Turkish Journal of Zoology, 31: 171-180 (2007)
Biometrical study of Artemia urmiana (Anostraca: Artemiidae) cysts harvested from the Urmia Lake (West Azerbaijan, Iran)
Alireza Asem*1, Nasrullah Rastegar-Pouyani1 and Naser Agh2
1) Department of Biology, Faculty of Science, Razi University, 67149 Kermanshah, Iran
2) Artemia and Aquatic Animals Research Institute, Urmia University, Urmia, Iran
Abstract:
Artemia urmiana has been reported from the Urmia Lake by Günther in 1899. The objectives of this study were to investigate the diameter of untreated and decapsulated cysts and chorion thickness in 26 stations of the Urmia Lake. One-Way ANOVA (Tukey Test, p<0.05) was used, for analyzing the data. Only 31 pair means of untreated cysts among 26 stations showed significant differences, but, on the other hand, 157 pair mean of decapsulated cysts among stations showed significant differences. Then there is high variation in cyst diameter among the different localities in the Urmia Lake. It has been shown that this lake consists of recognizable sections producing cysts with different hatching characteristics, which can be used for diverse aquacultural purposes.
Key Words: Artemia urmiana, Urmia Lake, Iran, untreated cyst, decapsulated cysts, chorion thickness, Hatching Efficiency
Introduction
The different populations of the Branchiopod Artemia, scattered over all temperate and tropical areas of the world, have become one of the most interesting models for systematic, taxonomic and evolutionary studies (Hontoria and Amat, 1992; Gajardo et al, 2002). The complex of species in the genus Artemia have wide variety of adaptive processes, mainly related to survival in extremely hyperhaline environments (Abreu-Grobois and Beardmore, 1982; Browne and Bowen, 1991; Hontoria and Amat, 1992). Van Stappen (2002) published an updated list, covering 600 Artemia site, and completed it with geographical coordinates and information about reproduction mode and species name. Bisexual populations of Artemia are distributed both in New World (A. franciscana Kellogg 1906, A. persimilis Piccinelli and Prosdocimi 1968) and Old World (A. urmiana Günther, 1899 A. sinica Cai 1989, A. tibetiana Abatzopoulos et al 1998, A. salina Linnaeus 1758). Schlöِsser made the first description of the brine shrimp, Artemia in 1755 from Lymington, UK (Kuenen and Bass-Becking, 1938). Günther (1899) described Artemia urmiana from the Urmia Lake as a bisexual species. This species is endemic to Urmia.
Taxonomists are still puzzled about the evolution and phylogenetic relationship of the Artemia species (Triantaphyllidis et al, 1997). Different methods have been used in order to characterize the species of the genus Artemia. The most relevant methods are comparison of biometrical and morphological characteristics, electrophoretic patterns of different allozymes and cross-fertility test and electron microscopic Survey of the morphology such as frontal knob and penis. (Abreu-Grobois and Beardmore, 1982; Mura, 1990; Hontoria and Amat, 1992; Triantaphyllidis et al, 1997; Torrentera and Belk, 2002).
Ionic composition of the habitat can produce ecological isolation and can result in morphological and biometrical differences (Bowen et l, 1985, 1988; Hontoria and Amat, 1992).In this paper we study biometrical variation of cyst from 26 different sampling sites in the Urmia Lake. We try to analyze their biometrical characteristics (diameter of untreated and decapsulated cysts and chorion thickness) using multivariate analysis.
Materials and Methods
Field study:
In this study we have chosen 26 stations in the middle, northern and southern parts of the Urmia Lake (Fig. 1). Four primary ecological factors were measured for each station: salinity (0.5 m from surface and 0.5 m from depth), PH (0.5 m from surface and 0.5 m from depth), depth and transparency, also date of sampling recorded (Agh., 2004)
Diameter of Untreated cysts:
Sample preparation
Prior to analysis, cyst samples containing a considerable amount of debris, which might interfere with the analysis, should be washed and cleaned. In any case, all salt or salty water should be removed before hydration.
