Hemolymph Of Last Instar Larvae Research Articles

Biochemical characterization of α- and β-glucosidases in alimentary canal, salivary glands and haemolymph of the rice green caterpillar, Naranga aenescens M. (Lepidoptera: Noctuidae)

The biochemical properties of α- and β-glucosidase in salivary glands, alimentary canal and haemolymph of Naranga aenescens larvae, one of the most damaging pests of the rice crop in Iran, were investigated. The specific activity of α-glucosidases were 3.88, 2.74 and 1.58 μmol/min per mg protein in the alimentary canal, salivary glands and haemolymph of last instar larvae, respectively. The specific activity of β-glucosidases were 1.27, 0.077 and 0.414 μmol/min per mg protein in the alimentary canal, salivary glands and haemolymph of last instar larvae, respectively. The optimal pH for α-glucosidases were 6.0, 6.0–8.0 and 6.0 and the maximum activity for β-glucosidases were obtained at pH 6.0, 5.0–7.0 and 5.0 in alimentary canal, salivary glands and haemolymph, respectively. The optimum temperatures for β-glucosidases were determined at 55°C in alimentary canal, 35–45°C in salivary glands and 55°C in haemolymph, whereas the α-glucosidases reached their optimum at 45°C in all three tissues. Effect of metal ions on the activity of α- and β-glucosidases showed that K+ (20 mM) and Mg2+ (10 and 20 mM) increased N. aenescens α- and β-glucosidases activities from salivary glands, while Ca2+ increased α- and β-glucosidases activities in haemolymph. In the presence of Fe2+, Mn2+, Hg+ and Zn2+ (10, 20 mM) and Hg2+ (20 mM), these enzymes from all tissues were completely inactivated. K m values were estimated for the α-glucosidases as 3.96, 0.547 and 3.084 mM and for β-glucosidases as 1.93, 1.014 and 1.93 mM in the alimentary canal, salivary gland and haemolymph, respectively. The zymogram analyses of N. aenescens crude extracts indicated the presence of at least two isoforms for α-glucosidase and one isoform for β-glucosidase.

VHDL, a larval storage protein from the corn earworm, Helicoverpa zea, is a member of the vitellogenin gene family

The hemolymph of last instar larvae of the corn earworm, Helicoverpa zea contains a blue very high-density lipoprotein (VHDL) that is selectively taken up into fat body prior to pupation. Its amino-terminal sequence was determined by Edman degradation, and used to design a degenerate primer for PCR amplification. With 5′ and 3′ RACE techniques, the entire cDNA coding for VHDL was amplified and sequenced. Conceptual translation reveals a 173 kDa protein that contains a 15 amino acid signal sequence immediately before the experimentally determined N-terminus of the mature protein. The protein contains a typical lipoprotein N-terminal domain, and shows high sequence similarity to vitellogenins from Lepidoptera and other insect species. VHDL mRNA was not detectable in adult H. zea, and antibodies raised against VHDL did not react with adult hemolymph or yolk proteins. Therefore VHDL, although a member of the vitellogenin gene family, seems to be distinct from the vitellogenin expressed in adult females.

Purification and cDNA Cloning of Lysozyme II from Cabbage Butterfly, Artogeia rapae Larvae

ABSTRACTLast instar larvae of cabbage butterflyArtogeia rapaerespond to injection of bacteria with a set of inducible antibacterial peptides/proteins. The inducible peptides/proteins are related to the known hinnavins (I and II) and lysozymes (I and II). The lysozyme II has been isolated by heat treatment, cation exchange, and reversed‐phase chromatography from immunized hemolymph of last instar larvae. The lysozyme II gene ofA. rapaewas isolated and its nucleotide sequence was determined by the RACE‐PCR from immunized fat body withE. coli.It has an open reading frame of 414 bp nucleotide corresponding to 138 amino acids including an 18 amino acid signal sequence. The molecular weight and the isoelectric point ofArtogeialysozyme II without a signal peptide were 13,649.38 Da and 9.11, respectively. It is great similarity withManducalysozyme among other lepidopteran.

