664 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 663–669
2. The questions
2.1. How many JHs are there? In some sense, the answer to this question depends on
how JH is defined. If we are restrictive and define JH as a farnesoid secreted by the CA, then the list is man-
ageable but growing. We are all familiar with the series from JH 0 to JH III with the addition of JHB3 and
methyl farnesoate see Riddiford, 1994 for details. To this list must be added two new products of the CA, both
hydroxy derivatives of JH III Darrouzet et al., 1997. At least one of these hydroxy derivatives has biological
activity in the Tenebrio wax test. More extensive assay- ing of the activity of such compounds needs to be done.
JH acid now appears to have some endocrine functions. In isolated prepupal abdomens of Manduca sexta, vitel-
logenin transcripts are absent. Once the abdomens have been switched to pupal commitment by injection of 20-
hydroxyecdysone, they require a further injection of JH II acid in order to become competent to produce vitellog-
enin transcripts in response to a further injection of methoprene. JH III acid, or other precursors of JH, will
not substitute for JH II acid Ismail et al., 1998. Ver- son’s gland in M. sexta becomes committed to secrete
pupal proteins early in the last larval instar before the first small commitment peak of ecdysteroid. In a recent
paper, Ismail et al. 2000 show that commitment in Ver- son’s gland depends both on a very low titre of ecdys-
teroid and on JH II acid.
Waiting in the wings for elucidation is the juvenile hormone of Hemiptera. This has been known for a very
long time to be different from the JH of other orders, but the structure has been a matter of controversy. It is
not worth reviewing the very long history of attempts to identify the hormone, but there is recent evidence that
may be useful. The membranes of follicle cells of Rhod- nius prolixus
bind JH I, but not JH II or JH III Ilenchuk and Davey, 1987, suggesting that the hormone might
be a form of JH I. Indeed, JH I has been identified from the hemolymph of the bean bug, Riptortus claveatus
Numata et al., 1992, although Kotaki 1993 was unable to detect JH I as being released from the CA of
this species in vitro. Kotaki 1996 has examined the JH- active compounds secreted in vitro by the CA of a stink
bug; the most active has a mobility in thin layer chroma- tography different from any known JH. Given the econ-
omic importance of this order, we ought to know more about its JH.
A glimpse of the complexity which may be emerging is provided by studies on the control of vitellogenesis in
the blowfly Phormia regina. In this insect, the CA secretes JHB3, JH III and methyl farnesoate in a parti-
cular ratio. While each of the hormones can support vit- ellogenesis on its own, the administration of the natural
cocktail is better than any of the compounds alone or in some other ratio Yin et al., 1995. These observations
raise a number of interesting questions, but they make it clear that it may not always be helpful to think of the
juvenile hormone of a particular species when a number of related compounds may be acting in concert to govern
a particular function. Of course, it is never clear when applying compounds to intact insects whether these com-
pounds are altered in some way before reaching their target receptors see below.
2.2. Are there other sources of JH in insects? Once more this depends on the definition of JH. If JH
is defined as a farnesoid secreted by the CA, then the answer is no. But there are clearly other sources of active
farnesoids in some insects. JH I was originally isolated from the abdomens of male pupae of Hyalophora cecro-
pia
Williams, 1956. The JH is synthesised by male accessory glands from JH acid secreted by the CA Peter
et al., 1981. The amounts synthesised and stored are substantial, and are transferred to the female Shirk et
al., 1980. The synthesis of JH by accessory glands is not confined to Lepidoptera. The male accessory glands
of mosquitoes synthesise JH I and III as well as JHB3 and methyl farnesoate, but not from JH acid. The syn-
thesis in this case is from acetate Borovsky et al., 1994a, raising the question of whether other tissues
might also constitute a source of the hormone. In Aedes, the ovary synthesises JH III from farnesoic acid
Borovsky et al., 1994b. If physiologically active farne- soids are secreted by tissues other than the CA, then
questions need to be raised about the interpretation of results based primarily on allatectomy. If a function dis-
appears upon allatectomy, it is probably safe to conclude that it is JH-dependent. But if a function persists, it may
not be safe to conclude that it is JH-independent. Do our assumptions about when JH is required for egg pro-
duction in mosquitoes need some further consideration?
