David S. Richard

Ecdysteroids regulate yolk protein uptake by Drosophila melanogaster oocytes

Juvenile hormones (JHs) are thought to drive the regulation of yolk protein uptake by ovaries in Drosophila melanogaster. However, the level of JH production in a mutant stock (ap(56f)) is depressed yet the flies are normally vitellogenic. The production of ecdysteroids by these ap(56f) ovaries in vitro is elevated above that of wild-type ovaries. The incubation of wild-type ovaries in the presence of 0.1mM JHB(3) increased ecdysteroid biosynthesis only during the first 18h following eclosion. Female Drosophila melanogaster undergo a pre-vitellogenic reproductive diapause when exposed to low temperature (11 degrees C) and a short-day photoperiod (L12:D12). The rate of ecdysteroid synthesis by the ovaries, but not JH production, increased within 12h of a temperature upshift to 25 degrees C from a basal level of 20+/-1pg/10 pair of ovaries/5h to a sustained level of 150+/-20pg/10 pair/5h. Vitellogenic oocytes were noted in all females within 12h of this temperature upshift. Diapause was also terminated by the injection of 1&mgr;g of 20-hydroxyecdysone into the abdomens of diapausing females as determined by an increase in ovary size, and the appearance of vitellogenic oocytes as compared to controls. These results are consistent with a revised model for the regulation of yolk protein uptake by ovaries in which ecdysteroids, and not JHs, play the prominent role.

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.

Vitellogenesis in diapausing and mutant Drosophila melanogaster: further evidence for the relative roles of ecdysteroids and juvenile hormones

Abstract The juvenile hormones (JHs) have long been believed to be key elements of the regulation of vitellogenesis in Drosophila melanogaster. This essential role for JH was challenged in Richard et al. (Journal of Insect Physiology 44 (1998)) in a novel model of the endocrine control of vitellogenesis. Further evidence supporting this proposed model and for understanding yolk protein (YP) production and uptake in JH-deficient conditions is presented here. Pre-vitellogenic diapause in the Canton-S strain was terminated within 4 days by the injection of 0.1 ng 20-hydroxyecdsyone; the application of 1 μg JH III failed to elicit a response suggesting once more that ecdysteroids may be the more important agent. Nevertheless, this dose of JH III did reverse the delay associated with the onset of reproductive development of the JH-deficient mutant ap56f in a manner consistent with the proposed role for JH of stimulating early YP synthesis by ovarian follicle cells. Similarly, JH III application to ap4 females also stimulated a degree of ovarian development. A high affinity JH III binding factor ( K D =1.5 nM ) in whole body extracts was quantified by equilibrium dialysis. Binding levels were greater in Canton-S females than in ap56f females though in ap56f binding could be stimulated within 18 h of eclosion by the application of 1 μg JH III. Ovaries from ap56f and Canton-S failed to produce any JH-like compounds. These data are discussed in the context of our model for the endocrine control of vitellogenesis in Drosophila.

Prothoracic gland function in diapause and non-diapause Sarcophaga argyrostoma and Calliphora vicina

Abstract The in vitro activation of isolated prothoracic glands from Sarcophaga argyrostoma (Robineau-Desvoidy) was accomplished with extracts of prepupal brains. The action of prothoracicotropic hormone (PTTH) could be mimicked by the use of cAMP analogues and a phosphodiesterase inhibitor, suggesting the involvement of a cyclic nucleotide-mediated secondary messenger system. This was further supported by the removal of calcium ions thereby lowering the effect of the PTTH extracts in vitro . Cyclic GMP and its derivatives had no effect on ecdysone synthesis. The PTTH extract from Sarcophaga was found to be equally effective at stimulating the production of ecdysone by isolated Calliphora vicina (Meigen) ring glands. The nature and time course of ring gland competency in diapause in both Sarcophaga and Calliphora was investigated. As the Sarcophaga population entered pupal diapause at 96 h after pupariation at 18°C, the prothoracic glands became refractory to PTTH, taking 24 h to lose their competency to respond to this hormone. The levels of ecdysone produced were below the limits of detection of the radioimmunoassay used. The glands could not be stimulated by 10 mM cyclic nucleotides indicating that the block of PTTH action occurred beyond the proposed stage of cyclic nucleotide mediation. The Calliphora population, which has a larval diapause, took 6 days to reach this refractory state. Their prothoracic glands continued to produce ecdysone at the basal rate throughout diapause and this basal rate could not be altered by the application of PTTH or cyclic nucleotides. Following a temperature increase from 11 to 25°C, all diapausing Calliphora had regained prothoracic gland competency within 24 h and had pupariated within 36 h. When isolated brain-ring gland complexes from diapausing Calliphora were incubated at 25°C, no recovery of ring gland competency was noted, indicating the requirement for in vivo reactivation after diapause. The levels of PTTH in pre-diapause and day-35 post-pupariation diapause brains of Sarcophaga were shown to be similar to those of non-diapause destined prepupal brains.

