Circadian changes in melatonin in the nervous system and hemolymph of the cabbage looper moth,Trichoplusia ni

Quantitative levels of melatonin and 5-hydroxytryptamine were measured over the scotophase in the protocerebrum, subesophageal ganglion, optic lobes, thoracic ganglia, and hemolymph of adult male cabbage looper moths, Trichoplusia ni, using HPLC with electrochemical detection. Melatonin levels were very low (s < 1 pmol) or undetectable during the photophase, but increased in all tissues during the dark. Lowest mean levels in the dark were observed in the optic lobes (0.3 to 0.7 pmols). Maximal mean levels in the protocerebrum (5.2 pmols) occurred in the early part of the scotophase, but in all other tissues (2.8 in the subesophageal ganglion; 9.5 in thoracic ganglia) and hemolymph (18 pg/l) maximal mean levels were observed later in the dark. Levels of 5-hydroxytryptamine in each tissue, in contrast, were higher than melatonin levels in the photophase, and in the protocerebrum and thoracic ganglia decreased during the dark, but in the optic lobes and subesophageal ganglion remained unchanged. Further, decreases in 5-hydroxytryptamine during the dark were significantly lower than the increased levels of melatonin, suggesting that active synthesis of 5-hydroxytryptamine was occurring. In a second experiment, when measured from individuals in three different photoperiods (618, 1212, 186 lightdark) maximum mean melatonin levels in the brain (protocerebral and subesophageal ganglia) peaked within the first 1.5 h of the dark and remained at measurable levels for the duration of the dark. In a third experiment, levels of melatonin in the brain and thoracic ganglia displayed rhythmicity in continuous dark conditions but not in continuous light, when compared with profiles obtained in a normal light dark regime.Abbreviations CNS Central nervous system - HPLC-ECD high performance liquid chromatography with electrochemical detection - MEL melatonin - 5HT 5-hydroxytryptamine - N-ac-SHT N-acetyl-5-hydroxytryptamine     

Molecular cloning and functional characterization of a GABA transporter from the CNS of the cabbage looper, Trichoplusia ni.

A cDNA encoding a GABA transporter in the caterpillar Trichoplusia ni has been cloned and expressed in baculovirus-infected insect cells. The cDNA contains an ORF encoding a 608-residue protein, designated TrnGAT. Hydropathy analysis of the deduced amino acid sequence suggests 12 transmembrane domains, a structure similar to that of all other cloned Na+/Cl(-)-dependent GABA transporters. The deduced amino acid sequence shows high identity with a GABA transporter (MasGAT) expressed in the embryo of Manduca sexta. Expression of TrnGAT mRNA was detected only in the brain. Sf21 cells infected with recombinant baculovirus exhibited a 20- to 30-fold increase in [3H]GABA uptake compared to control-infected cells. Several blockers of GABA uptake were used to determine the pharmacological profile of TrnGAT. Although most similar to mammalian neuronal GABA transporter GAT-1 in its kinetic properties, stoichiometry of ionic dependence and pharmacological properties, TrnGAT may be distinguished from mammalian GAT-1 by the inability of cyclic GABA analogues, such as nipecotic acid and its derivatives, to inhibit GABA uptake by the insect protein. The unique pharmacology of TrnGAT suggests that the GABA transport system in the lepidopteran CNS could be a useful target in the future development of rapidly-acting neuroactive agents used to control agriculturally-important insects.     

A novel chitin-binding protein identified from the peritrophic membrane of the cabbage looper, Trichoplusia ni