2.3. Are there non-farnesoids with JH activity in insects?
It has been known for many years that ecdysteroids from the ovary fulfil many of the functions of JH in adult
females. While JH is known to stimulate the synthesis of vitellogenin in many insects Wyatt and Davey, 1996,
ovarian ecdysteroids have this function in mosquitoes and some other Diptera Hagedorn, 1983. While we
may have originally viewed such an arrangement as spe- cific to Diptera, that view is no longer tenable, for Girar-
die and Girardie 1996 have shown quite clearly that ovarian ecdysteroid functions as a gonadotropin,
inducing vitellogenin synthesis in the fat body of Locusta
, a species previously regarded as depending exclusively on JH for the control of vitellogenin syn-
thesis. This may be more than a complicating factor: is
665 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 663–669
it not possible that JH and ecdysone might be operating at the same site and in the same way to induce vitellog-
enesis? Do these observations have anything to tell us about receptors for JH? There are some species for
which it is known that ecdysone is without any influence on vitellogenin synthesis. Thus in Rhodnius females,
which contain ecdysteroids from the ovary only after the peak of vitellogenesis is achieved, early treatment with
doses of ecdysone large enough to induce cuticle depo- sition has no effect on vitellogenin synthesis Wang,
1991.
There have also been persistent reports of peptides mimicking JH. The mechanism of at least one of these
is indirect, acting to limit the synthesis of JH esterase Hayakawa, 1992, a process which is known to be
directly stimulated by JH Venkataraman et al., 1994; Feng et al., 1999. The action of some of the other pep-
tide mimics seems less certain. In Rhodnius, for example, an uncharacterised neuropeptide from the brain
Barker and Davey, 1983 and JH I Gold and Davey, 1989 both have the capacity to stimulate protein syn-
thesis in the male accessory gland in vitro. There are also several reports of peptides stimulating the synthesis
of vitellogenin. The most recent involves Locusta ovary maturating parsin, which, in addition to its action on vit-
ellogenin synthesis via an ecdysiotropic action on the ovary Girardie and Girardie, 1996, may also act
directly on the fat body Girardie et al., 1998. In the case of the direct action on the fat body it is probably
the C terminal end of the peptide which is active, the N-terminal portion being the ecdysiotropin. In this case,
the peptide did not directly stimulate vitellogenin syn- thesis, but sustained it, either by reducing turnover or
enhancing the translation of mRNA. Several peptides have either stimulatory or inhibitory effects on vitellog-
enin synthesis, but where the action of these peptides has been characterised, it is evident that they act at the
level of translation, whereas JH acts on transcription see Wyatt and Davey, 1996 for a fuller discussion. Never-
theless, some closer examination of these actions might be illuminating in terms of the precise mode of action
of JH, and a knowledge of their effects serves to empha- sise the complexity of the events in which JH is interven-
ing.
The thyroid hormones, long known to influence vari- ous processes in insects, have recently been shown to be
rather precise mimics of the action of JH on follicle cells of the insect ovary. Thus, they stimulate cell shrinkage
and patency, T3 39,3,5-triiodo thyronine binds to the same receptor on locust follicle cells as JH III Davey
and Gordon, 1996; Kim et al., 1999, and T3 is taken up by follicle cells by receptor mediated endocytosis,
apparently via the JH receptor Davey, 2000. These observations raise a number of interesting possibilities,
not the least of which is the functional significance of thyroid hormones in insects Davey, 2000. At least as
interesting are the clues that it might present to us in terms of our search for JH receptors. While the nuclear
receptor mechanisms for thyroid hormones are well known, the membrane receptors are not at all well
characterised McNabb, 1992. The origin of the thyroid hormones in locusts appears to be the food Davey,
2000, and this raises an obvious question about sup- plementary sources of JH in other insects.
2.4. Is the product of the CA the effective hormone? It has been an implicit assumption underlying most
of our work that the product of the CA intervenes in development and physiology unchanged, and that any
metabolites are less active breakdown products. A little reflection, however, will reveal that this is unlikely on
a priori grounds. Thus, ecdysone is well known to be processed into the more active 20-hydroxy-ecdysone,
and thyroxine, the principal product of the thyroid gland, is deiodinated in the liver and other tissues to yield the
much more active T3.
