Showing papers in "Insect Molecular Biology in 2007"

A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti.

Abstract: Pyrethroids are commonly used as mosquito adulticides and evolution of resistance to these compounds is a major threat to public health. 'Knockdown resistance' to pyrethroids (kdr) is frequently caused by nonsynonymous mutations in the voltage-gated sodium channel transmembrane protein (para) that reduce pyrethroid binding. Early detection of kdr is critical to the development of resistance management strategies in mosquitoes including Aedes aegypti, the most prevalent vector of dengue and yellow fever viruses. Brengues et al. described seven novel mutations in hydrophobic segment 6 of domain II of para in Ae. aegypti. Assays on larvae from strains bearing these mutations indicated reduced nerve sensitivity to permethrin inhibition. Two of these occurred in codons Iso1011 and Val1016 in exons 20 and 21 respectively. A transition in the third position of Iso1011 encoded a Met1011 replacement and a transversion in the second position of Val1016 encoded a Gly1016 replacement. We have screened this same region in 1318 mosquitoes in 32 additional strains; 30 from throughout Latin America. While the Gly1016 allele was never detected in Latin America, we found two new mutations in these same codons. A transition in the first position of codon 1011 encodes a Val replacement while a transition in the first position of codon 1016 encodes an Iso replacement. We developed PCR assays for these four mutations that can be read either on an agarose gel or as a melting curve. Selection experiments, one with deltamethrin on a field strain from Santiago de Cuba and another with permethrin on a strain from Isla Mujeres, Mexico rapidly increased the frequency of the Iso1016 allele. Bioassays of F(3) offspring arising from permethrin susceptible Val1016 homozygous parents and permethrin resistant Iso1016 homozygous parents show that Iso1016 segregates as a recessive allele in conferring kdr. Analysis of segregation between alleles at the 1011 and 1016 codons in the F(3) showed a high rate of recombination even though the two codons are only separated by a ~250 bp intron. The tools and information presented provide a means for early detection and characterization of kdr that is critical to the development of strategies for resistance management.

Molecular characterization of the Aedes aegypti odorant receptor gene family.

Abstract: The olfactory-driven blood-feeding behaviour of female Aedes aegypti mosquitoes is the primary transmission mechanism by which the arboviruses causing dengue and yellow fevers affect over 40 million individuals worldwide. Bioinformatics analysis has been used to identify 131 putative odourant receptors from the A. aegypti genome that are likely to function in chemosensory perception in this mosquito. Comparison with the Anopheles gambiae olfactory subgenome demonstrates significant divergence of the odourant receptors that reflects a high degree of evolutionary activity potentially resulting from their critical roles during the mosquito life cycle. Expression analyses in the larval and adult olfactory chemosensory organs reveal that the ratio of odourant receptors to antennal glomeruli is not necessarily one to one in mosquitoes.

Gene transcription during exposure to, and recovery from, cold and desiccation stress in Drosophila melanogaster

Abstract: We exposed adult male Drosophila melanogaster to cold, desiccation or starvation, and examined expression of several genes during exposure and recovery. Frost was expressed during recovery from cold, and was up-regulated during desiccation. Desiccation and starvation (but not cold) elicited increased expression of the senescence-related gene smp-30. Desat2 decreased during recovery from desiccation, but not in response to starvation or cold. Hsp70 expression increased after 1 h of recovery from cold exposure, but was unchanged in response to desiccation or starvation stress, and Hsp23 levels did not respond to any of the stressors. We conclude that D. melanogaster's responses to cold and desiccation are quite different and that care must be taken to separate exposure and recovery when studying responses to environmental stress.

Female-biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori.

