Insect Biochemistry最新文献

Glutathione S-transferase (GST) isozymes were purified from the GG strain of Aedes aegypti, a strain having ≥4-fold higher total GST activity compared to the wild-type lab strain. Purification involved S-hexyl-glutathione affinity chromatography in high salt buffer, and GST specific elution with S-(p-bromobenzyl)-glutathione. Final purification was accomplished on DEAE-Sepharose. Two isozymes, GST-1b and GST-2 were purified using this procedure, and an additional isozyme, GST-1a, was partially purified. The GST-2 isozyme has one of the highest specific activities reported for a GST, with a specific activity of 739 μmol/min/mg using 1-chloro-2,4-dinitrobenzene (CDNB), and 16.4 μmol/min/mg using 3,4-dichloronitrobenzene (DCNB) as substrates. GST-2, GST-1a, and GST-1b were analyzed for amino acid composition and subjected to N-terminal sequencing. All three GSTs showed amino acid differences, especially among the nonpolar and polar amino acids. The amino acid composition of GST-1b was found to be more similar to GST 1-1 from Drosophila melanogaster than to GST-2 or GST-1a from Aedes aegypti. Only GST-2 gave N-terminal sequence data, raising the possibility that GST-1a and 1b are N-terminally blocked. The A. aegypti GST-2 showed amino acid sequence identity or similarity in all but one residue between residue numbers 31 through 41 compared to the D. melanogaster and Musca domestica GST 1-1 isozymes. The pattern of GST isozyme expression was analyzed in various tissues and stages of development of the GG and wild type strains using isozyme-specific antisera and substrates. GST-1a was constitutively overexpressed in all tissues examined in the GG strain compared to the wild type strain. The expression of GST-1b was similar in both strains for all tissues and developmental stages examined. GST-2 was constitutively overexpressed in head, thorax and abdomen, but was not detected in ovaries of the GG strain. These results suggest that elevated GST activity in the GG strain is due to constitutive overexpression of GST-2 and GST-1a. GST-1a, GST-1b and GST-2 apparently are the products of 3 independently regulated genes and appear to be expressed in a tissue-specific manner.

Antibodies raised against the synthetic hapten $\text{p-}\text{azobenzyl}$ phosphonate (ABP), which specifically cross react with phosphotyrosine, were used to detect phosphotyrosine-containing proteins in integument, fat body, and haemocytes of C. capitata white pupae. Immunofluorescence experiments demonstrated that fat body, integument, and haemocytes contained proteins phosphorylated in tyrosine residues, while other tissues, such as muscle, showed no fluorescence. To determine which polypeptides are recognized by ABP antibodies, an immunoblotting analysis was performed. The results revealed that the majority of the immunoreactive polypeptide with a $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ ranging between 35- and 130 kDa were detected in the integument of C. capitata. Three immunoreactive polypeptides seem to be common in tissues we have examined. 130 kDa is common in integument and fat body, 98 kDa in integument and haemocytes, and 93 kDa is common in haemocytes and fat body. Solubilization of integumental proteins in Tris-HCl buffer pH 7.2 containing either KCl, or NaCl, or urea, or swittergen shows that several phosphotyrosine-containing proteins form non-covalent links with other cuticular components and/or cytoskeleton. Furthermore, treatment of unsolubilized fraction with lysozyme, indicated that some N-acetylglucosamine-containing material prevents extraction with salt solution; this material may be chitin or some intracellular material. The possible contribution of phosphotyrosine containing proteins in the pupariation process is discussed.

The accumulation of transcripts of a cytoplasmic actin gene, the actin A3 gene, has been studied in relation to the silk production cycle in the two secretory regions of the silk glands of Bombyx mori. This accumulation was roughly similar in the two regions. The cellular amount of actin A3 mRNA decreased during the first period of the fourth moult, increased on and after the second period of this moult to reach a maximum at Day 5 of the fifth instar, then finally decreased. Actin biosynthesis almost completely ceased in the first period of the fourth moult and recovered in the second period of this moult. The accumulation of actin A3 mRNA parallels the development, in the silk gland cells, of the apical network of microfilaments which are implicated in silk secretion and which are disrupted at the beginning of the moult period and recorganized during the intermoult period. The accumulation of transcripts from actin A3 gene was compared to that of transcripts from silk protein genes and they were found to be asynchronous during the secretory cycle, indicating that mechanisms regulating expression of these two types of genes are different in the silk glands.

