Hemolymph Juvenile Hormone Research Articles

The regulation of the first peak of haemolymph juvenile hormone esterase activity during the last stadium of the cabbage looper, Trichoplusia ni

The role of the head and a juvenoid, epofenonane, in the regulation of the first peak of haemolymph juvenile hormone esterase activity was examined during the fifth (last) stadium of the cabbage looper, Trichoplusia ni. The topical application of epofenonane to unligated day-1 larvae brought about an average 36% increase in the haemolymph juvenile hormone esterase activity. Epofenonane treatment also resulted in a 2-fold increase in the juvenile hormone and α-naphthyl acetate esterase activity of the whole body of unligated larvae suggesting that the juvenoid causes an increase in esterase biosynthesis or activation. Ligation and starvation prevented the appearance of the first peak of haemolymph juvenile hormone esterase activity. Application of epofenonane to newly ligated larvae had no effect whereas it significantly increased the juvenile hormone and α-naphthyl acetate esterase activity of the haemolymph in larvae ligated for 24 h prior to the treatment. In newly starved larvae, epofenonane increased the haemolymph juvenile hormone and α-naphthyl acetate esterase activity above respective controls. In 24 h starved larvae, juvenile hormone esterase activity of the haemolymph increased in response to epofenonane, head extract, or a combination of head extract injection and epofenonane application whereas α-naphthyl acetate esterase activity increased only to head extract or to a combination of head extract injection and epofenonane application. Injection of head extracts of day-1, fifth-instar larvae into the same aged ligated larvae increased both the juvenile hormone and α-naphthyl acetate esterase activity in the haemolymph by about 3-fold 5 h later compared to the respective controls. This increase, however, was not reflected in a similar increase in the whole body esterase levels suggesting that head extract may have caused a release of esterases from tissues into the haemolymph. Injection of head extract from day-1 larvae into the same aged unligated larvae inhibited the normal increase in the haemolymph juvenile hormone esterase activity seen between day 1 and 2 and no inhibition was noted with injections of body extracts or bovine serum albumin. Head extract injection significantly decreased the juvenile hormone esterase activity in the whole body, and haemolymph of unligated and 24 h ligated larvae treated with epofenonane. The head-extract-induced inhibition of haemolymph juvenile hormone esterase activity was dose dependent and the esterase activity in the haemolymph was unaffected when incubated in vitro with head extract for a period of 4 days at 30°C.

Differences in juvenile hormone esterase activity between presumptive macropterous and brachypterous Gryllus rubens: Implications for the hormonal control of wing polymorphism

The developmental profile of haemolymph juvenile hormone esterase activity was compared between presumptive macropterous and brachypterous morphs of the cricket, Gryllus rubens. Activities were measured during the penultimate and last stadia, two stadia where winglength development is sensitive to both exogenous juvenile hormone and crowding. Ester hydrolysis was the exclusive or nearly exclusive route of juvenile hormone degradation in the haemolymph. Individuals of a genetic stock nearly pure-breeding for long wings exhibited substantially higher enzyme activity during the last stadium compared with individuals of a stock pure-breeding for short wings. High juvenile hormone esterase activity strongly cosegregated with long wings in F 1 interstock crosses and in F 2 backcrosses. Crickets of the macropterous stock whose winglength development was redirected to short wings by crowding exhibited low juvenile hormone esterase activity during the last stadium, similar to individuals of the brachypterous stock. Rates of juvenile hormone hydrolysis (epoxide hydrase plus esterase activity) were also higher in wingbud homogenates from last-stadium macropterous versus brachypterous females. In contrast, haemolymph enzyme activity was similar in presumptive macropterous and brachypterous morphs during the penultimate stadium. Results suggest that variation in haemolymph juvenile hormone esterase activity during the last stadium may play an important role in wingmorph determination in Gryllus rubens.

Developmental synchrony between Spodoptera littoralis (Boisd.) and its parasite Microplitis rufiventris Kok.

