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Home , Atlantic horseshoe crab, Bioturbation

Effect of Horseshoe Bioturbation on Food Availability and Feeding Efficiency of Mummichogs Within Intertidal Flats

Kimberly Ohnemus Skidmore College, Saratoga Springs NY Semester in Environmental Science 2012

In collaboration with: Dr. Kenneth Foreman Dr. Daniel G. Gibson III Marine Biological Laboratory, Woods Hole Ohnemus 2

Abstract Juvenile American horeshoe ( polyphemus) spend their first two years in intertidal flats, burrowing through and disturbing benthic particles. There has been noted feeding activity around burrowing crabs by the mummichog, Fundulus heteroclitus, suggesting some sort of beneficial interaction. In order to test the effect of bioturbation of feeding efficiency, mummichogs were placed in microcosms with varying crab population densities. Changes in body mass were analyzed along with changes in chlorophyll and the benthic community within each microcosm. It was determined that intermediate populations of crabs benefitted the primary productivity. Results about the effects of crab density on Fundulus feeding efficiency and nutrition were varied, but suggested that crab activity did have an effect on the ability of fish to locate and consume benthic food sources. This can be further investigated via more long-term and quantitative research. Key Words: Limulus, Bioturbation, Fundulus

Introduction There are many ways that fauna can alter the composition of their environments. Some organisms dig underground tunnels while others disturb just by moving aboveground. Bioturbation in aquatic is less studied than in terrestrial systems, but it has been determined that burrowing is the most influential form of bioturbation within beach and ecosystems (Little 2000). The American Horseshoe Crab (Limulus Polyphemus) is one of those burrowing species, and has been cited as one of the largest organisms physically altering soil in beach and estuary environments. Jackson et. al (2005) found that the majority of horseshoe crab bioturbation occurs during annual spawning, a time when adult crabs deposit eggs into the sediment and create noticeable changes in the topography and grain size characteristics of . Once eggs are laid, larvae hatch 14-30 days later and spend their first two years in estuaries, known as nursery habitats. These nursery areas are protected from surf and current that characterizes open ocean. Only when juveniles are larger and can tolerate currents stronger than 25mm s-1 (Meury and Gibson 1990) do they move further offshore. In estuarine habitats, juvenile horseshoe crab activity is greatest in around low . During this time juveniles begin to burrow through sand flat sediment, leaving behind them distinct trails of their activity (Rudloe 1981). Burrowing allows juvenile crabs a way of Ohnemus 3 obtaining benthic worms and bivalves that comprise their diet (Button 1984) while also offering protection from predators. Bioturbation caused by the burrowing of one crab may seem unimportant, considering the size of some estuaries, but it is important to consider the population size of juvenile horseshoe crabs in these areas. In one Cape Cod estuary alone, Carmichael et al. 2003 recorded 1.35x107 horseshoe crabs, roughly 12 juvenile crabs per square meter. In this experiment, I am interested in determining what the effect of such a high population of burrowing crabs can have on the food web and microbial structure of these nursery environments. In addition to limulus, estuary ecosystems are home to a wide range of organism occupying various trophic levels. One of these organisms is the mummichog: Fundulus heteroclitus. Fundulus are primarily omnivorous, put juveniles are found to be prefer rich food sources (Smith et al 2002). Fundulus have often been observed feeding in the wake of burrowing horseshoe crabs. This suggests that horseshoe crab bioturbation effects the ability for mummichogs to access protein rich benthic organisms. If this is true, I would expect to see that in areas of higher concentrations of horseshoe crabs, mummichogs would express characteristics of consuming a more nutritious diet. These characteristics include increased biomass as well as higher fat storage in liver tissue as well as other muscle and organ tissue. Methods Experimental Design The experiment I conducted consisted of three different treatments, each containing different populations of Limulus. There were treatments that had 0 crabs (treatment 1), 2 crabs (treatment 2) and 4 crabs (treatment 3). Because it is difficult to estimate population density of juvenile horseshoe crabs in the wild, I chose these densities based on Carmichael et all (2005) estimates, which suggest approximately 12 crabs per meter. Each treatment consisted of four repetitions. In two tanks, three juvenile Fundulus species were placed. The other two tanks were contained no fish, and were used as controls. A full diagram of the experimental design is presented in Figure 1.