Abstract: Olfaction plays an important role in the life history of insects, including key behaviours such as host selection, oviposition and mate recognition Odour perception by insects is primarily mediated by the large diverse family of odourant receptors (Ors) that are expressed on the dendrites of olfactory neurones housed within chemosensilla However, few Or sequences have been identified from the Lepidoptera, an insect order that includes some of the most important pest species worldwide We have identified 41 Or gene sequences from the silkworm (Bombyx mori) genome, more than double the number of published Or sequences from the Lepidoptera Many silkworm Ors appear to be orthologs of the 17 published tobacco budworm (Heliothis virescens) Ors indicating that many Or lineages may be conserved within the Lepidoptera The majority of the Or genes are expressed in adult female and male antennae (determined by quantitative real-time PCR analysis), supporting their probable roles in adult olfaction Several Or genes are expressed at high levels in both male and female antennae, suggesting they mediate the perception of common host or conspecific volatiles important to both sexes BmOrs 45–47 group together in the same phylogenetic branch and all three are expressed at moderate female-biased ratios, six to eight times higher in female compared to male moth antennae Interestingly, BmOrs19 and 30 appear to be expressed predominantly in female antennae, opposite to that of the published silkworm pheromone receptors BmOrs 1 and 3 that are specific to male antennae These results suggest that BmOr19 and 30 may detect odours critical to female behaviour, such as oviposition cues or male-produced courtship pheromones

Transcriptional analysis of insecticide resistance in Anopheles stephensi using cross-species microarray hybridization

Abstract: A large scale microarray (20k MMC1) from the African malaria vector Anopheles gambiae was used to monitor gene expression in insecticide resistant and susceptible strains of the Asian mosquito Anopheles stephensi. Heterologous hybridization at slightly reduced stringency yielded approximately 7000 significant signals. Thirty-six putative genes were differentially transcribed between the pyrethroid-resistant (DUB-R) and the susceptible (BEECH) strains. The expression profiles of selected transcripts were verified by real-time PCR. A gene putatively involved in the thickening of the adult cuticle showed the most striking up-regulation in DUB-R. A more specialized microarray containing 231 An. gambiae genes putatively involved in insecticide detoxification was used to further analyse classical insecticide resistance genes. Three glutathione S-transferase (GST) transcripts, one esterase and a cytochrome P450 were up-regulated in the resistant strain, while two peroxidases were down-regulated.

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

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

Cloning and interspecific altered expression of heat shock protein genes in two leafminer species in response to thermal stress

Abstract: Studies have demonstrated differences in temperature tolerance between two Liriomyza species, L. huidobrensis and L. sativae. To investigate whether the heat shock proteins (Hsps) in the two species have different expression profiles during temperature stress, we cloned hsp90, 70, 60, 40 and 20, and analysed their expression profiles across temperature gradients by real-time quantitative PCR and Western blotting. The results revealed that the number of TATA-box-like elements and A/T-rich insertion/deletions within the 5' UTRs of the hsps are different in the two species. The temperatures for onset (T(on)) or maximal (T(max)) induction of hsp expression in L. huidobrensis were generally 2.5-10 degrees C lower than those in L. sativae, and the T(on) were highly consistent with the temperature limits of the northern boundary of the range of these two leafminer species. These studies confirmed, in terms of gene expression levels, that L. huidobrensis is more cold tolerant than L. sativae, which is more heat tolerant, and suggest that the T(on) (or T(max)) of hsps can represent the differences in temperature tolerance of these two leafminer species, and may be used to determine their natural geographical distribution limits.

Biochemical and functional characterization of Salp20, an Ixodes scapularis tick salivary protein that inhibits the complement pathway.

Abstract: Ixodes ticks are vectors of several pathogens including Borrelia burgdorferi. Tick saliva contains numerous molecules that facilitate blood feeding without host immune recognition and rejection. We have expressed, purified, and characterized Ixodes scapularis salivary protein 20 (Salp20), a potential inhibitor of the alternative complement pathway that shares homology with the Isac protein family. When analysed by SDS-PAGE and size exclusion chromatography, Salp20 was approximately 48 kDa, more than double its predicted mass, primarily due N- and O-linked glycosylations. Recombinant Salp20 inhibited the alternative complement pathway by dissociating the C3 convertase, and partially protected a serum sensitive species of Borrelia from lysis by normal human serum. We propose that Salp20 facilitates tick feeding and possibly protects tick-borne pathogens from complement components.

