neuroendocrine control of hydromineral regulation in the american lobster homarus americanus h....

GENERAL AND COMPARATIVE ENDOCRINOLOGY 54, 20-34 (1984)

Neuroendocrine Control of Hydromineral Regulation in the American Lobster Homarus americanus H. Milne-Edwards, 1837

(Crustacea, Decapoda) 2. Larval and Postlarval Stages

G.~HARMANTIER,* M. CHARMANTIER-DAURES,* AND D. E. AIKEN**

*Centre de Physiologie des Inverte’bre’s, Universite’ des Sciences et Techniques du Languedoc, 34060 Montpellier Cedex, France, and **Fisheries and Environmental Sciences, Department of Fisheries and

Oceans, Biological Station, St. Andrews, New Brunswick EOG 2X0, Canada

Accepted February 10, 1983

Sodium regulation in Homarus americanus changes from isoionic in Stage III to slightly hyperionic in Stage IV, and this is paralleled by improved survival of Stage IV lobsters in dilute media. Bilateral eyestalk ablation converts Stage IV and V lobsters to isoionic sodium regulation, but implantation of Stage IV or V eyestalk neuroendocrine tissue restores their normal hyperionic regulation. These results indicate that sodium regulation is controlled from Stage IV by eyestalk neuroendocrine factors. It is suggested that these changes between stages are part of a true metamorphosis that occurs between the last larval stage (III) and the first postlarval stage (IV).

Although several studies have provided data on the range of salinity tolerance in larval crustaceans, the osmoregulatory ability of larvae has been compared to that of adults in only a few species of crustaceans: the isopod Cyathura polita (Kelley and Burbanck, 1972; 1976), and the decapods Rhithropanopeus harrisii (Kalber and Costlow, 1966), Cardisoma guanhumi (Kalber and Costlow, 1968), Callinectes sapidus, Libinia emargina ta, and Hepa tus epheliticus (Kalber, 1970), Sesarma retic- ulatum (Foskett, 1977), and Clibanarius vit- tutus (Young, 1979). Different patterns of larval osmoregulation have been described, but in each species the osmoregulatory ability almost always varies according to the larval stage, and occasionally even during the course of one stage. Moreover, the types of osmoregulation in larval forms often differ from those in adults.

The question is, are these different types of osmoregulation under hormonal control? Neuroendocrine control of hydromineral regulation exists in the adults of some species of crustaceans (see review by Kame-

moto, 1979). In general, in marine species maintained in dilute media, bilateral eyestalk removal results in an increase in total water content and a decrease in concentration of the principal ions in the hemolymph, and thus a decrease in the hemolymph osmotic pressure. The implantation of eyestalk tissue or injection of extracts of central nervous system generally restores normal ionic or osmotic regulation. More detailed information on this subject is given in the introduction of a companion paper dealing with juvenile Homarus americanus (Charmantier et al., 1984).

To our knowledge there has been only one study of the control of osmotic regulation in larvae: Kalber and Costlow (1966) studied the effect of eyestalk removal in the zoeal stages II to IV and in the megalops of R. harrisii. Except in diecdysial larvae in stage II, the larvae of this species normally hyperregulate in salinities between 10 and 30%0 and are slightly hyperosmotic or isosmotic between 30 and 40%0. Bilateral eyestalk ablation in metecdysial stage II or III zoeal larvae results in increased hyper-

20 0016~6480/84 $1.50 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

CONTROL OF OSMOREGULATION IN Homarus, 2 21

osmoticity in a salinity of 40%0 and isosmotic regulation in 30 to 10%0. In other words, eyestalkless larvae lose their ability to hyperregulate in dilute sea water, as do most of the juvenile or adult decapods that have been studied.

In juvenile H. americanus, eyestalk removal lowers the osmotic and ionic regulatory capability, but this is restored after implantation of eyestalk tissue (Charman- tier et al., 1984). We therefore tried the same type of experiment on the different larval stages of this species in order to com- pare the types of regulation and their neuroendocrine control during development.

The larvae of H. americanus pass through three or four stages (see Phillips and Sastry, 1980) first described by Herrick (1896). The fourth stage is considered by some to be the last larval stage. Others (for example, Neil et al. 1976) consider the molt between stages III and IV to be metamorphosis, stage IV thus becoming the first postlarval or juvenile stage. For these rea- sons, we have paid particular attention to stages III and IV.

The type of osmoregulation in adult H. americanus is known (Dall, 1970), as is the type of osmotic and ionic regulation in juveniles (Charmantier et al., 1984 and in preparation). The ranges of salinity tolerance are also known for the larvae of H. gammarus (Gompel and Legendre, 1927) and H. americanus (Templeman, 1936). In this latter species, Scarratt and Raine (1967) studied larval behavior in different salinities and Sastry and Vargo (1977) ex- amined the combined effects of temperature and salinity on survival and development.

