EFFECTS OF ULTRAVIOLET RADIATION ON DEVELOPING VARIEGATED
SEA URCHINS, LYTECHINUS VARIEGATUS
by
Eric Cary Tauchman
B.S., The University of Wisconsin—Madison, 2001
A thesis submitted to the Department of Biology College of Arts and Sciences The University of West Florida In partial fulfillment of the requirements for the degree of Master of Science
2008
The thesis of Eric Cary Tauchman is approved:
______Theodore C. Fox, Ph.D., Committee Member Date
______Wade H. Jeffrey, Ph.D., Committee Member Date
______Christopher M. Pomory, Ph.D., Committee Chair Date
Accepted for the Department/Division:
______George L. Stewart, Ph.D., Chair Date
Accepted for the University:
______Richard S. Podemski, Ph.D., Dean of Graduate Studies Date
ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Pomory, for presenting me with the opportunity to work on this thesis. He offered teachings and advice on all things science and many things not. I also had the most knowledgeable, available, and reasonable committee members a budding scientist could ask for—something I truly appreciate Drs.
Fox and Jeffrey. When one takes twice as long to complete this program as expected, focus wanders and new ideas pop up. The UWF biology and even chemistry faculty displayed wonderful patience and generosity of time and resources in abetting some less- than-entirely thought out ideas on where this research could go (some of them enough to have their names on this paper). I also did a bit of teaching during my tenure at UWF.
Human Anatomy and Physiology and Cell Biology were my homes away from home sometimes. I could not have been influenced by better mentors in teaching and life than
Mr. Davis and Dr. Pritchard. I’ve also made some great friends in my time at UWF
(aforementioned definitely not excluded). I am grateful for you. I think that’s all. . . . kidding. I will boldly put on paper that I owe my beautiful, new wife Jenny forever.
She stayed on my side for four years of grad school, then still married me. She’s also great at data entry and proofreading and knows more about UVR effects on sea urchin larvae than she ever wanted.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... iii
LIST OF TABLES ...... v
LIST OF FIGURES ...... vi
ABSTRACT ...... vii
INTRODUCTION ...... 1
METHODS ...... 8 A. Collection and Maintenance of Adults ...... 8 B. Gamete Extraction and Fertilization ...... 8 C. Ultraviolet Exposure ...... 9 D. Phytoplankton Culture for Larval Food ...... 10 E. Larval Maintenance ...... 10 F. Measurement of Morphological Effects ...... 11 G. Induction of Settlement ...... 12 H. Experiment 1 ...... 13 I. Experiment 2 ...... 14 J. Experiment 3 ...... 15
RESULTS ...... 18 A. Larval Condition ...... 18 B. Experiment 1 ...... 18 C. Experiment 2 ...... 21 D. Experiment 3 ...... 27
DISCUSSION ...... 32 A. Experimental Outcomes ...... 32 B. Effects on Larval Post-oral Arms ...... 33 C. Effects on Larval Settlement ...... 35 D. Implications of UVR Effects ...... 36
REFERENCES ...... 42
iv
LIST OF TABLES
1. Number of Lytechinus variegatus Larvae Transferred to Glass Bowls by Treatment to Examine Settlement Success in Experiment 2 ...... 12
2. Number of Lytechinus variegatus Larvae Transferred to Glass Bowls by Treatment to Examine Settlement Success in Experiment 3 ...... 13
3. Total Exposure in kJ m-2 Administered to Lytechinus variegatus Larvae in Experiment 1 for 0, 45, 90, or 135 Minutes...... 14
4. Experiment 1: Statistical Outcome of ANOVA for Linear Regression of Right and Left Post-Oral Arm Lengths of Lytechinus variegatus Larvae Exposed to UV Radiation for Different Lengths of Time at the Gastrula Stage ...... 21
5. Experiment 2: Statistical Outcome from One-Way ANOVA of Mean Left and Right Post-Oral Arm Lengths of Lytechinus variegatus Larvae Exposed to 30 Minutes of UV Radiation at the Blastula, Gastrula, or Pluteus Stage and an Unexposed Control ...... 24
6. Experiment 2: Fisher-Hayter Multiple Comparison Tests (Α = 0.01) for Differences in Mean Left and Right Post-Oral Arm Length by Day of Measure in Lytechinus variegatus Larvae Exposed to 30 Minutes of UV Radiation at the Blastula, Gastrula, or Pluteus Stage and an Unexposed Control. Different Letters Indicate Difference, A < B < C ...... 25
7. Experiment 3: Statistical Outcome from One-Way ANOVA of Mean Left and Right Post-Oral Arm Lengths of Lytechinus variegatus Larvae Following Exposure to Natural Sunlight with UV Filters of 280 nm, 320 nm, 395 nm or Completely Covered (Control) at the Gastrula Stage. No Statistically Significant Differences Are Present ...... 31
v
LIST OF FIGURES
1. Experiment 1: Right post-oral arm length (µm) of Lytechinus variegatus larvae exposed to UVR for different lengths of time at the gastrula stage by days following fertilization...... 19
2. Experiment 1: Left post-oral arm length (µm) of Lytechinus variegatus larvae exposed to UVR for different lengths of time at the gastrula stage by days following fertilization...... 20
3. Experiment 2: Mean right post-oral arm lengths (µm ±SE) of Lytechinus variegatus larvae exposed to UVR for 30 minutes at the blastula, gastrula, or pluteus stage and an unexposed control by days following fertilization...... 22
4. Experiment 2: Mean left post-oral arm lengths (µm ±SE) of Lytechinus variegatus larvae exposed to UVR for 30 minutes at the blastula, gastrula, or pluteus stage and an unexposed control by days following fertilization...... 23
5. Experiment 2: Mean percent settlement (±SE) of Lytechinus variegatus larvae 28 and 48 days following 30 minutes of UVR exposure at the blastula, gastrula, or pluteus stage and an unexposed control...... 26
6. Experiment 3: Mean right post-oral arm lengths (µm ±SE) of Lytechinus variegatus larvae following exposure to natural sunlight with UVR filters of 280 nm, 320 nm, 395 nm or completely covered (control) at the gastrula stage...... 28
7. Experiment 3: Mean left post-oral arm lengths (µm ±SE) of Lytechinus variegatus larvae following exposure to natural sunlight with UVR filters of 280 nm, 320 nm, 395 nm or completely covered (control) at the gastrula stage...... 29
8. Experiment 3: Mean percent settlement (±SE) of Lytechinus variegatus larvae 17 and 24 days following exposure to natural sunlight with UVR filters of 280 nm, 320 nm, 395 nm or completely covered (control) at the gastrula stage...... 31
vi
ABSTRACT
EFFECTS OF ULTRAVIOLET RADIATION ON DEVELOPING VARIEGATED SEA URCHINS, LYTECHINUS VARIEGATUS
Eric Cary Tauchman
Lytechinus variegatus larvae were used to examine the effects of ecologically relevant exposures of ultraviolet radiation (UVR) on larval morphology and settlement.
