The early phyllosoma stages of spiny lobster Panulirus echinatus Smith , 1869 ( Decapoda : Palinuridae ) reared in the laboratory

The early stages of the Panulirus echinatus were hatched and reared in the laboratory. Ovigerous females were captured in their habitat and carefully transported to the laboratory. Larvae were transferred in a recirculation water tank at a density of 10 larvae.L. The larvae were fed on Artemia and gonads of mussel Brachydonts sp. Microalgae Dunaliella viridis was added at a concentration of 150 x 10 cell.mL. Larvae and exuviae of each zoeal stage were preserved in an alcohol 70% + glycerin (1:1) solution. The phyllosomas moulted eight times; the intermoulting period of each instar averaged about 7 to 10 days. The main morphological changes of each appendage were described in detail, illustrated and compared with previous reports.


Introduction
The Brazilian spiny lobsters of the Panulirus genus have high economic value.The species P. argus (Latreille, 1804) and P. laevicauda (Latreille, 1817) are captured extensively, representing one of the most important fishery resources in the world (Phillips et al., 1980).For a long time another species, P. echinatus, was occasionally captured, however, an increase in their production has been observed (Santiago, 2001).Despite the economic significance that this latter species has represented in the fisheries, few studies concerning their biological aspects have been reported.
Information on the biology of phyllosomas is considered important and necessary to elucidate aspects related to larval biology and culture.The larval development of spiny lobster has been reported for specimens col-lected in the sea (Lewis, 1951;Baisre, 1964;Phillips and Sastry, 1980) and for those reared in the laboratory progressed through the metamorphic puerulus stage (Inoue, 1981;Kittaka and Kimura, 1989;Yamakawa et al., 1989;Matsuda and Yamakawa, 2000).The long larval period (about one year) is considered an important obstacle for phyllosoma culture research.The lack of detailed morphological description of some or all phyllosoma stages has also hindered studies of larval distribution and ecology.
Successful studies on culture from egg to the pueruli stage of Panulirus were achieved for P. japonicus (Von Siebold, 1824) (Kittaka and Kimura, 1989;Yamakawa et al., 1989) and P. longipes (A. Milne-Edwards, 1868) (Matsuda and Yamakawa, 2000).Some attempts at rearing phyllosoma larvae were reported for P. homarus gonads of Brachyodonts sp.Microalgae Dunaliella viridis Teodoresco 1906 were added daily in the culture system.
Samples of larvae were removed after each instar and fixed in 10% formalin solution for 24 hours and preserved in a glycerol+alcohol 70% (1:1) solution.The larvae were dissected under an optical binocular microscope.The illustrations of each larval instar, including appendages, were made using a camera lucida.
For the species P. homarus, information on the average length, number of setae in the apical segment of the 2 nd maxilla and number of the exopod setae of each appendage were based on the illustrations reported by Radhakrishnan and Vijayakumaran (1995).
The terminologies STAGES and INSTAR were used in order to make comparisons with other previously described species.Both terms were extensively used by the authors in descriptions of larval development of the spiny lobster.

On the larval culture
The larval hatching was closely related to the moon phases, in which the majority of the larval hatching was carried out on the first quarter and full moon, indicating that lunar cycle had direct influence in the life cycle in the laboratory.
The phyllosomas readily feed on Artemia nauplii in stages I to III.Then, the phyllosomas seemed to have difficulty in catching Artemia so other foods were tested.Some foods were initially well accepted by the phyllosomas, but later were rejected.This behaviour may be related to nutritional aspects, because many (Linnaeus, 1758) (Radhakrishnan and Vijayakumaran, 1995) and for P. ornatus (Fabricius, 1776) (Duggan and McKinnon, 2003) in which, 10 and 9 early instars were reared and described, respectively.More recently, Abrunhosa et al. ( 2004) described and compared the morphological characteristics of three early instars of P. laevicauda and P. echinatus, contributing to the identification of phyllosomas that occur on the Brazilian coast.These three early larval instars of P. echinatus are again reviewed in the present study.
The present study describes in detail and illustrates the 5 early stages and instars of each stage of the species P. echinatus in the laboratory.Morphological comparisons with larvae of its congeneric species, previously described, are briefly discussed.