Information presented in this paper is concerned with the neuroendocrine control of sodium regulation and water content in larval and postlarval H. americanus. So- dium (and chloride) ions are known to ac- count for most of the hemolymph osmotic pressure in many crustaceans, including H. americanus. Sodium concentration varies

in the same way as osmotic pressure in juveniles of this species (Charmantier et al., 1984), and it seems logical the situation is similar in larvae.

MATERIALS AND METHODS

Lobsters and rearing facilities. All larvae were obtained between April and August from lobsters held in the lobster culture facility at the Biological Station, St. Andrews, New Brunswick, Canada. A description of this facility is available in Aiken (1983). Larvae for each experiment were obtained from the same female. After hatching, larvae were transferred to 40-liter larval planktonkreisels (Huges et al., 1974) supplied with flow-through sea water at a salinity of 3 1%0. Tem- perature was maintained at 20”; photoperiod was natural but uncontrolled, and subject to light breaks during the dark phase. Larvae were fed three times a day with frozen adult brine shrimp (Artemia), except on the day before a hemolymph sample.

For experiments in dilute sea water, two rearing tanks were connected to a 60-liter reserve tank with a recirculating pump and aeration, the total volume of each rearing unit being 140 liters. Dilutions of sea water were by progressive addition of fresh tap water that had been passed through activated charcoal. Water temperature was established by the room temperature, about 18°C.

Operations. Both eyestalks were removed from individuals in stages III, IV, or V by severing the eyestalks at their articulation with fine forceps under a dissecting microscope. The implantation of two whole eyestalks into stage III or IV individuals caused 100% mortality, so we dissected out the neuroendocrine tissue and implanted it dorsolaterally in the hemocoel beneath the membrane separating thorax and abdomen (Fig. 1). Operated animals were held in full sea water for 1 day and transferred to the dilute medium. After 24 hr, a hemolymph sample was taken.

Titrations. For hemolymph samples, individuals were carefully dried between two sheets of filter paper and hemolymph was then drawn by inserting a glass capillary tube (Drummond microcaps of 0.5 or 1 ~1) dorsally between cephalothorax and abdomen. These volumes of hemolymph could usually be obtained from individuals in stage IV or V, but l-3 larvae were re- quired in stage III.

The sodium concentration in the media and hemolymph samples was measured on a Perkin-Elmer 303 atomic absorption photometer.

Water content of the animals was calculated from fresh and dry weights. For fresh weight, larvae were wiped and dried on filter paper to a constant weight (15-25 min according to stage). Dry weight was de- termined after drying the larvae to constant weight (4 hr in an oven at 8X).

CHARMANTIER, CHARMANTIER-DAURES, AND AIKEN

FIG. 1. Homarus americanus, stage IV. (A) Eyestalkless lobster (ES -) and eyestalkless then eyestalk-implanted lobster (EST). The arrow indicates the point at which eyestalk tissue was implanted. (B) and (C), transverse sections of the posterior part of the cephalothorax showing implanted eyestalks (ES) 2 days after their implantation; bc, branchial chamber; c, cuticle; ce, cells; dt, digestive tract; hp, posterior part of the hepatopancreas; m, muscle; o, ommatidia. Staining reaction:hematoxylin- picroindigocarmin.

RESULTS

Adaptation Time in Dilute Media

Individuals in stage III and IV were transferred directly from sea water (salinity 31%0, osmotic pressure 900 mOsm/kg, sodium concentration 409 mEq Na+fliter) to a dilute medium (17%0, 500 mOsm/kg, 254 mEq Na+/liter). The sodium content in hemolymph of groups of four to nine animals was followed for 24 hr (Fig. 2). After a sharp decrease during the first hour, and a slight overshoot, the hemolymph sodium content stabilized within 2 (stage III) or 6 hr (stage IV) and remained constant. In additional experiments we kept the animals

for 24 hr in each medium before sampling their hemolymph. This time is obviously long enough for osmotic adaptation.

There is a difference in the stabilized sodium hemolymph concentrations of stages III and IV in dilute sea water. While stage III larvae are isosmotic with the medium, stage IV lobsters are slightly hyperosmotic, and the difference between the medium and the hemolymph concentrations in stage IV is about 40 mEq Na+/liter, that is, 80-85 mOsm/kg.

Survival

Survival of intact larvae in dilute media. Five groups of 50 individuals in stage III,


t m Eq Na+/l

450 z d

1 ' 0 6 12 16 24

Time

FIG. 2. Change in hemolymph sodium content in stages III and IV after rapid transfer from sea water (31%0, 900 mOsm/kg, 409 mEq Na+fliter) to dilute sea water (17%0, 500 mOsm/kg, 254 mEq Na+/liter). Each point represents the mean value of determinations from 4 to 14 animals, with standard deviations. M: sodium concentration of the dilute medium.

and five similar groups in stage IV were progressively adapted and then held in one of five salinities: 31 (sea water), 20, 15, 12.5, and 10%0. The number of surviving animals was recorded daily until Day 19 (Fig. 3).

