Larval development and metamorphosis of the American lobster Homarus americanus (Crustacea, Decapoda): Effect of eyestalk ablation and juvenile hormone injection

GENERAL AND COMPARATIVE ENDOCRINOLOGY 70, 319-333 (1988)

Larval Development and Metamorphosis of the American Lobster Homarus americanus (Crustacea, Decapoda): Effect of Eyestalk

Ablation and Juvenile Hormone Injection

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

*Laboratoire de Physiologie des Inverttbrts, Universite’ des Sciences et Techniques du Languedoc, 34060 Montpellier Ctdex, France, and tFisheries and Environmental Sciences, Department of

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

Accepted December 18, 1987

Removal of eyestalks of Homarus americanus on different days and molting stages during larval development revealed that eyestalk tissue is involved in the larval and postlarval molting rhythm and in preparation for metamorphosis as early as the end of Stage II. Eyestalk removal in stages II and III reduced the duration of larval and postlarval stages. Eyestalk removal up to the end of Stage II delayed the completion of metamorphosis by one or two molts and caused additional development stages (designated IVa, IV’, and V’). In this study, the critical stage for eyestalk ablation to delay metamorphosis occurred at the end of molt stage D, of larval Stage II (the seventh day of development at 20”). Injection of juvenile hormone before the critical stage resulted in a few intermediate stage IV’ animals. This study demonstrates the involvement of eyestalk neuroendocrine tissue in the control of metamorphosis and investigates a possible involvement of juvenile hormone. 0 1988 Aca-

demic Press, Inc.

Control of metamorphosis has been studied in relatively few crustacean species and the results have been equivocal (Table 1). Except in Rhithropanopeus harrisii (Free- man and Costlow, 1980), eyestalk ablation usually reduces the length of the intermolt period in megalopa but not in the larval stages, and, in at least four of the seven species studied, eyestalk ablation resulted in extra larval stages and a consequent delay in metamorphosis.

In the opinion of Costlow (1963, 1966a, b, 1968) and Freeman and Costlow (1980), metamorphosis separates the megalopal stage from the first juvenile stage in brachyurans. But, as Le Roux noted (1980, 1984), in Anomura and Natantia the most important morphological and anatomical changes occur during the molt from the last zoeal stage to the megalopa, so this should be considered the metamorphosis molt.

Larval development of the American lob-

ster is easily completed under laboratory conditions and the changes associated with metamorphosis have been known since the work of Herrick (1896) and Hadley ( 1908). We previously described the morphological, anatomical, ethological, and physiological changes associated with metamorphosis in Homarus americanus (Charmantier et al., 1984a). Most of these metamorphic changes are either completed or well under- way during the fourth molt (from stage III, or last larval stage, to stage IV, the megalopa or first postlarval stage). The metamorphosis molt is therefore the fourth molt in this crustacean.

In an earlier study of hydromineral me- tabolism we discovered that if the eyestalks were bilaterally ablated in stage II the fourth molt would produce a lobster that was intermediate between stages III and IV (Charmantier et al., 1984b, 1985a). This suggested eyestalk involvement in the con-

319 0016~6480/88 $1 so Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

320 CHARMANTIER, CHARMANTIER-DAURES, AND AIKEN

TABLE 1 EFFECTOF BILATERAL EYESTALKABLATION ON DURATION OFINTERMOLTPEFUODAND OCCURRENCEOF

EXTRALARVAL STAGES IN VARIOUS CRUSTACEANS

Species Duration of intermolt period Extra

larval stages?

Palaemonetes pugiob

Palaemon macrodactylus’ Palaemonetes variansd

Callinectes sapidusef

Rhithropanopeus harrisiigf h

Sesarma reticulatumif

Pisidia longicornisi

No effect No effect Reduced duration of stage

preceding metamorphosis Reduced length of megalopal stage No effect Reduced length of zoeal and

megalopal stages Reduced length of megalopal stage;

no effect on zoeae Reduced length of megalopal stage;

no effect on zoeae

No Yes

Yes

No data Yes No data

Yes

No?”

a Small number of observations. e References: bHubschman, 1963; “Little, 1969; dLe Roux, 1984; Tostlow, 1963;fCostlow, 1968; gCostlow,

1966a; ‘Freeman and Costlow, 1980; i Costlow, 1966b; j Le Roux, 1980.

trol of metamorphosis and led to the present study in which we used eyestalk ablation to examine that control.