The first experiment investigated the effects of variable time lengths of exposure on gastrula larvae. The second experiment investigated the blastula, gastrula, and pluteus stages of larval development for susceptibility to damage from UVR. In the first two experiments, an artificial light source was used to provide UVR. The third experiment made use of light filters of 280nm, 320nm, and 395nm under natural sunlight to investigate which wavelengths were most deleterious to larvae. Measures to assess UVR effects were larval post-oral arm lengths and percent settlement. Larvae in Experiment 1 displayed significant reduction in left post-oral arm growth two days after exposure. In
Experiment 2, larvae in the blastula stage had a reduction in larval growth, while the gastrula stage, showed a significant reduction in percent settlement. No significant differences were seen between treatment groups in Experiment 3, likely because of the low dose rate on the overcast day of exposure. The experiments show that larvae have measurable morphological consequences and reductions in percent settlement from only a single exposure to ecologically relevant levels of UVR.
vii
INTRODUCTION
Ultraviolet (UVR) radiation is high energy radiation classified into three components based on wavelength. UV-C (200-280nm) is the highest energy component, but is completely absorbed by ozone and oxygen in the atmosphere and therefore is not an environmental concern (Smith & Baker, 1989; El-Sayed & Stephens, 1992). UV-B
(280-320nm) and UV-A (320-400nm) penetrate the atmosphere and are environmentally relevant (El-Sayed & Stephens, 1992; Karentz, Bosch, & Mitchell, 2004). At historically
―normal‖ ozone levels, ozone effectively absorbs most UVR radiation (El-Sayed &
Stevens, 1992; Smith & Baker, 1989; Solomon, 1990); however, stratospheric ozone levels are currently at, or near, an all-time low (Madronich, McKenzie, Bjorn, &
Caldwell, 1998). Ozone loss is due mainly to anthropogenic inputs of ozone-destroying halogens into the upper atmosphere that have occurred during the past several decades
(Roy, Gies, & Elliott, 1990; Smith et al., 1992; Solomon, 1990). Decreases in ozone have led to increases in the amount of (UVR) reaching Earth's surface (Adams & Shick,
2001; Bonaventura, Poma, Costa, & Matranga, 2005; Madronich, 1994), particularly the
UV-B component (Schroeder et al., 2005).
Exposure to UVR may result in several biological changes at the cellular level.
DNA is particularly susceptible to damage because it readily absorbs UV-B (Damkaer,
Dey, Heron, & Prentice, 1980; Epel, Hemela, Shick, & Patton, 1999). Common consequences of exposure are structural changes in DNA such as cyclobutane
1
pyrimidine (Boelen, Obernosterer, Vink, & Buma, 1999; Meador et al., 2002; Akimoto
& Shiroya, 1987), bonding between non-adjacent bases, and single strand breaks
(Karentz, Cleaver, & Mitchell, 1991; Karentz & Bosch, 2001). DNA damage may halt
RNA synthesis and block DNA replication (Kruhlak et al., 2007), which can lead to a reduction or loss of physiological activities (Huot, Jeffrey, Davis, & Cullen, 2000) such as growth and reproduction (Karentz & Lutze, 1990). Proteins and RNA are susceptible to damage from UVR (Herndl, Muller-Niklas, & Frick, 1993; Karentz, 1994). UVR can act as a catalyst in the chemical production of free radicals from components of seawater and cellular fluids, which result in further damage to biomolecules (Epel et al., 1999;
Karentz, 1994; Karentz & Bosch, 2001). Extensive mutagenesis leading to death has been attributed to UVR exposure (Smith & Baker, 1989). Detrimental effects of UVR exposure have been documented in a variety of organisms including DNA damage in bacterioplankton (Jeffrey et al., 1996), reduction of algal productivity (Smith et al.,
1992), cancers in humans (Maytin, Murphy, & Merril, 1993), and mortality of anuran embryos (Grant & Licht, 1995), shrimp larvae (Wubben, 2000), and brittlestars (Johnsen
& Kier, 1998).
Organisms have some ability to repair/avoid UVR damage. One common mechanism is visible light-dependent photoreactivation, which is an enzymatic repair system that corrects UVR damaged DNA (Damkaer & Dey, 1983; Karentz & Bosch,
2001). Photoreactivation can eliminate dimerization in DNA (Mitchell & Hartmann,
1990). This is accomplished by the binding of the enzyme photolyase to the dimer and its removal following exposure to visible light or long wavelength UV-A (Schroder et al., 2005). Another repair system is light-independent nucleotide excision (Sancar,
2
1996). Excision repair is a more complex process, but repairs a greater variety of DNA damage (Mitchell & Hartmann, 1990). This type of repair allows a cell to recognize and remove damaged DNA before reinserting the proper sequence (Karentz & Lutze, 1990).
Heat shock proteins (HSPs) are a group of proteins that are indispensable in the maintenance of cells (Nadeau, Corneau, Plante, Morrow, & Tanguay; 2001). Under normal conditions, HSPs perform such functions as helping maintain the proper configuration of polypeptides and enabling the translocation of synthesized proteins through cell membranes (Schlesinger, 1990). Nearly all cells are capable of increasing production HSPs in response to a wide range of environmental stressors (Trautinger,
Kindas-Mugge, Knobler, & Honigsmann, 1996) and are regarded as a principal cellular defense mechanism (Bonaventura et al., 2005). Their role in response to stress events, such as heat, chemical, or UVR exposure, is to bind to proteins thereby inhibiting denaturation (Mahroof, Zhu, & Subramanyam, 2005; Trautinger et al., 1996;
Schlessinger, 1990).
Mycosporine-like amino acids (MAAs) are nearly as cosmopolitan in their distribution as HSPs. They have been observed in most marine organisms (Dunlap &
Shick, 1998). MAAs absorb UVR (Dunlap & Shick, 1998) acting as "sunscreen" helping to block UVR damage. As an example, MAAs are obtained by adult sea urchins,
Strongylocentrotus droebachiensis, through diet and passed to gametes (Carrol & Shick,
1996). They reduce UVR-induced developmental delays (Adams & Shick, 1996) and abnormalities in the urchin such as blastoderm thickening and the formation of abnormal spicules (Adams & Shick, 2001).
3
In oceans and lakes UVR attenuates with water depth, thus decreasing harmful effects with increasing depth. The depth profile of attenuation depends on the chemical composition and the number of particles in the water column (Boelen et al., 1999). The primary UVR absorbers in water are various types of dissolved organic matter (Franklin
& Forster, 1997) including dissolved organic carbon (Crump, Lean, Berrill, Coulson, &
Toy, 1999) and other humic substances (Kirk, 1994). Even with attenuation, biological effects from UVR exposure have been detected in organisms residing as deep as 20-30m in the oceans (Jerlov, 1950; Karentz & Lutze, 1990) and at least 10m in the Gulf of
Mexico (Wilhelm, Jeffrey, Suttle, & Mitchell, 2002). This depth is well beyond that occupied by a myriad of benthic and planktonic species in both adult and larval stages.
