a delay in the nymph-adult transition, a prolonged prereproductive period, and a shortened reproductive period, which in turn resulted in slower embryonic development and fewer new-born nymphs. Interestingly, silencing of AcVg decreased the transcript level of AcVgR, but silencing of AcVgR resulted in increased transcript levels of AcVg. In addition, silencing of Vg/VgR had similar phenotypes between alate and apterous morphs, suggesting that the functions of these two genes are the same in the two wing morphs of the aphid. Our results demonstrate that Vg and VgR are involved in various aspects of aphid development and reproduction. Further studies on the synthesis of Vg could help to elucidate the reproductive mechanism and provide information that will be useful for developing new pest control strategies.

Entomotoxic proteins are produced by plants in defence against insect herbivory. Some carbohydrate-binding proteins exhibit strong insecticidal activity affecting the survival, growth, development and feeding behavior of phytophagous insects. The occurrence of entomotoxic lectins is well documented in the Amaryllidaceae, a plant family spread world-wide. In Chile, this family is represented by numerous species, many of which are also of high ornamental value. Protein extracts were obtained from bulbs of five different species of Chilean Amaryllidaceae. A dose-response assay was carried out with two important pests: the greenhouse whitefly Trialeurodes vaporariorum Westwood and the mealybug Pseudococcus viburni Signoret. The extracts were offered to insects in a liquid artificial diet for three days and the mortality was scored. The Phycella australis Ravenna extract caused the highest insecticidal activity (T. vaporariorum LC₅₀: 7200 µg/mL; P. viburni LC₅₀: 9500 µg/mL). Applied at 1000 µg/mL in the diet the P. australis extract did not repel feeding of these pests. A mannose-binding lectin isolated from the bulbs of P. australis proved to be moderately toxic for these pests (T. vaporariorum LC₅₀: 1127 µg/mL; P. viburni LC₅₀: 2320 µg/mL).

Esterase has been reported to be involved in malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel). However, the underlying molecular mechanism of the esterase-mediated resistance remains largely unknown in this species. Here, with the use of a strain selected for malathion resistance in the laboratory (MR), we found that two overexpressed α-esterase genes, namely BdCarE4 and BdCarE6, predominant in the adult midgut and fat body, function in conferring malathion resistance in B. dorsalis. Notably, these two genes were found to be mostly close to the esterase E3, which are usually implicated in detoxifying organophosphate insecticides. The transcript levels of BdCarE4 and BdCarE6 were investigated and compared between the MR and a susceptible (MS) strain of B. dorsalis. Both genes were significantly up-regulated in the MR strain, which was consistent with the enhanced esterase activity in the MR strain. However, no changes in either the coding sequence or gene copy number were observed between the two strains. Subsequently, heterologous expression combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE4 and BdCarE6 can probably detoxify malathion. Furthermore, RNA interference-mediated knockdown of each of these two genes significantly increased malathion susceptibility in the MR strain adults. In conclusion, these results expand our molecular understanding of the important role of α-esterases during the development of resistance to organophosphorous insecticides in B. dorsalis.

The generalist predator Chrysoperla externa (Neuroptera: Chrysopidae) is distributed in the Neotropical Region and presents a strong potential as a biological control agent due to its easily rearing, high voracity and tolerance to several pesticides. This species feeds on relevant pests such as aphids, whiteflies, trips and various lepidopterans. In Argentina, an indiscriminate chemical control with broad spectrum pesticides is still the first option for controlling pests causing resistance problems and reduction of beneficial organisms. Therefore, it would be desirable that predators or parasitoids develop tolerance or resistance as phytophagous pests for their maintenance in the agricultural ecosystems. The objective of this work was to evaluate the tolerance of C. externo to the pyrethroid cypermethrin by topical exposure in laboratory conditions. The symptoms evaluated were tremors, movement, knockdown, inability to walk due to being glued. Recovery from poisoning symptoms was also evaluated. Six different concentrations of the pesticide, including the full field one, were tested. The exposure method was by topical applications on third larval instar. A low toxicity on larvae at 24 h post-treatment was observed, even at the highest concentration evaluated. Most of treated individuals looked normal at 24 h post-treatment; those who were knocked down showed a complete recovery between 48 and 72 h post-treatment. C externa demonstrated a high tolerance to pyrethroids. This could be due to the activity involved in pyrethroids biotransformation (for example, esterases and mixed-function oxidases), or to a low sensitivity of the molecular target. Future studies should be addressed at biochemical and molecular levels to complete our knowledge about insecticide effects on this predator.

