Intersex females in the red claw crayfish , Cherax quadricarinatus ( Decapoda : Parastacidae )

Cherax quadricarinatus is a large freshwater crayfish species (Parastacidae) native of north-west Queensland and the Northern Territory of Australia. The species typically exhibits a gonochoristic sexual system, although in cultured populations various types of intersex individuals have been described as functional males. In the present study, the macroscopic morphology and the gonadal histology of one type of intersex are described and discussed. All intersexes having both pairs of genital openings (female and male openings) and lacking both appendix masculinae and red patches were functional females with normal ovaries and oviducts. From a histological point of view, they did not differ from normal females having previtellogenic and/or vitellogenic ovaries according to size. Rev. Biol. Trop. 55 (Suppl. 1): 25-32. Epub 2007 June, 29.

Cherax quadricarinatus (von Martens, 1868) is a large freshwater crayfish native of north-west Queensland and the Northern Territory of Australia which is intensively cultured for economic purposes in Australia and many other countries in southern Asia, North and South America and Africa (Lawrence andJones 2002, Edgerton 2005).Although many biological aspects related to the culture of C. quadricarinatus have been studied, including reproduction, growth and nutrition (Jones 1995a,b, 1997, Khalaila et al. 1999, Lawrence and Jones 2002, García Guerrero et al. 2003a,b, Karplus and Barki 2004, Shechter et al. 2005, among others), the sexual pattern of this species and its hormonal regulation is not fully understood.
C. quadricarinatus is considered a gonochoristic species with a bilaterally symmetrical reproductive system.In females, it consists of a pair of ovaries, oviducts and genital openings at the base of the third pereiopods, while in males a pair of testes, vasa deferentia and genital openings at the base of the fifth pereiopods are observed (Sagi et al. 1996).Males also present appendix masculinae at the base of the fifth pereiopods.An important male secondary sexual character is the soft red patch which is found on the outer surface of the propodus of the male claw giving to this species the common name of "red claw" crayfish (Thorn andFielder 1991, Karplus et al. 2003).In this species, a variable proportion of several types of intersex animals can be found in which both male and female genital openings occur in different combinations (Thorn and Fielder 1991, Medley and Rouse 1993, Sagi et al. 1996).Morphological, biochemical and endocrinological studies of intersexes have demonstrated that these individuals are functionally males with previtellogenic ovaries (e.g., Sagi et al. 1996, Khalaila and Sagi 1997, Khalaila et al. 1999, Sagi et al. 2002, Shechter et al. 2005).
According to some investigations done in our laboratory in which we observed some individuals having both pairs of genital openings being ovigerous "females" and hatching normal juveniles, we decided to carry out a macroscopic and histological analysis of the reproductive system of these intersex animals to achieve a better understanding of intersexuality in C. quadricarinatus.

MATERIALS AND METHODS
Female (n=33) and intersex (n=41) C. quadricarinatus purchased from a local dealer (San Mateo Farm, Buenos Aires, Argentina) selected for this study ranged between 19.6 and 47.3 mm carapace length and between 1.9 and 24 g body weight.All selected females presented their genital openings at the basis of the third pereiopods, while intersex individuals presented both genital openings at the base of the third and fifth pereipods, respectively (Fig. 1).None of them had appendix masculinae nor red patches.Crayfish were cold-anaesthetized at -20 °C during 15 min.The carapace was removed and the gonads were inspected recording their size, form and colour.Subsequently, they were quickly dissected and fixed in Bouin's solution for 4h at room temperature.Gonads were then sequentially passed through 90 % ethanol for 20 min, 96 % ethanol for 20 min, 96 % ethanol-buthylic alcohol (1:1 v/v) for 30 min and buthylic alcohol for 30 min, and embedded in paraffin.Sections (5-6 μm thick) were stained with haematoxylin-eosin.At least three slides for each crayfish were inspected under light microscope (López-Greco 1997).

