Impact of heat processing on the nutritional content of Gryllus bimaculatus (black cricket).

Insects are increasingly suggested as a potential novel solution to global nutrition challenges. However, limited research is available on the impact of processing methods on the nutritional content of edible insects. This trial examines the effect of heat processing on the nutritional profile of the black cricket, Gryllus bimaculatus. Adult black crickets were killed by freezing and then dried at either a low (45°C) or high (120°C) temperature followed by nutritional analysis of protein and micronutrient content. An additional set of samples was either freeze-dried or dried at 32, 45, 72 or 120°C followed by nutritional analysis of lipid content. Analysis showed that percentage protein content was significantly higher in crickets dried at 45°C, a difference of roughly 1% of the total weight. Similarly, calcium content was also significantly higher in crickets dried at 45°C, although no other measured micronutrients were affected. Additionally, the fatty acid content was significantly influenced by higher temperature processing. Freeze-drying black crickets conserved significantly more of the long-chain polyunsaturated fatty acids than drying at 120°C. Insects hold potential as a source of essential nutrients and fatty acids; however, consideration must be given to heat processing at high temperatures as this may affect the nutritional profile.

Structural and biochemical evaluation of Ceratitis capitata odorant-binding protein 22 affinity for odorants involved in intersex communication.

In insects, odorant-binding proteins (OBPs) connect the peripheral sensory system to receptors of olfactory organs. Medfly Ceratitis capitata CcapObp22 shows 37% identity and close phylogenetic affinities with Drosophila melanogaster OBP69a/pheromone-binding protein related protein 1. The CcapObp22 gene is transcribed in the antennae and maxillary palps, suggesting an active role in olfaction. Here, we recombinantly produced CcapObp22, obtaining a 13.5 kDa protein capable of binding multiple strongly hydrophobic terpene compounds, including medfly male pheromone components. The highest binding affinity [half maximal effective concentration (EC50) = 0.48 µM] was to (E,E)-α-farnesene, one of the most abundant compounds in the male pheromone blend. This odorant was used in cocrystallization experiments, yielding the structure of CcapOBP22. The monomeric structure shows the typical OBP folding, constituted by six α-helical elements interconnected by three disulphide bridges. A C-terminal seventh α-helix constitutes the wall of a deep, L-shaped hydrophobic cavity. Analysis of the electron density in this cavity suggested trapping of farnesene in the crystal structure, although with partial occupancy. Superposition of the CcapOBP22 structure with related seven-helical OBPs highlights striking similarity in the organization of the C-terminal segment of these proteins. Collectively, our molecular and physiological data on medfly CcapOBP22 suggest its involvement in intersex olfactory communication.

Use of the synergist piperonyl butoxide can slow the development of alpha-cypermethrin resistance in the whitefly Bemisia tabaci.

The development of insecticide resistance in insect pests of crops is a growing threat to sustainable food production, and strategies that slow the development of resistance are therefore urgently required. The insecticide synergist piperonyl butoxide (PBO) inhibits certain insect detoxification systems and so may delay the evolution of metabolic resistance. In the current study we characterized resistance development in the silverleaf whitefly, Bemisia tabaci, after selection with either a neonicotinoid (thiacloprid) or pyrethroid (alpha-cypermethrin) insecticide alone or in combination with PBO. Resistance development was significantly suppressed (> 60%) in the line selected with alpha-cypermethrin + PBO compared to the line selected with alpha-cypermethrin alone. RNA sequencing (RNAseq) analyses revealed an increase in frequency of a knock-down resistance mutation but no differentially expressed genes were identified that could explain the sensitivity shift. No significant difference was observed in the level of resistance between the thiacloprid and thiacloprid + PBO selected lines, and RNA sequencing (RNAseq) analyses revealed that the cytochrome P450 monooxygenase CYP6CM1, known to metabolize neonicotinoids, was significantly upregulated (>10-fold) in both lines. The findings of this study demonstrate that PBO used in combination with certain insecticides can suppress the development of resistance in a laboratory setting; however, the mechanism by which PBO supresses resistance development remains unclear.

Molecular cloning, characterisation and mRNA expression of the ryanodine receptor from the peach-potato aphid, Myzus persicae.

The peach potato aphid, Myzus persicae, is one of the most important agricultural pests of temperate climates. It is mainly controlled through the judicious application of insecticides; however, over time, aphids have developed resistance to many insecticidal classes. The recent introduction of synthetic diamide insecticides, with a novel mode of action, potentially offers new tools to control aphid populations. These diamides act on the ryanodine receptor (RyR), a large endoplasmic calcium release channel. In this study we have cloned cDNAs encoding the complete open reading frame of the RyR from M. persicae. The open reading frame is 15,306 base pairs long and encodes a protein of 5101 amino acids. The aphid RyR shares many of the features of other insect and vertebrate RyRs, including a highly conserved transmembrane region. However, unlike the other RyRs characterised to date, the M. persicae channel does not display alternative splicing at any stage of its developmental cycle, so it cannot generate functional variants of the channel.

Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata.

The Mediterranean fruit fly (or medfly), Ceratitis capitata (Wiedemann; Diptera: Tephritidae), is a serious pest of agriculture worldwide, displaying a very wide larval host range with more than 250 different species of fruit and vegetables. Olfaction plays a key role in the invasive potential of this species. Unfortunately, the pheromone communication system of the medfly is complex and still not well established. In this study, we report the isolation of chemicals emitted by sexually mature individuals during the "calling" period and the electrophysiological responses that these compounds elicit on the antennae of male and female flies. Fifteen compounds with electrophysiological activity were isolated and identified in male emissions by gas chromatography coupled to electroantennography (GC-EAG). Within the group of 15 identified compounds, 11 elicited a response in antennae of both sexes, whilst 4 elicited a response only in female antennae. The binding affinity of these compounds, plus 4 additional compounds known to be behaviourally active from other studies, was measured using C. capitata OBP, CcapOBP83a-2. This OBP has a high homology to Drosophila melanogaster OBPs OS-E and OS-F, which are associated with trichoid sensilla and co-expressed with the well-studied Drosophila pheromone binding protein LUSH. The results provide evidence of involvement of CcapOBP83a-2 in the medfly's odorant perception and its wider specificity for (E,E)-α-farnesene, one of the five major compounds in medfly male pheromone emission. This represents the first step in the clarification of the C. capitata and pheromone reception pathway, and a starting point for further studies aimed towards the creation of new powerful attractants or repellents applicable in the actual control strategies.

Identification and Expression Profile of Two Putative Odorant-Binding Proteins from the Neotropical Brown Stink Bug, Euschistus heros (Fabricius) (Hemiptera: Pentatomidae).

This work presents the first identification of putative odorant-binding proteins (OBPs) from a member of the Pentatomidae, i.e. the brown stink bug Euschistus heros (Fabricius), an important pest of soybean in Brazil. Antennae from both sexes of E. heros adults (12 days old and unmated) were used to construct a cDNA library, from which two transcripts encoding putative E. heros OBPs (EherOBPs) were identified. The expression levels of EherOBP1 and EherOBP2 were found to be higher in male antennae than in female and there was difference in expression in legs, wings, and abdomens of the two sexes. The histolocalization of EherOBP1 and EherOBP2 transcripts in antennae also showed a sexual dimorphism in the chemoreception system, with different expression sites in the antennal segments between males and females, occurring predominantly at the base of the sensillum. The implications of these findings for stink bug chemoreception are discussed.

The re-emergence of the bed bug as a nuisance pest: implications of resistance to the pyrethroid insecticides.

A global resurgence of bed bugs (Hemiptera: Cimicidae) has led to renewed scientific interest in these insects. The current bed bug upsurge appears to have started almost synchronously in the late 1990 s in Europe, the U.S.A. and Australia. Several factors have led to this situation, with resistance to applied insecticides making a significant contribution. With a growing number of insecticides (DDT, carbamates, organophosphates etc.) being no longer available as a result of regulatory restrictions, the mainstay chemistry used for bed bug control over the past few decades has been the pyrethroid insecticides. With reports of increasing tolerance to pyrethroids leading to control failures on the rise, containing and eradicating bed bugs is proving to be a difficult task. Consequently, several recent studies have focused on determining the mode of action of pyrethroid resistance in bed bug populations sourced from different locations. Correct identification of the factor(s) responsible for the increasing resistance is critical to the development of effective management strategies, which need to be based, wherever possible, on firm scientific evidence. Here we review the literature on this topic, highlighting the mechanisms thought to be involved and the problems currently faced by pest control professionals in dealing with a developing pandemic.

Expression of chemosensory proteins in the tsetse fly Glossina morsitans morsitans is related to female host-seeking behaviour.

Chemosensory proteins (CSPs) are a class of soluble proteins present in high concentrations in the sensilla of insect antennae. It has been proposed that they play an important role in insect olfaction by mediating interactions between odorants and odorant receptors. Here we report, for the first time, the presence of five CSP genes in the tsetse fly Glossina morsitans morsitans, a major vector transmitting nagana in livestock. Real-time quantitative reverse transcription PCR showed that three of the CSPs are expressed in antennae. One of them, GmmCSP2, is transcribed at a very high level and could be involved in olfaction. We also determined expression in the antennae of both males and females at different life stages and with different blood feeding regimes. The transcription of GmmCSP2 was lower in male antennae than in females, with a sharp increase in 10-week-old flies, 48 h after a bloodmeal. Thus there is a clear relationship between CSP gene transcription and host searching behaviour. Genome annotation and phylogenetic analyses comparing G. morsitans morsitans CSPs with those of other Diptera showed rapid evolution after speciation of mosquitoes.

Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid.

Genome annotation and comparative analyses of the odorant-binding proteins and chemosensory proteins in the pea aphid Acyrthosiphon pisum.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are two families of small water-soluble proteins, abundant in the aqueous fluid surrounding olfactory receptor neurons in insect antennae. OBPs are involved in the first step of olfactory signal transduction, carrying airborne semiochemicals to the odorant receptors and can be classified into three groups: Classic OBPs, Plus-C OBPs and Atypical OBPs. Here, we identified and annotated genes encoding putative OBPs and CSPs in the pea aphid Acyrthosiphon pisum using bioinformatics. This identified genes encoding 13 Classic and two Plus-C OBPs and 13 CSPs. Homologous OBP sequences were also identified in nine other aphid species, allowing us to compare OBPs across several aphid and non-aphid species. We show that, although OBP sequences are divergent within a species and between different orders, there is a high similarity between orthologs within a range of aphid species. Furthermore, the phylogenetic relationships between OBP orthologs reflect the divergence of aphid evolution lineages. Our results support the 'birth-and-death' model as the major mechanism explaining aphid OBP sequence evolution, with the main force acting on the evolution being purifying selection.

Identification of ion channel genes in the Acyrthosiphon pisum genome.

Aphids are major pests of crops, causing hundreds of millions of dollars worth of damage annually. Ion channel proteins are often the targets of modern insecticides and mutations in ion channel genes can lead to resistance to many leading classes of insecticides. The sequencing of the pea aphid, Acyrthosiphon pisum, genome has now allowed detailed in silico analysis of the aphid ion channels. The study has revealed significant differences in the composition of the ion channel families between the aphid and other insects. For example A. pisum does not appear to contain a homologue of the nACh receptor alpha 5 gene whilst the calcium channel beta subunit has been duplicated. These variations could result in differences in function or sensitivity to insecticides. The genome sequence will allow the study of aphid ion channels to be accelerated, leading to a better understanding of the function of these economically important channels. The potential for identifying novel insecticide targets within the aphid is now a step closer.

Cloning and characterisation of a prenyltransferase from the aphid Myzus persicae with potential involvement in alarm pheromone biosynthesis.

The majority of aphid species release an alarm pheromone with the most common component being the sesquiterpene (E)-beta-farnesene, sometimes accompanied by other sesquiterpenes or monoterpenes. The genes/enzymes involved in the production of these compounds have not been identified in aphids although some components of isoprenoid biosynthesis have been identified in other insect species. Here we report the cloning, expression and characterisation of a prenyltransferase from the aphid Myzus persicae which can act as a farnesyl pyrophosphate synthase or a geranyl pyrophosphate synthase to produce both sesquiterpenes and monoterpenes and hence could be responsible for the biosynthesis of the observed components of the alarm pheromones. In addition, the enzyme can utilise geranyl pyrophosphate to produce farnesyl pyrophosphate showing that the synthesis of the latter involves the sequential condensation of isoprenyl pyrophosphate units.

Identification of odorant-binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses.

The yellow fever mosquito Aedes aegypti is an important human health pest which vectors yellow fever and dengue viruses. Olfaction plays a crucial role in its attraction to hosts and although the molecular basis of this is not well understood it is likely that odorant-binding proteins (OBPs) are involved in the first step of molecular recognition. Based on the OBPs of Drosophila melanogaster and Anopheles gambiae we have defined sequence motifs based on OBP conserved cysteine and developed an algorithm which has allowed us to identify 66 genes encoding putative OBPs from the genome sequence and expressed sequence tags (ESTs) of Ae. aegypti. We have also identified 11 new OBP genes for An. gambiae. We have examined all of the corresponding peptide sequences for the properties of OBPs. The predicted molecular weights fall within the expected range but the predicted isoeletric points are spread over a wider range than found previously. Comparative analyses of the 66 OBP sequences of Ae. aegypti with other dipteran species reveal some mosquito-specific genes as well as conserved homologues. The genomic organisation of Ae. aegypti OBPs suggests that a rapid expansion of OBPs has occurred, probably by gene duplication. The analyses of OBP-containing regions for microsynteny indicate a very high synteny between Ae. aegypti and An. gambiae.

Mutations in DIIS5 and the DIIS4-S5 linker of Drosophila melanogaster sodium channel define binding domains for pyrethroids and DDT.