In eukaryotes, microRNAs (miRNAs) are small, conserved, noncoding RNAs that have emerged as critical regulators of gene expression. The oriental fruit fly Bactrocera dorsalis is one of the most economically important fruit fly pests in East Asia and the Pacific. Although transcriptome analyses have greatly enriched our knowledge of its structural genes, little is known about post-transcriptional regulation by miRNAs in this dipteran species. In this study, small RNA libraries corresponding to four B. dorsalis developmental stages (eggs, larvae, pupae and adults) were constructed and sequenced. Approximately 30.7 million reads of 18-30 nucleotides were obtained, with 123 known miRNAs and 60 novel miRNAs identified amongst these libraries. More than half of the miRNAs were stage-specific during the four developmental stages. A set of miRNAs was found to be up- or down-regulated during development by comparison of their reads at different developmental stages. Moreover, a small part of miRNAs owned both miR-#-3p and miR-#-5p types, with enormously variable miR-#-3p/miR-#-5p ratios in the same library and amongst different developmental stages for each miRNA. Taking these findings together, the current study has uncovered a number of miRNAs and provided insights into their possible involvement in developmental regulation by expression profiling of miRNAs. Further analyses of the expression and function of these miRNAs could increase our understanding of regulatory networks in this insect and lead to novel approaches for its control.

In the search for new methods of pest control, the potential of RNA interference (RNAi) is being explored. Because the gut is the first barrier for the uptake of double-stranded (ds)RNA, pyrosequencing of the gut transcriptome is a powerful tool for obtaining the necessary sequences for specific dsRNA-mediated pest control. In the present study, a dataset representing the gut transcriptome of the Colorado potato beetle (CPB; Leptinotarsa decemlineata) was generated and analysed for the presence of RNAi-related genes. Almost all selected genes that were implicated in silencing efficiency at different levels in the RNAi pathway (core machinery, associated intracellular factors, dsRNA uptake, antiviral RNAi, nucleases), which uses different types of small RNA (small interfering RNA, microRNA and piwi-RNA), were expressed in the CPB gut. Although the database is of lower quality, the majority of the RNAi genes are also found to be present in the gut transcriptome of the tobacco hornworm [TH; Manduca sexta (19 out of 35 genes analysed)]. The high quality of the CPB transcriptome database will lay the foundation for future gene expression and functional studies regarding the gut and RNAi.

The Bemisia tabaci Gennadius (Homoptera:Aleyrodidae) biotype complex is a key pest of several worldwide crops. The management and control of this pest has become difficult mainly due to its high reproductive rate and capacity to develop resistance to broad spectrum insecticides. In Argentina B. tabaci whitefly, causes economic losses in most areas of agricultural production. Eretmocerus mundus Mercet (Hymenoptera:Aphelinidae) is the most important parasitoid of B. tabaci and is commercialized as a biocontrol agent, mainly in Europe. Conservation of this biological control agent in Argentinean orchards requires the adoption of sustainable pest management practices due the negative impact of traditional pesticides on non-target organisms. Spirotetramat (Movento) belongs to a new class of pesticides that acts as a lipid biosynthesis inhibitor and claims to be selective towards natural enemies. The objectives of this work were 1) to evaluate the effectiveness of spirotetramat on eggs and nymphs of B. tabaci and 2) to determine the selectivity of spirotetramat towards E. mundus. Whitefly's eggs and nymphs (first nymphal settled instar) were exposed to the insecticide by foliar immersion whereas parasitoid adults (6 days old) were exposed to the insecticide by residual method for one hour, to simulate exposure of the parasitoid to the insecticide in the field. Lethal and sublethal effects of the insecticide were recorded daily. These preliminary studies have shown a high effectiveness of spirotetramat on the first nymphal instar of B. tabaci as well as a high selectivity for the pest in comparison to the parasitoid adults showing a low acute toxicity to them. These results suggest Movento could be included in Integrated Pest Management programs although more studies are required to complete its ecotoxicological profile.

As the second largest organ in insects, the insect midgut is the major tissue involved in the digestion of food and detoxification of xenobiotics, such as insecticides, and the first barrier and target for oral RNA interference (RNAi). In this study, we performed a midgut-specific transcriptome analysis in the oriental fruit fly, Bactrocera dorsalis, an economically important worldwide pest, with many populations showing high levels of insecticide resistance. Using high-throughput sequencing, 52 838 060 short reads were generated and assembled to 25 236 unigenes with a mean length of 758 bp. Interestingly, 34 unique sequences encoding digestion enzymes were newly described and these included aminopeptidase and trypsin, genes associated with Bacillus thuringiensis resistance and fitness cost. Second, 41 transcripts were annotated to particular detoxification genes such as glutathione S-transferases, carboxylesterases and cytochrome P450s, and the subsequent phylogenetic analysis indicated homology with tissue-specific and insecticide resistance-related genes of Drosophila melanogaster. Third, we identified the genes involved in the mechanism of RNAi and the uptake of double-stranded RNA. The sequences encoding Dicer-2, R2D2, AGO2, and Eater were confirmed, but SID and SR-CI were absent in the midgut transcriptome. In conclusion, the results provide basic molecular information to better understand the mechanisms of food digestion, insecticide resistance and oral RNAi in this important pest insect in agriculture. Specific genes in these systems can be used in the future as potential targets for pest control, for instance, with RNAi technology.

