Evolutionary Endocrinology of Juvenile Hormone Esterase in Gryllus Assimilis: Direct and Correlated Responses to Selection

Hemolymph juvenile hormone esterase (JHE) activity on the third day of the last stadium in the cricket, Gryllus assimilis, exhibited a significant response to selection in each of six replicate lines. Mean realized heritability was 0.26 +/- 0.04. The response was due to changes in whole-organism enzyme activity as well as to changes in the proportion of enzyme allocated to the hemolymph compartment. In vivo juvenile hormone metabolism differed between some lines selected for high vs. low enzyme activity. Only minimal differences were observed between lines with respect to hemolymph protein concentration or whole-cricket activity of juvenile hormone epoxide hydrolase, the other major JH-degrading enzyme. Dramatic correlated responses to selection, equal in magnitude to the direct response, were observed for JHE activity on each of three other days of the last juvenile stadium. In contrast, no correlated responses in JHE activity were observed in adults. This indicates that JHE activities throughout the last stadium will evolve as a highly correlated unit independent of adult activities and the evolution of endocrine mechanisms regulating juvenile development can be decoupled from those controlling adult reproduction. This study represents the first quantitative-genetic analysis of naturally occurring endocrine variation in an insect species.     

Regulation of juvenile hormone esterase gene expression in the tobacco budworm (Heliothis virescens)

The tissue distribution, developmental control, and induction of juvenile hormone esterase (JHE) mRNA was examined in Heliothis virescens using an 800-base pair fragment of a JHE cDNA clone. Northern hybridization analysis of poly(A)+RNA from fat body and integument of fifth stadium larvae indicated the presence of a single JHE mRNA species having an estimated length of 3 kilobases. On Day 2 of the fifth stadium (L5D2), basal JHE mRNA levels were 3-fold higher in the integument than the fat body, which correlated with the higher specific activity of the enzyme in the integument at this time. However, JHE mRNA levels in the fat body on Day 4 of the fifth stadium were 9-fold higher than on Day 2, while mRNA levels in the integument remained the same. This endogenous increase in JHE mRNA and activity in the fat body occurred at the time of peak hemolymph JHE activity. JHE mRNA was not detected in third stadium larvae which have very low levels of JHE activity. Treatment of L5D2 larvae with the juvenile hormone mimic epofenonane resulted in a 7- and 14-fold increase in the level of JHE mRNA in the integument and fat body, respectively. The mRNA induced in both tissues was of the same estimated length as the constitutively expressed message. The data indicate that the developmental regulation and induction of JHE can occur at the level of mRNA. There is evidence that the fat body secretes more JHE than does the integument and could be the major source of hemolymph JHE.     

In vivo and in vitro effects of epofenonane on juvenile hormone esterase activity in tissues of last stadium larvae of Trichoplusia ni

Topical application of the juvenoid, epofenonane, to last stadium postwandering larvae of Trichoplusia ni caused a precocious elevation of juvenile hormone esterase (JHE) activity that was tissue speific and time dependent. This increase in enzyme activity over controls was most dramatic in the hemolymph, whereas increases in the fat body were lower. Antibodies raised against JHE reacted on Western blots with a fat body and hemolymph protein present in epofenonane treated and untreated last stadium day 3 larvae. The abundance of this protein, which comigrated with JHE, closely coincided with the temporal increases in JHE catalytic activity that occurred in response to treatment in vivo with epofenonane.The presence of epofenonane (5–10,000 nM) in the medium at the start of fat body incubations failed to shift the temporal appearance of JHE activity or boost activity levels significantly over those of controls. If larvae were treated in vivo with epofenonane before fat body tissue was removed, only a small, but significant increase in JHE activity was found in vitro. The rate of enzyme secretion was insufficient to account for the rapid increases in enzyme activity that occur in the hemolymph in response to epofenonane, even though tissue held in vitro was deemed viable by monitoring lactate dehydrogenase activity in the medium, fat body intracellular ATP, and the incorporation of [³⁵S]methionine into fat body protein. Fat body tissue removed from various aged last stadium larvae released enzyme at different rates in vitro.     

