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PubMed2006

Phylogeny of the corticotropin-releasing factor family of peptides in the metazoa.

The corticotropin-releasing factor (CRF) family of peptides consists of four distinct paralogs found on separate chromosomes in vertebrates. Among invertebrates, the family has been relatively well characterized in the insects where at least 2 or 3 paralogs, a CRF-binding protein ortholog and a CRF receptor variant have been found. The conservation of structure and function of this system in insects imply that the CRF system evolved in ancestral species well before the Precambrian explosion. The CRF family peptides association with diuresis and feeding may have developed early in its evolution. However, CRF's role in the hypothalamo-pituitary-adrenal axis and regulation of the glucocorticoids in association with energy metabolism appears to have developed in the chordate lineage.

Amino Acid Sequence↗
PubMed1999

Conserved features at the 5 end of Drosophila R2 retrotransposable elements: implications for transcription and translation.

R2 non-LTR retrotransposable elements insert site-specifically into the 28S ribosomal genes of insects. The sequence of the 5' end of full-length R2 elements from thirteen species of Drosophila were compared. Sequences within the 5' untranslated region (5' UTR) revealed little to suggest the presence of a promoter. Protein translation initiates within the 5' UTR and requires the bypassing of a highly conserved termination codon preceding the single R2 open reading frame. This bypassing probably involves a conserved RNA secondary structure which brings a potential initiation codon into close proximity to this termination codon. The most highly conserved sequence within the 5' UTR has properties similar to internal ribosomal entry sites. Based on these findings, we propose that R2elements are co-transcribed with the 28S gene and are translated as part of a large ribosomal subunit.

5' Untranslated Regions↗
PubMedDate not supplied

Molecular cloning of a putative voltage- and cyclic nucleotide-gated ion channel present in the antennae and eyes of Drosophila melanogaster.

The amino acid sequence BCNG-1 (brain cyclic nucleotide gated 1, of the mouse), the first member of mamalian I(h) channels, was used to construct a set of polymerase chain reaction (PCR) primers from possibly conserved regions. Reverse transcription-PCR with Drosophila melanogaster mRNA yielded in a PCR product, which exhibited a high homology to BCNG-1. Using these PCR products to screen a D. melanogaster head cDNA library we isolated a cDNA encoding a member of a new class of putative voltage- and cyclic nucleotide-gated potassium channels from D. melanogaster. The most important features of the amino acid sequence predicted from the cDNA were a C-terminal cyclic nucleotide-binding region, an S4-voltage sensor and a putative potassium-selective pore-forming motif. The high homology of 51% to the sea urchin I(h) channel, which belongs to the same class of ion channels as BCNG-1, leads us to suggest that the Drosophila cDNA is the first insect member of a new class of hyperpolarization-activated and cyclic nucleotide-gated channels. As shown by in situ hybridization, a pronounced mRNA expression was detected in neuronal tissue, including sensory tissue like the compound eyes, and the olfactory and the auditory organs.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular characterization of a defensin in the IZD-MB-0503 cell line derived from immunocytes of the insect Mamestra brassicae (Lepidoptera).

The induction of anti-microbial peptides against Gram positive and negative bacteria in the IZD-MB-0503 cell line from the lepidopteran Mamestra brassicae is demonstrated, while no anti-fungal activity is detected. The identification of a defensin-like molecule active against Gram positive bacteria is described for the first time in Lepidoptera. This molecule shows between 43% and 59% homology with group A defensins from other dipteran and hymenopteran species.

Amino Acid Sequence↗
PubMed1992

Molecular biological approaches to the study of vectors in relation to malaria control.

To a large extent, control of malaria vectors relies on the elimination of breeding sites and the application of chemical agents. There are increasing problems associated with the use of synthetic insecticides for vector control, including the evolution of resistance, the high cost of developing and registering new insecticides and an awareness of pollution from insecticide residues. These factors have stimulated interest in the application of molecular biology to the study of mosquito vectors of malaria; focussing primarily on two aspects. First, the improvement of existing control measures through the development of simplified DNA probe systems suitable for identification of vectors of malaria. The development of synthetic, non-radioactive DNA probes suitable for the identification of species in the Anopheles gambiae complex is described with the aim of defining a simplified methodology which is suitable for entomologist in the field. The second aspect to be considered is the development of completely novel strategies through the genetic manipulation of insect vectors of malaria in order to alter their ability to transmit the disease. The major requirements for producing transgenic mosquitoes are outlined together with the progress which has been made to date and discussed in relation to the prospects which this type of approach has for the future control of malaria.

