Genotyping and Bio-Sensing Chemosensory Proteins in Insects.

Genotyping is the process of determining differences in the genetic make-up of an individual and comparing it to that of another individual. Focus on the family of chemosensory proteins (CSPs) in insects reveals differences at the genomic level across various strains and biotypes, but none at the level of individuals, which could be extremely useful in the biotyping of insect pest species necessary for the agricultural, medical and veterinary industries. Proposed methods of genotyping CSPs include not only restriction enzymatic cleavage and amplification of cleaved polymorphic sequences, but also detection of retroposons in some specific regions of the insect chromosome. Design of biosensors using CSPs addresses tissue-specific RNA mutations in a particular subtype of the protein, which could be used as a marker of specific physiological conditions. Additionally, we refer to the binding properties of CSP proteins tuned to lipids and xenobiotic insecticides for the development of a new generation of biosensor chips, monitoring lipid blood concentration and chemical environmental pollution.

Biotype expression and insecticide response of Bemisia tabaci chemosensory protein-1.

Chemosensory proteins (CSPs) are a group of small soluble proteins found so far exclusively in arthropod species. These proteins act in chemical communication and perception. In this study, a gene encoding the Type 1 CSP (BtabCSP1) from the agricultural pest Bemisia tabaci (whitefly) was analyzed to understand sequence variation and expression specificity in different biotypes. Sequence analysis of BtabCSP1 showed significant differences between the two genetically characterized biotypes, B and Q. The B-biotype had a larger number of BtabCSP1 mutations than the Q-biotype. Similar to most other CSPs, BtabCSP1 was more expressed in the head than in the rest of the body. One-step RT-PCR and qPCR analysis on total messenger RNA showed that biotype-Q had higher BtabCSP1 expression levels than biotype-B. Females from a mixed field-population had high levels of BtabCSP1 expression. The interaction of BtabCSP1 with the insecticide thiamethoxam was investigated by analyzing the BtabCSP1 expression levels following exposure to the neonicotinoid, thiamethoxam, in a time/dose-response study. Insecticide exposure increased BtabCSP1 expression (up to tenfold) at 4 and 24 h following 50 or 100 g/ml treatments.

Genome-wide identification of Thellungiella salsuginea microRNAs with putative roles in the salt stress response.

BACKGROUND: MicroRNAs are key regulators of plant growth and development with important roles in environmental adaptation. The microRNAs from the halophyte species Thellungiella salsuginea (salt cress), which exhibits extreme salt stress tolerance, remain to be investigated. The sequenced genome of T. salsuginea and the availability of high-throughput sequencing technology enabled us to discover the conserved and novel miRNAs in this plant species. It is interesting to identify the microRNAs from T. salsuginea genome wide and study their roles in salt stress response. RESULTS: In this study, two T. salsuginea small RNA libraries were constructed and sequenced using Solexa technology. We identified 109 miRNAs that had previously been reported in other plant species. A total of 137 novel miRNA candidates were identified, among which the miR* sequence of 26 miRNAs was detected. In addition, 143 and 425 target mRNAs were predicted for the previously identified and Thellungiella-specific miRNAs, respectively. A quarter of these putative targets encode transcription factors. Furthermore, numerous signaling factor encoding genes, defense-related genes, and transporter encoding genes were amongst the identified targets, some of which were shown to be important for salt tolerance. Cleavage sites of seven target genes were validated by 5' RACE, and some of the miRNAs were confirmed by qRT-PCR analysis. The expression levels of 26 known miRNAs in the roots and leaves of plants subjected to NaCl treatment were determined by Affymetrix microarray analysis. The expression of most tested miRNA families was up- or down-regulated upon NaCl treatment. Differential response patterns between the leaves and roots were observed for these miRNAs. CONCLUSIONS: Our results indicated that diverse set of miRNAs of T. salsuginea were responsive to salt stress and could play an important role in the salt stress response.

Lactobacillus for ribosome peptide editing cancer.

