Vie et milieu - Life and environment, 2017, 67 (1): 15-20
Aspects of life history of Artemia salina (Crustacea, Branchiopoda) from Algeria reared in different conditions of salinity M. Amarouayache*, M. H. Kara Marine Bioressources Laboratory, Marine Sciences department, Faculty of Sciences, Annaba University Badji Mokhtar, BP 230 Oued Kouba, Annaba 23008, Algeria * Corresponding author:
[email protected]
ARTEMIA SALINITY REARING CHOTT MAROUANE ALGERIA
ABSTRACT. – The brine shrimp Artemia (Crustacea, Branchiopoda) lives in hypersaline lakes distributed all around the world. It is widely used as live prey in larviculture for many advantages, especially easy operation, adequate size and nutritional quality. Many sites have been exploited for its cysts, and demand of aquaculturists remains increasing. The population of A. salina Linnaeus, 1758 from Chott Marouane, a huge saline lake (3900 km2) situated in the Sahara, was reared at 3 different salinities (40, 100 and 200 ppt) using baker’s yeast and a vitamin complex as food. Larval development, growth, adult’s size, reproductive traits and life span were studied in order to provide additional data for future management and a possible exploitation. At the highest salinity tested (200 ppt), growth was slower (0.19 ± 0.16 mm.day–1 vs 0.33 ± 0.18 mm.day–1 at 40 ppt) and adult’s size decreased significantly (P ≤ 0.05). This salinity was most advantageous for survival and life span. At lowest salinities, pre-adult stage was longest, reproductive traits were relatively rapid, with a precocious sexual maturity and a highest total offspring (24.8 ± 14.2 cysts/female). All the females reproduced by oviparity whatever the salinity.
INTRODUCTION In hypersaline biotopes, living conditions are harsh and few species have developed mechanisms of adaptation, like strong osmoregulation/osmoconformation, rapid growth and production of resting eggs or cysts (Hammer 1986). The brine shrimp Artemia Leach, 1819 (Branchiopoda, Anostraca) is the most characteristic of these mediums. The genus comprises seven known bisexual species and parthenogenetic populations, distributed on the five continents (Van Stappen 2002). Within the same species, populations which are geographically isolated have different physiological responses according to physicochemical conditions of the biotope, resulting in different phenotypes and behaviors (Amat 1980, Abatzopoulos et al. 2000, Baxevanis et al. 2004). Among abiotic factors that influence the life history of this Crustacean, salinity is one of the most important which affects its development and reproduction (Vanhaecke et al. 1984, Bowen et al. 1988, Amat et al. 1995, Barata et al. 1995). Several investigations on the effect of this factor on growth, survival and reproduction have been carried out on different species/populations (Wear & Haslett 1987, Brown & Wanigasekera 2000, De Los Rios 2001, El-Bermawi et al. 2004, Saygi 2004, Agh et al. 2008). The aim was to better understand the life history and/or to determine optimal conditions for inoculation of Artemia’s nauplii in culture ponds for aquaculture use. Thus, it is known that the latter remain the most important food in nurseries for numerous advantages, such as rapid hatching of cysts to obtain
this alive prey, the storage in the form of cysts, adequate size and fatty acid composition (Sorgeloos et al. 2001). Even though several thousand tons of Artemia cysts from different origins are harvested every year (Litvinenko et al. 2014), the market demand is increasing due to rapid aquaculture development, which is in favour of searching new cyst deposits. In Algeria, at least 24 hypersaline lakes, locally called Chotts or Sebkhas, have been found to harbour Artemia (Kara & Amarouayache 2012). Fourteen populations have been morphologically or genetically characterized, showing the presence of A. salina, and 2n and 4n parthenogenetic populations (Ghomari et al. 2011). Only three of them made the subject of some ecological and biological studies, Chott Marouane (Kara et al. 2004, Amarouayache et al. 2009), Sebkha Ez-Zemoul (Amarouayache et al. 2010, Amarouayache & Kara 2015) and El-Bahira Lake (Amarouayache & Belakri 2015). The results of these investigations led to the conclusion that the bisexual strain of Artemia from Chott Marouane (34°03’N-06°20’E), located in the Sahara in the Northeast of the country, was probably the most interesting for aquaculture exploitation for the quality of its cysts and nauplii (Kara et al. 2004), and the natural cyst production that was estimated in 2000 at 7.6 tons in its western part. This hypersaline lake is one of the largest in Algeria with 3900 km2 area. It is located at –31 m alt. from sea level and its maximum depth is of 9 m. Even if the climate is hyper-arid with temperatures often reaching 48 °C, the site does not completely dry being continuously fed by Oued Righ draining channel.
