Instar Fat Body Research Articles

Hidden antigens from third instar Hypoderma lineatum: Impact of immunization on larval survival in artificial infestations

Soluble fractions of Hypoderma lineatum third instar fat body, haemocytes and haemolymph were formulated with Quil A and used to immunize four groups of calves while a fifth group remained untreated. Calves received two subcutaneous injections of the soluble fractions, or adjuvant only delivered two weeks apart. Two weeks after the last injection the calves were exposed to 50 newly hatched larvae of H. lineatum which were placed on the skin and allowed to penetrate. Survival of larval stages was monitored by weekly palpation and collection of emergent third instars. Antibody responses to the immunogens were evaluated by immunoblots and following infestation antibody responses to first instar antigens were evaluated by an ELISA. Non-immunized calves and calves injected with adjuvant were all palpation positive for cattle grubs. In groups immunized with fat body, haemocyte and haemolymph components 100%, 33% and 33% were palpation positive for grubs respectively. First instar mortality, as reflected in palpable grubs, was high in the groups receiving injections with tissue components (99.3%, 95.1%, 95.8%, 83.9 and 80.4% mortality for those groups receiving fat body, haemocyte, haemolymph, adjuvant or control respectively). Second and third instar mortality was also higher in the immunized groups (100.0%, 91.7%, 91.7% for fat body, haemocyte, and haemolymph respectively) in comparison to the adjuvant only (14.0%) and unvaccinated (33.3%) groups. No viable flies emerged from pupae originating from larvae emergent from any of the immunized groups. Calves receiving the tissue extracts developed antibodies to several protein components following the second immunization which were still present 13 weeks post-infestation. Several proteins appeared to be common among the three tissue extracts and were recognized by antibodies from the immunized calves. All groups of calves became positive for antibodies to first instar antigens, although in some immunized calves the antibodies were transient.

Purification of the mRNAs encoding the subunits of larval serum proteins 1 and 2 of Drosophila melanogaster.

The messenger RNAs for the four subunits of larval serum proteins (LSPs) 1 and 2 were purified from third instar fat body total RNA by fractionating oligo(dT)-selected RNAs on the basis of size. Translations of RNA fractions in vitro showed that the LSP mRNAs were free of other fat body messenger activities, but they could not be completely resolved from each other. The molecular weights of the LSP mRNAs, estimated from their electrophoretic mobilities in 50% formamide/1.4% agarose gels, ranged from 1.0 X 10(6) to 1.35 X 10(6). These estimates are at least 20% larger than the size of RNA necessary to encode each LSP polypeptide.

Bombyx acid cysteine protease (BCP): hormonal regulation of biosynthesis and accumulation in the ovary

We previously reported purification of the cysteine protease from Bombyx eggs (BCP) and the occurrence of the enzyme in various tissues of this insect. In the present paper, we present a detailed analysis of stage-specific changes in activity of BCP between the fourth larval instar and pupal-adult development. A synthetic fluorescent peptide, carbobenzoxy-l-Phenylalanyl-l-Arginine4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), was used to assay proteolytic activity. When tissue extracts were treated with anti-BCP serum before assay of enzyme activity, most activity towards Z-Phe-Arg-MCA was removed from the extracts. Therefore proteolytic activity in the present experiments is due mainly to BCP. We used Western blot and Northern blot analyses to determine tissue and stage specific expression of the enzyme. In the 5th larval fat body and hemolymph, BCP activity dramatically increased at the time of spinning, returning to the basal level before ecdysis. Northern blot analysis showed that a 1.5 kilobase mRNA which hybridizes to BCPcDNA suddenly appears during this period. Similar results were obtained in 4th instar fat body. In pupal hemolymph and fat body, low basal activity of BCP was detected early (day 0 to day 3 after pupal ecdysis), followed by a pronounced increase to a maximum six days after ecdysis, before returning to the basal level. In ovariectomized female pupae, a significant amount of proteolytic activity accumulated in hemolymph, suggesting that the enzyme is synthesized in the fat body and transferred into the ovary along with vitellogenin. BCP activity increased three days after injection of 20-hydroxyecdysone into ligated pupae. Furthermore, putative BCPmRNA appeared in the fat body within 24 hours after injection. This increase was completely blocked by the administration of cycloheximide. The results suggest that, BCP is synthesized in extraovarian tissues such as fat body and ovarian follicle cells and accumulates in the ovary, thus representing a new class of yolk protein.