This study reviews newly discovered insect peptide point mutations as new possible cancer research targets. To interpret newly discovered peptide point mutations in insects as new possible cancer research targets, we focused on the numerous peptide changes found in the 'CSP' family on the sex pheromone gland of the female silkworm moth Bombyx mori. We predict that the Bombyx peptide modifications will have a significant effect on cancer CUP (cancers of unknown primary) therapy and that bacterial peptide editing techniques, specifically Lactobacillus combined to CRISPR, will be used to regulate ribosomes and treat cancer in humans.

An Expanded Survey of the Moth PBP/GOBP Clade in Bombyx mori: New Insight into Expression and Functional Roles.

We studied the expression profile and ontogeny (from the egg stage through the larval stages and pupal stages, to the elderly adult age) of four OBPs from the silkworm moth Bombyx mori. We first showed that male responsiveness to female sex pheromone in the silkworm moth B. mori does not depend on age variation; whereas the expression of BmorPBP1, BmorPBP2, BmorGOBP1, and BmorGOBP2 varies with age. The expression profile analysis revealed that the studied OBPs are expressed in non-olfactory tissues at different developmental stages. In addition, we tested the effect of insecticide exposure on the expression of the four OBPs studied. Exposure to a toxic macrolide insecticide endectocide molecule (abamectin) led to the modulated expression of all four genes in different tissues. The higher expression of OBPs was detected in metabolic tissues, such as the thorax, gut, and fat body. All these data strongly suggest some alternative functions for these proteins other than olfaction. Finally, we carried out ligand docking studies and reported that PBP1 and GOBP2 have the capacity of binding vitamin K1 and multiple different vitamins.

Interpopulational Variations of Odorant-Binding Protein Expression in the Black Cutworm Moth, Agrotis ipsilon.

A long-range migrant species of moth (Agrotis ipsilon) has served as a model to compare the expression profiles of antennal proteins between different continental populations. Our results showed that the American and French populations of the black cutworm moth, A. ipsilon, expressed the same odorant-binding proteins (OBPs), but apparently in different levels. Electrophoretic analysis of antennal protein profiles and reverse transcription polymerase chain reaction using RNA as a template showed significant differences between the two populations in the expression of antennal binding protein-X (ABPX) and general odorant-binding protein-2 (GOBP2). However, the two A. ipsilon populations showed no differences in RNA levels coding for pheromone binding proteins (PBPs), suggesting that the expression of generalist OBPs is population-specific and could be affected by specific odor and/or chemical changes in external environmental conditions. To support the role of ABPX and GOBP2 with expression, the role of ABPX and GOBP2 is discussed in regard to odor detection, memorization and/or degradation of toxic chemical insecticides.

Bioinformatic, genomic and evolutionary analysis of genes: A case study in dipteran CSPs.

Understanding physiological tasks may start by a good understanding of evolutionary traits in a given protein gene family. The successful completion of various genome projects is a key step forward for comparative analysis of similar and/or orthologous genes between species, measuring genetic relatednesses, studying evolutionary changes among different behaviors, helping to identify specifically conserved genes or genes that are variable enough to determine a new strain or organism. How evolutionary data can improve the understanding of a protein gene family is exploited here in the case of the insect Chemosensory Proteins (CSPs) as a guidance model. In this chapter, Diptera are presented not only as a huge diversity of appearances (phenotypes), behaviors and lifestyles, but also as major differences that are gene copy numbers, gene family members, polymorphisms and evolutionary rate variations, without any a priori assumption about the origin and function of the sampled gene family.

Curing piglets from diarrhea and preparation of a healthy microbiome with Bacillus treatment for industrial animal breeding.

