Showing papers in "Insect Molecular Biology in 2006"
TL;DR: It is suggested that an implied reduction in immune flexibility in bees reflects either the strength of social barriers to disease, or a tendency for bees to be attacked by a limited set of highly coevolved pathogens.
Abstract: Social insects are able to mount both group-level and individual defences against pathogens. Here we focus on individual defences, by presenting a genome-wide analysis of immunity in a social insect, the honey bee Apis mellifera. We present honey bee models for each of four signalling pathways associated with immunity, identifying plausible orthologues for nearly all predicted pathway members. When compared to the sequenced Drosophila and Anopheles genomes, honey bees possess roughly one-third as many genes in 17 gene families implicated in insect immunity. We suggest that an implied reduction in immune flexibility in bees reflects either the strength of social barriers to disease, or a tendency for bees to be attacked by a limited set of highly coevolved pathogens.
893 citations
TL;DR: There are some recent radiations in CYP6, CYP9 and certain CCE clades in A. mellifera that could be associated with the evolution of the hormonal and chemosensory processes underpinning its highly organized eusociality.
Abstract: The honeybee genome has substantially fewer protein coding genes (approximate to 11 000 genes) than Drosophila melanogaster (approximate to 13 500) and Anopheles gambiae (approximate to 14 000). Some of the most marked differences occur in three superfamilies encoding xenobiotic detoxifying enzymes. Specifically there are only about half as many glutathione-S-transferases (GSTs), cytochrome P450 monooxygenases (P450s) and carboxyl/cholinesterases (CCEs) in the honeybee. This includes 10-fold or greater shortfalls in the numbers of Delta and Epsilon GSTs and CYP4 P450s, members of which clades have been recurrently associated with insecticide resistance in other species. These shortfalls may contribute to the sensitivity of the honeybee to insecticides. On the other hand there are some recent radiations in CYP6, CYP9 and certain CCE clades in A. mellifera that could be associated with the evolution of the hormonal and chemosensory processes underpinning its highly organized eusociality.
653 citations
TL;DR: It is demonstrated in the horticultural pest, Epiphyas postvittana, that RNAi can be triggered by oral delivery of dsRNA to larvae, and Transcript levels of a larval gut carboxylesterase gene were reduced to less than half that of controls within 2 days of being fed EposCXE1 ds RNA.
Abstract: RNA interference (RNAi) or gene silencing is typically induced in insects by the injection of double-stranded RNAs (dsRNAs), short interfering RNAs, or through the use of hairpin constructs in transgenic insects. Here we demonstrate in the horticultural pest, Epiphyas postvittana (Lepidoptera: Tortricidae), that RNAi can be triggered by oral delivery of dsRNA to larvae. Transcript levels of a larval gut carboxylesterase gene (EposCXE1) were reduced to less than half that of controls within 2 days of being fed EposCXE1 dsRNA. Transcript levels of the pheromone binding protein gene (EposPBP1) were reduced in adult antennae by feeding larvae EposPBP1 dsRNA. Knockdown of EposPBP1 transcripts was observed for the first 2 days after adult eclosion but recovered to wild-type levels at 4 days posteclosion. The potential mechanisms involved in the initiation, movement and amplification of the silencing signal are discussed.
341 citations
TL;DR: Results demonstrate that the insulin pathway is a compelling candidate for pursing the relationship between diet and downstream signals involved in caste determination and differentiation in female honeybees.
Abstract: Female honeybees have two castes, queens and workers. Developmental fate is determined by larval diet. Coding sequences made available through the Honey Bee Genome Sequencing Consortium allow for a pathway-based approach to understanding caste determination. We examined the expression of several genes of the insulin signalling pathway, which is central to regulation of growth based on nutrition. We found one insulin-like peptide expressed at very high levels in queen but not worker larvae. Also, the gene for an insulin receptor was expressed at higher levels in queen larvae during the 2nd larval instar. These results demonstrate that the insulin pathway is a compelling candidate for pursing the relationship between diet and downstream signals involved in caste determination and differentiation.
310 citations
TL;DR: The report eventually is expected to shed light on the evolution of the hymenopteran genome within higher insects, particularly regarding the relative maintenance of conserved rDNA genes, related variable spacer regions and retrotransposable elements.