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Its salinity is of wide range, between 30 ppt at Oued Righ mouth in the South, where the Killifish Aphanius Nardo, 1832 occurs (Zemmouri 1991), and increases to saturation (360 ppt) in the summer at the Northern part, near the banks. The site is exploited by the national salt company ENASEL. Morphological study showed that the species of Chott Marouane was Artemia salina, which develops between winter and spring (Amarouayache et al. 2009). The aim of this work is to better understand the requirements and the tolerances of this population towards salinity. Thus, the wild population was found to withstand the high salinities of the medium from 257 to 320 ppt. Lower salinities were tested in this study to determine whether growth, survival and reproductive patterns could be enhanced. Such results might be helpful for future managements for both ecological and exploitation for aquaculture purposes. MATERIALS AND METHODS Artemia rearing: Artemia salina cysts have been collected in Chott Marouane (Northeastern part) in 2009. They have been cleaned, separated and stored according to Sorgeloos et al. (1986) protocol. A 0.2 g cyst sample has been incubated at standards conditions recommended by these authors. After 36 h, cysts hatched and nauplii were recovered for rearing after having attracted them towards a source of light. Three brines of 40, 100 and 200 ppt have been prepared using distilled water and non-iodized washed salt got from the site. Freshly hatched nauplii were immediately transferred, after count, in 1 L glass tank where they have been separately reared at a rate of 1000 ind.L–1 for each salinity, with three replicates. Temperature of water was maintained at 25 ± 1 °C using a bain-Marie, the photoperiod was of 16:8, oxygen was maintained at saturation using an aerator. Brine shrimps were fed with 85 % baker yeast and 15 % vitamin complex (Barnabé 1991). The latter’s composition was B1, B2, B5, B6, B8, B9, C, E vitamins, oligoelements copper, iron, iodine, magnesium, selenium and zinc, cod-liver oil, fatty acids (linoleic acid, gamma linoleic acid and docosahexanoic acid), amino-acids and carbohydrates. For the totality of tanks (3000 nauplii), the yeast was prepared by dissolving an amount of 0.15 g in a 50 mL tube with distilled tepid water (30 °C), which was gradually increased to 0.5 g at adult stage (50 adults) according to doses recommended by Coutteau et al. (1992). The yeast was daily distributed while the vitamin complex was added twice a week with the water renewing. Survival, growth and development: Survival was determined once the number of individuals decreased to allow direct calculation; it represents the percentage of living individuals in relation to the initial number. In order to take the measures, individuals have been anesthetized using diluted chloroform at the rate of one drop in 4 mL of water from the breeding medium (Amat 1980). From each tank, 10 individuals were measured, from the naupliar eye to the anus, under a compound microscope
equipped with an ocular micrometer (× 10), every 2 days until the first sexual maturity. Then they were rinsed with fresh water to reanimate them and placed back in their rearing medium. The growth rate was calculated by the average of size gained in mm per day. Each time that individuals were measured, the developing stages were determined according to morphologic criteria (number and shape of appendixes). These were considered at the moment when they were attained in 50 % of the population. They were grouped into Instars I, 12 h after hatching, at metanauplii (Instar I to IV), characterized by the appearance of the anus and beginnings of thoracopods, juveniles (Instar V to IX), characterized by the appearance of compound eyes and some thoracopods, pre-adults (X to XIV), characterized by the presence of 11 pairs of developed thoracopods and the beginning of the ovisac or hooks for future females and males respectively. Instar XV, Artemia are considered as adults, they are distinguished by the presence of an ovisac in females and a bifid penis and antenna in the shape of hooks in males (Sorgeloos et al. 1986). Females were considered as mature once 50 % of the population presented signs of reproduction, such as the presence of embryos or developed shell glands in the ovisacs (Amat 1982). The sizes (at the anus) of male and female adults were determined once the growth was completed (at death), and were compared using Student t-test; P values ≤ 0.05 were considered significant. Reproductive parameters: Thirteen couples of mature Artemia by tank, for the salinities of 100 and 200 ppt and 10 for the salinity of 40 ppt (due to high mortality rate), were reared separately in open boxes of 7 × 5 × 3 cm3 volume. Dead males were replaced. The presence of cysts or nauplii was daily checked in each box and water was renewed after their counting. Nine reproductive variables were determined according to Browne et al. (1984): total number of offspring per female, number of brood per female, time between broods, fecundity, which is the mean number of offspring per brood, percent offspring encysted per total offspring per female, pre-reproductive period, reproductive period, post-reproductive period and female life span. The results were statistically analyzed with a one-way ANOVA after checking normality and homogeneity of variances (Kolmogov-Smirnov test). A least significant differences test (LSD) was applied in cases where differences existed. Minitab 12 software was used for statistical analysis.
