JH regulation: binding protein and metabolism

625 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644 allatum of Manduca sexta, both in vitro and in vivo unpublished information. Preliminary data indicate that the USP proteins in the glands are represented by a group of isoforms that probably represent the phos- phorylation states of two primary translation products. In the last larval stadium, the diversity of these isoforms is greatest at wandering, while the highest concentration of USP in the corpora allata occurs several days later, during the premolt peak of circulating ecdysteroids. EcR proteins were also present in multiple isoforms, again probably as the result of multiple phosphorylation states of primary translation products. In contrast to USP, how- ever, the EcRs in the corpora allata show few differences in relative abundance or isoform diversity during the fifth larval stadium. Various other larval tissues were also assayed and were found to be considerably different from the corpora allata in their USP and EcR isoform profiles, and some of these tissues expressed several putative EcR isoforms that varied with developmental stage. These results suggest that USP in the corpora allata and other tissues may form functionally diverse, tissue- and stage-specific homo- andor heterodimers of the ecdysteroid receptor. In vitro analysis indicated that USP isoform diversity in the corpora allata is altered in response to changes in physiological ecdysteroid levels, while ecdysteroid- dependent changes in EcRs in the glands are limited to changes in abundance. Very preliminary studies indicate that JH may also influence USP isoform diversity in the corpora allata, although the effect may not be direct. An understanding of the diversity of EcR and USP isoforms in the corpora allata will provide the basis for further study of the complex interactions between the ecydys- teroidogenic and JH biosynthetic pathways, as well as of the possible regulation of putative JH receptors and their downstream targets in the glands. Specific phos- phorylation events and the EcR and USP isoform diver- sity they generate are thus undoubtedly at the core of stage-specific responses of tissues and organs to the ecdysteroid titer. This provides an explanation for how a relatively small number of translation products and a limited number of ligands can be utilized effectively in a large number of permutations. Thus the answer to the question originally posed by Carroll Williams and discussed at the First International Conference on the Juvenile Hormones in Lake Geneva, Wisconsin — Is JH the handmaiden of ecdysone? — may finally be within reach.

3. JH regulation: binding protein and metabolism

3.1. Nature of JHs From the structure of JH III, it is obvious that there are a number of challenges that insects face in using this compound as a hormone, i.e. the necessity to transport a highly lipophyllic molecule in an aqueous environment from its site of biosynthesis, through the insect hemo- lymph, to its site of action in target cells. JH biosynthesis and transport must therefore occur in what can be con- sidered a hostile environment, containing numerous esterases both outside and inside of the cell, capable of removing the conjugated methyl-ester of JH, and mem- brane bound epoxide hydrolases to degrade its stable C10,11 epoxide. Either of these metabolic events can significantly affect the pharmacology of JH and no doubt its biological action, discussed in more detail sub- sequently. Although these considerations of synthesis, transport and degradation are vital to JH function, the presence or absence of JH coordinated with changes in ecdysterioid levels at precise developmental time points is essential for normal growth, metamorphosis and reproduction. Insects have developed a unique system for the regulation of JH concentration which involves dynamic changes in two biochemical processes — JH biosynthesis and JH degradation. When JH biosynthesis levels are high, JH degradation is low, and when JH biosynthesis levels are low, JH catabolism is high. JH binding proteins play a critical role in these processes but at least in respect to the hemolymph, remain in molar excess in comparison to JH Goodman and Gilbert, 1978. The challenge of JH regulation is further compli- cated by the synthesis of multiple JH homologs and by multiple sites of biosynthesis in some insect species. The biological significance of these observations is conjec- tural. JH has been identified in approximately 100 insect species representing at least ten insect orders, with JH III being the predominant homolog Baker, 1990. It would be useful in the future, and in terms of under- standing the role of JH in arthropod development in gen- eral, to examine what JH homologs if any are found in the