Cloning and characterization of an ecdysone receptor cDNA from Locusta migratoria

To facilitate studies on the hormonal control of development in the migratory locust, Locusta migratoria, we have undertaken the cloning of cDNAs for nuclear hormone receptors. Sequences obtained by polymerase chain reaction (PCR) showed homology with receptor family members including the ecdysteroid receptor (EcR). A cDNA clone corresponding to the EcR fragment includes an open reading frame of 1622 nucleotides, predicting a 59 kDa protein showing clear homology with EcRs and distinct from other classes of nuclear receptors. Northern analysis revealed a major transcript of 9.2 kb. In fifth instar fat body, the transcript was most abundant at the end of the instar when ecdysone titres are highest. There was no obvious evidence of EcR regulation by a juvenile hormone analog. Although its role in development may be similar, the locust ecdysone receptor (LmEcR) is divergent from EcRs characterized from insects belonging to the dipteran and lepidopteran orders, presumably reflecting the more ancestral sequence in the relatively primitive locust.

Freeze tolerance in hermit flower beetle ( Osmoderma eremicola) larvae

Larvae of Osmoderma eremicola (Knoch)(Coleoptera: Scarabaeidae) were found to be freeze tolerant. Early (1–2.5 cm) and late (3–5 cm) larval stages survived freezing to −8.3°C for 96 h with 64% of body water as ice. Glycogen phosphorylase activity in the fat body was increased 6–7-fold during freezing, and glycogen stores were depleted in fat body (from 237 ± 1 to 57 ± 7 μmol/g fresh weight) and body wall (89 ± 4 to 40 ± 1 μmol/g fresh weight). However, no glycogen derived cryoprotectants (glycerol, sorbitol, glucose, fructose) were found to accumulate in tissues or haemolymph during freezing or acclimation at 9,0 and −5°C. Osmolatity (∼450 mOsmol) of haemolymph was constant during freezing and acclimation from 9.to −5°C, also suggesting the absence of low molecular weight cryoprotectants. Haemolymph trehalose levels never rose above 2.7 mM as detected by NMR. Total protein levels in fat body and body wall remained unchanged during acclimation. HPLC analysis showed increases in amino acid levels by 10–15 μmol/g fresh weight in the body wall of both early and late instar larvae during freezing to −8.3°C for 96 h, with consistent increases in glycine (7.4–8.7 μmol/g fresh weight), alanine (4.2–5.8 μmol/g fresh weight), glutamate (2.4 μmol/g fresh weight), and valine (0.8–1.6 μmol/g fresh weight). Smaller increases in glutamic acid (by 1 μmol/g fresh weight) and alanine (by 3.5 μmol/g fresh weight) were also seen in late instar fat body, whereas glycine levels remained unchanged and valine decreased slightly (by 1.4 μmol/g fresh weight) in this tissue. Proline levels, detected by NMR, ranged from 10 to 18 μmol/g fresh weight in fat body and 40–70 μmol/g fresh weight in haemolymph, making it the most abundant amino acid in fat body.

Structural features critical to the activity of an ecdysone receptor binding site

Two ecdysone-response elements from the hsp27 (hsp27 EcRE) and the Fbp1 (D EcRE) genes of Drosophila melanogaster were used as probes in a gel shift assay to investigate the interactions of the ecdysone receptor (EcR) with its cognate DNA response element. The source of EcR was a nuclear extract from the late third-larval instar fat body. The hsp27 and D EcREs share a sequence similarity at 12 positions over a 15bp region including an imperfect palindromic structure consisting of two pentamer half-sites separated by a single intervening nucleotide. We have shown that a short oligonucleotide containing this 11bp imperfect palindrome of the hsp27 EcRE and three flanking bp on each side is an efficient EcR binding site. Mutational analysis confirms that the integrity of both these half-sites as well as their 1bp spacing are critical for binding of the ecdysone receptor. The D EcRE behaved as a much weaker EcR binding sapacity equivalent to that of the hsp27 EcRE. These results have led us to propose the sequence PuG(G/T)T(C/G)A(N)TG(C/A)(C/A)(C/t)Py as a revised version of a previously proposed EcRE consensus sequence.

Synthesis of larval serum proteins 1 and 2 of Drosophila melanogaster by third instar fat body

Larval serum proteins (LSPs) 1 and 2 of Drosophila melanogaster have been shown to accumulate in third instar larval haemolymph ( Roberts et al., 1977). In the present paper the major third instar larval tissues have been assayed for LSP synthesis. Polypeptides radiolabelled in organ culture have been analyzed by SDS-polyacrylamide gel electrophoresis and by immunoelectrophoresis. Fat body was shown to be the major source of LSPs 1 and 2 and the two proteins were synthesized by all regions of third instar fat body.