Koichiro J. Yagi

Hormonal regulation of behavioural development in the honey bee is based on changes in the rate of juvenile hormone biosynthesis

Abstract In the adult worker honey bee (Apis mellifera L.), increases in the haemolymph titre of juvenile hormone underlie behavioural development, from nest duties to foraging. However, the physiological basis of juvenile hormone titre regulation was unknown. Using a radiochemical assay for juvenile hormone biosynthesis in vitro, we demonstrate that differences in juvenile hormone titres among bees performing different age-dependent tasks are a consequence of changes in rates of hormone synthesis by the corpora allata. Rates of juvenile hormone biosynthesis were low in newly emerged bees, 7–9-day-old nurse bees, and 14–15-day-old bees collected from the nest periphery, and high in foragers. Rates of biosynthesis were highly correlated with haemolymph titres of juvenile hormone measured in the same individuals. Corpora allata contained mostly methyl farnesoate, the immediate precursor of juvenile hormone, and released principally juvenile hormone III into the incubation medium, indicating no appreciable hormone storage. We also report similarities and differences in parameters of juvenile hormone biosynthesis between nurse bees and foragers that were found during the course of a detailed characterization of the radiochemical assay for adult worker honey bees. These results, coupled with the fact that it is possible to measure rates of juvenile hormone biosynthesis from individual bees, indicate that the radiochemical assay will be useful in further studies of hormonal regulation of bee behaviour.

Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: The search for a unifying arthropod endocrinology

It now appears that arthropods produce and release a wider variety of juvenile hormones (JH) and related compounds than previously thought. For instance, in the adult crayfish, Procambarus clarkii, the mandibular organs, the homologous structure to insect corpora allata (CA), release both farnesoic acid (FA) and methyl farnesoate (MF), the immediate precursors of JH III, but not JH III itself. In larvae of the cockroach Diploptera punctata, JH III production ceases during the last half of the 4th stadium, but the CA continue to produce and release FA throughout this period. The embryos of the same species also release JH III and a product that coelutes with MF on HPLC. In adult blowfly, Calliphora vomitoria, the CA release JH III bisepoxide and possibly the 6,7-epoxide, in addition to JH III. In the lepidopteran species Pseudaletia unipuncta, male CA produce and release JH acids I, II, and III as well as a product which we have tentatively identified as homo-(and/or) dihomo-FA. In the females, CA produce and release the three common JH homologues and a product that we believe is the esterified version of the male compound, homo/dihomo-MF. Although the release of JH precursors from their sites of synthesis might result in their conversion to the active hormone in peripheral tissues, there is only limited evidence for such a process. Studies on biological activities of these compounds and on the developmental changes in biosynthesis and its regulation should provide information necessary for the defining of these compounds as hormones or otherwise and should improve our understanding of the evolution of the JH biosynthetic pathway in the phylum Arthropoda.

Identification of release products of corpora allata of male and female armyworm moths, Pseudaletia unipuncta

Abstract The release of juvenile hormones (JH) by female and JH acids (JHA) by male corpora allata (CA) of Pseudaletia unipuncta was monitored in vitro using a radiochemical assay. Separation and quantification of the homologues was accomplished by HPLC and liquid scintillation counting of collected fractions. At emergence, CA of females maintained at 25°C, 16 h light:8 h dark (16L:8D) released only trace amounts of JH. On days 1–2, JH III remained virtually undetectable, but similar, low quantities of JH I and JH II were released. Between days 3 and 7, an increase in the release of each homologue was observed, the largest being for JH II. Similarly, the amount of JH released by CA of females maintained at 10°C, 12L:12D, increased as a function of age up to 25 days following emergence, with JH II again being the most abundant homologue. However, even after 25 days, the levels observed at 10°C were considerably less than those seen at 25°C. Under conditions of 25°C, 16L:8D, males exhibited a pattern of JHA release similar to that described for females, with the exception that JHA I rather than JHA II was generally the dominant homologue. CA from male moths held for 5–25 days at 10°C, 12L:12D, released little JHA III, but comparatively high and similar amounts of JHA I and II. However, contrary to the continuous age-related increase seen in females, JHA release peaked on day 10 and subsequently declined. We discuss these results in the context of ovarian development, pheromone production, calling behaviour and migration in armyworm females, and we argue that JH or JH acid regulates the maturation of the pheromonal communication system in males.

