Release of a juvenile hormone binding protein by fat body of the Indian meal moth, Plodia interpunctella,in vitro

When fat body of fifth instar larvae of Plodia interpunctella was cultured in vitro in a chemically defined medium, the tissue released a low mol. wt protein (FBBP) that binds juvenile hormone (JH). This FBBP has the same mol. wt, estimated by gel permeation chromatography, as the haemolymph JH binding protein. Furthermore, the FBBP protected JH from degradation by general esterases isolated from the haemolymph. Treatment of fat body with cycloheximide inhibited incorporation of [¹⁴C] leucine into the FBBP protein fraction and reduced the amount of FBBP released into the medium. We conclude that one source of the JH binding protein found in the haemolymph is the fat body.     

Haemolymph juvenile hormone esterase during the life cycle of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) esterase activity was found in the plasma of larvae, pupae and adults of wild-type tobacco hornworms, Manduca sexta. There was a single peak of plasma JH esterase activity approx. 28 h prior to ecdysis in each instar from the second through the fourth instar and a peak of activity prior to both wandering and pupation in the fifth (last) instar. JH esterase activity was high in newly formed male and female pupae but declined to minimal levels by day 1 of the pupal stage. For the remainder of the pupal period, activity was at background levels. JH esterase activity increased again in newly emerged, virgin male and female adults but declined and remained at a low level 1 day after emergence through death. Gel filtration analysis of larval, pupal and adult plasma resolved a single peak of JH esterase activity with an apparent molecular weight of 66,000. However, isoelectric focusing revealed three forms with isoelectric points of 5.5, 5.8 and 6.1. These isoelectric forms were also found in black and white mutants of last instar M. sexta and in purified JH esterase from wild-type larvae. The plasma JH esterase activity metabolized JH I 2–3 times faster than JH III and was sensitive to inhibition by octylthio-1,1,1-trifluoro-2-propanone and insensitive to O,O-diisopropyl phosphorofluoridate. Gel filtration, isoelectric focusing, substrate specificity and developmental studies suggest that the same JH esterases are found in the plasma of larvae, pupae and adults and appear to be different from general (α-NA) esterase.     

Interendocrine regulation of the corpora allata and prothoracic glands of Manduca sexta

Regulation of the haemolymph titres of ecdysteroids and the juvenile hormones (JH) during larval-pupal development of the tobacco hornworm, Manduca sexta, involves the interendocrine control of the synthesis of each hormone by the other. Temporal relationships between the ecdysteroid titre peaks in the fourth and early fifth larval instar and the increases in corpora allata (CA) activity at these times suggests that ecdysteroids are evoking the increases. Incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes and isolated CA from these stages with 20-hydroxyecdysone (20-HE) revealed that 20-HE stimulates CA activity and that it does this indirectly via the Br-CC. The resulting increase in the JH titre after the commitment (first) peak in the fifth instar stimulates the fat body to secrete a factor which appears to be the same as a haemolymph stimulatory factor for the prothoracic glands. This moiety acts as a secondary effector that modulates the activity of the prothoracic glands and thus the ecdysteroid titre. These findings together have begun to elucidate the mechanisms by which the principal developmental hormones in the insect interact to regulate postembryonic development.     

Characterization of hemolymph juvenile hormone esterase from Lymantria dispar

A major peak of juvenile hormone esterase (JHE) activity approaching 330 nmol JH III hydrolyzed/min/ml of hemolymph was observed during the last larval growth stage in Lymantria dispar. A smaller peak of JHE occurred 3–5 days after pupation. The gypsy moth JHE was purified from larval hemolymph using a classical approach. A specific activity of 766 units per mg of protein and a K_m of 3.6 × 10⁻⁷ M for racemic JH III and the (10R, 11S) enantiomer of JH II was determined for the purified enzyme. The 62 kDa esterase was insensitive to inhibition by O,O-diisopropyl phosphorofluoridate (DFP), or by phenylmethylsulfonyl fluoride (PMSF). Two forms of JHE isolated by RP-HPLC were indistinguishable by HPLC tryptic peptide mapping and share an identical N-terminal amino acid sequence. Polyclonal antisera raised against gypsy moth enzyme cross-reacted with JHE from Trichoplusia ni but not with JHE from Manduca sexta. A weak cross-reactivity was observed with JHE from Heliothis virescens. Forty amino acid residues of the N-terminus were placed in sequence. The N-terminal sequence of JHE from L. dispar showed little homology to the sequence of JHE from H. virescens. The immunological and structural data support the conclusion that markedly different esterases, which catalyze the hydrolysis of juvenile hormone, are present in the hemolymph of different Lepidoptera.     