RESULTS
Upon macroscopic and microscopic inspection, the gonads from females and intersex individuals presented an identical pattern.Both females and intersexes showed paired ovaries constituted by a modified H-shaped structure located within the cephalothorax, consisting of a pair of anterior lobes and a pair of posterior ovarian lobes.In the median part of the gonad, both ovarian lobes were very close and surrounded by the ovarian theca resembling an impair structure.A short and straight oviduct extended laterally from each posterior ovarian lobe toward the coxae of the third pereipods (Fig. 2).No evidence was observed of the presence of an atrophied male reproductive system (neither testicular tissue nor vasa deferentia).
According to their colour, immature ovaries were transparent to white, intermediate ovaries appeared as pale-orange, and mature ovaries were dark green, both in females and intersexes.From a histomorphological point of view, the ovary of C. quadricarinatus females and intersexes belongs to the "mandibulate type" (Krol et al. 1992, Ando andMakioka 1998).It is a sac-like structure where the proliferative zone, containing mainly oogonia, is located in the center of the gonad next to the ovarian lumen, while oocytes are surrounding the nests of oogonias in a more peripheral position.In immature ovaries, only germinative centers containing oogonia surrounded by primary vitellogenic oocytes and follicular

1A 1B 1C
cells were observed in the center, while in maturing and mature ovaries both primary and secondary oocytes were found (Fig. 2).The ovarian lumen was usually seen both in immature and mature ovaries.

DISCUSSION
The present study demonstrates the presence of intersex females in C. quadricarinatus, a species in which intersexuality has been described as different types of functional males (Sagi et al. 1996, Khalaila et al. 1999).These intersex females posses vitellogenic ovaries, being in accordance with the intersex "ovigerous" females observed.
According to the observations in previous and the present studies, we consider that more research is needed to clarify the patterns of intersexuality in C. quadricarinatus.In accordance with Sagi et al. (1996), we also observed other intersex crayfish in which one or the two appendix masculinae occurred, and these individuals were uni-or bilaterally males, although female genital openings were present, too.It seems that for being intersex males there must be at least one appendix masculina.Since the development of this secondary sexual character is under the hormonal control of the androgenic gland, it is necessary to present at least a unilateral male reproductive system that allows differentiating as a functional intersex male.Intersex males usually present one or two red claws in accordance with the role of the androgenic gland on the differentiation of this character, too (Sagi et al. 1997).
It has been stated that crustacean culture can be improved by selecting monosex stocks (Sagi et al. 1986, Curtis and Jones 1995, Lawrence and Morrissy 1997, Lawrence et al. 2000).In C. quadricarinatus, both males and females have a greater somatic growth in a monosex culture than in a mixed culture; moreover, isolated males have a significant higher growth rate than females (Curtis and Jones 1995).Therefore, the early identification of sex could be important to improve the growth rate of juvenile stages by means of a monosex culture, as well as to increase the probability of inducing the sexual reversion of both females and the described type of intersex juveniles to males, and the administration of sex-specific inducers of growth and reproduction.Our previous studies have demonstrated that secondary sexual characters in C. quadricarinatus appear early during the development, at stages VI-VIII for females, males and intersex females (Vazquez et al. 2004).
According to Hobbs (1974Hobbs ( , 1988) ) and Vogt (2002), the present Astacoidean taxa have evolved from a gonochoristic species; for the South American parastacids it has been proposed that intersexuality may be an adaptation for their burrowing life-style (these species dig deep and complex burrows where they live) (Rudolph and Almeida 2000).Burrowing crayfish species were classified by Hobbs (1942) into three general categories (primary, secondary and tertiary burrowers) according to the complexity of the burrow and the habitat where the species are distributed (Gherardi 2002).According to this criterion, South American species can be considered primary burrowers (with the exception of Samastacus spinifrons) and Cherax species tertiary burrowers.C. quadricarinatus burrows simple, not deep caves and they are not permanent occupants of the burrow.For this reason, in the case of C. quadricarinatus, we do not consider that intersexuality is related to its burrowing life-style.However, we can not discard that the presence of intersex individuals could be related to culture and/or laboratory conditions (e.g., density, sexual proportion, stress) since no evidence of intersexuality has ever been reported from animals captured from nature.
Further research must be carried out on the development of primary and secondary sexual characters of south hemisphere parastacids to understand the role of the habitat and/or social organization of the species and the underlying mechanism modulating the pattern of sexual differentiation.In addition, more evidence is needed to address the question if intersexuality is an ancestral or derived character among freshwater crayfish.

Fig. 1 .
Fig. 1.Cherax quadricarinatus in ventral view.A. Intersex female with female and male gonopores.B. Female with the corresponding gonopores at the basis of the third pereipods.C. Intersex female with male gonopores and eggs attached to the pleopod setae.