Tissue distribution and lipophorin transport of hydrocarbons and sex pheromones in the house fly, Musca domestica.

We investigated the relationship between epicuticular and internal hydrocarbons in the adult house fly, Musca domestica and the distribution of hydrocarbons, including the female sex pheromone component, (Z)-9-tricosene, in tissues. Internal hydrocarbons increased dramatically in relation to sexual maturation and were found in the hemolymph, ovaries, digestive tract, and fat body. (Z)-9-Tricosene comprised a relatively large fraction of the hydrocarbons in the female carcass and hemolymph, and less so in other tissues, while other hydrocarbons were represented in greater amounts in the ovaries than in other tissues. It therefore appears that certain hydrocarbons were selectively provisioned to certain tissues such as the ovaries, from which pheromone was relatively excluded. Both KBr gradient ultracentrifugation and specific immunoprecipitation indicated that > 90% of hemolymph hydrocarbons were associated with a high-density lipophorin (density = 1.09 g ml(-1)), composed of two apoproteins under denaturing conditions, apolipophorin I (approximately 240 kD) and apolipophorin II (approximately 85 kD). Our results support a predicted model (Chino, 1985) that lipophorin is involved in the transport of sex pheromone in M. domestica. In addition to delivering hydrocarbons and sex pheromones to the cuticular surface, we suggest that lipophorin may play an important role in an active mechanism that selectively deposits certain subsets of hydrocarbons at specific tissues.

Functional binding of a vertebrate hormone, L-3,5,3'-triiodothyronine (T3), on insect follicle cell membranes.

A vertebrate hormone, L-3,5,3'-triiodothyronine (T3), induces volume reduction in the follicle cells of Locusta migratoria and Rhodnius prolixus. The effect of T3 on locust follicle cells is inhibited by ouabain and by antibodies raised against a membrane binding protein for juvenile hormone (JH). [125I]-T3 binds to membrane preparations of vitellogenic follicles in a specific and saturable fashion, with a KD in the low nanomolar range. T3 and JH III exhibited equivalent abilities to compete for the T3 binding site. These findings strongly suggest that T3 and JH act via the same receptor in follicle cells.

Lipophorin of female Blattella germanica (L.): characterization and relation to hemolymph titers of juvenile hormone and hydrocarbons.

High density lipophorin (HDLp) from the hemolymph of the German cockroach, Blattella germanica (L.) (Family Blattellidae), has an apparent molecular weight of 670kDa, with an isoelectric point of 7.0 and a density of 1.109g/ml. It is composed of two subunits, apolipoprotein-I (212kDa) and apolipoprotein-II (80kDa), and consists of 51.4% lipid, 46.2% protein and 2.4% carbohydrate. Hydrocarbons constitute 42.2% of the total lipids which also contain diacylglycerol, cholesterol and phospholipid. Lipophorin is rich in the amino acids glutamic acid, aspartic acid, lysine, valine, and leucine. Specificity of a polyclonal antibody was demonstrated by Western blotting and Ouchterlony immunodiffusion: the antiserum recognized native HDLp and apolipoprotein-I, but not apolipoprotein-II, purified vitellin, or other hemolymph proteins. It also recognized a protein in the hemolymph of Supella longipalpa (Blattellidae) but did not cross-react with hemolymph proteins from Periplaneta americana (Blattidae) or Diploptera punctata (Blaberidae). An enzyme-linked immunosorbent assay was developed to measure the HDLp titer in the hemolymph of adult females. The titer of HDLp, a juvenile hormone binding protein, exhibited no clear relationship to the changing titer of juvenile hormone in hemolymph. The hemolymph titer of hydrocarbon, which is also carried by HDLp, showed some functional relation to the concentration of HDLp in the hemolymph. Because it concurrently serves multiple functions in insect development and reproduction, lipophorin titer might covary with the titers of lipid ligands that occur at high concentrations and require extensive shuttling through the hemolymph.

Site of synthesis, tissue distribution, and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs.

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.

The effect of substitutions at position three on the binding and bioactivity of proctolin in locust hindgut and oviduct.

A number of proctolin analogs modified at position three were analyzed for their relative binding affinities and biological activity on locust hindgut and oviduct. A decrease in chain length at this position (from Leu, Ile to Val) or an increase in hydrophobicity alone (Glu) or combined with a decrease in chain length (Val, Ser, Thr and Asp) decreased bioactivity but not necessarily binding. (Ser3)-proctolin had a higher affinity than proctolin for both hindgut and oviduct membranes but was less biologically active than proctolin in both tissues. Several other analogs bound with a similar affinity to proctolin but were significantly less biologically active, particularly on locust oviduct. These results suggest that the position three leucine of proctolin is more important for bioactivity than for binding in both oviduct and hindgut. The data also suggest the presence of two proctolin receptor subtypes on oviduct but not on hindgut membranes. Position three proctolin analogs may be useful in more precisely distinguishing these subtypes.

FMRFamide-like activity in the female locust during vitellogenesis.