Larval esterases of the southwestern corn borer, Diatraea grandiosella: Temporal changes and specificity

Disc electrophoresis was used to examine and characterize the esterases present in the fat body, haemolymph, and midgut of last stage larvae of the southwestern corn borer, Diatraea grandiosella. Significant temporal changes were observed in the pattern of the 4 major esterases of the fat body and 3 major esterases of the haemolymph. These changing profiles presumably relate, in part, to a requirement for the degradation of juvenile hormone (JH) in preparation for metamorphosis.The binding capacity of esterases present in the larval midgut towards JH I and three JH mimics (alkyl-3,7,11-trimethyl-2,4-dodecadienoates) was also examined. The midgut of last stage nondiapausing larvae was shown to contain a carboxylesterase which bound all three JH mimics. Another esterase which bound JH I, but not the mimics, was also present. An esterase with a similar electrophoretic mobility was detected in the haemolymph and integument. Since the JH I binding esterase did not bind the JH mimics, the mimics do not appear to synergize JH by inhibiting its ester hydrolysis.     

Characterization of a juvenile hormone binding lipophorin from the blowfly Lucilia cuprina

The larval haemolymph of the sheep blowfly Lucilia cuprina (Weidemann) contains a juvenile hormone binding protein with a K_d for racemic JH III of 33 ± 6 nM. The density of the binding sites is 212 ± 33 pmol/mg haemolymph protein. The binding protein is equally specific for JH III and methyl farnesoate. Some natural juvenoids were ranked for their ability to displace [³H]JH III with JH III > JH II > JH I > JH III > JH III diol > JHB₃ = no detectable displacement. These data, together with displacement studies for 14 synthetic juvenoids, indicate some characteristics of the JH binding cleft. The binding protein is a high density lipophorin (density = 1.15 g/ml) and has subunit molecular weights of 228 kDa (apolipophorin I) and 70 kDa (apolipophorin II). The N-terminal amino acid sequences of the subunits have no discernible homology to any previously sequenced protein. Lipophorin-specific immunocytochemical staining occurs in a subset of fat body cells.     

Uric acid levels during last larval instar of Manduca sexta, an abrupt transition from excretion to storage in fat body

Levels of uric acid in the whole body of the tobacco hornworm, Manduca sexta increased steadily for the 9 days of the fifth instar. However, concentrations in the haemolymph were lowest during the transition from the feeding stage to the wandering stage (days 3, 4), the time when there was a switch from uric acid excretion by the Malpighian tubule-hindgut system to storage in the fat body. Haemolymph volumes, determined for larvae between 2 and 6 days into the fifth instar by isotope dilution with [¹⁴C]-inulin, were used to calculate rates of incorporation of uric acid into Malpighian tubules and fat body of larvae injected with [¹⁴C]-uric acid. These labelling studies indicated that the Malpighian tubules ceased to remove uric acid from the haemolymph some time between the last 6 hr of day 3 of the fifth instar and the first 18 hr of day 4. At the same period, fat body removed significant quantities of uric acid from the haemolymph. The times of initial decreases and increases in levels of uric acid in haemolymph and fat body, respectively, indicated that storage in the fat body started before cessation of elimination via the Malpighian tubule-hindgut system.     

Apparent multiple catalytic sites involved in the ester hydrolysis of juvenile hormones by the hemolymph and by an affinity-purified esterase from Manduca sexta johannson (Lepidoptera: Sphingidae)

The esterases which metabolize juvenile hormone (JH) in some insects may be important in regulating the hormone titer. The JH ester-hydrolyzing activity (JHE) in the larval hemolymph of the tobacco hornworm (Manduca sexta) was found to be attributed to two forms of esterase with almost equivalent activity based on selectivity and kinetics of inhibition by two 3-substituted thio-1,1,1-trifluoropropan-2-ones and a phosphoramidothioate. Neither of the two forms were inhibited by diisopropyl phosphorofluoridate or iodoacetamide. Steady-state kinetics of JH II hydrolysis supported the inhibition studies and showed that the two forms were widely different in their affinity for JH II. The activity of the hemolymph was found to be bound selectively to an affinity column synthesized by the reaction of epoxy-activated Sepharose with 3-(4′-mercaptobutylthio)-1,1,1-trifluoropropan-2-one. This column offered a quantitative, one-step purification of JH esterase with a purification factor of ~800 and specific activity of ~573 nmol JH III hydrolyzed min⁻¹ mg protein⁻¹. The purified protein showed only a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of ~65,000. However, the purified enzyme apparently revealed the same two kinetic forms as the native enzyme, which indicates that two sites of the same protein are likely to be involved in JH hydrolysis.     

