Vertebrate-type Hormones in Crustaceans : Localization, Identification and Functional Significance

17 Pages·1993·6.4 MB·English

by  Milton Fingerman

ZOOLOGICAL SCIENCE 10: 13-29 (1993) © 1993Zoological SocietyofJapan
REVIEW
Vertebrate-type Hormones in Crustaceans: Localization,
Identification and Functional Significance
Milton Fingerman, Rachakonda Nagabhushanam
and Rachakonda Sarojini
Department of Ecology, Evolution, and Organismal Biology,
Tulane University, New Orleans,
Louisiana 70118, U.S.A.
mine, are not treated herein because (a) they are
INTRODUCTION
soubiquitiousin the animal kingdom thatconclud-
In a 1983 review of the literature relating to ingwhetheroneormoreofthemisvertebrate-type
invertebrate neuropeptides Greenberg and Price or invertebrate-type does not seem justified and
[49] referred to invertebrates as having two sets of (b) their presence and roles in crustaceans have
neuropeptides. One set they called "native been extensively reviewed in a recent publication
neuropeptides." These are the ones that were from this laboratory [40]. Where information is
originally isolated from invertebrates and have availableconcerningpossiblephysiologicalrolesof
been shown to have specific physiological or these vertebrate-type compounds in crustaceans,
biochemicalrolesintheseanimals. Incontrast, the that information will be presented also.
compounds that Greenberg and Price called In this review we shall use "hormone" as it has
"naturalized neuropeptides" are those originally been defined by Norman and Litwack [88]. They
discovered in vertebrates and later identified in stated that "any substance that operates at the
invertebrates by various techniques, including cellular level, generated either externally or inter-
chromatography,radioimmunoassay,andimmuno- nally, whichconveys to thatcell a message tostop,
cytochemistry. At the time Greenberg and start, or modulate a cellular process will come
Pricewrotetheirreviewverylittle information had under the purivew of modern endocrinology."
been obtained about what the physiological and Also, Norman and Litwack divided hormones into
biochemical roles of these "naturalized" com- three classes, based on the distance of action: (a)
pounds might be, and formany ofthem theirroles endocrine hormones, which are the classical hor-
remain obscure. Peptidesarenowknown tobe the mones such as lutenizing hormone that travel to
largestclassofneuroregulatorycompoundsin both distant target cells; (b) paracrine hormones, such
invertebrates and vertebrates [92]. The object of as the classical aminergic neurotransmitters (e.g.
this reivew is to present the more recent evidence acetylcholine), which travel only a short distance
for the presence in crustaceans of not only verte- to neighboring cellular targets; and (c) autocrine
brate-typeneuropeptidesbutofallvertebrate-type hormones, such asprostaglandins, that are synthe-
peptidergic, proteinaceous, steroid, and lipid- sized and released by the same cell on which they
derived hormones. However, the classical neuro- act.
transmitters, such as norepinephrine and dopa- Invertebrate nervoussystems, includingthoseof
crustaceans, are useful models for analyses of
Received September 14, 1992 neuronal functioning, including peptidergic
14 M. FlNGERMAN, R. NAGABHUSHANAM AND R. SaROJINI
neurotransmission. Technical improvements in maenas revealed the presence of substances that
such techniques as high performance liquid chro- appeared to be methionine enkephalin, leucine
matography,peptidesequencingandradioimmuno- enkephalin,andmethionineenkephalin-Arg6-Phe7
assay have greatly enhanced our ability to identify [58]. Likewise, Fingerman et al. [39] showed by
the vertebrate-type hormones in invertebrate tis- immunocytochemistry the presence of leucine en-
sues, even when these compounds are present in kephalin-like and methionine enkephalin-like
extremely low concentrations. material in the eyestalk of the fiddler crab, Uca
pugilator. Immunoreactivity to both substances
was apparent in the retinularcells, laminaganglio-
ENDORPHINS
naris, sinus gland, optic peduncle, the three
chiasms and medulla terminalis. However, in the
Localization and identification X-organ of the medulla terminalis, immunoreac-
The presence of substances in the mammalian tivity against only the methionine enkephalin anti-
brain that have opiate-like activity was clearly bodies, but not leucine enkephalin, was seen.