Animals↗
PubMed2002

Genetic and cytogenetic analysis of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae).

The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactmcera oleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomes including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubules of this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b) the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter as an accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence of toroid tips of the chromosome arms. The in situ hybridization of genes or DNA sequences to the salivary gland polytene chromosomes of B. oleae provided molecular markers for all five autosomes and permitted the establishment of chromosomal homologies among B. olea, B. tryoni and Ceratitis capitata. The heat shock response of B. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophila melanogaster.

Animals↗
PubMed2006

Olfactory responses in a gustatory organ of the malaria vector mosquito Anopheles gambiae.

The proboscis is an important head appendage in insects that has primarily been thought to process gustatory information during food intake. Indeed, in Drosophila and other insects in which they have been identified, most gustatory receptors are expressed in proboscis neurons. Our previous characterization of the expression of AgOR7, a highly conserved odorant receptor (OR) of the Afrotropical malaria vector mosquito Anopheles gambiae in the labellum at the tip of the proboscis was suggestive of a potential olfactory function in this mosquito appendage. To test this hypothesis, we used electrophysiological recording and neuronal tracing, and carried out a molecular characterization of candidate OR expression in the labellum of A. gambiae. These studies have uncovered a set of labial olfactory responses to a small spectrum of human-related odorants, such as isovaleric acid, butylamine, and several ketones and oxocarboxylic acids. Molecular analyses indicated that at least 24 conventional OR genes are expressed throughout the proboscis. Furthermore, to more fully examine AgOR expression within this tissue, we characterized the AgOR profile within a single labial olfactory sensillum. This study provides compelling data to support the hypothesis that a cryptic set of olfactory neurons that respond to a small set of odorants are present in the mouth parts of hematophagous mosquitoes. This result is consistent with an important role for the labellum in the close-range discrimination of bloodmeal hosts that directly impacts the ability of A. gambiae to transmit malaria and other diseases.

Animals↗
PubMed2002

Expression patterns of hedgehog, wingless, and decapentaplegic during gut formation of Gryllus bimaculatus (cricket).

We observed expression patterns of hedgehog (hh), wingless (wg), and decapentaplegic (dpp) during gut development of Gryllus bimaculatus (the cricket), a typical hemimetabolous insect, and compared with those observed in Drosophila, a typical holometabolous insect. Gryllus hh(Gbhh) and Gbwg are expressed in both foregut and hindgut, while Gbdpp is expressed only in the hindgut: at the boundaries between the small and large intestine, and between the large intestine and rectum. Although the expression patterns of Gbhh and Gbwg are essentially comparable to those observed in Drosophila, the expression pattern of Gbdpp differs from those of the Drosophila dpp.

Animals↗
PubMed1999

Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1). Functional and structural characterization.

Pheromone binding proteins (PBPs) are small proteins (17 kDa on average) present at high concentrations ( approximately 10 mM) in the sensillum lymph of Lepidoptera antennae, where they play a key role in the perception of pheromones. By expression in Escherichia coli, we have obtained large quantities (2-3 mg.L-1) of pure, soluble, Mamestra brassicae PBP1 (MbraPBP1). These quantities are compatible with the requirements of X-ray and NMR studies. The recombinant protein has been characterized by native-polyacrylamide gel electrophoresis, Western blotting, N-terminal sequencing, mass spectrometry, gel filtration, circular dichroism, and NMR. Moreover, the recombinant MbraPBP1 has been shown to be able to bind the specific pheromone and a structural analogue, Z11-16:TFMK (cis-11-hexadecenyl trifluoromethyl ketone), in displacement experiments. Our results on MbraPBP1 confirm and extend previous findings on PBPs. MbraPBP1 and two PBPs from different species have been found to exist as dimers under nondenaturing conditions. The CD and structural prediction data confirm a markedly helical structure for insect PBPs rather than the beta-barrel fold found in vertebrates odorant binding proteins. We have tentatively identified the location of the helices and the short beta-strands with respect to the binding site. Currently we have obtained small diffracting crystals of the recombinant MbraPBP1 and determined their space group and molecular content.

Amino Acid Sequence↗
PubMed1996

Inducible immune factors of the vector mosquito Anopheles gambiae: biochemical purification of a defensin antibacterial peptide and molecular cloning of preprodefensin cDNA.

