osmoregulation-lab - Osmoregulation in the Blue Crab,...

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Unformatted text preview: Osmoregulation in the Blue Crab, Callinectes sapidus BIO335A Animal Physiology Fall 2005 Dr. Henning Schneider 1 INTRODUCTION The homeostasis of salt concentrations in body fluids of animals is essential for maintaining normal body functions. Many organs and organ systems such as the nervous and the muscular system depend on specific concentrations of ions such as Na+, K+, Cl-, Ca2+ in- and outside of cells. The distribution of these ions represents the basis of normal electrical activity of neurons and muscles and maintains cell shape. Any imbalance would greatly impair the function of all cells of any organism. The permeability of cell membranes for ions depends on ion channels. Ion channels are controlled ionspecific pores that are regulated by hormones or neurotransmitters and the type and number of channels in cells determine their ability to maintain constant conditions inside a cell while the salt concentrations of extracellular fluids are changing. Aquatic invertebrate animals have developed different strategies to adjust to different salt concentrations in the water. Osmoregulators maintain a constant osmolarity of body fluids independent of environmental fluctuations of the water. In osmoconformers, salt concentrations of body fluids adjust to changing environmental salt concentrations and often the salt-concentrations inside osmoconformers is similar to that in the ambient medium. Many invertebrate marine species have developed strategies that include both regulatory and conforming types and are especially found in organisms that live in intertidal zones where the concentration of salts is fluctuating on a daily schedule. Such intertidal invertebrates migrate from marine environments into freshwater environments (upstream) during their life cycle and thus require specialized osmoregulatory organs that can cope with drastically changing ambient salt concentrations. The blue crab, Callinectes sapidus, is an intertidal invertebrate found along the Atlantic and Gulf coast of the US such as the Chesapeake Bay. These crabs maintain a relative constant hemolymph osmolarity of 800 mosmol even if ambient salt concentrations of the water are lower that 800 mosmol. However, if the ambient osmolarity increases above 800 mosmol, blue crabs become osmoconformers and the salinity of their body fluids increases with salinity of the sea water. The goal of this experiment is to measure how blue crabs acclimate to different salt concentrations. On the first day o this experiment you will set-up salt-water tanks that contain water of different ionic-strength and place crabs into these tanks. Blue crabs will be allowed to acclimate to the ionic strength of the seawater for one or two weeks. In the second lab session, you will draw hemolymph from animals, measure the osmolarities of body fluids of Callinectes using a vapor pressure osmometer. 2 MATERIALS AND METHODS Animals Blue crabs (Callinectes sapidus) were obtained from Gulf Specimens, FL, and maintained in artificial seawater in 10 gal. tanks at room temperature. Measurement of weight Measure the weight of crabs used at the beginning and the end of the experiment/lab. Preparation of artificial sea water of different ionic strength We have prepared artificial sea water of normal ionic strength that we call also 100% seawater, The ionic strength can be measured with a simple device. Your task on the first day of the lab is to set-up tanks of 100, 75, 50, 25 and 10 % seawater. Use provided buckets to fill tanks with appropriate amounts of 100% seawater and R.O. (reverse osmosis water). RO water and 100% seawater will be provided. After you have set-up the tanks measure the osmolarity with the osmometer (see below) and label the tanks appropriately. We have 10 crabs available and put two crabs into each sea water tank. Incubation of crabs in seawater of different osmolarities. Long-term adjustment (acclimatization): Each group will measure the osmolarity in crabs at 100, 75, 50, 25 and 10% seawater. In addition take a measurement of the water and compare with the crab hemolymph. Short-term adjustment (acclimation): To measure if the animals adjust to low seawater in short intervals, you will measure the osmolarity of animals after 20 min incubation in sea water of a different ionic strength. First, measure the osmolarity at long-term adjusted ionic strength, then place the animal in higher or lower (depends on salinity of water) for 20 – 30 min and measure the osmolarity of the hemolymph again. Compare acclimation data with the acclimatization data. General Mechanism of measuring osmolarity of solutions. The salt concentrations of solutions can be determined by measuring the lowering of the freezing point of salt solutions or by measurement of the vapor pressure depression using a thermocouple. The instrument measures the dew pint depression caused by salt in the solution. The osmometer is very accurate 0.00031°C and linear in the range from 100 mmol/kg and up. 3 1. Withdrawing saline from blue crabs Sampling of hemolymph will be performed using a 28gauge syringe. 