established in 1975 when Hughes et al. [57] pub- Usingthe mantidshrimp,Squillamantis, Marinoet
lished the structures of two such endogenous al. [77] showed that the subesophageal ganglia
opioids, the pentapeptides methionine enkephalin contain a peptide that is opioid-like because it is
and leucine enkephalin extracted from the porcine able to displace D-Ala2-D-Leu5-enkephalin (DA-
brain. The name "endorphin" is often used, aswe DLE) from rat brain membranes in a receptor
have done here, for the entire class ofcompounds binding assay, and gives positive results in the
endogenous in mammals that have morphine-like standardguineapigileum assayforopioids. Inthis
activity [43]. Since 1975, endorphins have been test, opioids inhibit the twitch response of the
found in several non-nervous organs ofvetebrates electrically stimulated ileum. Furthermore, the
such as the gut, adrenal medulla, and pancreas action of this opioid-like substance is inhibited by
[43]. The firstdemonstration ofthe presence ofan naloxone, an opioid receptor blocker. This pep-
opioid in an invertebrate came from a study with a tide, through use of proteolytic enzymes, gave
locust, Locusta migratoria. Radioimmunoassays evidence that it is synthesized as part of a larger
(RIA) showed its nervous system contains both inactive peptide and subsequently released in the
methionine enkephalin-like and leucine enkepha- active form. Because this opioid does not coelute
lin-like substances [50]. With respect to crusta- with methionineenkephalinorleucineenkephalin,
ceans, the first opioid study was done in 1981 by nor does it cross-react with antibodies to
Mancillas etal. [75]. Through immunocytochemis- methionine enkephalin or give positive results in
try they found leucine-enkephalin-like immuno- RIA for methionine enkephalin, leucine enkepha-
reactivity in all the retinular cells of the spiny lin, orbeta-endorphin, Marino etal. suggestedthis
lobster, Panulirus interruptus. In addition, such peptide from Squilla mantis is a "native" neuro-
immunoreactivity wasalsoapparent in nerve fibers peptide. Piccoli etal. [91], also using the DADLE
in chiasm 3 that pass from the medulla terminalis receptorbindingassay, foundopioid-like DADLE
to terminate in the medulla interna. No immuno- displacement with extracts of brain from Carcinus
reactivity against antibodies of another opioid, maenas and brains plus dorsal organ from Squilla
beta-endorphin, was found. Later, other mantis, suggestive of the presence of opioid pep-
crustaceans were similarly studied. Jaros et al. tides. In an abstract, Dircksen etal. [27] reported
[58], using the shore crab, Carcinus maenas, that leucine enkephalin-like immunoreactivity, as
obtained immunocytochemical evidence for a seen by light and electron microscopic immuno-
leucine enkephalin-like substance in the eyestalk, cytochemistry, was apparent in the pericardial
i.e. inthesinusgland,laminaganglionaris, medulla organs and their segmental nerve connections to
externa, medulla interna, and medulla terminalis. the thoracic ganglia of Carcinusmaenas and in the
In addition, high performance liquid chromatogra- pericardial organs of the crabs Cancer pagurus,
phy (HPLC) of sinus gland extracts of Carcinus Portunuspuber, and Maja squinado.
Vertebrate-type Hormones in Crustaceans 15
Carcinus maenas has also been used in other peptidases in the hemolymph ofthe crayfish,Asta-
studies involving enkephalins. Amiard-Triquet et cus fluviatilis, that rapidly degrade leucine en-
al. [1] by immunofluorescence showed the pre- kephalin.
sence of methionine enkephalin-like material in Another opiate first found in mammals is called
the hepatopancreas (midgut gland) of this crab. /3-endorphin. It is a 31-amino acid polypeptide.
The immunofluorescent material was located Sephadex G-50 column chromatography of ex-
mainly basally in hepatopancreas tubule cells of tracts of the hepatopancreas of the red swamp
crabs taken from nonpolluted water. However, crayfish, Procambarus clarkii, revealed three
in crabs from a polluted site or those exposed to peaks of immunoreactive /3-endorphin-like mate-
Cd, Pb, Cu or Zn for 1-3 weeks the immuno- rial. These immunoreactive peaks were identified
fluorescence was located mainly apically in these by immunocytochemistry and RIA [52]. Cells of
tubule cells. No immunoreactivity was seen in the the hepatopancreas of this crayfish contain en-
lumina of these tubules, which suggests that this zymes capable of degrading /?-endorphin [123].
immunoreactive materialiseithernotsecretedinto
thelumenorlosesitsimmunoreactivityupon being Functions
A
secreted. Also with Carcinus maenas, Dircksen Behavior
[26] found by immunocytochemistry that leucine The first study ofa possible role of an opioid in
enkephalin-like immunoreactive axons are present any crustaceans was that of Maldonado and
in the pericardial organs, neurohemal organs that Miralto [74] with Squilla mantis. They found that
are cardiostimulatory. These immunoreactive the defensive response, rapid flexure ofthe abdo-
axons enter the pericardial organs via segmental men, which can be experimentally induced by an
nerves 1, 3 and 7. No colocalization of this electrical shock, can be modified by morphine.
material with proctolin, FMRFamide or crusta- This drug increases the threshold current required
cean cardioactive peptide was seen. More recent- to elicit the response. The loss of sensitivity is
ly, both methionine enkephalin and leucine en- dose-related. Naloxone blocks the action of this
kephalin were found in extracts of the thoracic opioid;coinjectionofmorphineandnaloxonedoes
ganglion of Carcinus maenas [73]. Their presence not result in a loss of sensitivity.
was established by RIA, HPLC and sequence In a more recent study of locomotor activity of
analysis of the opioids in thoracic ganglion ex- Gecarcinuslateralis, Martinezetal. [79] foundthat
tracts. An immunocytochemical studyby Rotheet when the crabs were first placed into the activity
al. [94] of the eyestalks of Carcinus maenas re- monitoringchamberslocomotoractivityincreased.
