BULLETIN OF MARINE SCIENCE, 61(1): 73–80, 1997

EFFECT OF STARVATION ON THE FIRST LARVAE OF HOMARUS AMERICANUS (, NEPHROPIDAE) AND PHYLLOSOMAS OF VERREAUXI AND J. EDWARDSII (DECAPODA, PALINURIDAE)

Fernando A. Abrunhosa and Jiro Kittaka

ABSTRACT Food is one of the important factors controlling decapod larval culture, however, little is known about the effect of the starvation regimen on the physiological condition of the lar- vae. In the present study, the influence of starvation upon survival rate and the intermolt period was observed in the first instar of the American , Homarus americanus, the first instars of phyllosomas of the red rock lobster, Jasus edwardsii, and the green rock lobster, J. verreauxi. Larvae were reared in receptacles of 150 ml capacity filled with sea water and submitted to two feeding regimens: larvae were submitted to an initial period of starvation and larvae were submitted to an initial period of feeding. Larvae of H. americanus were cultured individually at 17 - 18°C, while phyllosomas were cultured at five larvae per receptacle at temperatures of 19 - 22°C and 16 - 17° C for J. edwardsii and J. verreauxi, respectively. No larvae succeeded in molting if completely starved or if they were fed after a prolonged starvation period. However, the showed a period of tolerance before food was introduced. The average interval between the first day of feeding and the first day of molting was relatively constant within each species: about 4, 10 and 12 d for H. americanus, J. edwardsii and J. verreauxi, respectively. The starvation tolerance period (50% survival) averaged about 5, 4 and 8 d for these species, respectively. The interval between the ending of starvation and the initial molting period were roughly equivalent although it was shorter in H. americanus than in the Jasus species. The beginning of molting in each species was delayed in accordance with prolonged days of starvation. The feeding period that allowed at least 50% of the larvae to molt to the 2nd instar was 1, 5 and 7 d for these species, respec- tively. Molting in each species began after a rather constant intermolt period (5, 12, 13 d, respectively), regardless of the length of the initial feeding period. These results indicate that the first instar phyllosoma of these Jasus spp. are less tolerant of starvation and require a longer feeding period to molt compared to Homarus larvae.

Decapod larvae usually hatch as zoea and begin to take plant or food. Food supply is considered critical for the successful culturing of such larvae. Food should be provided before the beginning of the feeding stage and the timing of the first feeding has to be syn- chronized just after hatching for Panulirus japonicus (Mikami and Takashima, 1993) and for Homarus americanus (Aiken and Waddy, 1995) or after metamorphosis from nauplius to first zoea stage of penaeid (Hudinaga, 1942; Shigueno, 1975) and naupliosoma to phyllosoma instar (e.g., genus Jasus spp. Kittaka, 1988; Kittaka et al., 1988). Recently, concepts of the “point-of-no-return” (PNR), which is the maximum period (days) of starvation prior to the first feeding which will allow the instar to molt to the next instar, and “point-of-reserve-saturation” (PRS), which is the minimum feeding time (depending on the species and, or, its rearing conditions, hours or days) during which nutrients are reserved to allow the instar to molt into the next instar, (differing for each larvae instar in a complete development series). The concepts have been introduced into larval culture tech- nology (Anger and Darwirs, 1981; Mikami et al., 1994). Estimation of the PNR is consid- ered to give us more practical information on allowable initial non-feeding time because our