Abstract: As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome, we expect our report eventually to shed light on the evolution of the hymenopteran genome within higher insects, particularly regarding the relative maintenance of conserved rDNA genes, related variable spacer regions and retrotransposable elements.
278 citations
TL;DR: A comparative analysis of honey bee with Drosophila melanogaster and Anopheles gambiae shows that although the basic components of the antioxidant system are conserved, there are important species differences in the number of paralogs.
Abstract: Antioxidant enzymes perform a variety of vital functions including the reduction of life-shortening oxidative damage. We used the honey bee genome sequence to identify the major components of the honey bee antioxidant system. A comparative analysis of honey bee with Drosophila melanogaster and Anopheles gambiae shows that although the basic components of the antioxidant system are conserved, there are important species differences in the number of paralogs. These include the duplication of thioredoxin reductase and the expansion of the thioredoxin family in fly; lack of expansion of the Theta, Delta and Omega GST classes in bee and no expansion of the Sigma class in dipteran species. The differential expansion of antioxidant gene families among honey bees and dipteran species might reflect the marked differences in life history and ecological niches between social and solitary species.
252 citations
TL;DR: Descriptions of groups of genes displaying major differences in transcript accumulation during the adult mosquito life are presented, including those regulated at each analysed time point and each biochemical pathway or biological processes in which they are involved.
Abstract: With their genome sequenced, Anopheles gambiae mosquitoes now serve as a powerful tool for basic research in comparative, evolutionary and developmental biology. The knowledge generated by these studies is expected to reveal molecular targets for novel vector control and pathogen transmission blocking strategies. Comparisons of gene-expression profiles between adult male and nonblood-fed female Anopheles gambiae mosquitoes revealed that roughly 22% of the genes showed sex-dependent regulation. Blood-fed females switch the majority of their metabolism to blood digestion and egg formation within 3 h after the meal is ingested, in detriment to other activities such as flight and response to environment stimuli. Changes in gene expression are most evident during the first, second and third days after a blood meal, when as many as 50% of all genes showed significant variation in transcript accumulation. After laying the first cluster of eggs (between 72 and 96 h after the blood meal), mosquitoes return to a nongonotrophic stage, similar but not identical to that of 3-day-old nonblood-fed females. Ageing and/or the nutritional state of mosquitoes at 15 days after a blood meal is reflected by the down-regulation of approximately 5% of all genes. A full description of the large number of genes regulated at each analysed time point and each biochemical pathway or biological processes in which they are involved is not possible within the scope of this contribution. Therefore, we present descriptions of groups of genes displaying major differences in transcript accumulation during the adult mosquito life. However, a publicly available searchable database (http://www.angagepuci.bio.uci.edu/) has been made available so that detailed analyses of specific groups of genes based on their descriptions, functions or levels of gene expression variation can be performed by interested investigators according to their needs.
180 citations
TL;DR: In this article, the complete nucleotide sequences of the mitochondrial genome (mitogenome) of the Korean hairstreak, Coreana raphaelis (Lepidoptera: Lycaenidae), were determined.
Abstract: We determined the complete nucleotide sequences of the mitochondrial genome (mitogenome) of the Korean hairstreak, Coreana raphaelis (Lepidoptera: Lycaenidae). The entire mitochondrial DNA (mtDNA) molecule was 15,314 bp long. The C. raphaelis genes were in the same order and orientation as the completely sequenced mitogenomes of other lepidopteran species, except for the presence of an extra copy of tRNA(Ser)(AGN). High similarity in primary sequence and secondary structure between the two tandemly located copies of the tRNA(Ser)(AGN) suggest a recent duplication of an original single tRNA(Ser)(AGN). The DHU arm of the two copies of tRNA(Ser)(AGN) formed a simple loop as seen in many other metazoan mt tRNA(Ser)(AGN). The putative initiation codon for the C. raphaelis COI gene appears to be a tetranucleotide, TTAG, found commonly in the sequenced lepidopterans. ATPase8, ATPase6, ND4L and ND6 genes, which are next to another protein-coding gene at their 3' end all had the sequences potential to form a hairpin structure, suggesting the importance of such a structure for precise cleavage of the mature protein-coding genes.