A novel midgut peritrophic membrane (PM) protein, TnPM-P42, was identified from the cabbage looper, Trichoplusia ni. TnPM-P42 was shown as a 42kDa protein by SDS-PAGE analysis and appeared to be associated with the PM throughout its entire length. In T. ni larvae, the midgut is the only tissue where TnPM-P42 could be detected during the feeding period of the larvae. TnPM-P42 has chitin-binding activity and is strongly associated with the PM, which is similar to the currently known peritrophin type PM proteins. However, TnPM-P42 represents a unique family of proteins distinctly different from the peritrophin type PM proteins in its sequence characteristics. TnPM-P42 does not contain the peritrophin domain which is present in all the currently known PM proteins, but instead has a chitin deacetylase-like domain. Sequence similarity search of the GenBank database did not result in identification of any known proteins with a significant overall sequence similarity to the TnPM-P42. However, expressed sequence tags (ESTs) from various arthropods were identified to code for proteins with high sequence similarities to TnPM-P42, indicating the presence of TnPM-P42 homologs in other arthropods. Consistent with the identification of various ESTs from arthropods, Western blot analysis demonstrated the presence of a TnPM-P42-like protein in the PMs from Heliothis virescens and Helicoverpa zea larvae. The sequence characteristics of TnPM-P42 indicate that TnPM-P42 represents a novel family of insect proteins. However, its biochemical and physiological functions require further investigation.     

Sulfhydryl-reagent alteration of soybean resistance to the cabbage looper, Trichoplusia ni

The hypothesis that the sulfhydryl reagent, N-ethylmaleimide, would function as an elicitor of alterable resistance in soybean (Glycine max) plants to Trichoplusia ni herbivory was tested experimentally under greenhouse conditions. This elicitory chemical, which allows receptor thiols to add across its carbon-carbon double bond, altered the resistance in one or more leaves of plants at one or more intervals after treatment; and thus yielded results supporting the hypothesis. Leaf dipping and soil application were both effective methods of treatment. Results support the interpretation that an elicitor may function in intact plants by altering the integrity of sulfhydryl groups in receptor macromolecules which are also involved in signaling a change in the plant's biosynthesis of characteristic defensive compounds such as phenylpropanoids including antifeedant and antibiotic flavonoids. Induced feeding non-preference by T. ni was highly correlated positively with the amount of glyceollins in the leaf.     

Electroretinogram components of the ocellus of the adult cabbage looper moth Trichoplusia ni

The electroretinogram (ERG) of the adult cabbage looper (Trichoplusia ni) ocellus has been studied by extracellular recording methods. Using white light stimulation, the ERG was found to have four components, two of which differ from those of ocelli previously studied. Here component 3 is an excitatory post-synaptic potential (EPSP) and component 4 is an excitatory spike discharge from the ocellar second-order neurons. The excitatory nature of these components has been verified by two experiments. In a light adaptation experiment decreased stimulus intervals caused a reduction in the number of excitatory spikes. In an experiment with the anticholinesterase tetraethylpyrophosphate (TEPP), treatment of the preparation abolished the excitatory spike discharge and reduced the magnitude of the EPSP.     

Molecular characterization of four midgut aminopeptidase N isozymes from the cabbage looper, Trichoplusia ni

Four aminopeptidase N (APN) isoforms, TnAPN1, TnAPN2, TnAPN3 and TnAPN4, were identified from the cabbage looper, Trichoplusia ni, by cDNA cloning. The deduced amino acid sequences of the four APNs indicate that TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are synthesized as pre-proteins of 110, 106, 114 and 108 kDa, respectively. Sequence features of the T. ni APNs include the presence of a signal peptide at their N-termini and a prepeptide at the C-termini for the GPI anchor, the zinc binding/gluzincin motif HEX2HX18E, the gluzincin aminopeptidase motif GAMENWG and the presence of glycosylation sites. After removal of the signal peptide and the C-terminal prepeptide, the predicted molecular weights of TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are 106, 102, 110 and 104 kDa, respectively. Enzymatic activity assays of various larval tissues showed that aminopeptidase activities were mainly localized in the midgut and the specific enzyme activity per mg of midgut tissue proteins was constant in T. ni larvae regardless of the composition of dietary proteins and amino acids. Both enzyme activity assays and RT-PCR analyses for the expression of the APN genes in T. ni larval tissues demonstrated that APN genes were expressed in Malphigian tubules in addition to the midgut, which was the first observation that APNs were also synthesized in insect Malphigian tubules. The finding of APN gene expression and enzyme activity in the Malphigian tubules indicated the biochemical and functional similarity of the insect Malphigian tubules to the mammalian counterpart, the kidney, in which APNs are known to play important functions.     