73 74 BULLETIN OF MARINE SCIENCE, 61(1): 73–80, 1997 common practice was to begin feeding immediately after hatching. PRS will also give us the possible time to stop feeding before each molt because feeding after some time of PRS is considered unnecessary. Such information will make it possible to improve the feeding schedule, because food such as Artemia nauplii and mussel flesh induces water quality deterioration (e.g., for phyllosomas of , Radhakrishnan and Vijayakumaran, 1995; Kittaka, 1995), thus helping to maintain water quality at a more satisfactory level and keep larvae in a healthy condition. Thus, starvation is not only an interesting subject for studies on digestive physiology but for critical measures in larval culture. Nevertheless, related studies are very few. The effect of starvation on larvae survival was examined for several decapod (spider , Hyas arenaeus (Anger and Darwirs, 1981), king crab Paralithodes camtchaticus (Kurata, 1959), tanner crab Chionescetes opilio (Kon, 1979), red frog crab (Minagawa, 1989). Recently, Mikami and Takashima (1993) and Mikami et al. (1994) studied effect of starvation on phyllosomas of the P. japonicus and a scyllarid sp. Anger et al. (1985) studied change in hepatopancreas of starved larvae of H. americanus in relation to the molting cycle. Phyllosomas are very difficult to culture (Kittaka, 1994). Therefore, knowledge of spe- cific PNR and PRS levels may help to improve method of culture phyllosomas of different species. The present paper reports on starvation effect on two different types of larvae: the first instar of phyllosomas of red rock lobster Jasus edwardsii (H. Milne Edwards, 1875), the green rock lobster of eastern Australia and northern New Zealand, J. verreauxi (H. Milne Edwards, 1851), and larvae of the American clawed lobster, H. americanus (H. Milne Edwards, 1837).

MATERIALS AND METHODS

The first larval instars of H. americanus, J. verreauxi and J. edwardsii were submitted to the follow- ing two contrasting feeding experiments: (1) an initial period of starvation and (2) an initial period of feeding. The larvae were collected after hatching and transferred to a 10-liter aquarium. The apparently healthy larvae were pipetted and transferred into transparent glass containers of 150 ml capacity filled with 100 ml of filtered and sterilized seawater. Each day, larvae in the two experiments were trans- ferred into newly prepared containers, and in accordance with the feeding regimen, they were fed with newly hatched Artemia nauplii at the density of 4 ind. ml-1. The containers were immersed partially into a water-bath with the temperature adjusted to 18 ± 1° C for H. americanus and 19° C (ranging from 19 to 22° C) for J. edwardsii. Phyllosomas of J. verreauxi were exposed to ambient temperature conditions in the laboratory, approximately 16 - 17° C. The photoperiod has an effect on larval development and survival of H. americanus for which the use of near normal light-dark day-lengths (LD 10:14 to LD 14:10) are recommended (Aiken and Waddy for H. americanus, 1995). For phyllosomas, the effect of light and/or day length under labora- tory conditions was not tested experimentally, but they showed a repetitive rotatory movement when exposed to light reflected in the culture receptacles. This behavior ceased in complete darkness. Thus, the larvae of H. americanus were exposed to laboratory day/night photoperiods while the phyllosomas were reared in darkness.

The PNR50 and PRS50 were estimated using the correlation between the number of larvae molting (at both treatments) and the initial feeding period or starvation period taken at each monitored group (Figs. 1,3). Experiments with H. americanus and J. edwardsii were carried out at Sanriku while those with J. verreauxi were done at Nemuro, Hokkaido, Japan. ABRUNHOSA AND KITTAKA: STARVATION IN LARVAE 75

LA RVA E OF H. AMERICANUS. In February of 1994 ovigerous females of the American lobster were placed in a tank with running seawater and constant aeration (salinity 35‰, temperature 14 - 18° C). They were fed mussels, Mytilus edulis, every 2 d. After hatching, the larvae were organized into several groups (6 - 10 individual/group) in accordance with the starvation treatment. The larvae were placed individually in the receptacles because cannibalism in H. americanus is related to their feeding behavior (Atema and Cobb, 1980). Experiment 1 - Larvae of H. americanus submitted to an initial period of starvation. Eight groups were formed with 10 larvae/group (10 replicates), in which they were starved for 0 (i.e., they were not starved) and 1, 2, 3, 4, 6, 8 and 10 d after hatching, except those groups starved 6, 8 and 10 d held 7, 6 and 6 larvae/group, respectively. After the starvation period larvae were fed Artemia nauplii. Experiment 2 - Larvae of H. americanus submitted to an initial period of feeding. Five groups were formed with 10 larvae/group (10 replicates), in which larvae were fed Artemia nauplii for 0 ( i.e., they were not fed) and 1, 2, 3 and 4 d, etc., before being starved, except those groups fed for 0 and 4 d held 50 and 8 individuals/group (i.e., 50 and 8 replicates, respectively).