Allatostatic regulation of juvenile hormone production in vitro by the ring gland of Drosophila melanogaster

A factor(s) extracted from the third instar larval brains of the dipteran species Sarcophaga bullata and Drosophila melanogaster causes a dose-dependent reduction of juvenile hormone (JHB3) biosynthesis by isolated ring glands in vitro. In situ, this factor is presumably neuronally transmitted from the brain to ring gland. The allatostatic effect of the brain factor is reversible in vitro and may be overcome partially by the JHB3 precursor farnesoic acid. Agents which act to increase the intracellular levels of cAMP (3-isobutyl-1-methylxanthine (MIX), forskolin, 8-benzoyl cAMP) all caused the reduction of JHB3 synthesis in vitro in a reversible manner. The inhibitory effect of increased levels of cAMP was overcome by the addition of farnesoic acid to the culture medium. The dependence of JHB3 synthesis on extracellular calcium was demonstrated by incubation of ring glands in the presence of the Ca2+ channel blocker lanthanum chloride, and in Ca2(+)-free medium containing EGTA. The inclusion of farnesoic acid abolished the zero Ca2+ effect completely. However, the Ca2+ ionophore A23187 inhibited JHB3 production in medium containing Ca2+, suggesting that elevated intracellular levels of Ca2+ also suppress JHB3 production. This latter inhibition could not be reversed completely by farnesoic acid.

Developmental regulation of juvenile hormone biosynthesis by the ring gland ofDrosophila melanogaster

SummaryThe synthesis in vitro of the putative dipteran juvenile hormone (JHB3) by ring glands isolated from third instarDrosophila melanogaster larvae was quantified by a radiochemical assay. The data indicate that JHB3 synthesis is developmentally regulated during the period prior to wandering until after puparium formation. The highest level of basal production occurred during the postfeeding stage, and synthesis declined after pupariation. Similar relative profiles of synthesis were obtained upon the addition of the JHB3 precursor, farnesoic acid, although the absolute levels of production were elevated considerably. Basal JHB3 production by brain-ring gland complexes in vitro was also highest during the postfeeding stage, although the synthetic rates were much lower than displayed by isolated ring glands. Further analysis revealed that methyl farnesoate, a JHB3 precursor, was synthesized by brain-ring gland complexes in significant quantity. A dual mechanism of brain-centered control of JHB3 biosynthesis is proposed.

Haemolymph ecdysteroid titres in diapause- and non-diapause-destined larvae and pupae of Sarcophaga argyrostoma

Abstract No differences were observed between the rates of development of larvae and pupae from diapause- and non-diapause-destined lines of Sarcophaga argyrostoma except that those destined for diapause have a longer post-feeding, wandering, larval phase associated with a lower haemolymph ecdysteroid titre, as measured by radioimmunoassay. Following pupariation, both cultures show a high haemolymph titre associated with larval/pupal apolysis. The developing culture displays an ecdysteroid peak at 72 h after pupariation which may be involved with pupal/adult apolysis and the initiation of pharate-adult development. This peak is reduced in the diapause-destined culture. Following the initiation of pharate adult development, there is a very large peak at 85–90 h. Those pupae entering diapause display very low titres as a result of the failure of the brain/prothoracic gland axis to release ecdysone. There are no quantitative or qualitative differences between the titres of specific ecdysteroids in the prepupae of the two lines as determined by reverse-phase high-performance liquid chromatography. A preliminary examination of the levels of free and conjugated ecdysteroids has provided the basis for proposing a mechanism of ecdysone metabolism in this insect.

Toxicity of cyanobacterial blooms from Scottish freshwaters

Abstract Cyanobacterial blooms collected from several Scottish freshwater lochs in 1981/82 were lethal to mice when tested by intraperitoneal bioassay. Toxic blooms were dominated by the cyanobacteria Microcystis aeruginosa, Aphanizomenon flos‐aquae and Anabaena flos‐aquae. Effects of M. aeruginosa bloom poisoning in mice resembled those of a toxic peptide from a clonal isolate with gross damage occurring to the liver. Toxicities of Scottish blooms can approximate to those of cyanobacterial blooms responsible for wild and domestic animal poisonings elsewhere in the world.