The developmental interaction between Spodoptera littoralis (Boisd.) and its endoparasite, Microplitis rufiventris Kok. were studied under controlled conditions of short (6 h light–18 h dark) and long (18 h light–6 h dark) photoperiods, 20 ± 1°C and 60 ± 5% r.h. The developmental times of both species were reduced under short-day photoperiod conditions. Photoperiod as well as parasitism markedly affected the number of moults of the host larvae and the developmental time of the parasite was correlated with its sex, the host's age and photoperiod. Juvenile hormone titres in the haemolymph of parasitized and unparasitized larvae were determined using the Galleria bioassay. The haemolymph juvenile hormone titres of parasitized larvae were significantly higher than those of unparasitized larvae. The high levels of juvenile hormone were recorded prior and subsequent to parasite emergence. Importantly, parasitism interferes with the normal development of the host larvae.

Synthesis and binding affinity of an iodinated juvenile hormone.

The synthesis of the first iodinated juvenile hormone (JH) in enantiomerically enriched form is reported. This chiral compound, 12-iodo-JH I, has an iodine atom replacing a methyl group of the natural insect juvenile hormone, JH I, which is important in regulating morphogenesis and reproduction in the Lepidoptera. The unlabeled compound shows approximately 10% of the relative binding affinity for the larval hemolymph JH binding protein (JHBP) of Manduca sexta, which specifically binds natural 3H-10R,11S-JH I (labeled at 58 Ci/mmol) with a KD of 8 X 10(-8) M. It is also approximately one-tenth as biologically active as JH I in the black Manduca and epidermal commitment assays. The 12-hydroxy and 12-oxo compounds are poor competitors and are also biologically inactive. The radioiodinated [125I]12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added 125I-labeled sodium iodide in acetone; however, synthesis using sodium iodide carrier to give the approximately 50 Ci/mmol radioiodinated ligand proceeds in higher radiochemical yield with fewer by-products and provides a radioligand which is more readily handled in binding assays. The KD of [125I]12-iodo-JH I was determined for hemolymph JHBP of three insects: M. sexta, 795 nM; Galleria mellonella, 47 nM; Locusta migratoria, 77 nM. The selectivity of 12-iodo-JH I for the 32-kDa JHBP of M. sexta was demonstrated by direct autoradiography of a native polyacrylamide gel electrophoresis gel of larval hemolymph incubated with the radioiodinated ligand. Thus, the in vitro and in vivo activity of 12-iodo-JH I indicate that it can serve as an important new gamma-emitting probe in the search for JH receptor proteins in target tissues.

Developmental endocrinology of larval moulting in the tobacco hornworm, Manduca sexta.

A larval moult in the tobacco hornworm, Manduca sexta, involves an endocrine cascade that begins with the release of a cerebral peptide hormone, the prothoracicotropic hormone (PTTH). The release of PTTH is gated, occurs during the scotophase and appears to be developmentally cued. In fourth instar Manduca larvae, PTTH release into the haemolymph occurs as a single burst over a few hours during the head critical period, i.e. the time during which the head (brain) is needed for the initiation of the moult to the fifth (last) instar. Released PTTH activates the prothoracic glands (PGs), and within a few hours the cumulative effect of this event results in a dramatic increase in the haemolymph ecdysteroid titre, which then elicits the moult. An assessment of the capacity of the corpora allata (CA) to synthesize juvenile hormone (JH) in vitro indicates that the above sequence of endocrine events begins only when JH synthesis has reached a nadir for the instar. Since CA activity is an indirect measure of the haemolymph titre of the hormone, it is conceivable that the developmentally cued release of PTTH is permissively controlled by a decreasing haemolymph titre of JH. With the increase in the ecdysteroid titre which marks the end of this endocrine cascade, the CA again become active, presumably to cause the increase in the JH haemolymph titre which directs the larval moult. This investigation has thus established the temporal and quantitative dynamics of the PTTH-PG axis that drive larval moulting and provides insight into the interendocrine regulatory relationships that may exist between the ecdysteroids and JHs. These possible relationships and the role of the brain in their regulation are discussed.