The role of juvenile hormone and juvenile hormone esterase in wing morph determination in Modicogryllus confirmatus

The role of juvenile hormone (JH) and juvenile hormone esterase (JHE) in regulating wing morph determination was studied in the cricket Modicogryllus confirmatus. JHE activities were significantly higher in nascent long-winged (LW) vs short-winged (SW) crickets during the latter half but not during the first half of the last stadium. The magnitude and direction of the activity differences were similar to those previously documented between wing morphs of the cricket, Gryllus rubens. In contrast, activities of general esterase, an enzyme or group of enzymes with no demonstrated role in regulating the JH titer in insects, showed no or only minor differences between morphs. The magnitude and direction of the JHE activity variation is consistent with a regulatory role for this enzyme in some aspect of wing dimorphism. However, the timing of the differences (exclusively during the last half of the last stadium) argue against a role in regulating wing length development per se. Single or multiple applications of juvenile hormone-III to nascent LW individuals during the first few days of the last stadium significantly redirected development from long to short wings. Multiple applications of acetone, by itself, also increased the production of short-winged adults. For most treatments, all individuals with shortened wings also had undeveloped flight muscles. These data suggest that JH may play a role in wing morph determination in M. confirmatus but that it affects a different aspect of the polymorphism from JHE.     

Multiple forms of juvenile hormone esterase active sites in the hemolymph of larvae of Trichoplusia ni

Kinetic analysis was performed on the juvenile hormone (JH) esterase activity in the hemolymph of feeding, last instar larvae of Trichoplusia ni (Lepidoptera: Noctuidae). When the results were analyzed by several different graphical and regression procedures, all approaches yielded the same conclusion that at least two forms of JH esterase active sites exist in the hemolymph. The apparent Km for one site for JH I, II and III was 8.5 X 10(-8) M, and 6.6 X 10(-8) M, respectively. The Km for the other site for JH I, II and III was 6.6 X 10(-7) M, 7.6 X 10(-7) M, 40 X 10(-7) M, respectively. When hemolymph JHE activity was subjected to high resolution isoelectric focusing (IEF), two distinct large peaks of JHE activity were observed, with pIs of 5.3 and 5.5, as well as a small peak at pI 5.1. Separate kinetic analysis of the JHE activity in each peak showed that only the higher Km active site for each substrate was present (in the 10(-7) M range). These data necessitate a change in the current model for JHE in T. ni, and some other insects, which states that a single active site is responsible for most or all of the JH esterase activity in vivo. The data also explain the different estimates of the Km of JHE in T. ni obtained by different laboratories. Studies on the purification of, and the development of inhibitors for, JHE esterase must consider the role of both JHE forms and sites in regulation of T. ni metamorphosis.     

Uptake of juvenile hormone esterase by pericardial cells of Manduca sexta

Immunohistochemical studies were conducted to determine tissue(s) which might be involved in the uptake of juvenile hormone esterase (JHE) from larval hemolymph. Purified JHE expressed by a recombinant baculovirus carrying the JHE gene from Heliothis virescens was injected into the hemolymph of second stadium larvae of Manduca sexta. Immunoreactive material detected with specific antibodies against the natural JHE purified by affinity chromatography from the hemolymph of H. virescens was localized only in the dorsal regions of whole larval mounts. Further immunohistochemical studies of whole and dissected larvae at the light and electron microscopic level showed the specific localization of JHE in pericardial cells. Western blot analysis confirmed the localization of injected JHE in pericardial cells and also indicated some apparent degradation of the incorporated JHE. Similar results were obtained with the JHE from H. virescens injected into larvae of H. virescens. These results indicate that pericardial cells are involved in the uptake of injected JHE from insect hemolymph and its degradation.     