Hemocyte diversity of the American bollwormHelicoverpa armigera

The hemocytes of the American bollwormHelicoverpa armigera (Hubner) were studied in the last instar larvae by phase contrast microscopy to investigate the differences among 14 populations, collected from ten locations, spread over a distance of approximately 3000 km in six different states of India. The cluster analysis of differential hemocytes showed as many as 11 clusters of populations at 5% and six clusters at 10% homogeneity level. The cluster analysis of hemocytes of seven populations from cotton crops showed five and three clusters at 5% and 10% homogeneity levels, respectively. Diversity of hemocytes of four populations from chickpea was wider than that from cotton. The differences in hemocytes of various populations may be due to space, time, host plant, insecticide use, other agroecological conditions, and insect genetic variabilityper se. The field populations ofH. armigera heavily treated with insecticides appeared to contain more spherulocytes at the expense of plasmatocytes and granulocytes. The treatment of 6-day-old larvae ofH. armigera with cypermethrin decreased plasmatocytes and granulocytes, and increased prohemocytes and spherulocytes in the hemolymph of last instar larvae. Thus, differential hemocyte counts may serve as an indicator of insecticide exposure and the diversity of insect populations.

Purification of Lysozyme from Hemolymph of Tobacco Cutworm,Spodoptera litura

ABSTRACTLysozyme is one of the components of the innate immunity of the insects. The lysozyme has been isolated by heat treatment, cation exchange, and reversed‐phase chromatography from immunized hemolymph of last instar larvae ofSpodoptera litura.The hemolymph immunized with the insect nonpathogen,Escherichia coliwas collected 2 days after the abdominal injection. The molecular weight ofSpodopteralysozyme was estimated to be about 15 kDa by SDS‐PAGE and it is great similarity withAgriuslysozyme, which recently purified from larval hemolymph ofAgrius convolvuli.

In vitro degradation of the Neb-Trypsin Modulating Oostatic Factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.

Comparative Analysis of Storage Proteins of the Fall Webworm (Hyphantria cunea, Drury)

Two kinds of storage protein (SP-1 and SP-2) were purified from the hemolymph of last instar larvae of Hyphantria cunea by ultracentrifugation, chromatofocusing, and ion-exchange chromatography. SP-1 and SP-2 each consist of six subunits and have the molecular mass of 79 KDa and 82.5 KDa, respectively. These showed different patterns of appearance in fat body and hemolymph, and different SDS-PAGE patterns in non-reducing gels. Their stability in citrate-phosphate or Tris-HCl buffers differs and their immunological charicteristics indicates that these are different storage proteins. Comparison with storage proteins of other species with respect to amino acid composition and N-terminal sequence, indicates that SP-1 can be classified as a methionine-rich storage protein but SP-2 cannot be regarded as a classical arylphorin.

Isolated Apolipophorin III from Galleria mellonella Stimulates the Immune Reactions of This Insect

Apolipophorin III (apoLp-III) was isolated from the haemolymph of last instar larvae of Galleria mellonella. The ultraviolet (u.v.) spectrum and the N-terminal amino acid sequence reveal high similarities with the apoLp-III from Manduca sexta. The protein is heat-stable. The molecular mass of apoLp-III was determined to be 18 077 Da using mass spectrometry. The heat treatment (90°C, 30 min) resulted in a pI shift from 6.6 for the non-heated to 6.1 for the heat-treated apoLp-III without change in the molecular mass, indicating that a conformational change might have been caused by the heat treatment, rather than covalent alterations. Intrahaemocoelic injection of pure apoLp-III into last instar G. mellonella larvae is followed by a dose-dependent increase of antibacterial activity in cell-free haemolymph of treated larvae 24 h after injection. Furthermore, pure apoLp-III enhances the phagocytic activity of isolated haemocytes in vitro. The newly discovered role of apoLp-III in inducing immune-related functions in insects is discussed in regard to the known features of this molecule in lipid metabolism. Arylphorin, another heat-stable protein in G. mellonella haemolymph, was likewise isolated in this study. The protein was identified by N-terminal protein sequencing, the sequence obtained exactly matches the known sequence data for this protein. © 1997 Elsevier Science Ltd. All rights reserved

Cosmic influences on the expression of a specific gene in the Colorado potato beetle: the diapause protein 1 gene.

