Artemia Investigation Report

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S L E 3 1 5 C O M PA R AT I V E A N I M A L P H Y S I O L O G Y

Artemia Research Investigation Physiological tolerance of brine shrimp (Artemia salina) nauplii to extreme salinities.

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Artemia: Background Artemia (Figure 1) inhabit salt and brine lakes worldwide, some of which are located in dry hot climatic zones. Their successful colonisation of these extreme habitats is made possible by their highly efficient osmoregulatory system. The absence of predators such as fish in these highly saline environments, often leads to the development of vast blooms of Artemia that can be commercially harvested. Various species are used extensively throughout the aquaculture industry, both in marine and freshwater systems. In fact the successful commercial production of many species of fish and crustaceans has only become possible since the development of advanced techniques for culturing Artemia. Considerable information exists regarding the culture, biology and nutritional value of Artemia. Artemia is principally a wild fishery resource and is harvested as cysts from saline lakes. Global production in 2011 was about 156 tonnes. Yearly harvests fluctuate widely depending on season and harvest intensity. Enough cysts must be left behind after each harvest event to ensure adequate replenishment of cysts the following year. Cyst quality is influenced by many factors including the collection site, strain, prevailing environmental conditions, methods of harvesting, and postharvest handling and processing techniques. Most processors have their own propriety methods for ensuring the highest quality product is produced for the end user, which is normally the aquaculturalist, although more recently developmental biologists have taken an interest in this species. Premium prices are obtained for high quality cysts and cyst price is strongly influenced by supply volume. Several parameters are routinely used to determine the quality of each batch of cysts and include: hatching percentage, hatching efficiency, hatching rate, hatching synchrony, hatching output and nutritional profile. These parameters are usually determined using standard methods of hatching. Deviation from “ideal” hatching procedures will usually result in lower quality cysts and poorer hatch rates. The final quality of the hatched Artemia is determined by the inherent quality of the cysts and the hatching technique used. An ideal environment for hatching cysts must be used to ensure the most efficient and cost effective use of the Artemia resource. The hatched nauplii generally need to be separated from their shell and from unhatched (non-viable) cysts as the shells and cysts are of no nutritional value. In fact unhatched cysts are highly resistant to digestion by animal digestive systems and sometimes cause digestive blockages and starvation of small larvae. There are various methods for concentrating/harvesting the hatched nauplii and separating them from the shells and cysts. One technique uses light to concentrate the nauplii to a particular mid- water location, followed by manual siphoning or pipetting into a separate vessel. Other methods use conical containers with a valve on the bottom to concentrate and harvest the nauplii. More recently magnetic separation of hatched nauplii from shells and cysts has become possible. Although Artemia are highly nutritious for many species of aquatic animals, their nutritional profile is not always suitable.

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Figure 1: Morphology of Artemia salina (Source: Atlas of Invertebrate Anatomy. D.T. Anderson Ed., Univ. NSW Press, 1996)

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Fortunately Artemia are a non-selective filter feeder and can ingest particles in the size range 1-40um, which means they can be enriched with a variety of foods in order to change their nutritional profile to suit the target species nutritional needs. Various enrichment media and techniques are available. Sometimes nauplii are too small for the target species to effectively feed and they need to be on-grown to larger juvenile and adult stages. This necessitates artificial feeding of the less well developed Artemia stages. Various commercial diets and methods are available, but all techniques are challenging to perform well and require vigilance and patience. While Artemia are capable of tolerating extremely variable salinities there is an energetic cost for living life on the edge, or outside an optimal range. Extreme salinities can influence metabolic processes in even the most highly salinity tolerant organisms, which subsequently influences survival potential. Artemia are strict osmoregulators and maintain their hemolymph osmolarity from 200 – 400mOsm/L over a wide range of external salinities (seawater osmolarity is ~1000 mOsm/L; salt lakes typically range from ~50 – 3000mOsM/L; freshwater is ~5 mOsm/L). So in salinities above ~400mOsm/L Artemia are hypo-osmoregulators, while at salinities lower than ~200mOsm/L they are hyper-osmoregulators. While the cuticle of the integument of Artemia offers some resistance to water movement, extreme osmolarity gradients still result in water and volume changes through osmosis. Powerful salt glands on the dorsal surface of the cephalothorax in nauplii and in the second maxilla of adults, that are rich in Na+ / K+ ATPase, are capable of maintaining ionic balance, albeit at an energetic cost, particularly at high salinity gradients. The exopodites (gills) also play an important role in osmoregulation, particularly in adults. In this investigation you will determine the survival time of hatched Artemia nauplii exposed to a broad range of extreme salinities, from freshwater (~0 g/L salt) to hyper- saline (300g/L salt) levels. You will find that nauplii will survive being transferred from seawater strength salinity to either of these extremes, but should come to appreciate that although they can survive, there is a metabolic cost of living life-on-the edge. Artemia nauplii rely on stored nutrients (i.e., are lecithotrophic or self-feeding) until they reach the second instar stage (usually about 7-10 hours post-hatch under standard conditions), after which they are ready to start feeding (i.e., have a developed digestion system). In the absence of food, survival time is dictated by the environment in which Artemia live. In metabolically demanding environments, where respiration and/or mechanisms of osmoregulation are compromised, survival time would be expected to be comparatively low. In your report, you will need to consider what physiological features enable Artemia to survive at such extremes and explain differences in survival time from metabolic/physiological perspectives. Artemia Research Investigation: An Overview Before commencing the experiment, you will first need to some preliminary reading and viewing using the resources mentioned below and study each of the steps provided in