The complete mitochondrial genome sequence of the Mormon cricket (Anabrus simplex: Tettigoniidae: Orthoptera) and an analysis of control region variability

Abstract: The Anabrus simplex is a swarming plague orthopteran found in western North America The genome is 15 766 bp in length and genome organization follows the ancestral insect gene arrangement atp6 lacked any readily identifiable stop codon Examination of mRNA secondary structure for this gene suggested a stem/loop-mediated mRNA post-transcriptional processing to liberate a mature atp6 mRNA with a complete stop codon produced by polyadenylation Comparison of similar protein with protein gene boundaries in other insect species reveal a general mechanism for mRNA excision and provide further supporting evidence for post-transcriptional mRNA processing in mitochondrial genomes The A + T-rich region, or control region, was sequenced for 55 A simplex individuals from 12 different populations Variance studies between these individuals show that the A + T-rich region contains significant phylogenetic signal to be used in population studies

The transformer gene in Bactrocera oleae: the genetic switch that determines its sex fate

Abstract: Transformer (tra) is the second gene of a regulatory cascade based on RNA splicing that determines sex in Drosophila melanogaster. Splicing of tra transcripts is regulated by the master gene Sex lethal and tra itself regulates splicing of the transcriptional regulator doublesex (dsx). We present the isolation and characterization of Botra, the olive fruit fly Bactrocera oleae orthologue to the Drosophila gene transformer. As in Drosophila, Botra transcripts are spliced in a sex-specific manner so that only females encode a functional polypeptide of 422 amino acids, whereas males encode presumably nonfunctional peptide(s). The identification of multiple TRA/TRA-2 binding sites within the Botra male-specific exons, suggests an autoregulation mechanism of tra, through TRA/TRA2 activities. The fundamental role of the TRA protein in sex determination of Bactrocera was investigated by RNA interference, where the introduction of Botra dsRNA into embryos resulted in complete transformation of XX flies into fertile males.

Tetracycline treatment influences mitochondrial metabolism and mtDNA density two generations after treatment in Drosophila

Abstract: Tetracycline is commonly used to clear Wolbachia from infected insects. Studies then compare specific biochemical and/or life-history traits between infected and uninfected individuals with the same genetic background. We investigated the potential for tetracycline to influence mitochondrial efficiency and mitochondrial (mt)DNA density two generations after treatment in Drosophila simulans. We observed that antibiotic treatment resulted in a decline in inorganic phosphate incorporated into ATP per mole of oxygen consumed (ADP:O ratio). Further, tetracycline treatment caused a significant increase in mtDNA density in naturally Wolbachia-uninfected but not in naturally Wolbachia-infected lines suggesting a dosage effect. These data suggest that the current practice of comparing Wolbachia-infected and Wolbachia-uninfected insects two generations after tetracycline treatment needs to be re-evaluated.

Microsatellite flanking region similarities among different loci within insect species.

Abstract: Although microsatellites are ubiquitous in eukaryota, the number of available markers varies strongly among taxa. This meta-analysis was conducted on 32 insect species. Sequences were obtained from two assembled whole genomes, whole genome shotgun (WGS) sequences from 10 species and screening partial genomic libraries for microsatellites from 23 species. We have demonstrated: (1) strong differences in the abundance of microsatellites among species; (2) that microsatellites within species are often grouped into families based on similarities in their flanking sequences; (3) that the proportion of microsatellites grouped into families varies strongly among taxa; and (4) that microsatellite families were significantly more often associated with transposable elements - or their remnants - than unique microsatellite sequences.

Relish regulates expression of antimicrobial peptide genes in the honeybee, Apis mellifera, shown by RNA interference

Abstract: Relationships of immune genes in adult honeybees (Apis mellifera) were investigated using RNA interference (RNAi). Quantitative RT-PCR was applied to estimate gene expression and the extent of gene silencing. Relish is a transcription factor and forms an important part of the IMD signalling pathway. The expression of the immune gene Relish was significantly reduced by RNAi (ca. 70%). The proposed regulation of antimicrobial peptide genes by Relish could be established for abaecin and hymenoptaecin. These two genes showed a reduction in gene expression to the same extent as Relish. However, the antimicrobial peptide gene defensin-1 was not affected which suggests defensin-1 is regulated by a different signalling pathway.

Ixodes ticks belonging to the Ixodes ricinus complex encode a family of anticomplement proteins.

Abstract: The alternative pathway of complement is an important innate defence against pathogens including ticks. This component of the immune system has selected for pathogens that have evolved countermeasures. Recently, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., 2000; J. Biol. Chem. 275, 18717-18723). Here, we isolated two different sequences, similar to Isac, from the transcriptome of I. ricinus salivary glands. Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins, called I. ricinus anticomplement (IRAC) protein I and II, are coexpressed constitutively in I. ricinus salivary glands and are upregulated during blood feeding. Also, we demonstrated that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses demonstrate that ticks belonging to the Ixodes ricinus complex encode a family of relatively small anticomplement molecules undergoing diversification by positive Darwinian selection.