Hemolymph juvenile hormone binding proteins of lepidopterous larvae: Enantiomeric selectivity and photoaffinity labeling

Juvenile hormone binding proteins (JHBP) from the hemolymph of late instar larvae of Heliothis virescens (Noctuidae), Lymantria dispar (Lymantriidae), Manduca sexta (Sphingidae), and Trichoplusia ni (Noctuidae) were purified by gel filtration by monitoring [ 3 H](10R,11S) JH I binding using a charcoal assay. Relative binding affinities of the four partially purified JHBPs for JH homologs and analogs were determined in competition experiments using unlabeled (10R,11S) JH I, (10S,11R) JH I, (10R,11S) JH II and (10S,11R) JH II, (±)JH III and (±)epoxyfarnesyl diazoacetate (EFDA) against the natural 3H-labeled JH I enantiomer. The gel-filtered proteins were photoaffinity labeled by irradiation at 254 nm in the presence of [ 3H]EFDA with and without protection by JH I. JH-specific binding proteins were located by fluorescence autoradiography. The versatility of [ 3H]EFDA as a photolabeling agent for insect JHBP is emphasized by its selective photoattachment to proteins of different JH homolog selectivity. This is the first report of enantiomeric selectivity for these proteins in which both the radioinert and radiolabeled hormones are optically active.

Physiological effects of parasitism by Apanteles congregatus in terminalstage tobacco hornworm larvae

The physiology of Manduca sexta (L.) larvae undergoes several modifications during the final stages of parasitism by the gregarious braconid wasp Apanteles congregatus (Say). Normal unparasitized larvae experience a drastic decline in the level of haemolymph juvenile hormone during the fifth instar that acts as a stimulus for metamorphosis to gegin. In contrast, the hormone titre of parasitized fifth-instar larvae remains high enough to inhibit metamorphosis of the host, probably due to increased rates of hormone biosynthesis combined with decreased degradation by juvenile hormone-specific esterase in the host. Although the haemolymph ecdysteroid titre rises to the normal prewandering level, it increases in the presence (as opposed to the normal absence) of juvenile hormone in parasitized fifth-instar larvae. In vitro assays of ecdysone 20-monooxygenase levels in homogenates of fat body and midgut show little activity exists during the latter phases of the fifth instar in conjunction with the major ecdysteroid peak coinciding with emergence of the wasps; as a consequence the prohormone ecdysone may not be efficiently converted to 20-hydroxyecdysone. After emergence, the lack of moulting of the host larva can be attributed to low titres of haemolymph ecdysteroid and ecdysteroid and ecdysone 20-monooxygenase. Parasitism also inhibits the growth, food consumption, and locomotor activity of terminal stage fifth-instar hosts. Debraining host larvae on day 4, however, causes the host to develop a hyperkinesis mimicking wandering, indicating the neural and muscular connections necessary for this behaviour are intact despite the normal inhibitory influence of the brain. Parasitism-specific proteins are detectable in the haemolymph of all terminal-stage hosts that are not seen in unparasitized larvae of the same age. The molecular weights of two of the most prominent proteins are 56 and > 120 kd, respectively, as determined by SDS gel electrophoresis. Haemolymph arylphorin titres also appear affected by parasitism.

Effects of injury to the neuroendocrine system of last-instar larvae of Galleria mellonella

Injury to 1-day old, last-instar Galleria mellonella larvae delays pupation and causes dramatic changes in the neurohormonal system. Injury increases brain allatotropic activity and consequently juvenile hormone titre. Appearances of the second, smaller peak of juvenile hormone and haemolymph juvenile hormone esterases are delayed by 1 day. Activity of juvenile hormone esterases is 40% higher in the injured insects as compared with controls. Injury to the host larvae, which were subsequently implanted with brains taken from donors treated in various ways, (injured, chilled or injured at the termination of cooling) results in increased host sensitivity to each kind of implant. The mechanisms involved in the injury response of Galleria larvae are discussed.