In sea water, 56% of the third stage and 62% of the fourth stage larvae survived and molted. The lowest salinities (10 and 12.5%0) were definitely lethal for the two stages since no larva was able to molt to the next stage. However, the individuals in stage IV survived longer than those in stage III, especially in 12.5%0. In 15%0, except for one individual which molted to stage IV, the stage III larvae did not survive more than 6 days. However, nearly 50% of stage IV lobsters survived until the end of the experiment, although only seven of them molted to stage V. A similar difference in survival rates exists for stages III and IV at 20%0, except that surviving stage IV animals molted to stage V at the same rate as control animals in sea water.

Survival after eyestalk ablation and im-

plantation. The rates of mortality in different stages ablated and then kept in sea water are given in Table 1. Mortality is highest during the first postoperative day and decreased thereafter. The l-day survival values give a reasonable indication of the trauma of eyestalk removal or implantation. After eyestalk removal, mortality is less in stage IV than in earlier stages. As already stated, survival after implantation of eyestalks is only possible if the eyestalk cuticle is first removed. Even so, the mortality rate is always high after this second operation. The implantation of eyestalk tissue into intact stage III larvae is very dif- ficult because of the small size of the animals, and the mortality rate is nearly 85% 1 day after the operation.

Survival of control and ablated lobsters in dilute media. After eyestalk ablation, the surviving larvae were progressively transferred along with controls to one of two dilute media (24 and 17%0) and held for 1 day. Compared to the controls, the eyestalkless larvae have a higher rate of mortality (Table 2), and it is increased by the implantation of eyestalk tissue.

Effect of eyestalk ablation on larval color. Lobsters in stages IV and V are normally a dark blue-grey color. However, a few hours after bilateral eyestalk ablation, this color changes to red. If eyestalk tissue is then implanted, the color rapidly (l-5 min) changes back to blue-grey. Histolog- ical sections of such implants appear normal (Fig. 1). However, in a small number of cases the recipients of the eyestalk implant remain red, possibly because the eyestalk tissue has been injured during the operation. These changes in color, re- sulting from changes in size of chromato- phores, are known to be under neuroendocrine control (see review in Kleinholz, 1976). They are useful in our experiments for they give a rapid indication of the success of the operation, especially in the case of implantation.

24 CHARMANTIER, CHARMANTIER-DAURES , AND AIKEN

50'

40 -

f E

'g 30-

% ii

3 20- E

i 10 -

O- Days

0 5 10 15 20 Time

FIG. 3. Effect of salinity on survival of stage III and IV H. americanus. Salinities of 31 (sea water), 20, 15, 12.5, and 10%0 are indicated on each curve. The number of molted animals (from III to IV and from IV to V) are indicated when necessary.

Sodium Regulation irt Intact Stages III and IV

In stage III, sodium regulation is nearly isoionic, which could be anticipated from the results of the adaptation-time experiment (Fig. 2). In contrast, in early stage IV, 1 or 2 days after the molt from stage III, sodium regulation is hyperionic except in the most dilute medium (~O%JO), which cor-

responds to a lethal salinity (Fig. 3). In the middle of stage IV (6 to 8 days after the molt), regulation remains slightly hyperionic, but less than at the beginning of the stage. These results are summarized in Fig. 4. This type of slightly hyperionic regulation in dilute sea water also exists in stage V (see following section and Fig. 6) and in juveniles (Charmantier et al., 1984 and in preparation).

TABLE 1 RATES OF MORTALITY OF STAGE III, IV, AND V Homarus americanus IN

SEA WATER AFTER DIFFERENT OPERATIONS

Stages and type of operation N Survivors Mortality

(%I Days

III ES- + IV ES- 250 60 76.0 7 IV ES- 305 183 39.7 1

100 67.0 3 IV EST (0 60 0 100 1

(2) 91 36 60.5 1 VES- 25 20 20 1 V ES+ (2) 42 20 52.4 1 V ES-, ES III+ (2) 50 12 76 1

III, ES IV+ (2) 46 7 84.8 1

Note. ES - , eyestalkless; ES r, eyestalks ablated then eyestalk tissue implanted; ES + , eyestalk implanted. (1) denotes implanted eyestalk with cuticle. (2) denotes implanted eyestalk tissue without cuticle.