Production and utilization of farnesoic acid in the juvenile hormone biosynthetic pathway by corpora allata of larval Diploptera punctata

The regulation of juvenile hormone III (JH III) biosynthesis and release by the corpora allata (CA) was studied in penultimate and final instar male and female larvae of Diploptera punctata using an in vitro radiochemical assay. Final instar female CA produced JH III (2 to 6 pmol.hr-1 per pair) only during the first 8 days of the 20-day stadium, but produced and released farnesoic acid (FA), a JH III precursor, (0.5 to 2.0 pmol.hr-1 per pair), during the entire stadium. FA was identified by cochromatography on TLC of both the product from CA incubations with [3H]FA standard as well as by esterification of biosynthesized FA to methyl farnesoate (MF). Biosynthesis of MF and JH III acid occurred at less than 0.25 and less than 2 pmol.hr-1 per pair, respectively. Because regression analysis of the time course of JH III release over 6 hr showed a constant rate of release after a lag period lasting up to 1.7 hr, rates of JH release of untreated CA (spontaneous) and FA-treated CA (stimulated) were determined by 2-hr sequential incubations of CA, following a 2-hr preincubation. FA-treated rates of JH III release were higher than spontaneous rates. In penultimate instars, the decline in the spontaneous rates during the latter half of the stadium was accompanied by a reduced degree of FA stimulation. In final instars, as the spontaneous rates declined between Days 2 and 6, the magnitude of the stimulation of JH III biosynthesis by FA increased, whereas in the second half of the stadium, the degree of stimulation was very low. Addition of exogenous FA to CA from late final instars increased FA production significantly more than JH III production. This apparent stimulation of FA biosynthesis is evident in CA with both high and low spontaneous rates of JH release and suggests that rate limitation of JH III biosynthesis in the final stadium occurs in part at the level of the o-methyl transferase which converts FA into MF.

Terminal stages in juvenile hormone biosynthesis in corpora allata of Diploptera punctata: Developmental changes in enzyme activity and regulation by allatostatins

Abstract The O -methyltransferase, which is responsible for the methylation of farnesoic acid in the corpora allata of Diploptera punctata , is a cytosolic enzyme. The activity of O -methyltransferase closely parallels JH biosynthesis in last instars and adult females. Because allatostatin 4 (AST 4) from D. punctata and callatostatin 5 (CAST 5) from Calliphora vomitoria can inhibit juvenile hormone biosynthesis, their effects on the activity of O -methyltransferase and epoxidase, the enzymes involved in the final two steps of juvenile hormone biosynthesis, were investigated in vitro . AST 4 can inhibit methyltransferase activity whereas CAST 5 stimulates it. AST 4 inhibits epoxidase activity slightly whereas CAST 5 inhibits it significantly (36%). Treatment of corpora allata with farnesoic acid (40 μM) can reverse the inhibitory effect of AST 4 and CAST 5 on JH release by corpora allata. Thus, allatostatins appear to exert their inhibitory effect on JH biosynthesis at least partially through inhibition of the activity of terminal enzymes. Two biosynthetic pathways for the conversion of farnesoic acid to JH may exist in corpora allata of D. punctata : the predominant pathway is farnesoic acid to methyl farnesoate, then to JH whereas the other, representing about 5–10% of total JH production, is farnesoic acid to JH III acid, then to JH.

In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.

Factors affecting the biosynthesis and release of juvenile hormone bisepoxide in the adult blowfly Calliphora vomitoria

Abstract The principal juvenile hormone produced by the corpus allatum of the adult blowfly Calliphora vomitoria has been identified as juvenile hormone bisepoxide. The precursor compounds, methyl farnesoate and juvenile hormone III are also synthesized and released from the isolated gland in vitro, but in small amounts (approx. 1% of the total radiolabelled products of the gland). In females given unrestricted access to sugar, water and meat immediately after eclosion, the rate of juvenile hormone bisepoxide production rises with the initiation of oocyte development, reaches high levels early in the first gonadotropic cycle and remains consistently high through successive cycles. Mature males on the same diet also produce juvenile hormone bisepoxide at high rates. Flies restricted to a sugar-and-water-only diet synthesize much lower levels of juvenile hormone bisepoxide (less than 50% of meat-fed flies). Partially-purified brain extracts of C. vomitoria contain material that is immunoreactive in an ELISA against the inhibitory neuropeptide, allatostatin 1, of the cockroach Diploptera punctata. This material is able to inhibit significantly the biosynthesis of juvenile hormone bisepoxide in flies, and also has strong inhibitory effects on juvenile hormone III biosynthesis by cockroach corpora allata in vitro. Cockroach allatostatins 1–4 at concentrations of 10−4–10−7 M have no significant effect on juvenile hormone bisepoxide release in the blowfly. Also tested at the same concentrations, and shown to have no significant effects on the synthesis and release of juvenile hormone-related compounds in either the blowfly or the cockroach were the vertebrate peptide Met5-enkephalin-Arg6-Gly7-Leu8 (YGGFMRGL) and its carboxyamidated analogue (YGGFMRGL-NH2). Similarly, the FMRFamide-related peptides, calliFMRFamides 1 and 5 and their non-amidated analogues, at concentrations of 10−4–10−6 M, had no effect on juvenile hormone bisepoxide biosynthesis and release. Forskolin (5 × 10−5 M) and 8-bromo-cAMP (10−4 M) appeared not to have an inhibitory influence on juvenile hormone bisepoxide release in the blowfly, suggesting that cAMP may not be the intracellular second messenger in this species. A presumed precursor of juvenile hormone bisepoxide, farnesoic acid, did not stimulate and increased production of juvenile hormone bisepoxide, but resulted in an increase in juvenile hormone III production.

