Zoeal stages of Hiplyra variegata (Rüppell, 1830) (Crustacea: Brachyura: Leucosiidae) reared in the laboratory and collected from plankton at Al-Kharrar creek, central Red Sea

Abstract The zoeal stages of Hiplyra variegata are described and illustrated for the first time. Zoea I was obtained from an ovigerous female, and the zoea II and zoea III stages were captured in the plankton (their identity was confirmed by rearing some of them from earlier stages). Comparisons are made with the only other species of the genus of which larvae are known, i.e. H. platycheir. The presence of a lateral spine on the carapace of zoea I is one of the most important characters that can be used for easy recognition of H. variegata. It is important to note that the third zoea of H. variegata has four small spines at each posterolateral margin of the carapace, in contrast to three spines in other species.


Introduction
The family Leucosiidae Samoulle, 1819 comprises about 476 species in some 66 genera (Ng et al. 2008;Naderloo and Apel 2012). Zoeal and megalopal stages of Leucosiidae are important components of the meroplankton (Quintana 1984). However, the larval morphology is only known for 66 species, including 15 genera; the relevant literature up to 2012 was listed by Al Haj (2012) and Hirose et al. (2012).
Leucosiidae larvae that are known can be distinguished from others by: (1) telson a more or less triangular plate with the posterolateral margins toothed, smoothly round, acute or square; (2) telson fork absent with three pairs of setae in all stages; (3) reduction or absence of the antenna, (4) endopod of the maxillule often unsegmented; (5) setae of the maxillar endopods numbering four or, more frequently three; (6) endopod of the second maxilliped unsegmented or incompletely segmented and with the setation reduced; (7) reduction of setation of the endopod of the first maxilliped; (8) pleon with five pleomeres in all stages (Rice 1980a(Rice , 1980bHirose et al. 2012).
Crabs of the family Leucosiidae are common in dredging-grounds on sandy bottoms (in littoral and sublittoral soft sediment), although some species can be found at depths of approximately 2500 m (Lebour 1928;Schembri 1982). They are one of the most diverse brachyuran families (Naderloo and Apel 2012). In females, the egg mass is not visible externally. Eggs are completely covered, protected and hidden by the large pleon which fits tightly under the carapace within a deeply excavated sternum (Lebour 1928;Ng et al. 2008).
Five species of leucosiid larvae have been identified and illustrated in detail in the central Red Sea to date. These are Hiplyra variegata, Leucosia anatum, Philyra sp. 1, Philyra sp. 2 (which might be P. granigera) and Arcania sp. (possibly A. brevifrons) (Al Haj 2012). Leucosiid larvae are among the most abundant brachyuran larvae in the northern and central Red Sea (Echelman and Fishelson 1990;Al Haj 2012) and their highest densities clearly appeared at inshore stations of the central Red Sea (Al Haj 2012). Galil (2009) re-examined the old genus Philyra and divided it into eight genera. Among those, Hiplyra comprises six species distributed in the Indo-West Pacific: H. elegans Gravier, 1920;H. longimana A. Milne Edwards, 1874;H. michellinae Galil, 2009;H. platycheir De Haan, 1841;H. sagitta Galil, 2009;and H. variegata (Rüppell, 1830). Recently a new species, H. ramli, was described from the Gulf of Oman by Naderloo and Apel (2012). Only H. variegata has been recorded from the Red Sea (Galil 2009).
Under laboratory conditions, Philyra and Hiplyra complete their larval development from zoea I to megalopal stages within at least 12 days, in two, two, three and four days, with respect to the individual larval stages (Hashmi 1968;Terada 1979Terada , 1984Krishnan and Kannupandi 1990;Ko 1996Ko , 2000Ko , 2001. The duration of the megalopal stages was recorded as being five days (Hashmi 1968;Krishnan and Kannupandi 1990;Ko 2000), while Quintana (1986a) found that the development of the megalopa to the early first crab stages took one month.
Hiplyra variegata is distributed in the western Indian Ocean, East Africa (Mombasa, Kenya), Red Sea, Gulf of Aden, Arabian Gulf and Gulf of Oman (Naderloo and Türkay 2012). This small-sized species was briefly described and illustrated by Rüppell (1830) from the Red Sea. A more elaborate description and illustration is given by Galil (2009).
For one species of Hiplyra, H. platycheir (De Haan, 1841) (as Philyra platycheir), the larvae have been described from material obtained by rearing and from plankton (Quintana 1986a(Quintana , 1986bKo 2000;Ghory and Siddiqui 2008;Sakhaie et al. 2010) from Korean, Iranian and Pakistani waters, respectively. The aim of the present study was hatching, rearing and describing the zoeal stages of H. variegata for the first time.