Mutations in the DIIS4-S5 linker and DIIS5 have identified hotspots of pyrethroid and DDT interaction with the Drosophila para sodium channel. Wild-type and mutant channels were expressed in Xenopus oocytes and subjected to voltage-clamp analysis. Substitutions L914I, M918T, L925I, T929I and C933A decreased deltamethrin potency, M918T, L925I and T929I decreased permethrin potency and T929I, L925I and I936V decreased fenfluthrin potency. DDT potency was unaffected by M918T, but abolished by T929I and reduced by L925I, L932F and I936V, suggesting that DIIS5 contains at least part of the DDT binding domain. The data support a computer model of pyrethroid and DDT binding.

DDT, pyrethrins, pyrethroids and insect sodium channels.

The long term use of many insecticides is continually threatened by the ability of insects to evolve resistance mechanisms that render the chemicals ineffective. Such resistance poses a serious threat to insect pest control both in the UK and worldwide. Resistance may result from either an increase in the ability of the insect to detoxify the insecticide or by changes in the target protein with which the insecticide interacts. DDT, the pyrethrins and the synthetic pyrethroids (the latter currently accounting for around 17% of the world insecticide market), act on the voltage-gated sodium channel proteins found in insect nerve cell membranes. The correct functioning of these channels is essential for normal transmission of nerve impulses and this process is disrupted by binding of the insecticides, leading to paralysis and eventual death. Some insect pest populations have evolved modifications of the sodium channel protein which prevent the binding of the insecticide and result in the insect developing resistance. Here we review some of the work (done at Rothamsted Research and elsewhere) that has led to the identification of specific residues on the sodium channel that may constitute the DDT and pyrethroid binding sites.

A comparative study of voltage-gated sodium channels in the Insecta: implications for pyrethroid resistance in Anopheline and other Neopteran species.

We report the complete cDNA sequence of the Anopheles gambiae voltage-gated sodium channel (VGSC) alpha-subunit isolated from mature adult mosquitoes. The genomic DNA contains 35 deduced exons with a predicted translation of

OS-D-like genes and their expression in aphids (Hemiptera: Aphididae).

It has been proposed that a family of small soluble binding proteins, variously named as chemosensory proteins, sensory appendage proteins, pherokines and OS-D-like proteins, are involved in insect chemoreception. These proteins are present in a wide range of insect species and have a characteristic four cysteine motif. We have cloned cDNAs and gene sequences encoding these proteins from a number of aphid species, the first report of such in the order Hemiptera and supporting the view that these genes predate the divergence of the Neoptera. In one aphid species, Megoura viciae, we have shown that the proteins are present primarily in adults with one protein being most abundant in antennae and legs. This supports the view that the proteins could be involved in chemoreception but our preliminary binding studies failed to detect binding to a range of compounds which are known to elicit an electrophysiological response by aphids.

Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover.

We have identified two mutations in the ace1 gene of Aphis gossypii that are associated with insensitivity of acetylcholinesterase (AChE) to carbamate and organophosphate insecticides. The first of these, S431F (equivalent to F331 in Torpedo californica), is associated with insensitivity to the carbamate insecticide pirimicarb in a range of A. gossypii clones. The S431F mutation is also found in the peach-potato aphid, Myzus persicae (Sulzer), and a rapid RFLP diagnostic allows the identification of individuals of both aphid species with a resistant genotype. This diagnostic further revealed the presence of S431 in several other pirimicarb-susceptible aphid species. The serine at this position in the wild-type enzyme has only been reported for aphids and provides a molecular explanation of why pirimicarb has a specific aphicidal action. A less specific insensitivity to a wide range of carbamates and organophosphates is associated with a second mutation, A302S (A201 in T. californica).

DNA methylation in insects.

Cytosine DNA methylation has been demonstrated in numerous eukaryotic organisms and has been shown to play an important role in human disease. The function of DNA methylation has been studied extensively in vertebrates, but establishing its primary role has proved difficult and controversial. Analysing methylation in insects has indicated an apparent functional diversity that seems to argue against a strict functional conservation. To investigate this hypothesis, we here assess the data reported in four different insect species in which DNA methylation has been analysed more thoroughly: the fruit fly Drosophila melanogaster, the cabbage moth Mamestra brassicae, the peach-potato aphid Myzus persicae and the mealybug Planococcus citri.

Characterization of acetylcholinesterases, and their genes, from the hemipteran species Myzus persicae (Sulzer), Aphis gossypii (Glover), Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood).

Gene sequences encoding putative acetylcholinesterases have been reported for four hemipteran insect species. Although acetylcholinesterase insensitivity occurs in insecticide-resistant populations of each of these species, no mutations were detected in the gene sequences from the resistant insects. This, coupled with a series of experiments using novel reversible inhibitors to compare the biochemical characteristics of acetylcholinesterase from a range of insect species, showed that the cloned cDNA fragments are unlikely to encode the hemipteran synaptic acetylcholinesterases, and there is likely to be a second ace locus.