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PubMed2003

A candidate olfactory receptor subtype highly conserved across different insect orders.

Candidate olfactory receptors of the moth Heliothis virescens were found to be extremely diverse from receptors of the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae, but there is one exception. The moth receptor type HR2 shares a rather high degree of sequence identity with one olfactory receptor type both from Drosophila (Dor83b) and from Anopheles (AgamGPRor7); moreover, in contrast to all other receptors, this unique receptor type is expressed in numerous antennal neurons. Here we describe the identification of HR2 homologues in two further lepidopteran species, the moths Antheraea pernyi and Bombyx mori, which share 86-88% of their amino acids. In addition, based on RT-PCR experiments HR2 homologues were discovered in antennal cDNA of the honey bee (Apis mellifera; Hymenoptera), the blowfly (Calliphora erythrocephala; Diptera) and the mealworm (Tenebrio molitor; Coleoptera). Comparison of all HR2-related receptors revealed a high degree of sequence conservation across insect orders. In situ hybridization of antennal sections from the bee and the blowfly support the notion that HR2-related receptors are generally expressed in a very large number of antennal cells. This, together with the high degree of conservation suggests that this unique receptor subtype may fulfill a special function in chemosensory neurons of insects.

Amino Acid Sequence↗
PubMed2006

Delta11 desaturases of Trichoplusia ni and Spodoptera littoralis exhibit dual catalytic behaviour.

The Delta(11) desaturases found in moths such as Spodoptera littoralis play a critical role in the biosynthesis of their sex pheromones. The ability to functionally express these enzymes in yeast has allowed one to study the transformation of long-chain fatty acyl substrates to their 11-ene products in greater mechanistic detail. In this article, we report on the detection and quantitation of a minor 11-hydroxylated byproduct (0.1% of total fatty acids), which is formed by the Delta(11) desaturases found in Trichoplusia ni and Spodoptera littoralis. The position of the hydroxyl group was determined by characteristic mass spectral fragmentation of the trimethylsilyl derivatives and is in accord with predictions based on previous mechanistic investigations of the Spodoptera Delta(11) desaturase. The level of 11-hydroxylation was insensitive to the mode of desaturase expression (constitutive vs. induced) and the presence or absence of a b5-fusion domain. Our findings suggest that in future, a search for hydroxylated products should be included in functional analyses of insect desaturase genes.

Animals↗
PubMed2004

Olfaction: mosquito receptor for human-sweat odorant.

Female Anopheles mosquitoes, the world's most important vector of Plasmodium falciparum malaria, locate their human hosts primarily through olfactory cues, but the molecular mechanisms that underlie this recognition are a mystery. Here we show that the Anopheles gambiae protein AgOr1, a female-specific member of a family of putative odorant receptors, responds to a component of human sweat. Compounds designed to activate or block receptors of this type could function as attractants for trapping mosquitoes or as insect repellents in helping to control Anopheles and other insect pests.

Animals↗
PubMed1996

Identification of insect cell lines by DNA amplification fingerprinting (DAF).

Fingerprint profiles were generated from twenty insect cell lines spanning the Orders, Lepidoptera, Diptera, Coleoptera and Homoptera employing DNA amplification fingerprinting (DAF) with arbitrarily selected primers. The fingerprint pattern is a stable characteristic of the cell line because high and low passages generated the same profile. In addition, insect hosts and homologous cell lines generated similar profiles. All cell lines could be distinguished from each other with the following exceptions: Plutella xylostella (BCIRL-PX2-HNU3) and Mamestra brassicae (IZD-MB-0503) produced identical patterns to Trichoplusia ni (TN-CL1). Also Spodoptera exigua (UCR-SE-1C) produced the same profile as Spodoptera frugiperda (SF9) and the parent cell line IPLB-SF21. All these cases of identical patterns are believed to be due to cross contamination or mislabelling of cultures. DAF will serve as an additional, valuable and reliable technique for the identification of insect cell lines.

Aedes↗
PubMed2005

Insect sex-pheromone signals mediated by specific combinations of olfactory receptors.

We describe two male-specific olfactory receptors (ORs) in the silk moth, Bombyx mori, that are mutually exclusively expressed in a pair of adjacent pheromone-sensitive neurons of male antennae: One is specifically tuned to bombykol, the sex pheromone, and the other to bombykal, its oxidized form. Both pheromone ORs are coexpressed with an OR from the highly conserved insect OR subfamily. This coexpression promotes the functional expression of pheromone receptors and confers ligand-stimulated nonselective cation channel activity. The same effects were also observed for general ORs. Both odorant and pheromone signaling pathways are mediated by means of a common mechanism in insects.