Further studies of the effect of L-canavanine on the tobacco hornworm, Manduca sexta.

Fifth instar Manduca sexta growth response to injected doses of canavanine was concentration-dependent over a range of 0·5 to 2·0 mg/g body weight. Twenty-four hr after injection of ¹⁴C-guanidinooxy-d,l-canavanine, M. sexta larvae incorporated approximately 3·6% of the labelled l-canavanine into protein of non-gut tissue. Adult M. sexta mortality was related to the level of injected canavanine over a range of 2 to 8 mg/g body weight. Injection of as little as 2 mg canavanine/g body weight caused hyperactivity in adult M. sexta. Arginine, able to negate the toxic effects of canavanine during larval growth, was only marginally capable of overcoming canavanine effects on larval-pupal ecdysis.     

Haemolymph protein patterns in developing tobacco hornworm larvae

Quantitative changes in haemolymph proteins from each physiological phase of the last three larval instars of the tobacco hornworm, Manduca sexta, were studied by means of disk electrophoresis. Twelve anodical migrating protein bands were found, six of which occurred only sporadically. Total protein concentration increased from pharate third instar to late fifth instar larvae, then decreased slightly in the pharate pupal stages. Some individual bands showed cyclic patterns within each instar similar to the overall cyclic pattern of total protein, whereas other bands showed different patterns or no pattern.     

The role of juvenile hormone in endocrine control of pigmentation in Manduca sexta

Spatial and temporal distribution of insecticyanin was studied in the fourth and fifth larval instars of Manduca sexta. The protein was distributed between the epidermis (62%), the hemolymph (37%) and the pericardial cells (0.5%). Hemolymph insecticyanin (HINS) was highest (0.6 mg/ml) in the very early fourth instar, gradually declining to 0.3 mg/ml. Levels in the fifth instar decreased after ecdysis (0.15 mg/ml), began to rise at wandering, and nearly doubled by the time of pupation. Titers of epidermal insecticyanin (EINS) followed the general growth patterns during the fourth and early fifth instar. At 76 hr after fifth instar ecdysis, titers of EINS dropped precipitously and then rose again to peak just after the wandering stage. Levels of EINS again rapidly declined and could not be detected after 180 hr. Ecdysteroids appear to shut off synthesis of EINS but this response is quantitatively modified in the presence of JH. Endocrine manipulation of the last larval-larval molt indicated that juvenile hormone (JH) acts quantitatively on EINS to induce a dose-dependent increase. The JH-induced increase can be as much as 4-fold, depending upon the body region.     

The substrate specificity of juvenile hormone esterase from Manduca sexta haemolymph

Haemolymph of $\text{Manduca sexta}$ fifth instar larvae contains a high esterase activity capable of hydrolyzing $\text{Hyalophora cecropia}$ C₁₈ juvenile hormone (JH). In an attempt to characterize the substrate specificity of the enzyme (s) involved, dilute haemolymph was incubated $\text{in vitro}$ with a series of JH-analogs. Ethyl esters with or without the 10,11-epoxide group were hydrolyzed readily but isopropyl esters and the (2Z)-isomer of JH were not affected. Indirect evidence was obtained for the hydrolysis of aziridine analogs of JH. Correlations of these results to biological activities are discussed.     

A Biochemical Evidence of the Inhibitory Effect of Diflubenzuron on the Metamorphosis of the Silkworm,Bombyx mori

Diflubenzuron (DFB) has been known to prevent metamorphosis of silkworm, Bombyx mori, from larval to pupal stage at low dose exposure. To explain this inhibitory action of DFB, a hypothesis was raised that DFB acts like juvenile hormone (JH) or DFB inhibits JH esterase to increase endogenous JH titer. A JH bioassay using isolated abdomen clearly indicates that DFB does not act as JH analog because DFB did not induce vitellogenesis in the isolated female abdomen, while endogenous JHs did significantly. General esterase activities in hemolymph were lower in DFB-treated fifth instar larvae than in the control larvae, but there was no difference between fat body esterase activities in both groups. Two hemolymph esterases (‘E1’ and ‘E2’) of the fifth instar larvae were separated and visualized by α-and β-naphthyl acetate. From in vitro incubation experiment, the cathodal esterase (‘E1’) was sensitive to DFB at its nanomolar range. Considering the fact that early fifth instar larvae have high level of JH esterase in the hemolymph, these results suggest that DFB inhibit larval to pupal metamorphosis by blocking JH degradation, which increases endogenous JH titer especially at the critical period when the larvae determine metamorphic development at the following molt.     

