619 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644
Table 1 Juvenile hormone content during the life history of the Cecropia silkworm
a
Stage Approximate juvenile
Approximate juvenile Approximate juvenile
hormone concentration of hormone concentraion per hormone activity per gram extract: Cecropia
insect or fragment: fresh weight: Cecropia
unitsgram extract Cecropia unitsanimal or
unitsgram animal or fragment
fragment fresh weight Unfertilized eggs
Minimum of: 165 0.042
8.151 Unfertilized eggs from alatectomized
“0” “0”
“0” 7-day embryos
Minimum of: 165 0.031
7.095 7-day embryos from allatectomized
Minimum of: 70 0.012
3.108 1st instar larvae newly hatched
Minimum of: 165 0.031
7.475 5th instar larvae mixed sex
35 4.162
0.571 Diapausing pupae
1 month old 20
5.780 1.468
Diapausing pupae 1 month old
20 6.300
1.114 Chilled pupae
6 months old “0”
“0” “0”
Chilled pupae 6 months old
“0” “0”
“0” 2-day old developing adults
“0” “0”
“0” 8-day old developing adults
“0” “0”
“0” 11-day old developing adults
“0” “0”
“0” 14-day old developing adults
“0” “0”
“0” 17-day old developing adults
“0” “0”
“0” 20-day old developing adults
125 25
13.70 22-day old developing adults
400 122.68
201.64 Adult 4 days
675 151.88
298.75 Adult 7 days
1000 178.30
418.00 Adult 7 days
125 17.50
11.63
a
Activity of extracts is expressed in Cecropia units. One Cecropia unit is equivalent to the juvenile hormone activity found in one milligram of extract obtained from the abdomens of seven-day old male Cecropia moths. Extractions of developing adults and adult moths were conducted
on the abdomens only and the activity noted is for the abdominal extract. From Gilbert and Schneiderman 1961b.
receptors. We simply could not summarize or do jus- tice to all the presentations made during the symposium
and chose topics we felt were important and with which we were personally involved. We apologize to those
individuals we did not cite since it is possible that their contributions may be even more important than those
summarized here.
2. Control of the corpora allata
Changes in the JH titer, which regulate the growth and development of immature insects and reproduction in
adults, are controlled precisely by various physiological and biochemical processes i.e. synthesis, degradation,
sequestration and secretion. Of these, the regulation of synthesis has generally been considered the most
important, and a large body of evidence for both stimu- latory allatotropic and inhibitory allatostatic control
of JH synthesis by the corpus allatum has accumulated from many years of studies in vivo.
2.1. Allatotropins ATs. Since the corpora allata are innervated by the axons of
both peptidergic neurosecretory cells and typical neurons located within the brain, the mechanisms stimulating and
inhibiting corpora allata function promised to be com- plex from the outset. Research in this area in the last
decade has confirmed this prediction. The primary focus of this work has been on regulatory peptides, termed
allatotropins and allatostatins. With regard to allatotrop- ins, despite a wealth of indirect evidence for the stimu-
lation of JH production by neural factors, particularly during larval development, only one had been isolated
and sequenced prior to this conference, an allatotropin from adult Manduca Mas-AT Kataoka et al., 1989.
This amidated tridecapeptide stimulates JH biosynthesis only by the corpora allata of adult Manduca. The gene
Mas-AT is expressed as three different mRNAs which differ from one another by alternative splicing and which
have been suggested to encode three distinct prohor- mones Taylor et al., 1996. Immunocytochemistry using
polyclonal antibodies to Mas-AT and in situ hybridiz- ation with riboprobes for its mRNAs have been used to
demonstrate that Mas-AT does exist in larvae, with its greatest abundance in two cells in the frontal ganglion
that project to the gut and in cells in the terminal abdominal ganglion Bhatt and Horodyski, 1999. Only
low levels were found in the brain in non-neurosecre- tory cells and subesophageal ganglion. This result con-
trasts with that of Z
ˇ itnˇan et al. 1995, who used anti- bodies to the peptide to demonstrate Mas-AT in a limited
number of cerebral neurons in the larva, plus axons in
620 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644
the corpora cardiaca and corpora allata. Thus, although Mas-AT has no apparent effect on the larval corpora
allata of Manduca, it must have some other unknown function at this stage. Interestingly, while Mas-AT does
stimulate adult corpora allata, its mRNAs were not detected in the prepupal, pharate adult or adult brain, but
were present in the pterothoracic and unfused abdominal ganglia of the pupa Bhatt and Horodyski, 1999. A
recent study by Lee et al. 1998 demonstrated the inhi- bition of midgut ion transport in Manduca fifth instars
by Mass-AT, an effect which appears to be species-spe- cific. It has another effect as well, exhibiting cardioacce-
leratory activity in the pharate adult, but not the larva Veenstra et al., 1994. These observations are illustra-
tive of a phenomenon observed with a number of other insect neuropeptides i.e. different effects which are both
stage- and tissue-specific. As was discovered with allato- statins, these effects can be species-specific as well
see below.