Alkadienes↗
PubMed1997

Electromechanical microinjection of pink bollworm Pectinophora gossypiella embryos increases survival.

We report and compare methods and apparatus for injection of pink bollworm, Pectinophora gossypiella (Saunders), embryos. Injection with an electromechanical device resulted in 59% survival of embryos. Previous techniques relying on a mechanical manipulator resulted in 8% survival. Pink bollworm (PBW) embryo injection technology is based in part on methods developed for injection and genetic transformation of Caenorhabditis elegans ([Maupas] Dougherty) and Drosophila melanogaster (Meigen) with substantial alterations in both method and apparatus to accommodate special conditions of PBW biology, behavior and morphology. The microinjection methodology described here has direct application to other difficult-to-inject insects and invertebrates.

Animals↗
PubMed2000

Organization, evolution and expression of a multigene family encoding putative members of the odourant binding protein family in the medfly Ceratitis capitata.

A multigene family encoding male specific serum polypeptides (MSSPs) that show significant structural similarity to the family of insect odourant binding proteins, has been characterized in the medfly Ceratitis capitata. This family comprises seven members classified in three subgroups, MSSP-alpha, MSSP-beta and MSSP-gamma. The genes of subgroups alpha and beta are clustered in tandem in a 35-kb genomic region, and present an exceptionally high degree of similarity not only in their coding but also in the surrounding regions, while the genes of the gamma subgroup are drastically divergent. Although MSSPs are predominantly expressed in the male fat body, detailed expression studies suggest that individual members of this family are expressed in a distinct sex- and tissue-specific manner.

Amino Acid Sequence↗
PubMed1994

The brain secretory peptides that control moulting and metamorphosis of the silkmoth, Bombyx mori.

Progress made toward the elucidation of molecular features of the prothoracicotropic hormone (PTTH) of the silkmoth Bombyx mori is reviewed. PTTH stimulates the prothoracic glands to synthesize and release ecdysone, and is therefore a key hormone for the regulation of insect moulting and metamorphosis. Bombyx PTTH is a 30 kDa homodimeric glycoprotein, whose carbohydrate moiety is not essential for the biological function. The Bombyx genome contains a single copy of the PTTH gene. PTTH is produced by four dorsolateral neurosecretory cells of brain. Another Bombyx brain peptide exerting prothoracicotropic activity to a heterologous moth Samia cynthia ricini but no activity to Bombyx has been identified and termed bombyxin. Bombyxin is a 5 kDa heterodimeric peptide that shows a high similarity to insulin in the amino acid sequence. The bombyxin gene structure also shows a high similarity with the insulin gene structure. The Bombyx genome contains more than 30 copies of the bombyxin gene. Bombyxin is synthesized by eight dorsomedial neurosecretory cells of brain.

Amino Acid Sequence↗
PubMed1999

Mitochondrial genes collectively suggest the paraphyly of Crustacea with respect to Insecta.

Complete sequences of seven protein coding genes from Penaeus notialis mitochondrial DNA were compared in base composition and codon usage with homologous genes from Artemia franciscana and four insects. The crustacean genes are significantly less A + T-rich than their counterpart in insects and the pattern of codon usage (ratio of G + C-rich versus A + T-rich codon) is less biased. A phylogenetic analysis using amino acid sequences of the seven corresponding polypeptides supports a sister-taxon status for mollusks-annelid and arthropods. Furthermore, a distance matrix-based tree and two most-parsimonious trees both suggest that crustaceans are paraphyletic with respect to insects. This is also supported by the inclusion of Panulirus argus COII (complete) and COI and COIII (partial) sequence data. From analysis of single and combined genes to infer phylogenies, it is observed that obtained from single genes are not well supported in most topologies cases and notably differ from that of the tree based on all seven genes.

Animals↗
PubMed1997

Role of the Drosophila EGF receptor in determination of the dorsoventral domains of escargot expression during primary neurogenesis.

