Regulation of methyl farnesoate production by mandibular organs in the crayfish, Procambarus clarkii: a possible role for allatostatins

Decapod crustaceans do not appear to produce juvenile hormone, but rather its immediate precursor, methyl farnesoate (MF). Both MF and its immediate precursor, farnesoic acid (FA) are produced by the mandibular organs (MO) in crustaceans. The MO are homologous to the insect corpora allata (CA), the site of juvenile hormone biosynthesis. However, the FGLamide allatostatin (ASTs) peptides, of which there are about 60 distinct forms reported from crustaceans, have previously been found to have no effect on MO activity in crustaceans. We have identified by immunocytochemistry the presence of FGLamide-like AST immunoreactivity in neurosecretory cells throughout the CNS as well as in neurohaemal structures such as the sinus gland and pericardial organs. The ASTs are likely delivered to the MO hormonally and/or by local neurohaemal release. Using MO from adult males, we have found wide variability between animals in the in vitro rates of MF and FA biosynthesis. Treatment with Dippu-ASTs has a statistically significant stimulatory effect on MF synthesis, but only in MO that are initially producing MF at lower rates. No effect on FA production was observed, suggesting that the FGLamide ASTs exert their effect on the o-methyl transferase, the enzyme responsible for the conversion of FA to MF.     

Relationship of claw form and exoskeleton condition to reproductive system size and methyl farnesoate in the male spider crab,Libinia emarginata

Summary

Methyl farnesoate (MF) expression and reproductive system size were compared in five representative groups of male L. emarginata selected from a sample collected in November. The groups differed from each other with respect to carapace size (small, intermediate and large), relative propodus size (small and large claw forms), and condition of the exoskeleton (abraded and unabraded). Large males with large claws and abraded exoskeletons had reproductive system indices which were significantly larger than any other group. The mandibular organs of these crabs also had significantly higher rates of methyl farnesoate synthesis in vitro. Hemolymph titers of methyl farnesoate were also highest in this group, but were not significantly different from the group with small carapaces, small claws and unabraded exoskeletons. Methyl farnesoate titers were significantly lower in all other groups of unabraded animals with small or large claws. These results suggest that methyl farnesoate may play a role in morphogenesis and reproduction in male L. emarginata.     

Bacterial degradation of strobilurin fungicides: a role for a promiscuous methyl esterase activity of the subtilisin proteases?

Abstract

Strobilurin fungicides are some of the most heavily used antifungal chemicals in agriculture. However, little is known of their fate in the environment. We have identified bacteria that slowly transform strobilurin fungicides via hydrolysis of a methyl ester group in the toxophore, rendering them non-fungicidal. A carboxypeptidase (subtilisin Carlsberg) was found to have this activity, albeit with low specific activity (2.4 × 10^?2 nmol s^?1 mg^?1), and to possess specificity towards an analog of the fungicidal isomer of commercial strobilurins. Substrate-docking studies using the known structure for subtilisin Carlsberg revealed a plausible explanation for both the activity and isomer specificity of this class of hydrolase. These findings suggest that the promiscuous strobilurin methyl esterase activity of the subtilisin-like carboxypeptidases may have a role in the environmental fate of the strobilurin fungicides.     

Induction of vitellogenesis by 17-hydroxyprogesterone and methyl farnesoate during post-reproductive period,in the estuarine crab Neohelice granulata

Adult female crabs (Neohelice granulata) were treated during the post-reproductive period with both 17-hydroxyprogesterone (17PG) and methyl farnesoate (MF). During the 4-week in vivo assay, animals were fed hormone-enriched (17PG or MF) pellets twice a week, at a dose of 2 nmol/g of body weight. Additionally, at the middle of the in vivo experiment, ovarian explants were incubated in vitro with each hormone at a concentration of 15 μM for 24 h. At the end of the in vivo assay, both the gonadosomatic index and the vitellogenic protein content (Vg) of crabs treated with either 17PG or MF were significantly higher (p < 0.05) than the values of the concurrent controls. In addition, the hemolymphatic vitellogenin level of both hormonally treated groups was higher. At the end of the in vitro assay, ovary explants incubated with 17PG had a significantly higher (p < 0.05) protein synthesis and content of Vg proteins than controls. Ovary explants incubated with MF had similar levels to controls (p > 0.05). The involvement of these hormones in the regulation of ovarian growth during the reproductive cycle of N. granulata is discussed.     