Role of trichomes in soybean resistance to cabbage looper, Trichoplusia ni

Role of leaf trichome density and length in Glycine max (L.) Merr. resistance to Trichoplusia ni (Hübner) was evaluated at different leaf positions on a relatively resistant soybean, plant introduction (PI) 227687, and a relatively susceptible cultivar, Davis. The uppermost (juvenile) leaf within both PI 227687 and Davis plants, which is densely covered with trichomes, was most resistant to T. ni larval feeding and survival. When these trichomes were shaven off, such leaves became as susceptible to T. ni larval feeding as unshaven TL3 and TL5 leaves (PI 227687) or all other unshaven leaves (Davis). Bioassays for antifeedants in ethyl acetate and hexane extractables from leaves at the different positions on PI 227687 and Davis plants showed that the resistance observed in the uppermost Davis leaf is attributable to trichomes (i.e., a morphological factor); whereas, in the uppermost PI 227687 leaf it involves both morphological (trichomes) and chemical factors, but the trichome parameter is dominant.

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Résumé L'influence de la densité et de la longueur des trichomes de Glycine max sur la résistance à Trichoplusia ni a été évaluée suivant la position des feuilles sur une lignée résistante (PI 227687) et sur un cultivar sensible (Davis).Les feuilles apicales (juvéniles) tant de PI 227687 que de Davis, recouvertes de trichomes denses, résistèrent mieux à l'alimentation larvaire et à la survie de T. ni.Quand ces trichomes étaient éliminés, ces feuilles ne résistaient pas plus à l'alimentation des larves de T. ni que les feuilles non rasées TL3 et TL5 de PI 227687 ou toutes les autres feuilles de Davis.Les tests avec des extraits dans l'acétate d'éthyle et l'hexane de feuilles provenant de différentes positions de PI 227687 et Davis, destinés à mettre en évidence des phagodissuadants, ont montré que la résistance observée chez les feuilles apicales de Davis était attribuable aux trichomes (c'est-à-dire à un caractère morphologique), tandis que chez les mêmes feuilles de PI 227687 elle impliquait à la fois des trichomes (morphologie) et des substances chimiques, mais avec une prédominance de l'influence des trichomes.

     

Surface morphology of peritrophic membrane formation in the cabbage looper,Trichoplusia ni

Summary The development of the peritrophic membrane in the cabbage looper, Trichoplusia ni, was investigated by scanning electron microscopy. The development of this membrane is characterized by a series of events suggested by the observations to be (1) secretion of material among and above the microvilli of the midgut epithelial cells, (2) maturation of this material into a randomly cross-linked fibrous matrix, and (3) aggregation of amorphous materials in and within the matrix. The membrane, possessing small discontinuities, remains intact in the midgut, but shows gross damage by the time it is passed from the insect, surrounding the feces.     

Approaches to the purification of the juvenile hormone esterase from the cabbage looper,Trichoplusia ni

Several approaches to the purification of juvenile hormone (JH) esterase from second-day last-instar larvae of Trichoplusia ni were taken, including: ammonium sulphate precipitation, polyethylene glycol precipitation, hydrophobic interaction chromatography, anion exchange chromatography, and gel filtration chromatography. The most successful procedure involved a combination of polyethylene glycol precipitation with anion exchange chromatography on DEAE Sephacel which yielded a 134-fold purification of juvenile hormone esterase. When this preparation was subjected to semi-preparative electrophoresis followed by isoelectric focusing on a polyacrylamide slab gel, a single band of apparently homogeneous enzyme was obtained. Juvenile hormone esterase activity was unstable after electrophoresis and isoelectric focusing. The stability of juvenile hormone esterase activity in a water solution is influenced by protein concentration and by agents protecting sulphydryl groups. The results of this study support the hypothesis that a single protein is responsible for the majority of the JH hydrolysis catalyzed by haemolymph from the larvae of T. ni used in this study.     