Understanding how variations in genetic sequences are conveyed into structural and biochemical properties is of increasing interest in the field of molecular evolution. In order to gain insight into this process, we studied the ecdysone receptor (EcR), a transcription factor that controls moulting and metamorphosis in arthropods. Using an in silico homology model, we identified a region in the lepidopteran EcR that has no direct interaction with the natural hormone but is under strong evolutionary constraint. This region causes a small indentation in the three-dimensional structure of the protein which facilitates the binding of tebufenozide. Non-Mecopterida are considered much older, evolutionarily, than Lepidoptera and they do not have this extended cavity. This location shows differences in evolutionary constraint between Lepidoptera and other insects, where a much lower constraint is observed compared with the Lepidoptera. It is possible that the higher flexibility seen in the EcR of Lepidoptera is an entirely new trait and the higher constraint could then be an indication that this region does have another important function. Finally, we suggest that Try123, which is evolutionarily constrained and is up to now exclusively present in Lepidoptera EcRs, could play a critical role in discriminating between steroidal and non-steroidal ligands.

The citrus red mite, Panonychus citri, is known for its ability rapidly to evolve resistance to insecticides/acaricides and to adapt to hosts that produce toxins. In this study, we constructed an unprecedented four gigabase pair transcriptome of P. citri, which was assembled into 64 149 unique transcripts, the functions of which were annotated by five public databases. A total of 116 unique transcripts were identified as representatives of potential involvement in the detoxification of xenobiotics. Genes recorded to encoding insecticide/acaricide target proteins were also obtained from the P. citri transcriptome. In order to explore novel candidate genes potentially involved in the pesticide detoxification of P. citri, we also constructed digital gene expression libraries of short-term transcriptome responses of P. citri to pesticides, which resulted in the identification of 120 unique transcripts potentially associated with insecticide/acaricide detoxification. Our study will facilitate molecular research on pesticide resistance in citrus red mites, as well as in other phytophagous mites.

In Argentina, transgenic soybean crop (Roundup Ready, RR) has undergone a major expansion over the last 15 years, with the consequent increase of glyphosate applications, a broad-spectrum and post emergence herbicide. Soybean crops are inhabited by several arthropods. Eriopis connexa Germar (Coleoptera: Coccinelidae) is a predator associated to soybean soft-bodies pest and have a Neotropical distribution. Nowadays, it is being considered a potentially biological control agent in South America. The objectives of this work were to evaluate the side-effects of glyphosate on larvae (third instar) and adults of this predator. Commercial compound and the maximum registered concentrations for field use were employed: GlifoGlex 48 (48% glyphosate, 192 mg a.i./litre, Gleba Argentina S.A.). The exposure was by ingestion through the treated prey (Rophalosiphum padi) or by drinking treated water during 48 h for treatment of the adult. The herbicide solutions were prepared using distilled water as solvent. The bioassays were carried out in the laboratory under controlled conditions: 23 +/- 0.5 degrees C, 75 +/- 5% RH and 16:8 (L:D) of photoperiod. Development time, weight of pupae, adult emergence, pre-oviposition period, fecundity and fertility were evaluated as endpoints. Larvae from glyphosate treatment molted earlier than controls. In addition, the weight of pupae, longevity, fecundity and fertility were drastically reduced in treated organisms. The reductions were more drastic when the treatments were performed at the third larval stage than as adult. The reproduction capacity of the predator was the most affected parameter and could be related to a hormonal disruption by glyphosate in the treated organisms. This work can confirm the deleterious effects of this herbicide on beneficial organisms. Also, it agrees with prior studies carried out on other predators associated to soybean pest, such as Chrysoperla externa (Neuroptera: Chrysopidae) and Alpaida veniliae (Araneae: Araneidae).