Processing of an inactive product of the CA, JH acid, to a more active form, JH I, by male accessory glands,
is, as made clear above, already established. In addition, perhaps the most provocative paper presented at JH VI
revealed that fat body and ovary, but not other tissues of locusts, processed injected labelled JH into unknown
compounds Couillaud et al., 1996. The fact that there are tissue specific metabolites raises questions about the
function of such metabolites. They may simply be degra- dation products, but equally, as is well known in ver-
tebrates, such products may contribute additional and significant biological activity Robel and Baulieu, 1994.
We would be wise to be alert to the possibility of such processing.
2.5. How does JH get into cells? It has been an overt assumption that because JH is
lipid soluble, it will pass through membranes into cells rather easily. Two aspects of that assumption have
always given me some difficulty. First, if the assumption is correct, then JH will tend to accumulate in mem-
branes, and will partition into the cytosol more sparingly. Second, given the specificity of the various receptors and
transporters now known to regulate the entrance of sub- stances into cells in general, it is at least possible, if not
likely, that the entrance of a compound as important as JH might be regulated in some way. Even for ecdysone,
which is also assumed to enter cells freely, there is recent work which suggests that the hormone may be selec-
tively accumulated by target cells. In ticks, the target of ecdysone is the salivary gland, and salivary gland cells
accumulate vastly more ecdysone than other tissues Lomas et al., 1998. The notion that steroids enter cells
666 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 663–669
by receptor-mediated endocytosis has been lurking in the background for many years Szego, 1984.
In vertebrates, the thyroid hormones are taken up by receptor-mediated endocytosis Di Liegro et al., 1987.
I have recently shown that rhodamine conjugated T3 enters the follicle cells of locusts by receptor-mediated
endocytosis, via a receptor which appears to be the mem- brane receptor for JH Davey, 2000. These observations
may or may not indicate that JH is also taken up by endocytosis. The functional significance of such a mech-
anism remains obscure. For T3, it is clear that the uptake results in the conversion of T3 to a much more active
molecule, but whether this is a functionally important phenomenon remains to be explored. The entire process
may be artifactual, induced by an attempt on the part of the cell to eliminate the T3 rather than capitalise on its
presence. Or, it may be simply a matter of the normal turnover of the receptor.
Nevertheless, there is at least the possibility that JH may be selectively accumulated by the cell. If this should
prove to be the case, there are several potential conse- quences. Perhaps the most intriguing is the possibility
that the concentration of hormone inside selected target cells may be considerably higher than the concentration
in the hemolymph. This might expand the range of potential receptors to proteins with a somewhat lower
affinity for the hormone than is customarily thought to be required.
At least one study has been conducted on JH uptake into cells. Mitsui et al. 1979 placed pieces of epidermis
from M. sexta in medium containing [
3
H]JH I and meas- ured the time course of uptake into epidermis. From the
data in the paper it is possible to calculate the concen- tration inside the cell at equilibrium, and it appears that
the cells accumulate JH to a level 10 times that in the medium.
2.6. How many modes of action are there? This is obviously a complex question, for JH is among
the most pleiotropic of hormones. The notion of an acti- vation or priming action, by which a target cell is pre-
pared to respond to a later regulative action of the hor- mone was first hinted at more than 25 years ago Pratt
and Davey, 1972, and the first experimental evidence for the phenomenon was provided for the patency
response of the follicle cells by Abu-Hakima and Davey 1975, who showed that follicles of Rhodnius which had
developed in the absence of JH were not able to respond to JH by becoming patent. This was later shown to be
the result of a failure to develop the JH membrane recep- tor and possibly the special JH-responsive isoform of
Na
+
K
+
ATPase Ilenchuk and Davey, 1987. The con- cept was later expanded to include the notion that the
activation might be independent of the regulative func- tion, as in the case of the stimulation of vitellogenin syn-
thesis by ecdysone in flies, which requires a prior exposure of fat body to JH Davey, 1994. The concept
has been developed to include a wide variety of effects Wyatt and Davey, 1996. Is priming really a distinct
mode of action from regulation? The fact that priming by JH is sometimes followed by regulation by a different
hormone supports this notion, although we have no clear idea about the mechanism. Perhaps the synthesis of
receptors is involved. While there is no doubt that the distinction between priming and regulation is useful in
conceptual terms, there is perhaps less certainty about its utility in thinking about receptors see below.