Yolk protein endocytosis by oocytes in Drosophila melanogaster: immunofluorescent localization of clathrin, adaptin and the yolk protein receptor

The process of yolk protein (YP) uptake by developing oocytes in Drosophila melanogaster has been investigated by immunofluorescent localization of the endocytosis proteins, clathrin, alpha-adaptin and the putative yolk protein receptor (YP receptor). Data suggests that YPs from the follicle cells are trafficked into the oocyte during early stages of vitellogenesis, and that hemolymph YPs are sequestered by nurse cells adjacent to the developing oocyte during late stages of vitellogenesis. Yolk proteins were immunolocalized to both follicle cells and nurse cells during these processes. Diapausing female Drosophila melanogaster undergo a pre-vitellogenic arrest of ovarian development associated with the absence of ovarian alpha-adaptin, clathrin and putative YP receptor. Diapause termination by transfer of whole animals from 11 degrees C to 25 degrees C, or by 20-hydroxyecdysone injection, results in the appearance of immunopositive material in the nurse cells for all three proteins between 12 h and 16 h post upshift and within four days of injection. Immunopositive material was not noted in the follicle cells during diapause termination. In vitro warming of diapausing ovaries, or incubation in the presence of 1 &mgr;M 20-hydroxyecdysone failed to initiate early vitellogenic development suggesting that diapause termination requires factor(s) external to the ovary. Western blotting analysis of extracts of 24 h post-eclosion wild type and ap(56f) females identified putative yolk protein receptor with a molecular weight of 208 kDa and clathrin with a molecular weight of 178 kDa.

Photomechanical movements in the trout retina following brief flashes of light

In two invertebrate species, L&go pealii (cephalopoda) and Procambarus clarkii (crustacea), it has been shown that the screening pigments of the eye will migrate to the light adapted position in response to brief flashes of light, even though the movement takes several minutes to complete (Daw and Pearlman, 1974; Olivo and Larson, 1978). The light flash thus initiates the response, which then continues in the dark. The present paper reports a similar phenomenon for the photomechanical movements in the retina of a vertebrate, the rainbow trout (S&no gairdneri). The experiments were performed on trout with body lengths between 20 and 26cm. obtained from the Howietoun fish farm, Bannockburn. Adaptation was carried out in grey tanks, 75 x 45 x 40 cm deep, provided with running water between 7” and 9°C. Because the photomechanical movements of trout show a clear diurnal rhythm (Douglas, 1980) all experiments started at 12.00 hr with an initial 2 hr period of dark adaptation, following which various light stimuli were presented. At various times after this stimulus fish were taken from the experimental tank, killed by a blow on the head, and the eyes removed and fixed in Bouins’ solution. Small parts of the retina were then taken from the centre of the back of the eye, embedded in Emix resin, and 2-5 pm sections cut and stained with Toluidine blue. Photomechanical movements of the cones and pigment epithelium were estimated from conventional cone and pigment indices (Ah, 1961). For the cone index the distance between the scleral end of the cone elipsoid and the external limiting membrane was measured, and for the pigment index the distance between the basement membrane and the outermost projections of the screening pigment. These distances were then expressed as a proportion of the total retinal thickness between the basement membrane and the external limiting membrane. For the cone index, measurements were always made on one of the identical members of a double cone, and three such measurements averaged. Since the screening pigment expands while the cone myoids contract in response to light, light adaptation results in high values of the pigment index and low values of the cone index. No measurements were made on the rods, because these could not be distinguished by the histological procedures used. Following the 2 hr dark adaptation period fish were exposed to light for 300, 30 or 0.0025 XC. The first two stimuli were obtained from a 60W tungsten bulb immediately above the tank, and the last from a stroboscopic flash (Rollei 140 RES). In the first two cases the illuminance at the bottom of the tank was 1292 lx, and the luminance of the tank walls ranged from 21 to 86 cd m*, measured with a UDT 40X optometer: in the last case the equivalent intensities at the peak of the flash, measured with a calibrated PIN silicon photodiode, were 3.1 log units higher. Fish were exposed also to continuous light from the 60W bulb, and to no light. In the latter case some fish were disturbed with a net at the time the light stimulus would have been given, in case activity in itself might influence photomechanical movements. Table 1 shows results for fish sampled 30 min after the onset of the light stimulus (or after the equivalent time for those that remained in the dark). Analysis of variance shows that there is a significant effect of treatments on both the cone and pigment indices (P < 0.001). Duncan’s New Multiple Range Test (Steel and Torrie, 1960) shows further that, for the cone indices, the two dark treatments do not differ significantly from each other, nor do the four light treatments differ between themselves, but the dark treatments differ significantly from the light treatments (P < 0.01). The same is true for the pigment indices at the 5 percent level. Thus none of the groups subjected to brief light flashes are significantly less light adapted than the group exposed to continuous light, although there is some indication in Table 1 that the pigment migration, as opposed to the cone migration, is slightly less complete following light flashes than it is for continuous light. Further fish were sampled at 5 min intervals in order to follow the time course of the photomechanical movements. The results are shown in Fig. 1. Here again there is some indication that continuous light may be more effective in causing pigment migration than brief exposures are. There is clearly no difference in the effect of the two types of stimulus on the cones. This figure also shows results for fish kept for 2 hr in the light and then subjected to 3OOsec of darkness: