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Hormones and excretion in locusts
GENERAL
APiD
COMPARATIVE
ENDOCRINOLOGY
HYDROMlNERAL
3, 289-298 (1972)
SUPPLEMENT
REGULATION Chairman-K.
Hormones
and
H-4 ANIMLS.
PART
G. DAVEU
Excretion
in Locusts
W. MORDIJE Depwtment
of
Zoology London,
und
Applied
Entomology,
Imperictl
College,
S. W. 7, United Kingdom
Normones present within the cerebral neurosecretory cell-corpus cardiacum complex exert both diuretic and antidiuretic effects upon the excretory system. The Malpighian tubules and rectum respond to the diuretic hormone, which increases fluid secretion by the tubules and reduces rectal reabsorption. The antidiuretic hormone increases rectal reabsorption but has little elect upon tubule function. Feeding is of prime importance in regulating the release of t’he diuretic hormone; the control of release of antidiuretic hormone is not yet understood. Locusts remain in water balance under a variety of environmental conditions by the regulation of their fecal water content. This regulation is exercised by the diuretic and antidiuretic hormones. The diuretic hormone may increase fluid secretion by Malpighian t,ubules by increasing the intracellular levels of cyclic 3’,5’-AMP. The nature and number of hormonal factors present in the cerebral endocrine system and the ventral nerve cord are discussed.
It is now well established that excretory and their interrelationships difficult to rlefunction in insects is under hormonal con- solve, a better understanding may come trol. In locusts the hormones involved are from looking at the actions of the diuretic released from the cerebral neurosecretory and antidiuretic hormones upon the Maicell-corpus eardiacum complex, and possibly pighian tubules and rectum separately. Once also from the neurosecretory organs present these different hormonal actions have been within the medial nervous system (Mordue, examined: their integrated effect in producing a flexible regulatory system, capable 1966,1967,1969,1970a; Cazal and Girardie, 1968; de Bessb and Cazal, 1968). An anal- of dealing with both water loading and ysis of the hormonal regulation of ex- water conserva,tion, should be made clear. cretion and water balance is made difficult by the facts that more than one type of 1, The Mazpighian tubu1es It has been known for some time that hormone is involved and that these can effect quite different target organs. A num- destruction of the medial neurosecretory ber of factors are involved in water balance cells of the pars intercerebralis produces a in locusts (Loveridge, 1967), the most im- marked increase in the blood volume of the locust (Girardie, 1964, 1970; Highnam et nZ. portant of which is fecal water loss (Norris, 1961). The Malpighian tubules and rectum 1965; Mordue, 1966). This operation is are primarily involved in regulating fecal strongly suggestive that a factor, which water, but other regions of the gut may possessesstrong diuretic activity, is proalso be involved in overall water balance duced by the neurosecretory cells. In viva measurements of excretion in both Locz~dn; (see also Berridge, 1970) . and Xchistocerca show that excretion is Since the roles played by the different hormones and target tissues are complex very much reduced following removal of 289
@ 1972 by Academic
Press, Inc.
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MORDUE
the neurosecretory cells (Mordue, 1966, 1967, 1969). This low level of excretion can be elevated by injections of corpus cardiacum extracts; the active principle is restricted to the storage lobe of the gland (Mordue and Goldsworthy, 1969). The storage lobe of the gland contains neurosecretory factors produced by the cerebral Extracts of the neurosecretory cells. glandular lobe of the corpus cardiacum have little effect upon secretion through the Malpighian tubules: there is a slight increase in the rate of secretion (Mordue and Goldsworthy, 1969). Corpus cardiacum hormones affect fluid secretion through the tubules in viva (Table 1) and also increase the rate of excretion of compounds such as urate (Mordue, 1969). This evidence for the prime importance of neurosecretory hormones in regulating tubule function is from studies on intact insects. However, using in vitro preparations, Cazal and Girardie (1968) have demonstrated in Locusta that a factor is present within the corpora cardiaca which reduces the rate of excretion through the Malpighian tubules. In locusts which have been deprived of their neurosecretory cells for a number of days, and thus have no diuretic hormone, the rate of excretion through the tubules is very slow and undoubtedly represents a basal secretory rate (Mordue 1966, 1969). Since the basal rate of secretion is so slow and the breakdown of the diuretic hormone so rapid (Sect. 3)) a factor whose principal action is to reduce TABLE RATES
OB FLUID
TUBULES
Starved Starved, then fed for 2.5 hr Starved + 0.5 pair corpora cardiaca Starved f 1.0 pair corpora cardiaca Starved (data from Phillips, 1964)
1
SECRETION
BY MALPIGHIAN
IN
LOCUSTS
INTACT
No.
Fluid secretion (/.J/hr * SE)
6 5
7.5 27.5
&- 0.7 f 1.7
5
14.2
+_ 0.8
5
23.0
f
1.0
9
7.7
*
1.2
tubule secretion may not be necessary. In their work, Cazal and Girardie (1968) have demonstrated the presence of a diuretic factor within the neurosecretory cells of the pars intercerebrales, which increases tubule secretion. Using whole corpora cardiaca they were unable to demonstrate the presence of this diuretic factor within the corpora cardiaca. Either the diuretic factor is absent or its effect is masked by the strong antidiuretic action of the whole gland. With in vivo measurements, brain extracts have no marked effect upon tubule function (Mordue and Goldsworthy, 1969) ; the diuretic effects of the pars intercerebralis can only be demonstrated in locusts which have been deprived of their neurosecretory cells for some time. The rates of fluid secretion in isolated preparations are much lower than those in intact animals. Phillips (1964), using a cannulation method, measured fluid secretion by Malpighian tubules in starved Schistocerca (the lack of food restricted contamination of the hindgut fluid by midgut fluid) at some 8 &‘hr. It is known that excretion in starved locusts occurs at a very low rate; indeed the rate is not much above that in animals without their neurosecretory cells (Mordue, 1966, 1969). Table 1 shows the rates of fluid secretion through tubules in cannulated in uivo preparations of starved Xchistocerca. The rates of secretion are in very close agreement with those of Phillips (1964). If the starved locusts are allowed to feed, there is a dramatic increase in the secretion of fluid by the Malpighian tubules. It is well established that feeding brings about the release of the diuretic hormone (Sect. 5). Moreover, a similar increase in fluid secretion occurs following the injection of extra,cts of corpora cardiaca, and this increase in fluid secretion is dose dependent (Table 1). Measurement of fluid secretion in normal animals is made very difficult by the flow into the hindgut of fluid from the midgut, as well as from the Malpighian tubules. However, in some maturing females, successful noncontaminat.ed measurements of fluid secretion have been made, and were as high as 3540 &‘hr. These maturing
EXCRETIOI’G
locusts have very active neurosecretory systems (Highnam and Luntz, 1970) and high rates of excretion as measured by amaranth excretion (Mordue, 1966, 1969). Any attempts to tie off the midgut. from the hindgut to prevent contamination by midgut fluid involve serious interference with the Malpighian tubules. The physiological actions of neurosecretions present within the ventral nerve cord are, for the most part, &ill obscure. de Bessb and Cazai (1968) have shown that a factor is present in extracts of the ‘(organes pbrisympathiques,” which reduces secretion by the NIalpighian tubules. However, the extent of the reduction in tubule function is not given, nor as yet is there any information concerning dose-response relationships of this antidiuretic factor. This attempt to show the physiological function of these ventral nerve cord neurosecretions is necessary, and is a worthwhile attempt to establish a link between cytological and physiological evidence for the role of the neurosecretory system of the ventral nerve cord. 2. Rectd
Function
The physiological mechanisms involved in rectal function in locusts are now well understood, as a result of the work of Phillips (1964, 1970), but some confusion st’ill exists as to the role played by hormones in the regulation of the secretory and reabsorptive mechanisms of the rectum. In Locusta and EW&tocercu, diuretic and antidiuretic hormones are present within the corpora cardiaca. Both hormones affect the rectum (Mordue, 1966, 1969, 1970a; Cazal and Girardie, 1968). Analysis of rectal function in viva is technically difficult and in consequence most work has been carried out using in. vitro preparations. In isotonic saline, inverted rect#a are capable of secreting some E-17 ~1 of fluid/hr from the recta lumen into the hemolymph (Mordue, 1966, 1969, 1970a; Phillips, 1970). This basal secretory rate can be altered by corpus carclia,cum factors. Extracts of the storage lobe of the corpora cardiaca, reduce rectal reabsorption; this reduction is dose dependent, but even at high concentrations
IN
LOCUSTS
291
of hormone the rectum still allows some fluids to pass into the hemolymph ( due 1969, 197Oa). The work of Cazal and Girardie (1968) ascribes only an antidiuretic effect to extracts of corpora cardiaca. The presence of this antidiuretic factor within the corpore cardiaca has been confirmed (Morduc, 1970a). In this latter study the effects oi whole corpora cardiaca and the separatec! storage and glandular lobes were analysed. The diuretic hormone, as outlined in Sect. 1, is effective in storage lobe extracts from both Locka and Schistocema. The a&i-, diuretic component is restricted to the glandular lobe and is more potent in Loczlsta than in Xchistocerca (Mordue, 197Oa). In Locusta whole corpora cardiaca exert an antidiuret.ic effect upon rectal function; whereas whole glands from Schistocercn exert a diuretic effect (Mordue, 1970a). Whole corpora rardiaca from the t,wo species have different quantitat.ive effects upon excretion through the Malpighian tubules (Mordue, 1967, 1970a). Antidiuretic effects of extracts of the neurohemal organs of the ventral nerve chain upon isolated recta> have been described (de Bess& and Canal, 1968; Cazal and Girardie, 1968). The quantitative data provided by Cazal and Girardie suggests that this antidiuretic effect is much weaker than that produced by the corpora cardiaca. However, it seems likely that the volume of neurosecretory substance present within the ventral nerve cord is much less than the amount present within the corpora cardiaca. The antidiuretic hormone of the ventral nerve cord may well be as potent as that found within the corpora cardiaca. 3. Hornzone Turnover Dose-response relat8ionships have been established for the diuretic hormone released from the corpora cardiaca of locusts (Mordue, 1966, 1969; Mordue and Goldsworthy, 1969). There exists a straight-line relat’ionship between increased excretion end the amount of corpus cardiacum hormone within the hemolymph. The lowest toncentration at which consistent and measurable increases in excret,ion can be obtained is at
292
W.