Sample hydration
The cyst diameter is normally determined on fully hydrated cysts, when the shape of the embryos becomes spherical. Generally cysts are fully hydrated after incubation at room temperature for at least 2 hr (Lavens and Sorgeloos, 1987). Nevertheless, some strains or batches may require longer periods to reach maximal hydration (Vanhaecke et al., 1980). Therefore, a small but representative cyst sample (a few hundreds of individual cysts are sufficient) is incubated in a small vial for at least 12 hr at room temperature (20-25°C). Freshwater can be used as incubation medium. In order to inhibit metabolic activity within the cysts, a few drops of lugol solution are added. Neither the addition of the lugol’s solution, nor the storage temperature has a significant influence on the final volume of the cysts (Vanhaecke et al., 1980).
Measurements and calculation
The maximum diameter of at least 100 cysts is determined under a microscope, equipped with a calibrated eyepiece. The cysts are measured at random to avoid biased results. The cysts that are measured are taken at random, so that no bias occurs towards e.g. bigger cysts. Cracked or hatching cysts, empty cyst shells, obviously not fully hydrated cysts etc. are not taken into account. Average value and standard deviation are calculated, and data are expressed in µm up to 1 decimal.
Diameter of decapsulated cysts:
Cysts are decapsulated according to the following procedure (Bruggeman et al., 1980): Wash and purify sample, if necessary (see above)
· Hydrate cysts by incubating them for 2h in freshwater in a tube with aeration from the bottom at room temperature (20-25°C)
· Bring hydrated cysts into a tube with a minimum of fresh water; equal volume of liquid bleach (NaOCl) and a few drops of NaOH (to increase pH and obtain a fast chemical reaction). The gradual decapsulation of the cysts can be followed under a stereoscopic microscope.
When decapsulation is completed (removal of cyst shell after 3-15 minutes, depending on concentration of chemicals and on ambient temperature), rinse thoroughly over 100-125 µm sieve to remove decapsulating chemicals. Prolonged exposure of decapsulated embryos to high concentration of NaOCl may result in gradual degradation of embryos (and hence problematic measurement) and should therefore be avoided. When the decapsulated cysts are uniquely used for biometrical measurement, the hatchability of the embryos is not an issue; when the batch of decapsulated cysts is also used for hatching, a more strict modus operandi has to be followed in order to maintain maximal viability of the embryos (Lavens and Sorgeloos, 1996).
· Take samples to measure minimum 100 embryos (see ‘Diameter of untreated cysts’). Atypical embryos (i.e. not fully hydrated cysts; hatched cysts, embryos with hatching membrane not completely filled with yolk mass) should not be taken into account.
Chorion thickness:
This value is calculated as follows:
chorion thickness = (Mean diameter of untreated cyst– Mean diameter of decapsulated Cyst)/2
Consequently, this value is presented without standard deviation. (Vanhaecke and Sorgeloos, 1980)
Statistical analysis:
Biometrical variation of cysts from different location in the Urmia lake was determined by One-Way ANOVA (Tukey test, p<0.05) and Hierarchical Cluster Analysis using the computer program SPSS 11.5.
Results
Some physico-chemical parameters for the 26 sampling locations are shown in Table 1. The diameter of the untreated and decapsulated cysts and chorion thickness are summarized in Table 2. Also these results are shown in Figure 2 and 3. The statistical comparisons of the results are given in Table 3 and Table 4. The biggest untreated cysts were found at N3-1 station (259.34±11.36µm) and the smallest size at M3-2 station (247.63±11.47µm). The Biggest decapsulated cysts were found at M2-2 (251.6±11.24µm) and smallest at N1-2 (231.29±10.43 µm) sire. The thickest chorion was observed in N4-1 (9.37µm) and the thinnest in the cyst chorion belongs to the cysts harvested from M3-2 (1.31µm). According to Tables 3 and 4, only 31 pair means of untreated cysts among 26 stations showed significant differences, but, on the other hand, 157 pair mean of decapsulated cysts showed significant differences.
With regard to Hierarchical Cluster Analysis, that used means of untreated and decapsulated cysts and chorion thickness, 26 stations are divided into six main groups. These groups form two clusters. First cluster has two categories that isolated in 20-25 span. Second cluster also has two categories that divided in 15 span (Figure 4).