PHYLLOSOMAS OF J. VERREAUXI AND J. EDWARDSII. After mating and oviposition in the laboratory, egg- bearing females were placed in a tank with running seawater and aeration (salinity 35‰, temperature 15 - 18° C). They were fed mussels every 2 d. Larvae of J. edwardsii that hatched out in March of 1994, and those of J. verreauxi in August of 1995 were used for the experiments. Unlike H. americanus, Jasus phyllosomas are not cannibalistic (Kittaka, 1994). Thus, these larvae were placed in transparent glass containers communally at 5 phyllosomas per container (4 and 5 replicates per group for J. edwardsii and J. verreauxi, respectively). Experiment 1- Phyllosomas submitted to an initial period of starvation. For J. edwardsii, ten groups were formed with 4 replicates (5 larvae/receptacle) in which larvae were starved for 0, 1, 2, ..., 10 d after hatching. For J. verreauxi, ten groups were formed with 5 replicates (5 larvae/receptacles), in which larvae were starved for 0, 1, 2, 3, ... , 10 d after hatching. After the starvation period all larvae were fed Artemia nauplii. Experiment 2 - Phyllosomas submitted to an initial period of feeding. The J. edwardsii phyllosomas were cultured initially in a recirculating water tank used for phyllosomas of the spiny lobster (Kittaka, 1994), and were fed Artemia nauplii. For an initial 8-d period, 20 phyllosomas were transferred daily into separate rearing receptacles (5/receptacle). Thus, 9 groups were formed and each group was fed for 0, 1, 2, 3, ... , 8 d, respectively. The same holding procedure was used for J. verreauxi. Eleven groups of 25 phyllosomas per group (5/receptacle) were fed for 0, 1, 2, 3, ..., 10 d, respectively.

RESULTS

LARVAE SUBMITTED TO AN INITIAL PERIOD OF STARVATION. Tolerance to initial period of starva- tion. No first instar of H. americanus succeeded in molting at entire starvation. The per- centage of molting to second instar was 100% for larvae starved 2 d and 80% for 3 d of starvation. There was a decrease in molting rate for groups starved longer than this period

(Fig. 1). PNR50 for this species was 6.9 d. The molting rate or phyllosomas was different between two species. In J. edwardsii relatively a high molting rate was 52 to 65% until 4 d of starvation, but it decreased drasti- cally on 5th day (10%) when just a single phyllosoma molted. No phyllosoma molted after the 6th day (Fig. 1). On the other hand, J. verreauxi showed more resistance to starvation. Larvae succeeded in molting with about 60 - 80% success after an initial 4 d of starvation

(Fig. 1). The PNR50 was 4.2 days for J. edwardsii and 6.5 days for J. verreauxi. 76 BULLETIN OF MARINE SCIENCE, 61(1): 73–80, 1997

Figure 1. Percentage of successful molting to the 2nd instar from the 1st instar larvae of Homarus americanus and phyllosomas of Jasus edwardsii and Jasus verreauxi submitted to an initial starvation period. Vertical bars indicate standard deviations.

DELAY OF MOLTING BY STARVATION. The interval between the first day of feeding and first day of molting was relatively constant within each species. However, this interval was progressively extended in the groups starved for a period of more than 4 d (Fig. 2a,b,c). For H. americanus (Fig. 2a) this interval ranged from 4 - 6 d, and 9 - 11 and 12 - 16 d for J. edwardsii and J. verreauxi, respectively (Fig. 2b,c).

LARVAE SUBMITTED TO AN INITIAL PERIOD OF FEEDING. For H. americanus a 100% molting rate was shown for groups fed daily and for an initial 3 and 4 d feeding (Fig. 3). Rates were 80 and 50% for groups fed for only the initial 2 d and 1 d, respectively. A molting rate of 80% was shown in J. edwardsii phyllosomas fed for the 5-d period (Fig. 3). However, success in molting, in this experiment, was relatively low for J. verreauxi, compared to J. edwardsii. The survival rate of J. verreauxi phyllosomas appeared relatively low compared to the re- sults obtained for H. americanus and J. edwardsii. This fact may be attributed to a lower Nemuro water quality (Kittaka and Onoda, 1995). Survival averaged about 60% in groups feeding for more than the initial 7-d period (Fig. 3). Larvae of H. americanus that fed every day began to molt on the 5th day. Such synchronized molting was observed in all groups (1- 5 d feeding), though the molting rate decreased in the group fed for only 1 d. Total mortality occurred in those submitted to entire starvation (Fig. 4a). Phyllosomas showed different intermolt periods with larvae beginning to molt in about 10 and 12 d in the case of unstarved larvae of J. edwardsii and J. verreauxi, respectively. This fact may be attributed to the different temperature in which each species was held. However, a similar synchronized initial period of molting (11 and 13 d) was observed for the species in the groups with succeeding molts of larvae, with delay occurring in those groups fed for 4 d for J. edwardsii

