Chaoxian Geng

DOPAMINERGIC CONTROL OF CORPORA ALLATA ACTIVITY IN THE LARVAL TOBACCO HORNWORM, MANDUCA SEXTA

The corpora allata (CA) of insects are innervated by axons of non-neurosecretory cerebral neurons, and of the various known neurotransmitters in the brain of the tobacco hornworm, Manduca sexta, only dopamine is detected in the CA by electrochemical detection HPLC. This neurotransmitter stimulates the biosynthetic activity of the CA in vitro for the first 2 days of the last larval stadium, but inhibits CA from day 3 through day 6, the beginning of the prepupal period. Stimulation of JH synthesis has previously been linked with an increase in the production of cyclic AMP (cAMP) in the CA, and dopamine stimulates the adenylyl cyclase system of CA from larvae early in the fifth stadium, while on day 6, its effect is inhibitory. These results suggest: (1) the existence in the CA of both D1- and D2-like dopamine receptors, which in vertebrates stimulate and inhibit, respectively, adenylyl cyclase; and (2) the developmental control of their expression. A potent D1 agonist, (+/-)-SKF 82958-HBr, did not stimulate JH biosynthesis by day 0 CA as expected, but appeared to inhibit it at a concentration of 10(-5)M. Thus the apparent D1-like receptor in Manduca CA may be pharmacologically distinct from vertebrate D1 receptors. The existence of D2-like receptors is supported by the finding that a vertebrate D2 receptor agonist, (+/-) PPHT-HCl, and an antagonist, eticlopride, have the predicted effects on JH acid biosynthesis and cAMP production by day 6 Manduca CA. However, the D1 agonist also significantly reduces JH acid biosynthesis and cAMP production, indicating that while the Manduca D2-like receptor is pharmacologically similar to the vertebrate D2, it shares some characteristics with D1 receptors. The developmental regulation of these receptors by ecdysteroids is suggested by the fact that when day 0 larvae are treated in vivo with exogenous ecdysone:20-hydroxyecdysone, the biosynthetic activity of the CA in vitro 24 h later is no longer stimulated by dopamine.

A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression

Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dalpha6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dalpha6, providing convincing evidence that Dalpha6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dalpha6 alone, co-expression of Dalpha6 with an additional nAChR subunit, Dalpha5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR.

Drosophila Rosa Gene, Which When Mutant Causes Aberrant Photoreceptor Oscillation, Encodes A Novel Neurotransmitter Transporter Homologue

The Drosophila receptor oscillation A (rosA) mutations, which cause electroretinogram (ERG) defects, including oscillations, were localized to the 24F4-25A2 region of chromosome 2L. Genomic fragments from this region, isolated from bacteriophage P1 clones, included those that detect transcriptional defects in rosA mutants in RNA blot experiments. One of these genomic fragments was used to screen a head cDNA library. The largest cDNA clone (3.6 kb) isolated was shown to rescue a rosA mutant in P element-germline transformation experiments. The ROSA protein deduced from the open reading frame in the 3.6 kb rosA cDNA is 943 amino acids long and is 36-41% identical to members of the superfamily of Na+/Cl(-)-dependent neurotransmitter transporters, with no indication of higher sequence identity to any one subgroup within the superfamily. RNA blot experiments revealed multiple transcripts in various developmental stages, the most abundant one being a 3.7 kb transcript, particularly in the adult head. Tissue in situ experiments identified the rosA transcript to be localized to many tissues, with higher levels of hybridization in the nervous system and digestive tract. The results demonstrate that the rosA gene encodes a novel Na+/Cl(-)-dependent transporter important for normal response properties of the photoreceptor.

Analysis of the biogenic amines in the central nervous system of the tobacco hornworm by high-performance liquid chromatography with 16-sensor electrochemical detection☆

A method was developed to analyze biogenic amines in extracts of the central nervous system of the tobacco hornworm, Manduca sexta by high-performance liquid chromatography with 16-sensor electrochemical detection (n-EC-HPLC). The amines, precursors, and metabolites were separated in two dimensions. The first dimension involves separation based upon retention time by reversed-phase HPLC, while the second dimension involves separation based upon the characteristic oxidation potentials achieved by n-EC. Biogenic amine identification was based upon maximum oxidation potential and peak height ratios in addition to retention time. The improved resolving power of this method allows for a simplified sample preparation procedure and simultaneous determination of a wide range of compounds, including phenylethylamine, catecholamines, indoleamines, and some of their precursors and metabolites.

Specific molecular alterations in the norpA-encoded phospholipase C of Drosophila and their effects on electrophysiological responses in vivo

A large number of mutants in the norpA gene, which encodes the phospholipase C (PLC) involved in Drosophila phototransduction, is available for the investigation of the effects of specific amino acid substitutions in PLC on biochemical and electrophysiological properties of these mutants. Of the 47 norpA mutants screened for PLC protein content, all but one (H43) displayed drastically decreased amounts of the protein suggesting that almost any mutational alteration has a deleterious effect on the integrity of the protein. Three new amino acids were identified in the catalytic domains X and Y that are important for PLC catalytic activity and the generation of photoreceptor responses (ERG). One of them was found substituted in H43, which showed a low specific PLC activity, a pronounced decrease in ERG sensitivity, and a wild‐type‐like response termination time. The response termination times obtained from three mutants was found to be approximately inversely proportional to the amount of PLC. In addition, we show that (i) the specific PLC activity is a key factor determining the photoreceptor sensitivity; (ii) the catalytic activity and response termination are separable functions of PLC; and (iii) a mutation in the putative Gα‐interacting C2 domain causes a preferentially strong defect in latency.

Biogenic amines in the brain of Manduca sexta during larval-pupal metamorphosis

Abstract 1. The extracts of brains (cerebral ganglion) of the tobacco hornworm larvae contain octopamine, dopamine and 5-hydroxytryptamine, but not norepinephrine via analysis using a 16-channel electrochemical array HPLC system. 2. A comprehensive daily analysis reveals the patterns of biogenic amine levels in the brain during larval—pupal metamorphosis. 3. Monoamine oxidase-based amine metabolites are not present at detectable levels. N -β-Alanyldopamine is the predominant metabolite of dopamine present in the brain during the postwandering phase of the last larval stadium.

Photoreceptor Degeneration and Ca2+ Influx through Light-Activated Channels of Drosophila

We discuss in this chapter the role of Ca2+ homeostasis in maintaining the structural integrity of photoreceptor cells in Drosophila. Both insufficient and excessive amounts of Ca2+ in photoreceptor cells appear to lead to cell degeneration. Because one of the two classes of light-sensitive channels in Drosophila photoreceptors is highly Ca2+-permeable, how well this class of channels functions can profoundly affect Ca2+ homeostasis. We will begin by reviewing Drosophila phototransduction, emphasizing what is known about the mechanism of activation of light-sensitive channels. We will then describe Ca2+ entry through light-sensitive channels and the presumed mechanisms by which too little and too much Ca2+ entry can both cause photoreceptor degeneration. We will conclude the chapter with discussions of two examples of mutations known to cause unregulated Ca2+ entry through light-sensitive channels, leading to massive photoreceptor degeneration.