RESULTS Survival and growth The survival rate at 40 ppt was of 10 % after 22 days and 0 % after 34 days. At 100 ppt, it was of 12.06 % after 29 days and 0 % after 38 days. At 200 ppt, the survival was higher with 22.22 % after 33 days and 0 % after 50 days. In general, males lived longer than females. The growth of individuals until adult stage at the three salinities is illustrated in Fig. 1. The calculated growth
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SALINITY EFFECT ON ARTEMIA FROM ALGERIA
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rate was of 0.33 ± 0.18, 0.25 ± 0.22 and 0.19 ± 0.16 mm.day –1 for individuals reared at 40, 100 and 200 ppt, respectively. Limits of size and the time of development of each larval stage according to salinity are given in table I. The longest developing stage was that of metanauplius (6-8 days) and development was as slow as the salinity was higher, especially for 200 ppt. Individuals reared at 40 ppt spent long time at pre-adult stage (6 days) in comparison with other salinities. Sexual differentiation was observed starting from a lower size of 4.56 mm at 200 ppt and a higher one (6.05 mm) at 100 ppt. Fig.1. – Growth (mm.day–1) of Artemia salina from Chott Marouane reared at Sizes of males and females reared at salinities 40, 100 and 200 ppt. salinities of 40, 100, 200 ppt are given in table II. The Student’s t-test Table I. – Sizes (mm) and ages (day) at different developing stages in Artemia salina from Chott shows that females were Marouane reared at salinities 40, 100 and 200 ppt. larger than males whatever the salinity (5.78 ≤ t ≤ 6.42, 40 ppt 100 ppt 200 ppt P ≤ 0.05). At the adults Size (mm) Age (day) Size (mm) Age (day) Size (mm) Age (day) Developing stages (males and females togethNauplii [0.45-0.67[ [0-1[ [0.45-0.74[ [0-1[ [0.45-0.72[ [0-1[ er), total lengths were of Metanauplii [0.67-1.64[ [1-7[ [0.74-1.59[ [1-9[ [0.72-1.68[ [1-9[ 6.92 ± 0.5, 6.71 ± 0.48 and 5.77 ± 0.5 mm, respectiveJuveniles [1.64-3.23[ [7-11[ [1.59-4.19[ [9-16[ [1.68-3.92[ [9-16[ ly for the 3 salinities menPre-adults [3.23-5.18[ [11-17[ [4.19-6.05[ [16-18[ [3.92-4.56[ [16-20[ tioned above. The ANOVA Adults (males and females) [5.18 [17 [6.05 [18 [4.56 [20 test showed that significant differences existed Table II. – Sizes of males, females and adults of Artemia salina between these lengths (F = 3.8; P ≤ 0.05). The least signiffrom Chott Marouane reared at salinities 40, 100 and 200 ppt icant difference test showed that adult’s lengths observed with comparison of both sexes (* significant difference between males and females sizes, P ≤ 0.05). Different superscript letters at 40 and 100 ppt did not differ, while that observed at in the same column indicate significant difference between 200 ppt was smaller (P ≤ 0.05). adults (P ≤ 0.05).
Reproduction and life span
Salinity (ppt) 40
100
200
Males
6.46 ± 0.18
6.33 ± 0.34
5.36 ± 0.28
Females
7.36 ± 0.28
7.04 ± 0.30
6.17 ± 0.33
Adults
6.92 ± 0.50
6.71 ± 0.48
5.77 ± 0.50b
6.42*
5.96*
5.78*
Size (mm)
Student t-test
a
a
In females reared at 40 ppt, 50 % became mature at a size of 6.79 mm after 18 days. For salinity of 100 ppt, 50 % became mature at a size of 6.75 mm after 20 days and at 5.25 mm after 23 days for the salinity of 200 ppt.