In the decade since the identification of Mas-AT, no allatotropin sequences were published for any other
species, and until recently, there was a dearth of work on other putative allatotropins. The exceptions are the
demonstration of allatotropic activity in the subeso- phageal ganglion of crickets Lorenz and Hoffmann,
1995, and some recent work with Galleria Bogus and Scheller, 1996. In the latter study, monoclonal anti-
bodies raised to a protein fraction of larval brains that stimulated JH II synthesis by corpora allata in vivo and
in vitro, were used to identify a single 20 kDa peptide by immunoblotting. Immunoreactivity was also observed
in two pairs of cerebral neurosecretory cells and in cells of the corpus cardiacum. Although the size of this puta-
tive allatotropin makes it an unlikely homolog of Mas- AT, a pair of immunoreactive median cerebral neuro-
secretory cells were observed with antibodies to Mas- AT. This leads to the suggestion that Mas-AT and Gal-
leria AT have common epitopes due to splicing from the same preprohormone.
A significant outcome of this conference is direct evi- dence from several different studies showing that Mas-
AT is a functional moiety in other Lepidoptera see Stay, this symposium. A stimulatory peptide in methanol
extracts of adult brains of Spodoptera frugiperida, the fall army worm, has been identified by Edman degra-
dation and mass spectrometry as Mas-AT Oeh et al., this symposium. Exposure of adult corpora allata to
synthetic Mas-AT results in a strong, dose-dependent and reversible stimulation of JH biosynthesis. The most
interesting observation of this study is that a synthetic Manduca allatostatin Mas-AS did not affect the rate of
JH production by Spodoptera glands but inhibited Mas- AT-stimulated synthesis. In the tomato moth, Lacanobia
oleracea, larval corpora allata can be stimulated and inhibited, respectively, by synthetic Mas-AT and Mas-
AS, in contrast to Manduca, where only Mas-AS is effective Audsley et al., this symposium. Furthermore,
a Mas-AT-like peptide has been identified by ELISA in extracts of larval brains of this species. Genes containing
sequences identical to those for Mas-AT and Mas-AS have been cloned in Pseudaletia unipunctata, the true
armyworm moth Truesdell et al., this symposium, and immunocytochemical analysis with antibodies to Mas-
AT have revealed immunoreactivity in the brain, abdominal ganglia, and corpora allata of Pseudaletia
adults. Finally, Mas-AT stimulates the corpora allata of honey bee larvae in a dose-dependent, reversible, and
stage-specific manner, suggesting that this peptide could be
a regulatory factor in Hymenoptera as well
Rachinsky et al., this symposium. 2.2. Non-neural allatotropins
If Mas-AT does not stimulate larval glands in Mand- uca, what does? Perhaps, as seen in Spodoptera, the
activity of one peptide is only detected in the presence of the other. Thus in Manduca, Mas-AT may only stimulate
allatostatin-inhibited glands. Alternatively, stimulation may occur as a result of the absence of the allatostatin
or the presence of an AT-like factor from another tissue source. It has been suggested for Diploptera that the
absence of allatostatins results in the stimulation of JH biosynthesis, and that certainly could suggest the pres-
ence of an allatotopin in certain experiments in vivo. Alternatively, the source of a stimulatory factor could
be other than neural tissues see Stay, this symposium. The ovaries have been indicated as a source of allatotro-
pin in Diploptera see Stay, this symposium, in Grillus bimaculatus Hoffmann et al., 1996, and the male
accessory sex gland in Drosophila Moshitsky et al., 1996. As reported at this conference, the sex peptide of
the medfly Ceratitis capitata stimulates the corpora allata to produce a lipoidal molecule related to JH
Moshitsky et al., this symposium. In Diploptera, the ovarian factor appears to be proteinaceous and water sol-
uble, with a molecular weight of less than 10 kDa Unnithan et al., 1998. Ovary-preconditioned medium
stimulates corpora allata activity in vitro, but only when the glands’ neural connections to the brain are severed,
further complicating the picture of gland control in this species.