It is in the planktonic larval stage that many benthic animals are most likely to be susceptible to damage from UVR radiation. Cells in larvae are rapidly dividing making them especially vulnerable to lasting DNA damage (Kaufmann & Paules, 1996).
They lack protective coverings (Adams & Shick, 2001; Schroder et al., 2005), and are largely at the mercy of currents to determine their position in the water column (Karentz
& Bosch, 2001).
The end of the larval stage commences with the drastic change from larval to juvenile form. After sufficient time in the plankton larvae become competent; that is they develop the capacity to undergo metamorphosis (Heyland & Moroz, 2006).
Metamorphosis is characterized by the loss of the larval form, behavior, and habitat and the transition to those of the juvenile (Hadfield, Carpizo-Ituarte, Del Carmen, & Nedved,
2001). In many marine invertebrates metamorphosis occurs in response to an external stimulus after sufficient development has occurred (Cameron & Hinegardner, 1974).
4
Settlement cues include a surface covered with a microbial film or algae (Hinegardner,
1969; Cameron & Hinegardner, 1974). Though metamorphosis is often perceived as a new beginning, carry over effects from larva to juvenile may exist (Pechenik, 2006).
Food limitation, toxin exposure, and salinity extremes in the larval stages of a variety of organisms can delay metamorphosis and diminish juvenile success (Pechenik, Gleason,
Daniels, & Champlin, 2001; Pechenik, Jarrett, & Rooney, 2002; Thiyagarajan, Pechenik,
Gosselin, & Qian, 2007; Meidal, Scheibling, & Metaxas, 1999).
The ultimate amount of damage done to an aquatic organism by UVR exposure is related to a combination of interacting factors including UVR total dose and UVR dose rate (Damkaer, Dey, & Heron, 1981), which is related to depth, time of day, water clarity, season, latitude as well as the rate of repair. Species of zooplankton tolerate UVR with no ill effects up to a certain threshold level where detrimental effects begin to appear (Damkaer et al., 1980). This threshold is likely determined by how effectively the rate an organism's repair mechanisms match the rate at which UVR damage occurs
(Damkaer et al., 1980; Damkaer et al., 1981). At a sustained lower exposure to UVR, repair mechanisms are able to fix damage. However, with a shorter duration, but increased intensity of exposure to UVR (amounting to the same total dose) detrimental effects appear (Damkaer et al., 1981). The level of UVR an organism can tolerate is thus dependent on its ability to either avoid or block UVR, and on the efficacy of its repair systems (Karentz, 1994).
Lytechinus variegatus, the variegated sea urchin, is found in the Atlantic and
Caribbean from North Carolina, USA through southern Brazil and is named for the variety of color patterns of the test and spines (Moore, Jutare, Bauer, & Jones, 1963). It
5
is found most commonly in association with sea grass beds, particularly Thalassia testudinum, which makes up a large part of its diet, though it is an omnivorous feeder
(Hill & Lawrence, 2003; Cobb & Lawrence, 2005). The life history of L. variegatus includes a planktonic feeding pluteus larva similar to many other echinoids (George,
Lawrence, & Lawrence, 2004). Larvae pass through a series of stages during which they progressively develop one to four pairs of larval arms, a complete digestive system, calcareous skeleton, and ciliated bands used for feeding and locomotion (McEdward &
Herrera, 1999). They require 9 to 37 days in the larval form before competence is reached and metamorphosis can occur (Mazur & Miller, 1971; McEdward & Herrara,
1999).
L. variegatus could be a model for bio-indication of environmentally detrimental levels of UVR in a subtropical-tropical ecosystem (Steevens, Slattery, Schlenk, Aryl, &
Benson, 1999), a geographical area much less examined in comparison with polar regions. This is of interest because geographical latitude may play a role (sun angle, intensity, seasonality) in UVR adaptation of organisms (Crutzen, 1992). Historical and current use of sea urchins as models in research make results from experiments readily comparable among times, locations, and species (Nacci et al., 2000).
As evidence of the detrimental effects of UVR continues to mount, it is imperative to have a complete understanding of how UVR interacts with organisms. This knowledge is of particular import in the larval stages of species as they are the basis of adult populations (Meidel et al., 1999), as well as an integral part of aquatic food webs.
Planktonic stages of marine invertebrates, including sea urchins, are sensitive to stressors
6
and are useful for ecological monitoring (King & Riddle, 2001; Abessa, Rachid, Ceci, &
Sousa, 2001).
Three experiments were carried out to investigate effects of UVR exposure on the development of L. variegatus larvae. The first experiment addressed the question of how different durations of UVR exposure at the gastrula stage affect larval arm length.
The second experiment addressed the questions of how exposure to UVR at different larval stages affects larval arm length and percent settlement. The third experiment addressed the questions of how exposure to different UVR wavelengths at the gastrula stage affects larval arm length and percent settlement. Experiments tested the hypothesis that UVR exposure has detrimental effects on developing L. variegatus larvae, and the effects will manifest themselves through decreased growth rates of larval arms and a reduction in percent settlement.
7
METHODS
Collection and Maintenance of Adults
Adult Lytechinus variegatus were collected by hand from St. Joseph's Bay,
Florida (29° 52.4' N, 85° 23.4' W). Animals were individually bagged in quart-sized
Glad-Lock® Zipper storage bags and placed in coolers for transport to laboratory facilities at the University of West Florida, Pensacola, Florida, USA. Six to ten animals were held per 38 L (10 gal) aquarium. They were acclimated to a water temperature of
22 °C and a salinity of 30‰ for not less than five days before use. Water quality was maintained using air-driven box filters filled with activated carbon and polyester filter fiber. The adults were fed daily to every other day with spinach or romaine lettuce.
Gamete Extraction and Fertilization
Gametes were extracted by a 3 mL injection of 0.5 M potassium chloride into the coelom through the peristomial membrane using a 10 ml syringe with a 22 gauge needle
(Strathmann, 1987), which caused spawning to commence. Multiple individuals were injected until 10 of each sex spawned. Sperm were collected and stored in transfer pipets at 4 °C until eggs were prepared for fertilization. Female urchins were placed inverted over 100 ml beakers filled with filtered (63 μm) seawater (30‰). The eggs were allowed to settle, filtered with a 125 μm sieve into a 1 L beaker, and rinsed twice with seawater.
Sperm were activated by adding approximately 20 μl to 10 ml of seawater (Mazur &
8
Miller, 1971). Sperm were diluted to prevent polyspermic fertilizations that can occur with high sperm density (Levitan, 2005). All activated sperm samples were then combined. The combined sperm solution (3 ml) was added to the egg solution (1 L).
Fertilization was confirmed by microscopic observation of the vitelline membrane. The beaker was covered with paper towels and larvae observed until the appropriate developmental stage was reached for a particular experiment.