Allatotropic and allatostatic activities in extracts of brain and the effects of Manduca sexta allatotropin and Manduca sexta allatostatin on juvenile hormone synthesis in vitro by corpora allata from the Eri silkworm, Samia cynthia ricini

Both allatotropic and allatostatic activities were found in crude extracts of brain from adult and larval Eri silkworm, Samia cynthia ricini, but it seems that allatotropic activity dominates in each stage. There was a high level of allatotropic activity in the crude extract of brain from newly emerged female adults, but allatostatic activity appeared in the bioassay when excessive amounts of crude extracts of brain were added. Crude extracts of brain from premoulting fourth‐instar larvae and from newly ecdysed fifth‐instar larvae exhibited allatotropic activities, whereas extracts of brain from the second and third day of the fifth‐instar larvae inhibited juvenile hormone (JH) release slightly. Allatotropic activity from the brains of adults and larvae stimulated both adult and larval corpora allata (CA) to synthesize JH. Manduca sexta allatotropin (AT) (Mas‐AT) and M. sexta allatostatin (AST) (Mas‐AST) also stimulated and inhibited both adult and larval S. cynthia ricini CA to synthesize JH, respectively. Higher concentrations of Mas‐AT (10^?4 or 10^?3 M) showed an inhibitory effect on adult CA. CA from newly emerged female adults were the most sensitive to inhibition by Mas‐AST, whereas CA from female pharate adults at about 6 h before adult emergence were the most sensitive to stimulation by Mas‐AT and S. cynthia ricini brain allatotropic activity. An extract of brain and Mas‐AT induced some of the non‐active female pharate adult CA at 12 h before emergence to synthesize a small amount of JH.     

Juvenile hormone analog binding in Manduca epidermis

Of a series of derivatives of JH I and methoprene, iodovinylmethoprenol (IVMA) proved most active (ed₅₀s of 3.2 pmol and 20 nM in the Manduca black larval assay and in the prevention of the 20-hydroxyecdysone-induced change to pupal commitment in the epidermis in vitro, respectively). When incubated with nuclei isolated from day 1 fifth instar abdominal epidermis, [¹²⁵I]IVMA bound specifically to two components with K_ds of 4 and 59 nM. This binding was competed by IVMA and methoprene but not by JH I; similar binding by [³H]JH I was not competed by IVMA or methoprene. Specific binding of IVMA was not found in pupally committed epidermis from wandering larvae, but it reappeared in pupal abdominal and thoracic epidermis but not in the wings. Culture of day 2 fifth instar epidermis with 20-hydroxyecdysone to cause pupal commitment caused the loss of the IVMA nuclear binders whereas culture in the absence of hormortes had no effect, indicating that 20HE in the absence of JH downregulates JH receptors.     

Juvenile hormone metabolism in the plasma,integument, midgut,fat body,and brain during the last instar of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) III esterase and JH III epoxide hydrolase activity was found in the integument, midgut, fat body, and brain during last instar development of the tobacco hornworm, Manduca sexta. JH esterase activity was primarily located in the cytosol in these tissues while the majority of the JH epoxide hydrolase activity was found in the microsomes. A prewandering (on day 3) and postwandering (on day 8) peak in plasma JH III esterase activity occurs in the last instar of gate I M. sexta. The JH esterase activity profile in integument, midgut, fat body, and brain followed a similar pattern to that of the plasma. The only exception to this was the absence of the postwandering, prepupal (on day 8) JH esterase peak in the fat body. The topical application of the juvenoid, (RS)-methoprene, failed to induce fat body JH esterase activity but increased activity in the plasma, integument, midgut, and brain in M. sexta prepupae. These results indicate that the source of plasma JH esterase activity is not always the fat body as previously hypothesized. The developmental profile of tissue JH epoxide hydrolase activity was also similar to that of JH esterase suggesting that both enzymes may be regulated partly by the same factors and that JH epoxide hydrolase may also have an important, previously unrecognized functional role in JH regulation and insect metamorphosis. Multiple isoelectric forms of tissue-specific JH esterases and JH epoxide hydrolases were found in integument, midgut, fat body, and brain. The JH esterases in these tissues had isoelectric points more acidic than that for plasma. Tissue α-naphthyl acetate esterase, developmental profiles, and inhibitor sensitivity to 3-(octylthio)-1,1,1-trifluoropropan-2-one differed significantly from that for JH esterase, suggesting that they represent different enzymes. ©1992 Wiley-Liss, Inc.     