2.3. Future research on allatotropins In summary, there are sufficient gaps in our knowl-
edge of insect allatotropins to indicate that there is both the opportunity and the need for more work in this area.
It is clear from the most recent studies that Mas-AT may be ubiquitous in Lepidoptera, much as the dipteran alla-
tostatin Dip-AS is in different insect orders. The mol- ecular basis for these similarities needs to be established.
The fact that Mas-AT has functions other than the stimu-
621 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644
lation of JH production mirrors the trend observed for the allatostatins see below and also deserves further
investigation. Is the allatotropic effect the primary func- tion of the molecule? Perhaps most importantly, the
interactions between allatostatins and allatotropins in the control of the corpora allata are fertile, but unexplored,
territory, and should lead to studies of their receptors and the downstream events controlled via the ligand-receptor
interaction. It is these paths that typify the most recent work on allatostatins.
2.4. Allatostatins AS In contrast to the slowly developing field of research
on allatotropins, there has been an explosion of research on allatostatins, which inhibit JH synthesis, beginning
with the seminal work of Stay and Tobe on the allatostat- ins of Diploptera punctata Stay et al., 1991. At the
time of the Sixth Conference on the Juvenile Hormones, 13 allatostatins had been identified in Diploptera, each
with a similar C terminus: YF-X-F-G-LI-NH
2
, with one terminating in isoleucine. All of the Diploptera allatosta-
tins appear to be produced in the same cell, and all inhibit JH synthesis in vitro but with different potencies,
ranging in concentration from an ED
50
of 10
211
M to 10
27
M Stay et al., 1996. Localization of allatostatin immunoreactivity in the nervous system of Diploptera
revealed numerous immunoreactive neurons in the brain and also in all other ganglia Stay et al., 1994; Stay, this
symposium. The arborization of the axons of cerebral allatostatin immunoreactive cells within the corpora car-
diaca suggested that allatostatins could be released from this neurohemal organ, and allatostatin I was found in
the hemolymph. Thus the corpora allata of Diploptera appear to be regulated by allatostatins delivered directly
to the gland via the axons of the lateral neurosecretory cells of the brain or indirectly via the hemolymph.
A Manduca allatostatin Mas-AS was purified and sequenced from pharate adult heads in 1991 Kramer et
al., 1991. Its sequence bears no homology to the YXFGLamide family of allatostatins, but it inhibits JH
biosynthesis reversibly in both adults and larvae, with an ED
50
value of 2–5 nM with corpora allata from day 0 fifth instars. An immunohistochemical demonstration
of Mas-AS revealed two groups of lateral neurosecretory cells in brains of last instars, and arborization of the
axons of one group Ib within the corpora allata Z ˇ itnˇan
et al., 1995. Another factor has been identified in Manduca which
also inhibits the corpora allata, but which does so in a non-reversible fashion Bhaskaran et al., 1990. This cer-
ebral factor, termed an allatinhibin, provides stable inhi- bition when corpora allata are treated with the factor in
vitro and then implanted into penultimate instar larvae Unni et al., 1993. Its structure is unknown.
2.4.1. Allatostatins in other species At the time of the last JH conference, 30 different
peptides belonging to the allatostatin family had been identified in several different insect species. Since that
time a considerable number have been added to the list see Stay, this symposium, and members of the allatos-
tatin family have been identified by immunoreactivity in Crustacea as well: crab, lobster, and crayfish Skiebe,
1999. Allatostatins with the typical C terminus sequence and an inhibitory effect on JH synthesis have been ident-
ified in two other cockroaches, Periplaneta americana Weaver et al., 1994 and Blatella germanica Belles et
al., 1994 and two crickets, Gryllus bimaculatus and Acheta domestica Lorenz and Hoffmann, 1995. Three
structurally similar allatostatins for which a functions has not yet been determined, also have been isolated
from the mosquito, Aedes aegypti Veenstra et al., 1997. In addition, structurally identical peptides were isolated
from the blowfly, Calliphora vomitoria, where they inhibited corpora allata activity Duve and Thorpe, 1994;
Duve et al., 1996 and from the honeybee, Apis melli- fera, where they did not Rachinsky and Feldlaufer,
2000. As reported at this conference, multiple allatos- tatic peptides have been isolated from the brains of the
stick insect Lorenz et al., this symposium and are identical to those in Diploptera and Schistocerca. How-
ever, while these peptides strongly inhibit JH synthesis in crickets, they have no effect on the corpora allata of
the stick insect, suggesting a primary function other than the inhibition of JH biosynthesis.