Larvae of the mosquito vector of human malaria, Anopheles gambiae, were inoculated with bacteria and extracts were biochemically fractionated by reverse-phase HPLC. Multiple induced polypeptides and antibacterial activities were observed following bacterial infection, including a member of the insect defensin family of antibacterial proteins. A cDNA encoding An. gambiae preprodefensin was isolated using PCR primers based on phylogenetically conserved sequences. The mature peptide is highly conserved, but the signal and propeptide segments are not, relative to corresponding defensin sequences of other insects. Defensin expression is induced in response to bacterial infection, in both adult and larval stages. In contrast, pupae express defensin mRNA constitutively. Defensin expression may prove a valuable molecular marker to monitor the An. gambiae host response to infection by parasitic protozoa of medical importance.

Amino Acid Sequence↗
PubMed2006

Detection of a gypsy-like sequence in the genome of the cat flea Ctenocephalides felis (Bouché 1835).

The gypsy virus of Drosophila melanogaster is the first identified retrovirus of insects. Its infectious properties have been reported, and it is able to cause diverse mutations of the phenotype in the fruit fly. As a typical endogenous retrovirus, it is transmitted vertically as part of the host genome, but horizontal transmission has also been shown for the D. melanogaster gypsy virus. Using polymerase chain reaction with degenerate primers designed from the gypsy element of Drosophila subobscura, a gypsy-like sequence was amplified from the genome of the cat flea Ctenocephalides felis in a pool of laboratory and field strains. High identities with other gypsy-like sequences of Drosophila spp. can be detected. In contrast to some of these gypsy sequences, the 410-bp fragment of C. felis possesses two deletions, which are localised in the env gene region. Because of these deletions and the lack of virus particles in C. felis, the criteria for host infectivity are not fulfilled. Nevertheless, the general detection of a gypsy-like sequence in C. felis and the capability of the ectoparasite to act as transmitter of viral diseases can be of importance for the cat flea's position in the epidemiology of diseases, which are accompanied by a higher rate of mutation.

Animals↗
PubMed2006

The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta.

Bacterial recognition in the lepidopteran insect, Manduca sexta, is mediated by pattern recognition proteins including Hemolin, Peptidoglycan recognition protein (PGRP) and Immulectin-2. These proteins bind to molecular patterns present on the surface of bacteria and trigger a protective response involving humoral and cellular reactions. Cellular mechanisms mediated by haemocytes include phagocytosis, encapsulation, and the formation of melanotic nodules. Here, we show that a non-pathogenic strain of Escherichia coli induces mRNA transcription and protein expression of Hemolin and PGRP but not Immulectin-2 in Manduca haemocytes. This upregulation can be effectively prevented (knocked-down) using RNA interference (RNAi) following injection of double-stranded (ds) RNA. Knock-down of Hemolin significantly decreased the ability of insects to clear E. coli from the haemolymph and caused a reduction in the number of free haemocytes. RNAi of Hemolin reduced the ability of haemocytes to engulf bacteria through phagocytosis and to form melanotic nodules in vivo. Importantly, washed haemocytes taken from RNAi-treated insects showed reduced ability to form microaggregates around bacteria in vitro. This shows that the immune function affected by RNAi knock-down of Hemolin is intrinsic to the haemocytes. In contrast, RNAi of PGRP had no effect on any of these cellular immune functions. These results demonstrate the vital role of Hemolin in Manduca cellular immune responses.

Animals↗
PubMed2006

Fat-body remodeling in Drosophila melanogaster.

The remodeling of the larval fat body is observed in many insects during metamorphosis, but little is known about the physiological importance or the regulation of this process. In Drosophila melanogaster, fat-body remodeling involves the dissociation of the fat body into individual fat cells, which persist throughout pupal development but are later removed by cell death in the young adult. Inhibition of fat-body dissociation is associated with pharate adult lethality and thus is likely to be an essential developmental event. As a start toward understanding the role of fat-body remodeling in the life history of insects, we carried out a detailed study of fat-body disassociation in D. melanogaster using fluorescent microscopy, and tested whether this process is mediated by hemocytes as proposed for fat-body remodeling in Sarcophaga peregrina. We identified and correlated stereotypic events in fat-body dissociation with developmental changes during metamorphosis, and have demonstrated by cell ablation studies that fat-body remodeling in D. melanogaster is a hemocyte independent process.

Animals↗
PubMed2004

Cloning, expression and functional characterisation of chitinase from larvae of tomato moth (Lacanobia oleracea): a demonstration of the insecticidal activity of insect chitinase.