Biosynthesis and titre of juvenile hormone during the first gonotrophic cycle in isolated and crowded Locusta migratoria females

Basal oöcyte length, corpus allatum volume and “ in vitro” juvenile hormone biosynthesis were measured in isolated and crowded Locusta migratoria females at selected times during the first gonotrophic cycle. Using gas chromatography-mass spectrometry with selected ion monitoring, the juvenile hormone titre in the haemolymph of isolated and crowded females was also determined 1 and 4 days after fledging. The rate of oöcyte growth was more rapid in isolated females and a significant ( P < 0.01) difference in mean length was apparent as early as 3 days after fledging. This early manifestation of a difference in rate of oöcyte growth was correlated with a difference in haemolymph juvenile hormone titre between isolated and crowded females. Whilst there was no difference in titre 1 day after fledging, by day 4 the juvenile hormone titre in isolated females was found to be approximately twice that in crowded females. There was no significant difference in the rates of juvenile hormone biosynthesis by corpora allata from isolated and crowded females on days 0 through to 6 after fledging. On day 8, however, the rates of juvenile hormone biosynthesis of corpora allata from isolated females were very high (mean value = 136 pmol/h/pair) and were significantly ( P < 0.002) greater than those of corpora allata from crowded females. Day 8 was also the point in the first gonotrophic cycle at which the difference in the mean basal oöcyte length in isolated and crowded females was at a maximum. The mean volume of corpora allata from isolated females was greater than that of corpora allata from crowded females at all points at which measurements were taken during the first gonotrophic cycle.

Juvenile hormone esterase activity in developmentally synchronous ultimate stadium larvae of the migrant insect, Anticarsia gemmatalis

Growth and development of cumulatively aged velvetbean caterpiller ( Anticarsia gemmatalis Hübner) larvae were characterized on the basis of weight, stadium and gate. Criteria were developed for the selection of synchronous ultimate (5th)-stadium larvae before they entered the ultimate stadium. Using synchronous larvae, precise determinations were made of haemolymph juvenile hormone III esterase activity, α-naphthyl acetate esterase activity and protein concentration. The first of two juvenile hormone III esterase activity peaks coincided with the time of maximal weight gain while the second peak coincided with formation of the prepupa. Juvenile hormones (5 × 10 −6 M) I, II and III were hydrolyzed at similar rates. The α-naphthyl acetate esterase activity and protein patterns differed from the pattern of juvenile hormone III esterase activity. A major peak of α-naphthyl acetate esterase activity was observed during the wandering phase of the penultimate and ultimate stadia. Haemolymph protein concentration increased to a peak, which coincided with the initiation of wandering, and then decreased through pupation.

Parasite regulation of host insect metamorphosis: a new form of regulation in pseudoparasitized larvae of Trichoplusia ni.

When eggs of Trichoplusia ni (lepidoptera) are stung by a parasitic wasp, Chelonus sp., the developing host larvae precociously initiate metamorphosis ten days later. Precocious initiation of metamorphosis occurs even in 'pseudoparasitized' stung hosts which contain no living parasites at the time of symptoms of host regulation by the parasite. In feeding, penultimate instar, pseudoparasitized hosts, the corpora allata activity, hemolymph juvenile hormone esterase activity, in vivo rates of juvenile hormone metabolism and changes in hemolymph protein composition all follow the pattern of the normal last instar. This and other evidence suggests the entire developmental pattern of the last larval instar is precociously expressed in penultimate instar, pseudoparasitized hosts. The cause of precocious expression of the developmental program leading to metamorphosis is a significant decrease in the critical size parameter that, in normal larvae, signals attainment of the last instar. The induction, in preultimate instar larvae, of the entire feeding stage developmental program leading to metamorphic commitment, using either biochemical, surgical or parasitic experimental probes, has not been previously reported. The results have important implications for the study of host-parasite endocrine interaction, of normal insect metamorphosis and even of human puberty.

