Shalom W. Applebaum

Sex-peptide activates juvenile hormone biosynthesis in the Drosophila melanogaster corpus allatum

Mating elicits two well-defined reactions in sexually matured females of many insects: reduction of receptivity and increased oviposition. These post-mating responses have been shown to be induced by factors synthesized in the reproductive tract of the adult male and transferred in the seminal fluid to the female during copulation. One of these factors, named sex-peptide (SP), has been identified in Drosophila melanogaster. Using an in vitro radiochemical assay, we show that synthetic sex-peptide considerably activates juvenile hormone III-bisepoxide (JHB3) synthesis in corpus allatum (CA) excised from Days 3 and 4 post-eclosion virgin females. Base levels are significantly lower at emergence (Day 0) than on subsequent days, and only weak stimulation is obtained on Day 1, while none is obtained on Day 2, where maximal basal synthesis occurs. The CA of mated females cannot be stimulated further for at least 7 days, but regain responsiveness by Day 10 after mating. Synthesis of JHB3 stimulated by SP in vitro persists for at least 4 h after removal of the peptide. Development of responsiveness of the CA to SP in vitro is compared with development of the post-mating reactions of sex-peptide injected virgin females. Our results suggest that the CA is a direct target for SP in vivo and that sexual maturity is established separately for the two post-mating reactions.

The kinetics of nutrient incorporation into body tissues of gilthead seabream ( Sparus aurata ) females and the subsequent effects on egg composition and egg quality

The interaction between essential dietary components and changes in tissue nutrient reserves, egg quality and egg composition, were studied from 60 d before and during the spawning of Sparus aurata broodstock. Fish were given isonitrogenous (550 g/kg dry weight) and isolipidic (100 g/kg dry weight) diets, based on protein and lipid extracts of squid meal. Diets differed in the levels of n-6 (10-30 mg/g dry weight) and n-3 (0-10 mg/g dry weight) essential fatty acids. The effects of these diets on biochemical and fatty acid composition of body tissues, and the subsequent effects on egg composition and egg viability were measured. Dietary essential fatty acids were mostly incorporated into the liver, ovaries, digestive tract and associated adipose tissues. The lipid composition of these tissues reached an equilibrium with dietary lipid composition within 15 d of feeding on any given diet. Muscle and gill cartilage tissues did not show any significant changes in their biochemical and fatty acid composition, even after 60 d feeding. Egg viability decreased significantly within 10 d of feeding the broodstock with a diet deficient in n-3 highly unsaturated fatty acids (n-3 HUFA). The levels of n-3 HUFA in both polar and neutral fractions of egg lipid were directly correlated with their levels in the broodstock diet. When the total amount of egg n-3 HUFA dropped below 17 mg/g dry weight, egg viability and larvae hatching rate decreased by 53% and 47% respectively. These results suggest that the biochemical composition of organs involved in S. aurata reproduction are highly sensitive to the nutritional value of the diet, which affects egg and larval quality rapidly.

Dissociation of ribosomal ribonucleic acid from silkmoth pupae by heat and dimethylsulfoxide: Evidence for specific cleavage points☆

Abstract Phenol treatment of wing epidermal tissue from Hyalophora cecropia silkmoth pupae yields RNA with ribosomal components having slightly lower sedimentation rates than those from rat liver. These are designated 26 s and 17s. Heating for three minutes at 50 or 60 °C abolishes the 26 s component, the material of which then appears quantitatively in the 17 s position in sucrose gradient analyses. Isolated 26 s RNA breaks down in the same manner, whereas native 17 s RNA is unaltered by brief heat treatment. Gradual degradation attributable to nuclease action, exhibited by both 26 s and 17 s components, is kinetically distinct from the sharp thermal dissociation. The thermal dissociation is inhibited by NaCl (0.4 m ) or magnesium ions. Similar conversion of 26 s RNA to a 17 s product is brought about by 80% dimethylsulfoxide at 0 °C. It is attributed to separation of base-paired secondary structure at points of pre-existing cleavage in the polynucleotide chain. The homogeneity of the dissociated product indicates specificity in the points of cleavage, which, it is suggested, may be due to nuclease action in the intact ribosome. RNA heavier than 26 s, demonstrated in the same preparations by pulse labeling and presumed to represent ribosomal precursor, is stable during brief heating.

