Brittlestar regeneration rate paper janice price and lee ann clements
Proceedings of The National Conference On Undergraduate Research (NCUR) 2006 The University of North Carolina at Asheville Asheville, North Carolina April 6 – 8, 2006 Hydrodynamic Stress and Its Effect on the Arm Regeneration Rate in the Brittlestar, Ophiophragmus filograneus Janice Price Department of Biology and Marine Science Jacksonville University 2800 University Blvd N. Jacksonville, Fl 32221. USA Faculty Advisor: Lee Ann Clements, Ph. D AbstractOphiophragmus filograneus is a burrowing brittlestar found in local seagrass beds. In this study we examined theeffects of hydrodynamic stress on regeneration of brittle stars when food was limited. We predicted that animals inwave-induced oscillatory flow would regenerate higher amounts of skeletal tissue relative to muscle mass than thoseexposed to laminar flow conditions. Thirty brittlestars with 2 arms removed were placed randomly into separatetanks with either oscillatory or laminar flow, and allowed to regenerate for 4 wks. Animals were fed for discretetime periods each week. Upon completion of the experiment, animals in the laminar flow tank regenerated a higherrelative proportion of skeletal tissue than those in the other treatment. The rate of soft tissue and skeletalregeneration under different hydrodynamics may influence the amount of secondary productivity available to highertropic levels.Keywords: Ophiophragmus filograneus, brittlestar, regeneration, hydrodynamic stressI. IntroductionSome of the energetic considerations of marine organisms is how they allocate the food they obtain for metabolism,reproduction, and growth. The allocation of this energy is determined by environmental influences and by theorganism’s needs of the energy budget, such as in growth. Some of the factors that affect growth are foodavailability, temperature, salinity, and predation. There are two types of predation: lethal, where the organism dies,or sublethal, where the predation causes the need to re-grow or regenerate lost parts. Sublethal, or partial predation,is a common phenomenon in the marine environment and has been examined in the recent past on organisms such asechinods, polycheates, and bivalves. Although sublethal predation is often overlooked, it maybe very important inaccounting for secondary productivity. Understanding environmental factors that affect regeneration is not onlyimportant in assessing the impact to the affected species, but also to their predators. A good study animal to investigate the regeneration in is Ophiophragmus filograneus an infaunal brittlestar fromthe family Amphiuridae. They are the dominant component of the benthic community in the low flow environmentsof seagrass beds and mud flats. The amphiurids are “burrowing animals” that are rarely found on the surface of thesubstratum. Their common name comes from the fact that these organisms burrow themselves into the sedimentusing their arms. They create a “semi-permanent burrow whose walls are reinforced with mucous” that containstheir disk or central unit of their body1. While their disk is submerged in the sediment, their arms are bothunderground and partly exposed at the surface, where the tips of the arms can search for food. They tend to be foundwithin the top 5 cm of the sediment, but they may burrow themselves up to 20 cm deep if they are threatened or indanger2. If the threat or danger is great enough these brittlestars will expose themselves briefly to move to anotherlocation where they will be less disturbed3. Brittlestars are considered both deposit and suspension feeders. They can obtain food from within their burrowor by sticking their arm tips on the sediment surface above their burrow or in the water that passes above theirburrow1. Brittlestars transfer the particles collected from these areas and pass them onto their mouths using their
tube feet, which are covered with sticky mucus, to collect individual sediment grains and food particles 1. When thebrittlestar collects particles from within its burrow or on the surface above its burrow, it is called deposit feeding.They also participate in passive suspension feeding, where the brittlestars are dependent on the flow of water to passfood particles over their burrows. This process should not be confused with filter feeding, which describes how thefood is obtained rather than the nature and location of the food 4. These organisms will ingest sediment and obtainnutrition from some part of it5. Regeneration is an important function for brittlestars because of the continual grazing upon their arms by fish,shrimp, crabs, stingrays, and other predators6. This ability is an important function not only for the brittlestars, butalso for the entire tidal ecosystem, which partially relies on the renewable food source it provides7. The regenerationof their arms occurs via absorption of nutrients from food, other body parts, or from the environment of theorganism8,6. Previous studies have been done to determine if hydrodyamics affect the regeneration of brittlestar muscle andskeletal tissues. While the regeneration was accurately measured in these studies, the feeding was not controlledwhich makes the interpretation of their results difficult. This study will test the