Ultraviolet Exposure
A full spectrum Xe-arc lamp (Oriel model 66021) was used to expose larvae to
UVR in Experiments 1 and 2. The exposure cuvette containing a batch of larvae was a covered, opaque acrylic cylinder with a UVR transparent acrylic bottom (14 cm diameter, 13 cm depth). An acetate filter was positioned between the lamp and cuvette, immediately below the cuvette, to filter out wavelengths shorter than 295 nm, which are not environmentally relevant (Kirk, 1994). Radiation from the lamp was directed to a mirror positioned 67 cm from the lamp and 22 cm below the cuvette where the light was reflected up through the cuvette. Water in the cuvette was gently stirred by airflow provided by two airline tubes (4 mm internal diameter) positioned on opposite sides of the cuvette just above the surface of the water creating a circular water motion to help equalize UVR exposure within a batch of larvae. The cuvette was immersed in a 24 °C water bath during UVR exposure to maintain constant temperature.
A Biospherical Instruments GUV 511C Radiometer was positioned midway in the seawater filled cuvette to measure the irradiance at 305, 320, 340, and 380 nm.
Irradiances by wavelengths were 15.28, 20.39, 38.39, and 49.54 μwatts cm-2,
9
respectively. Integrated UVR was measured using an International Light IL1400 UVB
Radiometer and was 75% of the noon time spring equinox in Pensacola, Florida, USA
(30° 25’ N, 87° 13’ W). In Experiment 3 larvae were exposed to natural sun light
(described below).
Phytoplankton Culture for Larval Food
The phytoplankton food source for larvae consisted of Nannochloropsis, a non- motile, small, green algae; and Rhodomonas, a flagellated, red algae (Hoff & Snell,
1987). Stock cultures of the phytoplankton genera were obtained from Aquatic
Ecosystems, Inc., and the Seahorse Source respectively. After initial inoculation, phytoplankton cultures were maintained in clear, 2 L plastic bottles. They were grown in f/2 nutrient media (Guillard, 1975) with air bubbled in to provide gases and agitation. A
12:12 light dark cycle was maintained using one cool-white fluorescent bulb (General
Electric, 15 Watt, 4100K).
Aliquots of each phytoplankton culture were centrifuged in 50 ml Falcon tubes for 5 min at 350 × g to remove media. Phytoplankton were then resuspended in clean seawater before being added to larval cultures in a ratio of 1:2
Nannochloropsis:Rhodomonas at a concentration of 25,000 phytoplankton cells ml-1.
Algal cell counts to determine concentration of food supply were made using a Spencer
Bright-Line Neubauer chamber.
Larval Maintenance
Following UVR exposure, each batch of larvae from the cuvette was maintained in separate plastic buckets (2.5 L) at a larval density of approximately 1 ml-1
10
(Hinegardner, 1969). All buckets were placed in a culture system consisting of a water bath (24°C) and a paddle system modified from Strathmann (1987) to stir the water inside the buckets providing agitation and aeration. Larvae obtain a functioning digestive tract and are thus capable of feeding at about 24 hours (Mazur & Miller, 1971).
Beginning at this time, they were fed every other day with the phytoplankton mix. After four to five days of culture approximately half of the water in each bucket was removed by siphoning and straining through 63 µm mesh netting, and was replaced with fresh filtered seawater.
Measurement of Morphological Effects
Larval cultures were examined daily by removing a 50 mL water sample via
Finnpipette Labsystems 4540 model 50 mL pipette. The water sample was split across two Petri dishes and all larvae were counted by observation through a stereomicroscope.
The approximate stage of development for each culture was categorized as blastula, gastrula, 2-arm pluteus, 4-arm pluteus, 6-arm pluteus, and 8-arm pluteus (McEdward &
Herrera, 1999). A stage of development for a bucket was considered reached when 50% of the larvae had reached that stage (George & Lawrence, 2002). Growth of larval structures was examined by haphazardly removing five larvae and placing each on a glass slide under a cover slip. Arm measures for all treatments and controls were taken as near to identical times in development as possible. Two measurements of right and left post-oral arms were made using an ocular micrometer at 100 x magnification.
Larvae in the 50 mL sample were then discarded. Larvae were measured through the 8- arm stage.
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Induction of Settlement
Larvae that were competent to settle possessed a readily visible rudiment. At this point, number of larvae in each culture bucket was estimated by counting larvae present in a 50 mL sample and multiplying by the volume (mL) of the bucket divided by 50 mL.
To examine settlement success, all surviving larvae were transferred to glass bowls (20 cm diameter, 7 cm depth), with one bowl for each bucket, by filtering via 63 μm mesh netting and resuspension (Experiment 2 – Table 1; Experiment 3 – Table 2).
Table 1
Number of Lytechinus variegatus Larvae Transferred to Glass Bowls by Treatment to Examine Settlement Success in Experiment 2
Replicate Control Blastula Gastrula Pluteus
1 1360 1660 2160 2160
2 1500 1220 1760 1760
3 1260 2240 1360 1360
4 480 940 1180 1180
5 1460 1260 2800 2800
6 1180 1900 1820 1820
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Table 2
Number of Lytechinus variegatus Larvae Transferred to Glass Bowls by Treatment to Examine Settlement Success in Experiment 3
Replicate Control 280nm Filter 320nm Filter 395nm Filter
1 620 500 660 360
2 540 420 740 640
3 480 260 580 640
4 620 500 680 660
Glass bowls contained 1 L of seawater collected from the natural environment
(collection location of adults) to provide the necessary settlement cues. Settlement was deemed successful when individuals obtained the juvenile appearance (Carpizo-Ituarte,
Salas-Garza, & Pares-Sierra, 2002). Percent settlement per bowl was calculated as
(number of juveniles per bowl / number of initial larvae per bowl) * 100.
Experiment 1
Experiment 1 examined the sensitivity of larvae to different times of UVR exposure. Adults were collected on July 3, 2007, and induced to spawn on July 25, 2007.
Treatments consisted of exposure times of 0, 45, 90, or 135 min. Total exposures are displayed in Table 3. Approximately 15,000 larvae at the gastrula stage were pipetted into 1 L of filtered seawater in the exposure cuvette for each exposure treatment. The
UVR lamp was turned on for the appropriate exposure time. Following exposure, larvae from each treatment (n = 1 batch of 15,000 per treatment) were evenly distributed into 13
six buckets, the volume of each brought to 2.5 L with filtered sea water, and placed in the culture system.
Table 3
Total Exposure in kJ m-2 Administered to Lytechinus variegatus Larvae in Experiment 1 for 0, 45, 90, or 135 Minutes.
Time (minutes) 305 nm 320 nm 340 nm 380 nm
0 0 0 0 0
45 0.41 0.55 1.04 1.34
90 0.83 1.10 2.07 2.67
135 1.24 1.65 3.11 4.01
Morphological measures of larval arms were made daily from July 27 to August
4, 2007. Percent settlement was not determined in this experiment. Data analysis for
Experiment 1 was conducted using linear regression with one replicate for each quantitative treatment level. ANOVA was used test regression slope = 0 for each day of measurement (α 0.01 considered significant to account for multiplicity).
Experiment 2
Experiment 2 examined the relative sensitivity of different larval stages to UVR.