Discussion
This study offers an opportunity to expand our knowledge on biometrical variations of A. urmiana from the Urmia Lake. Vanhaecke and Sorgeloos (1980) have done a comprehensive study on biometrical variation of Artemia strains from different geographical origins. According to their findings Artemia cysts could be categorized into three groups:
1. The smallest cysts belong to the Adelaid-strain and Artemia from San Francisco Bay area, including the SFB inoculated strain from Macau and Barotac Nuevo.
2. The parthenogenetic strains from China, France, Italy and India are characterized by large cysts.
3. Strains with cysts of intermediate size but with very thin chorion which is characteristic for A. franciscana from Chaplin Lake and Great Salt Lake.
Hontoria (1990), studying 14 A. franciscana populations, found a diameter ranging between 217 µm and 230 µm, with the exception of Great Salt Lake (Utah, USA) and Galera Zamba (Colombia) cysts, with 242 µm and 245 µm diameter. Zhenqiu et al, (1991) collected cysts from Xinjiana Uighur and Shandong. They showed that all of these samples belong to parthenogenetic populations and their cysts size is larger than those of bisexual species. Pilla and Beardmore (1994) reported diameter of untreated cysts for A. urmiana, A. sinica and Artemia sp. (RUS). Mean cyst size differ significantly between the three populations (p<0.001). Cysts of A. urmiana are bigger (265.82±15.85µm). Duncan's test for multiple comparisons of mean indicates that cyst diameters of Artemia sp. and A. sinica (230.05±15.14µm and 232.75±11.22µm) are also significantly different (p<0.01) from each other. Triantaphyllidis et al (1996) characterized two Artemia populations from Namibia and Madagascar. They showed that diameter of untreated cysts from these locality were 247.7±11µm, 285.9±11.6µm and for decapsulated cysts, 233.1±9.8µm, 246.2±11.7µm respectively. They suggested that the cysts from Namibia were significantly smaller compared with those from Madagascar. According to the results of Abatzopoulos et al (1998) A. tibetiana is the biggest ever recorded for both bisexual and parthenogenetic species (323±11.2µm and 230±14.6µm). Cohen et al (1999) found a diameter ranging between 246.1±21µm and 230.3±1µm from four Argentinean Artemia populations.
Comparing our results with those of other researchers it could be said that the cyst diameter of A. urmiana exhibits the wider range in the Artemia genus. Also, our results were different from that reported by Pilla and Beardmore (1994) with regard to the biometry of untreated cysts of A. urmiana. Abatzopoulos et al (2006a,b) reported diameter of untreated and decapsulated cysts and their thickest chorion for A. urmiana from the Urmia Lake:
1. A. urmiana cysts were collected from seven sites in the Urmia Lake. The mean value of the diameter of the untreated cysts ranged from 262.7 to 286.6 µm, decapsulated cysts from 258.6 to 274.4 µm and the thickest chorion ranged from 1.2 to 9.3 µm (Abatzopoulos et al 2006a).
2. In the other study A. urmiana cysts were harvested in three sites from the Urmia Lake. This study show that, the mean value of the diameter of the untreated cysts ranged from 249.8 to 280.7 µm, decapsulated cysts from 218.4 to 259.8 µm and the thickest chorion ranged from 2.7 to 15.6 µm (Abatzopoulos et al. 2006b).
These nine sites that have recently been studied conform to our stations, but the data don't correspond with some traits.
These differences can be attributed to seasonal fluctuations in physico-chemical parameters and food availability in different regions of the Urmia Lake (Abatzopoulos et al. 2006b) and also the differences can be observed in different times in a single site. But it should be noted that the pattern of population/species genetics determines ranges of the biometry of the cysts.
Our results suggest that all three cyst traits of A. urmiana show variation between 26 stations but a high variation was observed in diameter of decapsulated cysts and chorion thickness. Hatching efficiency (H.E.) refers to the number of nauplii hatched per gram dry weight of cysts. Typically high H.E. is possible due to the small diameter of cysts (high number per gram), therefore small diameter of cysts show someway high quality of cysts (Sorgeloos et al, 1978). According to the results, the Urmia Lake has high variation of diameter of cysts in different locality, so nowadays the brine shrimp of Artemia used as a useful aquatic animal in aquaculture and it may be proved of some economical value. It is suggested that a proper management in Artemia cyst harvesting should be focused on selecting localities that produce small cysts with high Hatching Efficiency.
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