TABLE 2 RATES OF MORTALW OF STAGE III, IV, AND V

Homarus americanus IN SALINITIES OF 17 AND 24%0

AFTER DIFFERENT OPERATIONS

Stage and types Mortality of operation N Survivors (%) Days

III c 200 142 29 2 IV c 80 75 6.3 2 IV ES- 60 51 15 1 IV EST 36 20 44.4 1 vc 22 21 4.6 1 V ES- 20 13 35 1 V EST 20 12 40 1

Note. C, control; ES - , eyestalkless; EST, eyestalks ablated then eyestalk tissue implanted.

Influence of Eyestalk Removal and Implantation on Sodium Regulation in Stages III, IV, and V

For these experiments, individuals were selected from the middle of each stage. Regulation was studied in dilute media in

m Eq NayI

200 300 400

Medium

FIG. 4. Change in hemolymph sodium concentration in stage III and IV H. americanus according to that of the medium. The isoconcentration line is shown. Each point is the mean value of measurements from 13 to 15 animals, with standard deviations. The sodium regulation in juveniles (Juv.) is indicated for comparison (from Charmantier et al., 1984).

which the previous experiment had shown regulation to be distinctly hyperionic, with maximum difference between the hemolymph and medium concentrations (for example, the two low salinities for the experiment in stage IV were 225 and 325 mEq Na+fliter, or 17 and 24%0). The results are given in Table 3 and Figs. 5 and 6.

Regulation in intact stage IV Lobsters is hyperionic. Regulation in stage IV lobsters after eyestalk ablation is isoionic just as it is in intact stage III larvae (Table 3, Fig. 5). Similarly, in eyestalkless stage III larvae that molt to stage IV, regulation remains isoionic (Table 3). Implantation of tissue from two eyestalks appears to partially re- store their regulatory ability, but as a group it remains less hyperionic than in the control group (Table 3, Fig. 5). However, if the results obtained from red- and blue-grey- implanted animals are separated, the effect of implantation becomes obvious: in red larvae (where we presume the implanted eyestalk tissue has been injured) regulation remains isoionic as in eyestalkless lobsters. On the contrary, in those larvae that have returned to a normal blue-grey color, the normal slightly hyperionic regulation is completely restored (Table 3). Statistical comparison of results shows significant differences between control and eyestalkless lobsters and also between red- and blue- grey implanted animals; on the contrary, no significant difference appears between eyestalkless and implanted red lobsters, and between control and blue-grey implanted lobsters (Table 4).

The same kind of experiment was done with stage V lobsters in 17%~ The results are similar to those found with stage IV (Ta- bles 3, 4; Fig. 6).

When eyestalks from stage IV lobsters are implanted into stage III larvae, a high rate of mortality results (Table 1) and the surviving larvae remain nearly isoionic (Table 3). When eyestalks from stage III larvae are implanted into destalked stage

26 CHARMANTIER, CHARMANTIER-DAURES, AND AIKEN

TABLE 3 INFLUENCEOFEYESTALKREMOVALORIMPLANTATIONOFEYESTALKTISSUE ON HEMOLYMPH SODIUM

CONCENTRATION OF STAGES III,IV, AND V Homarus americanus, IN Two DILUTE MEDIA

Stages Medium Hemolymph Medium Hemolymph

(mEq Na+/liter) (mEq Na+/liter) (mEq Na+/liter) (mEq Na+/liter)

III c III ES- + IV ES- IV c IVES-

IV EST

vc VES-

V EST

III, ES IV+ IV ES-, ES III+

339 326

325

348 + 11 (15) 239 238 1 7 (14) 331 + 6 (15) 232 230 ?I 7 (15) 365 k 9 (15) 274 2 11 (12) 324 e 10 (14) 229 2 9 (11)

315 + 15 (3) Y 225 214 I~I 25 (4) Y 348 + 22 255 -c 33

(9) 364 e 18 (6) d (9) 288 ” 28 (5) d

260 ? 5 (12) 217 IL 9 (9)

215 1 221 ? 18 (5) Y 244 -r 15

(12) 269 I~I 11 (7)d

240 I!Z 34 (5) 227 { 223 ? 14 (9:6v+3d)

Note. C, control; ES -, eyestalkless; EST, eyestalkless then eyestalk tissue implanted; ES + , eyestalk implanted; Y, red; d, dark. The number of titrations is in parentheses.

IV individuals, the recipients remain Injluence of Eyestalk Removal on isoionic (Table 3). Water Content

9

m Eq Na+/l

350 -

= 300-

E

* E

s

250 - o---ES-

A-.-ES

Water content of stage II, III, and IV lobsters was measured in salinities of 17 and 31%0 (three replicates of lo-20 lobsters for each stage and salinity) (Fig. 7). In general, lobsters in the dilute medium have a higher water content. Moreover, it is generally higher in stages II and III (about 82% in sea water) than in stage IV (71.5% in sea water).