Testis-specific expression of the beta2 tubulin promoter of Aedes aegypti and its application as a genetic sex-separation marker.

Abstract: Sex-specific expression of transgenes in pest insects enables novel genetic control strategies, based either on genetic sexing or the spread of transgenes through the germ-line, to be developed and then tested for implementation. We describe the isolation of the beta tubulin genes from the yellow fever mosquito, Aedes aegypti, and the identification of the particular beta2 tubulin gene which has expression confined to the testes. We demonstrate that the beta2 tubulin promoter of Ae. aegypti can direct the expression of a DsRed genetic marker in the testes and show that labelled sperm can be detected in inseminated spermathecae. The applications for this technology in the genetic control of Ae. aegypti are discussed.

Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in transgenic silkworms.

Abstract: RNA interference (RNAi)-mediated viral inhibition has been used in a few organisms for eliciting viral resistance. In the present study, we report the use of RNAi in preventing baculovirus infection in a lepidopteran. We targeted the baculoviral immediate early-1 (ie-1) gene in both a transformed lepidopteran cell line and in the transgenic silkworm Bombyx mori L. Constitutive expression of double-stranded RNA was achieved by piggyBac-mediated transformation of Sf9 cell line with a transgene encoding double-stranded ie-1 RNA (dsie-1). Strong viral repression was seen at early stages of infection but subsequent recovery of viral proliferation was observed. In contrast, the same transgene inserted into the chromosomes of transgenic silkworms induced long-term inhibition of B. mori nucleopolyhedrovirus infection, with nearly 40% protection compared with nontransgenic animals. Protection was efficient at larval stages after oral infection with occlusion bodies or hemocoel injection of budded viruses. Virus injected pupae also displayed resistance. These results show that heritable RNAi can be used to protect silkworm strains from baculovirus infection.

A technique for nucleic acid in situ hybridization to polytene chromosomes of mosquitoes in the Anopheles gambiae complex

Abstract: A sensitive, simple, and reproducible in situ hybridization technique for the detection and precise localization of specific nucleic acid sequences on chromosomes of members of the Anopheles gambiae complex is described. Modifications of the in situ hybridization technique are described that allow simultaneous hybridization of several probes with the chromosomes on a single slide and the multiple use of a single chromosome preparation for several different probes hybridized successively on the same slide. Examples are shown that illustrate the utility of the technique for localization of both single copy and repeated sequences in both polytenized euchromatin and centromeric heterochromatin.

piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids.

Abstract: We describe an efficient method for generating new piggyBac insertions in the germline of F 1 hybrid Tribolium castaneum derived from crosses between transgenic helper and donor strains. Helper strains carried single Minos elements encoding piggyBac transposase. The donor strain carried a single piggyBac element inserted into an actin gene, expanding the eye-specific, 3xP3-EGFP (enhanced green fluorescent protein) reporter expression domain to include muscle. Remobilization of the donor element is accompanied by loss of muscle fluorescence but retention of eye fluorescence. In a pilot screen, the piggyBac donor was remobilized in 84% of the hybrid crosses, generating hundreds of new lethal, enhancer-trap, semisterile and other insertions. The jumpstarter system described herein makes genome-wide, saturation insertional mutagenesis a realistic goal in this coleopteran species.

cDNA cloning of heat shock proteins and their expression in the two phases of the migratory locust.

Abstract: The high population density of insects is often a stress factor. Insects synthesize heat shock proteins (Hsps) in response to the impacts of stress through molecular chaperone activity. Locust solitary and gregarious phases occur at low and high population density, respectively. In this study, we compare the expression profiles of the Hsp genes in the two phases of the migratory locust in an attempt to examine the role of Hsps in adaptation to high density populations. The full length cDNAs of Hsp20.5, 20.6, 20.7, 40, 70 and Hsp90 of the migratory locust were cloned and sequenced. The expressional differentiation of the six Hsps in mRNA levels between solitary and gregarious locusts was observed. Results from real-time PCR indicate that the six Hsps are expressed throughout all developmental stages except in the early stage embryo. The expression levels of the six Hsps were significantly upregulated in gregarious locusts. The expressional variations among certain organs, such as the head, thorax and leg of fifth instar nymphs in gregarious locusts were also higher than those in solitary ones. These observations suggest that population density may be an important factor in determining Hsp expression in the locust.

Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L.

Abstract: Peptidase sequences were analysed in randomly picked clones from cDNA libraries of the anterior or posterior midgut or whole larvae of the yellow mealworm, Tenebrio molitor Linnaeus. Of a total of 1528 sequences, 92 encoded potential peptidases, from which 50 full-length cDNA sequences were obtained, including serine and cysteine proteinases and metallopeptidases. Serine proteinase transcripts were predominant in the posterior midgut, whereas transcripts encoding cysteine and metallopeptidases were mainly found in the anterior midgut. Alignments with other proteinases indicated that 40% of the serine proteinase sequences were serine proteinase homologues, and the remaining ones were identified as either trypsin, chymotrypsin or other serine proteinases. Cysteine proteinase sequences included cathepsin B- and L-like proteinases, and metallopeptidase transcripts were similar to carboxypeptidase A. Northern blot analysis of representative sequences demonstrated the differential expression profile of selected transcripts across five developmental stages of Te. molitor. These sequences provide insights into peptidases in coleopteran insects as a basis to study the response of coleopteran larvae to external stimuli and to evaluate regulatory features of the response.

Cytochrome P450s in Papilio multicaudatus and the transition from oligophagy to polyphagy in the Papilionidae

Abstract: Although substrate-specific CYP6B1 and CYP6B3 enzymes in Papilio polyxenes contribute to specialization on furanocoumarin-containing host plants, CYP6B4 and CYP6B17 enzymes in the polyphagous Papilio glaucus and Papilio canadensis have a broader range of substrates. Papilio multicaudatus, an oligophage with one furanocoumarin-containing host, is putatively ancestral to polyphagous Papilio species. Furanocoumarin-inducible CYP6B33-CYP6B37 and CYP6AB6 were characterized from this species. Heterologous expression of CYP6B33 revealed furanocoumarin metabolism resembling that of CYP6B4-CYP6B17 enzymes from P. glaucus and P. canadensis. Molecular models of CYP6B33 and CYP6B4 indicate that seven conserved aromatic side chains stabilize their hydrophobic catalytic sites and that a Lys484-Ser484 substitution enlarges the CYP6B4 active site pocket to increase the predicted distance between the substrate and reactive oxygen relative to CYP6B1. Loss of specialization in this lineage may have resulted from relatively few mutational changes, allowing acquisition of broader catalytic activities without loss of ancestral furanocoumarin-metabolizing activities.

Nuclear mitochondrial-like sequences in ants: evidence from Atta cephalotes (Formicidae: Attini).

Abstract: Nuclear mitochondrial-like sequences (numts) are copies of mitochondrial DNA that have migrated to the genomic DNA. We present the first characterization of numts in ants, these numts being homologues to a mitochondrial DNA fragment containing loci the 3′′ portion of the cytochrome oxidase I gene, an intergenic spacer, the tRNA leucine gene and the 5′′′ ′ portion of the cytochrome oxidase II gene. All 67 specimens of Atta cephalotes (Hymenoptera: Formicidae: Attini) investigated had these homologues, which are within two monophyletic groups that we called numt1 and numt2. Numt1 and numt2 sequences are less variable than mitochondrial sequences and released from the severe purifying selection constraining the evolution of mitochondrial genes. Their formation probably involved bottlenecks related to two distinct transfer events of ancient and fast evolving mitochondrial DNA fragments to comparative slowly evolving nuclear DNA regions.