Eyestalks of young H. americanus have been ablated as early as stage IV (Rao et al., 1973; Mauviot and Castell, 1976; Trider et al., 1979), but this was too late in development to yield information on the control of metamorphosis. Intermediate fourth stage individuals have also recently been observed by Snyder and Chang (1986) after eyestalk removal in stage II.

We bilaterally ablated the eyestalks of H. americanus at selected points during larval stages I, II, and III and assessed the effect on metamorphosis. We sought to determine whether it was possible to consistently produce additional intermediate stages before metamorphosis, and whether there is a critical stage during larval development after which eyestalk ablation has no effect on metamorphosis.

We also injected juvenile hormone into larval stages I, II, and III to determine if this would interfere with metamorphosis and result in the production of intermediate stages. The involvement of juvenile hormone in the control of insect larval development is well known (review in Wiggles-

worth, 1985) and is under investigation in crustaceans as well (reviews in Hertz and Chang, 1986; Laufer et al., 1987).

MATERIAL AND METHODS

Lobsters and facilities. Larvae were obtained between August and November from six different females held in the Lobster Culture Facility at the Bio- logical Station, St. Andrews, New Brunswick, Can- ada. Some larvae were from females held more than 6 months at 20” to accelerate embryonic development. Others were from ovigerous females that were held 2-3 months at 2-4” to delay embryonic development (Aiken and Waddy, 1985). When larvae were required, these ovigerous females were transferred to 11” for 2 days, then to 20” until hatch (l-3 weeks).

Larvae were reared in natural photoperiod in 40-liter larval tanks (Hughes et al., 1974). Seawater at 20” and 30-31%0 salinity was supplied to the tank and dis- charged. A different larval tank was used for each day of hatch so the different larval groups would be as homogeneous in their development as possible. All larvae were fed to slight excess twice daily with frozen adult brine shrimp (Artemia).

Eyestalk ablation. Eyestalks of individuals in stages I, II, and III were removed under a dissecting microscope by severing the stalk at the articulation with fine forceps. Eyestalk ablations were scheduled either by day of development (experiments l-3) or molt stage (experiments 4,5).

Each group of ablated larvae was held communally in a separate tank. In addition, 40 larvae ablated in the middle of stages II and III were held individually in

CONTROL OF LOBSTER METAMORPHOSIS 321

50-ml plastic compartments with screened bottom to allow water exchange. These were suspended in the circulating seawater of a larval tank.

To facilitate feeding by larvae in the communal tanks, water circulation was interrupted 3-4 times a day. This allowed food and larvae to settle on the bottom of the tank. Ablated larvae showed a distinct pref- erence for brine shrimp instead of other larvae, so can- nibalism was not a significant problem.

Animals were counted and examined daily until the fifth or sixth stage (fourth stage in the case of operation in stage I). Control larvae in experiment 1 were stocked at the standard density of 2000-3000 per tank. In subsequent experiments four categories of control larvae were used:

C-l : held in individual compartments, natural daylength, C-2: 2OOO/tank communally, natural daylength; C-3: 200/tank communally, natural daylength; C-4: 200/tank communally, constant darkness.

Control group C-4 was established to estimate the effect of vision on food capture ability and therefore development rate. All control groups were examined daily for survival and development. Examination of control group C-4 was done before the first feeding each day. Larval stage duration was determined either from individually reared lobsters and expressed as a mean with confidence intervals, or from communally reared lobsters and expressed as minimum and maximum values.

Molt stage determination. Larvae were classified as “postmolt” for the first 12-20 hr after the molt. Sub- sequent molt stages were determined according to the method developed by Drach and Tchernigovtzeff (1967) and adapted for the lobster by Aiken (1973) and Rao et al. (1973). Live animals were placed in a drop of seawater on a microscope slide and the posterior edge of the telson and the tip of pereiopod-4 were examined under a dissecting microscope for evidence of epider- ma1 retraction dactylus.

and the formation of new setae or a

Larvae that reached the middle of the larval stage but showed no epidermal retraction were classified stage C. If there was some indication of epidermal retraction, especially in the telson, the lobster was classified stage D,. Stage D, is characterized by retraction of the new setae and the new dactylus. Begin- ning and end of D, was estimated from the rate of setal retraction and the appearance of barbules at the tip of the dactylus (present only in late D1). In D,, maximum epidermal retraction is attained in telson and pereiopod, the epidermis at the edge of the telson becomes refringent, and barbules are well developed on the new dactylus. As stage D, lasts for only 2-5 hr, some con- fusion with the end of D, is possible.