most primitive insect orders, e.g. the Protura and Diplura. The higher homologs isoJH 0, JH I and JH II are found in the more advanced insect orders, i.e. the Lepidoptera, where in some cases multiple homologs are found in the same insect. In the cyclorrhaphous dip- terans, a new JH species, the bis-epoxide of JH III, was identified in Drosphila melanogaster, Calliphora vom- itoria, Phormia regina and Lucilia cuprina Richard et al., 1989; Yin, 1994; Yin et al., 1995. There is no evi- dence that this JH III bis-epoxide is a degradation pro- duct of JH III Moshitzky and Applebaum, 1995, and in fact, Yin et al. 1995 found that JH III, JH III bis- epoxide and methyl farnesoate had a synergistic effect on oogenesis in Phormia. Thus, the production of mul- tiple JH species has now been demonstrated in both the higher Diptera and the Lepidoptera. Methyl farnesoate is released from the corpora allata of a number of insect species Cusson et al., 1991b. Sparagana et al. 1984 also showed that JH acid was produced by the corpora 626 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644 allata of Manduca sexta beginning early in the last stad- ium Janzen et al., 1991, and the JH acid is the only product at the time of pupal commitment Bhaskaran et al., 1986; Janzen et al., 1991. JH acid acts as a pro- hormone and is converted to JH by imaginal discs Sparagana et al., 1985 and may have morphogenetic roles of its own see subsequent discussion. The de novo synthesis of JH apparently is not limited to the corpora allata since Borosvky et al. 1994 demonstrated the synthesis of JH III from acetate by the ovaries of the mosquito, Aedes aegypti. We must exercise caution and not limit our view as to what constitutes a “JH”. For example, as discussed elsewhere in this review, the Bhaskaran lab has demon- strated that JH acid is a hormone. In addition, Granger et al. 1995a discovered a polar JH acid ester synthe- sized and released from the corpora allata of Manduca, but its function is unknown. The Roe laboratory reported at this symposium that both hard and soft ticks have no detectable levels of the common insect JHs as measured by the Galleria wax bioassay, by SIM GC-MS and by radiobiosynthesis with a lower limit of detection in the low fg range. Yet ticks, like insects, have multiple life stages and instars and undergo metamorphosis and reproduction, all of which now appears to be controlled by a hormone other than the common insect JHs. The predominant JH-like molecule in the Crustacea is methyl farnesoate see Laufer, this symposium. It is clear from the few studies that have been conduc- ted, that the different JH homologs have different levels of biological activity. For example, JH I and JH II are more potent morphogenetically than JH III in a number of different biological assays using Manduca Truman et al., 1973; Nijhout and Riddiford, 1974; Fain and Rid- diford, 1975; Bhaskaran et al., 1986 but this may be due to the greater polarity of JH III, which makes it more prone to degradation Gilbert and Schneiderman, 1960; Gilbert and Schneiderman, 1961a,b. In addition, the overall peak levels of JH can vary greatly between spec- ies. For example, low concentrations of JH in the range of 10 ng per ml hemolymph elicit the status quo effect in most insects with the exception of the social Hymen- optera Rachinsky et al., 1990; Huang et al., 1994 and the cockroach, D. punctata Tobe et al., 1985. The func- tion of the multiplicity of JH products synthesized by the corpora allata sometimes simultaneously and other tissues and the significance of peak levels in different species are not fully appreciated. It could be argued that some of these products, i.e. JH III in Lepidoptera, methyl farnesoate in cockroaches or JH III in the higher flies, are evolutionary biochemical remnants. However, the small amount of work in this area suggests otherwise Yin et al., 1995 and references therein. More research is needed to provide an understanding of both the signifi- cance of JH blends and the differences in JH levels between species and between developmental stages in some species. Most JH identification and JH titering in the past has involved either hemolymph in the case of large insects, or the whole organism with small insects, e.g. Droso- phila. It is apparent now that this approach will not be adequate in the future. The function of JH cannot be defined by simply studying JH levels in hemolymph or in a whole insect homogenate. It is critical to our under- standing of the mode of action of JH to study JH at the level of individual tissues. In the past, this perspective has essentially been ignored, and at this juncture, surpris- ingly, we know almost nothing about the quantitative nature of JHs in individual tissues, with the exception of hemolymph. Careful quantitative analysis of JH in specific tissues during development using at least a few insect models is requisite for a real understanding of JH function. We should also confirm data obtained from in vitro assay systems by developing in vivo assays to study JH biosynthesis, transport, degradation and func- tion. It is necessary to prove that data from in vitro stud- ies are not artifacts of the incubation conditions. 