171 citations
TL;DR: Comparison of the sequences with those from Drosophila led to a proposed SP pathway for establishing the dorsoventral axis of honey bee embryos, and a framework for designing experimental studies of the roles of SPs and related proteins in embryonic development and immune responses of A. mellifera.
Abstract: We have identified 44 serine protease (SP) and 13 serine protease homolog (SPH) genes in the genome of Apis mellifera. Most of these genes encode putative secreted proteins, but four SPs and three SPHs may associate with the plasma membrane via a transmembrane region. Clip domains represent the most abundant non-catalytic structural units in these SP-like proteins −12 SPs and six SPHs contain at least one clip domain. Some of the family members contain other modules for protein–protein interactions, including disulphide-stabilized structures (LDLrA, SRCR, frizzled, kringle, Sushi, Wonton and Pan/apple), carbohydrate-recognition domains (C-type lectin and chitin-binding), and other modules (such as zinc finger, CUB, coiled coil and Sina). Comparison of the sequences with those from Drosophila led to a proposed SP pathway for establishing the dorsoventral axis of honey bee embryos. Multiple sequence alignments revealed evolutionary relationships of honey bee SPs and SPHs with those in Drosophila melanogaster, Anopheles gambiae, and Manduca sexta. We identified homologs of D. melanogaster persephone, M. sexta HP14, PAP-1 and SPH-1. A. mellifera genome includes at least five genes for potential SP inhibitors (serpin-1 through -5) and three genes of SP putative substrates (prophenoloxidase, spatzle-1 and spatzle-2). Quantitative RT-PCR analyses showed an elevation in the mRNA levels of SP2, SP3, SP9, SP10, SPH41, SPH42, SP49, serpin-2, serpin-4, serpin-5, and spatzle-2 in adults after a microbial challenge. The SP41 and SP6 transcripts significantly increased after an injection of Paenibacillus larva, but there was no such increase after injection of saline or Escherichia coli. mRNA levels of most SPs and serpins significantly increased by 48 h after the pathogen infection in 1st instar larvae. On the contrary, SP1, SP3, SP19 and serpin-5 transcript levels reduced. These results, taken together, provide a framework for designing experimental studies of the roles of SPs and related proteins in embryonic development and immune responses of A. mellifera.
164 citations
TL;DR: Two site‐specific integration mechanisms in the yellow fever mosquito, Aedes aegypti, are investigated, one of which was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31.
Abstract: Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germline transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species.
141 citations
TL;DR: Striking changes in gene number or genomic organization are identified for genes encoding glycolytic enzymes, cellulase, glucose oxidase and glucose dehydrogenases, glucose‐methanol‐choline (GMC) oxidoreductases, fucosyltransferases, and lysozymes in the honey bee and mosquito.
Abstract: Carbohydrate-metabolizing enzymes may have particularly interesting roles in the honey bee, Apis mellifera, because this social insect has an extremely carbohydrate-rich diet, and nutrition plays important roles in caste determination and socially mediated behavioural plasticity. We annotated a total of 174 genes encoding carbohydrate-metabolizing enzymes and 28 genes encoding lipid-metabolizing enzymes, based on orthology to their counterparts in the fly, Drosophila melanogaster, and the mosquito, Anopheles gambiae. We found that the number of genes for carbohydrate metabolism appears to be more evolutionarily labile than for lipid metabolism. In particular, we identified striking changes in gene number or genomic organization for genes encoding glycolytic enzymes, cellulase, glucose oxidase and glucose dehydrogenases, glucose-methanol-choline (GMC) oxidoreductases, fucosyltransferases, and lysozymes.
TL;DR: Of the 69 unique honey bee proteins found, 66 are also in Drosophila melanogaster, and there is over‐representation of genes implicated in the glycolysis pathway.