Titrations. For hemolymph samples, individuals were dried between sheets of filter paper and he-

molymph was then drawn by inserting a glass micropi- pet dorsally between cephalothorax and abdomen and into the heart. Osmotic pressure measurements were made with a Clifton nanoliter osmometer requiring 30 nanoliters of hemolymph.

Juvenile hormone injections. Juvenile hormone (C 18 JH, JH-I), supplied by Roussel-Uclaf, France, was dissolved in peanut oil for injection. Injected larvae in molt stage C of larval stages I, II, and III received either 1 l.~l of peanut oil (control animals) or 1 ~1 of solution containing 10 ug of JH. Injections were done through fine hand-drawn glass needles inserted into the abdominal muscle. Injected larvae (and a group of intact control larvae) were held in individual compartments and examined daily for survival and development. For animals that reached the fourth stage, morphometrics were determined according to a previously described protocol (Charmantier and Aiken, 1987).

RESULTS

Effect of Eyestalk Ablation on Development

Larval stages II and III in eyestalk ablated lobsters are similar to the same stages in intact animals. In contrast, three different types of larvae result when a destalked stage III lobster molts, depending on timing of ablation relative to larval and molt stage. We have designated these different types of destalked larvae as stages IVa, IV’, and IV (Fig. 1) (the stages are described in detail in Charmantier and Aiken, 1987). Stage IVa is intermediate between normal stages III and IV; stage IV is morphologically, biometri- cally, and behaviorally similar to intact stage IV. Stage IV’ has normal fourth stage behavior but differs from the normal stage IV in possessing abdominal dorsal spines and long exopodites on the pereiopods (Ta- ble 2). These larval characteristics disap- pear after the following molt, resulting in a normal stage V.

Intermediate stage IVa is larger than stage III and nearly as large as normal stage IV. The carapace length of 2&30 lobsters was 3.20 + 0.07 mm, 3.95 2 0.05, and 4.07 + 0.12 mm in stages III, IVa, and IV, respectively. However, stage IVa differs markedly from stage IV and retains several important characteristics of stage III (Table


FIG . 1. Control (c) stages III and IV, and eyestalkless (es-) stages IVa, IV’, and IV that result from eyestalk ablation in stage II.

2). Stage IVa appears to be a true intermediate stage, and when it molts it may be- come a normal appearing stage V or it may retain some of the characteristics of stage IV and be classified as stage V’. Stages IVa and IV’ were found infrequently among normal intact lobsters (approximately 3-5%0 stage IVa and l-2%0 stage IV’); rearing in constant darkness (control group C-4) did not increase this frequency.

Osmotic Regulation

“intermediate” stages and the physiological development characteristic of metamorphosis (Charmantier et al., 1981, 1984a, b), we compared osmotic regulation in a dilute medium in nonablated (intact) stages III, IVa, IV’, and IV. Seawater was diluted to an osmotic pressure of 500 mOsm/kg (about 17%0). Results are summarized in Fig. 2. Stages III and IVa were slight hyperosmo- conformers, while regulation in stages IV and IV’ was slightly hyperosmotic. No significant difference existed between stages

To assess the relationship between the III and IVa or between stages IV and IV’. morphological development of these However, the differences between stages

TABLE 2 MORPHOLOGICAL AND BEHAVIORALCHARACTERISTICSOFINTACTSTAGES III AND IV INHO~UW

americanus COMPAREDTOSTAGES IV’ AND IVa THATRESULTFROM EYESTALKREMOVAL

III IVa IV’ IV

Shape of body Bent Flagellum of antenna Short Propodite of pereiopod- 1 Short Exopodite of pereiopods Long Dorsal abdominal spines Present Lateral spines of telson Long Uropods Short Swimming appendages Exopodite Chelae swimming position Downward Food searching behavior Passive Agonistic behavior Normal

Bent Short Intermed Long/intermed Present Long Long Exopodite Downward Passive Normal

Straight Medium/long Long Variable Present Short Long Pleopods Forward Active Aggressive

Straight Long Long Short/absent Absent Short Long Pleopods Forward Active Aggressive


100 r

PI Ea II! ls! Stages

FIG. 2. Difference between the osmotic pressures of hemolymph and medium (HL-MD) in a medium at 500 mOsm/kg (about 17%0) in stages III, IVa, IV’, and IV. Each bar represents the mean value of determinations from 10 (III), 7 (IVa), 5 (IV’), and 12 (IV) animals, with confidence intervals.