Adults were collected on August 1, 2007 and induced to spawn on August 15, 2007. The treatment groups were exposure at the blastula, gastrula, or pluteus stage, and an unexposed control group. All groups of larvae were exposed for 30 min providing a total
14
exposure of 0.28, 0.37, 0.69, and 0.89 kJ m-2 at wavelengths of 305, 320, 340, and 380 nm respectively. After pre-exposure larval cultures had reached the appropriate stage, six replicate batches per stage of 2,500 larvae per batch were created. Each batch was pipetted into 1 L of filtered sea water in the exposure cuvette in random order and subjected to irradiation. Immediately following exposure a batch of larvae was transferred from the cuvette to a bucket in the culture system. The volume of culture buckets was then brought to 2.5 L.
Morphological measures of larval arms were made daily from August 16 to
August 24, 2007. Remaining larvae were presented with settlement cues on August 24.
Percent settlement was measured on September 12 and October 2, 2007. Data analysis for morphological measures was conducted using a one-way ANOVA with six replicates for each treatment for each day of measurement (α 0.01 considered significant to account for multiplicity). Data for percent settlement were rank transformed to correct for violations of ANOVA assumptions before performing one-way ANOVAs for two dates
(α 0.025 considered significant to account for multiplicity). Normality was tested using skew, kurtosis, D’Agostino and Pearson, and Anderson and Darling tests (D’Agostino,
Belanger, & D’Agostino, 1990; Anderson & Darling, 1954). Homogeneity of variance was tested using the Brown-Forsythe homogeneity of variance test (Brown & Forsythe,
1974). Significant ANOVA was followed with a Fisher-Hayter multiple comparison test
(Hayter, 1986).
Experiment 3
Experiment 3 examined the relative sensitivity of larvae to different wavelengths of UVR exposure under natural sunlight. Adults for this experiment were collected
15
August 1, 2007, and induced to spawn on September 22, 2007. Treatment groups were differentiated by broad band UVR cut-off filters. Group one was covered with a 280 nm filter, group two with a 320 nm filter, group three with a 395 nm filter, and group four wrapped in aluminum foil to provide an unexposed control. Approximately 2,000 larvae in the gastrula stage were pipetted into UVR-transparent whirlpak bags (four bags per treatment), containing 100 ml filtered seawater, placed in a circulating water bath at
23 °C, and exposed to natural sunlight for 5 hours at a roof-top location under largely overcast conditions on September 23, 2007. Exposures were 0.20, 1.44, 3.00, and 3.56 kJ m-2 at wavelengths of 305, 320, 340, and 380 nm, respectively, measured by a
Biospherical Instruments GUV 511C Radiometer. Following exposure, bags were emptied into randomly assigned plastic buckets, the volume brought to 2 L with filtered seawater, and placed in the culture system.
Morphological measures of larval arms were made daily from September 24 to
October 1, 2007. Larvae were presented with settlement cues on October 1. Percent settlement was measured on October 8 and October 15, 2007. Data analysis for morphological measures was conducted using a one-way ANOVA with four replicates for each treatment for each day of measurement (α 0.01 considered significant to account for multiplicity). Data for percent settlement was analyzed by one-way ANOVAs for two dates (α 0.025 considered significant to account for multiplicity). Normality was tested using skew, kurtosis, D’Agostino and Pearson, and Anderson and Darling tests
(D’Agostino et al., 1990; Anderson & Darling, 1954). Homogeneity of variance was tested using the Brown-Forsythe homogeneity of variance test (Brown & Forsythe,
16
1974). Significant ANOVA was followed with a Fisher-Hayter multiple comparison test
(Hayter, 1986).
17
RESULTS
Larval Condition
Larvae successfully fed on Rhodomonas and Nannochloropsis throughout the three experiments. Larvae appeared to preferentially feed on Nannochloropsis for the first day of feeding likely because its small size and immobility allowed for relative ease of capture. Larvae in all treatment groups in all experiments maintained the capacity for feeding as indicated by the presence of phytoplankton in the stomachs. They advanced through larval stages as described by Mazur and Miller (1971), George et al. (2004), and
McEdward and Herrera (1999). Competence was reached at about ten days in each treatment in all three experiments; timing was similar to observations of McEdward and
Herrera (1999). After exposure to cues apparently present in water native to the urchin collection site, metamorphosis to the juvenile form was observed in as little as three hours.
Experiment 1
Both right (Figure 1) and left (Figure 2) post-oral arm lengths increased over the time of measurement (nine days). A plateau in length was reached on Day 5 for both post-oral arms with little change in length from Day 6 to Day 10. A significant non-zero slope for the linear regression was detected on Day 3 for right post-oral arm length
(Table 4) indicating a difference across treatments. Order of right post-oral arm length by 18
treatment was control > 45 min exposure > 90 min exposure > 135 min exposure.
Beyond this day post-oral arm length and the quantitative treatment order were not aligned resulting in non-significant tests of slope for all other days of measurement. No statistically significant differences were detected for left post-oral arm length.
600
500
400
300 Arm Length (µm) Arm Length
Exposure=0 min _ _ _ Exposure= 45 min 200 Exposure= 90 min - - - - Exposure= 135 min
100 0 2 4 6 8 10 12
Day Post Fertilization
Figure 1. Experiment 1: Right post-oral arm length (µm) of Lytechinus variegatus larvae exposed to UVR for different lengths of time at the gastrula stage by days following fertilization.
19
600
500
400
300 Arm Length (µm) Length Arm
Exposure=0 min _ _ _ 200 Exposure= 45 min Exposure= 90 min - - - - Exposure= 135 min
100 0 2 4 6 8 10 12
Day Post Fertilization
Figure 2. Experiment 1: Left post-oral arm length (µm) of Lytechinus variegatus larvae exposed to UVR for different lengths of time at the gastrula stage by days following fertilization.
Table 4
Experiment 1: Statistical Outcome of ANOVA for Linear Regression of Right and Left Post-Oral Arm Lengths of Lytechinus variegatus Larvae Exposed to UV Radiation for Different Lengths of Time at the Gastrula Stage
Left Arm Right Arm Day F (1,2) P value Day F (1,2) P value
2 5.576 0.142 2 10.119 0.0862
3 66.47 0.0147 3 416.663 *0.002391
4 1.029 0.4172 4 1.013 0.4202
5 3.316 0.2102 5 1.242 0.3811
6 5.811 0.1375 6 0.9782 0.4269
8 0.9461 0.4333 8 1.7548 0.3164
10 2.973 0.227 10 1.517 0.3432
* P value indicates significant difference between treatments
Experiment 2
Both right (Figure 3) and left (Figure 4) post-oral arm lengths increased over the time of measurement (nine days). Mean arm lengths in larvae exposed during the gastrula and blastula stage were consistently closer together and less than mean arm lengths in control larvae and larvae exposed at the pluteus stage, which were similar to one another, leading to a highly significant treatment effect by the end of nine days
(Tables 5 and 6).