2.5. Structural allatostatins with other activities The fact that some of the allatostatins, as defined by
their structure, did not function as their nomenclature implied has led to the discovery of a wide range of
activities for these neuropeptides. Allatostatin-like pep- tides have frequently been found to affect muscle con-
tractility, particularly in the midgut. In Diploptera, where Dip-AS immunoreactivity and mRNA synthesis
have been found in endocrine cells of the midgut as well Yu et al., 1995, allatostatins act as potent inhibitors
of myogenic and proctolin-induced contractions of the midgut Stay et al., 1994. Allatostatins also inhibit gut
motility in moths Duve et al., 1997, in addition to cock- roaches and blowflies. Recently, a new member of the
YXFGLamide, or allatostatin, family, has been isolated and sequenced from the ventral nerve cord of Manduca
Davis et al., 1997. An antibody to cockroach allatosta- tin that recognizes the Manduca peptide was used to map
allatostatin immunoreactive neurons in the larval ner- vous system and revealed neuroendocrine cells in the
brain, abdominal ganglia and their respective neu- rohemal organs. By contrast, immunoreactive inner-
vations of the corpora allata were sparse, suggesting that this peptide does not regulate corpora allata
622 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644
activity. However, many thoracic motor neurons were immunoreactive,
indicating a myotropic or
anti- myotropic function in the larva. Interestingly, immuno-
reactivity disappeared during metamorphosis and did not reappear in the adult.
Ten peptides belonging to the YXFGLamide allatosta- tin family have been identified and sequenced in Schisto-
cerca Schoofs et al., 1997. These allatostatins, termed schistostatins, inhibit peristaltic movements of the ovi-
ducts. In the earwig, where Dip-AS 7 immunoreactivity is found predominantly in the recurrent and esophageal
nerves, the last abdominal ganglion and the proctodeal nerve, Dip-AS significantly and reversibly decreased
hindgut motility but had no effect on JH biosynthesis Rankin et al., 1998a. HPLC fractions of earwig brain
extract were found to contain Dip AS-like material by radioimmunoassay using antibodies to the cockroach
allatostatin, and these fractions also inhibited JH biosynthesis by cockroach corpora allata and earwig
hindgut motility Rankin et al., 1998b. The demon- strated presence of allatostatins in Diploptera hemocytes
Skinner et al., 1997 suggests that they may also play an unknown roles in hemolymph functions. In Blatella
germanica, allatostatins inhibit vitellogenin release by the fat body, presumably by inhibiting the process of
vitellogenin glycosylation Martin et al., 1996.
The existence of Mas-AS in other insects has also been suggested by the work of Jansons et al. 1996 and
by results presented at this meeting W.G. Bendena, M. Cusson., P. Koladich, M.D. Price, I.S. Jansons, P.
Truesdell, and S.S. Tobe, unpublished observations. Brain cDNAs have been found in Pseudalecia and Dro-
sophila which specify a neuropeptide that upon cleav- age, yields a peptide that is similar to Mas-AS.
Although numerous cells in the brains of these two species are immunoreactive to antibodies to Mas-AS,
synthetic Mas-AS does not affect JH biosynthesis in vitro by Drosophila ring glands and has limited ability
to inhibit Pseudalecia glands in vitro Jansons et al., 1996. The Drosophila peptide has no affect on JH
biosynthesis in vitro, when tested with ring glands from third instars and day 2 adults W.G. Bendena, personal
communication. Thus in both Drosophila and Pseuda- lecia, this peptide probably has a functional role differ-
ent from that in Manduca. Nevertheless, there is evi- dence for a cerebral allatostatin in Drosophila, based
on the work of Richard et al. 1990 and Altaratz et al. 1991. As previously noted, the larval corpora allata
of Lacanobia can be inhibited effectively by synthetic Mas-AS, and Mas-AS-like immunoreactivity has been
detected in the central nervous system, midgut and Mal- pighian tubules of this species Audsley et al., 1998
Audsley et al., 1999, this symposium. Its presence in sites other than the central nervous system suggests that
other roles may exist for this peptide. 2.6. Control of allatostatin titer
If allatostatins can exert their effects via the hemo- lymph, how are their hemolymph titers controlled?
