Linda S. Ross

Steroid Induction of a Peptide Hormone Gene Leads to Orchestration of a Defined Behavioral Sequence

At the end of each molt, insects shed the old cuticle by performing preecdysis and ecdysis behaviors. Regulation of these centrally patterned movements involves peptide signaling between endocrine Inka cells and the CNS. In Inka cells, we have identified the cDNA and gene encoding preecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which activate these behaviors. Prior to behavioral onset, rising ecdysteroid levels induce expression of the ecdysone receptor (EcR) and ETH gene in Inka cells and evoke CNS sensitivity to PETH and ETH. Subsequent ecdysteroid decline is required for peptide release, which initiates three motor patterns in specific order: PETH triggers preecdysis I, while ETH activates preecdysis II and ecdysis. The Inka cell provides a model for linking steroid regulation of peptide hormone expression and release with activation of a defined behavioral sequence.

Blood meal induces global changes in midgut gene expression in the disease vector, Aedes aegypti

Blood feeding is an essential developmental process for many arthropods and plays a significant role in disease transmission. Understanding physiological responses in the midgut is important because it is the primary site of blood meal digestion and pathogenic infection. Processes that occur in the midgut in response to a blood meal have been studied but are poorly understood. Here, we use cDNA microarrays to examine midgut gene expression on a global level in response to blood feeding to assist in unraveling these processes. We have developed Aedes aegypti microarrays consisting of clones obtained from an expressed sequence tag project. Individual clones were amplified by polymerase chain reaction and printed onto glass slides. These microarrays were used to study the effects of a blood meal on midgut gene expression over a 72-h time course. As a result, a number of genes involved in processes such as nutrient uptake and metabolism, cellular stress responses, ion balance, and PM formation, as well as a number of unknown genes were induced or repressed in response to a blood meal based on this microarray data.

Molecular cloning and characterization of the B subunit of a vacuolar H(+)-ATPase from the midgut and Malpighian tubules of Helicoverpa virescens.

Using the polymerase chain reaction (PCR) a 0.8-kb product was amplified from cDNA made from the midgut and Malpighian tubules of fifth instar larvae of Helicoverpa virescens. This 0.8-kb PCR product was then used to isolate a clone of the B subunit of the V-type ATPase from a cDNA library made from the same tissues. The cDNA clone encodes for a protein of 55 kDa which shows very high amino acid homology to other known B subunits of V-type ATPases. The transcript size of the B subunit in the midgut of H. virescens was 2.3 kb, and a transcript of identical size was also detected in the Malpighian tubules. Northern blot analysis revealed the presence of a homologous transcript of 2.6 kb in the midgut of Manduca sexta and PCR analysis also confirmed the presence of such a transcript in the Malpighian tubules and the nervous system of M. sexta, and in the midgut Malpighian tubules of Culex quinquefasciatus. The presence of the V-type ATPase in the Malpighian tubules of lepidopteran insects suggests that the transport of ions across the cell membrane in this tissue is also probably driven by a similar process as that observed in the midgut of these insects.

ISOLATION OF THE V-ATPASE A AND C SUBUNIT CDNAS FROM MOSQUITO MIDGUT AND MALPIGHIAN TUBULES

Using conserved amino acid sequences for the design of oligonucleotide primers, we isolated cDNA clones for two subunits of the V-ATPase from the midgut and Malpighian tubules of Aedes aegypti larvae. The 3.1 kb cDNA of the A subunit of the peripheral catalytic V1 sector codes for a protein of 68.6 kDa. The protein contains conserved motifs, including an ATP/GTP binding site, found in all other A subunits. Southern analysis using the A subunit as a probe suggests the presence of only a single copy of gene in the Aedes aegypti. The 0.85 kb cDNA of the c subunit of the membrane H+ conducting V0 sector codes for a protein of kDa. This protein has four transmembrane domains and contains a conserved glutamic acid that serves as the binding site for dicyclohexylcarbodiimide. Southern analysis using the c subunit as a probe suggests the presence of more than one related gene in the genome of Aedes aegypti. Pileup analysis of various A and c subunits shows that these subunits fall into distinct clusters, including one in which all arthropod proteins are clustered.