and 5 d for J. verreauxi (Fig. 4b,c). The PRS50 was 1, 4.6 and 6.7 d for H. americanus, J. edwardsii and J. verreauxi, respectively. ABRUNHOSA AND KITTAKA: STARVATION IN CRUSTACEAN LARVAE 77

Figure 2. Molting to the 2nd instar from the 1st instar larvae submitted to an initial period of starvation: a. Homarus americanus, b. Jasus edwardsii, c. Jasus verreauxi

DISCUSSION

The length of intermolt period was relatively stable for submitted to short periods of initial starvation, specifically within 3, 4 and 4 d for J. verreauxi, J. edwardsii and H. americanus, respectively. When starvation continued beyond these periods, the intermolt period was extended gradually, probably due to a lowering of digestive function or feeding activity. The intermolt period of the first instar for J. verreauxi, J. edwardsii and H. americanus

was 12, 10 and 4 d, respectively (Fig. 2). Mikami et al. (1994) reported PNR50 at 3.4 d for first phyllosomas of Panulirus japonicus with 6.18 d as the intermolt period for the first instar. Thus, tolerance to starvation is apparently higher for larvae of H. americanus com- pared to phyllosomas. Timing of molting from the first to second instar was relatively stable for animals submit- ted to an initial period of feeding of more than 6, 7 and 1 d for J. verreauxi, J. edwardsii and H. americanus, respectively. When feeding was stopped within these periods, delay or molting with mortality was distinct probably due to a deficiency of nutrients needed to molt 78 BULLETIN OF MARINE SCIENCE, 61(1): 73–80, 1997

Figure 3. Percentage of successful molting to the 2nd instar from 1st instar larvae of Homarus americanus and phyllosomas of Jasus edwardsii and Jasus verreauxi submitted to an initial feeding period. Vertical bars indicate standard deviations

to next instar. The PRS50 at only 1 d of feeding for H. americanus (Fig. 3) may suggest that they store sufficient egg yolk at the first instar. The possibility of such a food reserve was investigated for this species by Anger et al. (1985). They observed that after food depriva- tion the stored lipids in hepatopancreas were reduced markedly and that there was an irre- versible loss of the capability to absorb and store lipids in R-cells of the hepatopancreas after the PNR has been surpassed. Delay of the molting period and molting rate (Fig. 2a,b,c) may indicate that an optimal timing of feeding is needed for these species. Beyond these time limits, decreasing molting rate or mortality usually occurred. Such phenomena were observed also for first larvae of H. americanus when they were fed less than half of the food quantity normally given (Templeman, 1936). Complete larval development of phyllosomas was shown for J. edwardsii (Kittaka et al., 1988), J. verreauxi ( Kittaka et al., in press) and P. japonicus (Kittaka and Kimura, 1989). Sixteen, 168 and 2 individuals metamorphosed into pueruli from the initial 12,000, 1,690 and 20,000 first instars, respectively. Compared to the relatively high survival rate for J. verreauxi, the survival rates for J. edwardsii and P. japonicus were very low. Experiments on initial starvation showed a sudden decrease of survival rate at 10, 5 and 3 d for J. verreauxi, J. edwardsii (present study) and P. japonicus (Mikami et al. 1994), respectively. This may indicate that the first instar of J. verreauxi may retain more egg yolk as compared to J. edwardsii and P. japonicus.