MORDUE
levels of 0.04 glands/100 ~1 of hemolymph. In this work the corpora cardiaca used were taken from mature male locusts. Extracts of corpora cardiaca from immature locusts contain less diuretic hormone (Table 2). The variation in hormonal content of the neurosecretory complex with age, conditions of rearing, and stage of development is an important factor which must be considered when data from different groups of workers is compared. At present, it is impossible to define, with any degree of exactness, the hormonal complement of whole corpora cardiaca. The work of Cazal and Girardie (1968) well illustrates this point. They were able to demonstrate differences in the hormonal content of corpora cardiaca of locusts reared under different conditions of hydration and dehydration. Furthermore, injection of hemolymph from mature female locusts into locusts deprived of their neurosecretory cells produces an increase in excretion through the Malpighian tubules. However, blood from immature locusts hams little effect (Table 2). The responses of isolated rectal preparations to hormone-rich extracts also demonstrate the considerable variation in the amounts of hormone present within the neurosecretory complex of animals at different stages of development or in different states of hydration. Preliminary work with Locusta and Schistocerca suggests that under normal rearing conditions, the corpora cardiaca of immature and mature locusts contain approximately the same amount of antidiuretic hormone. Thus the TABLE
relative amounts of diuretic and antidiuretic hormones can differ markedly during development, and in some instances the amounts of hormones themselves may also alter. At the present time there is no evidence as to the levels of antidiuretic hormones within the locust hemolymph. In cockroaches these levels have been estimated (Mills and Nielsen, 1967). However, in other insects such as Rho&Gus and Carausius (Pilcher, 1970) there seems to be little variation in the diuretic complement of the appropriate endocrine glands. The hemolymph does, however, show marked variations in the amount of diuretic hormone present. The circulatory levels of diuretic hormone released from the corpora cardiaca in locusts have been measured and estimates made of the rate of breakdown in intact locusts (Mordue, 1966, 1969). In viva measurements of the titer of diuretic hormone in locusts show that it can vary between 0.040.4 pairs of corpus cardiacum-equivalents/l00 ,~l of hemolymph. These circulatory levels necessary to elicit diureisis in Malpighian tubules are very similar to the levels needed in Dysdercus (Berridge, 1966) and Carausius (Pilcher, 1970). In locusts, the circulatory levels of the diuretic hormone are altered by a large ra#nge of environmental stimuli which influence the activity of the cerebral endocrine system (Mordue et al. 1970). In locusts, breakdown of the hormone is rapid, and a sustained rate of excretion is only possible if there is continuous release of the diuretic factor. An amount of corpus cardiacum 2
THE EFFECTS OF CORPUS CARDIACUM EXTRACTSAND HEMOLYMPHFROM IMMATURE AND MATURE FEMALES UPON THF: EXCRETORYSYSTEMSIN IMMATURE FEMALE Schdocerca DEPRIVED OF THEIR NEUROSECRETORYCELLS FOR 5 Daysa Amaranth excreted (70 zk SE)
Donor Control (cauterised Immature females 0.2 pair corpora hemolymph, 75 Mature females 0.2 pair corpora hemolymph, 75 a At least
females)
Rectal reabsorbtion Whr 4 SE)
30 * 3
cardiaca ~1
54 t- 2 40 & 5
1 pair corpora
cardiaca
cardiaca ~1
75 * 2 45 i: 3
1 pair corpora
cardiaca
5 preparations
per test..
16.5
k 1.6
13.0
+ 1.3
5.0
+ 0.8
EXCRETION
hormone equivalent to 0.2 gland disappears from the hemolymph within l-l.5 hr. Obviously since there is a store of hormone within the corpus cardiacum, high circulatory titers can be maintained by an initial high release rate, followed by a rate of release equal to the rate of breakdown. These release rat’es are well within the capabilities of the neurosecretory complex (Mordue, 1966, 1969; Highnam et al. 1966; Highnam and Luntz, 1970). There is evidence from studies on Rhodnius and Carausius (Maddrell, 1964; Pilcher, 1970) that the Malpighian tubules are responsible, in part at least, for the inactivation and removal of the diuretic hormone from the hemolymph. In locusts, the Malpighian tubules are very rich in peptidase (Mordue, unpublished data) and are perhaps also involved in the inactivation of the hormone. 4 Nature of the Hormones
The preceding sections have been concerned with factors extracted from endocrine tissue and presumptive endocrine tissue and their effects upon excretory function. However, it is important to analyze these effects carefully to see which are truly hormonal and of sufficient quantitative response to be of importance to the overall excretion and water balance of the locust. Diuretic hormones have been described as being present within the terminal abdominal ganglion of the cockroach 1 (Mills, 1967), but these increase fluid secretion by the Malpighian tubules by only some 2 &‘hr. The blood volume of the cockroach is some 150~200 ~1 and consequently this diuretic effect can be of littie physiological importance to the insect. The effects of the antidiuretic and diuretic hormones in locusts are of a sufficiently quantitative nature to be of obvious significance to the overall water balance of I the locusts. The cerebral neurosecretory cell-corpus cardia.cum complex has been implicated in the regulat,ion of a considerable number of short- and long-term physiological, metabolic, and developmental events. However, the nature and number of hormones present are not yet resolved. The situation is further complicated by the fact that pharma-
IN
LOCUSTS
293
cologically active substances are known to be present within the corpora cardiaca (Colhoun, 1963). The use of whole-gland extracts of either corpora cardiaca or ventral nerve cord should be interpreted with caution until the effects can be shown to be hormonal and not pharmacological. Some of the confusion which exists among workers, as to the source and action of the hormones regulating excretory function, is in part due to the use of different test preparations, but also more importantip due to the use of whole-gland extracts which are known to contain more than one active principle (Mordue and Goldsworthy, 1969, unpublished data; Mordue, 197Oa; Goldsworthy and Mordue, unpublished data). Factors with different biological activities have been separated from the corpora cardiaca and ventral nerve cords oi other insects (Gersch ef: al. 1960, 1970; Natalizi et al. 1970; Goldbard et nl. 1970). As far as excretion in locusts is concerned, Mordue and Goldsworthy (4969) were able to separate a peptide (cr group of peptides) with diuretic activity from whole corpora cardiaca and storage lobes of the gland. More recent work has involved the use of Sephadex gel filtration to separate the different components of the corpora cardiaca. These separated components have been assayed for their diuretic and antidiuretic activity and also for their effects upon heart beat, lipid mobilisation, phosphorylase activation (trehalose factor) ~ and nitrogen metabolism (Mordue ant3 Goldsworthy unpublished data ; Goldsworthy and Mordue, unpublished data). Saline and methanol extracts of whoie glands or the separated lobes have been subjected to chromatography, and assay 04 the eluates allows a clear distinction to be made bet,ween the diuretic and antidiuretic hormones. The diuretic hormone is present in chromatographed extracts from mature locust,s, but is present in smaller amount,s in gland extracts from immature locusts. The antidiuretic hormone is found in eluates from both mature and immature locusts thus substantiating the results gained from experiments using saline extracts of whoic corpora cardiaca. The region of eluate eontaming t’he diuretic hormone also affects
294
W.