1. Carefully remove a blue crab from the tank 2. Insert the syringe through the membrane in the coax joint of the last walking leg that is now a walking leg. 3. Remove one drop (about 30 µl) of hemolymph from the animal. 4. Transfer the solution directly to the osmometer or store the sample in a 0.5 ml microcentrifuge tube. 5. If the hemolymph coagulates, spin the hemolymph in a microcentrifuge or add some octopamine. 2. Measuring Osmolarity The osmolarity of hemolymph will be measured with a calibrated Vapro 5520 Vapor pressure osmometer (Wescor, Fig. 1). Fig. 1 Shows the Vapro 5520 osmometer. The lever on the right (arrow) is used to open and close the vapor chamber. 4 2.1 Loading samples 1. rotate the sample chamber lever upward 2. pull the black sample slide out from the instrument until it comes to a stop. 3. use forceps to obtain a solute-free paper disc and place the disc into the central depression of the disc holder 4. Aspirate a 10 µl hemolymph sample with a pipettor and a clean tip 5. Pipette the 10 µl sample onto the solute-free paper disc without touching the filter paper with the tip. (use the pipettor guide if you don’t have a steady hand). 6. whether the sample droplet falls onto the disc of clings to the tip, please complete the next step. 7. with the plunger of the pipettor still held against the stop, lightly touch the pipette tip to the sample disc to press it flat against the holder. 8. The paper disc should appear fully saturated, with a slight liquid meniscus on its surface. 9. Gently push the sample slide into the instrument until it stops. 10. Immediately, turn the sample chamber lever (on the right side of the instrument) and rotated it clockwise into the “closed” position. 11. The closing of the lever starts the measurement cycle that takes about 75 seconds. 2.2 Measurement Cycle 1. During the measurement cycle the display screen shows “in process” and counts down the remaining time. 2. When the measurement is completed the display screen shows the osmolarity of the solution on the filter disc. 2.3 Clean sample holder 1. Remove the filter disc from the instrument immediately. 2. Rotate the chamber lever to the open position 3. Slide the sample slide out 4. Using a lint free tissue such as a cotton swab or lens paper, remove the wet disc and any remaining fluid. DO NOT USE FORCEPS for removing the filter since that may scratch the holder. 5. Clean the sample holder with a fresh cotton swab or lens paper. Leave no visible residue on the holder and use a cotton swab moistened with distilled water if necessary. 5 6. The sample holder should appear bright, shiny and perfectly dry before the next sample is loaded. 2.4 Clean Test If your group believes that the sample measurements are not working correctly because of some sort of contamination you can run a Clean Test (takes about 3 –4 min). 1. Measure the osmolarity of a 100 mM standard sample solution, 2. Before opening the chamber press SELECT to reveal the Function Menu. Press SELECT again to point the selection arrow at CLEAN TEST. 3. Press ENTER. The osmometer performs a second measurement of the sample and displays the difference of both measurements in 2 – 3 min. 4. If the displayed contamination level is 10 or more the thermocouple needs to be cleaned. ANALYSIS OF DATA Generate a graph showing the relationship between seawater osmolarity (x-axis) and hemolymph osmolarity (y-axis) of animals at different salinities of the long-term and short-term experiment and discuss the data. PREPARARTION OF LAB-REPORT Prepare a lab report including an Introduction, Material and Methods, Results and Discussion section. Provide also a short abstract and cited literature. Following the presentation of your data in the Results section discuss at least the following questions: • • • • • Are the blue crabs used in this lab osmoregulators or osmoconformers? What is the hemolymph osmolarity of animals at 100 % seawater? What is the hemolymph osmolarity of animals in diluted seawater? Does the hemolymph osmolarity change with salt water osmolarity ? Which other invertebrates show similar osmoregulatory activities as Blue crabs? 6 ...
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This note was uploaded on 03/22/2009 for the course MARS 1020 taught by Professor Binder during the Spring '08 term at University of Georgia Athens.

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