BACKGROUND: Primary neurogenesis in the central nervous system of insects and vertebrates occurs in three dorsoventral domains in each side of the neuroectoderm. Among the three dorsoventral domains of the Drosophila neuroectoderm, the medial and lateral columns express the zinc-finger gene escargot (esg), whereas the intermediate column does not. We studied esg expression as a probe to investigate the mechanism of neuroectoderm patterning. RESULTS: The effect of dorsoventral patterning genes on esg expression was studied. decapentaplegic, snail and twist were found to repress esg expression outside the neuroectoderm. The expression of esg in the intermediate column is normally repressed, but was de-repressed when the EGF receptor homologue (DER) activity was either elevated or reduced. A neurogenic enhancer of esg was identified, and was shown to be separable into a distal region that promotes ubiquitous expression in the neuroectoderm and a proximal region that represses the intermediate expression. CONCLUSIONS: decapentaplegic, snail, twist and an activator act through the distal region to initiate transcription of esg in the neuroectoderm. We propose that the combination of opposing gradients of DER and its ligand creates a peak of DER activity in the intermediate column where DER represses esg transcription through the proximal repressor region. These two kinds of regulation establish the early esg expression that prefigures the neuroectoderm patterning.

Animals↗
PubMed1998

Functionality and cell anchorage dependence of the African swine fever virus gene A179L, a viral bcl-2 homolog, in insect cells.

The African swine fever virus gene A179L has been shown to be a functional member of the ced9/bcl-2 family of apoptosis inhibitors in mammalian cell lines. In this work we have expressed the A179L gene product (p21) under the control of the baculovirus polyhedrin promoter using a baculovirus system. Expression of the A179L gene neither altered the baculovirus replication phenotype nor delayed the shutoff of cellular protein synthesis, but it extended the survival of the infected insect cells to very late times postinfection. The increase in cell survival rates correlated with a marked apoptosis reduction after baculovirus infection. Interestingly, prevention of apoptosis was observed when recombinant baculovirus infections were carried out in monolayer cell cultures but not when cells were infected in suspension, suggesting a cell anchorage dependence for p21 function in insect cells. Cell survival was enhanced under optimal conditions of cell attachment and cell-to-cell contact as provided by extracellular matrix components or poly-D-lysine. Since it was observed that cytoskeleton organization varied depending on culture conditions of insect cells (grown in monolayer versus grown in suspension), these results suggested that A179L might regulate apoptosis in insect cells only when the cytoskeletal support of intracellular signaling is maintained upon cell adhesion. Thus, cell shape and cytoskeleton status might allow variations in intracellular transduction of signals related to cell survival in virus-infected cells.

African Swine Fever Virus↗
PubMed2000

Purification and molecular cloning of chemosensory proteins from Bombyx mori.

Soluble low molecular weight acidic proteins are suspected to transport stimulus molecules to the sensory neurons within insect sensilla. From the antennae of Bombyx mori, we have purified and sequenced a protein (BmorCSP1) bearing sequence similarity to a class of soluble chemosensory proteins recently discovered in several orders of insects. Based on its N-terminal sequence, the cDNA encoding this protein has been amplified and cloned. Differential screening of a B. mori antennal cDNA library led to the identification of a second gene encoding a related protein (BmorCSP2), sharing 35-40% identity to BmorCSP1 and chemosensory proteins from other species. The predicted secondary structures of moth's, chemosensory proteins comprise alpha-helical foldings at conserved positions and a reduced hydrophobicity with respect to this novel family of chemosensory proteins.

Amino Acid Sequence↗
PubMed2006

Insights into social insects from the genome of the honeybee Apis mellifera.

Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A+T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A. mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A. mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement.

Animals↗
PubMed1990

Structural and immunological characterization of human cytomegalovirus gp55-116 (gB) expressed in insect cells.

The gene encoding the major envelope glycoprotein complex, gp55-116 (gB), of human cytomegalovirus (HCMV) was expressed at high levels in insect cells utilizing a recombinant baculovirus. The mature intracellular form of the insect-derived gp55-116 was a protein of Mr 150K which contained approximately 50K of N-linked oligosaccharides. The oligosaccharide linkages were almost exclusively endoglycosidase H-sensitive. The 150K protein was processed, presumably by proteolytic cleavage, to yield at least one of the previously defined cleavage products of the gp55-116. This processing step was significantly less efficient in insect cells than the analogous step in mammalian cells. Finally, the insect-derived gp55-116 was highly immunogenic in experimental animals and readily recognized by antibodies contained within HCMV-immune human serum, suggesting that this recombinant protein warrants further study as a potential HCMV subunit vaccine candidate.

Animals↗
PubMed2004

Identification of a chemosensory receptor from the yellow fever mosquito, Aedes aegypti, that is highly conserved and expressed in olfactory and gustatory organs.