MORDUE
the rate of beating of isolated heart prepa&ions, although the doses involved in the preparations are very different. In a similar manner, the antidiuretic fractions also affect heart beat. Both sets of fractions have an effect upon lipid mobilisation, but much smaller amounts are needed to elevate lipids than to have an effect upon excretion, i.e., less than 0.001 gland-equivalents/ 100 ~1 of hemolymph. The effects of neurohormones C and D upon the rectum and Malpighian tubules is confused. Neurohormone D has been shown to increase tubule function (Unger, 1965) and the equivalent factor extracted from locusts (Mordue and Goldsworthy, 1969) has a similar effect. However, Vietinghoff (1967) has shown that neurohormone D can reduce tubule function. These hormonal factors and others extracted from locust corpora cardiaca can influence a multiplicity of physiologica systems within insects. Thus it is important to distinguish carefully between true hormonal effects, of direct and indirect nature, and those effects which are of pharmacological significance only. From their elution characteristics and other properties, these biologically active peptides separated from locust corpora cardiaca are similar in many ways to the factors separated from cockroaches by Gersch et al. (1960, 1970) and Natilizi et al. (1970). They are very different in nature from the diuretic and antidiuretic hormones isolated by Goldbard et al. (1970) from the terminal abdominal ganglion of cockroaches. 5. Release of Hormone The release of many insect hormones is in response to feeding stimuli, and this also appea,rs to hold true for the release of the diuretic hormone. In Rhodnius the link between feeding and release of the diuretic hormone has been elucidated by Maddrell (1964). This insect is an intermittent feeder, each blood meal being of large size relative to the mass of the insect. The distension of the abdomen caused by the intake of such a large amount of fluid is monitored by proprioceptors, and their responses are relayed via the ventral nerve
cord to the thoracic neurosecretory cells, which contain the diuretic hormone. In locusts, and in many other insects, feeding is a much more frequent phenomenon; and the link between feeding and hormone release is necessarily more obscure and difficult to resolve. However, we do know that feeding brings about the release of diuretic and developmental hormones in locusts (Highnam et al. 1966; Hill et al. 1966; Mordue, 1966, 1969; Highnam and Luntz, 1970). Food deprivation results in a marked reduction in the release of the diuretic hormone; after a few days the level of excretion through the Malpighian tubules is only slightly faster than in animals deprived of their neurosecretory cells. If starved locusts are allowed to feed, within 2-3 hr there is a massive release of diuretic hormone from the neurosecretory cellcorpus cardiacum complex (Mordue, 1966, 1969; Table 1). In Dysdercus, which is also a frequent feeder, Berridge (1966) has been able to show that feeding brings about the relea,se of a diuretic hormone from the neurosecretory cells of the pars intercerebralis. A high titer of diuretic hormone causes a high secretion of fluid by the Malpighian tubules, and at this time rectal reabsorption is reduced and the animal suffers a depletion in body water. Nonfeeding periods are associated with low secretory rates of fluid by the tubules, and little or no diuretic hormone circulating within the hemolymph. A similar link possibly exists between feeding and the release of diuretic hormone from the cerebral neurosecretory system in Carausius (Pilcher, 1970). Few experiments have been specifically designed to examine the link between feeding and hormone release. Davey (1962) has demonstrated that stimulation of chemoreceptors on the la,brum brings about the relea,se of hormone from the corpora cardiaca. This release could be prevented by secretion of the labral nerve. In locusts, gross interference with the stomatogastric nervous system (especially that closely associated with the mouthparts and gut) by removing the frontal ganglion is reported to prevent the release of neurosecretion
EXCRETION
from the cerebral system, with the concomit,ant complete cessation in growth (Clark and Langley, 1963). However, the importance of this nervous connection between feeding and hormone release has to be anaIysed carefully. Hill et al. (1966) have shown that this operation prevents the normal passage of food through the gut, and in some instances feeding may be particularly abnormal. The emptying of t,he foregut has been show to depend upon an intact stomatogastric system (Davey and Treherne, 1963). ZvIeasurements of excretory rates and the effects upon egg production show that release of diuretic and other neurosecretory hormones can still occur in locusts deprived of their frontal ganglia (Mordue, 1966, 1969; Highnam et al., 1966). Removal of the frontal ganglion from maturing locusts results in the death of the animal within a few days (Hill et al., 1966). Death results from a restrictecl intake of water i&o the midgut (although fresh food may be present within the foregut) coupled with the continued relea.se of diuretic hormone. Since such operated animals can still release neuroseeretory hormones, the link between feeding stimuli and hormone release, in part at least, can lie within some part of the nervous system other than the frontal ganglion. Osmoreceptors are known to be present within the foregut of cockroaches and have been shown to be of prime importance in the regulation of food passage into the midgut (Treherne, 1962). Recently, Penzlin, (1971) has suggested that these osmoreceptors are involved in the release of the diuretic hormone from the corpus cardiacum. Certainly, factors exist within the cockroach corpus eardiacum which will increase excretion through the Malpighian t’ubules (Mordue, unpublished data). Removal of the frontal g,anglion in the cockroach is thought to interfere with the normal release of the diuretic hormone (Penzlin, 1971). In locusts the presense of osmoreceptors which can monitor the water content of the hemolymph has not been reported. The work of Highnam et al. (1965) does suggest that the osmolarity of the hemolymph can affect the release of neuro-
IN
LOCUSTS
2%
secretory hormones. However, injection oi distilled water or saline has no immed.iate effect upon hormone release as is judged. from excretion rates (Mordue, unpublished observations). It seems likely that any effect of change in hemolymph osmolarity upon the secretion of hormones would be of a long-term nature, i.e., occur over hours rather than minutes. Further, indirect evidence for this is that immature locusts can have high blood volumes but low rates of excret,ion (Mordue, 1969). The recent observations of Cirardie ~~~~~) that regions of the protocerebrum other than the pars intercerebralis nemosecretory cells may be involved in the release of a diuretic hormone in locusts is of particular importance. However; it must, be borne in mind that the marked increases in blood volume which occur following remova,l of these cells or the neurosecretory cells of the pars intercerebralis may ir, part be due t’o an abnormality in the secretion of the antidiuretic hormone as well as ithe prevention of secretion of the diuretic hormone. 6. Mode of Action of the Hormones The action of the hormones upon t,he excretory system is not at all meil understood. There is cytological evidence that> neurosecret’ory hormones may not necessariIy be transported by the hemolymph to their target organs, but may be transported directly via nerve axons (see drell, 1970 for review). However, p logical evidence in support of these bindings is not yet forthcoming and because technical difficulties iEvolved will be d to accumulate. It is disputable that axonal transport, without storage of hormone, could provide sufficient hormone to bring about even short-term physiological responses in the target tissues. The presence of a direct, neuroseeretory supply to the Malpighian tubules and rectum may ~~11 overcome any delay in response of the target tissues caused by the relatively slow elevation in the titer of circula-tory hormones to the threshold response levels. The local release of small amounts of hormone may well be important in initiating these events (see also Maddrell, 1969).
296
W.
MORDUE
In vitro studies (Mordue, 1970b, 1971) measuring dye incorporation into Malpighian tubules have shown that a number of compounds may affect this aspect of tubule function (Fig. 1). Maddrell et al. (1969) have reported an increase in Malpighian tubule function in response to 5-hydroxytryptamine; however, this compound is without effect upon locust tubules (Fig. 1). Adrenalin does increa,se tubule function, whereas acetylcholine reduces dye uptake. These data, when considered alongside the stimulatory effect of cyclic 3,‘5’-AMP, do suggest, albeit somewhat circumstantially, that tubule function may be increased by the cyclic compound in vivo. Further, support for this hypothesis is that the action of low concentrations of cyclic AMP are potentiated by Mg*+ (1W4 M) . Tubule preparations are unresponsive to 5’-AMP. The effects of corpora cardiaca hormones upon dye uptake are paralleled by cyclic 3’,5’BMP. The way in which the diuretic hormone
Adr
Ach
reduces rectal reabsorption and the action of the antidiuretic hormone in increasing reabsorption are not yet understood. 7. The Regulation
of Water
Balance
The daily water intake of a locust can be as much as 5-6 times the blood volume, i.e., in excess of 2000 ~1. The water loss through the cuticle and spiracles is insufficient to keep the locust in water balance (Loveridge, 1967). The main factor responsible is fecal water loss. In a detailed analysis of feeding, Norris (1961) was able to show that there is considerable flexibility in the amount of water lost in the feces. The water content of the feces immediately after feeding is very high and is considerably reduced when feeding stops. These observations accord well with the demonstration that feeding brings about the release of diuretic hormone in locusts (Mordue, 1966, 1969). In addition, it is known that locusts reared in crowded conditions eat more food (Norris, 1961) and have
5-HT
25 1 1 ??