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Collection Sites and Husbandry The horseshoe crabs and mummichogs used in this experiment were collected from the Chappaquiot in West Falmouth, Massachusetts in early November 2012. Limulus specimens were collected by hand in the hours preceding low tide, when the are believed to be the most active. Mummichogs were collected using minnow traps baited with canned cat-food and shellfish. Sediment samples were collected from the same location at low tide. Sediment was collected along with more organic mud, which was collected from areas around the salt marsh. In the weeks prior to the start of the experiment, both mummichogs and horseshoe crabs were fed once every 48 hours. Diet consisted of dried, sinking shrimp pellets. Feeding was suspended 48 hours before the experiment started in order to purge guts of any remaining food. Experimental Set-Up A series of twelve fiberglass flow-through tanks were used for treatments in this experiments. The tanks were approximately 0.3m2 in area. Each tank contained an average of 5 centimeters of sediment. The first 2 centimeters were an organic mud, which provided nutrients to each microcosm. The other 3 centimeters were lined with less organic sediment more commonly found in the topsoil of salt marshes. Prior to filling the tanks with sediment, the sediments were homogenized by hand in order to insure that any organisms within the sediment were evenly distributed. Above each tank a lighting structure was constructed using 70-80 watt grow light bulbs. Bulbs above each tank, providing both light and warmth to each microcosm and allowing for algal growth. Light levels averaged at 23 μE/m2s. Tanks were filled with un-chilled flowing from Woods Hole Harbor. Water flowed in at approximately 16°C at the start of the experiment. Due to changing season, temperature dropped to approximately 10°C by end of the second trial. Limulus specimens were placed into the appointed tanks approximately 24 hours before the fish specimens, allowing the crabs to acclimate to the new environment and temperature. Ohnemus 5

The experiment was run for a one-week period in mid-November. Fish were weighed and marked via subcutaneous injection (Lotrich and Meredith 1974) prior to being placed in their designated microcosms.

Chlorophyll Analysis Chlorophyll samples were collected both prior to and after the seven-day experimental run. Three samples were collected from each of the twelve microcosms, resulting in 36 total samples. Each sample was collected using a sediment core, and contained only the top 2cm of sediment. Samples were placed in 50mL centrifuge tubes. 25mL of chilled acetone were added to each chlorophyll sample. The samples were then sonicated at medium speed and left in the dark, on ice for a 24-hour period. Chlorophyll samples were then analyzed using a spectrophotometer. Chlorophyll was measured at wavelengths of 665 and 750 before and after being degraded with hydrochloric acid. From these measurements it became possible to calculate the concentration of chlorophyll in each treatment.

Benthic Samples Similar to the chlorophyll samples, benthic samples were taken both before and after the one-week trial period, in order to better understand possible food available to both the fish and horseshoe crabs placed in each microcosm. Samples were collected using a syringe core. Three random samples were collected from each of the twelve microcosms. These samples were then preserved using a formalin solution made with Rose Bengal dye, which dyed all living specimens red. The preserved samples were then analyzed using a flotation method. Samples were filtered through 300 μm and 1 mm sieves and analyzed under a dissection scope. Intermediate-sized benthic samples were added to 300mL of 40 wt. % Ludox solutions. Doing so simplified analysis by separating organic material in the benthos from denser inorganic sediments. Benthic organisms were identified by taxonomic : nematode , and (a subphylum of ). Organisms from each class were counted and recorded. Ohnemus 6

Oxygen-depth profiles of Sediment In order to discern that crab activity was actually affecting the sediment, as well as to better understand how that activity may affect the microbial community, profiles were created, measuring oxygen concentrations at various depths beneath the sediment/water interface. An oxygen microelectrode measured electric currents in picoamps (pA) in the water column above the sediment, and at 1mm intervals within the sediment. From this information we were able to create an oxygen profile of the sediment in control treatments as well treatments containing Limulus.