The reproductive tissues of the primitive insect Thermobia domestica synthesize several eicosanoids when incubated with exogenous arachidonic acid. The enzymatic system was characterized with respect to kinetic parameters, Ca²⁺ requirement and pH- and cofactor-dependence. Five monohydroxylated eicosatetraenoic acids (HETEs) were identified by mass spectrometry procedures, demonstrating the existence of the lipoxygenase pathway. Quantitative studies were performed by high-pressure liquid chromatography. It is confirmed that the 8-lipoxygenase activity remains the major pathway in tissues from males and from inseminated females. In female tissues, the amounts of metabolite depend on the number of spermatophores contained in the seminal receptacles. These data are in agreement with the hypothesis of a transfer of the enzyme from male to female during mating. The mechanisms involved are discussed in comparison with those of other insect species in which the synthesis of prostaglandins has been reported.

In a previous study it has been reported that dsp28 is induced during desiccation in Tenebrio larvae. During that study it was observed that in non-stressed larvae the concentration of dsp28 in hemolymph drops dramatically just prior to pupation. These results suggested that control of dsp28 synthesis is subject to environmental as well as hormonal cues. This study identifies a site of synthesis as fat body, as dsp28 was secreted into the medium during in vitro incubation of larval fat bodies. Using immunoelectrophoresis to determine protein concentration a developmental profile showing changes in levels of dsp28 in hemolymph during larval, pupal and adult stages of Tenebrio molitor was established. The concentration of dsp28 in larval hemolymph dropped from 4 to 5 mg/ml to 1.5 mg/ml just prior to pupation. This lower level was maintained until adults emerged when the concentration of dsp28 rose to prepupal levels again. Hormonal regulation is suggested since application of methoprene to newly-emerged pupae resulted in an increased incorporation of radiolabeled cysteine into dsp28.

In Manduca sexta, storage proteins accumulate during the final larval stadium for utilization during subsequent larval-pupal-adult transformations. Two cDNA clones (designated clone 119 and clone 201), that represent two distinct but related genes (42% sequence identity), were isolated from a cDNA library prepared from day 7 fifth instar larval fat body and found to encode two different storage proteins synthesized during the last larval instar. Northern blot analyses revealed that the two clones hybridize to 2.4 kb transcripts that are translated to 79 kDa protein products during in vitro translation experiments. Clone 119 encodes a methionine-rich storage protein, designated as SP1A, that shares 37% sequence identity with the Bombyx mori sex-specific storage protein SP1. Clone 201, on the other hand encodes a storage protein, designated as SP1B, that is more closely related to B. mori SP1 (63% identity), and is probably identical to the Manduca female-specific storage protein (FSP). Insert DNA from clone 201, but not clone 119, cross-hybridizes to that of FSP cDNA (Webb and Riddiford, Dev. Biol.130, 671–691, 1988a). Both storage proteins are synthesized only in the fat body and only during the fifth larval stadium, indicating tissue- and stage-specific expression of the two genes. Both genes exhibit sex-specific differences in expression. In the fifth larval stadium, the mRNAs for the SP1A and SP1B proteins begin to accumulate at about day 2 in the female fat body but appear 2 or 3 days later in the male fat body. In both sexes SP1A mRNA remains relatively high beyond the time when SP1B mRNA has already declined to low levels, suggesting differences in mRNA stability or expression. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens causes substantial increases in the levels of both mRNAs, whereas topical application of the juvenile hormone mimc, fenoxycarb, to feeding fifth instar larvae produces substantial declines in the mRNA levels, indicating hormonal effects at the transcriptional level. The data support the hypothesis that the expression of these M. sexta methionine-rich storage protein genes is stimulated by ecdysteroid and inhibited by juvenile hormone.

