New distribution record of the brine shrimp Artemia ( Crustacea , Branchiopoda , Anostraca ) in Tunisia

The genus Artemia Linnaeus, 1758 comprises a number of sexual species and several parthenogenetic populations. In the New World, only two sexual species are present: Artemia franciscana (Kellogg, 1906) and Artemia persimilis (Piccinelli and Prosdocimi, 1968). In the Old World, where both sexual and parthenogenetic populations occur, five sexual species have been described: Artemia salina (Leach, 1819), Artemia urmiana (Günther, 1890), Artemia sinica (Cai, 1989), Artemia sp. (Pilla and Beardmore, 1994) and Artemia tibetiana (Abatzopoulos et al., 1998).

Artemia is an anostracan crustacean, occurring in every continent except Antarctica (Triantaphyllidis et al. 1998). It is a typical inhabitant of inland salt lakes, coastal salt lagoons and solar saltworks (Persoone and Sorgeloos 1980). The brine shrimp is able to overcome the severe physiological demands imposed by these habitats, due to a set of various adaptations, the most salient of which is probably an interchangeable (diapausing cysts versus nauplii) life cycle. In fact, the life cycle of Artemia can begin as an embryo within a dormant cyst. Depending on environmental conditions, embryos can enter into diapause and arrested development for many years and are capable of surviving a very wide range of environmental conditions (Clegg and Trotman 2002). Artemia was first described from saltpans of Lymington, Hampshire (England) by Schlösser in 1755 (Kuenen and Baas-Becking 1938).
Leach (1819) named this taxon Artemia salina (Artom 1931), but Bowen and Sterling (1978) suggested the binomen Artemia salina to be restricted to the extinct population at Lymington (England) and use the binomen Artemia tunisiana to describe bisexual populations in the Mediterranean area. Browne (1988) confirmed that all strains sampled from the Mediterranean region are able to interbreed and should be classified as Artemia salina. Mura (1990) based on the comparison of the frontal knob morphology, using scanning electron microscopy, reported that in one hand there are no significant differences between Lymington and North African Artemia populations and on the other hand, North African populations cannot be separated from the Italian populations.
Lately, Barigozzi and Baratelli (1993) suggested to keep the binomen Artemia salina for the Italian populations, while the North African populations could be named Artemia tunisiana. The confusion continued until Triantaphyllidis et al. (1997) using Amplified Fragment Length Polymorphism (AFLP), confirmed that all strains from the Mediterranean Basin should be classified as Artemia salina, and that Artemia from Mediterranean Basin can be grouped in two subclusters (the Eastern Mediterranean basin group and the Western Mediterranean basin group).
However, the Western Mediterranean Basin shows the unfortunate event of the presence of the American species Artemia franciscana, causing a great change in the Artemia populations' biodiversity in this region (Amat et al. , 2007. This event was initially stated in Portugal (Hontoria et al. 1987in Amat et al. 2007, in France (Thiery and Robert 1992), in Spain, in Morocco and in Italia (Amat et al. 2007), but there are no information about the existence of the exotic brine shrimp A. franciscana in Tunisia.
The presence of Artemia in Tunisia was first reported by Seurat (1921) and Gauthier (1928) in Chott Ariana and Sabkhet Sidi El Hani, respectively. Later, Ben Abdelkader (1985), Sorgeloos et al. (1986), Romdhane (1994), Triantaphyllidis et al. (1998) and Romdhane et al. (2001) announced the occurrence of Artemia populations in 10 other sites. Since then, Artemia reported from the Tunisian sites was used as a biological material and as a reference for several scientists (e.g. Léger et al. 1986;Abatzopoulos et al. 2002;Bossier et al. 2004). From then no other Artemia sites have been announced in the review of the distribution of the genus Artemia in Tunisia.  In this work 49 Tunisian inland and costal saline lakes and saltworks were investigated, aiming to inventory the Artemia sites (Table 1 and Figure  1). The geographical coordinates were obtained by a GPS receiver (GARMIN GPS 12). Cysts were harvested from the shore of the sites, and adult specimens were collected with a plankton net (150 Ām mesh size). The mating behaviour, the presence or absence of males was recorded in situ and after laboratory culturing. For this purpose, nauplii obtained by cyst hatching were made to grow up in 2 l plastic container, with 90 g l -1 filtered and autoclaved sea water plus crude sea salt . Temperature was maintained at 24°C under 16 h light/8 h dark photoperiod. The animals were fed with the unicellular algae Chlorella sp. The medium was completely renewed twice a week with fresh microalgae cultures.