Material and Methods
Ovigerous females of P. echinatus were obtained at Iparana Beach (Caucaia/CE) in October 2001 by autonomous diving in the coastal shallow waters.They were transported to the laboratory in plastic baskets (10 L) containing local seawater.
In the laboratory the females were acclimated and then placed in a circular tank (cap.1000 L) with biological filter and constant aeration.
After hatching, the phyllosomas were carefully transferred to three transparent water recirculation tanks at a density of 60 larvae.L -1 .These tanks are similar to those used for culture of many species of spiny lobsters of the family Palinuridae (Kittaka, 1994).The tanks, which were confectioned with acrylic (70 cm Ø and 30 cm deeper), allow free circulation of flowing water and total visualization of the floating elements is possible.Salinity was maintained at 35‰.The larvae were fed Artemia nauplii for the 3 first instars and then, with phyllosomas failed to moult to the subsequent stage.This fact was more evident for larvae in the VIII instar.
The larvae moulted eight times, once at stages I to II, two times at stage III and four times at stage IV (Table 1).
There was not a specific day or night period in which the larvae moulted to the next instar.The moult of the phyllosomas from stage I to II and II to III ranged from 7 to 11 and 7 days, averaging in 9 and 7 days, respectively.For successive instars, it was not possible to determine accurately the intermoult period for later instars, because the phyllosomas were not reared individually.The intermoult average for each instar is shown in Table 1.
The largest mortality of the phyllosomas was on the 4 th day of rearing, when about 50% of the larvae died in both systems.After this period, low mortality rates were observed.

On gross morphology
The morphological features were fully described only for instar I.For the subsequent instars, only main morphological changes were reported.The morphological changes and the main characteristics of each stage are fully described in the Table 2.
Antenna (Figure 2b): Uniramous, 2-incompletely segmented, ending in a short curved spine and 2-3 simple setae, 2 simple setae in the distal region near to the apex.
Third Maxilliped: Endopod well developed, 5-segmented, segment distal with long setae rounding the distal margin and 2 long setae on the sub-distal margin; exopod with 3 pairs of long and plumose setae.
First and second Pereiopods: Endopod 5-segmented, completely developed, distal segment ending in a strong spine, dorsal and ventral sub-exopodal spines absent, coxal spine present but not elongated (disposition of setae as illustrated); exopod with 5 pairs of long and plumose setae.
Abdomen: Short, unsegmented with 3 setae and 1 spine at each ramous on the posterior portion.
First and second Pereiopods: Exopod with 9 pairs of long and plumose setae.
Fourth Pereiopod: More developed in relation to previous instar (as illustrated), lacking setae.
First and second Pereiopods: Exopod with 10 pairs of long and plumose setae.
First and second Pereiopods: Exopod with 11 pairs of long and plumose setae.
Antennule: Segmented, proximal segment longer than the distal one.
First and second Pereiopods: Exopod with 6 pairs of long and plumose setae.
Third Pereiopod: Exopod a little more developed compared to the first instar, lacking setae.
First and second Pereiopods: Exopod with 7 pairs of long and plumose setae.
Third Pereiopod: Exopod well developed compared to previous instars with 3 pairs of long and plumose setae.
First and second Pereiopods: Exopod with 8 pairs of long and plumose setae.
First and second Pereiopods: Exopod with 12 pairs of long and plumose setae.
Fourth Pereiopod: More developed in relation to previous instar (as illustrated), surpassing abdomen.Exopod more developed, lacking setae.
On the other hand, the distinct difference among other species is the presence of coxal spines on P. e chinatus, which is included in group B, according to Gurney, 1936and Baisre and Ruiz de Quevedo, 1982. According to Abrunhosa et al. (2004), phyllosomas of the Brazilian species, P. echinatus and P. argus, bear only the coxal spines while P. laevicauda exhibited both the coxal and sub-exopodal spines placing these species in different groups.
The Intermoult period appears to be distinct for palinurid species and may vary in accordance to the rearing conditions in which the larvae are submitted, such as, adaptation, type of food administered, density, water quality, and others (Kittaka and Ikegani, 1988;Kittaka et al., 1988;Kittaka and Ikegami, 1988;Kittaka and Kimura, 1989;Radhakrishnan and Vijayakumaran, 1995;Kittaka et al., 1998;Matsuda and Yamakawa, 2000;Sekine et al., 2000).Kittaka and Kimura (1989) rearing phyllosomas of P. japonicus, obtained an intermoult period of 6 to 7 days for instar I.However, a gradual increase in that period was observed for the subsequent instars.Matsuda and Yamakawa (2000) observed an intermoult period for the first two instars of P. longipes similar to P. echinatus of 7 to 10 days.But, for P. homarus, cultured individually, this period increased to 8 to 10 days.When P. homarus were submitted to mass culture, this period increased to 12 to 14 days (Radhakrishnan and Vijayakumaran, 1995).These facts may be related to the elevated density.
Feeding is considered a critical factor in the success of the phyllosoma culture.Delay or decrease of the amount of food requested by the larva may prolong the intermoulting period or cause death during the ecdyse (Abrunhosa and Kittaka, 1997).High mortality was observed in the instar IV of P. echinatus.This fact may be related to the nutritional requirement needed for moulting as many phyllosomas failed to moult to the subsequent stage.An interesting study on larval feeding of P. elephas revealed their ability to capture large-sized fish larvae (Kittaka and Abrunhosa, 1997).Presumably, fish larvae may compose the natural diet for phyllosomas in the sea.
First and second Pereiopods: Exopod with 13 pairs of long and plumose setae.
Fourth Pereiopod: Surpassing about 2 times the abdomen.
Fifth Pereiopod: Still bud and lacking setae.