The integrated pest management (IPM) premises claim for a joint use of beneficial organisms (parasitoids and predators) and selective pesticides. Botanical pesticides are being considered as selective pesticides. During the last 20 years of the 20th century, the plant family Meliaceae was identified as a promising source of compounds with insect-control properties. The Chinaberry tree, Melia azedarach L., is a deciduous tree native to Persia, India, and China, but has been cultivated in many countries including Africa, Australia, and the Americas. It is a source of tetranortriterpenoid compounds called meliatoxins, which have similar structures and insecticidal activities compared to azadirachtin. Eriopis connexa Germar (Coleoptera: Coccinelidae) is a native predator from the Neotropical Region. It is a beneficial organism associated to several agro-ecosystems, feeding on soft-bodies pest such as aphids, whiteflies and thrips. In horticultural crops, it has been observed to feed on worldwide important pest such as Bemisia tabaci Gennadius (Heteroptera: Aleyrodeidae) and Myzus persicae L. (Heteroptera: Aphididae), both of them considered as virus vectors. The objectives of this work were to evaluate the side-effects of M. azaderach (Meliaceas) extract on eggs, larvae and adults of Eriopis connexa. Extracts from unripe fruits (1%, w/w, obtained by the CEPROCOR, Cordoba-Argentina) were employed for ecotoxicological bioassays. The exposure method was by immersion for eggs and by ingestion of drinking water for adults. The botanical extract solutions were prepared using distilled water as solvent and a tensioactive (Tween 20) for topical application. The bioassays were carried out in the laboratory under controlled conditions: 23 +/- 0.5 degrees C temperature, 75 +/- 5% RH and 16:8 (L:D) photoperiod. Hatching, development time, adult emergence, pre-oviposition period, fecundity and fertility were evaluated as endpoints. No detrimental effects were observed in treated organisms irrespective of the exposure way and development stage. According to these preliminary results, M. azaderach fruit extracts can be compatible with E. connexa for pest control. However, future studies should be addressed to conclude about its toxicity on beneficial organisms.

Potato virus Y (PVY) is the type species in the potyvirus genus of the family potyviridae. This plant pathogenic virus is transmitted through plant sap inoculation by stem and core grafting and by at least 25 aphid species in a non-persistent manner. According to potato specialists in most parts of the world, PVY is currently considered as the most harmful virus in cultivated potatoes. This is also the case for potato production in Iran. In this project we investigated potato leaves that were collected in the Kurdistan province in Iran for the presence of PVY with use of different biochemical/molecular techniques as ELISA, RT-PCR and qPCR. The different PVY strains, including PVY-O, PVY-N, PVYN-TN, PVY-NWi, were determined by using a triplex RT-PCR. In conclusion, the results demonstrated the presence of PVY-NWi strains in the potato leaf samples from Kurdistan (Iran). The data are discussed in relation to prevalence of PVY strains in Iran.

The mortality pattern of workers involved in the production of stainless steel (SS) was studied from 1968 to 1984 in order to investigate a possible risk of lung cancer in relation to exposure to chromium compounds, polycyclic aromatic hydrocarbons, and silica. The role of heat exposure in mortality from cardiovascular diseases also was examined. The cohort was comprised of 4,227 workers. Complete individual job histories were provided by the company (UGINE SA). The smoking habits of 24 percent of the cohort members were known from the interview of workers still active during the data collection. The observed numbers of deaths were compared with the expected ones based on national rates with adjustment for age, sex, and calendar time (standardized mortality ratio, SMR). No significant excesses of lung cancer were observed among workers employed in the manufacture of ferroalloys (SMR = 0.68) and in the melting and casting of SS (SMR = 1.04), whereas a significant excess appeared among SS foundry workers (SMR = 2.29). This excess was higher and remained significant among workers with more than 30 years since first employment in the foundry area (SMR = 3.34). Among subjects exposed to heat, no excess was observed for all cardiovascular diseases or for ischemic heart diseases.

Aphids exhibit unique attributes, such as polyphenisms and specialized cells to house endosymbionts, that make them an interesting system for studies at the interface of ecology, evolution and development. Here we present a comprehensive characterization of the developmental genes in the pea aphid, Acyrthosiphon pisum, and compare our results to other sequenced insects. We investigated genes involved in fundamental developmental processes such as establishment of the body plan and organogenesis, focusing on transcription factors and components of signalling pathways. We found that most developmental genes were well conserved in the pea aphid, although many lineage-specific gene duplications and gene losses have occurred in several gene families. In particular, genetic components of transforming growth factor beta (TGFbeta) Wnt, JAK/STAT (Janus kinase/signal transducer and activator of transcription) and EGF (Epidermal Growth Factor) pathways appear to have been significantly modified in the pea aphid.