FIG. 1. Amaranth uptake by isolated agents. Adr, adrenalin; Ach, acetylcholine; phosphate; CC, corpora cardiaca; CON, tisted.
preparations of Malpighian 5-HT, 5-hydroxytryptamine; control. At least 5 preparations
tubules in the presence of various c3’,5’-AMP, cyclic adenosine monowere used for each concentration
EXCRETIOS
more active neurosecretory systems (Highnam and Haskell, 1964; Highnam and Luntz, 1970) than isolated locusts. It is of interest that crowded locusts, although eating more food than isolated locusts, lose considerably more water through their excretory systems than isolated locusts. Under other conditions, such as a food deprivation, the water content of the feces can be as low as 30% (Loveridge, 1967). Obviously the flexibility within the excretory system for varying fecal water loss is very great. During periods of water conservation, fecal wa,ter loss would be drastically reduced by the action of the antidiuretic hormone in increasing rectal reabsorption. Since this hormone has little effect upon the Malpighian tubules, excretory products can be secreted into the hindgut at basal rates or perhaps slightly increased above this rate. It seems likely that the basal secretory rate of the Nalpighian tubule is some 8-10 J/hr ; this is of course more than balanced by the basal rate of rectal reabsorption, which is some 15-17 ,J/hr. Under these conditions, the locust will remain in water balance ; the rate of rectal rea,bsorption will, of course, accommodate fluid flow from the midgut, into the highEat.. The antidiuretic hormone may well be of importance during the early part of adult life in enabling the locust t.o increase its blood volume. The blood volume of locusts is low at emergence and increases to a peak at about 10 days after emergence (Mordue! 1969; Hill et al., 1968). The increa’sed blood volume may be necessary to accommodate Iarge amounts of metabolites prior to and during reproductive development, without. bringing about large increases in the osmotic pressure of the hemolymph. The excess water ingested while obtaining sufficient foodstuffs for growth and development from a diet of fresh greens, is excreted under the action of the diuretic hormone. Studies on intact locusts suggest that a reduction in tubule secretion is brought about by the disappearance from the hemolymph of the diuretic hormone, rather than by the presence of an antidiuretic hormone. Rho&&us, Dysdercus, and Carawizcs a!so lack a hormone which
specifically reduces tubule secretion (Maddrell, 1964; Berridge, 1966; Pilcher, 1970). Diuresis is reduced by the breakdown of the diuretic hormone. ” ACKNOWLEDGMENT Part of this work was from the Science Research
b,? a grant
supported Couucil.
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GERSCH, M., FISCHER, F., UNGER, H., AND Kocxr~ H. (1960). 2. Nnturfo~sch. B 15, 31%x22. GERSCri, M., RICHTER, kr., B~~EM, G. A., ~\ND ST~~RZEBECHER, J. (1970). J, Insect Plu~sioi. 16, 1991-2013. GIRARDIE, A. (1964). J. Insect Piq&ol. 10, 59C9-609. GIFARDIE, A. (1970). C. R. Acad. Sei. 271, 504407. GOLDBARD, G. A., SAUER, J. R., AND MILLS. R. 1%. (1970). Comp. Cen. Phcmmcol. 1, 82-86. GOLDSWORTHY, G. J.; AP\‘D MORDIIE, W.. unpuSlished data. HIGHNAM, K. C., AND HASKEI,L, I?. T. (1964). i.
Insect Physiol. HIGHNAM, (1965). HIGXEAM: (1966). HIGHN.4M>
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C., HILL, L., AND GI~EI.~. D. J J. Zool. 147, 201-215. K. C., HILL., L., AXD MORDUE, W.
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K. c., AND LUNTZ, L%. J. (1970). &??'I Camp. Enclocrinol. 15, 31-38. HILL, L., MORDUE, W., AND HIGHNAX, EC. 6, (1966). J. Insect Physiol. 12, 1.197-1208. HILL, L., LUNTZ, A. J.: AND STEELE, P. :1. (196%).
J. Insect Psysiol. 14, l-20. LOVERIDGE, London. MADDRELL.
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S. H. P., PILCHER, D. E. M., AND B. 0. C. (1969). Nature (London) 222, 784-785. MILLS, R. R. (1967). J. Ezp. Biol. 46, 35-41. MILLS, R. R., AND NIELSEN, D. J. (1967). Gen. Comp. Endocrinol. ‘9, 380-382. MORDUE, W. (1966). Endocrinol. Exp. 5, 79-83. MORDUE, W. (1967). Gen. Comp. Endocrinol. 9, 475. MORDUE, W. (1969). J. Insect Physiol. 15, 273-285. MORDUE, W. (1970a). J. Endocrinol. 46, 119-120. MORDUE, W. (1970b). Gen. Comp. Endocrinol. 13, 521. MORDUE, W., AND GOLDSNORTHY, G. J. (1969). Gen. Comp. Endocrinol. 12, 360-369. MORDUE, W., AND GOLDSWORTHY, G. J., unpublished data. MORDUE, W., HIGHNAM, K. C., HILL, L., AND MADDRELL, GARDINER,
LUNTZ, A. J. (1970). Mem. Sot. Endocrinol. 18, 111-136. NATALIZI, G. M., PANSA, M. C., D’AJELLO, Q., CASAGLIA, O., BETTINI, S., AND FRONTALI, N. (1970). J. Insect Physiol. 16, 1827-1836. NORRIS, M. J. (1961). Bull. Entomol. Res. 51, 731-753. PENZLIN, H. (1971). J. Insect Physiol. 67, 559-573. PHILLIPS, J. E. (1964). J. Exp. Biol. 41, 69L80. PHILLIPS, J. E., (1970). Amer. 2001. 10, 41-36. PILCHER, D. E. M. (1970). J. Eqo. Biol. 53, 465-484. TREHERNE, J. E. (1962). In “Viewpoints in Biology” (J. D. Carthy, and C. L. Duddington, eds.), Vol. 1, pp. 201-241. Butterworths, London. UNGER, H. (1965). Zool. Jahrb. (Physiol.) 71, 710-717. QIETINGHOFF, U. (1967). Gen. Comp. Endocrinol. 9, 503.
DISCUSSION FRAENKEL: What is the relation between your corpus cardiacum (CC) factors, and the diuretic hormone of Mills, purported to be identical with bursicon and released from the last abdominal ganglion? MORDUE: The diuretic and antidiuretic hormones extracted from locust corpora cardiaca are different from those extracted by Mills and his co-workers from the terminal abdominal ganglion of cockroaches. Indeed, the factors so far extracted from the medial nervous system have only a slight effect upon excretion which may not be sufficient to be physiologically important. BROOKES: What is the effect of frontal ganglion removal on excretion? MORDUE: Removal of the frontal ganglion is a rather drastic operation and results in serious damage to many parts of the nervous system. However, its removal does prevent food from passing through the gut in a normal manner. The diuretic hormone is still released in maturing females following removal of the frontal ganglion. Thus, any nervous link which exists between feeding stimuli and hormone release, in part at least, must reside in some part of the nervous system other than the frontal ganglion. ENGELMANN: Amaranth is taken up by tissues other than the Malphigian tubules, and therefore it may be difficult to interpret the decrease of amaranth content in the hemolymph in terms of water excretion. I would like to ask whether you have information on amaranth uptake by other tissues as affected by CC factors. MORDUE: Some a.maranth is taken up by the pericardial cells, but their uptake within the time of the measurement of excretion is less than 2% of the total injection. I have not followed the effects of hormones upon this uptake. HELLER: It seems to me an interesting fact that insects have both an antidiuretic and a diuretic hormone, whereas in vertebrates, with the possible exception of fishes, no diuretic hormone has ever been found. Would you care to speculate whyeither in terms of metabolic situations or in the function of their excretory organs-a diuretic hormone has been evolved in insects? MORDUE: Many insects, both intermittent feeders, such as blood-sucking insects, and many continuous feeders, have diets which necessitate the intake of large amounts of water to obtain the requisite amounts of foodstuffs for growth and development. The presence of a diuretic hormone is important to enable such insects to dispose rapidly of this dietary water.