EVOLUTIONARY ENDOCRINOLOGY OF JUVENILE HORMONE ESTERASE: FUNCTIONAL RELATIONSHIP WITH WING POLYMORPHISM IN THE CRICKET,GRYLLUS FIRMUS

The existence, nature, and physiological consequences of genetic variation for juvenile hormone esterase (JHE) activity was studied in the wing-polymorphic cricket, Gryllus firmus. Hemolymph (blood) JHE activity was sixfold lower in nascent short-winged (SW) females, relative to nascent long-winged (LW) females during the last juvenile stadium (stage). Morph-associated genetic variation for JHE activity had two causes, variation in loci: (1) regulating whole-organism enzyme activity; and (2) controlling the degree to which JHE is secreted into the blood Reduced JHE activity in nascent SW-selected individuals was associated with reduced in vivo juvenile hormone catabolism. This suggests that variation in JHE activity during juvenile development may have important physiological consequences with respect to the regulation of blood levels of juvenile hormone and consequent specification of wing morph. This is the first definitive demonstration of genetic variation for hormonal metabolism in any insect and a genetic association between hormone metabolism and the subsequent expression of morphological variation (wing morph). However, we have not yet firmly established whether these associations represent causal relationships In contrast to the clear association between JHE activity and wing morph development, we observed no evidence indicating that variation in JHE activity plays any direct or indirect role in causing the dramatic differences in ovarian growth between adult wing morphs. Variation in JHE activity also does not appear to be important in coordinating the development of wing morph with the subsequent expression of reproductive differences between adult morphs. Finally genetic variation for the developmental profiles of JHE activity during juvenile and adult stages are remarkably similar in three Gryllus species. This suggests that genetic correlations between JHE activities during different periods of development, which underlie these activity profiles, have been conserved since the divergence of the three Gryllus species.     

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.     

Juvenile hormone esterase activity from Manduca sexta corpora allata in vitro

Juvenile hormone esterase (JHE) activity released by the corpora allata (CA) into incubation media (CA-JHE) was titered daily during the course of the last (fifth [V]) larval stadium of Manduca sexta. This CA-JHE activity was relatively low during the early last stadium up to the time of commitment (V4), then rose rapidly to a peak on V6. Activity declined sharply almost to precommitment levels by V8, before rising to a second peak on the first day of the pupal phase (P0). This pattern of activity is distinct from that of hemolymph JHE activity, which peaks just prior to wandering on V4 and again just prior to pupation (V9). Although the CA-JHE and hemolymph-JHE possess different temporal patterns of activity, isoelectric focusing, gel electrophoresis, and initial studies with selected inhibitors suggest that the enzymes responsible for the CA-JHE and hemolymph-JHE activities are similar, but not identical, in nature. Exposure of the V6 CA in vitro to JH II (0.1 μM) or fluoromevalonolactone (FMev; 0.1 mM) produced an approximate fivefold increase and 60% decrease in JH acid synthesis, respectively. Conversely, the same treatments resulted in an inhibition (JH II) and stimulation (FMev) of CA-JHE activity. These observations suggest that JH may be involved in the direct positive feedback regulation of postwandering larval CA and that the CA-JHE may also be integrally related to this positive feedback mechanism.     

Site of synthesis,tissue distribution,and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.     

A whole genome screening and RNA interference identify a juvenile hormone esterase-like gene of the diamondback moth,Plutella xylostella

Juvenile hormone (JH) plays a crucial role in preventing precocious metamorphosis and stimulating reproduction. Thus, its hemolymph titer should be under a tight control. As a negative controller, juvenile hormone esterase (JHE) performs a rapid breakdown of residual JH in the hemolymph during last instar to induce a larval-to-pupal metamorphosis. A whole genome of the diamondback moth (DBM), Plutella xylostella, has been annotated and proposed 11 JHE candidates. Sequence analysis using conserved motifs commonly found in other JHEs proposed a putative JHE (Px004817). Px004817 (64.61 kDa, pI = 5.28) exhibited a characteristic JHE expression pattern by showing high peak at the early last instar, at which JHE enzyme activity was also at a maximal level. RNA interference of Px004817 reduced JHE activity and interrupted pupal development with a significant increase of larval period. This study identifies Px004817 as a JHE-like gene of P. xylostella.     

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments.     