III and IVa on the one hand and IV and IV’ on the other, were highly significant (P < 0.001).

Effect of Eyestalk Ablation on Development Rate

Results were obtained from animals held both individually (Fig. 3) and communally (Table 3). In those held individually, eyestalk removal in the middle of stage II or III decreased the duration of the following stages as compared to controls (P < 0.05 for stage III and P < 0.001 for the fourth and fifth stages; see Fig. 3). In destalked fourth and fifth stage lobsters there was no significant difference between groups destalked during stages II and III.

Results from communally reared animals show the same trend (Table 3). Control group C-2 (2000 per tank) had the fastest rate of development, close to those found in C-l (held individually). Control group C-3 (200 per tank in natural photoperiod) and C-4 (200 per tank in continuous dark) both showed longer stage durations and slower rates of development.

Eyestalk ablation in stage I delayed the molt to stage II. However, stages II and III

were accelerated, so the total development time to fourth stage was relatively un- changed.

Eyestalk removal in stage II dramatically reduced development time to the fourth and sixth stages. All stage durations were decreased, including larval stage III and to a lesser extent stage II itself. However, when the operation was performed at the end of stage II, the molt to stage III was delayed by l-2 days, but the subsequent stages were shortened. The duration of stage III remained the same whether the lobsters molted to an intermediate stage IVa or to a normal stage IV.

The same results followed eyestalk removal in stage III: slightly reduced duration of stage III (except delayed molt if the operation occurred late in III) followed by decreased duration of fourth and fifth stages.

Effect of Eyestalk Removal on Types of Fourth and Fifth Stages

In experiment 1, 100 larvae were ablated at the end of stage II. Among the 29 surviv- ing stage III animals that molted, 55% molted to stage IVa and 45% molted to stage IV.

In experiments 2 and 3, 3950 larvae from two hatches were ablated on different days during stages I, II, and III. Results are summarized in Table 4 and Fig. 4. The mortality rate was 100% when eyestalks were removed in the first half of stage I, but it progressively decreased when the operation took place in stages II and III. In controls, survival to completion of fourth stage was 60-80% in communal rearing, 8&85% in individual rearing.

When eyestalks were removed by Day 4 (middle of stage II), 100% of the resulting third stage larvae molted to stage IVa. The proportion of IVa decreased when the operation was performed toward the end of stage II and dropped to zero when done in stage III. Proportions of stage IV were roughly the reverse of stage IVa. Day 7 proved to be the critical time: an operation


20-

15 -

2 2 10 -

5- z

;L

0’ I II 4 th

E St-In

/ ESC-,m

/

5 th Staqes

FIG. 3. Duration of larval and postlarval stages in control and destalked lobsters reared individually. Each bar indicates the mean value of measurements from 9 to 36 animals, with confidence intervals. Data from experiment 2. C, controls, ES’-‘& ES’-‘III, lobsters destalked in the middle of stages II or III.

100

a -1

o---o--o

Ea

\ ‘0 --0 .--.H’

\

\

\

: u 50 G

\ 0 / l \

\ / \

\ \

0 t--I-s 0 .- 7’

s~-+-A’“- t----r---y--_---=-, b”y--p

1 I I 1 1 J.-II 2 3 4 5 6 7 8 10 12 Days

A * / I II III Stages

FIG. 4. Percentage of the different categories of fourth stage lobsters occurring after eyestalk removal at different days of development in stages I, II, and III. Data from experiments 2 and 3.

100 c o---o- w-0

IPa ‘1, .

\ “\ :

/ lx

I I I I I I B C Do “I D2

A

II III

Moulting Stages

Larval Stages 1 ES(-)

FIG. 5. Percentage of the different categories of fourth stage lobsters occurring after eyestalk removal at different molting stages in larval stages II and III. Data from experiment 4.