While research on the synthesis of allatostatins and its control has not yet begun, some recent research explores
their degradation. It is now known that allatostatin con- centrations are affected by a susceptibility to degradation
Bendena et al., 1997. Incubation of Dip-As 7 or Dip- AS 9 with hemolymph for 30 min revealed two primary
catabolic cleavage steps: cleavage by a putative endopeptidase, yielding a C terminal hexapeptide, and
subsequent cleavage of this product by an amastatin- sensitive aminopeptidase, to yield the C-terminal penta-
peptide. Nevertheless, these hemolymph enzymes do not inactivate allatostatins since the C-terminal pentapeptide
core the minimal sequence necessary for the inhibition of JH synthesis remains Garside et al., 1997a. On the
other hand, membrane preparations of brain, gut and corpora allata cleave allatostatins at the C-terminus, also
in a two-step process, completely inactivating the pep- tide Garside et al., 1997b. On the basis of information
provided by the prior two studies, Nachman et al. 1999 have successfully synthesized pseudopeptide mimetic
analogs of Dip-AS resistant to degradation by hemo- lymph and tissue-bound peptidases, by increasing steric
hindrance of the degradative enzymes. As reported at this meeting, these analogs show varying resistance to
catabolism, and all inhibit JH biosynthesis in vitro Nachman et al., 1999; Garside et al., this symposium.
Injection of mated Diploptera females with some of the analogs inhibited JH biosynthesis significantly by sub-
sequently explanted corpora allata, as well as basal oocyte growth in vivo. This approach should allow more
critical studies of the physiological processes modulated by allatostatins, since allatostatic activity was retained
in the analogs.
2.7. Allatostatin receptors Recent work on allatostatins has focused on their
receptors, since the kind and number of receptors present in the corpora allata or any other tissue with a specific
response to an allatostatin would control the timing, dur- ation, and strength of the response. Early work by Cus-
son et al. 1991a demonstrated by photoaffinity labeling the presence of two different putative receptors in
Diploptera glands. A later study, which employed an in vitro binding assay in addition to the photoaffinity assay,
identified a 37 kDa receptor for both Dip-AS-5 and Dip- AS-7 in adult female brains Yu et al., 1995. This recep-
tor had a single K
d
of 9 ×
10
210
M for Dip-AS-5, but two for Dip-As-7 1.5
× 10
29
and 3.8 ×
10
29
M, sug- gesting that two receptor sites exist for Dip-AS-7. The
affinity of the brain binding site for Dip-AS-5 was higher than that for Dip-AS-7, corresponding to the order of
623 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644
their potencies in vitro. In the corpora allata, a single K
d
was obtained for an AS-7 receptor 7.2 ×
10
210
M. A subsequent structure-activity study, using synthetic anal-
ogs of Dip-ASB2 Dip-AS-2, again revealed two recep- tor types in the corpora allata, based on the biphasic
inhibitory response of the corpora allata to some of the ASB2 analogs Pratt et al., 1997. These authors propose
that the C-terminal portion of allatostatins contains, in addition to the “message” portion of the neuropeptide
that part responsible for a full effect, a significant amount of “address” information binding affinity for the
receptor. They conclude that a pentapeptide is likely to be the smallest fully active allatostatin structure. Diver-
gent evolution of receptor types is proposed to occur together with the evolution of multiple allatostatins from
a common ancestor see below. The recognition of shorter allatostatic peptides by one receptor, and of
larger peptides by the other, would allow for cross-talk between the two types of receptors, for the congruence
of the “message” of all allatostatins, and for a divergence of function. Cloning and sequencing of allatostatin
receptors is clearly the next step in sorting out the basis of the ubiquitous occurrence but varied functions of the
multiple allatostatins.