Methyl farnesoate controls adult male morphogenesis in the crayfish, Procambarus clarkii

Adult male crayfish Procambarus clarkii exist in two morphotypes. They continue to molt as adults, switching between Form Is and Form IIs. Form Is are primary reproductive types, with large chelae and spines on the ischiopodites of the third and fourth pair of walking legs. Form IIs are non-reproductive types with smaller chelae and no spines on the ischiopodites. We investigated the hormonal control of these transitions in two ways, by eyestalk ablation and by methyl farnesoate (MF) treatments. Eyestalk ablation accelerates molting and increases MF levels in the blood. MF is a hormone that regulates both reproduction and morphogenesis. MF concentrations were determined in two ways. The hemolymph samples were extracted first, then purified, using normal phase HPLC. The fractions containing MF were collected and analyzed for MF concentration, utilizing both internal and external standards by GC/MS. The other hemolymph samples were analyzed from individual animals by HPLC. The concentrations of ecdysteroids were determined by radioimmunoassay. In the control animals, 4 out of 4 untreated Form I males molted into Form II, while 6 out of 7 Form IIs molted into Form Is. Eight of 8 ablated Form Is molted into Form IIs as expected, while 5 of 5 ablated Form IIs molted into Form IIs, instead of Form Is. MF treatment of intact animals resulted in 6 of 7 Form Is becoming Form IIs and 5 of 6 Form IIs becoming Form IIs. These results were highly significant in comparison of Form I and IIs in each treatment (eyestalk intact, eyestalk ablated and eyestalk intact with MF) by a chi square analysis, P = 0.006, P < 0.0005, and P = 0.013, respectively. MF premolt blood levels suggested that Form IIs were produced in the presence of 1.3 ng/ml MF, while Form Is result from MF levels less than 0.5 ng/ml. Since both eyestalk ablation and MF treatment resulted in the failure of Form IIs becoming Form Is, it was concluded that the control of morphogenesis of primary reproductives (Form Is) depends on a low level of MF prior to the molt, while Form IIs are formed in the presence of increased levels of MF.     

Methyl farnesoate and juvenile hormone III in normal and precocene treated embryos of the ovoviviparous cockroach Nauphoeta cinerea

Summary

Titers of juvenile hormone III and methyl farnesoate, its unepoxidized precursor, were measured throughout embryonic development using a gas-chromatographic method and it was revealed that both substances are undetectable before dorsal closure. Thereafter they both reach similarly high concentrations (800 ng/g) until a few days before hatching, when their titers begin to decrease. Application of the precocene analogue, 7-ethoxy-6-methoxy-2,2-dimethyl-chromene, to egg-cases at dorsal closure results in very low or undetectable titers of juvenile hormone III, depending on the dose applied, whereas methyl farnesoate is seen to reach high levels similar to those seen in the controls. The severe developmental disturbances observed suggest that juvenile hormone III is very important for normal development and formation of the 1st instar larva.     

Assay methods for methyl farnesoate esterases in crustaceans

Methyl farnesoate (MF) is a sesquiterpenoid that is synthesized by crustaceans and is structurally similar to the insect juvenile hormones. MF is metabolized to farnesoic acid by carboxylesterases present in crustacean tissues. In order to investigate the biological significance of MF metabolism, we have developed two rapid methods for measuring MF esterase activity. The first method is a radiochemical partition assay that utilizes an authentic substrate. The [³H]MF partition assay was used to evaluate the spectrophotometric esterase substrates methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-pentylthioacetothioate (PENTAT) for use with crude and partially purified samples of MF esterases from lobster hepatopancreas (midgut gland). The spectrophotometric method is less specific for MF esterases than the partition assay but is less time consuming and nonradioactive, and it provides kinetic information. HEPTAT and PENTAT were suitable for rapid screening of chromatographic fractions for MF esterase activity. Arch. Insect Biochem. Physiol. 36:115–128, 1997. © 1997 Wiley-Liss, Inc.     