Abstract: Honey bee (Apis mellifera L.) queens mate early in life and store sperm for years. Male bees likely contribute significantly to sperm survival. Proteins were extracted from seminal vesicles and semen of mature drones, separated by electrophoresis, and analysed by peptide mass fingerprinting. Computer searches against three databases, general species, honey bees and fruit flies, were performed. Spectra were used to query the recently generated honey bee genome protein list as well as general species and fruit fly databases. Of the 69 unique honey bee proteins found, 66 are also in Drosophila melanogaster. Two proteins only matched honey bee genes and one is a widespread protein lost from the fly genome. There is over-representation of genes implicated in the glycolysis pathway. Metabolism-associated proteins were found primarily in the seminal vesicle. Male accessory gland proteins as identified in Drosophila rarely had orthologs among proteins found in the honey bee. A complete listing of gel spots chosen including honey bee genome matches and Mascot searches of MALDI-TOF results with statistics is in the Supplementary table. MALDI-TOF spectra and more complete Mascot peptide mass fingerprinting data are available on request. Supplementary figs 1-3 show the stained protein gels.
TL;DR: It is demonstrated that the major sites of adult mosquito CPR expression are oenocytes, mid‐gut epithelia and head appendages, and high CPR expression was also evident in Drosophila oenocyte indicating a general functional role in these insect cells.
Abstract: We describe an in vivo model for investigation of detoxification mechanisms of the mosquito Anopheles gambiae, important for the development of malaria control programmes. Cytochrome P450s are involved in metabolic insecticide resistance and require NADPH cytochrome P450 reductase (CPR) to function. Here we demonstrate that the major sites of adult mosquito CPR expression are oenocytes, mid-gut epithelia and head appendages. High CPR expression was also evident in Drosophila oenocytes indicating a general functional role in these insect cells. RNAi mediated knockdown drastically reduced CPR expression in oenocytes, and to a lesser extent in mid-gut epithelia; the head was unaffected. These flies showed enhanced sensitivity to permethrin, demonstrating a key role for abdominal/mid-gut P450s in pyrethroid metabolism, aiding the development of insecticides.
TL;DR: The genome sequences of two of the major pathogens of honey bees, the bacterium Paenibacillus larvae and the fungus Ascosphaera apis are reported to provide a contrast with pathogenic, benign and freeliving relatives.
Abstract: Genome sequences offer a broad view of host–pathogen interactions at the systems biology level. With the completion of the sequence of the honey bee, interest in the relevant pathogens is heightened. Here we report the genome sequences of two of the major pathogens of honey bees, the bacterium Paenibacillus larvae (causative agent for American foulbrood disease) and the fungus Ascosphaera apis. (causative agent for chalkbrood disease). Ongoing efforts to characterize the genomes of these species can be used to understand and mitigate the effects of two important pathogens, and will provide a contrast with pathogenic, benign and freeliving relatives.
TL;DR: It is suggested that desatF is a crucial enzyme for female pheromone biosynthesis and courtship behaviour in D. melanogaster, which led to a dramatic decrease in female dienes and increase in monoenes paralleled with an increase in copulation latency and a decrease in courtship index and copulation attempts by the males.
Abstract: Drosophila melanogaster shows sexually dimorphic cuticular hydrocarbons, with monoenes produced in males and dienes produced in females. Here we describe a female-specific desaturase gene, desatF. RNAi knock-down led to a dramatic decrease in female dienes and increase in monoenes paralleled with an increase in copulation latency and a decrease in courtship index and copulation attempts by the males. The desatF gene was also expressed in females from D. sechellia, rich in dienes, but not D. simulans, which produce only monoenes. When hydrocarbons were feminized in D. melanogaster males by targeted expression of the transformer gene, the expression of desatF occurred. These results strongly suggest that desatF is a crucial enzyme for female pheromone biosynthesis and courtship behaviour in D. melanogaster.
TL;DR: A new member of the immulectin family, immUlectin‐4 (IML‐4), is reported, which was detected in thefat body of control larvae and was induced in the fat body when larvae were injected with bacteria.
Abstract: Insect C-type lectins function as pattern recognition receptors in innate immunity. In the tobacco hornworm Manduca sexta, we have previously isolated three C-type lectins named immulectins, which are involved in innate immune responses. Here, we report a new member of the immulectin family, immulectin-4 (IML-4). IML-4 mRNA was detected in the fat body of control larvae and was induced in the fat body when larvae were injected with bacteria. Recombinant IML-4 bound to bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and the binding activity was not affected by addition of calcium or EGTA. IML-4 agglutinated bacteria and yeast, and agglutination of Escherichia coli by IML-4 was concentration- and calcium-dependent. IML-4 also enhanced haemocyte encapsulation and melanization.