vealed immunoreactivity to both methionine en- FK33824, astable methionine enkephalin analog,
kepahlin and leucine enkephalin in the sinusgland markedly enhances this initial activity; but nalox-
(more leucine enkephalin than methionine en- one blocks this excitatory action of the opioid.
kephalin being present in this neurohemal organ); In studies with another crab, Chasmagnathus
only leucine enkephalin in the medulla terminalis, granulatus, Lozada et al. [72] found that this crab
lamina ganglionaris, medulla externa and medulla assumes a defensive posture, with both chelae
interna; and only methionine enkephalin in the extended and the body elevated, when an electric
chiasm between the medulla interna and medulla shock, 50 Hz, one second duration, and ofat least
terminalis. In addition, by HPLC analysis a sub- 8V, is given. Morphine produces a dose-
stance that coelutes with leucine enkephalin was dependent reduction in the sensitivty of this crab
identified in sinus gland extracts. to the electrical shock. When naloxone was co-
With another crab, the land crab, Gecarcinus injected with morphine no reduction in sensitivity
lateralis, Leung et al. [70] by HPLC and RIA occured, which suggests that opioid receptors are
analyses found a methionine enkephalin-like sub- indeed involved in this reduction of sensitivity to
stancein theeyestalk, and methionineenkephalin- theelectricshock. Inotherbehavioral studieswith
like and leucine enkephalin-like substances in the this crab [10, 117], it was found that a danger
brain. Colleti-Previero et al. [22] found three stimulus in the form ofa passing shadow elicits an
16 M. FlNGERMAN, R. NaGABHUSHANAM AND R. SaROJINI
escape response that habituates after repeated produce black pigment dispersion in both intact
stimulation, andthatthishabituationappearstobe Ucapugilator and in isolated legs [93]. Additional
mediated by endorphins. Support for this sugges- evidence for the involvement of opioid-like pep-
tion that habituation is the result of endorphin tidergicsubstancesincrustaceancolorchangeswas
release is the observation that after an habituation provided by Martinez et al. [80], who found that
session there is an analgesiceffectonthe defensive although injection of the stable methionine-
response of this crab to an electrical shock. enkephalin analog FK 33 824 alone into
B Pigmentary effectors Gecarcinus lateralis has no effect on the chroma-
Crustaceans have two types ofpigmentaryeffec- tophores, whereas coinjection of this analog with
tors: (a) chromatophoreswhich are responsiblefor eyestalk extract strongly potentiates the black pig-
color changes and (b) the retinal pigments, which ment-dispersingandredpigment-concentratingac-
control the amount of light impinging on the tions of the eyestalk extract. This potentiation is
rhabdom. Translocation of the pigments in the blocked by naloxone,
chromatophores and at least in the distal and (b) Distal retinalpigment
reflecting pigments is clearly regulated by neuro- Norepinephrine produceslightadaptationofthe
hormones, distal retinal pigment of Uca pugilator whereas
(a) Chromatophores dopamine induces dark adaptation [62, 64], pre-
The physiology of crustacean chromatophores sumably by stimulating release respectively of the
hasbeen reviewed recently [38]. In severalspecies light-adapting and dark-adapting neurohormones.
dual control of the chromatophores by pigment- Methionine enkephalin, like dopamine, produces
dispersing and pigment-concentrating neurohor- dark adaptation of this pigment, but leucine en-
mones has been demonstrated. In this laboratory kephalin has no effect on this pigment [63].
we have been particulary intersted in identifying C Bloodglucose
the neuroregulators that control the release of The crustacean hyperglycemic hormone (CHH)
crustacean neurohormones. In the fiddler crab, is found in the sinus gland. In several crustaceans
Ucapugilator, 5-hydroxytryptamine stimulates re- CHH release has been found to be triggered by
lease of red pigment-dispersing hormone while 5-hydroxytryptamine [40]. Recently Liischenetal.