Regulation of juvenile hormone synthesis in wild-type and apterous mutant Drosophila

Juvenile hormone (JH) is a major regulator of insect development and reproduction and its titer is determined largely by central nervous system regulation of JH synthesis by the corpora allata. To establish the basis for a molecular genetic dissection of the neuroendocrine system responsible for modulating JH titer, a radiochemical assay was utilized to examine JH synthesis in vitro by the isolated corpus allatum as well as the regulation of this synthesis by brain extracts of wild-type and apterous mutant Drosophila melanogaster females during reproductive maturation. JH production by glands of wild-type females increases in parallel with the progress of ovarian maturation, the major product of the adult corpus allatum being juvenile hormone 3 bis-epoxide (JHB3). Gland activity appears to be regulated by both the availability of JH precursors and the level of terminal oxidase(s) in the JH biosynthetic pathway. The brain contains an allatostatic factor, that is transmitted to the glands via nervous connections. Allatostatin production in the brain appears to be positively regulated by JHB3. Adult corpora allata from the mutants ap4 and ap56f synthesize very low levels of JH; additionally, brains of ap56f homozygotes lack allatostatic activity.

Identification of a diuretic hormone of Locusta migratoria

We have isolated a peptide from brains and corpora cardiaca of Locusta migratoria which is immunologically related to the diuretic hormone of Manduca sexta. We determined its structure as a 46 amino acid linear peptide with 43-50% identity to the M. sexta hormone. Moreover, we showed that the new peptide functions as a diuretic hormone in L. migratoria, stimulating urine production by Malpighian tubules and elevating levels of cAMP in tubules.

Drosophila melanogaster sex peptide stimulates juvenile hormone synthesis and depresses sex pheromone production in Helicoverpa armigera

Previous studies demonstrate that virgin female adult Helicoverpa armigera (Lepidoptera: Noctuidae) moths exhibit calling behaviour and produce sex pheromone in scotophase from the day after emergence, and that mating turns off both of these pre-mating activities. In the fruit fly Drosophila melanogaster, a product of the male accessory glands, termed sex peptide (SP), has been identified as being responsible for suppressing female receptivity after transfer to the female genital tract during mating. Juvenile hormone (JH) production is activated in the D. melanogaster corpus allatum (CA) by SP in vitro. We herein demonstrate cross-reactivity of D. melanogaster SP in the H. armigera moth: JH production in photophase virgin female moth CA in vitro is directly activated in a dose-dependent manner by synthetic D. melanogaster SP, and concurrently inhibits pheromone biosynthesis activating neuropeptide (PBAN)-activated pheromone production by isolated pheromone glands of virgin females. Control peptides (locust adipokinetic hormone, AKH-I, and human corticotropin, ACTH) do not inhibit in vitro pheromone biosynthesis. Moreover, SP injected into virgin H. armigera females, decapitated 24 h after eclosion, or into scotophase virgin females, suppresses pheromone production. In the light of these results, we hypothesize the presumptive existence of a SP-like factor among the peptides transmitted to female H. armigera during copulation, inducing an increased level of JH production and depressing the levels of pheromone produced thereafter.

Comparative studies on proteolytic enzymes of Tenebrio molitor L.

Abstract 1. 1. Three distinct proteolytic components of Tenebrio molitor larvae are differentiated on the basis of selective inhibition by specific trypsin inhibitors: an endopeptidase—“Tenebrio trypsin”—and two exopeptidases—carboxypeptidase B and amino-tripeptidase. 2. 2. The exo- and endopeptidase activities are separated by column chromatography on ECTEOLA-cellulose, Tenebrio trypsin eluting freely. The latter is further purified by absorption on CM-cellulose and subsequent gradient elution. 3. 3. Sulphydryl or chelating compounds activate, stabilize and partially reactivate the exopeptidase activity. 4. 4. The relative proteolytic and esterolytic activities of both bovine and Tenebrio trypsins are similar, and esterolysis of carbobenzoxy-tyrosine nitrophenyl ester is completely inhibited by “crystalline soybean trypsin inhibitor”; this is in contrast to chymotrypsin. The stages of enzymatic hydrolysis of polylysine are strikingly similar for both trypsins.