High-throughput farming of animals for an essential purpose such as large scale health and production of hogs is a challenge for the food industry in the modern world. The problem is that the breeding of livestock for fast growth or high yields of meat is often associated with illness and microbial infection that develop under the breeding conditions. Piglet diarrhea is most common pig disease, leading to heavy mortality and thereby economic loss. We proved that chemical drugs can relieve the symptoms of diarrhea in ill piglets, but they do not treat the underlying cause, i.e. significantly altered bacterial gut flora. Using Illumina sequencing of fecal DNA, we showed that the bacterial gut flora of piglets treated with antibiotics remain close to the ill conditions. However, using Illumina sequencing of fecal DNA from piglets treated with a specific Bacillus (Bacillus subtilis Y-15, B. amyloliquefaciens DN6502 and B. licheniformis SDZD02) demonstrated the efficiency of natural bioproducts not only on curing diarrhea, but also on beneficial bacteria to re-establish in the piglet gut. We therefore propose a new natural "medicine" to be explored by the world farm animal agriculture industry, particularly for sustainable improvement of swine livestock production and health.

Comprehensive History of CSP Genes: Evolution, Phylogenetic Distribution and Functions.

In this review we present the developmental, histological, evolutionary and functional properties of insect chemosensory proteins (CSPs) in insect species. CSPs are small globular proteins folded like a prism and notoriously known for their complex and arguably obscure function(s), particularly in pheromone olfaction. Here, we focus on direct functional consequences on protein function depending on duplication, expression and RNA editing. The result of our analysis is important for understanding the significance of RNA-editing on functionality of CSP genes, particularly in the brain tissue.

Cartography of odor chemicals in the dengue vector mosquito (Aedes aegypti L., Diptera/Culicidae).

This study was aimed to identify the chemical compounds of Aedes aegypti that can be potentially used to develop pheromone-based vector control methods. In this study, we compared the chemical compounds collected from the organs of mosquitoes at different developmental stages in the life cycle. We also compared the composition and amount of extracts from the different tissues of male and female adult mosquito. Interestingly, we found large amount of C17-C20 ethyl and methyl esters in the wings of female and antennae of male mosquito. We also found that isopropyl esters, dodelactone, octadecenoic acid and medium-chain fatty acid increase drastically during the late larval stage (L4). Old adult mosquitoes showed remarkable increase in production of C16:1 and C18:1 methyl esters, as a first example of chemical signatures specifically associated with aging in the animals. This knowledge may open the ground to find new behaviorally-important molecules with the ability to control Aedes specifically.

Author Correction: Cartography of odor chemicals in the dengue vector mosquito (Aedes aegypti L., Diptera/Culicidae).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multiple functions of an odorant-binding protein in the mosquito Aedes aegypti.

To prevent spreading of deadly diseases, populations of mosquitoes can be controlled by interfering with their chemical communication system. Odorant-binding proteins, recently shown to be required for olfaction, represent interesting targets for such purpose. Here we describe the ligand-binding properties and the unusual tissue expression of odorant-binding protein 22 from the repertoire of Aedes aegypti. Best ligands are molecules with two aromatic rings connected by a short rigid chain. The protein is expressed not only in sensory organs, such as the antennae and proboscis, but also in the male reproductive apparatus and transferred to the spermathecs of females. This suggests an additional function for this protein as pheromone carrier, analogously to vertebrates' urinary and salivary proteins as well as some insect chemosensory proteins. Antiserum against odorant-binding protein 22 also stained the edges and sensilla of spiracles, indicating a third, still unknown, role for this protein.

Lactobacillus for ribosome peptide editing cancer

This study reviews newly discovered insect peptide point mutations as new possible cancer research targets. To interpret newly discovered peptide point mutations in insects as new possible cancer research targets, we focused on the numerous peptide changes found in the ‘CSP’ family on the sex pheromone gland of the female silkworm moth Bombyx mori. We predict that the Bombyx peptide modifications will have a significant effect on cancer CUP (cancers of unknown primary) therapy and that bacterial peptide editing techniques, specifically Lactobacillus combined to CRISPR, will be used to regulate ribosomes and treat cancer in humans (AU)

Differential expression of cytochrome c oxidase subunit III gene in castes of the termite Reticulitermes santonensis.