Farnesoic acid and methyl farnesoate production during lobster reproduction: possible functional correlation with retinoid X receptor expression.

Farnesoic acid (FA) and methyl farnesoate (MF) are juvenile hormone-related compounds secreted by the mandibular organ (MO) of crustaceans and play an important role in stimulation of ovarian maturation. To better understand how the MO activity influences female reproduction by secretion of FA and MF, the biosynthesis and release of these two compounds were measured in vitro by the incorporation of l-[(3)H-methyl]methionine into MF and [2-(14)C]acetate into FA by the MO of Homarus americanus. The production of FA is 7.5 times that of MF, and most FA and MF synthesized remained within the gland, and was not released into the surrounding medium. Most FA and MF were synthesized in the anterior fan-fold region of the MO. The rates of biosynthesis of FA and MF were stage-related, with maximal production occurring during secondary vitellogenesis (i.e. stages 4 and 5). A potential juvenoid receptor, retinoid X receptor (RXR), HaRXR, was characterized using PCR cloning techniques. HaRXR belongs to the nuclear hormone receptor superfamily and its deduced amino acid sequence shares a high homology to other RXRs of crustaceans, insects, and vertebrates. Transcripts of HaRXR can be detected in many tissues, and significant high expression level was detected in the MO, especially in the anterior fan-fold region. Expression of HaRXR was also related to reproductive stage, and maximal level of expression was observed at stage 4, in which secondary vitellogenesis is occurring. Changes in transcript level of HaRXR and the rates of FA/MF biosynthesis in the female reproductive cycle indicate that HaRXR and FA/MF may play important roles in crustacean reproduction.

In vitro juvenile hormone biosynthesis in adult virgin and mated female brown-banded cockroaches, Supella longipalpa

Abstract The in vitro radiochemical assay for juvenile hormone biosynthesis by pairs of corpora allata has been employed in the adult female brown-banded cockroach, Supella longipalpa . [2- 14 C]acetate and the methyl moiety of l -[methyl- 3 H]methionine were incorporated into juvenile hormone in a 9:1 ratio. The corpora allata of 9-day mated females release juvenile hormone at a linear rate for 5 h and maximal release rates occur at l -methionine concentrations greater than 30 μM. The juvenile hormone homologue was identified as juvenile hormone-III by HPLC analysis of the radiobiosynthesized corpora allata product. The release of juvenile hormone by the corpora allata closely parallels oocyte development in both mated and virgin females over two gonotrophic cycles. The corpora allata exhibit rapid, nonoverlapping cycles of activity. Mating further activates the corpora allata and accelerates ovulation. The depression of corpora allata activity when the basal oocytes exceed a critical volume of 0.9 mm 3 suggests a negative feedback mechanism.

The radiochemical assay for juvenile hormone biosynthesis in insects: problems and solutions.

Measurement of juvenile hormone (JH) production using the radiochemical assay (RCA) for JH biosynthesis and release is usually a reliable and precise technique. However problems with radiolabeled precursors and misunderstanding of the data, the techniques and the calculations have contributed towards uncertainty with respect to published experimental results. Problems with the purity of [methyl-3H]-methionine or determination of its specific radioactivity have had detrimental effects on the reliability of results using the RCA. Proper control procedures and the use of 14C/3H-double-label RCA can be useful in detecting irregularities in the experimental results, and in determining contributing factors to any problems. The use of [methyl-14C]-methionine and an awareness of normally expected RCA values can also assist the researcher in checking the validity of results. The radiolabeled methyl moiety of methionine is incorporated into JH without discrimination relative to unlabeled methyl methionine, by the o-methyl transferase. However unexpected preferential incorporation of the [methyl-14C]- vs. [methyl-3H]-moiety into JH occurs, but is only evident at concentrations of radiolabeled methionine outside the normal range of the RCA. Changes in radioactive precursor formulation have no effect on the RCA.