Brine Shrimp
The common name of brine shrimp is Artemia that seldom reaches more than 1/2 in. (1.3 cm) in length and is commonly used for fish food in aquariums. Brine shrimp, which are not closely related to true shrimp, can be found almost everywhere in the world in inland saltwaters, although they are completely absent from oceans. They can live in water having several times the salinity of seawater, but they can also tolerate water having only one tenth the marine salt concentration. Brine shrimp usually occur in huge numbers and can be seen in vast windblown lines in the Great Salt Lake. Their absence from the sea has been explained by their vulnerability to attack by predators and the absence of the latter in their inland saline habitat.
Artemia is a well known genus as one member, sometimes identified as a hybrid species Artemia salina × nyos, is sold as a novelty gift, most often under the marketing name Sea-Monkeys.
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| Family: | Artemiidae Grochowski, 1895 |
| Genus: | Artemia |
Species: Artemia franciscana
Fig: Brine shrimp
Life cycle
Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours. Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average.
Ecology
Wild brine shrimp eat microscopic planktonic algae. Cultured brine shrimp can also be fed particulate foods including yeast, wheat flour, soybean powder or egg yolk. Brine shrimp can tolerate varying levels of salinity. A common biology experiment in school is to investigate the effect of salinity levels on the growth of these creatures. The preferred level of salinity is about 30–35 ppt (parts per thousand).
Nutritional benefits
The nutritional properties of newly hatched brine shrimp make them particularly suitable to be sold as aquarium food as they are high in lipids and unsaturated fatty acids (but low in calcium)..
Threatened
Artemia monica, the variety commonly known as Mono Lake brine shrimp, are found only in Mono Lake, Mono County, California.Despite there being trillions of these creatures in Mono Lake, it was felt that rising levels of salinity and sodium hydroxide concentration of the lake would endanger them because of the increase in pH. Sea-Monkeys® are in no way harmful to humans or the environment. If they somehow find their way into natural water ways or sewer lines, they simply will not be able to survive outside of the formula
Lethality assay
The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity.
Preparation of the bioassays
The tests were conducted in multiwell plates in filtered (0.45 μm pore diameter) and sterilized seawater (final volume 5 ml). Each of the extracts for each species was tested at 1000, 100 and 10 μg of extract per ml. The concentrations were obtained by transferring the corresponding volume from the stock solution to different wells for evaporation. After evaporation, 5 ml of seawater were added to each well with gentle shaking to ensure that the compounds diffused adequately in the aqueous solution. Four replicates were used for each treatment and control. The control was performed by adding the solvent used to dissolve the extracts in the assays, and it was allowed to evaporate. Before the assays, the average time of appearance of the first free nauplii and the subsequent developmental stages was calculated. The first cysts hatched approximately after 12 h of incubation (average time); the maximum percentage of instar II (55.33%) appeared 12 h later (24 h after the start of incubation). All the tests were performed in a temperature-controlled room at 28°C, under a continuous light.
Brine shrimp lethality bioassay
The brine shrimp lethality bioassay was used to predict the cytotoxic activity [15],[19] of the n-hexane, carbon tetrachloride, chloroform, and aqueous fractions from methanolic crude extracts. For the experiment, 4 mg of each of the extracts was dissolved in dimethylsulfoxide (DMSO) and solutions of varying concentrations (400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 µg/ ml) were obtained by the serial dilution technique using simulated seawater. The solutions were then added to the pre-marked vials containing 10 live brine shrimp nauplii in 5 ml simulated seawater. After 24 h, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted. The mortality endpoint of this bioassay was defined as the absence of controlled forward motion during 30 s of observation. [20] From this data, the percent of lethality of the brine shrimp nauplii for each concentration and control was calculated. An approximate linear correlation was observed when logarithm of concentration versus percentage of mortality [21] was plotted on the graph paper and the values of LC 50 were calculated using Microsoft Excel 2003 [Figure 1]. Vincristine sulphate was used as positive control.
Result and Discussion
With the exception of aqueous fraction, all the other fractions of D. indica leaves were active against most of the tested organisms Table 1. The average zone of inhibition produced by the n-hexane, carbon tetrachloride, and chloroform fraction was ranged from 6-8 mm, 7-8 mm, and 6-7 mm, respectively, at a concentration of 400 µg/ disc. Against the Escherichia More Details coli, only chloroform fraction was active (zone of inhibition was 7 mm) and carbon tetrachloride fraction exhibited highest antimicrobial activity compared to other solvent fractions. In both the cases of bacteria and fungi, the zone of inhibition was found to be 6-8 mm.
The LC 50 values obtained from brine shrimp lethality bioassay [Table 2] and [Table 3] were 1.94, 4.46, 2.13, and 5.13 µg/ml for n-hexane (HX), carbon tetrachloride (CT), chloroform (CF), and aqueous (AQ) fraction, respectively. Compared to positive control (vincristine sulphate, VS, LC 50 0.52 µg/ml), all the fractions tested showed good brine shrimp larvicidal activity. Again the crude extracts resulting in LC 50 values less than 250 µg/ml were considered significantly active and had the potential for further investigation. [22] The cytotoxic activity exhibited by the solvent fractions was promising and this clearly indicates the presence of potent bioactive compounds.
Table: 01
Table: 02
Table: 03
Conclusion
The antimicrobial and cytotoxic activities of various fractions of D. indica leaves, found in this study, may explain some of the traditional medicinal uses of this plant. These could be of particular interest in relation to find out its unexplored efficacy and can be a potential source of chemically interesting and biologically important drug candidates.
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