The pea aphid (Acyrthosiphon pisum) is the first whole genome sequenced insect with a hemimetabolic development and an emerging model organism for studies in ecology, evolution and development. The insect steroid moulting hormone 20-hydroxyecdysone (20E) controls and coordinates development in insects, especially the moulting/metamorphosis process. We, therefore present here a comprehensive characterization of the Halloween genes phantom, disembodied, shadow, shade, spook and spookiest, coding for the P450 enzymes that control the biosynthesis of 20E. Regarding the presence of nuclear receptors in the pea aphid genome, we found 19 genes, representing all of the seven known subfamilies. The annotation and phylogenetic analysis revealed a strong conservation in the class of Insecta. But compared with other sequenced insect genomes, three orthologues are missing in the Acyrthosiphon genome, namely HR96, PNR-like and Knirps. We also cloned the EcR, Usp, E75 and HR3. Finally, 3D-modelling of the ligand-binding domain of Ap-EcR exhibited the typical canonical structural scaffold with 12 alpha-helices associated with a short hairpin of two antiparallel beta-strands. Upon docking, 20E was located in the hormone-binding groove, supporting the hypothesis that EcR has a role in 20E signalling.

Tebufenozide, a dibenzoylhydrazine insect growth regulator, was applied topically (1 microg/insect) on female adults of the German cockroach Blattella germanica and its effects on ovarian measurement and proteins contents were investigated during the first gonadotrophic cycle (0, 2, 4 and 6 days). Dissection of treated females showed a clear reduction in oocyte numbers and volume of the basal oocyte. The ovarian proteins content was also significantly reduced. Behavioral tests demonstrated that treatment of 6-days-old females with tebufenozide caused in 80% of the males an increase in numbers of antennal contacts, but there was no effect on the numbers of wing raisings. In contrast, it was striking that in 20% of the males there was a total loss of antennal contacts and wings raisings in response to a calling treated female. These behavior effects are probably caused by a reduced production of contact sex pheromone in the females and may help in explaining the lower ovary reproductive parameters.

Methoxyfenozide is an ecdysone agonist, the most new class of insect growth regulators. Most studies of the toxicity of ecdysone agonists on lepidopteran pests have been conducted during the larval stages, and little has been published regarding its sublethal effects in the surviving individuals. In this study, the lethal and sublethal effects of methoxyfenozide on the fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) were assessed. For this, fifth instars of this pest were continuously fed, until pupation, with artificial diet containing 0.24 and 0.35 mg of active ingredient/kg diet, which correspond to the LC10 and LC25 values, respectively, for this insecticide. Larval mortality reached 8% and 26% in the low and high concentration groups, respectively, on the seventh day of the experiment. A progressive larval mortality of 12% for the LC10 and 60% for the LC25 was observed before pupation. Treated larvae exhibited lower pupal weights, higher pupal mortality, presence of deformed pupae, and more deformed adults than untreated larvae. The incorporation of methoxyfenozide into the diet had a significant effect on the timing of larval development. Both male and female treated larvae lived about seven days longer than the controls for both concentrations tested. Finally, S. frugiperda adults that resulted from fifth instars treated with methoxyfenozide were not affected in their sex ratio. Our results suggest that the combination of lethal and sublethal effects of methoxyfenozide may have important implications for the population dynamics of the fall armyworm.

Diflubenzuron (Dimilin 25WP) is an insecticide of the class of benzoylphenylurea (BPUs) and is widely used in the control of insect pest in agriculture and forestry. Typically, these chemistries prevent the moulting process by inhibiting chitin formation and thereby causing abnormal cuticular deposition and abortive moulting in insect and crustacean species. In a previous study, we have shown that diflubenzuron could cause a modification in the lamellar ultrastructural, particularly in the membranous layers, in the non-target organism, the caramote prawn or triple-grooved shrimp Penaeus kerathurus (Forskal, 1775) (Decapoda, Peneidae). Therefore, the aim of this study was to evaluate under laboratory conditions the potential side-effects of diflubenzuron on the biochemical composition of the cuticle of treated P. kerathurus. Diflubenzuron was added to the rearing seawater at a final concentration of 1 microg/L) and newly-ecdysed adult shrimps were exposed for 10 days, i.e. until stage C during the molting cycle. The most typical treatment symptoms were a significantly reduced thickness of both the principal and membranous layers, and reduced amounts of chitin in the cuticle. These symptoms help in explaining the structural alterations observed in treated cuticles, and confirm the primary mode of action of diflubenzuron to inhibit chitin biosynthesis.