The control of oviposition in the locust involves the expulsion of eggs from the lateral oviducts, a process believed to be under neurohormonal control. In this paper we have attempted to identify this putative hormone. Immunohistochemical staining of the brain retrocerebral complex and suboesophageal ganglion of Locusta migratoria with antiserum against FMRFamide revealed a number of FMRFamide-immunopositive cells. FMRFamide-like immunoreactivity was present in median neurosecretory cells and lateral neurosecretory cells of the protocerebrum. Other FMRFamide-immunoreactive cells were detected in the deutocerebrum and tritocerebrum. Immunoreactive cell processes were observed in the mushroom bodies, the central body, the optic lobes, and in the axon tracts leaving the pars intercerebralis and tritocerebrum. FMRFamide-like material was also seen in the circumoesophageal commissures. Further FMRFamide-like material was present in cell bodies of the suboesophageal ganglion. FMRFamide-like staining activity changed dramatically during the oviposition cycle in mature adult females. The median neurosecretory cells stained lightly immediately after oviposition and remained pale until the third day, when staining of perikarya and axon tracts increased. The staining intensity decreased on days 4 and 5. The titre of FMRFamide-like material in the hemolymph increased during the vitellogenic cycle but plummeted after oviposition. A single band of FMRFamide-like material was evident on immunoblot following sodium dodecyl sulphate-polyacrylamide gel electrophoresis of adult female hemolymph. The approximate molecular weight of this molecule was 8,000. Gel permeation chromatography of hemolymph revealed a FMRFamide-immunoreactive fraction with a molecular weight of 8,000. This fraction possessed myotropic activity when applied to the locust oviduct.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like molecules in the beetle Tenebrio molitor.

Immunocytochemical staining of the nervous system of larva, pupa, and adult stage of Tenebrio molitor with anti-insulin serum demonstrated insulin-like peptides in the protocerebrum, corpora allata, and suboesophageal ganglion. During pupal development, marked changes in staining intensity of the protocerebral cells were detected. The staining pattern suggests release of insulin-like peptides early on day 0 and again on day 3 of the stadium. Injections of anti-insulin at these times caused significant delays in the timing of pupal/adult ecdysis. An immunoblot of haemolymph from day-3 pupae revealed a 6.5-kDa insulin-like molecule. These results suggest that the prothoracicotropic hormone of T. molitor is an insulin-like molecule.

Hemolymph Insulin-Like Peptides (ILP) Titers and the Influence of ILP and Mammalian Insulin on the Amino Acid Incorporation in the Mantle Collar In Vitro in Helisoma (Mollusca).

In Helisoma duryi, the periostracum is the outermost organic layer of the shell and it is secreted by the mantle collar. Addition of porcine insulin (0.1 µg/ml) to the incubation medium increases the incorporation of labeled amino acids in the mantle collar in vitro. The immunoblotting technique revealed two immunoreactive insulin bands with a molecular weight of 16 and 7 kDa in the hemolymph. Partial purification of insulin-like peptides from the hemolymph by gel filtration chromatography showed that only one fraction containing approximately 7 kDa polypeptide stimulated the incorporation of amino acids into the mantle collar as well as into the periostracum in a dose-dependent manner. In laboratory populations of Helisoma, snails with two different shell growth rates can be recognized: fast and slow. Hemolymph titers of insulin-like peptide are low in fast-growing snails (2.3 +/- 0.25 mIU/ml) and higher in slow-growing snails (7.8 +/- 0.46 mIU/ml). When a piece of shell at the edge is removed, a structurally identical new piece is formed within either two days (fast regeneration) or a longer period of seven days (slow regeneration). Hemolymph titers of insulin-like peptide undergo fluctuations during the period of shell regeneration, but a general pattern can be recognized. The titers are low when the shell deposition rate is high and vice versa. We suggest that the insulin-like peptide in the hemolymph is involved in shell growth or shell regeneration.

Insulin-like immunoreactivity and molting in Rhodnius prolixus.

Two prominent cells were observed when fifth stage Rhodnius female larval brains were stained with anti-insulin serum. The staining intensity of these cells varied during the instar, being lowest on Day 1 and Days 5 and 6 after feeding. Injection of anti-insulin serum into 5th stage larvae immediately after feeding and on Days 4 and 5 in females and on Days 5 and 6 in males prevented molting. Control antiserum had no effect on the molting process. Injections at other times during the instar had no effect unless serum was injected just prior to ecdysis. Control or anti-insulin serum injected at this time disrupted normal ecdysis. These results are discussed in terms of the control of the developmental program of the insect.

A FMRFamide-like peptide is associated with the myotropic ovulation hormone in Rhodnius prolixus.

The protocerebral neurosecretory cells previously shown to be the source of the myotropin controlling ovulation in Rhodnius prolixus react in an immunocytochemical assay using an antiserum against FMRFamide. When the same antiserum was injected into fed mated females at the appropriate time the timing of oviposition was delayed, but the total number of eggs developed was unaffected compared to controls injected with pre-immune rabbit serum. The titer of FMRFamide-like peptide (assayed by RIA) in the hemolymph of mated and virgin females was found to fluctuate with the egg laying cycle, and to reflect earlier determinations of the titer of myotropic activity. Western blots of SDS-PAGE revealed a FMRFamide-immunoreactive peptide of approximately 8.5 kDa in both hemolymph and extracts of the ovulation hormone cells.