21
700
600
500
400 Arm Length (µm) Arm Length Control 300 - - - - Blastula Gastrula
200 _ _ _ Pluteus
100 0 2 4 6 8 10
Day Post Fertilization
Figure 3. Experiment 2: Mean right post-oral arm lengths (µm ±SE) of Lytechinus variegatus larvae exposed to UVR for 30 minutes at the blastula, gastrula, or pluteus stage and an unexposed control by days following fertilization.
700
600
500
400 Arm Length (µm) Arm Length Control 300 - - - - Blastula Gastrula _ _ _ 200 Pluteus
100 0 2 4 6 8 10
Day Post Fertilization
Figure 4. Experiment 2: Mean left post-oral arm lengths (µm ±SE) of Lytechinus variegatus larvae exposed to UVR for 30 minutes at the blastula, gastrula, or pluteus stage and an unexposed control by days following fertilization.
Table 5
Experiment 2: Statistical Outcome from One-Way ANOVA of Mean Left and Right Post- Oral Arm Lengths of Lytechinus variegatus Larvae Exposed to 30 Minutes of UV Radiation at the Blastula, Gastrula, or Pluteus Stage and an Unexposed Control
Left Arm Right Arm Day F (3,20) P value Day F (3,20) P value
1 1.78 0.183 1 2.27 0.1119
2 12.39 *0.00008 2 10.57 *0.00023
3 6.6 *0.0028 3 4.46 0.0149
4 6.01 *0.00431 4 3.69 0.0291
5 3.37 0.039 5 7.18 *0.0019
6 3.18 0.0462 6 5.08 *0.0089
7 4.73 0.0119 7 4.04 0.0212
9 8.44 *0.000802 9 5.41 *0.00683
* P value indicates significant difference between treatments
Table 6
Experiment 2: Fisher-Hayter Multiple Comparison Tests (Α = 0.01) for Differences in Mean Left and Right Post-Oral Arm Length by Day of Measure in Lytechinus variegatus Larvae Exposed to 30 Minutes of UV Radiation at the Blastula, Gastrula, or Pluteus Stage and an Unexposed Control. Different Letters Indicate Difference, A < B < C
Right Post-oral Arm Left Post-oral Arm Day Control Blastula Gastrula Pluteus Day Control Blastula Gastrula Pluteus
1 A A A A 1 A A A A
2 B A AB B 2 B A AB B
3 B A AB B 3 B A AB AB
4 A A A A 4 B A AB AB
5 B A AB AB 5 A A A A
6 B AB A AB 6 A A A A
7 A A A A 7 B AB A AB
9 B AB A AB 9 C AB A BC
A pattern of control and blastula exposed larvae being close together in mean percent settlement; and gastrula and pluteus exposed larvae being close together in mean percent settlement is evident at both 28 and 48 days (Figure 5). A large amount of variation in percent settlement reduced the power of the statistical tests. At 28 days after fertilization, no statistically significant difference in mean percent settlement occurred between treatments (One-Way ANOVA F (3,20) = 2.50, P = 0.208). After 48 days, mean percent settlement of unexposed control larvae was significantly greater compared to
25
larvae exposed at the gastrula stage (One-Way ANOVA F (3,20) = 2.66, P = 0.014), based on Fisher-Hayter MCP outcome). No significant differences in mean percent settlement occurred between other treatments.
7 Control PluteiPluteus 6 GastulaGastrulae BlastulaBlastulae
5
4
3 Percent Settlement Percent
2
1
0 29 2831 37 4839 41
Day Post Fertilization
Figure 5. Experiment 2: Mean percent settlement (±SE) of Lytechinus variegatus larvae 28 and 48 days following 30 minutes of UVR exposure at the blastula, gastrula, or pluteus stage and an unexposed control.
26
Experiment 3
Both right (Figure 6) and left (Figure 7) post-oral arm lengths increased over the time of measurement (nine days). No statistically significant differences were detected for either mean left or right post-oral arm lengths among treatments for all days (Table
7). Mean percent settlement varied between 20 - 50% (Figure 8) with no significant differences among treatments on either day of measure (Day 17: One-Way ANOVA F
(3,12) = 0.92, P = 0.46; Day 24: One-Way ANOVA F (3,12) = 0.99, P = 0.43).
27
550
Control 500 _ _ _ 280 nm 320 nm
450 - - - - 395 nm
400
350 Arm Length (µm) Length Arm
300
250
200 0 2 4 6 8 10
Day Post Fertilization
Figure 6. Experiment 3: Mean right post-oral arm lengths (µm ±SE) of Lytechinus variegatus larvae following exposure to natural sunlight with UVR filters of 280 nm, 320 nm, 395 nm or completely covered (control) at the gastrula stage.
550
Control 500 _ _ _ 280 nm 320 nm
450 - - - - 395 nm
400
350 Arm Length (µm) Arm Length
300
250
200 0 2 4 6 8 10
Day Post Fertilization
Figure 7. Experiment 3: Mean left post-oral arm lengths (µm ±SE) of Lytechinus variegatus larvae following exposure to natural sunlight with UVR filters of 280 nm, 320 nm, 395 nm or completely covered (control) at the gastrula stage.
Table 7
Experiment 3: Statistical Outcome from One-Way ANOVA of Mean Left and Right Post- Oral Arm Lengths of Lytechinus variegatus Larvae Following Exposure to Natural Sunlight with UV Filters of 280 nm, 320 nm, 395 nm or Completely Covered (Control) at the Gastrula Stage. No Statistically Significant Differences Are Present
Left Arm Right Arm Day F (3,12) P value Day F (3,12) P value
2 3.27 0.059 2 1.32 0.312
3 0.73 0.555 3 3.47 0.051
4 0.72 0.561 4 1.45 0.278
5 0.81 0.513 5 0.53 0.670
6 1.19 0.354 6 1.34 0.306
8 0.52 0.675 8 0.37 0.779
9 1.00 0.426 9 1.14 0.373
70 Control 280 nm 60 320 nm 395 nm
50
40
30 Percent Settlement Percent
20
10
0 17 24 15 16 20 23 24 Day Post Fertilization
Figure 8. Experiment 3: Mean percent settlement (±SE) of Lytechinus variegatus larvae 17 and 24 days following exposure to natural sunlight with UVR filters of 280 nm, 320 nm, 395 nm or completely covered (control) at the gastrula stage.
DISCUSSION
Experimental Outcomes
In Experiment 1, L. variegatus larvae had treatment-dependent diminished growth of the right post-oral arm on the second day following UVR exposure. This could imply damage done to larvae was not detectable one day after irradiation, and larvae were then able to repair and compensate for damage after only one more day. No change was found for left post-oral arms. Asymmetrical arm development has been observed in
Paracentrotus lividus embryos exposed to UVR (Schroder et al., 2005). The possibility exists that the effect may have been observable on more days with increased replication as there was only a single replicate in the present study leading to low power of the statistical test. Even a one day disadvantage in nine available to the larvae to prepare for juvenile life could be costly in terms of larval success and survival. However, lack of replication leaves the conclusion uncertain. The experiment was successful in providing an estimate for the appropriate exposure length for Experiment 2.