APiD
COMPARATIVE
ENDOCRINOLOGY
HYDROMlNERAL
3, 289-298 (1972)
SUPPLEMENT
REGULATION Chairman-K.
Hormones
and
H-4 ANIMLS.
PART
G. DAVEU
Excretion
in Locusts
W. MORDIJE Depwtment
of
Zoology London,
und
Applied
Entomology,
Imperictl
College,
S. W. 7, United Kingdom
Normones present within the cerebral neurosecretory cell-corpus cardiacum complex exert both diuretic and antidiuretic effects upon the excretory system. The Malpighian tubules and rectum respond to the diuretic hormone, which increases fluid secretion by the tubules and reduces rectal reabsorption. The antidiuretic hormone increases rectal reabsorption but has little elect upon tubule function. Feeding is of prime importance in regulating the release of t’he diuretic hormone; the control of release of antidiuretic hormone is not yet understood. Locusts remain in water balance under a variety of environmental conditions by the regulation of their fecal water content. This regulation is exercised by the diuretic and antidiuretic hormones. The diuretic hormone may increase fluid secretion by Malpighian t,ubules by increasing the intracellular levels of cyclic 3’,5’-AMP. The nature and number of hormonal factors present in the cerebral endocrine system and the ventral nerve cord are discussed.
It is now well established that excretory and their interrelationships difficult to rlefunction in insects is under hormonal con- solve, a better understanding may come trol. In locusts the hormones involved are from looking at the actions of the diuretic released from the cerebral neurosecretory and antidiuretic hormones upon the Maicell-corpus eardiacum complex, and possibly pighian tubules and rectum separately. Once also from the neurosecretory organs present these different hormonal actions have been within the medial nervous system (Mordue, examined: their integrated effect in producing a flexible regulatory system, capable 1966,1967,1969,1970a; Cazal and Girardie, 1968; de Bessb and Cazal, 1968). An anal- of dealing with both water loading and ysis of the hormonal regulation of ex- water conserva,tion, should be made clear. cretion and water balance is made difficult by the facts that more than one type of 1, The Mazpighian tubu1es It has been known for some time that hormone is involved and that these can effect quite different target organs. A num- destruction of the medial neurosecretory ber of factors are involved in water balance cells of the pars intercerebralis produces a in locusts (Loveridge, 1967), the most im- marked increase in the blood volume of the locust (Girardie, 1964, 1970; Highnam et nZ. portant of which is fecal water loss (Norris, 1961). The Malpighian tubules and rectum 1965; Mordue, 1966). This operation is are primarily involved in regulating fecal strongly suggestive that a factor, which water, but other regions of the gut may possessesstrong diuretic activity, is proalso be involved in overall water balance duced by the neurosecretory cells. In viva measurements of excretion in both Locz~dn; (see also Berridge, 1970) . and Xchistocerca show that excretion is Since the roles played by the different hormones and target tissues are complex very much reduced following removal of 289
@ 1972 by Academic
Press, Inc.
290
W.
MORDUE
the neurosecretory cells (Mordue, 1966, 1967, 1969). This low level of excretion can be elevated by injections of corpus cardiacum extracts; the active principle is restricted to the storage lobe of the gland (Mordue and Goldsworthy, 1969). The storage lobe of the gland contains neurosecretory factors produced by the cerebral Extracts of the neurosecretory cells. glandular lobe of the corpus cardiacum have little effect upon secretion through the Malpighian tubules: there is a slight increase in the rate of secretion (Mordue and Goldsworthy, 1969). Corpus cardiacum hormones affect fluid secretion through the tubules in viva (Table 1) and also increase the rate of excretion of compounds such as urate (Mordue, 1969). This evidence for the prime importance of neurosecretory hormones in regulating tubule function is from studies on intact insects. However, using in vitro preparations, Cazal and Girardie (1968) have demonstrated in Locusta that a factor is present within the corpora cardiaca which reduces the rate of excretion through the Malpighian tubules. In locusts which have been deprived of their neurosecretory cells for a number of days, and thus have no diuretic hormone, the rate of excretion through the tubules is very slow and undoubtedly represents a basal secretory rate (Mordue 1966, 1969). Since the basal rate of secretion is so slow and the breakdown of the diuretic hormone so rapid (Sect. 3)) a factor whose principal action is to reduce TABLE RATES
OB FLUID
TUBULES
Starved Starved, then fed for 2.5 hr Starved + 0.5 pair corpora cardiaca Starved f 1.0 pair corpora cardiaca Starved (data from Phillips, 1964)
1
SECRETION
BY MALPIGHIAN
IN
LOCUSTS
INTACT
No.
Fluid secretion (/.J/hr * SE)
6 5
7.5 27.5
&- 0.7 f 1.7
5
14.2
+_ 0.8
5
23.0
f
1.0
9
7.7
*
1.2
tubule secretion may not be necessary. In their work, Cazal and Girardie (1968) have demonstrated the presence of a diuretic factor within the neurosecretory cells of the pars intercerebrales, which increases tubule secretion. Using whole corpora cardiaca they were unable to demonstrate the presence of this diuretic factor within the corpora cardiaca. Either the diuretic factor is absent or its effect is masked by the strong antidiuretic action of the whole gland. With in vivo measurements, brain extracts have no marked effect upon tubule function (Mordue and Goldsworthy, 1969) ; the diuretic effects of the pars intercerebralis can only be demonstrated in locusts which have been deprived of their neurosecretory cells for some time. The rates of fluid secretion in isolated preparations are much lower than those in intact animals. Phillips (1964), using a cannulation method, measured fluid secretion by Malpighian tubules in starved Schistocerca (the lack of food restricted contamination of the hindgut fluid by midgut fluid) at some 8 &‘hr. It is known that excretion in starved locusts occurs at a very low rate; indeed the rate is not much above that in animals without their neurosecretory cells (Mordue, 1966, 1969). Table 1 shows the rates of fluid secretion through tubules in cannulated in uivo preparations of starved Xchistocerca. The rates of secretion are in very close agreement with those of Phillips (1964). If the starved locusts are allowed to feed, there is a dramatic increase in the secretion of fluid by the Malpighian tubules. It is well established that feeding brings about the release of the diuretic hormone (Sect. 5). Moreover, a similar increase in fluid secretion occurs following the injection of extra,cts of corpora cardiaca, and this increase in fluid secretion is dose dependent (Table 1). Measurement of fluid secretion in normal animals is made very difficult by the flow into the hindgut of fluid from the midgut, as well as from the Malpighian tubules. However, in some maturing females, successful noncontaminat.ed measurements of fluid secretion have been made, and were as high as 3540 &‘hr. These maturing
EXCRETIOI’G
locusts have very active neurosecretory systems (Highnam and Luntz, 1970) and high rates of excretion as measured by amaranth excretion (Mordue, 1966, 1969). Any attempts to tie off the midgut. from the hindgut to prevent contamination by midgut fluid involve serious interference with the Malpighian tubules. The physiological actions of neurosecretions present within the ventral nerve cord are, for the most part, &ill obscure. de Bessb and Cazai (1968) have shown that a factor is present in extracts of the ‘(organes pbrisympathiques,” which reduces secretion by the NIalpighian tubules. However, the extent of the reduction in tubule function is not given, nor as yet is there any information concerning dose-response relationships of this antidiuretic factor. This attempt to show the physiological function of these ventral nerve cord neurosecretions is necessary, and is a worthwhile attempt to establish a link between cytological and physiological evidence for the role of the neurosecretory system of the ventral nerve cord. 2. Rectd
Function
The physiological mechanisms involved in rectal function in locusts are now well understood, as a result of the work of Phillips (1964, 1970), but some confusion st’ill exists as to the role played by hormones in the regulation of the secretory and reabsorptive mechanisms of the rectum. In Locusta and EW&tocercu, diuretic and antidiuretic hormones are present within the corpora cardiaca. Both hormones affect the rectum (Mordue, 1966, 1969, 1970a; Cazal and Girardie, 1968). Analysis of rectal function in viva is technically difficult and in consequence most work has been carried out using in. vitro preparations. In isotonic saline, inverted rect#a are capable of secreting some E-17 ~1 of fluid/hr from the recta lumen into the hemolymph (Mordue, 1966, 1969, 1970a; Phillips, 1970). This basal secretory rate can be altered by corpus carclia,cum factors. Extracts of the storage lobe of the corpora cardiaca, reduce rectal reabsorption; this reduction is dose dependent, but even at high concentrations
IN
LOCUSTS
291
of hormone the rectum still allows some fluids to pass into the hemolymph ( due 1969, 197Oa). The work of Cazal and Girardie (1968) ascribes only an antidiuretic effect to extracts of corpora cardiaca. The presence of this antidiuretic factor within the corpore cardiaca has been confirmed (Morduc, 1970a). In this latter study the effects oi whole corpora cardiaca and the separatec! storage and glandular lobes were analysed. The diuretic hormone, as outlined in Sect. 1, is effective in storage lobe extracts from both Locka and Schistocema. The a&i-, diuretic component is restricted to the glandular lobe and is more potent in Loczlsta than in Xchistocerca (Mordue, 197Oa). In Locusta whole corpora cardiaca exert an antidiuret.ic effect upon rectal function; whereas whole glands from Schistocercn exert a diuretic effect (Mordue, 1970a). Whole corpora rardiaca from the t,wo species have different quantitat.ive effects upon excretion through the Malpighian tubules (Mordue, 1967, 1970a). Antidiuretic effects of extracts of the neurohemal organs of the ventral nerve chain upon isolated recta> have been described (de Bess& and Canal, 1968; Cazal and Girardie, 1968). The quantitative data provided by Cazal and Girardie suggests that this antidiuretic effect is much weaker than that produced by the corpora cardiaca. However, it seems likely that the volume of neurosecretory substance present within the ventral nerve cord is much less than the amount present within the corpora cardiaca. The antidiuretic hormone of the ventral nerve cord may well be as potent as that found within the corpora cardiaca. 3. Hornzone Turnover Dose-response relat8ionships have been established for the diuretic hormone released from the corpora cardiaca of locusts (Mordue, 1966, 1969; Mordue and Goldsworthy, 1969). There exists a straight-line relat’ionship between increased excretion end the amount of corpus cardiacum hormone within the hemolymph. The lowest toncentration at which consistent and measurable increases in excret,ion can be obtained is at
292
W.