TL;DR: Proteomics tools are used to identify the biochemical alterations that occur in the head of the cricket Nemobius sylvestris when it is driven to water by the hairworm Paragordius tricuspidatus and it is found that the parasite produces molecules from the Wnt family that may act directly on the development of the central nervous system (CNS).
Abstract: Despite increasing evidence of host phenotypic manipulation by parasites, the underlying mechanisms causing infected hosts to act in ways that benefit the parasite remain enigmatic in most cases. Here, we used proteomics tools to identify the biochemical alterations that occur in the head of the cricket Nemobius sylvestris when it is driven to water by the hairworm Paragordius tricuspidatus. We characterized host and parasite proteomes during the expression of the water-seeking behaviour. We found that the parasite produces molecules from the Wnt family that may act directly on the development of the central nervous system (CNS). In the head of manipulated cricket, we found differential expression of proteins specifically linked to neurogenesis, circadian rhythm and neurotransmitter activities. We also detected proteins for which the function(s) are still unknown. This proteomics study on the biochemical pathways altered by hairworms has also allowed us to tackle questions of physiological and molecular convergence in the mechanism(s) causing the alteration of orthoptera behaviour. The two hairworm species produce effective molecules acting directly on the CNS of their orthoptera hosts.
TL;DR: A proteolytic activity profile from fifth larval instar to new pupae of the lepidopteran Helicoverpa armigera is detailed, suggesting an essential role for cathepsin L in larval moulting.
Abstract: Moulting is an essential process of insect development but little is known about cysteine proteases in the process. Here, we detail a proteolytic activity profile from fifth larval instar to new pupae of the lepidopteran Helicoverpa armigera. At fifth to sixth instar moulting, the activities were significantly higher than those in non-moulting stages, and were inhibited by the cysteine protease inhibitor, 2S, 3S-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64), or by the cathepsin L-selective inhibitor CLIK148. Further, a 1513 bp cathepsin L cDNA (Har-CL) was isolated from the H. armigera larval cuticle and epidermis layer. Har-CL gene expression, which is correlated closely with ecdysone, was higher during larval moulting. Injection of E-64 or CLIK148 resulted in delayed fifth to sixth instar moulting, suggesting an essential role for cathepsin L in larval moulting.
TL;DR: Full length cDNAs encoding two acetylcholinesterases (AChEs) were cloned and characterized from the German cockroach, Blattella germanica, showing that Bgace1 is predominantly transcribed and its transcript is found in almost entire region of inter or motor neurones including the cell bodies and axonal/dendritic branches.
Abstract: Full length cDNAs encoding two acetylcholinesterases (AChEs; Bgace1 and Bgace2) were cloned and characterized from the German cockroach, Blattella germanica. Sequence analyses showed that both genes possess all the typical features of ace, and that Bgace1 is orthologous to the insect ace1 whereas Bgace2 is to the insect ace2. Transcript level of Bgace1 was significantly higher (c. 10 fold) than that of Bgace2 in all 11 tissues examined, suggesting that Bgace1 likely encodes a predominant AChE. Multiple AChE bands were identified by native polyacrylamide gel electrophoresis and isoelectricfocusing from various tissue preparations, among which ganglia produced distinct two major and two minor AChE bands, indicative of the presence of at least two active AChEs. B. germanica AChEs appeared to be mainly localized in the central nervous system as demonstrated by histochemical activity staining, together with quantitative analysis of Bgace transcripts. Fluorescence in situ hybridization of the 1st thoracic ganglion confirmed that Bgace1 is predominantly transcribed and further showed that its transcript is found in almost entire region of inter or motor neurones including the cell bodies and axonal/dendritic branches. Bgace2 transcript is found only in the subset of neurones, particularly in the cell body. In addition, certain neurones were observed to express Bgace1 only.
TL;DR: The honey bee queen and worker castes are a model system for developmental plasticity and reproduction and a first genome‐based initiative to provide an annotated framework for trends in gene regulation during female caste differentiation and reproduction is provided.