Genetic exchange in 2La inversion heterokaryotypes of Anopheles gambiae

Abstract: In the malaria vector Anopheles gambiae, alternative arrangements of chromosome 2 (2La and 2L+(a)) vary in relative frequency along clines of aridity, suggesting the action of natural selection on targets within the inversion. Our long term goal of detecting such targets depends in part on the level of genetic exchange between arrangements. Accordingly, we estimated recombination rates on 2L from the backcross progeny of 2La/+(a) heterokaryotypes and as a control, from 2L+(a) homokaryotypes. In homokaryotypes, the recombination rate was uniform at ~2.0 centimorgans per megabase (cM/Mb). In heterokaryotypes, recombination within the rearranged region was reduced to < 0.5 cM/Mb, with slightly higher but nevertheless reduced levels (< 1.0 cM/Mb) flanking the rearrangement. Yet, gene exchange was recorded between nearly all markers, including those very near the distal inversion breakpoint. These results suggest that reduced recombination is a necessary but not sufficient mechanism for genetic isolation between alternative arrangements, and that the targets of natural selection can be identified against the different chromosomal backgrounds.

A cytoskeletal actin gene in the mosquito Anopheles gambiae.

Abstract: Five actin genes have been identified in the mosquito Anopheles gambiae, and a constitutively expressed actin gene has been chosen for detailed analysis. We have physically mapped and sequenced this gene and six associated cDNAs, including translated coding regions, as well as the 5' and 3' flanking sequences. Analysis of stage-specific RNA shows this gene to be present in all stages of mosquito development and in an established A. gambiae cell line, thus indicating a cytoskeletal actin. In the sequence of the translated coding region and in pattern of expression, this gene is very similar to the cytoskeletal actin genes of Drosophila melanogaster, and in sequence, equally similar to the Artemia cytoskeletal actin gene 403 (99.2% identity among the three amino acid sequences). Sequencing of this A. gambiae actin gene (designated act1D for its location in chromosome division 1D) and selected cDNAs shows that it possesses three alternative leader sequences; thus the gene appears to have three alternative promoters. These promoters should ultimately prove useful in the production of transgenic constructs for constitutive expression.

Major ampullate spidroins from Euprosthenops australis: multiplicity at protein, mRNA and gene levels.

Abstract: Spider dragline silk possesses extraordinary mechanical properties. It consists of large fibrous proteins called spidroins that display modular structures. It is known to consist of two proteins: the major ampullate spidroin (MaSp) 1 and MaSp2. This study analyses MaSp sequences from the nursery-web spider Euprosthenops australis. We have identified a previously uncharacterized MaSp2 sequence and a new MaSp-like spidroin, which display distinct homogenous submotifs within their respective Gly-rich repeats. Furthermore, a group of MaSp1 cDNA clones show unexpected heterogeneity. Genomic PCR identified several MaSp1 gene variants within individual spiders, which suggests the presence of a gene cluster in E. australis. Finally, the evolution of spidroin genes is discussed in relation to phylogenetic analysis of nonrepetitive C-terminal domains from diverse species.

Molecular cloning of a GABA receptor subunit from Laodelphax striatella (Fallén) and patch clamp analysis of the homo-oligomeric receptors expressed in a Drosophila cell line.

Abstract: A cDNA encoding a gamma-aminobutyric acid (GABA) receptor subunit was cloned from the small brown planthopper Laodelphax striatella. The L. striatella GABA receptor subunit was found to have high amino acid sequence similarity to the bd-type splice variant of the Drosophila GABA receptor Rdl subunit and several other GABA receptor subunits, with identities of over 70%. The cDNA was inserted into the expression vector pAc5.1-lac-Hygro. Clonal cell lines stably expressing homo-oligomeric L. striatella GABA receptors were generated by transfecting the vector into D.mel-2 cells. Expression of functional GABA receptors in the cell lines was demonstrated by whole-cell patch clamp recordings. GABA induced inward currents with an EC(50) value of 29 microM and a Hill coefficient of 1.7. The GABA-evoked responses reversed close to the Nernst equilibrium potential for chloride ions. The amplitudes of agonist-induced currents were found to be in the order muscimol (100 microM) >/= GABA (100 microM) > isoguvacine (100 microM) > cis-4-aminocrotonic acid (CACA) (100 microM) > 5-(4-piperidyl)-3-isoxazolol (4-PIOL) (1 mM). Antagonists such as fipronil (100 nM), 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) (100 nM), dieldrin (100 nM) and SR95531 (gabazine) (1 microM) suppressed GABA-induced currents. The functional expression of a GABA receptor from an agricultural pest presents a unique opportunity to discover new molecules active at this important target site.