norepinephrine stimulates release of black pig- [73] and Rothe et al. [94] provided evidence that
ment-dispersing hormone, and dopamine stimu- synthetic leucine-enkephalin, endogenous se-
lates release of red pigment-concentrating hor- quenced leucine-enkephalin isolated from thoracic
mone and black pigment-concentrating hormone. ganglia of Carcinus maenas, and purified leucine-
Methionine-enkephalin, but not leucine- enkephalin-like material (not yet sequenced) from
enkephalin, was found to stimulate black and red sinus glands of Carcinus maenas inhibit CHH
pigment concentration in intact Ucapugilator, but release. Injection into intact Uca puglilator of
not in isolated legs [93]. Methionine-enkephalin synthetic leucine-enkephalin or either the sinus
also stimulated the release of black and red pig- gland or thoracic gland material from Carcinus
ment-concentrating hormones from isolated eye- maenas results in a decrease in the hemolymph
stalks [13, 93]. The opioid antagonist, naloxone, glucose concentration. Also, eyestalksofCarcinus
blocked the effects of methionine-enkephalin in incubated in the presence of synthetic leucine
intact crabs and on isolated eyestalks. Methionine enkephalin release less than the basal level of
enkephalin,liketheclassicalaminergicneurotrans- CHH as compared with eyestalks incubated in
mitters, has no direct effect on these chromato- saline alone. Injection into intact fiddler crabs of
phores, as evidenced by the lack of effect on the naloxone prior to injection of leucine-enkephalin
chromatophores in isolated legs. Presumably, blocks the hypoglycemic action of this opioid. In
therefore, these compounds exert their effect only contrast, leucine-enkephalin does not affect the
indirectly, by stimulating release of the appropri- hemolymph glucose concentration in eyestalkless
ate specific chromatophorotropic neurohormone. fiddler crabs, which is consistent with the hypoth-
CHH
However, surprisingly, /^-endorphin was found to esis that the enkephalin isactingby reducing
Vertebrate-type Hormones in Crustaceans 17
output from the sinus glands in the eyestalks. immunoreactive to arginine vasotocin/arginine
vasopressin antibodies. Such neuronswere seen in
brains of the sea louse, Gnorimosphaeroma rayi,
HYPOTHALAMIC AND HYPOPHYSIAL
and the crab, Hemigrapsus sanguineus, but not in
HORMONES AND NEUROPHYSIN
the brain or subesophageal ganglion of Procam-
barus clarikii or the crab, Helice tridens. Mizuno
Localization and identification and Takeda [83] also looked for oxytocin-like
In what appears to have been the first effort to immunoreactivity in the nervous systems of these
identify a "naturalized" peptide in a crustacean, four crustaceans, but none was seen. In a pre-
Martin and Dubois [78] by use of an immuno- liminary study done earlier Takeda et al. [108]
fluorescence technique demonstrated the pres- examined only isopod central nervous systems by
ence of a somatostatin-like substance in the immunocytochemistry with these neurohypophy-
brain, subesophageal ganglion, and ventral nerve sial hormone antibodies. Arginine vasotocin/argi-
cord of the isopod, Porcellio dilatatus. However, nine vasopressin immunoreactivity was apparent
no localization of this somatotropin release- with Ligiaexotica, Porcellioscaber, andArmadilli-
inhibiting hormone-like material was apparent in dium vulgare, but no oxytocin immunoreactivity
the sinus glands. Van Herp and Bellon-Humbert was apparent.
[120], using the prawn, Palaemon serratus, pro- A human somatotropin-like substance was iden-
vided immunocytochemical evidence for neuro- tifiedbyRIAinthe hemolymph andextractsofthe
physin-like and arginine vasopressin-like sub- stomach and hepatopancreas ofPalaemon serratus
stancesintheorganofBellonci,acomponentofthe [113]. The amountsinthe three tissuesvariedwith
eyestalk. However, whereas the neurophysin-like the moltingcycle, being highest in all three tissues
material was present at all times, the vasopressin- during proecdysis.
like substance was present only at ecdysis. Later,
Van Deijnenetal. [119] did an immunocytochemi- Functions
cal study of the eyestalk of the crayfish, Astacus Few attempts have been made to identify possi-
leptodactylus, with antibodies against melanocyte- ble roles in crustaceans of hypophysial com-
stimulating hormone, vasotocin, and oxytocin. pounds. Sarojini et al. [99] found that oxytocin
The immunoreactivity they observed to antivaso- elvates the hemolymph glucose concentration in
tocin was the most widespread while that to anti- the freshwater crab, Barytelphusa cunicularis and
oxytocinwastheleastwidespread. Thesinusgland the prawn, Macrobrachium kistnensis. The gly-
reacts positively to antimelanocyte-stimulating cogen levels in the hepatopancreas, muscles and
hormone and antivasotocin. In addition, cells or integument of both species showed concomitant
fibersreactive: (a)toantivasotocinareinchiasms 1 decreases, which suggests that these glycogen
and 2, the medulla externa, medulla interna, the stores were hydrolyzed to provide the glucose that
medulla terminalis neuropil and X-organ of the appeared in the hemolymph. Later, Mattson and
medulla terminalis, (b) to antialpha-melanocyte- Spaziani [82] reportedthatlysinevasopressin, argi-
stimulating hormone are in the lamina ganglio- nine vasopressin, vasotocin, and oxytocin mimic
naris, chiasm 1, medulla externa, medulla interna the action of molt-inhibiting hormone, inhibiting
and X-organ of the medulla terminalis; and (c) ecdysteroid production by Y-organs of the crab,
antioxytocin in the neuropils ofthe medulla exter- Cancerantennarius. Charmantieretal. [17] found
na and medulla terminalis. In a related study, that larval and postlarval lobsters, Homarusamer-