Another dichotomy has arisen between membrane effects and nuclear effects. There is no doubt that there
are effects of JH which are mediated by or even occur entirely within the cell membrane. We are moving
towards a more complete description of the events in the membrane of follicle cells. We had earlier shown that
JH binds to a membrane protein and by a cascade involv- ing the activation of protein kinase C, a 100 kDa protein
is phosphorylated see Wyatt and Davey, 1996 or Davey, 1996 for detailed references. It is now clear that the 100
kDa peptide is the
α -subunit of the Na
+
K
+
ATPase, and, moreover, high resolution gels reveal that there are two
isoforms of the α
-subunit, one of which is preferentially phosphorylated in the presence of JH Davari, 1999.
The involvement of other signalling elements such as a G protein, phospholipase C and diacylglycerol between
the receptor and protein kinase C remains hypothetical. This is not a complete description of the events mediated
by the membrane receptor, for the development of pat- ency involves a good deal more than the shrinkage of
the cell: there are changes in the cytoskeleton and rearrangements of cell junctions Davey, 1996.
There have been suggestions of other membrane receptors governing the synthesis of proteins in male
accessory glands. Yamomoto et al. 1988 produced evi- dence suggesting that the in vitro stimulation of protein
synthesis by JH in male accessory glands of Drosophila required both Ca
++
and protein kinase C. There has been some preliminary evidence of binding of a photoaffinity
analogue of JH III to membranes of fat body and brain of Locusta Sevala et al., 1995.
There may be other modes of action in addition to those at the membrane and in the nucleus. There are
several reports of cytosolic binding proteins see Wyatt and Davey, 1996 for detailed references, but there has
been no function attached to the binding. Whether these represent functional receptors remains to be seen. Most
current models of JH action assume the existence of a cytosolic binding protein in order to transport the JH to
the nucleus.
An interesting potential cytosolic mechanism has recently been described in a marine annelid in which JH
and relatives are capable of inducing larvae to settle and metamorphose. The mode of action involves the acti-
667 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 663–669
vation of protein kinase C Bigger and Laufer, 1999, but the initial action is apparently cytosolic, since the
juvenoids were capable of directly activating purified cytosolic kinase C rather than acting to stimulate mem-
brane bound kinase C. The concentrations necessary to achieve these results were an order of magnitude larger
than what is normally regarded as physiological, but once the possibility of accumulation of JH by target cells
has been raised, these concerns may not be so great.
There is also the possibility that JH might affect mito- chondria directly. The various attempts to demonstrate
a direct effect have been reviewed, and we concluded that the definitive experiments necessary to examine the
question have yet to be done Wyatt and Davey, 1996. Given the parallels between JH and the thyroid hor-
mones, it would be useful to do such experiments. Thy- roid hormones have been known for some time to stimu-
late mitochondrial respiration, but that has always been assumed to be an indirect effect. Recently, however,
there have been clear reports that 3,5-diiodothyronine can increase the metabolism of isolated mitochondria at
doses in the low nanomolar range Leary et al., 1996.
However, it is that group of effects which involve some interaction with the genome which are central to
the action of JH. It is clear that the priming or activating actions are likely to be of this type, and they can only
complicate the design and interpretation of experiments. The regulatory actions of JH that proceed via the nucleus
are of two broad types. There are those that require an interaction with ecdysone, and those that are the result
of independent action.
The requirement for ecdysone is clearest and most complete in the metamorphic actions of JH, for JH
actions can only be expressed in the context of moulting controlled by ecdysone. This is a system of growing
complexity. There is no doubt that one of the functions of JH in metamorphosis involves preventing the ecdy-
sone-controlled switch in developmental pathway from larval to adult as made clear by Riddiford 1996. This
will involve some interaction between ecdysone and JH, and given the timing of the switch, the strategy of docu-
menting the effects of JH on the earliest ecdysone-con- trolled events e.g. Hiruma et al., 1999; Zhou et al.,
1998 is likely to be productive. It is, however, likely that there will also be connections between the actions
of JH and ecdysone not only in terms of its action on metamorphosis, but also in its action on vitellogenin syn-
thesis. The involvement of both JH and ecdysone in the control of vitellogenin synthesis is now clear in a number
of species see above.