Relative hemolymph juvenile hormone binding capacity during larval, pupal and adult development in Manduca sexta

Hemolymph from larval, pupal and adult Manduca sexta was analyzed for juvenile hormone binding protein activity by a hydroxylapatite adsorption assay. During each of the larval instars studied, the relative binding capacity followed a cyclical fluctuation with the early part of each instar displaying relatively lower capacities than the midpart of the instar. Binding levels declined in the period preceding ecdysis to the next instar or stage. Analysis of total hemolymph protein indicated that the binding titers generally followed the increase in hemolymph protein concentration. During the pupal and adult stages, sexual dimorphism was observed, with females displaying significantly more binding per unit hemolymph than males. The highest levels of binding at any time during development were observed in the adult female hemolymph 36 hr post eclosion. Adult male hemolymph showed no corresponding increase during the same period.

Substituted thiotrifluoropropanones as potent selective inhibitors of juvenile hormone esterase

A series of 27 substituted thio-1,1,1-trifluoropropanones was synthesized by reacting the corresponding thiol with 1,1,1-trifluoro-3-bromopropanone. The resulting sulfides were screened as inhibitors of hemolymph juvenile hormone esterase and α-naphthyl acetate esterase activity of the cabbage looper, Trichoplusia ni, electric eel acetylcholinesterase, bovine trypsin, and bovine α-chymotrypsin. The presence of the sulfide bond increased the inhibitory potency on all of the enzymes tested when compared with compounds lacking the sulfide. In general, the compounds proved to be poor inhibitors of chymotrypsin and moderate inhibitors of trypsin. By varying the substituent on the sulfide, good inhibitory activity was obtained on α-naphthyl acetate esterase, acetylcholinesterase, while some of the compounds proved to be extremely powerful inhibitors of juvenile hormone esterase. The most powerful inhibitor tested was 3-octylthio-1,1,1-trifluoro-2-propanone, with an I 50 of 2.3 × 10 −9 M on JH esterase. This compound showed a molar refractivity similar to that of the JH II backbone, was not toxic to T. ni, and was moderately toxic to mice, with a 48-hr LD 50 of >750 mg/kg. It effectively delayed pupation when applied to prewandering larvae of T. ni, as expected for a JH esterase inhibitor. Thus, some members of this series are promising for evaluating the role of JH esterase in insect development. The series also indicates that, by varying the substituent on the sulfide moiety, potent “transition-state” inhibitors can be developed for a wide variety of esterases and proteases.

Temporal variations of hemolymph esterase activity and juvenile hormone titers during ovocyte maturation in Acheta domesticus (Orthoptera)

Using in vitro methods, juvenile hormone (JH) esterase activity and α-naphthylacetate esterase activity were determined in the hemolymph during the first reproductive cycle of the house cricket, Acheta domesticus. Biochemical properties of the hemolymph JH esterase were studied. α-Naphthylacetate esterases increased during the first gonotrophic cycle:peaks of their activity could be observed concomitant with peaks of JH esterase activity. The fluctuations in JH esterase activity correlated with those of hemolymph JH titers. The results are discussed.