Mattson and Spaziani [82], using arginine vaso- icanus, grow more rapidly when injected with
pressin antiserum in an RIA with eyestalk extract human somatotropin. This hormone did not bring
ofthecrab, Cancerantennarius, foundvasopressin- on precocious molting activity, but the specimens
like peptides are indeed present. that received the hormone became longer and
MizunoandTakeda[84]usedimmunocytochemi- weighed more than the controls. The hormone
stry to identify in several crustaceans neurons appeared to stimulate tissue growth without in-
18 M. FlNGERMAN, R. NAGABHUSHANAM AND R. SaROJINI
fluencing the water content of these test indi- tive to both. It is nowrecognized that both gastrin
viduals. and cholecystokinin belong to a single peptide
Zukowska-Arendarczyk [127] tested ovine folli- family called the gastrin/cholecystokinin (G/
cle-stimulating hormone and luteinizing hormone CCK) family. Notonlydogastrin andcholectysto-
onthesandshrimp, Crangoncrangon. Bothintact kinin function as vertebrate gastrointestinal hor-
and eyestalk ligated females were used. Eyestalk mones, but both are also present in vertebrate
ligation was used to eliminate any influence ofthe nervous systems, probably functioning there as
gonad-inhibitinghormone ofthe sinusgland inthe neurotransmitters. By RIA Larson and Vigna [67]
eyestalk. However, the responses when these showed the presence of G/CCK-like material in
gonadotropinswere injectedwere qualitativelythe the digestive tract of Cancer magister (the highest
same among the ligated and non-ligated indi- concentration occurring in the stomach), and also
viduals, although the ligated individuals did show in the digestive tractof Upogebiapugettensis. This
accelerated vitellogenesis and oocyte growth as materialwasnot detected inthe hepatopancreasof
compared with the non-ligated shrimp. Follicle- Cancernorin the digestive tractsoftwo barnacles,
stimulatinghormonecausesthenumberofsomatic Balanus sp. and Pollicipes polymerus. G/CCK-
cellsintheovarytoincrease butdoesnotaffectthe like material of Cancer magister was analyzed by
oogonia or oocytes. In contrast, luteinizing hor- Sepahdex G-50 chromatography [66] from the
mone affects only the germinal epithelium; the stomach, hemolymph and carapace. Three
oogonia increasing in number and size and the immunoreactive molecular forms of G/CCK-like
oocytes undergoing accelerated vitellogenesis, but peptides were found.
the somatic cells are not affected. So, both hor- Scalise etal. [102], using immunocytochemistry,
mones stimulate this ovary, but in different ways. provided supporting evidence for the presence of
Although chorionic gonadotropin is not an G/CCK-like material in the stomach of Cancer
hypophysial hormone, in mammals it isfunctional- magister. This material appears to be synthesized
ly similar to luteinizing hormone, and this for this in the gastricepithelial cells and then secreted into
reason is treated here. Bomirski and Klek- the procuticle which lines the stomach lumen.
Kawihska [9] found that human chorionic gonado- Bellon-Humbert et al. [6] reported that by
tropin stimulates oogenesis in Crangon crangon. immunocytochemistry and with an antibody to
Similarly, Souty and Picaud [106] found that hu- cholecystokinin 8 they observed that neurosecre-
man chorionic gonadotropin stimulates vitel- tory cells in the medulla externa and medulla
logenesis by the fat body in the isopod, Idotea terminalis X-organ of Palaemon serratus are
balthica. Maruo et al. [81], using RIA and a immunoreactive to this antibody.
radioreceptor assay, found a human chorionic Van Deijnen et al. [119] who, as mentioned
gonadotropin-like substance in the stomach and above, used a wide variety ofmammalian antisera
hepatopancreas of the lady crab, Ovalipes ocella- to test for "naturalized' peptides in eyestalks of
tus. However, when these investigators tested this Astacus leptodactylus found immunoreactivity
substance from thecrabstomachin amouseuterus against a gastrin (C-terminal) antibody in the
bioassay, this substance exhibited no biological medulla externa nd medulla terminalis X-organ.
activity. They [119] also used an antibody to cholecystoki-
nin that was raised against the middle portion of
the molecule which does not crossreact with gas-
GASTRIN/CHOLECYSTOKININ
trin. This anticholecystokinin revealed the pre-
sence of reactive cells in the lamina ganglionaris,
Localization and Identification medulla externa, medulla interna, medulla termi-
Gastin and cholecystokinin are peptides that nalis (both in the X-organ and the rest of the
have the same five amino acids at their carboxyl ganglion), and also in the sinus gland. By im-
end [68]. Consequently, antisera raised against munofluorescence and RIATurrigianoand Selver-
eitherofthese peptidesgenerallyare immunoreac- ston [114] showed G/CCK-like material is in the
Vertebrate-type Hormones in Crustaceans 19
input nerve and neuropil of the stomatogastric does not stimulate secretion of either gastric acid
ganglion, commissuralganglia, brain andeyestalks orpancreaticamylasein the rat. Thissuggeststhat
Panulirus interruptus. the crab material while sufficiently like vertebrate
Favrel et al. [37], by immunocytochemistry, G/CCK to react with the antisera is nevertheless
showed the presence of G/CCK-like peptides in sufficientlydifferent so asto be incapable ofelicit-
the eyestalk andstomach ofPalaemonserratus. In ing a response in the rat. Working with Palaemon
the eyestalk, cells in the medulla externa and serratus, Favrel and Van Wormhoudt [36] found
medulla terminalis X-organ and the sinus gland that injection of gastrin 17 results in increased
were labelled. In the stomach, epithelial cells and incorporation ofleucine into proteins ofthe hepa-
the cuticle were labelled also. Like with Cancer topancreas.