Table III. – Mean (± SD) of reproductive parameters in Artemia salina from Chott Marouane reared at salinities 40, 100 and 200 ppt. A: Total offspring; B: Number of broods; C: Time between broods (day); D: Fecundity (offspring/brood); E: % oviparity; F: Pre-reproductive period (day); G: Reproductive period (day); H: Post-reproductive period (day); I: Female life span (day); N: effective. Significant differences were tested with ANOVA and LSD (P ≤ 0.05). Different superscript letters in the same column indicate significant difference. Parameters A
B
C
D
E
F
G
H
I
40 (N = 10)
24.8a ± 14.2
1.8a ± 0.7
1.7a ± 0.7
17.1a ± 7.8
100
26.0a ± 1.4
3.2ab ± 1.7
0
28.0a ± 1.6
100 (N = 30)
14.7b± 6.7
1.5a ± 0.7
2.0ab ± 1.2
13.7a ± 7.4
100
28.4b ± 1.3
2.3a ± 2.4
0.2a ± 1.2
30.4b ± 3.2
200 (N = 30)
16.9ab ± 11.7
2.3b ± 1.4
2.6b ± 1.3
12.1a ± 6.7
100
29.0b ± 2.0
4.6b ± 4.9
0.8a ± 2.1
33.6c ± 5.3
Salinity (ppt)
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Reproductive parameters are given in table III. Total number of offspring was higher at the lower salinity with 24.8 ± 14.2 cysts, while the number of broods 2.3 ± 1.7 was higher at 200 ppt. Even if at first sight, fecundity decreased from 17.1 to 12.08 cysts/brood with the increase of salinity, no significant differences were found (F = 1.83; P > 0.05). The time between broods, time of pre-reproduction, reproduction time, post-reproduction time and life-span were longer at the highest salinity of 200 ppt. All the females reproduced by oviparity, whatever the salinity of the medium. DISCUSSION The highest survival rate in reared Artemia salina from Chott Marouane was obtained at 200 ppt. It decreased with the decrease of salinity. The wild population was found to live in high salinities (254-328 ppt) at which it should be well adapted (Amarouayache et al. 2009). These results corroborate those of Browne & Wanigasekera (2000) for the Larnaca (Cyprus) A. salina population and of El-Magsodi et al. (2005) for Abu Kamash (Libya) population which survived better at 180 ppt. On the contrary, El-Bermawi et al. (2004) found that the survival rate of Egyptian A. salina populations reared at salinities of 35, 80 and 120 ppt were higher than those observed at 150 and 200 ppt, while parthenogenetic ones survived better at higher salinities of 150 and 200 ppt. According to Vanhaecke et al. (1984), geographic isolation of Artemia populations in specific biotopes with different temperatures and salinities could result, in response, to different tolerance limits for these abiotic conditions. Works on the growth of Artemia are not numerous (Abreu-Grobois et al. 1991, Correa-Sandoval et al. 1994, El-Bermawi et al. 2004). These latter authors described the Egyptian population growth using von Bertalanffy model and reported that bisexual ones had a lower growth rate than parthenogenetics. In this study, salinity affected the growth rate of A. salina from Chott Marouane which decreased from 0.33 to 0.19 mm.day-1. Saygi (2004) also found that salinity had a negative influence on the growth of a parthenogenetic population from Kalloni in Greece. Generally, values obtained herein were higher than those obtained by Correa-Sandoval et al. (1994) in Mexican and San Francisco Bay populations of A. franciscana (0.16 and 0.18 mm.day–1, respectively), which means that A. salina from Chott Marouane grows faster than these populations. Adults were significantly smaller at 200 ppt salinity which corroborates observations of Amat (1980) who concluded that size was negatively correlated with this parameter. Thus, brine shrimps consume more energy for osmoregulation, when salinity is higher, which prevents them to attain larger size. Sizes obtained herein are close to those observed in wild population at the lowest salin-
ity of the site (near 230 ppt) (Amarouayache et al. 2009) and those of other Mediterranean populations of A. salina from Sardinia (Mura 1993) and Egypt (El-Bermawi et al. 2004). Females became mature at lengths that are close to those of wild population with 6 mm observed at 254 ppt, but larger than the 4.5 mm observed between 287 and 328 ppt (Amarouayache et al. 2009). The age at which 50 % of females became mature varied between 18 and 23 days and increased with salinity contrary to the conclusions of Amat (1982). This author found that females attained their first sexual maturity earlier when salinity was higher. However, in this study, individuals spent few time (2-4 days) at the pre-adult stage at 100 and 200 ppt in comparison with time (6 days) at 40 ppt. All the females reproduced by oviparity in this study, regardless of the salinity, even though it is known that Artemia can switch both modes of reproduction oviparity and ovoviviparity within the same population. According to Barata et al. (1995), oviparity is the preferential mode of reproduction in bisexual populations. Ovoviviparity is generally borrowed for the first broods, and then, females become oviparous when conditions of the medium become harsh, like increase of salinity and decrease of oxygen level inducing hemoglobin biosynthesis in Artemia body, which is an essential component for elaboration of cyst chorion (Amat 1982). On the other hand, the capacity to produce cysts is linked to genetic factor, especially the degree of heterozigosity of individuals; the most heterozygote females would be more resistant to adversity and able to produce more resting eggs in the long term in order to insure the perenniality of the population (Gajardo & Beardmore 1989). In general, Mediterranean populations of Artemia live in ephemeral ponds that fill in winter and spring and dry in summer, and should be oviparous (Barata et al. 1995). Among them, the species A. salina was adapted to cold temperatures (15-25 °C) (Browne et al. 1988) and intolerant to low salinities (