3.2. JH esterase So far, only two definitive, primary pathways have been described for the metabolism of JH, i.e., hydrolysis of the methyl ester of JH by soluble esterases and hydration of the C10,11 epoxide by microsomal epoxide hydrolases Fig. 1. The most recent comprehensive reviews on JH metabolism are those of Hammock 1985, Roe and Venkatesh 1990 and de Kort and Granger 1996. It is at least theoretically possible that the JH bis-epoxide from the higher Diptera is a degra- dation product of JH, although there are no current data to support this view. Conjugation of the epoxide of JH is also possible, but again there is no evidence that this is an important in vivo metabolic pathway in insects. The general assumption has been that JH is metabolized by JH esterase to JH acid, and JH acid is then metabol- ized by JH epoxide hydrolase to JH acid diol. This will be discussed in more detail later; however, it should be noted, that this metabolic sequence is tenuous. Finally, there are a number of reports in the literature of highly polar JHs andor JH metabolites Halarnkar et al., 1993; Lassiter et al., 1994; Granger et al., 1995a; Kallapur et al., 1996; Debernard et al., 1994; review of Roe and Venkatesh, 1990. For the most part, their structures are unknown. The Roe laboratory showed at this meeting that the water soluble JH metabolites produced in the Lepidoptera are not all alike; the metabolite of JH III produced in vivo by last stadium cabbage loopers was different from the phosphate diol synthesized in vivo by last stadium Manduca Halarnkar et al., 1993. Until the structures of these water soluble JH metabolites are 627 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644 Fig. 1. A model for JH metabolism. Bp, binding protein. From M. Roe, unpublished information. known, other primary pathways for the degradation of JH cannot be ruled out. Most of the research on JH metabolism has focused on hemolymph esterases and the interaction of JH ester- ase with hemolymph JH binding proteins. The hypoth- esis by Sanburg et al. 1975 noted that there were two classes of esterases involved in the metabolism of JH, JH specific esterase and “general” a-naphthyl acetate esterase. As will be discussed below, there is no question that insects have a JH-specific esterase, and that this esterase is involved in reducing the JH titer. However, with all of the research conducted on the metabolism of JH by JH esterases, the significance of non-specific esteratic action is still not clear. It is assumed that most of the JH in insects is bound to JH binding proteins and is in that way protected from the non-specific esterases with low binding affinities Touhara and Prestwich, 1993; Touhara et al. 1993, 1995. This conclusion is based in large measure on research with hemolymph, rather than with other tissues where the interaction of JH with binding proteins, JH receptors, metabolic enzymes and JH synthetic processes must be considered. One definitive study with Manduca has even shown that JH levels fell prior to the increase in JH esterase activity Baker et al., 1987. The general conclusion reached based on the research utilizing hemolymph is that since insects have a JH-specific esterase and high affinity, hemolymph binding proteins, JH esterase is the only esterase important in JH degradation. Whether this con- clusion is warranted and applicable to all tissues and insects in general is not known. Evidence for the substrate specificity of JH esterase and its functional role in the reduction of the JH titer is clear. Potent and highly selective transition state analog inhibitors of hemolymph JH esterase have been synthe- sized Hammock et al. 1982, 1984; Linderman et al., 1987; Roe et al., 1997. Detailed structureactivity stud- ies and molecular modeling of the interaction of these trifluoromethylketones with JH esterase from the hemo- lymph of Trichoplusia ni have shown that the active site of JH esterase is hydrophobic, with a molecular volume almost identical to that of JH Hammock et al., 1984. There is also a hydrophobic site near the active site of the enzyme which apparently accommodates the C-3 methyl branch of JH III Linderman et al., 1987. Mim- icry of the JH backbone is a critical factor in inhibition of the esterase Hammock et al., 1984; Roe et al., 1990. Interestingly, the trifluoromethylketones mimicking the alpha,beta unsaturation of JH were poorer