Chitinases are vital to moulting in insects, and may also affect gut physiology through their involvement in peritrophic membrane turnover. A cDNA encoding chitinase was cloned from larvae of tomato moth (Lacanobia oleracea), a Lepidopteran pest of crops. The predicted protein contains 553 amino acid residues, with a signal peptide of 20 a.a. Sequence comparison showed 75-80% identity with other Lepidopteran chitinases. L. oleracea chitinase was produced as a functional recombinant enzyme in the yeast Pichia pastoris. A fusion protein containing chitinase joined to the N-terminus of snowdrop lectin (GNA) was also produced, to determine whether GNA could deliver chitinase to the haemolymph of Lepidopteran larvae after oral ingestion. The purified recombinant proteins exhibited similar levels of chitinase activity in vitro. Both proteins were highly toxic to L. oleracea larvae on injection, causing 100% mortality at low dose (2.5 microg/g insect). Injection of chitinase prior to the moult resulted in decreased cuticle thickness. The recombinant proteins caused chronic effects when fed, causing reductions in larval growth and food consumption by up to 60%. The oral toxicity of chitinase was not increased by attaching GNA in the fusion protein, due to degradation in the larval gut, preventing GNA acting as a "carrier".

Amino Acid Sequence↗
PubMed2004

Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera.

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are encoded by a single mRNA in the suboesophegeal ganglion (SG) and are responsible for induction of embryonic diapause in Bombyx mori and sex pheromone biosynthesis in lepidopteran insects. PBAN cDNA analyses revealed that the DH-like peptide is present in several species that have a pupal diapause. However, the function of the DH-like peptide remains unknown. In the present study, we cloned the cDNA encoding DH-PBAN in Helicoverpa armigera utilizing the rapid amplification of the cDNA ends method. The nucleotide se quence analysis revealed that the longest open reading frame of this cDNA encodes a 194-amino acid precursor protein that con tains a 33-aa PBAN, a 24-aa DH-like peptide, and three other neuropeptides, all of which have a common C-terminal pentapeptide motif FXPR/KL ( X=G, T, S). A homology search showed that H. armigera DH-like and PBAN are highly homologous to those from other insects. Northern blot analysis demonstrated a single message RNA corresponding to the size of Har-DH-PBAN cDNA from pupal SG with significantly higher expression in the SG of nondiapause pupae than diapausing pupae. Western blot analysis showed DH-like peptide expression from SG of both males and females. When DH-like peptide was injected into nondiapause larvae and pupae, it did not induce diapause, but rather efficiently broke pupal diapause in H. armigera. The ED(50) of DH to terminate pupal diapause is 20 pmol/pupae. The other four FXPRLamide neuropeptides from the DH-PBAN polyprotein precursor have cross activity for diapause termination. These observations therefore suggest a potential role for these FXPRL family peptides in promoting continuous development in several noctuid species. The high expression of this gene in pharate adults and adults indicates that the FXPRL family peptides may have multiple physiological functions.

Amino Acid Sequence↗
PubMed2005

Simple sequence repeat-based consensus linkage map of Bombyx mori.

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an F(1) male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an F(1) female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of approximately 3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated.

Animals↗
PubMedDate not supplied

Pleiotropy, epistasis and new QTL: the genetic architecture of honey bee foraging behavior.

The regulation of division of labor in social insects, particularly in the honey bee (Apis mellifera L.), has received considerable attention from a number of biological subdisciplines, including quantitative and behavioral genetics, because of the high complexity of the behavioral traits involved. The foraging choices of honey bee workers can be accurately quantified, and previous studies have made the foraging behavior of honey bees one of the best studied naturally occurring behavioral phenotypes. Three quantitative trait loci (QTL) have been identified that influence a set of foraging variables, including the concentration of nectar collected and the amount of pollen and nectar brought back to the hive. This study extends previous genetic investigations and represents the most comprehensive investigation of the genetic architecture of these foraging variables. We examined the effects of markers for the three established QTL and for one further candidate gene (Amfor), in two reciprocal backcross populations. These populations were also used to carry out two new QTL mapping studies, with over 400 Amplified Fragment Length Polymorphism (AFLP) markers in each. We detected a variety of effects of the genetic markers for the established QTL and the candidate gene, which were mostly epistatic in nature. A few new QTL could be detected with a variety of mapping techniques. Our results add complexity to the genetic architecture of the foraging behavior of the honey bee. Specifically, we support the hypotheses that pln1, pln2, pln3, and Amfor are involved in the regulation of foraging behavior in the honey bee and add some new factors that deserve further study in the future.

Animals↗