Partial characterization of the pathway from propionate to acetate in the cabbage looper,Trichoplusia ni

Experiments were performed to characterize the metabolism of propionate to acetate in the cabbage looper Trichoplusia ni and correlate the results with vitamin B₁₂ levels. Fourth and fifth instar larvae contain 2–4 pg vitamin B₁₂/mg dry wt whereas pupae and adults do not contain detectable amounts. In vivo studies as a function of time in larvae, pupae and adults gave evidence that [2-¹⁴C]propionate was converted to 3-hydroxypropionate and then to acetate, which subsequently labeled Krebs cycle intermediates. Radioactivity from [1-¹⁴C]propionate was recovered only in the propionate and 3-hydroxypropionate fractions, and not in acetate or Krebs cycle intermediates, suggesting that carbon 1 of propionate was lost as carbon dioxide and that carbons 2 and 3 of propionate were retained during conversion to acetate. The enzymes of this pathway were located entirely in the mitochondrial fraction. Cyanide inhibited the metabolism of propionate to 3-hydroxypropionate and acetate in mitochondrial preparations, whereas carbon monoxide did not. [2,3-¹⁴C]Acrylic acid was metabolized to 3-hydroxypropionate, which is consistent with a dehydrogenase converting propionate to acrylate which is then hydrated to 3-hydroxypropionate and then oxidized and decarboxylated to acetate.     

Isolation and some properties of components of nuclear polyhedra from the cabbage looper, Trichoplusia ni

Nuclear polyhedra obtained from diseased cabbage looper, Trichoplusia ni, were digested with sodium carbonate-saline buffer, pH 11.0. The dissolved polyhedra formed 3 general zones when subjected to density gradient centrifugation. The slowest sedimenting component (Zone 1) had an ultraviolet absorption curve typical of protein and a sedimentation coefficient of 11 S. Capsids, 310 × 40 nm, were located in Zone 2. Virus particles were found in 1–3 bands (Zone 3); those with envelopes measured 300 × 72 nm, and those without envelopes measured 300 × 33 nm. Virus preparations stained with phosphotungstic acid at pH 7.0 exhibited extensive disruption whereas preparations stained at pH 3.0 did not. Virus particles in the sodium carbonate-saline-digested polyhedra had a sedimentation coefficient of 1228 S. Virus particles isolated by high speed centrifugation had a sedimentation coefficient of 1530 S.     

Hydrolysis of sex pheromone by antennal esterases of the cabbage looper, Trichoplusia ni

Esterases were isolated from chemosensory sensilla on the antennaeof Trichoplusia ni (Hübner). The disc gel electrophoreticpatterns of these esterases from males and females were similar;however, more bands were observed in the antennae than in 8other tissues examined. Most of the esterases detected in the100,000 g supernatant of the antennal preparation could be dissociatedfrom the 100,000 g membrane pellet. Esterases from both maleand female antennae hydrolyzed the sex attractant, (Z)-7-dodecen-l-olacetate, more rapidly than did the legs, fat body or Malpighiantubules. The enzymes primarily responsible for pheromone catabolismwere less sensitive to paraoxon, eserine and p-(hydroxymercuri)benzoatethan those hydrolyzing 1-napthyl acetate. This suggested thata major portion of the observed pheromone-hydrolytic activitywas due to acetylesterases. Measurement of pheromone hyrolysisin sections of disc gels that contained separated antennal orabdominal body wall esterases revealed 2 peaks of activity withboth tissues; however, the rate of pheromone hydrolysis by theabdominal esterases was slower than that of the antennae. Thesignificance of these findings is discussed in relation to thepossibility of antennal esterases having a functional role inthe olfactory process of males of T. ni.     

