Copeia, 2003(2), pp. 397–401

Behavioral Responses of Mosshead and Woolly to Increasing Environmental Hypoxia

JASON V. WATTERS AND JOSEPH J. CECH JR.

Behavioral responses of the rocky intertidal mosshead ( globi- ceps) and woolly sculpin () to increasing environmental hypoxia (low

PO2) were observed in a seawater aquarium with a depth gradient. The experimental design allowed observation of the response of these to encroaching envi- ronmental hypoxia. We observed three behaviors by which the fish dealt with hyp- oxia: emergence from substrate (EFS), first avoidance response (FAR), and aquatic surface respiration (ASR). With respect to EFS and FAR, mosshead sculpins were

more labile in their responses than woolly sculpins. However, the mean PO2 at which ASR was initiated was statistically indistinguishable between . We suggest that the differences we observed represent alternate behaviors by which these related species deal with the trade-off imposed by the simultaneous needs to avoid preda- tion and optimize respiration. We further suggest that these alternate behaviors have

evolved around a conserved physiological constraint, the PO2 at which ASR must be performed.

ALIFORNIA’S rocky coast tidepool habitats congeners in the intertidal habitat (Gibson, C may rarely achieve homeostasis during low 1999). For example, Yoshiyama (1981) noted tide (Martin, 1995). that live in tidepools that the highest proportion of mosshead scul- may face challenges such as increasing environ- pin () was found in elevated mental hypoxia caused by high respiring bio- wave-swept tidepools in the mid-intertidal zone, mass and poor wind mixing during these low whereas a large proportion of woolly sculpin tide events (Morris and Taylor, 1983; Yoshiyama (Clinocottus analis) was found in a broader ver- et al., 1995). Many fish perform aquatic surface tical intertidal range (high intermediate and respiration (ASR), the inspiration of surface (1– low tidepools), with small individuals occurring 2 mm) water that equilibrates with atmospheric in the highest of habitable tidepools. Higher air, to deal with aquatic hypoxia (Gee et al., tidepools should experience more extreme hyp- 1978, Kramer, 1983, 1987). Additionally, several oxia than low tidepools because they are the intertidal species are capable of breathing air first to be removed from wave action at low tide (Martin, 1991, 1995, Yoshiyama and Cech, and the last to be reinundated with oxygenated 1994). Both of these respiratory behaviors take water when the tide returns. Because they in- advantage of the comparative O2 richness of at- habit tidepools that are more likely to become mospheric air and may enhance survival and hypoxic, small woolly sculpins are therefore maintenance in hypoxic water when access to likely to have evolved physiological tolerance or the surface is available (Beittinger and Pettit, behavioral mechanisms to counteract decreased 1984; reviewed by Graham, 1997; Martin and oxygen availability. Presumably, these mecha- Bridges, 1999). nisms would evolve to limit the exposure of Tidepool dwelling sculpins of the genus Cli- these fish to aerial predators. Conversely, moss- nocottus represent an assemblage of fishes that head sculpin accustomed to relatively high-oxy- may routinely experience environmental hyp- gen environments would be forced to deal with oxia (Yoshiyama, 1981). Field measurements in- hypoxia less often. Thus, we hypothesized that dicate that the dissolved oxygen in tidepools at mosshead sculpin would be more sensitive to low tide can decrease to nearly zero in only a decreased oxygen levels and act to avoid hyp- few hours (Truchot and Duhamel-Jouve, 1980; oxia at a higher partial pressure of oxygen

Morris and Taylor, 1983). Such fluctuating dis- (PO2) than woolly sculpin. solved oxygen levels can be a strong force in the Our objective was to compare the responses selection of physiological tolerances and/or the of the intertidal mosshead sculpin and woolly shaping of respiratory behaviors employed to sculpin to increasing environmental hypoxia. deal with respiratory hardship. Differences in The respiratory behavior of these species has the ability to cope with low dissolved oxygen, be not been quantitatively described and may shed they physiological or behavioral, can explain light on their distribution differences over in- the differential distribution of closely related tertidal microhabitats. Relating the distribution,

᭧ 2003 by the American Society of Ichthyologists and Herpetologists 398 COPEIA, 2003, NO. 2 behavior, and physiology of intertidal fishes to the unique environmental conditions they en- counter offers insight into their evolutionary ecology (Gibson, 1999) because adaptive differ- ences in behavior and/or physiology should re- flect the differences in selective pressures faced by the two species.