Aedes aegypti is a highly anthropophilic mosquito responsible for the transmission of dengue and yellow fever around the world. Like other mosquitoes, the biting and host preference behaviors of this disease vector are largely influenced by its sense of smell, which is presumably facilitated by G protein-coupled receptor signaling cascades. Here, we report the identification and characterization of AaOr7, the first candidate odorant receptor from Ae. aegypti. This receptor displays extremely high primary amino acid conservation with AgOr7 another candidate odorant receptor identified in the Aftrotropical malaria vector, Anopheles gambiae as well as several previously identified candidate odorant receptors in Drosophila melanogaster and other insects. Its transcript is expressed in adult chemosensory tissues and during several stages of Ae. aegypti development. Within the adult olfactory system, AaOr7 protein is found specifically within most antennal and maxillary palp sensilla, as well as in a subset of proboscis sensilla. These results are consistent with a role for AaOr7 in olfaction and gustation supporting the hypothesis that AaOr7 and its orthologs may be of general importance to chemosensory processes throughout the lifetime of an insect.

Aedes↗
PubMed1998

Parallel molecular genetic pathways operate during CNS metamorphosis in Drosophila.

Insect metamorphosis provides a valuable model for studying mechanisms of steroid hormone action on the nervous system during a dynamic phase of functional remodeling. The Drosophila Broad Complex (BRC) holds a pivotal position in the gene expression cascade triggered by the molting hormone 20-hydroxyecdysone (20E) at the onset of metamorphosis. We previously demonstrated that the BRC, which encodes a family of zinc-finger transcription factors, is essential for transducing 20E signals into the morphogenetic movements and cellular assembly that alter the CNS from juvenile to adult form and function. We set out to examine the relationship of BRC to two other genes, IMP-E1 and Deformed (Dfd), involved in the metamorphic transition of the CNS. Representatives of the whole family of BRC transcript isoforms accumulate in the CNS during the larval-to-pupal transition and respond directly to 20E in vitro. IMP-E1 is also directly regulated by 20E, but its induction is independent of BRC, revealing that 20E works through at least two pathways in the CNS. DFD expression is also independent of BRC function. Surprisingly, BRC and DFD proteins are expressed in distinct, nonoverlapping subsets of neuronal nuclei of the subesophageal ganglion even though both are required for its migration into the head capsule. This suggests that the segment identity and ecdysone cascades operate in parallel to control region-specific reorganization during metamorphosis.

Animals↗
PubMed2002

In situ hybridization to the Rdl locus on polytene chromosome 3L of Anopheles stephensi.

We are interested in generating a Y-autosome translocation of the Resistance to dieldrin (Rdl) locus in the malaria vector mosquito Anopheles stephensi Liston (Diptera: Culicidae), for use in sterile insect release. To ensure stability of the system, a recombination suppressing inversion can also be induced which encompasses the Rdl locus. As a first step, here we report the cloning of fragments of the Rdl gene from both An. stephensi and An. gambiae Giles using degenerate primers in the polymerase chain reaction. These fragments encode the second membrane-spanning region of the gamma-aminobutyric acid receptor and show high levels of both nucleotide and predicted amino acid identity to other Rdl-like receptors. They confirm that, as in all other arthropod species examined, dieldrin resistance in An. stephensi is associated with replacement of alanine302, in this case with a serine. In situ hybridization of the Rdl probe to polytene chromosomes of An. stephensi localizes the gene to the left arm of chromosome 3 (3L) in region 45C. Rdl localization will enable us to identify chromosomal rearrangements encompassing the Rdl locus and help anchor the genome sequence of An. gambiae to the polytene map.

Amino Acid Sequence↗
PubMed2000

Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis.

Wounding in multicellular eukaryotes results in marked changes in gene expression that contribute to tissue defense and repair. Using a cDNA microarray technique, we analyzed the timing, dynamics, and regulation of the expression of 150 genes in mechanically wounded leaves of Arabidopsis. Temporal accumulation of a group of transcripts was correlated with the appearance of oxylipin signals of the jasmonate family. Analysis of the coronatine-insensitive coi1-1 Arabidopsis mutant that is also insensitive to jasmonate allowed us to identify a large number of COI1-dependent and COI1-independent wound-inducible genes. Water stress was found to contribute to the regulation of an unexpectedly large fraction of these genes. Comparing the results of mechanical wounding with damage by feeding larvae of the cabbage butterfly (Pieris rapae) resulted in very different transcript profiles. One gene was specifically induced by insect feeding but not by wounding; moreover, there was a relative lack of water stress-induced gene expression during insect feeding. These results help reveal a feeding strategy of P. rapae that may minimize the activation of a subset of water stress-inducible, defense-related genes.

Animals↗