Diapause protein 1 from the Colorado potato beetle, Leptinotarsa decemlineata, belongs to the family of insect storage hexamers. The protein accumulates in the hemolymph of last instar larvae and in adults reared under short-day (diapause-inducing) conditions. The protein disappears during metamorphosis, but remains in the hemolymph during diapause. The gene which encodes diapause protein 1 encompasses about 9 kb of chromosomal DNA and is composed of 5 exons, separated by 4 introns. The gene is expressed in last-instar larvae and in adults reared under short-day conditions. The messenger RNA for diapause protein 1 occurs only in the fat body. Gene expression is higher in the fat body from short-day adults than from last-instar larvae. Transcription is suppressed after topical application of pyriproxyfen, a JH-analog. The messenger RNA of the protein (approximately 2.3 kb) contains genetic information of a pre-protein of 702 amino acids, including a signal peptide of 17 amino acids and protein of 685 residues. The function of diapause protein 1 is unknown, but it is utilized during post-diapause development, probably for regeneration of the flight muscles.

Storage proteins are present in the hemolymph from larvae and adults of the Colorado potato beetle.

The protein composition of larval and adult hemolymph from the Colorado potato beetle, Leptinotarsa decemlineata, was investigated and some abundant, high molecular weight proteins were identified and characterized. Diapause protein 1, which occurs in the hemolymph of last instar larvae and short-day adults, appeared to be a storage protein. This protein dissociated into two bands due to the high pH used in nondenaturing gels. Its quaternary structure was established by chemical crosslinking. It appeared to be a hexamer. Diapause protein 1 is composed of approximately 82,000 subunits. The amino acid composition and N-terminal sequence of this protein has been determined. Specific antibodies against diapause protein 1 have been developed. Topical application of 1 microgram pyriproxyfen, a juvenile hormone analog, to last instar larvae and short-day adults suppressed the appearance of this protein in the hemolymph. Pyriproxyfen prematurely induced vitellogenin, when applied to last instar larvae. A larval specific protein was also identified in the hemolymph. Its temporary appearance in the hemolymph of last instar larvae, its subunit composition (M(r) approximately 82,000) and its suppression by pyriproxyfen suggests that this protein is a storage protein as well.

Vitellogenesis in the gypsy moth,Lymantria dispar(L.): Characterization of hemolymph vitellogenin, ovarian weight, follicle growth and vitellin content

Summary Gypsy moth vitellin (Vt, greater than 500, 000 daltons) was partially purified from pupal ovarian follicles by high-salt extraction and analyzed by non-SDS- and SDS-PAGE. A large-molecular-weight, female-specific, pupal hemolymph protein (vitellogenin, Vg) had electrophoretic and immuno-staining properties similar to Vt. A slightly larger female-specific hemolymph protein larval-Vt) was found in last-instar female gypsy moth larvae. Vt, pupal-Vg and larval-Vg were recognized on Western blots by a rabbit polyclonal antiserum made against L. dispar Vt. The apoprotein subunits of Vt were 180,000 (apo-Vt180), 165,000 (apo-Vt165), and 38,000 daltons (apo-Vt38). Apo-Vg of similar sizes as the apo-Vt (apo-Vg180, apo-Vg165, and apo-Vg38) were recognized in larval and pupal hemolymph by the anti-Vt antisera. Apo-Vg began to accumulate in the hemolymph of last-instar larvae after day 2 and were not detected by Coomassie-or immuno-staining prior to this time. This indicates that synthesis of Vg must be initi...