Social insects such as termites live in colonies in which cooperation is assumed by all individuals developing into castes to which specific tasks are allocated. Little has been reported about molecular aspects underlying termite caste-specific gene expression. Genetic regulation has recently been hypothesized to govern caste-specific traits and physiology in social insects. Cytochrome c oxidase (COX) has been shown to be an interesting candidate for expression study in insects. We used the cytochrome c oxidase subunit III gene (COXIII) that was cloned from mRNA in a lower termite, Reticulitermes santonensis De Feytaud (Isoptera; Rhinotermitidae). The full-length cDNA encodes a protein of 262 amino acids that shows high degree of homology with other insects COXIIIs. Reverse transcriptase-PCR and real-time PCR were performed to compare gene expression between larvae, workers, nymphs and soldiers. Analyses performed on head cDNAs revealed that COXIII is differentially expressed between castes. The level of COXIII is caste-regulated with an increase in workers (approximately 1.9-fold) and nymphs (approximately 2.8-fold) and a decrease in soldiers (0.8-fold) compared to the expression level in larvae (1.0-fold). These results may emphasize the physiological importance of COX in the termite brain at different developmental stages.

Sequence variation of Bemisia tabaci Chemosensory Protein 2 in cryptic species B and Q: New DNA markers for whitefly recognition.

Bemisia tabaci Gennadius biotypes B and Q are two of the most important worldwide agricultural insect pests. Genomic sequences of Type-2 B. tabaci chemosensory protein (BtabCSP2) were cloned and sequenced in B and Q biotypes, revealing key biotype-specific variations in the intron sequence. A Q260 sequence was found specifically in Q-BtabCSP2 and Cucumis melo LN692399, suggesting ancestral horizontal transfer of gene between the insect and the plant through bacteria. A cleaved amplified polymorphic sequences (CAPS) method was then developed to differentiate B and Q based on the sequence variation in exon of BtabCSP2 gene. The performances of CSP2-based CAPS for whitefly recognition were assessed using B. tabaci field collections from Shandong Province (P.R. China). Our SacII based CAPS method led to the same result compared to mitochondrial cytochrome oxidase-based CAPS method in the field collections. We therefore propose an explanation for CSP origin and a new rapid simple molecular method based on genomic DNA and chemosensory gene to differentiate accurately the B and Q whiteflies of the Bemisia complex around the world.

Biotype Characterization, Developmental Profiling, Insecticide Response and Binding Property of Bemisia tabaci Chemosensory Proteins: Role of CSP in Insect Defense.

Chemosensory proteins (CSPs) are believed to play a key role in the chemosensory process in insects. Sequencing genomic DNA and RNA encoding CSP1, CSP2 and CSP3 in the sweet potato whitefly Bemisia tabaci showed strong variation between B and Q biotypes. Analyzing CSP-RNA levels showed not only biotype, but also age and developmental stage-specific expression. Interestingly, applying neonicotinoid thiamethoxam insecticide using twenty-five different dose/time treatments in B and Q young adults showed that Bemisia CSP1, CSP2 and CSP3 were also differentially regulated over insecticide exposure. In our study one of the adult-specific gene (CSP1) was shown to be significantly up-regulated by the insecticide in Q, the most highly resistant form of B. tabaci. Correlatively, competitive binding assays using tryptophan fluorescence spectroscopy and molecular docking demonstrated that CSP1 protein preferentially bound to linoleic acid, while CSP2 and CSP3 proteins rather associated to another completely different type of chemical, i.e. α-pentyl-cinnamaldehyde (jasminaldehyde). This might indicate that some CSPs in whiteflies are crucial to facilitate the transport of fatty acids thus regulating some metabolic pathways of the insect immune response, while some others are tuned to much more volatile chemicals known not only for their pleasant odor scent, but also for their potent toxic insecticide activity.

Molecular characterization and evolution of pheromone binding protein genes in Agrotis moths.