TABLE 3 DURATION(DAYS)OF LARVALAND POSTLARVAL STAGES OFINTACTAND EYESTALKS Homarus americanus

Controls C-lb C-2” c-3 c-4

ES( - )d IE 11s IIM IIE 111s IIIM IIIE

I

3.0 + 0.2 2.5-4 2.5-4 2.5-4

4-5.5

II

4.9 2 0.2 4-5 47 47

3.5-4 3-4.5 4-5 5-7

III

5.6 + 0.3 5-5.5 6-7 6-7

4.5-8 3-5 3-6 4-6 4-6 5-6 7-8

IVa-IV” V”

15.3 2 0.7 16.8 k 0.7 15-17 15-17 16-19 17-20 16-19 18-20

5-7 7-8 5-7 7-9 5-7 7-9 6-8 7-10 6-9 7-10 7-8 8-10

I to IVth

12-16 12-14 13-17 13-17

12-17 lo-14 l&14 12-15 lo-13 lo-13 13-15

I to VIth

4348 42-47 46-56 47-56

23-26 24-27 2428 25-29 25-29 27-30

a Includes intermediate forms. b Means and confidence intervals for this group only. ’ For this and subsequent groups, numbers are minimum and-maximum days duration. d ES( -) eyestalk ablated; S, start of stage; M, middle of stage; E, end of stage. 9

on Day 7 resulted in nearly equal proportions of stages IVa and IV. Stage IV’ ap- peared only when the operation was performed at the end of stage II or beginning of stage III. Similar results were obtained under both individual and communal conditions.

Stages IV and IV’ molted to normal stage V. Of stage IVa, 70-80% molted to stage V and M-30% to stage V’ (Table 5). No relationship to day of operation could be detected.

In experiments 4 and 5 the molt stage of the animal at time of eyestalk ablation was

related to subsequent development pat- terns. In experiment 4, 827 larvae in stages II and III were ablated at different molting stages. Results are summarized in Table 6 and Fig. 5. Eyestalk ablation in larval stage II resulted in 100% stage IVa if done before the end of molt stage D,. The proportion of IVa decreased when the operation was performed in D, and D, and dropped to zero when ablation was done immediately after the molt to stage III. Proportions of stage IV were roughly reciprocal. Stage IV’ resulted only from ablations done in molt stage D, and D, of larval stage II and molt

TABLE 4 RELATIONSHIPBETWEEN STAGEANDDAYOFEYESTALKREMOVAL ANDTHETYPEOF STAGE IV LOBSTER

THAT RESULTS

Operation stage: I I I I II II” II II III III” III

Day: 0 1 2 3 4 5 6 7 8 10 12

Operated larvae (N) 500 500 200 500 450 450 200 250 400 400 100 Mortality at IV (%) loo loo 98 98 83 71 75 71 60 62 28 Incidence (%) of IVa 100 100 100 91 90 55 0 0 0

IV’ 0 0 0 1 4 3 7 1 0 IV 0 0 0 8 6 42 93 99 100

Note. Data from experiments 2 and 3. a Pooled data from individual and communal holding conditions.


TABLE 5 RELATIONSHIP BETWEEN STAGE AND DAY OF EYESTALK REMOVAL AND THE TYPE OF STAGE V LOBSTER

THAT RESULTS FROM STAGE IVa

Day operated in stage II: 4 5 6 7 Total

Stage IVa (ZV) 76 115 45 40 276 Mortality at stage V (%) 53 49 31 53 48 Incidence of stage V (%) 69 81 84 79 79 Incidence of stage V’ (%) 31 19 16 21 21

Note. Data from experiments 2 and 3.

stages A and B of larval stage III. This suggested a critical molt stage for the delay of metamorphosis.

To identify this critical stage, 151 larvae in stage II were ablated at different care- fully selected premolt stages (experiment 5). Results are summarized in Table 7. Eye- stalk removal up to the beginning of molt stage D, resulted in 100% stage IVa. Oper- ations at the end of D, and in D2 resulted in progressively decreasing incidence of stage IVa, increasing incidence of stage IV, and a small proportion of stage IV’.

Effect of Juvenile Hormone on Development Rate and Types of Fourth Stage

A total of 95 larvae were injected with oil or JH solution. Their survival at fourth stage is given in Table 9.

JH injection performed in stage C of larval stages I, II, and III did not significantly affect the duration of the larval development up to the fourth stage (Table 8).

The effects of oil and JH injections on morphometrics of fourth stage are summa-

rized in Table 9. The morphometrics of intact and oil-injected animals were comparable at stage IV. Most stage I and II larvae that were injected with JH developed into normal stage IV postlarvae, but some retained the long exopodites and dorsal abdominal spines characteristic of intermediate stage IV’. The incidence of stage IV’ depended on the larval stage at the time of JH injection, being respectively 33, 15, and 0% after injection in stages I, II, and III.