Coordinate induction of electrophoretic variants of juvenile hormone esterase

Major and minor electrophoretic variants of juvenile hormone esterase (JHE) were found in the hemolymph of last instar larvae of Trichoplusia ni , both before and after metamorphic commitment. The average ratios of activity of the two major forms were similar during both last stadium peaks in activity. Immunological analysis showed that the hemolymph concentration of JHE during this stadium paralleled the level of enzymatic activity, and no putative higher molecular weight, inactive forms were detected. Immunological analysis provided the first evidence of relatedness of major and minor forms. After hormonal stimulation, the concentration of the two major forms increased concomitantly and by a similar proportion, suggesting that charge variation, at least for these two major forms, is not a point of hormonal or developmental regulation of JHE.

Storage proteins of the fall webworm, Hyphantria cunea drury

AbstractTwo storage proteins, storage protein‐1 (SP1) and storage protein‐2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8‐day‐old 7th instar larvae).SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively.Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4‐day‐old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.

A larval specific lipoprotein: Purification and characterization of a blue chromoprotein from Heliothis zea

The green color of the hemolymph of last instar larvae of the corn earworm, Heliothis zea (Boddie), is caused by the appearance of a blue chromoprotein. This protein (Mr approximately 560,000) is composed of 4 subunits (Mr approximately 150,000). It contains 8.4% lipid and has an equilibrium density of 1.26 g/ml. This protein is absent during all early larval stages, in pupae and in adult animals, but is a major hemolymph protein in 5th instar larvae. Although its physiological function remains unclear, this blue chromoprotein represents the first member of a new class of larval-specific insect lipoproteins.

Age dependent changes in the binding and hydrolysis of juvenile hormone in the haemolymph of last instar larvae of Galleria mellonella

Binding and hydrolysis of juvenile hormone I (JH) and cleavage of four 1-naphthyl esters were determined in the haemolymph of Galleria mellonella throughout the last larval instar. JH binding per ml haemolymph parallels changes in the protein content with a maximum at the onset of spinning at 120 hr (152 mg protein/ml; binding of 20 n-mole JH/ml) and a drop at the pupal ecdysis. Activity of JH esterase(s) exhibits two maxima: first between cessation of feeding and the start of spinning at 96–120 hr (hydrolysis of 65 n-mole JH/ml/min) and second just before pupal ecdysis at 168 hr (52 n-mole JH/ml/min). Correlations between these changes and available data on the JH content in caterpillars indicate that JH binding protein and JH esterase(s) may play a role in clearing JH from its target tissues rather than in regulating its amounts. Fluctuations in the hydrolysis of 1-naphthyl esters of acetate (max. 4.5 μ-mole/ml/min), butyrate (max. 21 μ-mole/ml/min), palmitate (max. 0.5 μ-mole/ml/min), and laurate (max. 0.4 μ-mole/ml/min) reflect primarily the feeding activity of caterpillars and follow a very different course than changes in the potential to hydrolyze JH.

Effects of the anti hormone-hormone mimic ETB on the induction of insect juvenile hormone esterase in [formula omitted][formula omitted

Juvenile hormone (JH) esterases can be artificially induced to appear in the hemolymph of last instar larvae of the lepidopterous insect Trichoplusia ni (Noctuidae) by topical treatment with JH I, JH II, or dihomo branched juvenoids. ETB (ethyl-4-[2-(t-butylcarbonyloxy) butoxy] benzoate; ZR-2646) at high doses is a weak inducer of JH esterase (JHE). However, at doses of ETB that induce only low levels of JHE activity, ETB will block the JHE induction caused by the dihomo juvenoid epofenonane and at higher doses will reduce the induction caused by JH I or JH II. ETB is not a JHE inhibitor; rather, it appears to be acting as a JH agonist/antagonist in normal larvae and in isolated abdomens. These effects of ETB on JHE induction may illustrate a new mode of action of anti-JH's.