Pheromone-binding proteins (PBPs) are soluble transporter proteins that increase the capture and the solubilization of pheromone molecules in the lymph surrounding the olfactory receptors. A polymerase chain reaction-based method was used to identify PBP genes in Agrotis species for an evolutionary genomic study of noctuid moth PBPs. From genomic DNA we determined the structure of different PBP genes in the two closely related species, Agrotis ipsilon and A. segetum. In all, we clearly identified four genes (Aips-1, Aips-2, Aseg-1 and Aseg-2) that represent two distinct PBP orthology groups. We found that the four genes have the same exon-intron structure and that they comprise three exons and two introns but differ in length mainly in the second intron. The three exons of Aseg-2 and Aips-2 have the same lengths but both intron 1 and intron 2 differ in length between the genes. In contrast, Aips-1 and Aseg-1 show dissimilarity only in the length of intron 2. Interestingly, introns 1 and 2 are inserted in the same positions in the Aips-1, Aips-2, Aseg-1 and Aseg-2 genes. These findings show that the Agrotis PBP genes have common ancestry and probably originate from gene duplication before the speciation of ipsilon and segetum. We found that expression of Aips-1/Aseg-1 and Aips-2/Aseg-2 is antennal-specific, but expression is not restricted to the male antennae.

Increased expression of CSP and CYP genes in adult silkworm females exposed to avermectins.

We analyzed 20 chemosensory protein (CSP) genes of the silkworm Bombyx mori. We found a high number of retrotransposons inserted in introns. We then analyzed expression of the 20 BmorCSP genes across tissues using quantitative real-time polymerase chain reaction (PCR). Relatively low expression levels of BmorCSPs were found in the gut and fat body tissues. We thus tested the effects of endectocyte insecticide abamectin (B1a and B1b avermectins) on BmorCSP gene expression. Quantitative real-time PCR experiments showed that a single brief exposure to insecticide abamectin increased dramatically CSP expression not only in the antennae but in most tissues, including gut and fat body. Furthermore, our study showed coordinate expression of CSPs and metabolic cytochrome P450 enzymes in a tissue-dependent manner in response to the insecticide. The function of CSPs remains unknown. Based on our results, we suggest a role in detecting xenobiotics that are then detoxified by cytochrome P450 anti-xenobiotic enzymes.

Molecular evidence of RNA editing in Bombyx chemosensory protein family.

Chemosensory proteins (CSPs) are small scavenger proteins that are mainly known as transporters of pheromone/odor molecules at the periphery of sensory neurons in the insect antennae and in the producing cells from the moth female pheromone gland. Sequencing cDNAs of RNA encoding CSPs in the antennae, legs, head, pheromone gland and wings from five single individual adult females of the silkworm moth Bombyx mori showed that they differed from genomic sequences by subtle nucleotide replacement (RDD). Both intronless and intronic CSP genes expressed RDDs, although in different rates. Most interestingly, in our study the degree of RDDs in CSP genes were found to be tissue-specific. The proportion of CSP-RDDs was found to be significantly much higher in the pheromone gland. In addition, Western blot analysis of proteins in different tissues showed existence of multiple CSP protein variant chains particularly found in the pheromone gland. Peptide sequencing demonstrated the occurrence of a pleiad of protein variants for most of all BmorCSPs from the pheromone gland. Our findings show that RNA editing is an important feature in the expression of CSPs and that a high variety of RDDs is found to expand drastically thus altering the repertoire of CSP proteins in a tissue-specific manner.

Structure of Bombyx mori chemosensory protein 1 in solution.

Chemosensory Proteins (CSPs) represent a family of conserved proteins found in insects that may be involved in chemosensory functions. BmorCSP1 is expressed mainly in antennae and legs of the silkworm moth Bombyx mori and was cloned from antennal cDNA. Here we report the determination of the structure of Bombyx mori CSP1 (BmorCSP1) by NMR. The overall fold of BmorCSP1 is globular and comprises six alpha-helices. These helices span residues 10-14, 17-27, 35-49, 57-72, 75-85, and 92-100. The internal hydrophobic sides of the helices are formed mostly by leucine and isoleucine residues and, therefore, well suited to constitute a binding site for hydrophobic ligands.