After eyestalk removal, the water content of stage IV lobsters increases to about 75% or more in sea water or in a dilute me-

2oov , dium (Fig’ 7)’ D~SC”SS~ON , mEqNa+/l

200 250 300

Medium Adaptation and Survival

FIG. 5. Change in hemolymph sodium concentration In the American lobster, the time re- of stage IV lobsters according to that of the medium auired for adantation to a dilute medium is in control (C), eyestalkless (ES -), and eyestalkless then eyestalk implanted (EST) animals. The isocon-

about 2 hr in siage III and 6 hr in stage IV.

centration line is shown. Each point is the mean value It is between 12 and 24 hr in juveniles of measurements from 9-15 animals, with standard (Charmantier et al., 1984), and about 75 hr deviations. in adults (Dall, 1970). Adaptation time is


TABLE 4 STATISTICAL COMPARISON (t TEST) OF THE EFFECT OF EYESTALK REMOVAL OR IMPLANTATION ON SODIUM

REGULATION IN STAGE IV AND V Homarus americanus

Medium (mEq Na+/liter) Stages C/ES - C/EST ES-/EST EsT(d)/ES+)

325 IV 225 IV 215 V

325 IV 225 IV 215 V

** ** **

C/ES I

** ** **

ns ns *

C/EST(d)

ns ns ns

*

ns *

ES --/EST(r)

ns ns ns

* ** **

ES -/EST(d)

** ** **

Note. ns, not significant; *P < 0.05; **P < 0.01; C, control; ES - , eyestalks ablated; ES+, eyestalk ablated then eyestalk tissue implanted; (r), red color; (d), dark color.

therefore size dependent. In larvae of other species, adaptation time is similar to that of the young stages of H. americanus. Kalber and Costlow (1966, 1968) and Kalber (1970) suggested adaptation time was about 1 hr for larvae of R. harrisii and 2 hr for C. guanhumi, C. sapidus, and L. emarginata. Foskett (1977) found an adaptation time of 1 hr in larvae of S. reticulaturn. This author stressed the physiological and ecological importance of rapid adaptation of hemo-

1 mEqNa’/l

ES- \r+d r d,

E3Z

FIG. 6. Sodium concentration in the hemolymph in a 17%0 medium in control (C), eyestalkless (ES-), and eyestalkless then eyestalk-implanted (EST) stage V lobsters. In the last group (EST), values from red (r), dark grey (d), and all (r + d) are indicated. Each bar represents the mean value of measurements from 5 to 12 animals, with standard deviations.

lymph osmotic pressure to changes in the salinity of the medium. This may be partic- ularly true in the case of larval Homarus, which are planktonic and thus exposed to sudden changes in salinity following heavy rainfall. These rapid changes also require the existence of intracellular osmotic adaptation, especially in the isoionic stages such as stage III.

Salinity affects the survival of young H. americanus. A salinity of 12.5%0 is lethal for stages III and IV. The rate of molting to the following stage increases as salinity increases from 15 to 20%~; the molt from stage IV to stage V is delayed in 15%0 but occurs normally in 20%0. In all salinities, but especially in those in the range of 12.5 to 20%0, survival is better in stage IV than stage III. These results agree with those of Gompel and Legendre ( 1927)) Templeman ( 1936)) Scarratt and Raine (1967), and Sastry and Zeitlin-Hale (1977). Templeman (1936), who also worked at the St. Andrews Biological Station, noted that the minimum salinity for larval development was about 19%0 and that salinities between 21 and 28%0 were only slightly less favorable than 31%0, the mean salinity in St. Andrews.

Because of their small size, larval stages of Homarus do not survive operations well, and the high rate of mortality increases with additional operations performed on indi-


: l

. . . . . rl ..* . . . . . . . . . . . . . . 0 9.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JllLj c

. . . . . . . . . . . . . . . . . . . . . Lx& Y Yi

17%0 31

& .t: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E

‘?60 FIG. 7. Water content of stage II, III, and IV lobsters in two media (31 and 17%0) according to the

stage, and in stage IV in control animals (C) and j after eyestalk removal (ES the mean value of measurements from 10 to 20 animals. The bar indicates the

Each point represents general mean.

vidual animals. As a result, the implantation of eyestalk tissue may improve the survival of destalked juveniles (Charmantier et al., 1984) but it increases the mortality of larvae.

Osmotic and Ionic Regulation

The type of sodium regulation changes during early development. As sodium is one of the two main osmotic effecters in the hemolymph, we assume that it is regulated in the same way that osmotic pressure is regulated. This has been confirmed by many studies with crustaceans and by our own results in juveniles of this species (Charmantier et al., 1984).