Given the requirement for ecdysone, the discovery that JH binds to USP Jones and Sharp, 1997 is pro-
vocative. There are several unusual aspects to this bind- ing. First the affinity of USP for JH is in the high
micromolar range. While this is unusual, until it is clear what the intracellular concentrations of JH might be, we
ought not to eliminate the possibility on these grounds alone. The finding by Hiruma et al. 1999 that JH down
regulates at least one form of USP casts some doubt on the significance of the binding, since ligands normally
increase the expression of their receptors. The facts that some farnesoids bind to FXR, an orphan receptor of ver-
tebrates and that FXR has a high degree of homology to EcR is also intriguing, although JH III and methop-
rene failed to activate FXR Forman et al., 1995. Simi- larly, methoprene acid has been shown to bind to RXR,
which is the homologue of USP, although once more JH II and other JH active compounds did not Harmon et
al., 1995.
There is thus a good deal of evidence to suggest that one of the nuclear actions of JH is closely associated
with the way in which ecdysone exerts its effects, and may interact in some way with the ecdysone receptor
complex. But what of the other nuclear actions which do not involve ecdysone? A conceptual model for the
way in which JH might induce the synthesis of a parti- cular protein in this case vitellogenin has been
presented by Wyatt and Davey 1996, who emphasise that the model is speculative and highly simplified. Per-
haps the most useful system for studying the action of JH, leading perhaps to the identification of some of the
molecular events within the nucleus, is the stimulation of JH esterase synthesis. This is under study in at least
two laboratories Venkataraman et al., 1994; Feng et al., 1999. One system involves a cell line, and thus the tim-
ing of events can be precisely measured. The JH esterase begins to appear within about 30 min of the adminis-
tration of JH: there is no sign of a lag or priming phase. In addition, no other hormones are needed Feng et al.,
1999. Does this represent a mode of action which is distinct from that which requires the joint action of ecdy-
sone? That question remains open. In addition, our pre- occupation with the status quo actions of JH during
development ought not to blind us to the large body of evidence, based on simple and clear experiments, that
JH may also encourage the appearance of larval charac- ters rather than simply suppress the appearance of adult
characters Wigglesworth, 1970. As is made clear by Jones 1995, there are some larval developmental events
which JH appears to stimulate.
Once the presumed nuclear receptors have been acti- vated, there is a myriad of downstream events which
can come into play ranging from the production of an exportable protein to the proliferation of neuroblasts, for
which polyamine synthesis appears to be an essential step in the transduction pathway Cayre et al., 1997.
2.7. How many receptors are there? This is a question that can be answered only by ident-
ifying the receptors. The bewildering array of effects of JH, and its range of modes of action might argue for a
668 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 663–669
range of receptors. However, it is worth remembering that many steroids are equally pleiotropic, and that a var-
iety of events are affected as the result of interaction with a single receptor type. We have generally assumed
that membrane receptors for JH are likely to be entirely different molecules from nuclear receptors, and even
cytosolic receptors must surely argue for more than one type of receptor. Nuclear receptors with their DNA bind-
ing domains and zinc fingers are unlikely to be compat- ible with membrane receptors with their transmembrane
domains. In addition observations such as those outlined above concerning the requirement for JH B3, JH III and
methyl farnesoate in specific proportions for normal vit- ellogenin synthesis in blowflies Yin et al., 1995 might
be regarded as a powerful argument for a galaxy of receptors.
However, vertebrate steroids also have membrane effects Wehling, 1997. While rather few of the mem-
brane receptors have been characterised, a recent study shows that transfecting cDNA for nuclear estrogen
receptors into hamster ovary cells results in a single tran- script and expression of the receptor in both membrane
and nuclear fractions of the cell. The membrane-bound receptors were functional as determined by their ability
to stimulate adenyl cyclase and inositol phosphate pro- duction via associated G proteins Razandi et al., 1999.
The nuclear receptors do not possess transmembrane domains. This is not definitive evidence, but it is an
encouraging sign.
3. Conclusion