magister, Sephadex G-50 fractionation of eyes- Turrigiano and Selverston [114] found that elec-
talks, stomach and hemolymph ofPalaemonserra- trical stimulation of the stomatogastric input
tus revealed multiple molecular forms ofG/CCK- nerve, which contains G/CCK immunoreactive
like material. The quantity of G/CCK-like pep- fibers, results in the release of G/CCK-like pep-
tides, as measured by RIA, in the eyestalks tide into the stomatogastric ganglion of Panulirus
showed no significant variation with the molting interruptus. Also, application of cholecystokinin
cycle, butinthe hemolymph theconcentrationwas to this ganglion results in increases in the spike
highest, duringlateproecdysis,justbeforeecdysis; frequency and number of spikes per burst of the
and for the stomach concentration peaks were pyloric rhythm and also activates the gastric mill.
apparent during postecdysis and during proecdy- In view of these results, these investigators sug-
sis. Van Wormhoudt et al. [121], using shrimps, gested that in this lobster G/CCK-like peptide
Penaeusjaponicus and Penaeusstylirostris, and an functions as a neuromodulator in the stomatogas-
RIA for G/CCK-like peptides found, after frac- tric ganglion. When Penaeus japonicus and
tionation on a Sephadex R G-50 SF column, four Penaeus stylirostris are fed, the amount of G/
molecular forms of these peptides in the CCK-like material in their hemolymph increases
hemolymph of each shrimp. The amount of each [121]. This increase indicates that these peptides
form varies with the species. Four G/CCK-like may help regulate the digestive process. Also, in
peptides that cross-reacted with a specific C- Panulirus interruptus the hemolymph concentra-
terminal G/CCK antiserum were also isolated tionofG/CCK-likepeptideincreasesafterfeeding
from extracts of the stomach of the lobster, (up to fourfold), and the effect lasts about four
Nephrops norvegicus, by Favrel et al. [35]. Their hours [115]. Concomitant with this increase is an
molecular weights are in the range 1000-2000 increaseingastricmill activity. Also, thecholecys-
daltons. These four peptides show very weak tokinin antagonist, proglumide, when injected
crossreactivity with human gastrin, about 0.03%. within the first two hours after feeding, causes a
Amino acid analysis showed none of these pep- prolonged decrease in gastric mill activity.
tides has the same five terminal amino acids at the Sedlmeier and Resch [103] tested the efficacyof
carboxyl end as human gastrin and cholecystoki- gastrin and cholecystokinin in vitro, using the
nin, these five being necessary for full im- hepatopancreas of the crayfish, Orconectes limo-
munoreactivity. Instead they had only three or sus. Both gastrin and cholecystokinin were found
four of the requisite amino acids. to stimulate release of amylase and proteases into
the incubation medium. Favrel et al. [35], using
Functions three of the four G/CCK-like peptides they iso-
Because of the established roles of gastrin and lated from extracts of the stomach of Nephrops
cholecystokinin as regulators of digestion among norvegicus, found that two of the three stimulate
vertebrates, studies of the possible roles of G/ protease secretion in vitro from the hepatopan-
CCK-likepeptidesincrustaceanshavecenteredon creas of Orconectes limosus.
similar functions. Larson and Vigna [66] reported
that G/CCK-like material from Cancer magister
20 M. FlNGERMAN, R. NAGABHUSHANAM AND R. SAROJINI
muscleofthislobsterin vitro, butgutextractshave
INSULIN
no such effect. However, injection of these in-
sulin-like peptides from the hepatopancreas does
Localization and identification not affect the glucose concentration in the
Although early studies [41, 53, 69] showed that hemolymph of Homarus americanus [98]. High
injected mammalian insulin does not affect the glucose concentrations in the hemolymph did not
hemolymph glucose concentration in crustaceans, stimulate release of insulin-like peptides into the
interest in the possible presence and roles of hemolymph, nor did hepatopancreas extracts
insulin-like peptides in crustaceans continues. speed the rate of glucose clearance from the
Davidson et al. [25] reported that the hepatopan- hemolymph. Thesefindingsareconsistentwiththe
crease ofCarcinusmaenas showsinsulin-like activ- earlierresultsfrom several laboratories mentioned
ity in a mouse diaphragm glycogen synthesis test, above [41, 53, 69] that mammalian insulin has no
and that some cells in the hepatopancreas, but not effect on the hemolymph glucose concentration in
in the gut, of Carcinus maenas and Homarus crustaceans. This crustacean insulin-like material
gammarushave stainingcharacteristicsofmamma- presumably functions to regulate glycogenesis in
lian insulin-secretory pancreatic islet beta cells. muscle cells, but has no role in hemolymph gluco-
But Davidson et al. [25] cautioned about hastily stasis. Insulin does not stimulate release of amy-
drawing firm conclusions when using light micro- lase or proteinase from the isolated hepatopan-
scopic staining features and histochemical staining creas of Orconecteslimosus [103]. Consistentwith
reactionsdevisedformammaliancellson tissuesof the results ofSanders [96, 97] are the observations
invertebrates. Likewise, Falkmer [34] had earlier ofBaker and Carruthers [4, 5] that porcine insulin
found in a study with the use of light microscopy stimulates glucose transport into muscle cells of
that such insulin-producing cells are absent from the barnacle, Balanus nubilis. Baker and Car-
the gut ofthe blue crab, Callinectes sapidus, also. ruthers also found with these muscle cells that
Sanders [96], using an RIA and guinea pig insulin lowers the cytosolic ionized calcium level,
anti-insulin serum showed that the hepatopan- reduces the cyclic AMP content and increases the
creas, gut andhemolymphofHomarusamericanus cyclic GMP content. These observations are con-
contain insulin-like peptides. The hepatopancreas sistent with the earlier observations ofBittar etal.