Endocrine regulation of reproduction and diapause in the boll weevil,Anthonomus grandis Boheman

A review of the literature for the hormonal control of reproduction and diapause in Coleoptera is presented. The role of juvenile hormone and juvenile hormone esterase (JHE) in the control of the different life history strategies of the boll weevil are examined. Elevated levels of hemolymph JHE were found to be positively correlated with survival throughout the winter in South Texas population of weevils. Winter weevils were examined for hemolymph vitellogenin (Vg) and their subsequent survival was monitored. The majority of weevils surviving beyond ten weeks had no hemolymph Vg. We conclude that elevated hemolymph JHE and the absence of Vg are good predictors of survivors in the South Texas population. The hormonal basis of diapause termination was examined using hormone treatment and implant therapy. We were unsuccessful in our attempt to induce post-diapause reproductive development with all of our treatments. Only weevils given access to a food source were capable of reproductive development. Our recent experimental findings in the control of Vg synthesis and uptake in the boll weevil are reviewed. Arch. Insect Biochem. Physiol. 35:455–477, 1997. © 1997 Wiley-Liss, Inc.     

Stage-specific production and release of juvenile hormone esterase from the ovary of Diploptera punctata

Juvenile hormone esterase (JHE) is a catabolic enzyme that specifically degrades juvenile hormone (JH) and has been identified in hemolymph and tissues in both larvae and adults of numerous insect species. This study investigates the presence of JHE in ovaries of the viviparous cockroach, Diploptera punctata, and the in vitro release of JHE from these ovaries during the first gonadotrophic cycle. JHE is released in vitro from maturing basal (most posterior) follicles and from follicle cells isolated from oocytes during the short period of time between spermatophore release and chorion formation. Enzyme release is dependent upon the presence of calcium in the medium. This released ovarian JHE appears to be larger than and to display ionic characteristics that are different from the isolated hemolymph and fat body JHEs. In addition, JHE activity measured in homogenates of whole ovaries and subsequently oviposited basal oocytes increases dramatically following spermatophore release, coincident with a previously described decline in JH titer in the ovary. A likely role for ovarian JHE is the site-specific degradation of JH in and around the oocyte prior to fertilization and embryonic development.     

Jhe in Gryllus assimilis: cloning, sequence-activity associations and phylogeny

The 458 amino acid sequence of a mature JHE protein from the cricket Gryllus assimilis was identified after isolating the partial cDNA sequence encoding this protein from a fat body and midgut cDNA library. This hemimetabolan JHE sequence shows over 40% amino acid similarity to the known JHE sequences of several holometabolous insects. It also includes previously determined peptide sequences for G. assimilis JHE as well as two other motifs associated with JHE enzymes in holometabolous insects. The predicted molecular weight of the protein agrees with that of the JHE previously purified from G. assimilis. Partial genomic sequence encoding the Jhe contains two large (1330 and 2918 bp) introns. No coding DNA sequence variation was observed over a 1293 bp region between selected lines differing six to eight-fold in hemolymph JHE activity. However, a 19 bp indel was found in one of the introns; the insertion was strongly associated with elevated hemolymph activity, both in the selected lines and in the F₂ progeny of crosses between them. Phylogenetic analyses localised the G. assimilis JHE to a clade containing dipteran and coleopteran JHEs, with lepidopteran JHEs occurring in a separate clade.     

Precocious expression of the final larval instar developmental pattern in larvae of Trichoplusia ni pseudoparasitized by Chelonus spp

The present study has used a number of electrophoretic approaches to analyze the proteins in normal Trichoplusia ni larvae and those pseudoparasitized by Chelonus spp. A number of feeding-stage, hemolymph proteins appear or increase dramatically only during the final larval stadium. Other proteins highly abundant only during the penultimate stadium disappear or decrease dramatically during the final stadium. The comparative protein profiles of penultimate instar, pseudoparasitized larvae are very similar to those of last instar larvae. These changes in hemolymph proteins are seen on gels resulting from electrophoresis, isoelectric focusing (wide range and narrow range Ampholine and very narrow range Immobiline gels) and SDS-disc electrophoresis. It is concluded that the entire last instar developmental pattern of protein gene products is occurring precociously in pseudoparasitized larvae.