Juvenile hormone esterases of Lepidoptera II. Isoelectric points and binding affinities of hemolymph juvenile hormone esterase and binding protein activities

The juvenile hormone esterase (JHE) and juvenile hormone binding protein (JHBP) activities from the last larval instar of 14 species of Lepidoptera (Pieris rapae, Colias eurytheme, Danaus plexippus, Junonia coenia, Hemileuca nevadensis, Pectinophora gossypiella, Spodoptera exigua, Trichoplusia ni, Heliothis virescens, Orygia vetusta, Ephestia elutella, Galleria mellonella, Manduca sexta andEstigmene acrea) were analyzed by analytical isoelectric focusing (IEF). While the multiplicity and isoelectric point of these proteins varied, all of them were mildly acidic (pI 4.0–7.0), and a large number of the species possessed only a single JHE and/or JHBP activity. The Michaelis constants (Km's) of the whole hemolymph JHE activities from selected species for JH III were in the range of 10−7M. The equilibrium dissociation constantKd of the JHBP was determined by Scatchard analysis for selected species as well, with the majority of species having aKd near 10−7M. This information is consistent with JHE acting as a scavenger for JH at various times during development and relying entirely on mass action to remove JH from its protective JHBP complexes. The JHBP should limit nonspecific binding and thus facilitate the rapid transport of the intact hormone through-out the hemocoel. These data indicate that the species currently used in the study of the developmental biology of the Lepidoptera are biochemically similar to a variety of other species in this order.

Influence of temperature and carbon dioxide concentration on juvenile hormone titre and dependent parameters of adult worker honey bees ( Apis mellifera L.)

It is known that juvenile hormone plays an important role in the regulation of labour division and of the different life spans, and that the microclimate of the bee hive is characterized by its high CO 2 concentration and its varying temperature depending on the presence of brood. We have investigated the influence of microclimates characteristic of breeding and broodless areas on the juvenile hormone titre in the haemolymph and whole body extracts, on the corpora allata in vitro activity, on the degradation of juvenile hormone and on the dry weight of the hypopharyngeal glands using bees of known ages. A microclimate of 35°C and 1.5% CO 2, as observed in the breeding area, induces a rapid and pronounced increase in the juvenile hormone titre. On the other hand, this titre remains low in bees kept at 27°C and 1.5% CO 2, a microclimate associated with broodless combs. Rates of juvenile hormone synthesis by corpora allata in vitro were found to be extremely low, even in the presence of farnesenic acid, and not related to the juvenile hormone titre. In vitro incubation of juvenile hormone in haemolymph revealed no degradation while injected juvenile hormone was found to be degraded and taken up by the gut at rates only weakly correlated with the juvenile hormone titre. We propose a hypothetical model for the regulation of the juvenile hormone titre as well as the course of labour division by the varying microclimates observed in the bee hive.

The haemolymph juvenile hormone esterase of Manduca sexta (L.)—inhibition and regulation

A number of compounds were screened as potential juvenile hormone esterase (JHE) inhibitors, of which O- ethyl-S- phenyl phosphoramidothiolate was found to be the most potent ( I 50 = 4 × 10 −9 M). Gel filtration of the haemolymph resolved only one peak of enzyme activity capable of rapidly hydrolyzing juvenile hormone (JH). JHE titres were monitored during the last instar in wild type and black mutant larvae of Manduca sexta (L.). In wildtype larvae the increase in JHE activity was most rapid during the first 18 h and then became a function of weight until mid-day three. Although the prewandering peak of the JHE activity occurred on day three in both strains, the JHE activity peak was greatly reduced in black mutant and allatectomized larvae. JHE activity was induced in freshly ecdysed final stadium larvae by juvenoid applications. When pharate fifth stadium larvae were either neck- or abdomen-ligated, or when freshly ecdysed larvae were starved, the levels of JHE activity remained low and could not be induced by juvenoids. Thus, JH may complement the action, or stimulate the production of an unknown factor(s) responsible for increasing JHE activity during the prewandering phase. Both strains also possessed a second smaller JHE activity peak of identical size just prior to pupation (day eight). Just prior to pupation the haemolymph JHE activity can be induced by epofenonane to higher than normal levels in black mutant and in allatectomized larvae, as well as in untreated insects, suggesting that JH directly stimulates the second JHE peak.