affect of hydrodynamic forces on thearm regeneration on the O. filograneus with controlled feeding for both laminar and oscillatory treatments. Theproposed hypothesis is that in a wave dominated system the brittlestars will regenerate a higher amount of skeletalmass to muscle tissue than in a low flow system. Studies performed on sea urchins show that they grow thickertests than animals in a low flow system, so it is plausible to predict that the brittlestars in the wave dominated systemshould regenerate a higher amount of skeletal mass9.II. Methodology:The collection site for this experiment was a sea grass bed in the Banana River located near Melbourne, Fl(28 12’N, 80 37’W). Specimens were collected by digging the sediment up from the sea grass beds and then siftingit through a 0.15 cm sieve. The specimens were placed in a bucket filled with seawater from the collection site andwere brought back to the Jacksonville University Wet Lab. A 4 cm deep layer of sediment from the collection sitewas placed into each of the fifty-gallon tanks that were filled to a depth of 18 cm of seawater with salinity between18-20 ppt. The sediment was allowed to settle over night and the brittlestars were held in aerated seawater prior tothe start of the experiment. Both tanks have the same dimensions of 2.43 m x 30.5 cm x 30.5 cm and are constructed from 1.9 cm thickplexiglas. The first tank was set up as “flume tank” in order to produce laminar, uni-directional flow, using sodastraws that have been glued together to act as columators (Figure 1). The second tank was set up to mimic a coastalenvironment with an oscillating flow of waves (Figure 2). A paddle made out of plexigas was moved back and forthby a rod connected to a motor, which produced a wave frequency of 72 λ/min. Two damping devices, one behindthe motor and the other at the far end of the flume, were used to reduce reflection of waves and ensure thatwavelength and frequency remained constant. Each brittlestar was anesthetized with isotonic MgCl2 diluted 1:1 with natural seawater. Then each of thespecimens had two non-adjacent arms amputated near the central disk using a scalpel. After amputation each of thespecimens were randomly assigned to one of the treatments, for a total of 30 specimens in each treatment. Thebrittlestars were then allowed to regenerate for four weeks. Salinity, temperature, and water chemistry were monitored 3 times a week. On those days each of the tank motorswas stopped and the specimens were allowed to feed on 0.250 g of finely crushed tetra-min® fish flakes for 30 min.In order to prevent continued feeding, a 1 cm layer of sediment was added to both tanks and allowed to settle for 10min before the motors were turned back on. Since these brittlestars are deposit or suspension feeders, covering thefood with sand removed the possibility of differential feeding between the two tanks. After four weeks all of the specimens were removed from the tanks and re-anesthetized in MgCl2 in order toamputate the regenerated arm portion of the animals. The regenerated and non-regenerated portions were placedinto separate pre-weighed aluminum pans and the wet weight (WW) of each of these portions were determined.Then the pans were placed into a drying oven where they were allowed to dry for 24 hours at 70 C. After dryingwas complete, both portions were weighed to obtain the dry weight (DW=total amount of tissue and skeletonregenerated). Then they were placed into a muffle furnace at 400°C for 4 hrs, which was sufficient to burn the tissueoff of the skeletal material to obtain the ash weight (AW=total amount of skeleton regenerated). The ash free dryweight (AFDW=total amount of tissue regenerated) was calculated by subtracting the ash weight from the dryweight. These calculations were used to analyze and determine the effect of hydrodynamic stress from the wavetank versus the laminar flow of the flume tank when food was controlled.
III. ResultsThe temperature and salinity of the wave and flume tank stayed consistent throughout the duration of the study(Table 1). The pH remained fairly constant with a range of 8.11 to 8.12 ± .08. A comparison of the chemistry of thewater shows that the ammonia, nitrite, and nitrate remained consistent throughout the duration of the study (Table2). There was no significant difference between the body weights of the flume and wave tank which shows that thecontrol of the biomass of all the animals was similar and their was no biased toward one treatment or another(p=0.509 WW, p=0.852 DW, p=0.784 AW and p=0.833 AFDW) (Figure 5). However there was a significantdifference between each measurement of the arm weights and their regeneration of tissue. The flume tank had thehighest overall regeneration (WW and DW), skeletal (AW), and muscle mass (AFDW) regenerated between the twotreatments. The wet and dry regenerated weights of the flume animals are highly significant when compared to theregenerated weights of the wave animals (p=0.0004 WW, p=0.002 DW). The flume tank animals also regenerated asignificantly higher amount of AW and AFDW (p=0.011 AW, and p=0.010 AFDW). Figure 1 Schematic of the flume tank producing a laminar unidirectional flow of water. Figure 2 Schematic of the wave tank producing an oscillating flow of waves at 72rpm.