inhibitors than the sulfur containing OTFP 1-octylthio-1,1,1-trifluoro- propan-2-one or acetylenic trifluromethylketones Linderman et al., 1989. This was explained when 19 F- NMR studies showed that trifluoromethylketones are hydrated and tetrahedral in structure in aqueous solution, and also tetrahedral in structure in an esteratic active site Linderman et al., 1988, existing either as a hydrate or adduct to the catalytic serine Roe et al., 1997. The hydration rate is an additional critical factor in JH ester- ase inhibition and can in some cases have precedence over JH mimicry Roe et al., 1997. Trifluoromethylke- tones, when bound to Sepharose, proved to be an excel- lent affinity matrix for the purification of JH esterase Abdel-Aal and Hammock, 1986 and has greatly advanced our understanding of its protein chemistry and molecular biology. Most recently, an homology model of JH esterase from Heliothis virescens was constructed Thomas et al., 1999 that confirmed earlier molecular structureactivity studies showing that the JH esterase active site is highly specific for JH binding Hammock et al., 1984; Linderman et al. 1987, 1989; Roe et al., 1990. Two approaches have been used to determine the sig- nificance of JH esterase in the regulation of JH titer and insect development. The trifluoromethylketone, OTFP- sulfone 1-octyl [1-3,3,3-trifluoropropan-2,2-dihydroxy] 628 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644 sulfone demonstrated persistent, essentially complete selectivity for JH esterase versus a-naphthyl acetate esterases in last instars of the cabbage looper Roe et al., 1997. The application of OTFP sulfone to last instars and virgin female adults of T. ni delayed meta- morphosis and initiated egg production, respectively Roe et al., 1997, demonstrating the importance of JH esterase in the regulation of JH titer. Hammock et al. 1990 also showed that recombinant baculovirus expressing insect JH esterase could reduce JH levels in the Lepidoptera. These anti-JH effects of JH esterase provided a model system to demonstrate the usefulness of the baculovirus expression system as a novel insecti- cide. Although these studies show clearly that JH ester- ase has a significant role in the regulation of JH titer, it does not mean that the metabolism of JH by esterase results in a loss of biological activity, e.g. JH acid can act as a hormone or pro-hormone see subsequent discussion. There may also be other functions for JH esterase in insects. For example, the highly selective JH esterase inhibitor, OTFP, was shown by Anspaugh et al. 1995 and Devorshak and Roe unpublished observation to be an excellent inhibitor of insecticide resistance-associated esterases from the Colorado potato beetle, Leptinotarsa decemlineata, and the tufted apple budmoth, Platnota idaeusalis, respectively. In the tufted apple budmoth, structureactivity studies with trifluor- omethylketones showed that the optimum inhibitor of resistance-associated esterases was OTFP, as was also the case in similar studies conducted with T. ni Hammock et al., 1984. We must take care not to assume that the only function of JH esterase is JH metab- olism. Numerous studies have measured JH esterase activity both in hemolymph as well as other tissues during insect development Vince and Gilbert, 1977; reviewed by Hammock, 1985; Roe and Venkatesh, 1990. The corre- lation of JH esterase activity with developmental changes has been used extensively in the literature to argue for a role of JH esterase in the regulation of embryogenesis Roe et al., 1987; Share et al., 1988, lar- val development and metamorphosis reviewed by Ham- mock, 1985; Hanzlik and Hammock, 1988; Roe and Venkatesh, 1990; Jesudason et al., 1990, reproduction Lessman and Herman, 1984; Venkatesh et al., 1987, diapause Kramer and de Kort, 1976; Mane and Chip- pendale, 1981; Bean et al., 1983, and other processes. Jesudason et al. 1990 showed that both the peak levels of JH esterase activity as well as the timing of peak activity were regulated precisely in each stadium of Manduca see also Vince and Gilbert, 1977. The peak levels of JH esterase activity were positively correlated with insect size from the first through the last instar. Delineating the mechanism by which JH esterase activity is regulated relative to the animal’s size and for an indi- vidual tissue, should be of general interest to biochem- ists. Lassiter et al. 1996 found that a mechanism was in place for the tissue specific, differential expression of JH esterase in Culex quenquefaciatus adults during reproduction. This has been observed for other insect species during larval development see Roe and Venka- tesh, 1990. Our understanding of the mechanisms regulating JH esterase activity is meager considering that this is one of the critical unanswered questions in