2.8. Effect of allatostatins on the JH pathway Where in the pathway of JH biosynthesis does the
cockroach allatostatin act? Early work by Pratt et al. 1991 demonstrated that the addition of exogenous far-
nesol or mevalonate reversed the allatostatic inhibition of JH biosynthesis in vitro, suggesting that the target of
the allatostatin existed prior to mevalonate kinase activity. Recent work has shown that the allatostatin is
a more effective inhibitor of JH biosynthesis in glands that utilize glucose or amino acids as their carbon source,
rather than acetate Sutherland and Feyereisen, 1996. These results demonstrate further that either the transport
of citrate across the mitochondrial membrane the citratemalate shuttle andor the cleavage of citrate to
yield cytoplasmic acetyl-CoA ATP-citrate lyase are the probably targets of the allatostatin, i.e., the first commit-
ted steps in the synthesis of JH III.
2.9. Evolution of allatostatins The molecular evolution of the allatostatin precursor
in cockroaches has been analyzed recently using the gen- omic DNA sequences of the preproallatostatin precursor
for four species of cockroach Belles et al., 1990, as well as previously published sequences of two others
Donly et al., 1993; Ding et al., 1995. Phylogenetic analysis using parsimony revealed that the allatotropin
sequences in these species were generated through a pro- cess of duplication of a single gene, similar to the situ-
ation with the FMRFamides. This occurred before the divergence of these species from each other during evol-
ution and led to intragene families of peptides. A deter- mination of the physiological significance of such diver-
sity is the next step.
2.10. Neurotransmitter effects on corpus allatum activity
If functional allatotropinsallatostatins cannot be identified in certain cases, such as the lack of Manduca
allatotropin to stimulate larval glands, what else might regulate JH biosynthesis? Neurotransmitters are strong
candidates, since the axons of non-neurosecretory neu- rons also innervate the corpora allata. There is evidence
in both Manduca and Diploptera that neurotransmitters are regulatory factors, and this area of research deserves
further attention. Octopamine, a primary insect neuro- transmitter, was shown a decade ago to stimulate JH
biosynthesis in Locusta Lafont-Cazal and Baehr, 1988 and later was found to have the same effect in honey
bee adults Kaatz et al., 1994 and larvae Rachinsky, 1994. In Diploptera Thompson et al., 1990 and the
cricket Gryllus bimaculatus Woodring and Hoffmann, 1994, JH production is inhibited by octopamine. Dopa-
mine was subsequently found to affect JH synthesis in vitro by the corpora allata of adult female Blatella germ-
anica, and furthermore, its effect could be either stimu- latory or inhibitory, depending on the stage of the female
within the ovarian cycle Pastor et al., 1991. At the pre- vious JH conference, dopamine was reported to have an
effect on the corpora allata of Manduca sexta as well, and in a stage-specific manner during larval-pupal meta-
morphosis Granger et al., 1996. Early in the last larval stadium, dopamine stimulates both JH biosynthesis in
vitro and cAMP production. After day 2, when a small ecdysteroid peak was observed prior to the commitment
peak Wolfgang and Riddiford, 1986, its effect on both JH and cAMP production was inhibitory. The source of
dopamine affecting gland function has not yet been identified, but like the allatostatin, it has been found by
immunocytochemistry in both the brain and corpora allata Granger, unpublished information. When the
corpora allata were screened for neurotransmitter content by electrochemical detection HPLC Granger et al.,
1996; T.C. Sparks, personal communication, the only biogenic amine detected was dopamine.
There is one recent line of research that implicates dopamine in the regulation of larval development, poss-
ibly through an interaction with the corpora allata. Para- sitism of the armyworm, Pseudaletia separata, by the
wasp Cotesia kariyai elevates levels of dopamine in the hemolymph and nerve cord, slows normal development,
and delays pupation Noguchi et al., 1995. These effects are caused by a biogenic peptide which is elevated in the
hemolymph of the host Noguchi and Hayakawa, 1996. Although the mechanism by which pupation is affected
624 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 617–644
has not yet been elucidated, persistent levels of JH are known to delay pupation in Lepidoptera. Thus, it is poss-
ible that the elevated levels of dopamine may be affect- ing gland function. In the armyworm, Mamestra bras-
sicae, significantly higher levels of dopamine have been found in the hemolymph and central nervous system of
diapause-destined pupae
Noguchi and Hayakawa,
1997, and it would be of interest to know the primary site of dopamine action in this system as well.