In Experiment 2, L. variegatus larvae had reduced growth in left and right post- oral arm lengths following exposure to environmentally relevant levels of UVR, the extent of which was dependent on the developmental stage at time of exposure. Once detected, the growth deficiencies in the post-oral arms of larvae exposed at an early stage were observed during the entire span of the larval life.
32
In Experiment 3, L. variegatus larvae received a similar total UVR exposure as in the second experiment, but at a much reduced exposure-rate leading to no observable differences between treatment groups suggesting dose-rate dependence relative to effects. Reciprocity is the condition in which total UVR exposure, regardless of the rate at which it is received, is the determining factor leading to UVR-related damage. The absence of reciprocity in this experiment has also been observed in other sea urchin species (Anderson, Hoffman, Wild, Bosch, & Karentz, 1993) and other animals (Lesser and Barry, 2003; Damkaer et al., 1980; Damkaer et al., 1981).
Effects on Larval Post-oral Arms
The earlier larval stages appear to be the most susceptible to UVR damage with regard to post-oral arm growth. Sea urchin embryos contain pre-skeleton cells that lie in the epithelium of the blastula before moving into the blastocoel (Ettensohn, 1990;
Harky, Klueg, Sheppard, & Raff, 1995). Though the organism is small, the positioning of the cells on the surface in the early blastula would reduce the protection from UVR that even a few cell layers affords the later ingressed primary mesenchyme cells leading to effects manifesting from exposure at an early stage of development. Bonaventura,
Poma, Russo, Zito, and Matranga (2006) showed that UVB is more detrimental to early sea urchin embryos of Paracentrotus lividus than more advanced stages. The reduced post-oral arm growth of larvae exposed to UVR early in development could be a result of the necessity of larvae to repair cellular damage before growth is possible.
Reduction in growth could also be a consequence of resource allocation. Larvae manufacture heat shock or stress proteins in response to UVR (e.g., Bonaventura et al.,
33
2006). When expressed, heat shock proteins act by binding to other proteins to inhibit denaturation caused by exposure to a stressor such as heat, chemicals, or UVR (Mahroof et al., 2005; Trautinger et al., 1996). The increased manufacture of proteins would require larval resources possibly drawing from those which would be allocated to growth in the absence of UVR stress. Protein synthesis is a major contributor to metabolic changes in response to stress (Guppy, Fuery, & Flanigan, 1994). The energetic cost of protein synthesis increases with increased protein upregulation (Pace & Manahan, 2005).
The onset of the ability to transcribe HSPs varies with the species in sea urchins.
Arbacia lixula is able to synthesize HSP70 at the 32-64 cell blastomere stage, but
Paracentrotus lividus and Sphaerechinus granularis do not have the capacity until after the blastula emerges from the vitelline membrane (Guidice et al., 1986). This delay until hatching, with regards to a 21 kDa HSP, was also seen in L. pictus and
Strongylocentrotus purpuratus (Infante, Akhayat, Rimland, & Infante, 1986). Howlett,
Miller, and Schultz (1983) observed Arbacia punctulata first transcribing HSP proteins at 64-128 cells. The timing of transcription of HSP would have ramifications as to the organism’s ability to protect itself via HSP upregulation, and whether or not it has the option of reallocating resources from growth to increasing production of proteins. DNA repair mechanisms could also slow the growth process as they would be additional energetic expenses.
Reduction in arm length is of biological interest as echinoid larval arms form the basis of larval ciliated bands. Ciliated bands are ciliated structures that produce water currents enabling both feeding and locomotion (Strathmann, 1971; McEdward, 1984).
Clearing rates, the ability of larvae to obtain food, are directly proportional to the lengths
34
of the ciliated bands (McEdward & Herrera, 1999). Urchin larvae exhibit phenotypic plasticity in response to food availability. Larvae tend to grow longer arms, thus increasing ciliated band length, in instances of food paucity (Hart & Strathmann, 1994).
Little rudiment development occurs until feeding structures have completely developed and clearing rates near their maximum (Strathmann, 1971), so delays in post-oral arm development could delay rudiment development.
Effects on Larval Settlement
While reduction of post-oral arm growth was most notable when larvae were exposed to UVR at an earlier stage of development, the effect of UVR on percent settlement was most pronounced when larvae were exposed to UVR at a later stage of development. Perhaps structures pertinent to metamorphosis were not present in the early stages, so were not exposed to UVR, or they were able to be repaired by the time competence was reached. Possibly, if metamorphic structures are damaged later in development more time in the plankton than normally occurs would be needed to repair the damage. The implication is that a single, noteworthy exposure to UVR at the right time of larval development has repercussions on settlement, thus leading to possible effects on the adult population. The pattern of later stages being more susceptible to stress has also been observed in the sea urchin Sterechinus neumayeri, where larvae are more sensitive to metals when exposure occurs in the pluteus stage than the blastula
(King & Riddle, 2001).
Larvae and adults can be affected by the environment separately from one another. Effects in the larvae that transcend metamorphosis, such as percent settlement
35
in L. variegatus larvae in the present experiment, are known as latent effects and have been documented in other species (Pechenik, 2006; Pechenik et al., 2001). Post- settlement survival and juvenile growth rates are reduced by salinity stress to larvae of polychaete worms (Pechenik et al., 2001). Growth rate of juvenile barnacles can be reduced due to delayed metamorphosis (Thiyagarajan et al., 2007). Starving early larval prosobranch gastropods (Crepidula fornicata) for short time periods reduced juvenile growth rates by up to one third even when larval growth ultimately reached that of unstarved controls (Pechenik et al., 2002).
Implications of UVR Effects
Detrimental effects due to UVR have been documented in a range of urchin species. Considerable research has been conducted on the sea urchin Sterechinus neumayeri, a species with an Antarctic circumpolar distribution (Karentz et al., 2004) where the greatest global reduction in ozone occurs (Smith et al., 1992). In this species,
UVR exposure has been linked to irregular and delayed development, including deviations from normal mitotic processes, abnormal cleavages, and other aberrant embryological morphologies (Anderson et al., 1993; Karentz et al., 2004). DNA damage and increased mortality have also been documented (Lesser, Lamare & Barker, 2004).
Cellular abnormalities in Sterechinus neumayeri display a dose-rate dependent response to UVR exposure (Anderson et al., 1993).
Urchin species ranging beyond the Antarctic "ozone hole" are also susceptible to damage from UV radiation. In Stongylocentrotus droebachiensis harvested from the
Gulf of Maine, ecologically relevant levels of UVR caused developmental delays,
36
morphological abnormalities, and diminished survival (Adams & Schick, 2001; Lesser
& Barry, 2003). Under similar conditions, significant DNA damage in the form of cyclobutane pyrimidine dimers linked to apoptosis was also observed in S. droebachiensis (Lesser, Kruse & Barry, 2003). A more temperate species of urchin,
Paracentrotus lividus, displayed developmental anomalies and additionally showed elevated levels of HSP 70 expression in response to UVR exposure (Bonaventura et al.,
2006). Some types of UVR damage/response are likely to be general in most species of urchins; however, some responses to UVR seem to be species-specific (Keller et al.,
1997).