Abstract: The honey bee queen and worker castes are a model system for developmental plasticity. We used established expressed sequence tag information for a Gene Ontology based annotation of genes that are differentially expressed during caste development. Metabolic regulation emerged as a major theme, with a caste-specific difference in the expression of oxidoreductases vs. hydrolases. Motif searches in upstream regions revealed group-specific motifs, providing an entry point to cis-regulatory network studies on caste genes. For genes putatively involved in reproduction, meiosis-associated factors came out as highly conserved, whereas some determinants of embryonic axes either do not have clear orthologs (bag of marbles, gurken, torso), or appear to be lacking (trunk) in the bee genome. Our results are the outcome of a first genome-based initiative to provide an annotated framework for trends in gene regulation during female caste differentiation (representing developmental plasticity) and reproduction.
TL;DR: It appears the kdr‐his mutation had multiple evolutionary origins, but that the k dr mutation may have had a single origin, and the impacts of these findings on resistance management are discussed.
Abstract: House flies were collected from four dairies in Maine, New York, North Carolina, and Florida, where high levels of resistance to permethrin have been documented. Regions of two genes, CYP6D1 and Vssc1, having alleles that confer resistance to permethrin (and other pyrethroids) were analysed from individuals at each collection site. The combinations of resistance alleles for Vssc1 and CYP6D1 were highly variable between each state. The resistance allele CYP6D1v1 was found at a high frequency (0.63-0.91) at all sites. Individuals homozygous susceptible for CYP6D1 were very rare and detected only at the dairy in Maine. In addition to the typical Vssc1 mutation responsible for resistance, kdr (L1014F), we also identified individuals with a L1014H mutation. Although house flies homozygous for the L1014H mutation had a lower level of resistance to permethrin, compared to L1014F, the H1014 resistance allele was frequently detected. No individuals with the super-kdr allele (M918T + L1014F) were detected from the field collections. The intron 3 bp downstream of the kdr mutation was found to be extremely variable, providing an opportunity to reconstruct a phylogeny of Vssc1 alleles. Based on this analysis it appears the kdr-his mutation had multiple evolutionary origins, but that the kdr mutation may have had a single origin. The impacts of these findings on resistance management are discussed.
TL;DR: This work sequenced the entire mt genome of the small pigeon louse, Campanulotes bidentatus compar, and part of the mt genomes of nine other species of lice, from six families and the three main suborders of the order Phthiraptera.
Abstract: The arrangement of genes in the mitochondrial (mt) genomes of most insects is the same, or near-identical, to that inferred to be ancestral for insects. We sequenced the entire mt genome of the small pigeon louse, Campanulotes bidentatus compar, and part of the mt genomes of nine other species of lice. These species were from six families and the three main suborders of the order Phthiraptera. There was no variation in gene arrangement among species within a family but there was much variation in gene arrangement among the three suborders of lice. There has been an extraordinary number of gene rearrangements in the mitochondrial genomes of lice!
TL;DR: Results suggest that MdGluCl‐α assists in the expression of MdRdl when the two are coexpressed, and a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdam expression became shorter.
Abstract: Ligand-gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate-gated chloride channel (MdGluCl-alpha) and a gamma-aminobutyric acid (GABA)-gated chloride channel (MdRdl), were cloned from house-flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand-gated channels using two-electrode voltage-clamp electrophysiology. It was found that L-glutamate and GABA activated the MdGluCl-alpha homo-oligomers with an EC(50) value of 30 microM and the MdRdl homo-oligomers with an EC(50) value of 101 microM, respectively. Both channels were chloride ion-permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as gamma-hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin, than the MdGluCl-alpha channel. MdGluCl-alpha required only 1-2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4-7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl-alpha was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl-alpha assists in the expression of MdRdl when the two are coexpressed.
TL;DR: The results open up the possibilities of elucidating salivary gland–parasite interactions and generating transgenic mosquitoes refractory to parasites.