Changes in Fish Biomass and Liver Indexes In order to analyze how successful Fundulus were at accessing and consuming food in each treatment, I analyzed changes in liver mass and fat content. Livers are typically the first tissue to express changes in fat content when fish are exposed to new and different diets (Logan et al 2006). Fish were humanely sacrificed at the end of each weeklong trial using MS-222 compound dissolved in seawater. Fish were weighed and a qualitative gut content analysis was performed. The livers of each fish was then extracted and weighed, and a hepatosomatic index was created. Fish livers were then dried for 48 hours and ground into powder in order to analyze the lipid content of the tissue. Dried, ground samples were pre-weighed and placed into 1.5mL centrifuge tubes. A 2:1 chloroform-methanol was then added to the tissue. Samples were placed on a shaker table for a period of 45 minutes, in order to allow the chloroform solution to contact all tissue cells, extracting any lipids from the protein. After shaking, samples were centrifuged at 15,000 RPMs for 8 minutes. Supernatant was pipetted off of the solid protein pellet and dried. The extracted lipids were then weighed, and their mass was compared to overall liver mass for each fish.

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Results

Oxygen Profiles From the three electrode profiles taken in undisturbed tanks as well as in tanks containing Limulus, we were able to compile an oxygen profile measuring percent oxygen in the sediment beneath the sediment/water interface, as shown in Figure 2. Oxygen was depleted sooner in tanks containing no Limulus than in tanks containing Limulus. Tanks containing zero crabs were depleted of oxygen approximately 5mm beneath the sediment/water interface, while tanks containing two crabs had oxygen present until 7mm beneath the interface.

Chlorophyll Figure 3 reflects how chlorophyll levels in each treatment changed before and after each weeklong trial. In the control treatment, chlorophyll increased from 1.6 to 2.0 μg/mL during week one, decreasing by only 0.1μg/mL during week two. In zero-crab trials, there was a slight decrease in chlorophyll after one week before increasing from 1.09 to 1.76μg/mL in following the second trial. In two crab treatments, chlorophyll levels increased for both fish and non-fish treatments. Treatments occupied by four crabs showed varied results. In non-fish treatments, chlorophyll decreased from 3.01μg/mL during week one to 2.85μg/mL after the first trial, before increasing during the second week to 3.44μg/mL. Treatments occupied by 4 fish and 3 crabs saw no noticeable change in chlorophyll after the first week, but an increased from 163μg/mL to 1.99μg/mL in the second. When we analyzed the percent increase over the two-week period, it was obvious that the largest change occurred in intermediate treatments, containing two crabs (Figure 4). This phenomenon occurred when fish were both present and absent from the treatment. Treatments containing fish also had higher overall increases in chlorophyll than those containing crabs but no fish activity.

Mass Changes Ohnemus 8

Fish mass was lost during each trial in all treatments (Table 1 A and B). Overall the least amount of mass loss was measured in the second treatment for both trial one (Figure 5) and trial 2 (Figure 6). In trial one, fish occupying two-crab treatment only lost 12% of body mass, while fish in zero-crab and four-crab treatments lost 29% and 31% of body mass respectively. In treatment two, fish in treatment two lost 17% of body mass, compared to 47% in treatment one and 29% in treatment three.