We have accomplished gene transfer into embryos of Locusta migratoria, the African migratory locust. Freshly oviposited eggs were injected with circular or linear plasmids containing the Drosophila hsp70 promoter and the choramphenicol acetyltransferase (CAT) reporter gene (hsp-cat), or with circular plasmid containing the Drosophila copia promoter fused to CAT (copia-cat). Southern blot analysis showed that the hsp-cat plasmid persisted extrachromosomally for at least 8 days after injection. There was no evidence for plasmid replication. Transient expression from the introduced promoters was determined by monitoring CAT enzyme activity. After injection of hsp-cat, activity was detected at varying levels in 6–8% of day 3 and day 9 embryos. Embryos injected with copia-cat, assayed on day 3, had a greater frequency but no higher level of expression. The described gene transfer system is promising for analysis of other promoters, including those of Locusta.

A cDNA for a midgut trypsin induced by a blood meal has been cloned and sequenced from the mosquito Aedes aegypti. The 862 base sequence codes for a 257 amino acid protein, which is presumably a trypsin precursor, since the sequence of purified mosquito trypsin begins at residue 26, immediately following an arginine residue in the precursor. The amino terminal 25 amino acids in the precursor are composed of a putative 15 amino acid signal peptide and a 10 amino acid activation peptide. Compared to vertebrate trypsinogens, the putative mosquito trysinogen lacks the four acidic amino acids immediately preceding the basic amino acid at the activation peptide cleavage site, suggesting the mechanism of its activation may be different from the activation of vertebrate trypsinogens. However, since a trypsinogen has not yet been isolated from the mosquito, these conclusions are tentative. The deduced amino acid sequence is homologous to that of other trypsins in those residues around the catalytic triad, and in several residues which are found only in trypsins. However, the sequence of the specificity pocket in mosquito trypsin, KESPC, differs from that found in other trypins, KDSC. The Asp is thought to bind the basic residue of the substrate, and the Glu in the mosquito trypsin may serve the same role. The changes in trypsin protein and mRNA levels following a blood meal indicate that an important component of the regulation of trypsin synthesis is at the transcriptional level.

The concentrations of 5-hydroxytryptamine (5-HT), N-acetyl-5-hydroxytryptamine (NAS), 5-hydroxyindoleacetic acid (5-HIAA) (HPLC-EC) and tryptophan (Trp) were determined in the cerebral ganglia and ventral nerve cord (VNC) of both sexes of two acridids. In both species, the maximum values for all compounds appeared in the brain region, the converse occurring for Trp levels. 5-HT and its metabolites followed in both acridids the same tendency, males showing higher values than females. These male-female differences were markedly significant for NAS and 5-HIAA concentrations in Paracinema tricolor, and for 5-HT concentrations in Oedipoda caerulescens. Both acridids differed for brain concentrations of such compounds. Thus, P. tricolor showed the maximum values for NAS and 5-HIAA and O. caerulescens for 5-HT levels. Furthermore, in both species NAS represented about 75–90% of both catabolic products considered as a whole. The results support the idea of a coexistence of N-acetylation and oxidative deamination in both acridids, with higher importance of the 5-HT N-acetyl derivative. A differential 5-HT turnover between sexes is also suggested.

The effects of two plant phototoxins (xanthotoxin and harmine) and three plant phenols (quercetin, ellagic acid, and juglone) on detoxification enzymes were studied in the polyphagous cabbage looper, Trichoplusia ni, and the oligophagous black swallowtail, Papilio polyxenes. In P. polyxenes, glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitrobenzene (CDNB) were 1840 and 1750 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut and fat body, respectively. Dietary xanthotoxin (0.1% fw) increased the activity 2.5 and 2.9-fold in the midgut and fat body, respectively. Xanthotoxin-conjugating GST activity was absent in both tissues. In T. ni, GST activity, 513 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut, was increased almost twofold by dietary xanthotoxin and harmine. Plant phenols effectively inhibited in vitro GST and Se-independent glutathione peroxidase (GPOX) activities in a dose-dependent manner in the two species. Both GST and GPOX of P. polyxenes were 2-fold less sensitive to phenol inhibitors than T. ni. GST inhibition differed according to the nature of the inhibitor in P. polyxenes. Quercetin is competitive with CDNB and is non-competitive with respect to GSH. In contrast, inhibition by ellagic acid is non-competitive with CDNB and competitive with GSH. Juglone showed competitive inhibition with both GSH and CDNB.