Mosquitoes are vectors of many human diseases and cause environmental nuisances. Due to their Large geographical distribution and abundance, Culex pipiens and Culiseta longiareolata represent the most important mosquito species in ALgeria and many countries in the world. They are generally controlled by conventional insecticides. The high reproductive ability and development of insecticide resistance of mosquito species are Leading to a search for novel molecules. As so, Vectobac that is based on the insecticidal toxins of Bacillus thuringiensis isroelensis (Bti), may provide economical control of mosquito larvae. In this study, we tested the activity of Vectobac (WDG) at different concentrations, ranging between 2 and 30 microg/L, against the different Larval stages (L1-L4) of Cx. pipiens and C. longiareolata. Bioassays were done on newly moulted Larvae under Laboratory conditions according to the guidelines of WHO. In Cx. pipiens, mortality was scored already after 24 h, and in addition developmental effects were observed like inhibition of growth with incomplete moulting and failure of adult emergence. From a dose-response curve LC50 were calculated: 9.22 microg/L for L1 and 35.33 microg/L for L4. For C. longiareolata, the respective LC50's were 6.05 microg/L and 23 microg/L for L1 and L4. The results are discussed in relation to the susceptibility of the two mosquito species tested, lethaL doses, direct toxicity, and effects on growth and development.

Lectins are carbohydrate-binding proteins that contain at least one carbohydrate binding domain which can bind to a specific mono- or oligosaccharide. These proteins are widely distributed in plants. However, over the last decade evidence is accumulating that lectins occur also in numerous fungi belonging to both the Ascomycota and Basiodiomycota. Rhizoctonia solani is known to be an important pathogen to a wide range of host plants. In this study, isolates of R. solani from different anastomosis groups have been screened for the presence of lectin using agglutination assays to detect and quantitate lectin activity. The evaluation included determination of the lectin content in mycelium as well as in sclerotia. The amount of lectin in the sclerotia was higher than in the mycelium of the same strains. The R. solani strains with the highest amounts of lectin have been selected for cultivation, extraction and purification of the lectin.

Captopril is an inhibitor of angiotensin converting enzyme (ACE) used against arterial hypertension in mammals. The aim of this study was to evaluate captopril in the mealworm Tenebio molitor (Coleoptera: Tenebrionidae). The drug was diluted in acetone and administered topically (10 microg/3microl) on newly emerged adult females. The biochemical composition (proteins, lipids, carbohydrates, nucleic acids) and the ecdysteroid amounts of ovaries were determined at day 4 following treatment corresponding to the end of the vitellogenesis process. Biochemical analyses revealed that captopril applied in vivo reduced both total protein, lipid and carbohydrate amounts of ovaries, respectively. In addition, measurement of ecdysteroids by an enzyme-immunoassay indicated a significant decrease in ovarian ecdysteroid amounts. As a consequence, the reduction of reproductive capacity is possibly related to ecdysteroid production. Further experiments are needed to determine the action mechanism of captopril and the functions of ACE in insects.

In order to complete previous studies conducted on Blattella germanica, three insecticides from different groups were evaluated: boric acid, an inorganic compound, benfuracarb, a carbamate, and halofenozide, a non-steroidal ecdysone agonist. Boric acid (8.20%, LD50) and benfuracarb (2%, LD50) were incorporated into the diet and orally administrated to newly emerged adults of both sexes, while halofenozide (0.33%, LD50) was applied topically. Hydrocarbons extracts was made on bidistilled pentane from control and treated series sampled 6 days following treatment. Extracts was analyzed by gas chromatography. Data showed that cuticular profiles of control and treated series were qualitatively similar with thirteen major compounds; however, significant quantitative differences were noted. Boric acid seemed to feminize the cuticular profile in males with a significant reduction of the two first cuticular compounds detected. Halofenozide and benfuracarb reduced cuticular compounds in both sexes.

The objective of this work was to evaluate the effect of two conventional insecticides commonly used in soybean crops of Argentina, endosulfan (Endosulfan 35 Glex, 105 ppm) and cypermethrin (Glextrin 25, 75 ppm) and two insecticides belonging to the new insecticides' generation, methoxyfenozide (Inteprid SC 24%, 144 ppm) and spinosad (Tracer 48%, 120 ppm), on eggs and larvae of Chrysoperla externa (Hagen) by dipping and topical application, respectively, under laboratory conditions. After egg treatment, the percentage of egg hatching, duration of the embryonic period and survival of first-instar larvae were evaluated. In the third-instar larvae assay, the survivals of the larvae and pupae, as well as eggs fertility were recorded. Endosulfan and cypermethrin were highly toxic towards eggs and larvae with mortality rates ranging from 80% to 100%. Toxicity of spinosad was intermediate causing short-term effects, but it did not cause any long-term effects. Methoxyfenozide was harmless to eggs and larvae of C. externa, showing good potential for use in integrated pest management in soybean crops. The selectivity of these insecticides and their inclusion in IPM programs is discussed.