In the current set of experiments, phytoplankton were provided in abundance to eliminate the prospect of food limitation being a factor affecting growth. The effects of
UVR would likely be compounded by the reduced ability of larvae to phenotypically respond to food limitations. Larvae with shorter arms would be less able to capture food in a more natural, food-limited setting. Strongylocentrotus droebachiensis larvae required roughly twice as much developmental time under food limitation, and their development was incomplete (Meidel et al., 1999). Under conditions of food shortage, larvae experience a delay in rudiment development (Hart & Strathmann, 1994) causing settlement delays, which can lead to reduced fitness in juvenile and adult invertebrates
(Pechenik, 2006).
In a natural setting where the entire ecosystem is exposed to UVR, difficulty in larvae obtaining food might be further exacerbated by damage to phytoplankton.
Phytoplankton, having little or no control over position in the water column and short generation time, are highly susceptible to UVR damage (Karentz & Bosch, 2001). Upon
37
exposure to UVR, DNA damage occurs (Meador et al., 2002), photosynthesis is reduced
(Cullen, Neale, & Lesser, 1992; Smith et al., 1992), carbon fixation declines (Karentz &
Bosch, 2001; Kirk, 1994), and organismal growth and the ability to complete the cell cycle diminishes (Gieskes & Buma, 1997).
A combination of reduced arm growth and depleted food extends the time period of the echinoid larval stage (Pedrotti & Fenaux, 1993). Extension of the larval stage would increase the amount of time larvae are susceptible to predation and other causes of mortality that are already a great obstacle to survival of planktonic organisms
(Morgan, 1995). Though the capacity to delay metamorphosis has been cited as an adaptive advantage offering the opportunity to bypass unsuitable habitat (Hadfield et al.,
2001), in a setting such as Saint Joe Bay where L. variegatus occurs, this extended time would more likely allow for inadvertent, current-mediated migration out of the suitable habitat the bay provides to a less suitable or entirely inhospitable environment.
Adult L. variegatus exhibit a covering response partly based on exposure to UVR
(Sharp & Gray, 1962). If behavioral responses to UVR also exist in larvae, such as swimming away from the surface, any reduction in arm length could handicap avoidance efforts as it would reduce swimming potential (Emelet, 1991). The larval population may be further impacted by deleterious UVR effects on gametes. Since urchins are broadcast spawners, gametes are exposed to environmental UVR before fertilization takes place. The slight negative buoyancy of eggs in seawater would perhaps spare them exposure to the most powerful UVR doses, but they lack any UVR protective covering
(Schroder et al., 2005). Sperm, however, would be positioned throughout the water
38
column. Exposure to UV-B radiation decreases sperm motility along with fertilization success (Au et al., 2002).
Damage from UVR in echinoderms is not limited to echinoids. In the asteroid
Marthasterias glacialis, UVR inactivates sodium channels in the egg cell membrane increasing polyspermic fertilizations (David, Moreau, Vilain, & Guerrier, 1985). UVR exposure produced significant damage to Psilaster charcoti embryos in Antarctica
(Bosch, Komatsu, Murakami, & Krakowski, 2001). The adult stage may also be susceptible to UVR. In the holothuroid Stichopus japonicas, and other species, the body wall can undergo a process known as melting, the dissociation of catch-connective tissues, in response to UVR exposure (Zhu et al., 2008). Ophioderma brevispinum, an ophiuroid, experienced higher mortality upon exposure to natural sunlight off the coast of North Carolina (Johnsen & Kier, 1998).
Impacts of UVR on larvae, or reproductive potential, have been documented in a variety of other phyla. Corals display a number of different negative effects from UVR exposure. UVR induces bleaching, which is the expulsion of the zooxanthellae symbiont necessary for coral health (Glynn, 1996). Bleaching is immediately harmful to the coral and, if it survives, can reduce reproduction for a considerable time after recovery
(Torres, Armstrong, & Weil, 2008). Colonies of Acropora cervicornis transferred to shallower depth, leading to increased UVR exposure, showed a marked decrease in gonads per polyp (Torres et al., 2008). Planula larvae of the coral Porites asteroides preferentially settle in areas of reduced UVR radiation (Gleason, Edmunds, & Gates,
2006). UVR can have significant impacts on food web structure in part because of differential sensitivity of planktonic organisms (Belzile et al., 2006). A survey of 53
39
lakes in Patagonia indicated that richness and diversity of plankton declined when average water column UVR reached ten percent of surface radiation (Marinone et al.,
2006).
Fish may have larval stages that are susceptible to UVR damage. Catla catla larvae have a dose-rate dependent reduction in survival due to UVR stress (Sharma,
Mittal, & Chakrabarti, 2008). Embryos of Atlantic cod exposed to a UVR equivalent of
10m depth in the Gulf of Maine experience increased mortality concomitant with increased levels of cyclobutane pyrimidine dimers (Lesser, Farrell, & Walker, 2001).
The detrimental impacts of UVR are not always in the form of direct damage. Rainbow trout have reduced resistance to a bacterium and a trematode when exposed to UVR
(Markkula, Karvonen, Salo, Valtonen, & Jokinen, 2007). Juvenile salmon modify behavior in the form of shade seeking, which decreases feeding, in the presence of UVR
(Holtby & Bothwell, 2008).
In the present three experiments larvae received only a single exposure of UVR to allow more precise control of amount and dose rate. This would allow for repair of
UVR damaged structures without further damage created by the next day's UV radiation that would be present in the ocean. Additionally, larvae were provided with clean, filtered sea water during exposures and subsequent development. In nature the larvae could encounter a number of chemical contaminants, such as polycyclic aromatic hydrocarbons released from human activity which increase toxicity when exposed to
UVR, posing an additional stress to organisms (Steevens et al., 1999).
Ultraviolet radiation is a parameter impacting and stressing shallow water and terrestrial ecosystems. The World Meteorological Organization/United Nations
40
Environmental Programme "Scientific Assessment of Ozone Depletion: 2006" estimates the middle latitude ozone levels are 3% below pre-1980 values and that this will persist through the first half of the 21st century. That estimate assumes the continued decline in the use anthropogenic ozone depleting chemicals. In this study, and others, it is apparent that ecologically relevant levels of UVR have direct impacts on organisms in the environment and that the relatively unprotected larvae are often more susceptible than adults of the same species. While it is possible to approximate natural settings in the laboratory, it is impossible to include all factors that may be pertinent to a study in such a fashion. The aquatic ecosystem is complex, and the ways organisms interact and behave with regard to UVR—avoidance, repair mechanisms, timing of spawning, and others are difficult to quantify, but since increased levels of UVR will continue to affect these ecosystems for some time to come, every effort should be made to investigate its impact.
41
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