Functional characterization of a glutamate/aspartate transporter from the mosquito Aedes aegypti

SUMMARY Glutamate elicits a variety of effects in insects, including inhibitory and excitatory signals at both neuromuscular junctions and brain. Insect glutamatergic neurotransmission has been studied in great depth especially from the standpoint of the receptor-mediated effects, but the molecular mechanisms involved in the termination of the numerous glutamatergic signals have only recently begun to receive attention. In vertebrates, glutamatergic signals are terminated by Na+/K+-dependent high-affinity excitatory amino acid transporters (EAAT), which have been cloned and characterized extensively. Cloning and characterization of a few insect homologues have followed, but functional information for these homologues is still limited. Here we report a study conducted on a cloned mosquito EAAT homologue isolated from the vector of the dengue virus, Aedes aegypti. The deduced amino acid sequence of the protein, AeaEAAT, exhibits 40–50% identity with mammalian EAATs, and 45–50% identity to other insect EAATs characterized thus far. It transports l-glutamate as well as l- and d-aspartate with high affinity in the micromolar range, and demonstrates a substrate-elicited anion conductance when heterologously expressed in Xenopus laevis oocytes, as found with mammalian homologues. Analysis of the spatial distribution of the protein demonstrates high expression levels in the adult thorax, which is mostly observed in the thoracic ganglia. Together, the work presented here provides a thorough examination of the role played by glutamate transport in Ae. aegypti.

Identification, functional characterization and expression of a LAT type amino acid transporter from the mosquito Aedes aegypti

We isolated two cDNAs from the mosquito Aedes aegypti, an L-amino acid transporter (AeaLAT) and a CD98 heavy chain (AeaCD98hc). Expression of AeaCD98hc or AeaLAT alone in Xenopus oocyte did not induce amino acid transport activity. However, co-expression of AeaCD98hc and AeaLAT, which are postulated to form a heterodimer protein linked through a disulfide bond, showed significant increase in amino acid transport activity. This heterodimeric protein showed uptake specificity for large neutral and basic amino acids. Small acidic neutral amino acids were poor substrates for this transporter. Neutral amino acid (leucine) uptake activity was partially Na+ dependent, because leucine uptake was approximately 44% lower in the absence of Na+ than in its presence. However, basic amino acid (lysine) uptake activity was completely Na+ independent at pH of 7.4. Extracellular amino acid concentration could be the main factor that determined amino acid transport. These results suggest the heteromeric protein is likely a uniporter mediating diffusion of amino acids in the absence of ions. The AeaLAT showed high level expression in the gastric caeca, Malpighian tubules and hindgut of larvae. In caeca and hindgut expression was in the apical cell membrane. However, in Malpighian tubules and in midgut, the latter showing low level expression, the transporter was detected in the basolateral membrane. This expression profile supports the conclusion that this AeaLAT is a nutrient amino acid transporter.

Molecular cloning and functional expression of a proline transporter from Manduca sexta

We report the molecular cloning of a L-proline transporter, MasPROT cDNA and its splice variants MasPROT.16 and MasPROT.2 from the central nervous system of Manduca sexta. Sequence analysis revealed that MasPROT belongs to a family of high affinity Na+/Cl- dependent neurotransmitter transporters. The deduced amino acid (aa) sequence of 556 aa having an estimated molecular mass of 58.9 kDa is predicted to have 12 putative transmembrane domains (TMD) and a characteristic large extracellular loop between TMD3 and TMD4. Sequence comparison to other members of the family indicates that it falls into the glycine-proline transporter subfamily. Transiently expressed MasPROT cDNA in Xenopus oocytes exclusively transported proline. Northern analysis shows that it is expressed predominantly in central nervous system, however, low levels are present in midgut, hindgut and Malpighian tubules. Two mRNA transcripts of sizes 3.6 and 8 Kb were found in all tissues except hindgut, where only a smaller transcript exists. RT-PCR and Southern blot analysis revealed the presence of MasPROT transcripts in flight muscles but not in leg muscles. Our preliminary data suggests that this transporter is an insect homologue of mammalian proline transporters. MasPROT.16 is a short splice variant encoding for 174 amino acids and shares 138 amino acids from the N terminus of MasPROT. MasPROT.2 is a long splice variant that contains six introns that coincide precisely with the previously mapped exon/intron boundaries of the members of this superfamily.