The Mediterranean flour moth, Ephestia Kuehniella Zeller (Lepidoptera: Pyralidae), is an important pest in stored products worldwide, and is one of the major pests in flour mills in Algeria. Because environmental consideration, alternative approaches to neurotoxic insecticides, as well as safe, effective, and sound integrated pest management strategies are developed pest control agents such as the insect growth regulator (IGRs). Among these IGRs, the bisacylhydrazine derivatives are nonsteroidal ecdysterold agonists that mimic the action of moulting hormones and induce a precocious and incomplete moult in several insect orders. In topical bioassays using the pupae of E. kuehniella, three ecdysteroid agonists: RH-5849, the first bisaclhydrazine ecdysone agonist and two analogs, RH-5992 (tebufenozide) and RH-0345 (halofenozide), were evaluated on the reproduction under laboratory conditions. In a first series of experiments, the efficacy of these compounds was tested. These compounds exhibited insecticidal activity and the duration of pupal development was reduced with a dose-response relationship. Among the three tested compounds, tebufenozide (LD50 = 0.005 microg) appeared the most potent ecdysteroid agonist against E. kuehniella (RH-5849: LD50 = 0.05 microg and RH-0345: LD50 = 5.10 microg). In a second series of experiments, the effects of the ecdysone agonists (LD50) were investigated on the reproduction. Data showed that the three compounds affected growth of ovaries as evidenced by morphometric measurements of the ovaries from newly emerged adult females. In addition, the thickness of the chorion from basal oocytes was reduced only by RH-5992 and RH-0345. However, electron microscopic observations revealed that the three compounds had no significant effect on the fine structure of chorion. Finally, measurements of ovarian ecdysteroids' production by an enzyme immunoassay showed an increase in the hormonal amounts recorded in treated series compared to control series.

RH-0345 (halofenozide), a bisacylhydrazine derivative, is a nonsteroidal ecdysteroid agonist that mimics the action of the moulting hormones, while KK-42, an imidazole compound, is a potent inhibitor of ecdysteroid biosynthesis. Recent experiments with Tenebrio molitor suggested that the reduction of ecdysteroid titer, leading to a reduction of reproductive capacity, is due to a direct and rapid action of KK-42 on ecdysterold biosynthesis. Moreover, RH-0345 could partly restore the effects on reproductive events induced by KK-42. On the other hand, RH-0345 was found to affect growth and development of ovaries in a manner similar to that 20-hydroxyecdysone (20-E) and increased the ecdysteroid production. Therefore, the present study evaluates KK-42 applied topically (10 microg/insect) in combination with RH-0345 or 20-E in order to obtain more information on the mode of action of this compound on reproduction in T. molitor. In a first series of experiments, the compounds were assayed on morphometric measurements of freshly laid eggs. Results showed that both the weight and the volume of eggs increase significantly in series treated by KK-42 followed by 20-hydroxyecdysone as compared to controls and treated series by KK-42 followed by RH-0345. Data from enzyme immunoassay measurements revealed that KK-42 applied before 20-E was found to reduce significantly the amounts of free ecdysteroids in eggs comparatively to the others series.

Blatta germanica is the more prevalent cockroach species in Algeria. In the present study, we tested the effect on reproduction in B. germanica of two insect growth regulators, RH-0345, a benzoylhydrazine analogue that mimics the action of 20-hydroxyecdysone, and methoprene, one of the most commercially important juvenile hormone analogues, and a novel carbamate insecticide, benfuracarb. The compounds were applied topically (10 and 20 microg/insect for RH-0345, and 1 and 10 microg/insect for methoprene) or orally administrated (at 2% for benfuracarb) on newly emerged females and evaluated on reproductive events during the adult life (2, 4 and 6 days). Treatment with RH-0345 and benfuracarb reduced significantly the number of oocytes, the size and the volume of the basal oocyte during the experimental period. Methoprene distorted the ovarian development since it caused a significant reduction in the number of oocytes at 2, 4 and 6 days for the two tested doses, and an increase in oocyte size at 2, 4 and 6 days with 1 microg and a decrease with 10 microg. In a second series of experiments, the effects of these compounds were assayed on the ovarian proteins. Data from biochemical analysis revealed that RH-0345 and benfuracarb reduced the ovarian amounts of proteins, while treatment with methoprene increased it during the sexual maturation.