MORDUE
levels of 0.04 glands/100 ~1 of hemolymph. In this work the corpora cardiaca used were taken from mature male locusts. Extracts of corpora cardiaca from immature locusts contain less diuretic hormone (Table 2). The variation in hormonal content of the neurosecretory complex with age, conditions of rearing, and stage of development is an important factor which must be considered when data from different groups of workers is compared. At present, it is impossible to define, with any degree of exactness, the hormonal complement of whole corpora cardiaca. The work of Cazal and Girardie (1968) well illustrates this point. They were able to demonstrate differences in the hormonal content of corpora cardiaca of locusts reared under different conditions of hydration and dehydration. Furthermore, injection of hemolymph from mature female locusts into locusts deprived of their neurosecretory cells produces an increase in excretion through the Malpighian tubules. However, blood from immature locusts hams little effect (Table 2). The responses of isolated rectal preparations to hormone-rich extracts also demonstrate the considerable variation in the amounts of hormone present within the neurosecretory complex of animals at different stages of development or in different states of hydration. Preliminary work with Locusta and Schistocerca suggests that under normal rearing conditions, the corpora cardiaca of immature and mature locusts contain approximately the same amount of antidiuretic hormone. Thus the TABLE
relative amounts of diuretic and antidiuretic hormones can differ markedly during development, and in some instances the amounts of hormones themselves may also alter. At the present time there is no evidence as to the levels of antidiuretic hormones within the locust hemolymph. In cockroaches these levels have been estimated (Mills and Nielsen, 1967). However, in other insects such as Rho&Gus and Carausius (Pilcher, 1970) there seems to be little variation in the diuretic complement of the appropriate endocrine glands. The hemolymph does, however, show marked variations in the amount of diuretic hormone present. The circulatory levels of diuretic hormone released from the corpora cardiaca in locusts have been measured and estimates made of the rate of breakdown in intact locusts (Mordue, 1966, 1969). In viva measurements of the titer of diuretic hormone in locusts show that it can vary between 0.040.4 pairs of corpus cardiacum-equivalents/l00 ,~l of hemolymph. These circulatory levels necessary to elicit diureisis in Malpighian tubules are very similar to the levels needed in Dysdercus (Berridge, 1966) and Carausius (Pilcher, 1970). In locusts, the circulatory levels of the diuretic hormone are altered by a large ra#nge of environmental stimuli which influence the activity of the cerebral endocrine system (Mordue et al. 1970). In locusts, breakdown of the hormone is rapid, and a sustained rate of excretion is only possible if there is continuous release of the diuretic factor. An amount of corpus cardiacum 2
THE EFFECTS OF CORPUS CARDIACUM EXTRACTSAND HEMOLYMPHFROM IMMATURE AND MATURE FEMALES UPON THF: EXCRETORYSYSTEMSIN IMMATURE FEMALE Schdocerca DEPRIVED OF THEIR NEUROSECRETORYCELLS FOR 5 Daysa Amaranth excreted (70 zk SE)
Donor Control (cauterised Immature females 0.2 pair corpora hemolymph, 75 Mature females 0.2 pair corpora hemolymph, 75 a At least
females)
Rectal reabsorbtion Whr 4 SE)
30 * 3
cardiaca ~1
54 t- 2 40 & 5
1 pair corpora
cardiaca
cardiaca ~1
75 * 2 45 i: 3
1 pair corpora
cardiaca
5 preparations
per test..
16.5
k 1.6
13.0
+ 1.3
5.0
+ 0.8
EXCRETION
hormone equivalent to 0.2 gland disappears from the hemolymph within l-l.5 hr. Obviously since there is a store of hormone within the corpus cardiacum, high circulatory titers can be maintained by an initial high release rate, followed by a rate of release equal to the rate of breakdown. These release rat’es are well within the capabilities of the neurosecretory complex (Mordue, 1966, 1969; Highnam et al. 1966; Highnam and Luntz, 1970). There is evidence from studies on Rhodnius and Carausius (Maddrell, 1964; Pilcher, 1970) that the Malpighian tubules are responsible, in part at least, for the inactivation and removal of the diuretic hormone from the hemolymph. In locusts, the Malpighian tubules are very rich in peptidase (Mordue, unpublished data) and are perhaps also involved in the inactivation of the hormone. 4 Nature of the Hormones
The preceding sections have been concerned with factors extracted from endocrine tissue and presumptive endocrine tissue and their effects upon excretory function. However, it is important to analyze these effects carefully to see which are truly hormonal and of sufficient quantitative response to be of importance to the overall excretion and water balance of the locust. Diuretic hormones have been described as being present within the terminal abdominal ganglion of the cockroach 1 (Mills, 1967), but these increase fluid secretion by the Malpighian tubules by only some 2 &‘hr. The blood volume of the cockroach is some 150~200 ~1 and consequently this diuretic effect can be of littie physiological importance to the insect. The effects of the antidiuretic and diuretic hormones in locusts are of a sufficiently quantitative nature to be of obvious significance to the overall water balance of I the locusts. The cerebral neurosecretory cell-corpus cardia.cum complex has been implicated in the regulat,ion of a considerable number of short- and long-term physiological, metabolic, and developmental events. However, the nature and number of hormones present are not yet resolved. The situation is further complicated by the fact that pharma-
IN
LOCUSTS
293
cologically active substances are known to be present within the corpora cardiaca (Colhoun, 1963). The use of whole-gland extracts of either corpora cardiaca or ventral nerve cord should be interpreted with caution until the effects can be shown to be hormonal and not pharmacological. Some of the confusion which exists among workers, as to the source and action of the hormones regulating excretory function, is in part due to the use of different test preparations, but also more importantip due to the use of whole-gland extracts which are known to contain more than one active principle (Mordue and Goldsworthy, 1969, unpublished data; Mordue, 197Oa; Goldsworthy and Mordue, unpublished data). Factors with different biological activities have been separated from the corpora cardiaca and ventral nerve cords oi other insects (Gersch ef: al. 1960, 1970; Natalizi et al. 1970; Goldbard et nl. 1970). As far as excretion in locusts is concerned, Mordue and Goldsworthy (4969) were able to separate a peptide (cr group of peptides) with diuretic activity from whole corpora cardiaca and storage lobes of the gland. More recent work has involved the use of Sephadex gel filtration to separate the different components of the corpora cardiaca. These separated components have been assayed for their diuretic and antidiuretic activity and also for their effects upon heart beat, lipid mobilisation, phosphorylase activation (trehalose factor) ~ and nitrogen metabolism (Mordue ant3 Goldsworthy unpublished data ; Goldsworthy and Mordue, unpublished data). Saline and methanol extracts of whoie glands or the separated lobes have been subjected to chromatography, and assay 04 the eluates allows a clear distinction to be made bet,ween the diuretic and antidiuretic hormones. The diuretic hormone is present in chromatographed extracts from mature locust,s, but is present in smaller amount,s in gland extracts from immature locusts. The antidiuretic hormone is found in eluates from both mature and immature locusts thus substantiating the results gained from experiments using saline extracts of whoic corpora cardiaca. The region of eluate eontaming t’he diuretic hormone also affects
294
W.