containsthehighestconcentration ofimmunoreac- [7] that insulin stops sequestration ofsoldium ions
tive material while the eyestalk has none. Frac- in muscle cells of the barnacles, Balanus nubilus
tionation of hepatopancreas, hemolymph and gut and Balanus aquila, and also increases the activity
on a Sephadex G-50 column revealed molecular of the guanylate cyclase system. Insulin injected
heterogeneity of the insulin-like material. The into these cells produces an increase in the rate of
hepatopancreas contains the highest proportion of sodium efflux and injection of guanosine triphos-
highmolecularweightinsulin-likematerial, thegut phate into muscle cells pre-exposed to insulin also
and hemolymph having relatively more of the produces an increase in sodium efflux.
lower molecular weight insulin-like material. In
view of these results, Sanders [96] hypothesized
SUBSTANCE P
that a large insulin-like peptide is synthesized in
the hepatopancreas, secreted into the gut where it
is broken down to smaller units, reabsorbed into Localization and identification
the hepatopancreas and then secreted into the In the vertebrate central nervous system sub-
hemolymph. stance P apparently serves as a neurotransmitter
that mediates sensory input, e.g. transmission of
Functions pain impulses to the brain. By immunocytoche-
Sanders [97], using Homarus americanus, found mistry substance P-like material was found in the
that bovine insulin, hepatopancreas extract, and eyestalk of Panulirus interruptus [75]. This im-
hemolymph induce glycogenesis in abdominal munoreactivity material is in the lamina ganglio-
Vertebrate-type Hormones in Crustaceans 21
naris, medulla externa, medulla interna, medulla
CALCITONIN and CALCITONIN
terminalis (in the X-organ aswell asthe restofthis
GENE-RELATED PEPTIDE
ganglion) and sinus gland. In eyestalks of Uca
pugilator, substance P-like immunoreactivity was
seen in the retinular cells, lamina ganglionaris, Localization and identification
medulla externa, medulla interna, sinus gland and Calcitonin is asmall polypeptide secreted bythe
optic peduncle [39]. ultimobrachial glands in jawed fishes, amphibians,
Goldberg et al. [46, 47] by immunocytochemis- reptilesandbirds; andinmammalsbytheso-called
try identified cells immunoreactive to substance C cells of the thyroid gland. As a hormone,
P antibody in the stomatogastric nervous systems calcitonin produces hypocalcemia in vertebrates.
ofPanulirus interruputs, Homarus americanus and Calcitonin is also present in the vertebrate central
Cancer borealis. In these three decapods the nervous system, presumablyfunctioningthere as a
neuropil ofthe stomatogastric ganglion is strongly neuromodulator. The gene that codes for calcito-
immunoreactive, but none of the stomatogastric nin also produces another polypeptide, called cal-
ganglion somata showed any immunostaining. citonin gene-related peptide (CGRP). This sub-
Also, in the connective ganglia some somata and stance is present in central and peripheral nervous
the neuropil were positively immunoreactive. systems of vertebrates, where it appears to func-
Marder [76] notedthatthestomatogastricganglion tion as a neuromodulator, and in several other
of Cancer irroratus shows the same pattern of regionsofthevertebratebody, includingtheheart,
substance P-like immunoreactivity as do the spleen and urogential tract as well [89].
stomatogastric ganglia in these other three de- Bellon-Humbert et al. [6] by use of immuno-
capods studied by Goldberg et al. [46, 47]. cytochemistry and antibodies against human, pig
Sanderman et al. [95] by immunocytochemistry and salmon calcitonins demonstrated the presence
demonstrated substance P-like immunoreactivity ofacalcitonin-like substance in the medullatermi-
in cells of the brains of the crab, Leptograpsus nalis X-organ of Palaemon serratus. Arlot-
variegatus, and the crayfish, Cherax destructor. Bonnemains et al. [3] by RIA with anticalcitonin
Four large neurons in the brain of each of these antibodies from salmon showed the presence of a
crustaceans are immunoreactive, two in the proto- calcitonin-like polypeptide in the hemolymph of
cerebrum and two in the deutocerebrum. These Palaemon serratus. The amount of calcitonin-like
paired immunoreactive protocerebral cells are, material in the hemolymph varieswith the molting
more specifically, in the dorsal cell clusters that lie cycle, beinghighestduringpostecdysis, and lowest
on each side of the median protocerebrum. Each during intermolt The hemolymph calcium level,
of these cells extends through the ipsilateral and however, was maximal during proecydsis and
contralateral olfactory lobes to terminate among lowest during postecdysis.