In Tunisia, temporary saline lakes represent 29% of the total wetland area, they are formed by 54 Sabkhas (representing 22% of the total wetland area) and 17 chotts (representing 7% of the total wetland area). During our investigation, 21 different locations characterized as temporal or ephemeral catchments (Table 2), distributed over different hydrogeographical zones, were identified as a refuge of Artemia (in this work, there are no Artemia cysts or adult specimens harvested during our visit to Megrine saltwork. Artemia reported in this site was based on the literature and on the cyst collected on 1998 and stored in our laboratory).
The in situ study of all the sites where adult Artemia were found showed the presence of male and female individuals. This result was confirmed by the laboratory culture (Table 3).
The presence or the absence of Artemia at a particular site can have several explanations. Artemia cysts can be naturally dispersed over long distances by becoming attached to the feathers or after surviving passage through the digestive system of wading birds  or being carried by wind. They were also deliberately inoculated into salt pans for salt production improvement or for aquaculture purpose (Van Stappen 1996). In those habitats which desiccate completely, salinity fluctuations are extreme. High salinity or desiccation kills adult Artemia populations and hence can be the primary factor driving seasonality. However, the prime abiotic factor determining the presence of Artemia in these biotopes is high salinity. Although Artemia is restricted to hypersaline biotopes, other factors such as temperature, ionic composition and biotic interactions also play an important role in the patterns of its distribution (Van Stappen 2002). Lenz (1987) observed that zooplankton population dynamics are influenced by abiotic factors (salinity, temperature and nutrients concentrations) and by biological interaction (predation, competition and grazers). Moreover, the same author identified two critical factors that determine the population dynamics of Artemia: 1) habitat conditions allowing the survival of the animals throughout the year, and 2) predictability of the seasonality of the environment.
It is well known that the brine shrimp Artemia can be found in water with different physicochemical characteristics, because of their physiological adaptation to the hostile biotopes. In fact, Artemia occurs in the evaporation ponds at salinity levels from 80 to 220g l -1 and in rare occasions at salinity up to 340 g l -1 (depending on the strain and/or species) (Camargo et al. 2004). Van Stappen (2002) reported that no optimum can be clearly defined for salinity of the Artemia environment, for physiological reasons this optimum must be situated towards the lower end of the salinity range, as higher ambient salinity requires higher energy costs for osmoregulation. Moreover, Abatzopoulos et al. (2006) signaled the existence of the parthenogenetic populations in temporary brackish-hypersaline water bodies (lagoon around Urmia Lake, Iran), with salinities as low as 10 g l -1 . During this investigation Artemia was found at different salinities ranging from 60 to 330 g l -1 and different conductivities from 82.3 to 242 ms cm -1 (Table 3). Apart from salinity, temperature also affects the distribution pattern of Artemia (Vanhaecke et al. 1987). The maximum temperature that Artemia populations tolerate has repeatedly been reported to be close to 35 °C. However, this tolerance threshold is straindependent (Van Stappen 2002). In fact, Vanheacke et al. (1984) reported that substantial strain differences exist with regard to the resistance for high temperatures: Artemia salina and Artemia parthenogenetica strains did not survive temperatures exceeding 30°C, while 5-10% survival was observed for Artemia franciscana from Great Salt Lake (Utah, USA) and San Francisco Bay (San Francisco, USA) cultured at 34°C. In our survey Artemia was found at different temperatures ranging from 14 to 26.1°C and lethal temperatures of 30-34°C were never reported at the water surface of the visited lakes (Table 3). Vos (1979) reported that the nauplius growth decreases and the overall appearance of adults deteriorate with pH values below 7.0, and he concluded that the optimum pH for Artemia growth ranges from 8.0 to 8.5. In this study different pH values were reported ranging from 7.25 to 8.47 (Table 3).
According to Amat et al. (2005Amat et al. ( , 2007 A. franciscana was proven to be a very successful colonizer, which out-competes the endemic Old World bisexual or parthenogenetic species. More efforts, such as morphological characterization of adult specimens, cultured under standard conditions in order to avoid environmental influences, or biological marker techniques (mitochondrial DNA, polymorphic microsatellite markers) must be invested to characterize autochthonous populations, in order to assess if bisexual Artemia present in Tunisian sites belongs to Artemia salina or to the invasive American brine shrimp Artemia franciscana evidence by Amat et al. (2005Amat et al. ( , 2007 in the Western Mediterranean region.   ----------------