Values of PNR50 were more than 3 d for these spiny lobster species. Generally, phyllosomas are very sensitive to water quality, particularly the variation of microflora (Igarashi et al., 1990, 1991). Although the effects of food quantity and feeding regimen have not been investigated for phyllosomas, excess feeding may not be necessary for them. ABRUNHOSA AND KITTAKA: STARVATION IN CRUSTACEAN LARVAE 79

Figure 4. Molting to the 2nd instar from the 1st instar larvae submitted to an initial feeding period: a. Homarus americanus, b. Jasus edwardsii, c, Jasus verreauxi

The concept of PSR will be applicable also for the culture of the final instar phyllosomas. Usually the phyllosomas (final instar) stop feeding a few days before the metamorphoric molt to the puerulus, presumably because drastic physiological change occurs in their di- gestive organs. Mortality often occurred at late-instar final phyllosomas due to an intestine disease, which seemed to be derived from the atrophy of the intestine before metamorpho- sis.

LITERATURE CITED

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______. 1995. Culture of phyllosomas of spiny lobster and its application to studies of larval recruitment and aquaculture. Crustaceana 66 : 258-270. ______and K. Kimura. 1989. Culture of the Japanese spiny lobster Panulirus japonicus from egg to juvenile stage. Nippon Suisan Gakkaishi 55: 963-970. ______, K. Ono and J. D. Booth (in press) Complete development of the green rock lobster Jasus verreauxi from egg to juvenile. ______and S. Onoda. 1995. Effect of water quality in Zostera zone on larval development of marine invertebrates. Pages 29-33 in The Tenth International Symposium on Okhotsk Sea, Sea Ice & Peoples, February 5-8, Monbetsu, Hokkaido, Japan. 380 p. ______, M. Iwai, and M. Yoshimura. 1988. Culture of hybrid of spiny lobster genus Jasus from egg stage to puerulus. Nippon Suisan Gakkaishi 54: 413-17. Kon, T. 1979. Ecological studies on larvae of belonging to the genus . I. The influence of starvation on the survival and growth of the zuwai crab. Nippon Suisan Gakkaishi 45: 7-9. Kurata, H. 1959. Studies on the larva and the post-larva of Paralithodes camtschatica, I. Rearing of the larvae, with special references to the food of zoea. Bull. Hokkaido Reg. Fish. Res. Lab. 20: 76-83. [In Japanese with English abstract]. Hudinaga, M. 1942. Reproduction development and rearing of Penaeus japonicus Bate. Jap. J. Zool. 10: 305-93. Igarashi, M. A., J. Kittaka and E. Kawahara. 1990. Phyllosoma culture with inoculation of marine bacteria. Nippon Suisan Gakkaishi 56: 1781-6. ______, S. F. Romero and J. Kittaka. 1991. Bacteriological character in the culture water of penaeid, homarid and palinurid larvae. Nippon Suisan Gakkaishi 57: 2255-60. Mikami, S. and F. Takashima. 1993. Effects of starvation upon survival, growth and moulting inter- val of the larvae of the spiny lobster Panulirus japonicus (Decapoda, Palinuridae). Crustaceana 64 :137-142. ______, J. G. Greenwood and N. C. Gillspie. 1994. The effect of starvation and regimes on survival, growth and the moulting interval of cultured Panulirus japonicus and Thenus sp. phyllosomas (Decapoda; Palinuridae & Scyllaridae). Crustaceana 68: 160-9. Minagawa, M. 1988. Influence of the starvation on growth, feeding success and external morphology of the Ranina ranina (Crustacea, Decapoda, Raninidae) Suisanzoshoku 36: 227-230. [In Japanese with English abstract]. Radhakrishnan, E. V. and M. Vijayakumaran. 1995. Early larval development of the spiny lobster (Linnaeus, 1758) reared in the laboratory. Crustaceana 68: 151-9. Shigueno, K. 1975. Shrimp culture in Japan. AITP (Assoc. Int. Tech. Promot.). Tokyo. 153 p. Templeman, W. 1936. The influence of temperature, salinity, light and food conditions on the sur- vival and growth of the larvae of the lobster Homarus americanus. J. Biol. Bd. Can. 2: 487-97.

DATE ACCEPTED: October 15, 1996.

ADDRESS: Research Institute for Marine Biological Science, Research Institutes for Science and Technology, The Science University of Tokyo, Onnemoto 74, Nemuro, Hokkaido 087-01, Japan.