Fred C. Baker

In vivo fluctuation of JH, JH acid, and ecdysteroid titer, and JH esterase activity, during development of fifth stadium Manduca sexta

Abstract Juvenile hormone (JH), JH acid, and ecdysteroid titer, and JH esterase activity, were measured in hemolymph from synchronous last stadium larvae of Manduca sexta. JH and JH acids were identified and quantified by GC-MS: JH I and II (and the corresponding acids) were the predominant JH homologs detected in males or females. Maximum levels of JHs and JH acids were observed just following ecdysis to the fifth (last) stadium (day 0, 0 hr) and at the prepupal stage (day 6–day 7). JH titer (≥ 1 ng JH I or II/ml) was higher than JH acid titer (∼0.7 ng JH I acid or JH II acid/ml) in very early fifth stadium larvae. However, this was reversed at the prepupal stage when higher titers of JH acids than JH were observed. JH acid titer began to rise prior to JH titer at the prepupal stage. JH esterase activity rose significantly only after JH or JH acid titers had begun to decline; maximum JH esterase activity was observed at day 3 and day 8. Ecdysteroid titer (measured by RIA) decreased during the last larval molt to a low level by day 0 (0 hr) and to undetectable levels at day 0 (12 hr) of the fifth stadium, by which time JH and JH acid levels had also declined substantially. Just prior to wandering, a small ecdysteroid peak was noted and a slightly elevated level of ecdysteroid was maintained for a further 2 days before a surge in ecdysteroid titer occurred at the prepupal stage, in synchrony with JH and JH acid titer maxima. There was no sexual dimorphism in timing or magnitude of JH, JH acid, and ecdysteroid titer or JH esterase activity.

Methyl farnesoate and its role in crustacean reproduction and development

Studies with Libinia emarginata suggest that methyl farnesoate (MF), a product of the mandibular organs (MOs), may be a crustacean juvenile hormone. In order to better understand the significance of this compound in crustacean physiology, we first investigated the presence of MF in other decapods. MF was synthesized and secreted by MOs from all species tested. However, large differences in the level of MF secretion were observed between species and also between individuals of a species. For example, the level of secretion by MOs from L. emarginata was 100-fold greater than that observed in MOs from Homarus americanus. Analysis of hemolymph from these two species by GC-MS indicated comparable differences in the amount of MF present. Differences in the level of MF secretion by MOs from individuals of a species appear to reflect the physiological roles of this compound. For example, a close relationship was seen between MF secretion and gametogenesis in females of L. emarginata. Finally, treatment of lobster larvae with seawater containing MF caused a small but significant delay in their metamorphosis when compared with untreated larvae. These data suggest that MF affects reproduction in a manner similar to the effects of JH on insects, and may also have effects on the development of crustacean larvae. Taken together, these data support the classification of MF as a crustacean JH.

Development and application of a radioimmunoassay for the juvenile hormones.

Abstract A radioimmunochemical method for the quantification of juvenile hormones from hemolymph and whole body extracts is described. Rabbit polyclonal antiserum developed against a JH III-bovine thyroglobulin conjugate displayed minimal cross-reactivity with juvenile hormone metabolites including juvenile hormone acids, juvenile hormone diols and analogs but substantial cross-reactivity between juvenile hormone homologs. Minimum sensitivity of the assay toward racemic juvenile hormone III was 65 pg. The degree and relative order of cross-reactivities for juvenile hormones I, II and III varied according to the identity of the radioligand used. A method for isolating juvenile hormones from whole body and hemolymph for radioimmunoassay was developed utilizing organic solvent extraction followed by thin-layer chromatography. Noninterfering dyes were used to bracket the position of the hormone on thin-layer chromatography plates. Hemolymph extracts known to contain no JH did not interfere with the assay when this procedure was employed. Radioimmunoassay analysis of hemolymph samples containing known amounts of JH and corrected for recovery yielded the expected results. Quantification of total juvenile hormone in whole body and hemolymph extracts of Manduca sexta was in good agreement with total mass of JH determined by a GC/MS method.

IDENTIFICATION OF THE JUVENILE HORMONE FROM THE CRICKET, TELEOGRYLLUS COMMODUS, AND JUVENILE HORMONE TITRE CHANGES

Abstract In the cricket, Teleogryllus commodus , eggs, haemolymph of 7th and 8th (last)-larval instars, and haemolymph of adults of both sexes contain only juvenile hormone III. While in the male the hormone titre is independent of previous mating experience, juvenile hormone concentration in haemolymph taken from females 36–38 hr after mating (an event which is followed by oviposition) is at a level 5 times higher than that of virgin females. Based on data gleaned from several research groups the identification of juvenile hormone III as the exclusive juvenile hormone in the Order Orthopteroidea is discussed.