the field of JH metabolism. At least two parameters are presumed to be important in the regulation of JH esterase activity–JH esterase biosynthesis and degradation. It appears that one of the main sources of hemolymph JH esterase activity is the fat body Wing et al., 1981. Several studies have shown that JH esterase activity can be increased by the addition of JH and JH mimics at the time of pupal com- mitment through pupation in last stadium Lepidoptera Jones et al., 1981; Jones and Hammock, 1983; Venka- tesh and Roe 1988, 1989. However, prior to commit- ment, JH esterase activity is somewhat, but not absol- utely, refractory to JH. Venkatesh and Roe 1988, 1989 found that an inhibitory factor found only in the insect head reduced JH esterase activity but not a-naphthyl acetate esterase activity in the hemolymph and other tissues of T. ni and Manduca, respectively see also Vince and Gilbert, 1977. Inhibition was dose responsive and occurred even when insects were treated with a juvenoid. Jones et al. 1981 characterized a JH esterase induction factor in T. ni in experiments using isolated abdomens. Venkatesh and Roe 1989 found that it occurred only in the hemolymph of ligated abdomens and not in unligated insects. Both hemolymph JH ester- ase and a-naphthyl acetate esterase were increased, and the increase in JH esterase activity occurred only in the hemolymph and not in a whole abdomen homogenate. While there is currently insufficient information on the regulation of JH esterase activity to develop a reasonable hypothesis, a recent step toward such an hypothesis was taken by Jones et al. 1998, personal communication who showed that the JH esterase gene from the cabbage looper has a composite promoter consisting of an AT- rich motif TATA box, an initiator motif for transcrip- tion and at least one additional binding site for transcrip- tional factors. One other interesting characteristic of JH esterase is its biochemical stability in vitro. The half-life of JH esterase activity in T. ni hemolymph diluted in phosphate buffer containing 0.01 phenylthiourea at room tem- perature is approximately 1 week Roe and Hammock, unpublished information. However, in prepupal T. ni hemolymph, the half life for JH esterase in vivo is less than 12 h and in Manduca 90 min Ichinose et al., 1992. The rapid appearance and disappearance of JH esterase are important factors in the regulation of JH titer during the processes of commitment and metamorphosis. When radiolabeled JH esterase is injected into T. ni larvae, it 629 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644 appears to accumulate in the pericardial cells Booth et al., 1992; Ichinose et al., 1992, suggesting that this may be the primary site of inactivation, but whether this is a mechanism common to other insect species is conjec- tural. Bonning et al. 1997 discovered that the injected radiolabelled JH esterase enters the pericardial cells by receptor-mediated endocytosis, in association with a putative heat shock cognate protein HSC and then undergoes lysosomal degradation. We have much more to learn about this process. How specific is the mech- anism for JH esterase inactivation? Is this process found in other insects or used for the regulation of other pro- teins enzymes? Is JH esterase degradation itself regu- lated and by what means? With the availability of excel- lent biochemical and molecular probes for studying JH esterase, e.g. specific antibodies, recombinant enzyme, affinity ligands, etc., JH esterase would be a good model system to study the functional inactivation of a protein in insects. Considering that we know so little about protein degradation in insects, this would be a worthwhile goal for the future. 3.3. JH epoxide hydrolase The general assumption has been that JH epoxide hydrolase has a secondary role in the metabolism of JH acid Roe and Venkatesh, 1990. However, measure- ments of JH III epoxide hydrolase activity in vitro from last stadium T. ni Kallapur et al., 1996 revealed levels equivalent to that of JH III esterase activity. When 10R,11S-JH II was injected at physiological levels, as high as 40 of the known primary metabolites of JH metabolism was JH diol. Kallapur et al. 1996 also found that larval T. ni epoxide hydrolase preferred JH II to JH II acid at physiological cencentrations. Hal- arnkar et al. 1993 found that the major in vivo metab- olite of JH I in last stadium Manduca was the phosphate conjugate of JH diol, suggesting that JH esterase may be unimportant in the metabolism of this homolog. The Roe laboratory also demonstrated at this symposium that most of the metabolism of JH in vivo in last stadium T. ni results in a water soluble metabolite, although the structure is not yet known. Chromatography, enzyme derivatization and FAB-MS analysis showed that this