Material and methods
An ovigerous female specimen of H. variegata was collected by hand dredge from a sublittoral soft sediment habitat on 28 February 2012 for rearing and describing the zoea I stage. Other stages (zoea II and zoea III) were collected from Al Kharrar creek, located between latitudes 22°45ʹ and 23°00ʹ N and longitudes 38°45ʹ and 39°00ʹ E, northwest of Rabigh city and 169 km north of Jeddah, central Red Sea in March 2012 using a plankton net of 342 µm mesh size.
The planktonic samples (400 larvae) were sorted in a plastic container (2 l). Active larvae (20) from each zoeal stages of H. variegata were separated and reared in 800 ml bowls under laboratory conditions. This allowed them to grow and metamorphose into the next zoeal stages (Quintana 1986b).
The ovigerous crab was kept in a small aquarium (200 ml); the seawater was changed every day until hatching took place. The larvae hatched on 2 March 2012. Soon after hatching, the healthy and actively swimming larvae (100 larvae) were removed and separated into five large bowls (20 larvae in 800 ml of seawater). The temperature was kept constant at 27°C, salinity at 37‰ and photoperiod at 12 h light and 12 h dark (Al Haj and Al Aidaroos 2014). The larvae were subjected to moderate aeration and fed with the rotifer Brachionus sp., together with algae.
Some randomly selected active larvae and planktonic samples were kept and preserved in formaldehyde 4% for dissection. The dissection was done in polyvinylelactophenol using a WILD stereo-microscope. Drawings of appendages were made using a camera lucida attached to an Olympus BH-2 microscope equipped with differential interference contrast (DIC).
As suggested by Clark et al. (1998) at least five replicates of each appendage were drawn to determine any variations. The individual zoeal stages were described and fully illustrated. The sequence of the zoeal descriptions is based on the malacostracan pleomere plan from anterior to posterior. Setal armature of appendages was described from proximal to distal segments and in order of endopod to exopod. The long antennular aesthetascs, and the long plumose natatory setae of the first and second maxillipeds were drawn truncated (Clark et al. 1998). Figures were made to scale with aid of a camera lucida and ocular micrometer.
Measurements (nearest to 0.01 mm) were based mainly on five specimens of each larval stage. A micrometer was used for measurement of carapace length (CL) from the base of the rostral spine to the posterolateral carapace margin.
The female of Hiplyra variegata was deposited in the collection of the Senckenberg-Museum (Frankfurt, Germany) under the catalogue number SMF 40833.

Results
The larvae hatched on 2 March 2012 after three days of collection. High mortality rates were observed in zoeal hatching. After three days, all larvae had died. Zoea I was compared with zoea I captured in the plankton and all characters were confirmed. From the collected plankton sample, live H. variegata larvae of each stage were isolated and transported to bowls (20 larvae in 800 ml of sea water) with medium aeration. After one day, some of the zoea I and zoea II had metamorphosed into zoea II and zoea III respectively. The successfully moulted zoeal stages had the same characters as larvae captured in the plankton. Zoea III did not successfully moult and thus did not reach the megalopa stage.
Telson (Figure 4c): round and triangular plate with 3 small spines at each posterolateral margin; posterior margin concave, with 3 pairs of serrulate setae arranged in a single row, innermost 2 setae approximately twice longer than outer seta, telson forks absent.

Discussion
Recently, through the work of Galil (2009), the taxonomy of Hiplyra species has changed. Hiplyra platycheir is considered an exclusively eastern Asian species while, for the Indian Ocean material, a new species H. sagitta Galil, 2009 has been introduced. Therefore, the larval descriptions hitherto published under the name of P. platycheir certainly belong to two species. Only Ko (2000) and Quintana (1986aQuintana ( , 1986b refer to H. platycheir proper, while specimens from the Gulf (Sakhaie et al. 2010) and Pakistan (Ghory and Siddiqui 2008) presumably refer to H. sagitta Galil, 2009. The morphological features of the first zoeal stage of H. variegata correspond to those that define the zoeas of Leucosiidae according to Rice (1980aRice ( , 1980b and Hirose et al. (2012).
Characteristics of zoea I of H. variegata reared for the present study differ somewhat from those described for H. platycheir (Ko 2000 [in Korea]), presumed H. sagitta (Sakhaie et al. 2010 [in Iran]) and H. aff. platycheir (presumably also H. sagitta Galil 2009 or something different from that) (Ghory and Siddiqui 2008 [in Pakistan]) ( Table 1). One of the important characters of H. variegata that can be used to distinguish it from related species is the presence of a lateral spine on the carapace which is absent in other species.
There are some differences in the morphology of zoea I of H. platycheir reported by Ko (2000) and the presumed H. sagitta described by Sakhaie et al. (2010) and by Ghory and Siddiqui (2008). The number of aesthetascs of the antennule and the distal segment of the endopod of the first maxilliped are different. The specimens from the Gulf have four aesthetascs and one seta, while in the Pakistan specimens there are two aesthetascs and two setae (three aesthetascs and one seta in H. variegata), also the Gulf specimens have only four setae on the endopod of the first maxilliped while five setae are present in the other specimens (Table 1). Hirose et al. (2012) mention that the types of setae in each structure (such as the maxillule, maxilla, and the first and second maxilliped) are the main distinguishing characteristics of the four species of Persephona (Leach, 1817), but there are no large differences between the setae types of the four species of Hiplyra, except for the basial endite of the maxillule in zoea II (Tables 1, 2).
The lateral swollen protuberance of the carapace appears in zoea II of H. platychira (Ko 2000). Also the numbers of aesthetascs and seta of the antennule were different in the case of H. variegata (the present study) (6 + 1 versus 5) and also the plumose setae of the exopod (scaphognathe) maxilla have different numbers (12 versus 9) ( Table 2).
from Pakistan. It is important to note that zoea III of H. variegata has four small spines at each posterolateral margin, in comparison with three spines in the other species. The dorsomedial setae on the first pleomere of the Pleon can be used to distinguish between the different presumed species (three in Korea and Japan, four in the presumed H. sagitta from Pakistan) and H. variegata (five in the Red Sea). Also the numbers of aesthetascs of the antennule and plumose setae of the exopod (scaphognathite) of the maxilla were different in the two species of Hiplyra (Table 2). These differences could also point towards the Pakistan specimens not belonging to the presumed H. sagitta at all, but to another unknown species.

Disclosure statement
No potential conflict of interest was reported by the authors.