Long-Term Effects of Salinity on Bone Mass and Ion Levels in Blood Serum of the Killifish Fundulus heteroclitus

Kenneth SchlichtingDepartment of Biological Sciences, York College

Introduction: The killifishes, Fundulus heteroclitus, have a unique ability to adapt to a wide range of salinities but are most commonly found in full strength seawater (Perschabacher et al. 1990). Short term effects of fresh water adaptation have been studied but little research has been done to investigate the long term physiological differences seen in fish inhabiting fresh water for long periods of time (Marshall et al. 1999). Killifish are exposed to extremes in salinity on an almost daily basis and could have ways of dealing with this change. To fill the gap in knowledge, I propose to capture wild killifish and adapt them to differing degrees of salinity, ranging from fresh to full strength sea water. They will remain at this salinity for 5 months before they are collected and prepared so that mean skeletal ash weights can be compared (Grogan and Rehnberg 1997). Knowledge obtained from the comparison will indicate if the fish are using their boney structures as a reservoir for salts essential to life but normally present in reduced quantities in fresh water environments. Along with information collected from skeletal remains, blood serum levels will be analyzed for Na+ and Cl- concentrations at the time of harvesting. They will be compared to fish living in full strength sea water to determine if salt concentrations in blood serum are affected during long exposures to fresh water.

Review of Literature:

• Killifish have shown a change in Na+ and Cl-

concentrations in blood serum shortly after being moved to a different salinity (Marshall et al. 1999)

• Na+ and Cl- uptake and secretion have been studied for up to 30 days. This research shows that rapidly adapted fish start to compensate for their new environment after several hours but this research stopped short of determining whether prior ion levels were ever reached (Marshall et al. 1999)

• Bone deterioration has been documented in some non-osmoregulating fish that were living in below optimal salinity levels (Glowacki et al. 1986)

Collect fish from Long Island’s Great South Bay (n=400)

Seawater (35 ppt) 11 ppt23 ppt Freshwater (0 ppt)

Allow 5 months at constant salinity

Take blood sampleRemove soft tissue via double blind process

Measure osmolality of serum and Na+ and Cl– concentrations2

Incinerate remaining skeletal structure at 575°C

Weigh ash and compare groups using ANOVA test

Place each group of 25 fish in an aquarium containing 1 of 4 different salinities

Literature Cited

Glowacki, J., Cox, K.A., O’Sullivan J., Wilkie, D., and Deftos, L.J. Glowacki, J., Cox, K.A., O’Sullivan J., Wilkie, D., and Deftos, L.J. 1986. Osteoclasts can be induced in fish having an acellular bony 1986. Osteoclasts can be induced in fish having an acellular bony skeleton. skeleton. Proceedings of the National Academy of Sciences of the Proceedings of the National Academy of Sciences of the United States of America United States of America 83(11):4104-4107.83(11):4104-4107.

Grogan, S. and Rehnberg, B. 1997. Ash content of pellets and fecal-Grogan, S. and Rehnberg, B. 1997. Ash content of pellets and fecal-urinary wastes from a great-horned owl (urinary wastes from a great-horned owl (Bubo virginianusBubo virginianus) and a red-) and a red-tailed hawk (tailed hawk (Buteo jamaicensisButeo jamaicensis): Implications for raptor mineral ): Implications for raptor mineral budgets. budgets. Journal of the Pennsylvania Academy of Science Journal of the Pennsylvania Academy of Science 70(3): 70(3): 123-125.123-125.

Marshall, W.S., Emberley, T.R., Singer, T.D., Bryson, S.E., and Marshall, W.S., Emberley, T.R., Singer, T.D., Bryson, S.E., and McCormick, S.D. 1999. Time course of salinity adaption in a strongly McCormick, S.D. 1999. Time course of salinity adaption in a strongly euryhaline estuarine teleost, euryhaline estuarine teleost, Fundulus heteroclitusFundulus heteroclitus: A multivariable : A multivariable approach. approach. The Journal of Experimental BiologyThe Journal of Experimental Biology 202: 1535-1544. 202: 1535-1544.

Perschabacher, P.W., Aldrich, V., and Strawn, K. 1990. Survival and Perschabacher, P.W., Aldrich, V., and Strawn, K. 1990. Survival and growth of the early stages of gulf killifish in various salinities. growth of the early stages of gulf killifish in various salinities. Progressive Fish-CulturistProgressive Fish-Culturist 52(2): 109-111. 52(2): 109-111.

Mentor: Dr. Bradley RehnbergMentor: Dr. Bradley Rehnberg

Objectives:

• To determine if Fundulus heteroclitus uses its skeletal structure as a salt reserve in freshwater conditions.

• To determine if osmoregulation keeps Na+ and Cl- concentrations the same in fresh and saltwater adapted fish after 5 months.

Methods: Expected Results For Ash Weights

0

0.05

0.1

0.15

0.2

0.25

0 11 23 35

Salinity (ppt)

Weig

ht

of

ash

(g

)

Ash Weight

Expected Na+ and Cl- concentrations in Blood Serum

020406080

100120140160

0 11 23 35

Salinity (ppt)

Ion

co

nc.

mm

ol/L-1

Cl- conc.

Na+ conc.

Adult Female Fundulus heteroclitus

Bone ash will also be analyzed for calcium levels1

Expected Results:

• Fish reared in fresh water would yield the lightest ash weights, while those from full strength seawater would have the heaviest ash weights.

•Fish living in fresh water will exhibit lower Na+ and Cl- concentrations in their blood serum when compared to fish from the seawater group.

•Fish reared in fresh water would also show lower calcium levels in bone than fish reared in seawater conditions.

1 Calcium levels will be measured with flame absorbance spectroscopy.

2 Na+ and Cl- will be measured by sending a blood sample out to a lab equipped to test for electrolyte concentrations.

Fig 1. Graph adapted from Marshall et al. 1999 with expected data added

Fig 1

Fig 2

Fig 3

Na+ concentration in Blood Serum