Table 1 comparison of salinity and temperature between the flume and wave tank. Salinity (ppt) Temperature (*C) Mean std dev Mean std devFlume tank 18.57 0.85 24.68 0.32Wave tank 18.79 0.89 24.64 0.31Table 2 comparison of ammonia, nitrite, and nitrate between the flume and wave tanks. Ammonia (mg/L) Nitrite (mg/L) Nitrate (mg/L) mean std dev mean std dev mean std devFlume tank 0.09 0.03 0.01 0.04 0 0Wave tank 0.09 0.03 0.03 0.05 0 0 0.2500 0.2000 0.1500 Weight (g) 0.1000 0.0500 0.0000 Wave Flume Wave Flume Wave Flume Wave Flume Wet Weight Dry Weight Ash Weight Ash-Free Dry Weight Figure 3 Mean wet, dry, ash, and ash-free dry body weights ( 1 SE) between the wave (n=28) and the flume tank (n=26).
0.0350 ** 0.0300 0.0250 ** 0.0200 Weight (g) * = 0.01 0.0150 * * <0.002 0.0100 0.0050 ** ** * * * * 0.0000 Wave Flume Wave Flume Wave Flume Wave Flume Wet Weight Dry Weight Ash Weight Ash-Free Dry WeightFigure 4 Mean number of wet, dry, ash, and ash-free dry weight of the regenerated arms ( 1 SE) between the wave (n=28) and the flume tank (n=26).IV. DiscussionMany factors can influence the feeding process of marine suspension and deposit feeders, such as action and size ofparticles, environmental conditions (such as light, oxygen, and salinity), and hydrodynamic stress. The naturalenvironment and its hydrodynamic stress is a major factor that can cause physical changes in some animals. Some ofthe physical changes that could occur are that the brittlestars may build up their muscle mass instead of growinglonger arms. This was found to be true with the infaunal brittlestar Hemipholis elongate, which regenerated shorterand lighter arms than the control group when it was subjected to wave motion10. Clements et al demonstrated thatbrittlestars living in the shallower parts of sea grass beds regenerate a greater amount of skeletal regeneration thanthose in deeper beds6. This may have resulted from the increased wave action during low tide. Although, theregeneration rate of the soft tissue in each arm did not differ between the two sites, the animals in the shallower bedsshowed a three fold greater skeletal regeneration in the arms than those regenerated in the deeper beds6. Previous undergraduate studies have also evaluated the effects of hydrodymamic stress and how it affects the rateof skeletal regeneration in brittlestars. Benjamin J. Bryant, from Jacksonville University, subjected two groups of theO. filograneus to different unidirectional flow rates, one adjusted for high flow (~3 cm/sec) and the other for lowflow (~1.5 cm/sec). The results of this study was that the brittlestars in the high flow tank had a much lower DW andAFDW than the animals in the low flow tank, but they regenerated a significantly higher amount of AW than theanimals in the low flow tank11. During this experiment it was also observed that the animals in the high flow tankwere less active and were not feeding as much as the specimens in the lower velocity flume. This means that theylimited the exposure time to the treatment, which reduces the amount of energy required to keep their arms extendedin the water column, but it also limits the amount of nutrients they could acquire from the surrounding environment.The regeneration process is not only affected by the environment the organisms inhabit, but it also affected bylimiting factors, including available nutrients. The metabolic energy acquired from nutrient intake is partitioned between all of the organism’s metabolicexpenditures: growth (including reproduction and regeneration), specific dynamic action (SDA) the cost to digestthe food, feeding, waste products, routine activity and migration 12. The initial SDA is positively correlated to meal
size, so the larger the meal that is ingested by the organism the higher the energetic cost of digestion; however, thiseffect is made affordable by the larger return of energy available for other life processes 12. Although the ophiuridshave an adequate reserve of energy in their disk and remaining arms to regenerate without the ingestion of outsidenutrients, it has been observed that fed organisms deposit more skeletal material than starved individuals 13,4. Another undergraduate study tested the effects of food rationing on the arm regeneration in O. filograneus. Thefirst part of this study was performed without wave motion and the brittlestars were divided into three groups basedon feeding frequency: low ration groups were fed only 1 day a week, medium ration groups were feed 2 times aweek, and the high ration groups were feed three times a week. The results of the first part of the study showed thatthe total regenerated amount increased as the feeding increased14. The second part of the study compared theregeneration between the low flow environments to a wave-induced environment. Each of the tanks was