the lateral somata ofthe olfactory lobe, not in the Using RIA, a radioreceptor assay and anti-
neuropil. The cellsfrom each side ofthe brain are salmon and antihuman calcitonin antibodies, Fun-
mirror images of each other. Each deutocerebral chereau-Peron et al. [42] found a calcitonin-like
cell runs only ipsilaterally, terminating within the peptide in the eyestalks of Nephrops norvegicus,
neuropil ofthe olfactory lobes. Homarus americanus, Palaemon serratus, and
Penaeus vanamei. In addition, other organs of
Functions Nephrops were examined. The hepatopancreas of
Substance P was found to increase the amounts this lobster contains a higher concentration of
ofamylase andproteasesreleasedin vitrofrom the calcitonin-like peptide than any of the eyestalks
hepatopancreas of Orconectes limosus [103]. examined from these four species. The foregut of
Nephrops also contains a high concentration, like-
wise higherthaninthese eyestalks, butlessthanin
the hepatopancreas. This calcitonin-like material
of Nephrops that was detected by the antisalmon
22 M. FlNGERMAN, R. NaGABHUSHANAM AND R. SaROJINI
calcitonin antibodies exists in two forms. One has kilodalton protein with an amino acid composition
a molecular weight of 4500 daltons, somewhat very similar to those ofthe 17.5 kilodalton human
higherthanvertebratecalcitonins. Salmoncalcito- calcitonin precursor and the 22 kilodalton calcito-
nin is 3432 daltons, and human calcitonin is 3418 nin-like molecule isolated earlier from the
daltons. However, in addition to this 4500 dalton hemolymph and hepatopancreas ofNephrops nor-
molecule, a higher molecular weight calcitonin- vegicus by Van Wormhoudt and Fouchereau-
like substance was also found. The latter was Peron [122]. However, in Callinectes there is also
found by gel filtration to have a molecular weight some immunoreactive material of relatively low
greater than 5,000 daltons. molecular weight. These high molecular weight
Graf et al. [48], using extracts of an entire substancesof22and27.2 kilodaltons likelycorres-
amphipod, Orchestiacavimana, in an RIAfound a pond to the one found by Funchereau-Peron etal.
calcitonin-like substance, the concentration of [42] in Nephrops that was larger than 5,000 dal-
which changes with the molting cycle. The maxi- tons, whereas the low molecular weight material
mum amount is present at ecdysis, while the least from Callinectes likely corresponds to the 4,500
is found early in intermolt. The concentration dalton calcitonin-like compound found by
increases markedly late in proecdysis. The least is Fouchereau-Peron et al. in Nephrops [42].
seen early in intermolt.
Arlot-Bonnemains et al. [2] with an RIA and Functions
human anti-CGRP antibody found immunoreac- Therolesofcalcitonin andCGRPin crustaceans
tive material in several tissues ofNephrops norve- have not been examined extensively. In a studyof
gicus, includingthe hepatopancreas, foregut, eyes- Palaemon serratus by Arlot-Bonnemains et al. [3]
talk, brain andheart. Ofthe tissuesexamined, the they found that injection of salmon calcitonin
hepatopancreas has the highest concentration of during preecdysis does not alter the hemolymph
this CGRP-like material, followed by the foregut, calcium concentration. Sellem et al. [104] investi-
whichledtheseinvestigatorstosuggestthe CGRP- gated the effect of salmon calcitonin on the con-
like substance may function in crustaceans as a centrationofcalcium inthe hemolymphofOrches-
regulator of gastrointestinal function. tia cavimana. They found that the calcium level
Sasayama et al. [100] demonstrated by im- decreased when the calcitonin was injected during
munocytochemistry with antibodies to salmon cal- intermolt and early proecdysis but had no effect
citonin and rat CGRP the presence of cells im- during late proecdysis or duringpostecdysis. Nor-
munoreactive to both antibodies in the central mally, the hemolymph calcium level is high during
nervous system ofArmadillidium vulgare. CGRP proecdysis because ofcalcium resorption from the
immunoreactive cells are present in the brain and old cuticle, and is low during intermolt. As
circumesophageal connectives while calcitonin mentioned above, Graf et al. [48] found that
immunoreactive cells are in the brain only. More calcitonin-like material is at its lowest concentra-
recently, Cameron andThomas [14] byuseofRIA tion in Orchestia cavimana at the start ofintermolt
showed the presence of salmon calcitonin-like and increases rapidly during proecdysis to peak at
material in several tissues, including the hepato- ecdysis. So, the exogenous salmon calcitonin was
pancreas, brain, heart and hemolymph ofthe blue effective when the endogenous calcitonin-like
crab, Callinectes sapidus. The hepatopancreas activity was low, but ineffective when the endog-
containsthe highestconcentrationofimmunoreac- enous activity was high.
tive material. The concentration of immunoreac-
tive material in the hemolymph varies with the MISCELLANEOUS PEPTIDES
moltingcycle, beinghighest duringproecdysis, but
the calcitonin-like material in the other tissues
does not vary with the molting cycle. The im- Localization and identification
munoreactive material in the hemolymph and A few "naturalized" peptides have been the
hepatopancreas consists to a great extent ofa 27.2 subject of only one or two investigations. These