MATERIALS AND METHODS Animals.—Experiments were conducted during summer and fall of 1996. Mosshead sculpin (n ϭ 10) and woolly sculpin (n ϭ 10) were cap- tured from rocky intertidal habitat in Horse- shoe Cove at the Bodega Marine Reserve in Bo- Fig. 1. The experimental aquarium. Nitrogen was dega Bay, California. Animals were captured by introduced to the tank via the diffuser. Each sampling dipnetting bailed or unbailed tidepools during tube was attached to a three-way stopcock for water low tides. All fish used in the experiment were analysis. The sides and back of the aquarium were taken from tidepools of intermediate tidal ele- insulated with aluminized bubble wrap (not shown), vation (0.5–1.2 m above mean low water). Tide- and a grid was drawn on the front of the aquarium pool elevations were estimated by following the to record location of fish (not shown). tide down and recording the time that each tidepool surface was at sea level. Then, the pre- dicted tide elevations were calculated for the tubes (Tygon Microbore) were spaced along the relevant time for each pool. The calculation for length of the aquarium, 20 cm apart. They sam- the tide height at the time sea level and tide- pled water from just above the substrate and ran pool surface level matched is considered to be up, out of the tank. The end from which water the tidepool tidal elevation (P. Connors pers. was collected hung below the bottom of the out- comm.). Immediately after capture, animals side of the aquarium, allowing water sampling were placed in a 19-liter bucket containing fresh to occur without disturbing the fish. Each water seawater and transferred to holding aquaria at sampling tube was fitted with a blunt needle, the University of California, Bodega Marine three-way stopcock, and syringes for sampling Laboratory (BML). Aquaria were supplied with water without atmospheric contamination. The a natural photoperiod (38Њ19ЈN) and a constant PO2 of sampled water was measured (nearest seawater flow (10–12 C, 32 ppt salinity). mm Hg) using a Radiometer PHM 71/D616/ were maintained on a diet of chopped mussels, E5046 oxygen analyzer system. Figure 1 graph- live brine shrimp, chopped squid, and algae. Af- ically depicts the experimental tank. ter experimentation, fish (no mortalities) were A dissolved oxygen gradient along the aquar- weighed, measured (standard length) and re- ium’s long axis was created by slowly bubbling leased at their capture site. nitrogen into the deep end of the aquarium. Nitrogen was allowed to diffuse slowly out of a Experimental aquarium.—A 98-liter glass aquari- porous teflon tube (PTFE Tubing, IMPRA Inc., um was situated with one end elevated on an Arizona) at a rate that caused minimal surface indoor BML wet table with natural lighting. The disturbance and was controlled by eye or by us- bottom of the aquarium was thus inclined and ing a gas flow meter (Manostat). covered with small cobble for substrate. Three sides of the aquarium were insulated with a jack- Experimental protocol.—The species to be tested et of bubble-wrap sheet, and temperatures were in an individual experiment was chosen ran- maintained via a continuous spray of ambient domly by flipping a coin, and an individual fish seawater between the aquarium walls and the was quickly netted from one of the holding insulating jacket. The front (long side) and top aquaria and placed into the experimental of the aquarium were left unwrapped for obser- aquarium. Once released into the test aquari- vations and a grid of 3.5 ϫ 4 cm rectangles was um, most fish immediately sought cover among drawn on the front to fix the ’s location the rocks of the substrate. The fish were allowed on the slope and bottom. Before each experi- to acclimate to the normoxic conditions for at ment, fresh seawater was added to the aquarium least 40 min before experiments began. Forty to a depth of 24.5 cm at the deep end and 5 minutes appeared to be enough time for fish cm at the shallow end. Five water-sampling placed in the aquarium to ‘‘settle down’’ and WATTERS AND CECH—SCULPIN BEHAVIOR DURING HYPOXIA 399