Abstract: Malaria sporozoites invade the mosquito salivary glands and wait in the salivary duct until the next blood feeding. The mechanisms of the process and molecules involved in the salivary gland invasion remain largely unknown. To establish a robust salivary gland-specific transgene expression in Anopheles stephensi, we obtained a salivary gland-specific promoter for a gene encoding anopheline antiplatelet protein (AAPP). The aapp promoter is a female salivary gland-specific and blood meal-inducible strong promoter. Using this promoter, we generated a transgenic An. stephensi expressing abundant Discosoma sp. red fluorescent protein (DsRed) in the distal-lateral lobes of the glands, where the sporozoites invade preferentially. These results open up the possibilities of elucidating salivary gland-parasite interactions and generating transgenic mosquitoes refractory to parasites.
TL;DR: Study of the honey bee provides a model for understanding nuclear receptor function in the adult brain and identification of matched orthologous nuclear receptors in the two genomes reveals the fundamental set of nuclear receptors required to ‘make’ an endopterygote insect.
Abstract: The Drosophila genome encodes 18 canonical nuclear receptors. All of the Drosophila nuclear receptors are here shown to be present in the genome of the honey bee (Apis mellifera). Given that the time since divergence of the Drosophila and Apis lineages is measured in hundreds of millions of years, the identification of matched orthologous nuclear receptors in the two genomes reveals the fundamental set of nuclear receptors required to 'make' an endopterygote insect. The single novelty is the presence in the A. mellifera genome of a third insect gene similar to vertebrate photoreceptor-specific nuclear receptor (PNR). Phylogenetic analysis indicates that this novel gene, which we have named AmPNR-like, is a new member of the NR2 subfamily not found in the Drosophila or human genomes. This gene is expressed in the developing compound eye of the honey bee. Like their vertebrate counterparts, arthropod nuclear receptors play key roles in embryonic and postembryonic development. Studies in Drosophila have focused primarily on the role of these transcription factors in embryogenesis and metamorphosis. Examination of an expressed sequence tag library developed from the adult bee brain and analysis of transcript expression in brain using in situ hybridization and quantitative RT-PCR revealed that several members of the nuclear receptor family (AmSVP, AmUSP, AmERR, AmHr46, AmFtz-F1, and AmHnf-4) are expressed in the brain of the adult bee. Further analysis of the expression of AmUSP and AmSVP in the mushroom bodies, the major insect brain centre for learning and memory, revealed changes in transcript abundance and, in the case of AmUSP, changes in transcript localization, during the development of foraging behaviour in the adult. Study of the honey bee therefore provides a model for understanding nuclear receptor function in the adult brain.
TL;DR: Using a custom made microarray, the expression profile of the detoxification genes in adults, larvae and pupae of the malaria vector A. gambiae was determined and the information this data provides on putative functions of the mosquito detoxification enzymes is discussed.
Abstract: The diverse habitats and diets encountered during the life cycle of an Anopheles mosquito have necessitated the development of extensive families of detoxification enzymes. Expansion of the three detoxification enzyme families (cytochrome P450s, carboxylesterases and glutathione transfereases), has occurred in mosquitoes compared with Drosophila, however, very little is known regarding the developmental expression of theses genes. Using a custom made microarray we determined the expression profile of the detoxification genes in adults, larvae and pupae of the malaria vector A. gambiae. The expression of approximately one quarter of these genes was developmentally regulated. The expression profile of each of these genes and the information this data provides on putative functions of the mosquito detoxification enzymes is discussed.
TL;DR: Gene control in B. mori ASGs differs from that in Drosophila salivary glands, despite both tissues undergoing PCD in response to 20E at pupal metamorphosis, results indicate that EcR‐A and usp‐2, but not EcR'B1 or usp'1, may be components of the ecdysone receptor complex.
Abstract: Programmed cell death (PCD) in Bombyx mori anterior silk glands (ASGs) is triggered by 20-hydroxyecdysone (20E). We examined the expression profiles and effects of 20E on 11 transcription factor genes in the fifth instar to determine whether they demonstrate the hierarchical control seen in Drosophila PCD. Results indicate that EcR-A and usp-2, but not EcR-B1 or usp-1, may be components of the ecdysone receptor complex. Up-regulation of E75A, BHR3, and three BR-C isoforms, but not E75B, appeared to be associated with the induction of PCD. βFTZ-F1 was not expressed during PCD execution. Thus, gene control in B. mori ASGs differs from that in Drosophila salivary glands, despite both tissues undergoing PCD in response to 20E at pupal metamorphosis.