Inhibition and substrate specificity of the haemolymph juvenile hormone esterase of the cabbage looper, Trichoplusia ni (Hübner)

Twenty compounds, including juvenile hormones, juvenoids and selected esters, were screened as potential competitive inhibitors of haemolymph juvenile hormone esterase activity in the cabbage looper, Trichoplusia ni (Hübner). The natural juvenile hormones (I, II, and III) provided the greatest inhibition with virtually all of the other compounds being relatively inactive. Several O,O- dialkyl-O-p- nitrophenyl phosphates were also examined as potential as enile hormone esterase inhibitors of which the O,O- di-n- pentyl analogue provided the greatest inhibiton. An examination of the kinetics of juvenile hormone esterase demonstrated K m s of 4.0 × 10 −7 M and 2.5 × 10 −7 M for juvenile hormone I and III, respectively. Lineweaver-Burk plots indicated that ZR-448, a methyl ester dienoate juvenoid, was acting as a competitive inhibitor while O- ethyl-S- phenyl phosphoramidothiolate behaved as an irreversible inhibitor. Liquid chromatography was used to partially purify the juvenile hormone esterase from crude haemolymph. Both crude and partially purified juvenile hormone esterase hydrolyzed juvenile hormone I > III > hydroprene > methoprene, and both displayed identical K m values for juvenile hormone III. These results suggest that the juvenile hormone esterase of T. ni is highly selective for the naturally occurring juvenile hormones.

Seasonal variation in hemolymph juvenile hormone of adult monarchs (Danaus p. plexippus: Lepidoptera)

The Galleria wax test was used to quantify juvenile hormone (JH) activity in the hemolymph of adult monarch butterflies captured in the field at monthly intervals. In animals of both sexes captured in June and July, high levels of hemolymph JH activity were found. By contrast, both sexes had generally lower JH titers from August through January. These results are in general agreement with the state of the JH-sensitive reproductive tract components during these months. Nevertheless, there were notable exceptions, including males in February and September and females in October. During these months relatively high levels of JH were obtained for some hemolymph samples, while in general, reproductive tract development during these periods was variable. This result suggested that temperature, photoperiod, nutrition, flight, JH sensitivity, or some other variable may be important in the triggering of JH-induced reproductive tract growth during these periods. Verification procedures for the assay itself and for the biological material included construction of dose responses for JHI, JHII, JHIII, and Ayerst JH (AJH) (i.e. 16 isomers of JHI), tests for hemolymph JH activity inhibition, effect of allatectomy on JH titer, and comparison of the JH activity of field- and laboratory-collected hemolymph.

Effects of parasitism by Apanteles congregatus on the endocrine physiology of the tobacco hornworm Manduca sexta

The hemolymph juvenile hormone (JH) titers of Manduca sexta larvae parasitized by Apanteles congregatus were determined using the black Manduca bioassay and found to be significantly higher than titers of normally fed or starved unparasitized fifth instar larvae. In vitro measurements of hemolymph esterase activity indicated that JH-specific esterase activity was reduced in parasitized larvae, contributing to the high JH titer. Analysis of host ecdysteroid titers by radioimmunoassay showed the presence of an ecdysteroid peak during parasite emergence from the host that was similar to the prewandering peak seen in unparasitized larvae. Host prothoracic gland activity is apparently necessary for emergence, as indicated by the failure of parasites to emerge from hosts that were neck or abdomen ligatured early in the fifth instar. The onset of emergence in intact hosts was accelerated by the infusion of ecdysone prior to the normal expected time of emergence. In addition to causing in vivo endocrinological changes, parasitism also markedly affected host epidermal responsiveness to exogenous 20-hydroxyecdysone (20-HE). When terminal stage fourth instar larvae were neck ligatured and injected with 20-HE prior to parasite emergence, the host epidermis failed to synthesize new cuticle. Sensitivity increased after the onset of emergence, and larval molting was elicited with large doses of 20-HE similar to molting of preterminal stage hosts with first instar parasites. These findings were interpreted as indicating that parasitism interferes with the normal molting response to exogenous ecdysteroids.