show no outward signs of stress such as in- TABLE 1. MEAN Ϯ SE WITH (n)SEAWATER PO2S (MM creased respiration rate. After the acclimation HG) AT WHICH MOSSHEAD SCULPIN AND WOOLLY SCUL- PIN IRST ESPONDED TO RADUALLY NCREASING YP period, initial seawater PO2 was checked in both F R G I H - the deep and shallow ends of the aquarium, and OXIA IN A LABORATORY AQUARIUM. the experiment was started if initial PO2s were Ͼ 87% air saturation (135 mm Hg). During the Species EFS PO2 FAR PO2 ASR PO2 experiment, PO2 was monitored to assess the Mosshead 37 Ϯ 6 (10) 37 Ϯ 6 (10) 26 Ϯ 2 (10) level of aquatic hypoxia and to identify the PO2s Woolly 27 Ϯ 2 (10) 26 Ϯ 2 (10) 23 Ϯ 2 (10) at which fish exhibited respiratory distress be- Abbreviations: EFS: Emergence from Substrate, FAR: First Avoidance haviors. Each measurement episode entailed Response, ASR: Aquatic Surface Respiration. sampling the water for PO2 (mm Hg) nearest the fish’s location, and recording the time, the fish’s grid location, and behavioral notes. ly sculpins (Table 1). The FAR was also ob-

Three respiratory distress behaviors were re- served at a slightly higher PO2 for mosshead corded: emergence from the substrate (EFS), sculpins (Welch’s test, P ϭ 0.0572) than for first avoidance response (FAR), and ASR. EFS woolly sculpins. The small sample size resulted was the action by which a fish first relinquished in low power (power ϭ 0.3517 and 0.3641 for its hiding spot among the cobble substrate. Al- EFS and FAR, respectively), and taken alone, though it was difficult to quantify without dis- these results are unconvincing. However, the turbing the fish, it was obvious that the fish’s variance in PO2 of response among mosshead gill ventilation frequency increased as seawater sculpins (EFS SD ϭ 19.12; FAR SD ϭ 18.20) was

PO2 decreased. The FAR constituted the first much higher than the variance in PO2 of re- movement across at least one grid line toward sponse among woolly sculpins (EFS SD ϭ 6.82; shallower water and higher PO2. Thus, there is FAR SD ϭ 7.51) for both EFS and FAR (Le- a subtle distinction between EFS and FAR, in vene’s test, P ϭ 0.0145 and 0.0272, respectively). that EFS did not require a fish to move toward In contrast, the PO2s at which both species first a less hypoxic region of the aquarium. ASR was showed the most pronounced respiratory dis- the first active inspiration of the water surface, tress behavior, ASR, were statistically indistin- when the fish placed its mouth into the surface guishable (Student’s t-test, P ϭ 0.1711). The var- meniscus. Seawater PO2s at the site (along the iance in PO2 of response for ASR was also in- grid gradient) of the initiation of each behavior distinguishable between species (mosshead SD were recorded. ϭ 7.56; woolly SD ϭ 5.55; Levene’s F test, P ϭ We compared the lengths and weights of 0.2394). mosshead and woolly sculpins used in the ex- There were no differences in the lengths and periment with a Student’s t-test. Water temper- weights of fish of the two species used in the ature at which mosshead and woolly sculpins experiment (Student’s t-test for length P ϭ 0.66; were tested was compared using a Student’s t- for weight P ϭ 0.57). The average length and test. Because we hypothesized that mosshead weight of mosshead sculpins in the experiment sculpins would act to avoid hypoxia at higher was 60 mm (SD ϭ 8.9) and 5.7 g (SD ϭ 2.1).

PO2s than small woolly sculpins, we used a one- Woolly sculpins averaged 58 mm in length (SD tailed statistical analysis on the behavioral data ϭ 7.4) and 4.7 g (SD ϭ 1.7). All fish were tested (Zar, 1996). Data for EFS, FAR, and ASR were at similar temperatures (Student’s t-test P ϭ checked for equality of variances using Levene’s 0.81). Water temperatures ranged from 9.3 C to test for variances and the means for each spe- 12.6 C in the experiment. cies for each behavior were tested against each other using either Welch’s test (in the case of DISCUSSION unequal variances) or a Student’s t-test in JMP for Windows version 4 (SAS Institute, Cary, NC, Kramer (1987) postulated that a fish would 2000). Statistical tests were evaluated at a signif- engage in ASR when it could acquire more ox- icance value of P ϭ 0.05. ygen at the water surface than it could from aquatic respiration. In this manner, a fish be- RESULTS haves optimally with respect to respiration. If the fish in our experiment were respiring opti- Mosshead and woolly sculpins responded dif- mally, they would have been expected to switch ferently to encroaching environmental hypoxia. to ASR much earlier (higher PO2) than they did EFS was observed at a slightly higher though because of the higher availability of oxygen in not significantly different PO2 for mosshead the surface layer of the water (Burggren, 1982). sculpins (Welch’s test, P ϭ 0.062) than for wool- Behavioral decisions, however, are made based 400 COPEIA, 2003, NO. 2