Hepatosomatic Index and Liver Lipid Composition Lipid extractions were not successful, and did not yield accurate results. Wild specimens had a liver to body mass ratio of 3.71%. In treatment 3, the four-crab treatment, liver mass composed 4.8% of overall fish body mass, the highest of all treatments (Figure 7). Fish in treatment one had the lowest liver: body mass ratio, only 2.8% but increased in treatment two to 4.04%. Trial two had slightly different results (Figure 8). Liver to body mass ratio between trials one and two were unchanged at 2.9% for each treatment. Treatment 3 had a lower hepatosomatic ratio, only 1.8%. Benthic Counts We saw a decrease in all phyla identified within benthic samples (Figures 9-13), with nematodes and decreasing the most dramatically. Figure 14 shows the percent decrease of nematodes in each treatment, over a two-week period. In control treatments, nematodes decreased 6% in zero crab treatments, but remained unchanged in 2 crab control treatments. In the four crab control treatment, nematode numbers decreased by 25% over the two week period. Treatments containing fish and crabs saw a larger decrease in the nematode concentrations over the two-week, two-trial period. In Treatment 1, containing zero crabs, there was a 43% decrease in nematode concentrations. Treatment 2 saw a 76% decline in nematodes and treatment 3 saw a 37% decrease. Overall, it appears that treatment 2, the two-crab treatment, saw the larges decrease in nematodes over the two-week experimental period. There was a similar trend seen with annelid concentrations across treatments, as represented in Figure 15. In zero crab controls, annelid concentrations were unchanged, Ohnemus 9 but decreased by 51% in two crab controls and 41% in three crab controls. Treatments containing fish experienced a 50% decrease in annelids in treatments with no crabs present. Treatment 2 had a 32% decrease in annelids while treatment three experienced the largest decrease, 76% over a two-week period. Annelids decreased the most dramatically in treatment 3, as opposed to nematode concentrations, which decreased the most significantly in the intermediate treatment.

Discussion This experiment produced a myriad of results, all of which suggest that activity by Limulus affects trophic structure of intertidal flats by encouraging algal growth and making food more available for secondary consumers, such as Fundulus. Similar results were found in Winkel and David’s 1985 report, which found that bioturbation similarly increases the ability of other predatory organism, in their case the water mite’s, ability to sequester food. Data from the oxygen microelectrode prove that bioturbation is in fact occurring in the microcosms, and that oxygen is being mixed deeper into the sediment. This allows for an increase in benthic populations by increasing available habitat. Deeper oxygen penetration also may have an effect on the microbial structure of these intertidal ecosystems by determining what types of microbial processes can occur within the sediment. are a main source of primary productivity in aquatic ecosystems, especially these intertidal flats, where relatively shallow water allows for deeper light penetration. In this experiment we saw what appears to be an intermediate species effect. Treatments containing two crabs encouraged algal growth more than treatments containing double that concentration. Because the two-crab treatments contain concentrations of Limulus similar to what one would expect to find in the wild, this suggests that current crab populations are beneficial to the growth of primary productivity in these nursery habitats. Doubling that population may increase disturbance to a point where algae cannot grow as effectively, disrupting the base of many local food webs. The effects of Limulus bioturbation were not as conclusive when analyzing the effect it had on fish nutrition and feeding efficiency. The first trial showed promising results that supported my original hypotheses that higher concentration of Limulus promoted fish Ohnemus 10 growth. The second trial did not mirror these results; instead fish occupying environments with large Limulus populations stored less fat in liver tissue, signaling poor nutrition. Reasoning for these odd results may in fact be due to the food originally available to fish in the microcosms. Benthic analysis shows that in treatment 3, containing fish and four crabs, there were fewer nematodes, and annelids decreased rapidly within the first week. This could have resulted simply in a lack of food availability to fish during the second trial, which would explain why fish were unable to put on liver mass and had a lower overall liver mass to body mass ratio. The aspect of competition was always a factor considered in the designing in this experiment, since Limulus and Fundulus both feed on benthic mesofauna. Competition was avoided by placing Limulus of certain ages in the microcosms. All crabs used in this experiment were approximately in their 6th or 7th instar (20-25mm prosomal width). According to Carmichael et al (2009) horseshoe crabs at this particular instar are mostly consuming particulate organic matter (POM) and are only beginning to consume small benthic such as nematodes. Fundulus on the other hand are mostly consuming annelids (Smith et al. 2002), which are larger and easier to find within the sediment than the smaller nematodes. Due to these circumstances, I assume that any competition between Limulus and Fundulus was minimal. Due to the wide variety of results obtained involving bioturbation and feed efficiency, it is difficult to draw any definite conclusions from data collected during this experiment. It appears that current population density of juvenile Limulus are beneficial to promoting primary productivity as well as food availability for mummichogs and other secondary consumers feeding on benthic organisms. This interpretation can only be confirmed through further analysis of more long- term experiments. Experiments run for 6 months to a 1-year may yield more detailed and consistent results. Performing similar experiments in the wild, using cages throughout intertidal flats may also be beneficial, as the benthic community may remain more consistent and the test sites would be exposed to accurate environmental conditions such as and available light. Performing more in-depth gut content analyses could also yield more accurate and consistent results. Doing so would allow us a more quantitative way of analyzing which fish Ohnemus 11 were feeding on protein sources and which were feeding on benthic algae. It may also be possible to perform stable isotope analysis on whole mummichogs after performing more long-term experiments, similar to Carmichael et al.’s 2009 study. Doing so would allow us to better understand if fish subjected to certain concentrations of Limulus place differently within the food web of their environment. Population density of the American horseshoe crab is important not only to the of intertidal flats, but also to the commercial fishing and biomedical industries. In 2008 Industrial Economics, Incorporated released an assessment stating that in 2006 over 1.8 millions pounds of American Horseshoe Crabs were fished from the Eastern United States, down from 2.5 million three years earlier. Overfishing of horseshoe crabs is a concern in many local areas, and could have a detrimental effect on these fragile intertidal ecosystems. While this study begins to show that increasing populations may be also detrimental, it suggests that by maintaining current juvenile Limulus we can sustain healthy coastal ecosystems while continuing supplying the demand of the fishing industry for adult horseshoe crabs that have already been allowed the opportunity to spawn.