fed 3 timesa week for a one-hour interval each time. The motor of the wave tank was turned off during this time period toallow equal opportunity for feeding. The animals in the control tank fed during the one-hour food break andperiodically afterwards; however, the animals in the wave tank were only observed feeding during the break, butstopped after the wave generator was turned back on13. The results the second part of the study showed that theanimals in the animals in the low flow tank regenerated a significantly higher amount of DW, AW, and AFDW 13.When comparing the second part of the study to the first part, the total amount of regeneration in the low flow tankwas almost equal to the medium ration groups, while the wave tank was equivalent to that in the low ration groups 13.However, in the wave tank there was more skeletal regeneration than that in the low ration feeding group, whichimplies that something other than food limitation could have effected the regeneration and that perhaps the presenceof hydrodynamic stress caused for the allocation of limited energy to skeletal regeneration. This would havesupported the hypothesis first stated by Clements et al4, but there was an unequal amount of regeneration betweenthe control and wave treatments so no conclusions were confirmed. The results of this study were contradictory to the hypothesis that in the wave dominated system the brittlestarswould regenerate a higher amount of skeletal tissue to muscle mass than in a low flow system. The flume tank notonly regenerated a higher amount of muscle mass, but also regenerated a higher amount of skeletal mass. Aprobable reason for this could be that the food does not affect the amount of skeletal regeneration that occurs in ahydrodynamic situation, but rather that the regeneration rate of the organisms has to do with the hydrodynamicsitself. Future work could be done to study the amount of hydrodynamic stress, or lift and drag that is created, thatthe arms of these brittlestars could withstand in their natural environment.V. AcknowledgementsI would like to thank Dr. Lee Ann Clements and Dr. Daniel McCarthy for their encouragement and guidancethroughout this research.VI. References 1. Woodley, J.D. (1975). The behavior of some amphiurid brittle-stars. J. of Exp. Mar. Biol. Ecol. 18:29-46. 2. Stancyk, Stephen E. (1970). Study on the biology and ecology of ophiuoids at Cedar Key, Florida. MS thesis. University of Florida. Gainesville, Fl, USA. pp. 11-66. 3. Lawrence, J. (1987). A Functional biology of echinoderms. The Johns Hopkins University Press. p.340. 4. Vogel, Steven. 1994. Life in moving fluids. New Jersey: Princeton University Press. 5. Levinton, Jeffrey S. (2001). Marine biology: function, biodiversity, ecology. Second ed. New York: Oxford University Press. 6. Clements, L.A., S. Bell, and J.P. Kurdziel. (1994). Abundance and arm loss of the infaunal brittlestar Ophiophragmas filograneus (Echinodermata: ophiuridea), with an experimental determination of regeneration rates in natural and planted seagrass beds. Mar. Biol. 121:97-104. 7. Stancyk, S.E., H.M. Golde, P.A. Pape-Lindstrom, W.E. Dobso. (1994). Born to lose I. Measures of tissue loss and regeneration by the brittlestar Microphiophlis gracillma (Echinodermata: ophiuridea). Mar. Biol. 118: 451-262 8. Dobson, W. E. (1988). Early post-autonomy tissue regeneration and nutrient translocation in the brittlestar Microphiopholis gracillima (Stimpson) (Echinodermata: Ophiuroidea). Ph.D. Dissertation, University of South Carolina, Columbia, SC, USA. 9. Lawerence, John. A Functional Biology of Echinoderms. (Baltimore: The John Hopkins University, 1987), 3.
10. McAlister, Justin Scott. (1998). Effects of variable water motion on regeneration of the infaunal brittlestar, Hemipholis elongate (Say, 1825) (Echinodermata: ophiuridea). M.S. Dissertation. University of South Carolina, Colombia, SC, USA.11. Bryant, Benjamin. (1999). Effects of hydrodynamic stress on the skeletal regeneration rate of the infaunal brittlestar Ophiophragmus filograneus (Ehinodermata: ophiuridea) Undergraduate research, Jacksonville University. Jacksonville, Fl, USA.12. Alsop, D.H. and C.M. Wood. (1997). The interactive effects of feeding and exercise on oxygen consumpsion, swimming performance and protein usage in juvenile rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 200:2337-2346.13. Dobson, William E., Stephen E. Stancyk, Lee Ann Clements, and Richard M. Showman. (1991). Nutrient Translocation during Early Disc Regeneration in the Brittlestar Microphiopholis gracillima (Stimpson) (Echinodermata: Ophiuroidea). Bio. Bull. 180: 167-184.14. Remily, Elizabeth Renee”. (2000). Environmental influences on arm regeneration in the brittlestar, Ophiophragmus filograneus. Undergraduate research, Jacksonville University, Jacksonville, Fl, USA.