In this paper, we determined some physico-chemical properties like osmotic pressure, pH and electrical conductivity of the hemolymph from caterpillars of Spodoptera littoralis (Lepidoptera: Noctuidae) during the last larval instar. It was of interest that we observed an increase in osmotic pressure with the increase in age in the last instar that may concur with the start of histolysis at metamorphosis. These physicochemical properties were then compared to those of Grace's and modified Grace's tissue culture medium. In addition, concentrations of the cations Na, K, Ca and Mg, and the anions Cl, NO3, PO4 and SO4 were determined in the insect hemolymph of S. littoralis. The cations K and Mg reached high values with a percent of about 52% of the total amount of cations. The concentration of sodium was low. The total sum of the anions consisted about 56 meq/1, and this allows to neutralise about 45 % of the total cations.

The toxicity of botanical origin compounds such as two acetonic fractions of the seed kernels of the Meliacea Trichilia havanensis Jacq with insecticide properties (azadirone (F12) and the mixture F18 [1,7+3,7-di-O-acethylhavanensin (4:1)], three insecticides commercially available (imidacloprid, natural pyrethrins+PBO, triflumuron) and phloxine B, were tested in the laboratory. Topical bioassays using third instar and newly emerged adults of the lacewing Chrysoperla carnea (Stephens) at the maximum field recommended rate in Spain for commercials and at 1,000 ppm of active ingredient for T. havanensis acetone fractions and phloxine-B, were carried out. Imidacloprid and triflumuron were very toxic to third instar larvae inhibiting adult emergence, being the rest of insecticides harmless. Fecundity and fertility were not affected by the non-toxic compounds. Concerning adults, only imidacloprid and natural pyrethrins killed them significantly 24 hours after treatment. Phloxine B, triflumuron and T. havanensis fractions were harmless and did not cause any effect on fecundity and fertility with the exception of triflumuron, which reduced considerably the egg hatch. It can be concluded that T. havanensis acetonic fractions and phloxine B were non-toxic to larvae and adults of C. carnea when treated topically, whereas triflumuron, natural pyrethrins and imidacloprid affected one or more of the evaluated parameters under our conditions.

Some ecdysteroid agonists (RH-0345, RH-5849, RH-5992) were tested topically (10 microg/female) on newly emerged adult females of mealworms under in vivo conditions. In a first series of experiments the compounds were assayed on ovarian ecdysteroid production measured at day 2 and 4 following treatment, corresponding to the beginning and the end of vitellogenesis. All tested compounds resulted in a significant increase in ovarian ecdysteroid amounts. RH-5992 and RH-0345 were more active than RH-5849. In a second series of experiments, the activity of the compounds was investigated on ovarian proteins. The ecdysteroid agonists caused a significant reduction of protein amounts in the ovaries, and electrophoretic separation of ovary extracts from treated series showed that one or two bands were missing. These observations may an interference with the vitellogenesis process.

In this study, the effects of two insect growth regulators (KK-42 and RH-0345), applied either alone or in combination, were evaluated on the ecdysteroid production in vitro. Integument explants from the abdominal sternites of newly ecdysed pupae of mealworms, Tenebrio molitor, were cultured and the amounts of ecdysteroids, released into the culture medium, were determined by an enzyme immunoassay (EIA) after various intervals of incubation. In combined treatments, explants were cultured for the first two days in a medium added with the first compound and then transferred in new medium containing the second compound. EIA measurements showed that RH-0345, either alone or followed by KK-42, resulted in higher amounts of ecdysteroids as compared to controls. In contrast, KK-42 alone caused a significant reduction. But, when KK-42 was followed by RH-0345, the ecdysteroid amounts were equal to controls. In another series of experiments, the effects on cuticle secretion were examined. Only in the case that explants were treated with RH-0345, either alone or followed by KK-42, new cuticle synthesis was observed.

The secondary effects of conventional insecticides on the environment, vertebrates and beneficial organisms have caused a move to the use of more target-specific chemicals such as insect growth regulators. Halofenozide (RH-0345) is a novel nonsteroidal ecdysteroid agonist that induces a precocious and incomplete moult in several insect orders. In the present study, the insecticidal activity of a commercial formulation of halofenozide (23% EC) was evaluated at different concentrations ranging between 8 and 32 micrograms/l. Treatment was made on newly moulted fourth-instar larvae of the mosquito, Culex pipiens (Diptera: Culicidae), and mortality was scored until adult emergence. Results showed that death in the larval stage was relatively important. Moreover, treatment caused a significant reduction in the length of the larval and pupal stage. In a second series of experiments, halofenozide was tested at two doses (LC50 = 12.58 micrograms/l and LC90 = 28.58 micrograms/l) and the ecdysteroid amounts during the fourth instar were evaluated in an enzyme immunoassay. We noted that halofenozide did not shifted the moment of the single larval ecdysteroid peak, but it caused an increase of the titre values. Finally, the compound at the highest concentration was found to increase significantly the cuticle thickness recorded at day 4 following treatment.