product of JH metabolism was not the phosphate-diol found in Manduca. Lassiter et al. 1995 showed that JH III epoxide hydrolase was the predominant route of in vitro metabolism throughout the last stadium of the southern house mosquito, Culex quinquefasciatus, with two peaks in whole animal JH epoxide hydrolase activity occurring near the time of commitment and pupation. Three lepidopteran epoxide hydrolases have now been cloned and sequenced: GenBank Accession Number U46682 from Manduca eggs Wojtasek and Prestwich, 1996; U73680 from fat body of T. ni, and AF03542 from the gut of T. ni Harris et al., 1999. These epoxide hyrolases demonstrated very high homology and con- tained the exact catalytic triad Asp, Glu, His as that of mammalian microsomal epoxide hydrolases. Although there is evidence from differential centrifugation studies that insects have a soluble epoxide hydrolase Roe and Venkatesh, 1990, this has not been confirmed by sequence analyses. However, based on Southern blot analysis, it is likely that additional epoxide hydrolases are present in T. ni that have not yet been sequenced. The mechanism of JH epoxide hydration involves a nucleophilic attack by the active site Asp-residue of the least substituted oxirane carbon of JH which produces an enzyme-substrate adductester intermediate Linderman et al., 1995; Roe et al., 1996. 18 O labeling studies using microsomal epoxide hydrolase from T. ni in a multiturn- over reaction, showed clearly the exclusive addition of the label to the C10 position of JH. The enzyme-sub- strate adduct is broken by the hydrolysis of water which restores the Asp-carboxylate and produces JH diol. In this scheme, the oxygen that is added to the C10 position comes from aspartate and not from water, unlike the situ- ation for JH esterase. Until recently, there have been no effective inhibitors of insect epoxide hydrolase. Com- pounds designed to mimic a polarized and ionic tran- sition state were poor epoxide hydrolase inhibitors, while aliphatic epoxides like 10,11-epoxy-11-methyldode- canoate MEMD, Roe et al., 1996 demonstrated an I 50 of 80 µ M. In these studies, mimicry of the JH backbone appeared to be an important factor in JH epoxide hydro- lase inhibition, as was the case for JH esterase. The Lind- erman and Roe laboratories described this symposium S-MEMD with an I 50 of 12 nM. This is the most potent insect JH epoxide hydrolase inhibitor to date. Interest- ingly, this compound contains the unnatural enantiom- eric configuration of JH. One explanation may be that the S-enantiomer results in a more stable ester intermedi- ate as compared to the R configuration, resulting from a conformational change in the epoxide hydrolase. It may be necessary in the future to modify the methyl-ester of S-MEMD to reduce its metabolism by esterases in vivo. The in vivo inhibition of JH epoxide hydrolase is critical to understanding the significance of these enzymes in the regulation of JH titer and insect development. As mentioned earlier, mimicry of the JH backbone is important in JH epoxide hydrolase inhibition. Also, recombinant fat body epoxide hydrolase expressed in Sf9 cells using a baculovirus expression system was highly selective for JH III versus cis- and trans stilbene oxide Harris et al., 1999. These results should be viewed with caution, however, since it is expected that steric hindrance near the active site, which might occur with JH II, would reduce JH metabolism see also Debernard et al., 1998. As mentioned earlier, JH epox- ide hydrolase from T. ni metabolized JH faster than JH acid, and the major in vivo metabolite of JH I in last stadium Manduca was the phosphate diol. Jesudason et 630 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644 al. 1990 showed that changes in JH epoxide hydrolase activity were positively correlated with changes in JH esterase activity during the last stadium of Manduca. By contrast, Lassiter et al. 1995 demonstrated two peaks of JH epoxide hydrolase activity during the last stadium of C. quinquefasciatus, but no changes in JH esterase activity during the same period. These data argue that at least one endogenous function of this enzyme is JH degradation. Anspaugh et al. this symposium reported that baculovirus expressing recombinant fat body epox- ide hydrolase from T. ni was more virulent in last stad- ium cabbage loopers than the wild-type virus. The use of epoxide hydrolase recombinant baculoviruses as well as stable genomic transformations in Drosphila and novel EH inhibitors, will be useful in the future to increase our understanding of the roles of this enzyme in JH regulation. 