DISCUSSION

Effect of Eyestalk Removal on Development Rate

As previously noted (Charmantier et al., 1985a), subsequent larval development is dramatically accelerated when eyestalks are ablated in stages II and III. Snyder and Chang (1986) also demonstrated accelerated development in larval stages II and III, and others (Rao et al., 1973; Mauviot and Castell, 1976; Trider et al., 1979) have reported acceleration in the postlarval stages.

In most species, eyestalks do not influ-

TABLE 6 RELATIONSHIP BETWEEN MOLT STAGE AT TIME OF EYESTALK ABLATION IN STAGES II AND III AND

INCIDENCE OF THE DIFFERENT TYPES OF METAMORPHOSIS SUBSEQUENTLY OBTAINED

Larval stage at operation Molt stage at operation Number of ablated lobsters Mortality at Stage IV (%) Incidence (%) of stage IVa

stage IV’ stage IV

Note. Data from experiment 4.

II II II II II III A/B C JAI D, D* A/B 200 140 148 123 96 120 87 90 77 72 54 50

100 100 100 70 25 0 0 0 0 6 9 3 0 0 0 24 66 97


TABLE 7

RELATIONSHIP BETWEEN MOLT STAGE AT TIME OF EYESTALK ABLATION IN STAGE II AND INCIDENCE OF THE DIFFERENT TYPES OF METAMORPHOSIS SUBSEQUENTLY OBTAINED

Molt stage at ablation C& Ablated larvae (N) 32 21 25 57 16 Mortality at stage IV (%) 69 67 56 56 44 Incidence (%) of stage IVa 100 100 100 44 22

stage IV’ 0 0 0 4 11 stage IV 0 0 0 52 67

Note. Data from experiment 5.

ence molting frequency until after metamorphosis (Costlow 1963, 1966b, 1968; Le Roux, 1980; see Table 1). R. harrisii is the only species in which eyestalk removal reduced the length of the zoeal as well as the postmetamorphic megalopal stage (Free- man and Costlow, 1980).

Effect of Eyestalk Removal on Metamorphosis

Eyestalk removal before a critical point in larval development resulted in additional or intermediate stages. Concern that sight deprivation might have impaired larval food gathering ability and therefore indirectly induced intermediate stages was ruled out: control larvae reared in constant dark (group C-4) yielded very few intermediate stages whereas up to 100% of eyestalk ablated larvae were intermediate stages, here called IVa, IV’, and V’. In Fig. 2 and Table 2 these stages are compared to normal stages III and IV both physiologically and morphologically. In low salinity media

TABLE 8 MEANS AND 95% CONFIDENCE INTERVALS OF THE

DURATION IN DAYS FROM INJECTION TO MOLT TO FOURTH STAGE IN CONTROL AND JH-INJECTED

LARVAE OF Homarus americanus

Stage at Injection

I II III

Control + oil 16.3 +- 1.4 11.1 + 0.8 2.6 + 0.6 Injected + JH 16.3 z!z 1.0 11.5 + 0.7 3.2 2 0.5

Note. Number and percentage of survivors at fourth stage as in Table 9.

stage III larvae nearly osmoconform, while stage IV are able to slightly hyperregulate (Charmantier et al., 1984a, b). Stage IVa retained the morphological features and the osmoregulatory capabilities of a larval lobster and required one to two additional molts to lose its larval features and attain the postlarval condition. We therefore consider stage IVa to be an additional larval stage, intermediate between III and IV.

Stage IV’, in contrast, is more of a postlarval lobster. Although it retains a few morphological features of the larval lobster, the behavioral and osmoregulatory abilities are postlarval. We consider it to be a postlarval lobster which has retained some of the morphological characters of the previous larval stage.

It appears that these two forms are a con- sequence of the time of ablation relative to a critical point in larval development. If ablation occurs well before the critical point, complete metamorphosis is delayed by one to two molts (stages IVa, V’). If ablation occurs close to the critical point, the lobster undergoes physiological metamorphosis but retains some larval morphology (stage IV’).

Our initial experiments indicated that this critical point in larval development occurred on the 7th day at 20” (end of stage II) (Charmantier et al., 1985a). Subsequent experiments refined this estimate to the end of molt stage D, in larval stage II (II-Dr). Why then did approximately 20% of those ablated in stage II-D, molt to IVa instead of the expected stage IV? These are probably


the result of incorrect molt staging. Late stage D1 is easily confused with early D,. Twenty percent of those classified as D2 could have been in late D1 and as a result not able to complete all the morphological changes.