Antennal esterase cDNAs from two pest moths, Spodoptera littoralis and Sesamia nonagrioides, potentially involved in odourant degradation

Abstract: Rapid degradation of odours after interaction with olfactory receptors is a critical step of the signal reception process. However, the implied mechanisms are still largely unknown in vertebrates as well as in insects. Involvement of odourant-degrading enzymes in odourant degradation within the antennae has been shown in some insect species and, in particular, esterases could play a key role in degradation of sex pheromones from Lepidoptera. Using a PCR-based strategy, we isolated cDNAs encoding two new esterases from two moths which used acetates as pheromone compounds: the Egyptian armyworm Spodoptera littoralis and the Mediterranean corn borer Sesamia nonagrioides. In antennae, both transcripts were clearly restricted to olfactory sensilla, suggesting their involvement in the degradation of odourant acetate components.

Isolation and extreme sex-specific expression of cytochrome P450 genes in the bark beetle, Ips paraconfusus, following feeding on the phloem of host ponderosa pine, Pinus ponderosa.

Abstract: We have identified cDNAs and characterized the expression of 13 novel cytochrome P450 genes of potential importance in host colonization and reproduction by the California fivespined ips, Ips paraconfusus. Twelve are of the Cyp4 family and one is of the Cyp9 family. Following feeding on host Pinus ponderosa phloem, bark beetle transcript levels of several of the Cyp4 genes increased or decreased in males only or in both sexes. In one instance (IparaCyp4A5) transcript accumulated significantly in females, but declined significantly in males. The Cyp9 gene (Cyp9T1) transcript levels in males were > 85 000 x higher at 8 h and > 25 000 x higher at 24 h after feeding compared with nonfed controls. Transcript levels in females were approximately 150 x higher at 24 h compared with nonfed controls. Cyp4G27 transcript was present constitutively regardless of sex or feeding and served as a better housekeeping gene than beta-actin or 18S rRNA for the real-time TaqMan polymerase chain reaction analysis. The expression patterns of Cyp4AY1, Cyp4BG1, and, especially, Cyp9T1 in males suggest roles for these genes in male-specific aggregation pheromone production. The differential transcript accumulation patterns of these bark beetle P450s provide insight into ecological interactions of I. paraconfusus with its host pines.

Molecular identification of rickettsia‐like microorganisms associated with colonized cat fleas (Ctenocephalides felis)

Abstract: Cat fleas (Ctenocephalides felis) from eight commercial flea colonies from various regions of the USA were examined by selective PCR amplification, and subsequent restriction digest analysis and Southern hybridization of PCR products, for the presence of a rickettsia-like organism (ELB agent). These flea colonies were either started with fleas from one supplier (EL Labs), in which ELB agent was first identified, or were started with fleas from stray cats and dogs and later came into contact with ELB-infected fleas. Infection rates in the colonies ranged from 43% to 93%. The successful propagation of ELB agent in these colonies may be due to efficient trans-stadial and transovarial transmission. While ELB agent has recently been identified in blood from human murine typhus cases, attempts to infect mammalian cells and SCID mice with flea isolates were unsuccessful.

Identification and molecular characterization of a novel protein Saglin as a target of monoclonal antibodies affecting salivary gland infectivity of Plasmodium sporozoites

Abstract: Molecular mechanisms underlying the interaction between malarial sporozoites and putative receptor(s) on the salivary glands of Anopheles gambiae remain largely unknown. In previous studies, a salivary gland protein of ~100 kDa was identified as a putative target based on recognition of the protein by a monoclonal antibody (mAb) 2A3 that caused a >/= 70% reduction in the average number of sporozoites per infected salivary gland when fed to mosquitoes. Using affinity purification we purified the target of this mAb from extracts of female A. gambiae salivary glands and it was found to be a novel protein by tandem mass spectrometric analysis. Biochemical and molecular characterization of the 100 kDa protein showed that this molecule, designated Saglin, exists as a disulphide-bonded homodimer of 50 kDa subunits. The ability to form homodimers was retained even in the recombinant Saglin expressed in mammalian cells (HEK293). The amino acid sequence of Saglin contains a signal peptide suggesting that Saglin is a secreted protein. If Saglin is indeed involved in the process of invasion of A. gambiae salivary glands by sporozoites of Plasmodium, it could provide a novel target for future investigations aimed at interruption of malaria transmission.