Discussion
In the laboratory, phyllosoma larvae moulted eight times comprising five larval stages considering the developmental criteria of description adopted by the authors in previous studies, Inoue (1978Inoue ( , 1981) ) for P. japonicus, Radhakrishnan and Vijayakumaran (1995) for P. homarus, Matsuda and Yamakawa (2000) for P. longipes and Coutures, (2000) for palinurid species.
The term instar, which corresponds to each moulting of the phyllosoma, was used for palinurid and scyllarid species by Kittaka (1994), Booth and Phillips (1994) and Kittaka and Abrunhosa (1997).In general, the number of setae increased in the exopods of the phyllosoma appendages.In the case of P. echinatus, an additional pair of setae arises on each successive larval moulting (Table 2).Such morphological change appears to be a characteristic of the palinurid species, in which each instar could be considered a larval stage.In this case, comparisons with other species will become easier to observe.
Morphologically, the phyllosomas of P. echinatus are very similar to other palinurid species.At stage I the mean cephalic length is relatively large compared with eat large food items: fish larvae, chaetognaths and hydromedusae.In the current study, the phyllosomas were fed with gonads of mollusk Brachyodonts sp. from instar III and this kind of food has demonstrated to be suitable until instar VIII but it was not effective for instar IX because failure moult was observed on many phyllosomas in the tanks.These facts suggest that further research is required about the feeding and nutrition of P. echinatus.
Water quality is another decisive factor in the success of the phyllosoma culture.Phyllosomas with good activity and free of contaminations by protozoa and bacteria depend on rigid control of the water.Any negligence in the water quality can completely affect the larviculture (Radhakrishnan and Vijayakumaran, 1995).Intensive monitoring of the water quality was accomplished during the culture of P. echinatus.The results of phyllosoma culture may be considered satisfactory compared with those reported previously (Kittaka, 1988;Kittaka et al., 1988;Kittaka and Ikegami, 1988).

Table 1 . Intermoulting period, accumulative days and maim characteristics of each stage for phyllosomas of Panulirus echinatus reared in the laboratory. Stage Intermoult (Mean days) Accumulative days Main characteristics
segmented; exopod of the 1 st and 1 st and 2 nd pereiopods with 13 pairs of setae.*Nophyllosoma succeeded in moulting to the next instar.

Table 2 .
Cephalic length, number of setae of the 2nd maxilla and exopod of maxillipeds and pereiopods of Panulirus echinatus reared in the laboratory.

2 nd maxilla (Distal seg. ) Pairs of setae in the exopods 3 rd maxill.
*The culture finished at the first instar of the stage V (nine instars were reared.)