Is There Extraovarian Synthesis of Vitellogenin in Penaeid Shrimp

Extraovarian synthesis of vitellogenin (Vg), has been reported for several crustaceans, mainly in the subepidermal adipose tissue (SAT) or the hepatopancreas (HEP). The precise site(s) of Vg synthesis in penaeid shrimp is hitherto unknown and was investigated in a large local species Penaeus semisulcatus de Haan. Protein synthesis was determined in SAT and HEP tissue pieces incubated in vitro. Incubations were at 25{deg}C for eight hours in an oxygen enriched atmosphere, under sterile conditions in a physiological medium, containing 14C-leucine. At the end of the incubation period, tissue homogenates and medium samples were analyzed for de novo protein synthesis. Total protein synthesis was determined by trichloroacetic acid precipitation. Specific vitellin (Vt) synthesis was determined by radioimmunoprecipitation with a polyclonal Vt-specific antiserum. Characterization of other de novo synthesized proteins was carried out by fluorography from polyacrylamide gels. Subepidermal adipose tissues removed from females at all stages of ovarian development did not synthesize Vt-specific proteins, in spite of the fact that total protein synthesis levels were high. The major protein synthesized de novo in the SAT of males and females is a protein with an identical electrophoretic mobility as hemocyanin in polyacrylamide gels. In vitro protein synthesis in HEP tissues was low compared to SAT or ovary systems. Vt-specific de novo synthesized protein was identified in HEPs from early vitellogenic females, but constituted less than 15% of total protein synthesis. We have previously shown that ovarian tissues from vitellogenic females incubated in vitro exhibited high levels of protein synthesis, an average of 38% of which is Vt-specific (Browdy et al., 1990, J. Exp. Zool. 255:205-215). The calculated Vt synthesis rates in ovaries were up to 23 times higher than in HEP. We conclude that the extraovarian contribution to vitellogenesis in P. semisulcatus is low.

Common functional elements of Drosophila melanogaster seminal peptides involved in reproduction of Drosophila melanogaster and Helicoverpa armigera females.

Sex peptide (SP) and Ductus ejaculatorius peptide (Dup) 99B are synthesized in the retrogonadal complex of adult male Drosophila melanogaster, and are transferred in the male seminal fluid to the female genital tract during mating. They have been sequenced and shown to exhibit a high degree of homology in the C-terminal region. Both affect subsequent mating and oviposition by female D. melanogaster. SP also increases in vitro juvenile hormone (JH) biosynthesis in excised corpora allata (CA) of D. melanogaster and Helicoverpa armigera. We herein report that the partial C-terminal peptides SP(8-36) and SP(21-36) of D. melanogaster, and the truncated N-terminal SP(6-20) do not stimulate JH biosynthesis in vitro in CA of both species. Both of these C-terminal peptides reduce JH-III biosynthesis significantly. Dup99B, with no appreciable homology to SP in the N-terminal region, similarly lacks an effect on JH production by H. armigera CA. In contrast, the N-terminal peptides - SP(1-11) and SP(1-22) - do significantly activate JH biosynthesis of both species in vitro. We conclude that the first five N-terminal amino acid residues at the least, are essential for allatal stimulation in these disparate insect species. We have previously shown that the full-length SP(1-36) depresses pheromone biosynthesis in H. armigera in vivo and in vitro. We now show that full-length Dup99B and the C-terminal partial sequence SP(8-36) at low concentrations strongly depress (in the range of 90% inhibition) PBAN-stimulated pheromone biosynthesis of H. armigera. In addition, the N-terminal peptide SP(1-22), the shorter N-terminal peptide SP(1-11) and the truncated N-terminal SP(6-20) strongly inhibit pheromone biosynthesis at higher concentrations.

Studies on the midgut amylase activity of Tenebrio molitor L. larvae

Abstract The optimal conditions for in vitro amylase activity of the midgut of Tenebrio molitor L. larvae were determined as pH 5·4, 25°C for 10 min reaction, and 1% starch. The larval enzyme solution (LES) amylase, while stable for at least 20 hr at 5°C, loses 75 per cent of its activity on dialysis at this temperature, 25 per cent of its activity when kept at 25°C for 1 hr, and all its activity when kept at 35°C for 30 min. LES amylase is slightly activated by Ca ++ and Cl − , resembling in this respect α-amylase, and is inhibited by Hg ++ , Cu ++ , and ascorbic acid, which inhibit β-amylase. Both column chromatography and paper electrophoresis demonstrate the acidic nature of the enzyme. The soybean trypsin inhibitors and other soybean protein fractions assayed either activate LES amylase or exhibit self-amylase activity. It is suggested that the growth impairment of Tenebrio molitor larvae on raw soybean meal might be attributed to the sum of proteolytic inhibition on the one hand, and to amylolytic stimulation on the other, resulting in a metabolic imbalance more pronounced than that caused by proteolytic inhibition alone. Better carbohydrate utilization might result in reduced food consumption, which is correlated with the energy requirements of the organism, thereby lowering absolute protein consumption even more.