These experiments show that eyestalk ablation before the critical stage delays, but does not prevent, metamorphosis in larval American lobsters. Similar delays in metamorphosis following eyestalk ablation have been noted in four other species: R. harrisii (Costlow, 1966a), Sesarma reticulatum (Costlow, 1966b), Palaemon macrodactylus (Little, 1969), and Palaemonetes varians (Le Roux, 1984). R. harrisii is of par- ticular interest in that Costlow (1966a) demonstrated a critical stage on Day 3, during zoeal stage III.

We did find small numbers of stages IVa and IV’ with intact eyestalks among the control animals. Such extra or intermediate forms were described by Williamson (1905) for Homarus gammarus and Templeman (1936) for H. americanus. Wells and Spra- gue (1976) reported intermediate forms during a study of the effect of crude oil on larval development in H. americanus.

Judging from descriptions and drawings, the stage reported by Williamson corre- sponds to our stage IV’, and that of Tem- pleman to our stage IVa. Templeman reported an incidence of 1 to 8%, and Wells and Sprague (1976) noted that incidence increased from 1.5 to 11.8% after exposure to crude oil. All these authors considered their occurrence to be a result of such unfavor- able conditions as pollutants, insufficient food, or inadequate temperature and salinity. This is corroborated by our finding of up to 10% of these stages among larvae ex- posed to brine from a potash mine (Char- mantier et al., 1985b).

Effect of Juvenile Hormone on Development Rate and Metamorphosis

Injected JH-I had no effect on the duration of the larval period up to the fourth

stage. When injected during molt stage C of larval stages I and II, i.e., before the critical stage, a few intermediate stage IV’ were produced, and the highest incidence occurred after injection in larval stage I.

The effect of JH or JH analogs on larval development of crustaceans has so far been studied only by immersion. In the cirriped Balanus galeatus (Gomez et al., 1973; Ra- menofsky et al., 1974) and the cladoceran Daphnia magna (Templeton and Laufer, 1983) these substances failed to delay metamorphosis, although they did alter larval development slightly. In another cirriped, Elminius modestus, JH analogs induced size and morphological abnormalities, with formation of intermediate nauplius-cypris stages and unattached adults (Tighe-Ford, 1977).

In the decapod R. harrisii the JH mimics methoprene and hydroprene reduced larval survival and increased the duration of zoeal development but did not inhibit metamorphosis (Christiansen et al., 1977a, b). How- ever, Costlow (1977) noted “morphological abnormalities” in megalopae after exposure to methoprene. In H. americanus, Hertz and Chang (1986) found that immersion of larvae in solutions of JH-III pro- longed the time to metamorphosis and resulted in slight changes in morphometrics of postlarvae, which however did not retain any larval features. The difference between their results and those reported in this paper could be related to the kind of JH used or to the methods of JH administration (immersion in their case, injection in ours).

In our experiment the retention of larval features after JH injection could also have been a teratological effect of high hormone dose, but this seems unlikely since there is no comparable effect when the injection is performed in larval stage III, after the critical point.

Endocrine Control of Metamorphosis

The method by which eyestalk tissue controls metamorphosis is still unknown.



In Palaemonetes varians, Le Roux (1984) found that eyestalk removal on Day 1 of zoeal stage II shortened the stage preceding metamorphosis. He felt that because of this accelerated development there was insufficient time for completion of the morphological changes associated with metamorphosis, and suggested that metamorphosis was controlled by a balance between MIH and the molting ecdysteroids. Our results do not support such an hypothesis. Eyestalk ablation in stage II does reduce the length of stage III, but the effect on metamorphosis is entirely dependent on the molt stage during stage II at which the ablation is done. Although the duration of stage III is the same in either case, the effect on metamorphosis is completely different.

Snyder and Chang (1986) also obtained intermediate stages after ablating eyestalks from stage II Homarus larvae, but they concluded that eyestalk tissue is not involved in mediating the morphological as- pects of lobster metamorphosis. They as- cribed the abnormal larval development to the poor quality of frozen Artemia they used as food, but this ignores the fact that there were no intermediate stages among their control larvae that were also fed frozen Artemia. Furthermore, the molt stages of their animals at time of ablation were not known. In our experiments (Tables 6, 7; Fig. 5) molt stage at time of ablation had a significant effect on the type of intermediate stage that resulted. This supports an hypothesis of eyestalk involvement in the control of metamorphosis and the existence of a critical point at the end of stage Di. We suspect that if food does influence the proportions of intermediate stages, it does so by influencing the activity of the eyestalks.