3.3.1. JH binding protein JHBP JH binding proteins were first described by Whitmore and Gilbert 1972 and most recently reviewed by de Kort and Granger 1996. Three types of hemolymph binding proteins have been described in insects. The first fully characterized JHBP was from Manduca and has a low molecular weight of approximately 30, 000 Kramer et al., 1976; see also Goodman and Gilbert, 1978. This binding protein has subsequently been found in several other lepidopterans and consists of a single polypeptide and a single JH binding site per protein molecule. The lepidopteran JHBP is a minor hemolymph component comprising less that one percent of total hemolymph pro- tein. A second, high molecular weight binding protein, lipophorin, was found in the hemolymph of L. decemli- neata, Periplaneta americana and Locusta migratoria de Kort and Koopmanschap 1986, 1987, 1989. Lipo- phorin is the predominant hemolymph protein and the main hemolymph carrier of lipids in the insect. It con- tains about 40–50 lipid and is composed of two apop- roteins–apolipophorin I and II at 250 and 80 kDa, respectively. Because the hemolymph lipophorin con- centration is high, a high concentration of JH binding sites are available. The third class of JH binding protein is a 566 kDa hexameric protein first described from L. migratoria by Koopmanschap and de Kort 1988. The native protein contains 15 lipid, has 6 JH binding sites, and is composed of 77 kDa subunits. Although there are three categories of JH binding proteins, they can be gen- erally characterized as having high affinity for JH, but little affinity for JH degradation products like JH acid or JH diol, and high enantioselectivity for the natural occurring JHs. The presence of a hemolymph JHBP offers several advantages to insects in respect to the use of JH as a hormone. Because of the lipophilicity of JH, JHBP pro- vides an effective mechanism for downloading JH into the aqueous hemolymph and most likely reduces the problem of JH sequestration in lipophillic membranes. Similarly, JHBP reduces non-specific sequestration into liphophillic compartments or lipid-aqueous interfaces and facilitates the uniform circulation of JH to all of the insect tissues. These proteins may also be storage sites for JH. Binding protein may be an important factor in downloading JH from the corpora allata to the hemo- lymph, and from the hemolymph to target tissues. The reverse may be important for the tissue clearance of JH and JH degradation in the hemolymph. At physiological concentrations of JH, only a fraction of the high affinity JH binding sites in the hemolymph are occupied by JH Goodman and Gilbert, 1978. Most of the research to date on JH transport and degradation has focused on JH binding protein and JH esterase in a single tissue, hemolymph Fig. 1. For example, we know little about intracellular JHBPs see subsequent discussion of JH receptors and the role of binding proteins in general in the transport of JH from hemolymph to the cellular JH receptor. The same is true for JH metabolism. Although extensive studies have been conducted on hemolymph JH esterase, the signifi- cance of cellular JH esterases, JH epoxide hydrolases and polar JH metabolites remains unresolved. This is a considerable concern since JH analogs that were once thought to be JH metabolites are now found to act as JH prohormones or hormones. Certainly the view that JH esterase is the most important factor in JH inactivation needs to be re-examined. We can no longer assume that JH acid, which has a low affinity for JH binding protein, is devoid of biological activity or cannot be recycled back to JH. We also must be careful in extrapolating data from studies on hemolymph to other insect tissues. A case in point is the critical work of the Prestwich lab- oratory Touhara and Prestwich, 1993; Touhara et al., 1995 showing on strictly kinetic grounds that hemo- lymph binding protein in Manduca effectively protected JH from JH epoxide hydrolase and probably non-spe- cific esterases but not JH esterase. This suggests that JH acid is the primary metabolic route for JH in Mand- uca, and that epoxide hydrolase is important in the metabolism of JH acid. We must remember, however, that an insect is not a homogenous reaction mixture, that epoxide hydrolases are intracellular, and that the interac- tion of hemolymph JH binding proteins with intracellular binding proteins and the JH receptors has not been defined. As a consequence, we should not be surprised that Halarnkar et al. 1993 found that the predominant in vivo metabolite of JH I in last stadium Manduca is JH diol not JH acid, or JH acid, diol. It now appears likely that JH esterase is not the only factor important in JH turnover and that as with JH titering, the study of insect hemolymph alone will not be sufficient to describe JH action. Our attention must shift to other tissues, and the model proposed by Touhara and Prestwich 1993 and Touhara et al. 1995 should be expanded to the 631 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644 whole organism Figs. 1 and 2. It may also be useful to reexamine the organ culture experiments of Hammock et al. 1975 and the in vivo approaches, e.g. Slade and Zibitt 1972 and others see reviews by Hammock, 1985; Roe and Venkatesh, 1990. Since these older approaches more closely approximate the organism as a whole, they have less possibility of leading us in direc- tions and to conclusions that may have little to do with the insect’s physiology. 