Regulation is isoionic in stage III and slightly hyperionic in stages IV and V. So, the type of adult regulation (Dall, 1970), which is identical to juvenile regulation (Charmantier et al., 1984 and in preparation) seems to be acquired at stage IV, except in the lowest salinities (10%0) where stage IV animals are still nearly isoionic while juveniles are slightly hyperionic. The change in type of regulation is associated with a drop in water content from 82% in stage III to 71.5% in stage IV. One or two days after the molt from III to IV, the reg-

ulation is more hyperionic than in the middle of stage IV. Similar variations have been shown in the larvae of R. harrisii (Kalber and Costlow, 1966) and C. guanhumi (Kalber and Costlow, 1968) in which osmotic pressure increases before and just after the molt. These authors suggest cor- relation between the increased osmotic pressure of the hemolymph and the molting process, since a hypertonic internal medium would favor uptake of water at molt. However, we did not find any increase in the hemolymph sodium content in stage III, even at the end of this stage. In larvae of S. reticulaturn, Foskett (1977) found no consistent tendency toward increased hy- perregulation before ecdysis. So, it appears the mechanisms of water intake at molt in larval and postlarval stages involve more than an osmotic gradient between the ex- ternal and internal media.

From the few published comparisons of osmoregulation in larval and adult decapods (Kalber and Costlow, 1966, 1968; Kalber, 1970; Hubschman, 1975; Foskett, 1977; Young, 1979), and from our results with H. americanus, it appears the type of osmotic and ionic regulation changes during young stages of development, that


the different patterns of larval ionoregulatory and osmoregulatory abilities vary according to the species, and that the adult type of regulation appears either suddenly or progressively according to the species at different times during development. In H. americanus, the young lobster appears to shift from a larval to juvenile and adult type of regulation at stage IV. An interesting hypothesis has been proposed by Foskett (1977) to correlate the ecology of young stages with changes in their ionoregulatory and osmoregulatory abilities, at least in species living in estuarine and coastal wa- ters. In these areas the water mass is often separated into two layers: a deeper on- shore-flowing layer of sea water, and a surface offshore-flowing layer of dilute sea water. The isosmotic larvae could remain at the surface in relatively low salinities, while the higher-density hyperosmotic postlarval stages would be adapted to seek the bottom in normal seawater salinity. This hypothesis is compatible with the ecology of the young stages of H. americanus since the isoionic stage III larvae are planktonic while the slightly hyperionic regulating stage IV and V lobsters become benthic during stages IV and V.

Neuroendocrine Control

How are changes in the type of regulation controlled during larval and postlarval life? As far as we know, the only previous work on this subject was by Kalber and Costlow (1966) with larvae of R. harrisii. They found the effect of eyestalk ablation on osmotic regulation varies according to stage and time of operation. In particular, while normal metecdysial zoeae in stage II are hyperosmotic in media ranging from 5 to 30%0, eyestalkless larvae in the same stage are isomotic in these salinities. The eyestalk influence on the regulation is similar in stage III but seems to disappear in stage IV.

In H. americanus, eyestalk removal causes sodium regulation in stages IV and

V to shift from slightly hyperionic to isoionic while water content increases. The neurohormonal nature of the control of sodium regulation is confirmed by eyestalk implantation which quickly restores the type of regulation of intact animals. These results are similar to those obtained with larger juveniles (Charmantier et al., 1984), except that the effect of eyestalk ablation is expressed more quickly in stages IV and V than in older juveniles. Except for this difference, the type of neuroendocrine control found in large juveniles seems identical to that first seen in stage IV.

In stages IV and V, eyestalk ablation also alters the color of the animals, which appears to be under neuroendocrine control as in other crustacean adults (Kleinholz, 1976) and larvae (Broth, 1960; Costlow and Sandeen, 1961; Hubschman, 1963). The re- sulting color changes can be used as a test of the success of the operation, especially in the case of implants of eyestalk tissue.

These results, combined with the fact that regulation shifts from an isoionic type in stage III to a slightly hyperionic type in stage IV, suggest two hypotheses: (1) se- cretion of a neurohormone that controls sodium regulation begins in stage IV; and (2) the effector organs become functional between stage III and stage IV.

The first hypothesis is generated by the results of operations in stage IV: eyestalk ablation converts stage IV lobsters to isoionic regulation, and this is not changed by implantation of stage III eyestalk tissue. Thus, the eyestalk in stage IV releases a factor that induces a slightly hyperionic regulation, while the eyestalk in stage III does not. Work with juveniles suggests this factor is secreted in the “brain” and released from the sinus glands (Charmantier et al., 1984). A histological study of eyestalk development during the larval stages of H. americanus (Pyle, 1943) showed that the sinus gland, which first appears in stage III, is thin during this stage. It increases in size in stage IV but lacks the staining reac-


tion which exists in the adult gland. There- fore, only the increase in size of the sinus gland can be related to the functional evo- lution of the eyestalk in stage IV. However, eyestalk ablation in stage IV accelerates proecdysial preparation and causes preco- cious initiation of ecdysis (Rao et al., 1973). So, these anatomical and physiological results and our own findings suggest the eyestalk neuroendocrine complex becomes functional in stage IV.

On the other hand, implantation of stage IV eyestalks into intact stage III larvae does not change their sodium regulation. This suggests that the effector organs are not yet functional. In larvae of the freshwater shrimp Palaemonetes kadiakensis, Hubschman (1975) found that the ability to survive in higher salinities (i.e., withstand osmotic stress) increases gradually with successive larval stages and might be related to progressive gill development. In H. americanus, the gills undergo important changes during the molt from stage III to stage IV. If we assume that in lobsters, as in other species of crustaceans, the gills are a major site of ion and water exchange, their morphological change could also ex- plain the change in the type of regulation.

According to our hypothesis, control of sodium regulation by neurohormone secre- tion begins in stage IV. This control might be a consequence of the control of water content (see Charmantier et al., 1984), or it might be due to a factor that maintains a high level of sodium in the hemolymph when the animals are placed in dilute media. This type of neuroendocrine regulation of sodium has been established in adults of some crustacean species. Injec- tion of eyestalk or central nervous system extracts in the crayfish Procambarus clarkii increases the hemolymph sodium concentration (Kamemoto et al., 1966). In Uca pugilator maintained in dilute media, the hemolymph sodium concentration de- creases after eyestalk removal, and increases with injection of eyestalk extract

(Heit and Fingerman, 1975). We found similar responses in juvenile H. americanus (Charmantier et al., 1984). These experiments suggest the existence of a sodium regulating hormone that increases hemolymph sodium concentration. As discussed in the companion paper dealing with juvenile H. americanus (Charmantier et al., 1984), this factor could act on the sodium flux (Ehrenfeld and Isaia, 1974; Davis, 1979) and/or on the activity of Na+ K+ ATPase (Kamemoto and Tullis, 1972).

Metamorphosis

The concept of metamorphosis was orig- inally based mainly on morphological changes (Snodgrass, 1956), but it was en- larged by Passano (1961) to include physiological and behavioral changes as well. Passano defined metamorphosis as “. . . a pronounced change in form at a particular point in the animal’s life. . . .” Costlow (1968) also emphasized control mechanisms. The results reported here demon- strate major physiological differences between stages III and IV in H. americanus, differences indicative of metamorphosis.

Several aspects of metamorphosis have been studied in H. americanus (Table 5). The morphological changes are the most obvious: stages I to III have a typical larval morphology, whereas a stage IV lobster closely resembles the adult (there are some allometric differences; see Lang et al., 1977). One of the most important ecological changes commences during stage IV and in- volves the transition from a planktonic, typically larval, to a benthic, typically adult, existence. This transition includes morphological and behavioral changes (ap- pendage morphology, locomotory behavior, reaction to light, etc.) as well as the biochemical and physiological changes men- tioned above.

There seems little question that metamorphosis occurs during the development of H. americanus. However, there is lack of agreement on the exact timing of meta-


TABLE 5 MAJOR CHANGES ASSOCIATED WITH METAMORPHOSIS IN Homarus americanus

Morphological and anatomical changes Change in general body shape between III and IV ....................................... Development of antennae, antennulae, chelipeds in IV ................................... Changes in swimmeret system from the larval stages to IV ................................ Loss of functional exopodites in chelipeds and pereiopods in IV ........................... Changes in mouthparts (especially mandibles and third maxilliped) and digestive tract from III

to1v.. ............................................................................ Appearance of true statocysts in IV .................................................... Appearance of eyestalk sinus gland in III; increase in size of sinus gland in IV ..............

Ecological and behavioral changes Percentages of different stages in the plankton ........................................... Change from planktonic to benthic habitat during IV and V ............................... Change in swimming behavior from III to early IV ....................................... Change in behavior when experimentally released at the surface or at the bottom, from III

during IV to V ..................................................................... Change in movements of thoracic exopodites, and endopodites and in abdominal swimmerets

from III to IV ...................................................................... Changes in reaction to light within inside each larval stage, during IV and V ................ Changes in reaction to hydrostatic pressure during IV and V .............................. Change in diet from III to IV and V .................................................... Increased survival rates in IV and V, compared to II and III, when communally reared ......

Biochemical and physiological changes Decrease in carbohydrate content during III, IV, V. Decrease in lipid content from larval

stages to IV and V. Increase in ash and chitin content from larval stages to IV and V ...... Decrease in 0:N ratio from larval stages to IV and V .................................... Ammonia excretion rates: increase from I to IV and decrease in V ........................

donotchange ................................................ Weight specific respiration rates: increase from I to IV and decrease in V ..................

decreasefromIV ....................................... Growth, exponential during the larval life, slows from IV ................................. Ionic regulation changes from isosmotic in III to hyperosmotic in IV ....................... Neuroendocrine control of regulation appears in IV ......................................