2.11. Neurotransmitter receptors in the corpus allatum Dopamine exerts its effects at the cell membrane via
G protein-coupled receptors associated with cAMP Kababian, 1992. The stage-specific effects of dopam-
ine on JH biosynthesis by Manduca corpora allata are mirrored by dopamine effects on cAMP production
Granger et al. 1995b, 1996. This argues for 1 a direct effect of dopamine, rather than indirect via the release
of endogenous allatotropinsallatostatins, and 2 the existence of D1-like and D2-like dopamine receptors, the
members of these families of receptors stimulating and inhibiting cAMP production. The results of a pharmaco-
logical characterization of the dopamine receptors of the Manduca corpora allata were presented at this meeting
Granger et al., this symposium. On the basis of those results, it appears that the D2-like receptors bears con-
siderable similarity to those of vertebrates, while the D1- like receptors is unusual in its limited recognition of a
wide range of D1 and D2 receptor agonists and antagon- ists active in vertebrates. In this respect, the receptor is
strikingly similar to a D1-like receptor isolated and sequenced in Drosophila Gotzes et al., 1994; Sugamori
et al., 1995.
2.12. Second messenger systems In Diploptera, the effect of octopamine on corpora
allata activity is paralleled by a concomitant effect on cAMP levels Thompson et al., 1990, while diacylgly-
cerol and 1,4,5-inositol triphosphate appear to be involved in transduction of the allatostatin signal
Rachinsky et al., 1994. Mas-AT stimulates inositol phosphate IP production in both male and female adult
Manduca corpora allata Reagan et al., 1992, and it has been proposed that the allatotropin stimulates JH pro-
duction via an increase in 1,4,5-inositol triphosphate IP
3
, leading to an increase in intracellular calcium. High levels of intracellular calcium correlate with
biosynthetically active glands in Manduca Allen et al., 1992. Dopamine, by contrast, functions via cyclic AMP
as its second messenger Granger et al., 1996. The mechanism by which Mas-AS inhibits corpora allata
activity in Manduca remains conjectural. 2.13. Interrelationship between neuropeptides and
neurotransmitters in the control of the corpus allatum The possible interrelationship between a neuro-
transmitter and a neuropeptide in the control of JH biosynthesis is not a new observation, since it was shown
several years ago that both octopamine and Dip-AS down-regulate JH biosynthesis in Diploptera Thompson
et al., 1990; Stay et al., 1994. Mechanisms proposed for the inhibition of JH synthesis by octopamine in Diplop-
tera include roles as 1 a neuromodulator of JH syn- thesis, regulating ion channels or releasing allatostatin
from terminals within the corpora allata; and 2 a neuro- hormone directly affecting JH biosynthesis. Thus, the
relationship of dopamine to Mas-AS in inhibiting the corpora allata of Manduca larvae and adults is a critical
consideration in the regulation of gland activity in this species.
2.14. Other factors affecting JH biosynthesis Calcium is another factor impacting on the ability of
the corpora allata to synthesize JH, and considerable work has been done in the last decade demonstrating the
central and critical role of this ion in both Diploptera and Manduca Rachinsky et al., 1994. There is a need
for further information about the role of calcium in the transduction of neuropeptide and neurotransmitter sig-
nals to the corpora allata, as well as of other intracellular events that would modulate the response.
One way to modulate the response of the glands to regulatory ligands is by controlling the kind andor num-
bers of their receptors. Ecdysteroid receptors are known to exist in the nuclei of Manduca corpora allata
Bidmond et al., 1992, and there is preliminary evidence that ecdysteroids control the switch in the responsiveness
of the corpora allata to dopamine in Manduca, thus gov- erning the occurrence andor numbers of D1- and D2-
like receptors Granger et al., 1996. Thus, the ecdys- teroids are strong candidates to figure in the overall
scheme of the control of corpora allata function, but there are surprisingly few clues as to the mechanism by
which this class of insect hormone is involved. With this decade’s accumulation of information about ecdysteroid
receptors as well as transcription factors Henrich et al., 1999, the stage is set for the investigation of the poss-
ible control of corpora allata activity by ecdysteroids at the genomic level.
The ecdysteroid receptor is a heterodimeric complex of an ecdysteroid receptor protein EcR and ultraspira-
cle protein USP Yao et al., 1993; Thomas et al., 1993. While there have been no studies of the abundance of
EcR-USP complexes or of their isoform diversity in the corpora allata, R. Rybczynski, P. Moshitsky, S. Apple-
baum and L.I. Gilbert reported at this conference on the expression of EcR and USP gene products in the corpus
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