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the experimental design so as to better understand the process (for example take a look at Brine shrimp hatchery HD https://www.youtube.com/watch?v=vc9ZuzCSepQ ). You are provided with a stepwise experimental procedure to enable you to successfully execute the experiment. Most of the required equipment will be provided to you; it is your responsibility to make sure you collect the equipment kit from your local campus or contact me early to make alternative arrangements. Extensions will not be granted due to problems associated with collecting the equipment kits. Some basic equipment will also need to be provided by you, just a few things that are usually found around home. After you complete the experiment you need to record your data in the “Artemia Investigation Web Application” (which has a semi-automated collation function to compile the class dataset) located on the SLE315 home page in cloud. You will use this class dataset to write an individual scientific report (worth 50% overall for the unit) which you will submit into the Assessment box as a Word document. During the experiment you are required to take photographs of the main phases of the procedure (including at least one with you in the photograph) and compile into your report in the methods section. Photographs must be logically ordered with appropriate captions and labels. Please refer to the timetable for the key dates associated with this assignment Useful References and links Aquatic community website: Raising and Growing Large Brine Shrimp

http://www.aquaticcommunity.com/fishfood/largebrineshrimp.php (accessed 27/05/2015)

Artemia- We Live to Die video; located on cloud under Resources echo link. Brine shrimp hatchery HD; https://www.youtube.com/watch?v=vc9ZuzCSepQ (accessed

27/05/2015) Gajardo, G.M. and Beardmore, J. A. (2012). The brine shrimp Artemia: adapted to critical

life conditions. Review Article. Frontiers in physiology, 185 (3). 1 – 7. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381296/ (accessed 27/05/2015)

Gulbrandsen, J. (2001). Artemia swarming- Mechanisms and suggested reasons. Journal of Plankton Research, 23: 659-669.

Hoff, F.H. & Snell, T.W. (1989). Plankton culture manual. Florida Aquafarms Inc. Florida, 126p.

Lavens and Sorgeloos (1996). Manual on the production and use of live feed for aquaculture. FAO Fisheries Technical Paper, No 361. Rome, FAO. pp 107-136.

Medina, G., Goenaga, J., Hontoria, F., Cohen G., & Amat F. (2007). Effects of temperature and salinity on pre-reproductive life span and reproductive traits of two species of Artemia (Branchiopoda, Anostraca) from Argentina: Artemia franciscana and A.persimilis. Hydrobiologia. 579:41–53.

Nambu, F., Tanaka, S. & Nambu. Z. (2007). Inbred Strains of Brine Shrimp Derived from Artemia franciscana: Lineage, RAPD Analysis, Life Span, Reproductive Traits and

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Mode, Adaptation, and Tolerance to Salinity Changes. Zoological Science 24(2):159- 171.

Tolomei, A., Burke, C.A., Crear, B. & Carson, J. (2003). Bacterial decontamination of on- grown Artemia. Aquaculture 232 (1-4): 257-371.

Treece, G.D. (2000). Artemia production for marine larval fish culture. Southern Regional Aquaculture Centre, SRAC Publication No 702.

Wikipedia link: http://en.wikipedia.org/wiki/Brine_shrimp (accessed 27/05/2015)