Acknowledgements This research project is the culmination of a semester’s worth of experiences in both the lab and the classroom. And would have been impossible without the help of many members of the MBL Ecosystems Center staff. Thank you to Ken Foreman for his constant guidance and input as well as Dan Gibson for inspiration and his unfailing and extensive knowledge of horseshoe crab physiology and behavior. Jimmy Nelson was extremely helpful with laboratory processes and was indispensible in this process. Immense thanks to the assitants, Rich McHorney, Carrie Harris and Alice Carter for their constant guidance both in an out of the laboratory.

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Literature Cited

Botton M, Haskin H (1984) Distribution and feeding of the horseshoe crab, Limulus polyphemus, on the off New Jersey. Fish Bull 82:383–389

Carmichael, R. H., Rutecki, D., & Valiela, I. 2003. Abundance and population structure of the Atlantic horseshoe crab Limulus polyphemus in Pleasant Bay, Cape Cod. Marine Ecology Progress Series, 246: 225-239.

Carmichael, R.H, Gaines, E., Sheller, Z., Tong, A., Clapp, A., and Ivan Valiela. 2009. Diet composition of juvenile horseshoe crabs: implications for growth and survival of natural and cultured stocks. Biology and conservation of horseshoe crabs. Pp 531-534.

Chellappa, S., Huntingford, F. A., Strang, R. H. C., & Thomson, R. Y. 2006. Condition factor and hepatosomatic index as estimates of energy status in male three‐spined stickleback. Journal of Fish Biology, 47(5): 775-787.

Industrial Economics Incorporated, 2008. Economic assessment of mid-Atlantic horseshoe crab and dependent fisheries including a qualitative discussion of the potential effects of addendum iv. Pp 10-26.

Jackson, N. L., Nordstrom, K. F., & Smith, D. R. 2005. Influence of waves and horseshoe crab spawning on beach morphology and sediment grain‐size characteristics on a sandy estuarine beach. , 52(5): 1097-1108

Little, C., 2000. The Biology of Soft Shores and Estuaries. Oxford University Press, New York. Pp 20-23.

Logan, J., Haas, H., Deegan, L., & Gaines, E. 2006. Turnover rates of nitrogen stable isotopes in the salt marsh mummichog, Fundulus heteroclitus, following a laboratory diet switch. Oecologia, 147(3): 391-395.

Lotrich, V. A., & Meredith, W. H. 1974. A technique and the effectiveness of various acrylic colors for subcutaneous marking of fish. Transactions of the American Fisheries Society, 103(1): 140-142.