Fig. 2. A graphical model depicting the hypothesized relationship between perceived risk, water PO2, and aquatic surface respiration (ASR) behavior in tidepool-inhabiting sculpins. At low levels of perceived risk, fish are expected to perform ASR as needed to optimize respiratory oxygen intake. At high levels of perceived risk, fish are expected to perform ASR as demanded by some physiological threshold PO2 below which the behavior must be performed. At some threshold level of risk, fish are expected to put off ASR until physio- logical demands provide motivation to perform the behavior. on complex interactions between perceived risk related species. Although our small sample sizes and physiological demands (Smith and Kramer, resulted in low statistical power, it appears that 1986; Lima and Dill, 1990). The perceived woolly and mosshead sculpins deal differently (high) risk in this study resulted from the ex- with encroaching environmental hypoxia. In- posed, illuminated conditions in the experi- deed, our data suggest that these two species mental aquarium and the presence of the ob- have evolved different behavioral responses server who was visible to the fish. We placed around a shared physiological constraint (iden- severe physiological demands on the fish by low- tical limiting PO2s). Thus, even with a physio- ering the aquatic PO2 in the experimental tank. logical constraint that limits the ability to re- Limiting PO2s presumably occur where oxygen spire in hypoxic water, the two species are able demand is such that metabolic requirements to respond to similar changes in dissolved oxy- take precedence over the need to seek cover, gen while minimizing different levels of preda- and they, therefore, represent a proximate con- tion risk. For example, the high variance in the straint on the distribution of these fishes in the response of mosshead sculpins suggests that this tidepool environment (Beittinger and Pettit, species is inclined to deal with hypoxia through 1984). Additionally, Yoshiyama et al. (1995) in- subtle behavioral changes. Variation in response dicated that intertidal sculpins were reluctant to in this species would be expected because of the emerge for aerial respiration in laboratory more biologically diverse, lower elevation tide- aquaria suggesting that the fish perceive a high- pools in which it lives. These tidepools contain risk situation. Our experiment allowed us to ex- cover and allow mosshead sculpins to optimize amine one side of the trade-off between per- respiration through subtle behavioral changes ceived risk of predation and metabolic require- such as moving about to locate and take advan- ments. By creating an environment in which the tage of areas where dissolved oxygen remains fish would otherwise choose not to perform high, while still remaining concealed from pred- ASR, we noninvasively determined these limit- ators. The higher elevation pools in which wool- ing PO2s for two species of sculpin (Fig. 2). ly sculpins are found have less cover and rep- Our experimental design also made it possi- resent a ‘‘higher risk’’ environment, for which ble to observe the behaviors that may arise as a the less variable ‘‘sit-and-wait’’ behavior for cop- result of different selective pressures in closely ing with environmental hypoxia is well suited. WATTERS AND CECH—SCULPIN BEHAVIOR DURING HYPOXIA 401