RXR isoforms and endogenous retinoids in the fiddler crab, Uca pugilator

The pleiotropic effects of circulating ecdysteroids in the adult fiddler crab, Uca pugilator, during molting, regeneration, and reproduction are mediated by a limited number of receptor proteins. We hypothesize that hormonal effects in vivo may be the result of complex interactions between at least two receptor heterodimer conformations that differentially respond to multiple ecdysteroid/retinoid signals. Two splicing variants of the fiddler crab retinoid-X-receptor (UpRXR) differ from one another by the addition of a 33 amino acid insert in the ligand-binding domain. We show here that the ecdysteroid receptor in the fiddler crab (UpEcR) behaves differently depending upon the UpRXR isoform with which it is partnered. The two UpRXR variant partners for UpEcR confer slightly different responses in the binding of Ponasterone A (PA)—a naturally occurring ecdysteroid in the blood of Uca. UpRXR can bind 9-cis retinoic acid (9cRA) as well as terpenoids. 9cRA and the naturally occurring terpenoid, methyl farnesoate, influence the binding of PA to UpEcR/UpRXR dimers. Endogenous retinoids are found in the blastema of regenerating limbs of Uca and they (plus blood-borne terpenoids) may add additional levels of differential response by target tissues. Thus, the two sets of heterodimers tested here may represent different dynamic complexes whose properties are defined by the specific heterodimeric subunits involved and the specific ligands available.     

Induction of male production in clones of Daphnia pulex by the juvenoid hormone methyl farnesoate under short photoperiod

The juvenile hormone, methyl farnesoate (MF), and its analog insecticides have been used successfully to induce the production of males in cladocerans under long-day conditions in the laboratory. However, without hormone addition Daphnia do not usually produce male offspring under long photoperiods, while short photoperiods are a stimulus for the induction of males. We used 21 clones of Daphnia pulex differing in their propensity to produce males under short-day conditions to test whether the treatment with MF would result in an additive effect of shifting the sex ratio towards males. Contrary to our expectations, clones with a high tendency of male production showed a reduced sex ratio in response to MF treatment under short-day conditions, but clones that produced normally few males or did not produce males were stimulated by 700 nM MF to produce up to 40% males. We suggest that the endocrine disruptive effect of MF or juvenile hormone analogs in the field may depend on the clonal composition of the cladoceran population and on the natural photoperiod. This may affect the seasonal occurrence of sexual reproduction and eventually cause a mismatch between the presence of males and ephippial females.     

Correlation among corpus allatum volume,cell size,and juvenile hormone biosynthesis in ovariectomized adult Blattella germanica

The corpora allata (CA) of both intact and ovariectomized Blattella germanica adult females exhibited a high degree of bilateral symmetry in the rate of juvenile hormone (JH) biosynthesis, the mean size of CA cells, and gland volume (81.3%, 98.3%, and 100% respectively with less than a twofold difference between the two glands in CA pairs). This permitted us to split each CA pair randomly, measure JH biosynthesis in one gland, and dissociate the other gland into a cell suspension in which the size of CA cells was measured. In ovariectomized females, changes in CA volume and the spontaneous and farnesoic acid (FA)-stimulated rates of JH biosynthesis, measured from the same glands, were well correlated (r = 0.78, for both correlations). Similarly, the mean volume of CA cells in one gland increased in relation to increases in both the spontaneous and FA-stimulated rates of JH biosynthesis by the contralateral member of the pair (r = 0.83 and r = 0.91, respectively). Concurrent changes in CA cell size and activity suggest that in the CA of B. germanica cellular growth and degradation are involved in the regulation of JH biosynthesis.