1-3 2 2,4-6 2, 6

7-9 6 10

I1 -15 16, 17 6. 16

18

6, 19, 20 16 18 I, 21 22

23, 24 23, 24 23, 24 25 23 25 25 This study This study

Note. The larval and juvenile stages are indicated by Roman figures. The arabic numbers refer to the authors indicated below. * Herrick, 1896. I0 Pyle, 1943. I9 MacMillan al., 1976. et 2 Hadley, 1909. *I Templeman, 1937. 20 Laverack al., 1976. et 3 Heckman, 1978. z2 Templeman, 1939. 21 Williams, 1907. 4 Davis, 1973. I3 Templeman and Tibbo, 1945. 22 Sastry and Zeitlin-Hale, 1977. 5 Davis and Davis 1973. I4 Scarratt, 1964. 23 Capuzzo and Lancaster, 1979a. 6 Neil al., 1976. et I5 Rogers al., 1968. et 24 Capuzzo and Lancaster, 1979b. 7 Factor, 1977. l6 Hadley, 1908. 25 Logan and Epifanio, 1978. 8 Factor, 1978a. z7 Herrick, 1911. 9 Factor, 1978b. I8 Ennis, 1975.

morphosis. In recent studies, the so-called “first postlarval” or “first juvenile” stage has been given as stage V (Ennis, 1975; Sastry and Zeitlin-Hale, 1977; Capuzzo and Lancaster, 1979a, b) or stage IV (Neil et al., 1976; Factor, 1977, 1978a, b; Logan and Epifanio, 1978). Actually, some of the changes result from the molt separating

stages III and IV (especially morphological,anatomical, and some physiological changes) while others are not completed until the end of stage IV or during stage V (primarily ecological, behavioral, and some physiological changes).

Our results showed that two related physiological changes occur in stage IV: (1)


neuroendocrine control of sodium regula- Charmantier, G., Charmantier-Dam-es, M., and Aiken,

tion appears; and (2) sodium regulation D. E. (1984). Neuroendocrine control of hydro-

shifts from isoionic to slightly hyperionic. mineral regulation in the American lobster Horn-

In other words, the juvenile (and probably UYUS americanus H. Milne-Edwards, 1837 (Crus- tacea, Decapoda): 1. Juveniles. Gen. Camp.

adult) type of regulation and neuroendo- Endocrinol., 53, 8- 19. crine control first annears in stage IV. This Charmantier, G., Charmantier-Daures, M., and Aiken,

demonstrates the ir&ortance of the transition from stage III to stage IV in this an- . - imal .

studies indicate the metamornhosis molt In our opinion, results from various

I

D. E. Influence of temperature on osmotic pressure and ionic content of hemolymph in the Amer- ican lobster Homarus americanus H. Milne-Ed- wards, 1837 (Crustacea, Decapoda), in preparation.

occurs between stages III and IV. There- fore, stage III should be considered the last larval stage and stage IV the first post larval. Since benthic behavior is not firmly established until stage V, this could be considered the first juvenile stage, stage IV being called megalopa as proposed by Wells and Sprague (1976). This seems to be a reasonable solution to the problem of termi- nology of the young stages of the American lobster.

ceans. In “Metamorphosis” (W. Etkin and L. I. Costlow, J. D., Jr. (1968). Metamorphosis in crusta-

Gilbert, eds), p. 3-41. Meredith, New York. Costlow, J. D., Jr., and Sandeen, M. I. (1961). The

appearance of chromatophorotropic activity in the developing crab, Sesurmu reticulutum. Amer. 2001. 4, 191.

Dall, W. (1970). Osmoregulation in the lobster Hom- urus americanus. J. Fish. Res. Board Cunad. 27, 1123-1130.

Davis, W. J. (1973). Development of locomotor patterns in the absence of peripheral sense organs and muscles. Proc. Nutl. Acud. Sci. USA 70, 954-958.

Davis, C. W. (1979). Neuroendocrine control of sodium balance in the fiddler crab, Ucu pugilutor. Diss. Abst. Znt. B. Sci. Eng. 39, 3169. ACKNOWLEDGMENTS

This study was carried out at the Biological Station, Davis, W. J., and Davis, K. B. (1973). Ontogeny of a St. Andrews, New Brunswick, Canada. We thank simple locomotor system: role of the periphery in Susan Waddy, Kay Stephen, Eugene Henderson, the development of central nervous circuitry. Debbie Martin-Robichaud, and the numerous other Amer. Zool. 13, 409-425.

staff members who assisted in the conducting of this Ehrenfeld, J., and Isaia, J. (1974). The effect of liga- work. turing the eyestalks on the water and ion perme-

abilities of Astucus leptoductylus. J. Comp. Phy-

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