MORDUE
the rate of beating of isolated heart prepa&ions, although the doses involved in the preparations are very different. In a similar manner, the antidiuretic fractions also affect heart beat. Both sets of fractions have an effect upon lipid mobilisation, but much smaller amounts are needed to elevate lipids than to have an effect upon excretion, i.e., less than 0.001 gland-equivalents/ 100 ~1 of hemolymph. The effects of neurohormones C and D upon the rectum and Malpighian tubules is confused. Neurohormone D has been shown to increase tubule function (Unger, 1965) and the equivalent factor extracted from locusts (Mordue and Goldsworthy, 1969) has a similar effect. However, Vietinghoff (1967) has shown that neurohormone D can reduce tubule function. These hormonal factors and others extracted from locust corpora cardiaca can influence a multiplicity of physiologica systems within insects. Thus it is important to distinguish carefully between true hormonal effects, of direct and indirect nature, and those effects which are of pharmacological significance only. From their elution characteristics and other properties, these biologically active peptides separated from locust corpora cardiaca are similar in many ways to the factors separated from cockroaches by Gersch et al. (1960, 1970) and Natilizi et al. (1970). They are very different in nature from the diuretic and antidiuretic hormones isolated by Goldbard et al. (1970) from the terminal abdominal ganglion of cockroaches. 5. Release of Hormone The release of many insect hormones is in response to feeding stimuli, and this also appea,rs to hold true for the release of the diuretic hormone. In Rhodnius the link between feeding and release of the diuretic hormone has been elucidated by Maddrell (1964). This insect is an intermittent feeder, each blood meal being of large size relative to the mass of the insect. The distension of the abdomen caused by the intake of such a large amount of fluid is monitored by proprioceptors, and their responses are relayed via the ventral nerve
cord to the thoracic neurosecretory cells, which contain the diuretic hormone. In locusts, and in many other insects, feeding is a much more frequent phenomenon; and the link between feeding and hormone release is necessarily more obscure and difficult to resolve. However, we do know that feeding brings about the release of diuretic and developmental hormones in locusts (Highnam et al. 1966; Hill et al. 1966; Mordue, 1966, 1969; Highnam and Luntz, 1970). Food deprivation results in a marked reduction in the release of the diuretic hormone; after a few days the level of excretion through the Malpighian tubules is only slightly faster than in animals deprived of their neurosecretory cells. If starved locusts are allowed to feed, within 2-3 hr there is a massive release of diuretic hormone from the neurosecretory cellcorpus cardiacum complex (Mordue, 1966, 1969; Table 1). In Dysdercus, which is also a frequent feeder, Berridge (1966) has been able to show that feeding brings about the relea,se of a diuretic hormone from the neurosecretory cells of the pars intercerebralis. A high titer of diuretic hormone causes a high secretion of fluid by the Malpighian tubules, and at this time rectal reabsorption is reduced and the animal suffers a depletion in body water. Nonfeeding periods are associated with low secretory rates of fluid by the tubules, and little or no diuretic hormone circulating within the hemolymph. A similar link possibly exists between feeding and the release of diuretic hormone from the cerebral neurosecretory system in Carausius (Pilcher, 1970). Few experiments have been specifically designed to examine the link between feeding and hormone release. Davey (1962) has demonstrated that stimulation of chemoreceptors on the la,brum brings about the relea,se of hormone from the corpora cardiaca. This release could be prevented by secretion of the labral nerve. In locusts, gross interference with the stomatogastric nervous system (especially that closely associated with the mouthparts and gut) by removing the frontal ganglion is reported to prevent the release of neurosecretion
EXCRETION
from the cerebral system, with the concomit,ant complete cessation in growth (Clark and Langley, 1963). However, the importance of this nervous connection between feeding and hormone release has to be anaIysed carefully. Hill et al. (1966) have shown that this operation prevents the normal passage of food through the gut, and in some instances feeding may be particularly abnormal. The emptying of t,he foregut has been show to depend upon an intact stomatogastric system (Davey and Treherne, 1963). ZvIeasurements of excretory rates and the effects upon egg production show that release of diuretic and other neurosecretory hormones can still occur in locusts deprived of their frontal ganglia (Mordue, 1966, 1969; Highnam et al., 1966). Removal of the frontal ganglion from maturing locusts results in the death of the animal within a few days (Hill et al., 1966). Death results from a restrictecl intake of water i&o the midgut (although fresh food may be present within the foregut) coupled with the continued relea.se of diuretic hormone. Since such operated animals can still release neuroseeretory hormones, the link between feeding stimuli and hormone release, in part at least, can lie within some part of the nervous system other than the frontal ganglion. Osmoreceptors are known to be present within the foregut of cockroaches and have been shown to be of prime importance in the regulation of food passage into the midgut (Treherne, 1962). Recently, Penzlin, (1971) has suggested that these osmoreceptors are involved in the release of the diuretic hormone from the corpus cardiacum. Certainly, factors exist within the cockroach corpus eardiacum which will increase excretion through the Malpighian t’ubules (Mordue, unpublished data). Removal of the frontal g,anglion in the cockroach is thought to interfere with the normal release of the diuretic hormone (Penzlin, 1971). In locusts the presense of osmoreceptors which can monitor the water content of the hemolymph has not been reported. The work of Highnam et al. (1965) does suggest that the osmolarity of the hemolymph can affect the release of neuro-
IN
LOCUSTS
2%
secretory hormones. However, injection oi distilled water or saline has no immed.iate effect upon hormone release as is judged. from excretion rates (Mordue, unpublished observations). It seems likely that any effect of change in hemolymph osmolarity upon the secretion of hormones would be of a long-term nature, i.e., occur over hours rather than minutes. Further, indirect evidence for this is that immature locusts can have high blood volumes but low rates of excret,ion (Mordue, 1969). The recent observations of Cirardie ~~~~~) that regions of the protocerebrum other than the pars intercerebralis nemosecretory cells may be involved in the release of a diuretic hormone in locusts is of particular importance. However; it must, be borne in mind that the marked increases in blood volume which occur following remova,l of these cells or the neurosecretory cells of the pars intercerebralis may ir, part be due t’o an abnormality in the secretion of the antidiuretic hormone as well as ithe prevention of secretion of the diuretic hormone. 6. Mode of Action of the Hormones The action of the hormones upon t,he excretory system is not at all meil understood. There is cytological evidence that> neurosecret’ory hormones may not necessariIy be transported by the hemolymph to their target organs, but may be transported directly via nerve axons (see drell, 1970 for review). However, p logical evidence in support of these bindings is not yet forthcoming and because technical difficulties iEvolved will be d to accumulate. It is disputable that axonal transport, without storage of hormone, could provide sufficient hormone to bring about even short-term physiological responses in the target tissues. The presence of a direct, neuroseeretory supply to the Malpighian tubules and rectum may ~~11 overcome any delay in response of the target tissues caused by the relatively slow elevation in the titer of circula-tory hormones to the threshold response levels. The local release of small amounts of hormone may well be important in initiating these events (see also Maddrell, 1969).
296
W.
MORDUE
In vitro studies (Mordue, 1970b, 1971) measuring dye incorporation into Malpighian tubules have shown that a number of compounds may affect this aspect of tubule function (Fig. 1). Maddrell et al. (1969) have reported an increase in Malpighian tubule function in response to 5-hydroxytryptamine; however, this compound is without effect upon locust tubules (Fig. 1). Adrenalin does increa,se tubule function, whereas acetylcholine reduces dye uptake. These data, when considered alongside the stimulatory effect of cyclic 3,‘5’-AMP, do suggest, albeit somewhat circumstantially, that tubule function may be increased by the cyclic compound in vivo. Further, support for this hypothesis is that the action of low concentrations of cyclic AMP are potentiated by Mg*+ (1W4 M) . Tubule preparations are unresponsive to 5’-AMP. The effects of corpora cardiaca hormones upon dye uptake are paralleled by cyclic 3’,5’BMP. The way in which the diuretic hormone
Adr
Ach
reduces rectal reabsorption and the action of the antidiuretic hormone in increasing reabsorption are not yet understood. 7. The Regulation
of Water
Balance
The daily water intake of a locust can be as much as 5-6 times the blood volume, i.e., in excess of 2000 ~1. The water loss through the cuticle and spiracles is insufficient to keep the locust in water balance (Loveridge, 1967). The main factor responsible is fecal water loss. In a detailed analysis of feeding, Norris (1961) was able to show that there is considerable flexibility in the amount of water lost in the feces. The water content of the feces immediately after feeding is very high and is considerably reduced when feeding stops. These observations accord well with the demonstration that feeding brings about the release of diuretic hormone in locusts (Mordue, 1966, 1969). In addition, it is known that locusts reared in crowded conditions eat more food (Norris, 1961) and have
5-HT
25 1 1 ??