So the question remains-how do the eyestalks regulate metamorphosis in crustaceans? Do their neuroendocrine products exert a direct control over metamorphic changes, or is their influence expressed indirectly through control over the produc-

tion of or tissue response to a juvenalizing factor?

Costlow (1966a, b) proposed the direct approach. For the brachyurans R. harrisii and S. reticulatum he suggested that prior to the critical period the eyestalks release a factor, different from MIH or the ecdysones, that controls the changes associated with metamorphosis. This hypothesis was subsequently accepted by Little (1969) to explain the responses of the natantian Palaemon macrodactylus. Both authors stress the basic difference between this mechanism and that in insects, where ablation of corpora allata causes precocious (rather than delayed) metamorphosis (Wig- glesworth, 1934, 1936, 1940a, b, 1948).

If this is the mechanism utilized by Homarus, our data suggest the eyestalks must release this substance in a brief pulse toward the end of stage II. We found that ablation in stage I or stage II before the critical point produced intermediate stage IVa, a result that seems incompatible with continuous release of a metamorphosis factor over this same period.

Eyestalk ablation in Homarus delays metamorphosis but does not block it. This suggests that substances involved in metamorphosis continue to be released after ablation, but at a much slower rate. If so, the site of production must be outside the eyestalk complex, possibly in the “brain.” Given the undeveloped state of the sinus gland at this stage (Pyle, 1943), the site of release within the eyestalk complex is also an enigma.

An alternative hypothesis involves juve- nilizing factors, which would bring the control of crustacean metamorphosis closer to that in insects. Substances with juvenile hormone activity have been isolated from adult Homarus (Schneiderman and Gilbert, 1958), and methyl farnesoate, a likely pre- cursor of JH that is secreted by the mandibular organs (Laufer et al., 1985, 1986a; Borst et al., 1985) has been detected in


adults of several decapod species, including H. americanus.

The production of JH-like compounds by mandibular organs in adult Libinia emarginata is regulated by eyestalks (Laufer et al., 1986b, 1987), but these results, like those of previous studies (Payen and Cost- low, 1977; Hinsch, 1981), associate the activity of JH with reproduction. However, if JH-like substances are also produced during earlier stages of decapod development they might also be responsible for the juvenilization seen during the first three larval stages of Homarus. Under normal conditions the juvenilization would cease with the fourth (metamorphosis) molt, and our experiments suggest the eyestalks are involved in this change. If, starting late in stage II, the eyestalks begin releasing (but not producing) a factor that nullifies the ju- venilizing effect of these hypothetical JH- like substances, eyestalk removal during stages I and II would tend to prolong the juvenilization and delay metamorphosis. This is precisely the effect we obtained. Also consistent with this hypothesis is the fact that JH injection in stage III (after the critical stage) did not interfere with metamorphosis.

On the other hand, injection of JH before the critical stage should have resulted in intermediate larval stages. In our experiments injected JH did not induce the appearance of the true intermediate stage IVa, but it did cause larval characteristics to be retained in up to one-third of postlarvae (stage IV’). These results resemble those in R. harrisii (Costlow, 1977) and Elminius modestus (Tighe-Ford, 1977) in which abnormal or intermediate forms were obtained. A confirmation of the involvement of JH in the control of metamorphosis of Homarus would therefore require additional injections of various JH compounds at different doses and the successful titra- tion of JH-like substances during larval development.

Neither of these hypotheses can be con- firmed or rejected on the basis of work reported here. These experiments have ex- panded our understanding of the role of eyestalk tissue in the control of metamorphosis and defined the critical stage at which this eyestalk control is expressed.

ACKNOWLEDGMENTS

This study was carried out at the Biological Station, St. Andrews, New Brunswick, Canada, and supported by grants from the National Research Council of Can- ada and the Minis&e Francais des Relations Ex- terieures and a grant from NATO-Conservatoire Na- tional des Arts et Metiers. Susan Waddy, Michael Ea- gles, Wilfred Young-Lai, and others at the Biological Station assisted in this study. We thank Professor Pi- erre Lasserre for loan of the osmometer, the Scientific Division of Roussel-Uclaf for providing the JH, and Professors G. Vernet and J.-P. Trilles for comments on the manuscript.

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Larval development and metamorphosis of the American lobster Homarus americanus (Crustacea, Decapoda): Effect of eyestalk ablation and juvenile hormone injection