3.4. Comparative regulation by JH and JH-like compounds other arthropods The consensus view has been that the insect model for the regulation of metamorphosis and reproduction applies to the Arthropoda in general. This view has been heightened by work during the last ten years on the Crus- tacea. Apparently, crustaceans have the same biosyn- thetic pathway as insects for the synthesis of farnesyl pyrophosphate and methyl farnesoate MF [the pre- sumed JH of the Crustacea Laufer, this symposium], but unlike insects, do not make the C10,11 epoxide of juvenile hormone Smith and Sedlmeier, 1990; Charm- antier et al., 1991; Cusson et al., 1991a; Chang et al., 1993; Laufer et al., 1993; Chang, 1993; Homola and Chang, 1997. Crustacean methyl farnesoate is synthe- sized by the mandibular organ Fig. 2 with the rate of synthesis controlled by the peptidic mandibular organ inhibitory hormone from the X-organsinus gland XOSG complex. Ecdysteroids are also present in the Crustacea and are synthesized by the Y-organ and con- trolled by the peptidergic molt inhibitory hormone. Although the details are still under investigation, and there is some variability between species, MF promotes the production of ecdysone and reduces the duration of the intermolt period. Evidence was presented in these Fig. 2. Hormones involved in the regulation of arthropod development. From M. Roe, unpublished information. proceedings by the Laufer laboratory that MF also can act as a “status quo” hormone, as JH does in insects. MF in adult Crustacea is believed to initiate the synthesis of the yolk protein precursor in the hepatopancreas or other tissues, just as JH controls vitellogenesis in many adult female insects. The general assumption has been that the chelicerates, and more specifically ticks, regulate their metamorphosis and reproduction in a manner similar to insects. How- ever, in comparison to our knowledge of insect endocrin- ology, and even that of Crustacea, there is a dearth of data on tick endocrinology Fig. 2. The retrocerebral organ complex RCO and associated brain neurosecre- tory cells of the tick synganglion have been hypothesized to be the counterpart of the corpus cardiacum-corpus allatum complex of insects and the XOSG complex of the Crustacea reviewed by Sonenshine, 1991. How- ever, the function of the RCO is unknown and no neuro- endocrine products have been traced to this complex. It has been hypothesized that the synganglion peri- neurium can serve as a neurohemal organ. In addition, the lateral segmental organs located in the lateral nerve plexus between the pedal nerves have been assumed to be the site of JH biosynthesis. This conclusion is based primarily on the abundance of smooth endoplasmic retic- ulum in these glands and the histological similarity of these organs to the insect corpora allata. Ticks do pro- duce ecdysteroids, most likely from the epidermis, and probably under the control of the synganglion Dees et al., 1984; Sonenshine et al., 1985; Schriefer et al., 1987; Zhu et al., 1991; Lomas et al., 1997. It has been gener- ally accepted that ticks also synthesize JH and that JH is used in ticks to regulate nymphal-adult metamorphosis and egg maturation reviewed by Obenchain and Galun, 1982; Sonenshine, 1991. However, the evidence has been mostly pharmacological, i.e., the topical application 632 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644 of JHs, JH mimics and anti-JHs, and the results from these experiments have been equivocal. It was reported at this symposium that no JH could be detected in eggs, larvae, nymphs and adults of the hard tick, Dermacentor variabilis, by the Galleria wax test bioassay; no JH could be detected in the hemolymph of adult D. varia- bilis and the adult soft tick, Ornithodoros parkeri, by GCMS; and no JH, MF or farnesol was synthesized by the synganglion or any other tissues of nymphs and adults of these two species Roe et al., 2000; Neese et al., 2000. These results are not the final word on the subject of JH in ticks but do question whether the com- mon insect JHs have a role in tick development and per- haps in chelicerates in general. Certainly, the processes of metamorphosis andor reproduction are regulated by a hormone, but the structure of this hormone and its origin remain conjectural.

4. Future research and speculation