FIG. 1. Amaranth uptake by isolated agents. Adr, adrenalin; Ach, acetylcholine; phosphate; CC, corpora cardiaca; CON, tisted.
preparations of Malpighian 5-HT, 5-hydroxytryptamine; control. At least 5 preparations
tubules in the presence of various c3’,5’-AMP, cyclic adenosine monowere used for each concentration
EXCRETIOS
more active neurosecretory systems (Highnam and Haskell, 1964; Highnam and Luntz, 1970) than isolated locusts. It is of interest that crowded locusts, although eating more food than isolated locusts, lose considerably more water through their excretory systems than isolated locusts. Under other conditions, such as a food deprivation, the water content of the feces can be as low as 30% (Loveridge, 1967). Obviously the flexibility within the excretory system for varying fecal water loss is very great. During periods of water conservation, fecal wa,ter loss would be drastically reduced by the action of the antidiuretic hormone in increasing rectal reabsorption. Since this hormone has little effect upon the Malpighian tubules, excretory products can be secreted into the hindgut at basal rates or perhaps slightly increased above this rate. It seems likely that the basal secretory rate of the Nalpighian tubule is some 8-10 J/hr ; this is of course more than balanced by the basal rate of rectal reabsorption, which is some 15-17 ,J/hr. Under these conditions, the locust will remain in water balance ; the rate of rectal rea,bsorption will, of course, accommodate fluid flow from the midgut, into the highEat.. The antidiuretic hormone may well be of importance during the early part of adult life in enabling the locust t.o increase its blood volume. The blood volume of locusts is low at emergence and increases to a peak at about 10 days after emergence (Mordue! 1969; Hill et al., 1968). The increa’sed blood volume may be necessary to accommodate Iarge amounts of metabolites prior to and during reproductive development, without. bringing about large increases in the osmotic pressure of the hemolymph. The excess water ingested while obtaining sufficient foodstuffs for growth and development from a diet of fresh greens, is excreted under the action of the diuretic hormone. Studies on intact locusts suggest that a reduction in tubule secretion is brought about by the disappearance from the hemolymph of the diuretic hormone, rather than by the presence of an antidiuretic hormone. Rho&&us, Dysdercus, and Carawizcs a!so lack a hormone which
specifically reduces tubule secretion (Maddrell, 1964; Berridge, 1966; Pilcher, 1970). Diuresis is reduced by the breakdown of the diuretic hormone. ” ACKNOWLEDGMENT Part of this work was from the Science Research
b,? a grant
supported Couucil.
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DE BESSB, K., AND CAZAL, M. (1968). C. R. &xx& sci. 266, 615-618. CAZAL, M., AND GIRARDIE, it. (1968). f. lmxt Physiol. 14, 655-668. CLARK, IX. U. AND LAKGLEY, P. A. (1963). J. insect
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COLHOUN, E. H. (1963). In “Advances in Insect. Physiology” (J. W. L. Beament, J. E. Treherne, and V. B. Wigglesworth, eds.), Vol. I, pp. l-46. Academic Press, New York. DSYEY, x. G. (1962). J. I,nsect Physiol. 8, 20%&S. DAVBY; K. G., AND TREIIERNE, J. E. (1963). /.
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GERSCH, M., FISCHER, F., UNGER, H., AND Kocxr~ H. (1960). 2. Nnturfo~sch. B 15, 31%x22. GERSCri, M., RICHTER, kr., B~~EM, G. A., ~\ND ST~~RZEBECHER, J. (1970). J, Insect Plu~sioi. 16, 1991-2013. GIRARDIE, A. (1964). J. Insect Piq&ol. 10, 59C9-609. GIFARDIE, A. (1970). C. R. Acad. Sei. 271, 504407. GOLDBARD, G. A., SAUER, J. R., AND MILLS. R. 1%. (1970). Comp. Cen. Phcmmcol. 1, 82-86. GOLDSWORTHY, G. J.; AP\‘D MORDIIE, W.. unpuSlished data. HIGHNAM, K. C., AND HASKEI,L, I?. T. (1964). i.
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S. H. P., PILCHER, D. E. M., AND B. 0. C. (1969). Nature (London) 222, 784-785. MILLS, R. R. (1967). J. Ezp. Biol. 46, 35-41. MILLS, R. R., AND NIELSEN, D. J. (1967). Gen. Comp. Endocrinol. ‘9, 380-382. MORDUE, W. (1966). Endocrinol. Exp. 5, 79-83. MORDUE, W. (1967). Gen. Comp. Endocrinol. 9, 475. MORDUE, W. (1969). J. Insect Physiol. 15, 273-285. MORDUE, W. (1970a). J. Endocrinol. 46, 119-120. MORDUE, W. (1970b). Gen. Comp. Endocrinol. 13, 521. MORDUE, W., AND GOLDSNORTHY, G. J. (1969). Gen. Comp. Endocrinol. 12, 360-369. MORDUE, W., AND GOLDSWORTHY, G. J., unpublished data. MORDUE, W., HIGHNAM, K. C., HILL, L., AND MADDRELL, GARDINER,
LUNTZ, A. J. (1970). Mem. Sot. Endocrinol. 18, 111-136. NATALIZI, G. M., PANSA, M. C., D’AJELLO, Q., CASAGLIA, O., BETTINI, S., AND FRONTALI, N. (1970). J. Insect Physiol. 16, 1827-1836. NORRIS, M. J. (1961). Bull. Entomol. Res. 51, 731-753. PENZLIN, H. (1971). J. Insect Physiol. 67, 559-573. PHILLIPS, J. E. (1964). J. Exp. Biol. 41, 69L80. PHILLIPS, J. E., (1970). Amer. 2001. 10, 41-36. PILCHER, D. E. M. (1970). J. Eqo. Biol. 53, 465-484. TREHERNE, J. E. (1962). In “Viewpoints in Biology” (J. D. Carthy, and C. L. Duddington, eds.), Vol. 1, pp. 201-241. Butterworths, London. UNGER, H. (1965). Zool. Jahrb. (Physiol.) 71, 710-717. QIETINGHOFF, U. (1967). Gen. Comp. Endocrinol. 9, 503.
DISCUSSION FRAENKEL: What is the relation between your corpus cardiacum (CC) factors, and the diuretic hormone of Mills, purported to be identical with bursicon and released from the last abdominal ganglion? MORDUE: The diuretic and antidiuretic hormones extracted from locust corpora cardiaca are different from those extracted by Mills and his co-workers from the terminal abdominal ganglion of cockroaches. Indeed, the factors so far extracted from the medial nervous system have only a slight effect upon excretion which may not be sufficient to be physiologically important. BROOKES: What is the effect of frontal ganglion removal on excretion? MORDUE: Removal of the frontal ganglion is a rather drastic operation and results in serious damage to many parts of the nervous system. However, its removal does prevent food from passing through the gut in a normal manner. The diuretic hormone is still released in maturing females following removal of the frontal ganglion. Thus, any nervous link which exists between feeding stimuli and hormone release, in part at least, must reside in some part of the nervous system other than the frontal ganglion. ENGELMANN: Amaranth is taken up by tissues other than the Malphigian tubules, and therefore it may be difficult to interpret the decrease of amaranth content in the hemolymph in terms of water excretion. I would like to ask whether you have information on amaranth uptake by other tissues as affected by CC factors. MORDUE: Some a.maranth is taken up by the pericardial cells, but their uptake within the time of the measurement of excretion is less than 2% of the total injection. I have not followed the effects of hormones upon this uptake. HELLER: It seems to me an interesting fact that insects have both an antidiuretic and a diuretic hormone, whereas in vertebrates, with the possible exception of fishes, no diuretic hormone has ever been found. Would you care to speculate whyeither in terms of metabolic situations or in the function of their excretory organs-a diuretic hormone has been evolved in insects? MORDUE: Many insects, both intermittent feeders, such as blood-sucking insects, and many continuous feeders, have diets which necessitate the intake of large amounts of water to obtain the requisite amounts of foodstuffs for growth and development. The presence of a diuretic hormone is important to enable such insects to dispose rapidly of this dietary water.