Analysis and purification of acyl coenzyme A thioesters by reversed-phase ion-pair liquid chromatography

Abstract A rapid method is described for the analysis of mixtures of short-chain acyl coenzyme A thioesters by reversed-phase ion-pair chromatography on LiChrosorb RP-8 and μBondapak C 18 columns. The technique is applicable to separation of CoASH, acetyl-CoA propionyl-CoA, and 3-hydroxy-3-methylglutaryl-CoA, as well as dicarboxylic acids and several nucleotides commonly used as cofactors for biosynthetic reactions. The method was utilized on a preparative scale for purification of 3-hydroxy-3-ethylglutaryl-CoA from CoASH and 3-hydroxy-3-ethylglutaric acid. The counterion employed was tetrabutylammonium (phosphate), pH 5.5, in various methanol:water mixtures. Elution profiles and retention values of compounds were influenced by the concentration of counterion and mass of injected sample. Tetrabutylammonium ions could be removed from effluent by ionexchange chromatography on Amberlite IR-120 resin.

The identification of an aphid juvenile hormone, and its titre in relation to photoperiod

ABSTRACT. Whole body extractions from larval and adult apterous forms of Megoura viciae, and from adult Aphis fabae, were analysed for the known insect juvenile hormones (JHs) by a gas chromatography‐mass spectrometric method. Low levels of JH III were detected in both aphid species, the first identification of a juvenile hormone from an homopteran insect. Although the mean titre in adult M. viciae is higher in long‐day than in short‐day reared insects (0.12±0.03 v. 0.04±0.01 ng/g), titres were variable and measurements overlapped. The results are discussed in the context of the hormonal control of aphid polymorphism and the question of identity of homopteran and hemipteran JH.

Detection of only JH III in several life-stages of Nauphoeta cinerea and Thermobia domestica

We have conducted a reinvestigation into both the identification and quantification of juvenile hormone (JH) from several developmental stages of the cockroach, Nauphoeta cinerea, and the firebrat, Thermobia domestica, using a gas chromatography-mass spectrometric (GC/MS) method. We detected only JH III in these animals in contrast to prior studies in which JH I, II, and/or III had been reported using a different scheme relying on HPLC purification and subsequent GC/MS analysis under chemical ionization (CI) conditions. Very high levels (approximately 800 ng/g) of JH III were found in N. cinerea embryos at stages after dorsal closure whereas first stadium nymphs and female penultimate stadium nymphs contained only low levels (approximately 1 ng/g and approximately 7 ng/ml respectively); in adult females at the stage of rapid oocyte growth approximately 150 ng JH III per ml of hemolymph was measured. T. domestica nymphs and egg laying adults contained only low levels (approximately 1 ng/g) of JH III. The results emphasize the caution which must be used in interpreting results of procedures for analysis of JH at parts-per-billion levels, and also enforce prior observations that the higher JH homologs are not present except in the Lepidoptera.

The juvenile hormone titre during the penultimate and ultimate larval stadia of Trichoplusia ni

Abstract The juvenile hormone titre in penultimate and last-stadium larvae of Trichoplusia ni (Hubner) was determined by a method utilizing gas chromatography-mass spectrometry with selected ion monitoring. Juvenile hormone II was the predominant homologue detected; highest levels were only ⋍400 pg/g. The juvenile hormone titre was high during the fourth stadium and during apolysis of the last stadia then declined to barely detectable levels just before cocoon spinning, and peaked again during the prepupal stage. Prepupal levels of this hormone were similar to those detected in 4th and 5th larval stadia suggesting an important biological role for juvenile hormone in the prepupa. Starvation of day-1, 5th stadium larvae or inhibition of in vivo juvenile hormone esterase activity with O-ethyl-S-phenyl-phosphoroamidothioate (EPPAT) resulted in higher levels of juvenile hormone in mid last-stadium larvae. T. ni larvae undergoing precocious development caused by parasitization with Chelonus sp. showed greatly reduced levels of juvenile hormone II compared to control larvae, but contained a considerable level of juvenile hormone III. Separate analysis of the host carcass and the parasite larva supported the hypothesis that the hymenopterous parasite was the major source of the juvenile hormone III detected.

Biosynthesis of the sesquiterpenoid, capsidiol, in sweet pepper fruits inoculated with fungal spores

Abstract Capsicum frutescens fruits inoculated with spore suspensions of Monilinia fructicola incorporated 1–4% of sodium acetate-[2- 14 C] or RS -mevalonolactone-[2- 14 C] into the phytoalexin, capsidiol. Labelled capsidiol was characterized by GC-RC, TLC-RC, gel chromatography (in conjunction with liquid scintillation counting) and GC-MS. The mode of incorporation of sodium acetate-[1,2- 13 C 2 ] into capsidiol, as indicated by the pattern of 13 C- 13 C coupling from 13 C NMR data, supports the hypothesis that the angular methyl group of the capsidiol skeleton arises by migration from the C-10 position of a eudesmane-type intermediate.

Influence of the ovaries on JH titer in Teleogryllus commodus

Abstract Mating and availability of substrate were found to have a dramatic effect on the level of egg production and juvenile hormone III (JH III) titer in Teleogryllus commodus. Mated females with egg laying opportunity produced twice as many eggs, and exhibited an 8-fold increase in JH titer, as compared with virgin females or mated females forced to retain most of their eggs due to the absence of substrate for egg deposition. Denervation of the ovaries, or nerve cord transection, also caused accumulation of eggs and lowered the JH level, suggesting a feedback mechanism in which the state of egg depletion of the ovaries determines the level of JH secretion by the corpora allata (CA). Results from ovariectomy of mated or virgin animals suggest that the feedback mechanism includes inhibitory humoral factors secreted by the ovary.