Meury, T. W., Gibson, I. I. I., & Daniel, G. 1990. Force generation in juvenile Limulus polyphemus: effects on mobility in the intertidal environment. Bulletin of marine science, 47(2): 536-545.

Revsbech, N. P., Jorgensen, B. B., Blackburn, T. H., & Cohen, Y. 1983. Microelectrode Studies of the Photosynthesis and O_2, H_2S, and pH Profiles of a . Limnology and Oceanography, 1062-1074.

Rudloe, A. 1981. Aspects of the biology of juvenile horseshoe crabs, Limulus polyphemus. Bulletin of marine science, 31(1): 125-133.

Smith, K.J., Taghon G.L., and Kenneth W. Able. 2002. Trophic linkages in marshes: ontogenetic changes in diet for young-of-the-year mummichog Fundulus heteroclitus.

Winkel, E.H. and C. Davids. 1985. Bioturbation by cyprinid fish affecting the food availability for predatory water mites. Oecologia (Berlin). 67: 218-219.

Tables and Figures Figure 1: Experimental Design

Table 1: Changes in fish mass over the course of trial one (A) and trial two (B) Ohnemus 13

Figure 2. Percent oxygen composition of sediment beneath the sediment/water interface

Figure 3. Changes in chlorophyll concentrations across treatments

Figure 4. Percent increase in chlorophyll after a two-week period across treatments

Figure 5. Percent of original body mass lost during trial one across treatments

Figure 6. Percent of original body mass lost during trial two across treatments

Figure 7. Liver mass to body mass ratio for Fundulus in treatment one

Figure 8. Liver mass to body mass ratio for Fundulus in treatment two

Figure 9. Decrease in nematodes (A), annelids (B) mollusks (C) and (D) across treatments

Figure 10. Percent decrease in nematodes after two-week experimental period

Figure 11. Percent decrease in annelids over two-week experimental period

Figure 1. Experimental Design Rep 1 Rep 1 3 Fish 3 Fish Rep 2 Rep 2 0 Crabs 2 Crabs Rep 1 Rep 1 No Fish No Fish Rep 2 Rep 2

Rep 1 3 Fish Rep 2 4 Crabs Rep 1 No Fish Rep 2

Table 1A. Changes in average fish mass over the course of trial one

Treatment Starting Mass (g) Ending Mass (g) Difference(g) Pre Experiment 1.30 1.30 0 0 Crabs 1.48 0.91 -0.57 Ohnemus 14

2 Crabs 1.18 1.02 -0.16 4 Crabs 1.51 1.11 -0.40

Table 1B. Changes in average fish mass over the course of trial two Treatment Starting Mass (g) Ending Mass (g) Difference (g) Pre Experiment 1.43 1.43 0 0 Crabs 1.8 0.96 -0.84 2 Crabs 1.4 1.16 -0.95 4 Crabs 1.2 0.88 -0.47

Figure 2. Percent oxygen composition of sediment beneath the sediment/water interface

Oxygen Concentration

0% 20% 40% 60% 80% 100% 0

-1

-2

-3

-4 Crabs -5 No Crabs

-6 Depth I(mm) Depth beneath sediment surface -7

-8 Ohnemus 15

Figure 3. Changes in chlorophyll concentrations across treatments Ohnemus 16

Figure 4. Percent increase in chlorophyll after a two-week period across treatments

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Figure 5. Percent of original body mass lost during trial one across treatments

Figure 6. Percent of original body mass lost during trial two across treatments

Figure 7. Liver mass to body mass ratio for Fundulus in treatment one Ohnemus 18

Figure 8. Liver mass to body mass ratio for Fundulus in treatment two

Figure 9. Decrease in nematodes (A), annelids (B) mollusks (C) and crustaceans (D) across treatments Ohnemus 19

A.

. B

C. Ohnemus 20

D.

Figure 10. Percent decrease in nematodes after two-week experimental period Ohnemus 21

Figure 11. Percent decrease in annelids after two-week experimental period