In this way, the woolly sculpin may be able to LIMA,S.L.,AND L. M. DILL. 1990. Behavioral decisions wait out the low tide and avoid exposure to ae- made under the risk of predation: a review and pro- rial predators altogether. spectus. Can. J. Zool. 68:619–640. MARTIN, K. L. M. 1991. Facultative aerial respiration in an intertidal sculpin, Clinocottus analis (Scorpaen- ACKNOWLEDGMENTS iformes: ). Physiol. Zool. 64:1341–1355. ———. 1995. Time and tide wait for no fish: intertidal We thank C. Myrick, J. May, M. Danley, and fishes out of water. Environ. Biol. Fish. 44:165–181. M. O’Farrell for fish collecting assistance, the ———, AND C. R. BRIDGES. 1999. Respiration in water BML staff (especially K. and E. Uhlinger) for and air, p. 54–78. In: Intertidal fishes, life in two logistical support, R. Yoshiyama for useful dis- worlds. M. H. Horn, K. L. M. Martin, and M. A. cussions and comments, J. Roessig for editorial Chotkowski (eds.). Academic Press, San Diego, CA. assistance, and G. Nevitt for comments on a MORRIS, S., AND A. C. TAYLOR. 1983. Diurnal and sea- manuscript draft. Research support was provid- sonal variation in physico-chemical conditions with- ed in part by a University of California (UC) in intertidal rock pools. Estuar. Coast. Shelf Sci. 17: 339–355. President’s Undergraduate Fellowship (to JVW) SMITH, R. S., AND D. L. KRAMER. 1986. The effect of and by a UC Agricultural Experiment Station apparent predation risk on the respiratory behavior grant (3455-H) to JJC. We also thank P. Connors of the Florida gar (Lepisosteus platyrhicus). Can. J. for tidepool height data. Animals were collected Zool. 64:2133–2136. under California Scientific Collecting Permit TRUCHOT,J.P.,AND A. DUHAMEL-JOUVE. 1980. Oxygen 801058-01 and kept under the guidelines of UC and carbon dioxide in the marine inter tidal envi- Davis Animal Care and Use Protocol 7342. ronment: diurnal and tidal changes in rock pools. Respir. Physiol. 39:241–254. YOSHIYAMA, R. M. 1981. Distribution and abundance LITERATURE CITED patterns of rocky intertidal fishes in central Califor- BEITTINGER,T.L.,AND M. J. PETTIT. 1984. Comparison nia. Environ. Biol. Fish. 6:315–332. of low oxygen avoidance in a bimodal breather, Er- ———, AND J. J. CECH JR. 1994. Aerial respiration by petoichthys calabricus, and an obligate water breather, rocky intertidal fishes of California and Oregon. Percina caprodes. Environ. Biol. Fish 11:235–240. Copeia 1994:153–158. ALPEY CHALK SWALD BURGGREN, W. W. 1982. ‘‘Air gulping’’ improves blood ———, C. J. V ,L.L.S ,N.M.O ,K. oxygen transport during aquatic hypoxia in the K. VANESS,D.LAURITZEN, AND M. LIMM. 1995. Dif- goldfish, Carassius auratus. Physiol. Zool. 55:327– ferential propensities for aerial emergence in inter- 334. tidal sculpins (Teleostei: Cottidae). J. Exp. Mar. GEE, J. H., R. F. TALLMAN, AND H. J. SMART. 1978. Re- Biol. Ecol. 191:195–207. actions of some great plains fishes to progressive ZAR, J. H. 1996. Biostatistical analysis. Prentice Hall, hypoxia. Can. J. Zool. 56:1962–1966. Upper Saddle River, NJ. GIBSON, R. N. 1999. Methods for studying intertidal fishes, p. 7–25. In: Intertidal fishes, life in two ( JVW) CENTER FOR ANIMAL BEHAVIOR, ࡆ DE- worlds. M. H. Horn, K. L. M. Martin, and M. A. PARTMENT OF ENTOMOLOGY,UNIVERSITY OF Chotkowski (eds.). Academic Press, San Diego, CA. CALIFORNIA,DAVIS,CALIFORNIA 95616; AND RAHAM G , J. B. 1997. Air-breathing fishes: evolution, ( JJC) DEPARTMENT OF WILDLIFE,FISH, AND diversity, and adaptation. Academic Press, San Di- CONSERVATION BIOLOGY,UNIVERSITY OF CALI- ego, CA. FORNIA,DAVIS,CALIFORNIA 95616. E-mail: KRAMER, D. L. 1983. Aquatic surface respiration in the fishes of Panama: distribution in relation to risk of ( JVW) [email protected]; and ( JJC) hypoxia. Environ. Biol. Fish. 8:49–54. [email protected]. Send reprint requests to ———. 1987. Dissolved oxygen and fish behavior. JJC. Submitted: 27 March 2002. Accepted: 15 Ibid. 18:81–92. Oct. 2002. Section editor: S. J. Beaupre.