Screening for Bacillus thuringiensis crystal proteins active against the cabbage looper, Trichoplusia ni

Toxicity tests were performed to find among Cry1 and Cry2 Bacillus thuringiensis crystal proteins those with high activity against the cabbage looper. Tests were performed with neonate larvae on surface-contaminated artificial diet. The crystal proteins found to be toxic were, from higher to lower toxicity: Cry1Ac, Cry1Ab, Cry1C, Cry2Aa, Cry1J, and Cry1F (LC50 of 1.14.1, 3.4-4.4, 12, 34, 87, and 250 ng/cm2, respectively). Cry1B, Cry1D, and Cry1E can be considered nontoxic (LC50 higher than 2500 ng/cm2). Cry1Aa was moderately toxic to nontoxic, depending on the source (LC50 of 420 ng/cm2 from PGS and 8100 ng/cm2 from Ecogen). In vitro binding assays with trypsin-activated 125I-labeled Cry1Aa, Cry1Ab, and Cry1Ac crystal proteins and brush border membrane vesicles from midgut larvae showed a direct correlation between toxicity and binding affinity. Heterologous competition experiments indicated that Cry1Aa and Cry1F bind, though only at very high concentrations, to the Cry1Ab/Cry1Ac shared high-affinity binding site.     

Establishment of a cell line from embryos of the cabbage looper,Trichoplusia ni (hubner)

Summary A new cell line was developed from 3-d-old embryonated eggs of the cabbage looper,Trichoplusia ni, and has been designated IPLB-TN-R². It contains a variety of morphological cell types, including myoblastlike, neuroblastlike, and epithelial-like cells. Chromosome analysis revealed typical lepidopteran chromosomes. Isozyme characterization showed patterns similar to two other cabbage looper cell lines (TN-368 and IAL-TND1) in the case of five enzymes but differed from these two lines for two other enzymes. Virus infectivity tests revealed the line is highly susceptible toAutographa californica nuclear polyhedrosis virus, but no cytopathology was observed after inoculation with several other lepidopteran viruses.     

Hydrocarbon accumulation and lipid biosynthesis during larval development in the cabbage looper,Trichoplusia ni

Larvae of the cabbage looper, Trichoplusia ni, were analyzed for the accumulation and biosynthesis of cuticular and internal hydrocarbon at closely spaced and accurately timed intervals during the fourth and fifth stadia. Large differences in the incorporation of [1-¹⁴C]acetate into hydrocarbon were observed at different times during larval development. Much higher incorporation was observed during feeding stages as compared to wandering stages, while lowest rates of biosynthesis occurred just prior to ecdysis. Fourth stadia wanderers accumulated increased amounts of internal hydrocarbon, which is apparently used to cover the newly forming cuticle. During the fourth to fifth stadium moult insects lost all cuticular hydrocarbon that was present on the old cuticle (about 8 μg/insect) and had about 8 μg/insect on the surface of the newly exposed cuticle. During the fourth stadium incorporation of [1-¹⁴C]acetate into total lipid declined between feeding and wandering stages from 24% of injected radiolabel to 7%. Similar decreases in lipid biosynthesis were observed between feeders and wanderers in fifth stadium larvae with the greatest decrease found in the triacylglycerol fraction. These results document dramatic changes in the accumulation and biosynthesis of hydrocarbon and other lipids during larval development.     

Modulatory effects of octopamine and serotonin on male sensitivity and periodicity of response to sex pheromone in the cabbage looper moth,Trichoplusia ni

The pheromone-mediated flight behavior of male cabbage looper moths in a sustained-flight tunnel and random activity exhibited during scotophase were observed after males were treated with octopamine or serotonin (5 hydroxytryptamine). Octopamine induced a hypersensitivity to the olfactory signal, resulting in a significant lowering of the pheromone dose that elicited peak levels of response. Octopamine, however, did not affect the circadian rhythmicity of response to pheromone. In contrast, serotonin disrupted the circadian rhythmicity of response, resulting in a high percentage of males exhibiting random activity and response to pheromone throughout the entire 8 h scotophase instead of during the normal peak period during the latter part of the scotophase. Serotonin did not effect a decrease in the dose of pheromone eliciting peak response. In addition, at the highest dosages tested octopamine and serotonin induced opposite postures associated with a paralysis that occurred when males attempted to take flight to the pheromone.