TL;DR: The putative fat body transcriptome is analysed based on homology to other gene products with known functions available in the public domain and the immune‐related products, reproductive function related yolk proteins and milk‐gland protein, iron metabolism regulating ferritins and transferrin, and tsetse's major energy source proline biosynthesis are described.
Abstract: Tsetse flies (Diptera: Glossinidia) are vectors of pathogenic African trypanosomes. To develop a foundation for tsetse physiology, a normalized expressed sequence tag (EST) library was constructed from fat body tissue of immune-stimulated Glossina morsitans morsitans. Analysis of 20,257 high-quality ESTs yielded 6372 unique genes comprised of 3059 tentative consensus (TC) sequences and 3313 singletons (available at http://aksoylab.yale.edu). We analysed the putative fat body transcriptome based on homology to other gene products with known functions available in the public domain. In particular, we describe the immune-related products, reproductive function related yolk proteins and milk-gland protein, iron metabolism regulating ferritins and transferrin, and tsetse's major energy source proline biosynthesis. Expression analysis of the three yolk proteins indicates that all are detected in females, while only the yolk protein with similarity to lipases, is expressed in males. Milk gland protein, apparently important for larval nutrition, however, is primarily synthesized by accessory milk gland tissue.
TL;DR: An improved molecular model for the CYP6AB3 protein based on this biochemical characterization and the recently defined mammalian CYP3A4 crystal structure provides insight into the remarkable substrate specificity of this protein.
Abstract: The parsnip webworm, Depressaria pastinacella, a specialist on two genera in Apiaceae, feeds exclusively on the furanocoumarin-containing reproductive structures of its host plants. This caterpillar relies principally on cytochrome P450-mediated detoxification for coping with the high concentrations of furanocoumarins in its diet. A cDNA encoding the furanocoumarin-inducible P450 CYP6AB3 from this species was coexpressed with house-fly NADPH P450 reductase in baculovirus-infected Sf9 cells and tested for binding and metabolism of the six furanocoumarins typically encountered in host plant tissues. Only imperatorin and bergapten bind in close proximity to the catalytic haem and only imperatorin is metabolized (V(max) and K(m) of 2.412 pmol/min per pmol P450 and 94.28 microm, respectively). Purification of the imperatorin metabolite by normal phase HPLC and characterization of its structure by MS-MS analysis indicate that CYP6AB3 initially epoxidizes the carbon-carbon pi-bond on the isoprenyl side chain on imperatorin. An improved molecular model for the CYP6AB3 protein based on this biochemical characterization and the recently defined mammalian CYP3A4 crystal structure provides insight into the remarkable substrate specificity of this protein.
TL;DR: An oligoarray analysis was conducted to determine the differential expression of genes due to phenobarbital exposure in Drosophila melanogaster (w1118 strain) third instar larvae, and 17 genes were observed to be induced with increased expression by a statistical analysis of microarrays approach.
Abstract: An oligoarray analysis was conducted to determine thedifferential expression of genes due to phenobarbitalexposure in Drosophila melanogaster ( w 1118 strain)third instar larvae. Seventeen genes were observed tobe induced with increased expression by a statisticalanalysis of microarrays approach with a q ≤ 0.05. At q ≤ 0.12, four more genes ( Cyp12d1, DmGstd4 , and twogenes with unknown function) were found to be up-regulated, and 11 genes with unknown function werefound to be down-regulated. Fifteen of these genes, Cyp4d14, Cyp6a2 , Cyp6a8 , Cyp12d1 , Cyp6d5 , Cyp6w1 ,CG2065, DmGstd6, DmGstd7 , Amy-p/Amy-d, Ugt86Dd ,GC5724, Jheh1, Jheh2 and CG11893, were verified usingquantitative real time polymerase chain reaction. Someof these genes have been shown to be over-transcribedin metabolically DDT-resistant Drosophila strains.Keywords: P450, GST, carbohydrate metabolism,oxido-reductase.Introduction Phenobarbital (PB) treatment has a variety of effects onmammals: (1) reduces frequency of epileptic seizures(Baumann, 2001; Hernandez