Haemolymph juvenile hormone esterase activity in synchronous last instar larvae of the cabbage looper, Trichoplusia ni

Weight and time of moult during the last instar of the cabbage looper (Trichoplusia ni) were examined and used to select last instar larvae that had similar rates of development. Haemolymph protein content and titres of haemolymph esterases hydrolyzing juvenile hormone I, juvenile hormone III, and α-naphthyl acetate were monitored during the last instar using these closely timed larvae. Juvenile hormone I and juvenile hormone III esterase profiles were very similar and differed markedly from the α-naphthyl acetate esterase and protein content profiles. Two major peaks of juvenile hormone esterase activity were observed, one before ecdysone release and the other just prior to pupal ecdysis. Juvenile hormone I was hydrolyzed 15 times faster than juvenile hormone III when assayed at 5 × 10^?6 M.     

Characterization and the developmental role of plasma juvenile hormone esterase in the adult cabbage looper,Trichoplusia ni

Juvenile hormone (JH) esterase was characterized from the plasma of adult females of the cabbage looper, Trichoplusia ni, and compared with that present in 4th and 5th instar larvae. Ester hydrolysis was the principal route of JH metabolism. Gel filtration of plasma resolved a single peak of JH esterase which was distinct from that of the α-naphthyl acetate (α-NA) esterase activity. The JH esterase apparent molecular weight was 62,000 in prepupae and virgin, female adults and 69,000 in 2-day-old 4th instar larvae. Broad range isoelectric focusing of plasma of prepupae and adults resolved a major peak of activity at pH 5.5 with a minor peak of activity at pH 6.1 and in 4th instar larvae at pH 5.45 and 5.8, respectively. By this method JH esterase was resolved from the α-NA esterase activity. The plasma of prepupae and adults metabolized JH I at about twice the rate of JH III. JH esterase activity from adult plasma was more stable than the α-NA esterase activity. Adult JH esterase activity was insensitive to inhibition by O,O-diisopropyl phosphorofluoridate in contrast to that of the α-NA esterase activity. Mated females oviposited 8 times more eggs than virgin females to 10 days after emergence. The total haemolymph protein content of virgin females remained high throughout the period of study whereas mated females showed a significant decline beginning on day 4. JH esterase activity remained unchanged in virgins whereas it declined drastically in mated females. The α-NA esterase activity declined to low levels shortly after emergence in both groups. JH and α-NA esterase activity was not affected by the application of the juvenoid, (RS)-methoprene. The present study provides evidence of a functional role for JH esterase in JH metabolism and reproduction in adult T. ni. JH esterases in the adult were identical to that of prepupae by the methods described above.     

Utilization of fatty acids by Pieris brassicae reared on artificial and natural diets

The fatty acid composition of Pieris brassicae was measured from larvae reared on four different diets. Pieris can alter the composition of fatty acids in the diet through selective incorporation and synthesis. Oleate is preferentially accumulated on artificial diets (15·9 per cent in diet, 43·8 per cent in neutral lipid (NL) of fifth instar larvae), but not equally on natural diets (18·1 per cent in Brassica napus, 25·6 per cent in the NL of fifth instar larvae). Incorporation of linolenate appears to depend on the concentration of both linolenate and linoleate in the diet. With dietary levels of 35·7% linolenate and 32·2% linoleate, fifth instar larvae contain 12·2 and 16·0 per cent, respectively, of these acids. With 45·8% linolenate and 12·5% linoleate in the diet, fifth instar larvae contain 44·1 and 11·6 per cent of these acids, respectively, in the NL. Palmitoleate is actively synthetized on the artificial diets; with trace amounts of dietary palmitoleate, fifth instar larvae have 9·3 per cent of this acid in the NL. Pieris regulates the uptake of linoleate from the diet at the intestinal wall as was shown by linoleic acid-1-¹⁴C, and is unable to convert dietary linoleate to any of the 18-carbon analogues. The female apparently accumulates linolenate into egg phospholipids on the artificial diet, but in general the fatty acid composition of the eggs resembles that of the fat body.