Homing ability of adult cardinalfish is affected by elevated carbon dioxide
The levels of carbon dioxide (CO2) predicted for the oceans by the end of this century have recently been shown to impair olfactory discrimination in larval fishes. However, whether this disruption extends to olfactory-mediated behaviour in adult fishes is unknown. In many fishes, adult survival and reproduction can be critically dependent upon navigation to home sites. We tested the effects that near-future levels of CO2 (550, 700 or 950 ppm) have on the ability of adult five-lined cardinalfish, Cheilodipterus quinquelineatus, to home to their diurnal resting sites after nocturnal feeding. Cardinalfish exposed to elevated CO2 exhibited impaired ability to distinguish between odours of home- versus foreign-site conspecifics in pair-wise choice experiments. A displacement experiment demonstrated that fish from all CO2 treatments displayed a 22–31% reduction in homing success compared with control fish when released at 200 m from home sites. While CO2-exposed cardinalfish released directly back onto home sites exhibited similar site fidelity to control subjects, behaviour at home sites was affected, with CO2-exposed fish exhibiting increased activity levels and venturing further from shelter. This study demonstrates that the potential disruption of chemosensory mechanisms in fishes due to rising CO2 levels in the ocean extend to critical adult behaviours.
KeywordsClimate change Ocean acidification Navigation Apogonidae Cheilodipterus quinquelineatus
Many aquatic organisms display remarkable homing behaviour, capable of transversing vast distances while navigating to return to specific sites (Tesch 1967; Dittman and Quinn 1996; Luschi et al. 1996). Possessing home sites can contribute to the overall fitness of an individual, with benefits derived from familiarity with local food resources, shelter, and the location of mates, competitors and predators (Shapiro 1986; Noda et al. 1994; Brown and Dreier 2002). While homing is often contingent upon imprinting of sensory cues associated with natal sites during early development (Scholz et al. 1976; Lohmann et al. 2008), adults may also regularly home to non-natal sites used for feeding (Dalpadado et al. 2000; Heyman et al. 2001), breeding (Dawbin 1966; Broderick et al. 2007), and shelter (O’Gower 1995; Karnofsky et al. 1989). Homing in fishes is likely achieved with the aid of a range of sensory cues depending on the required navigational distance. While migratory species transversing great distances are thought to rely on celestial orientation and detection of geomagnetic fields (Quinn 1980; Klimley 1993), navigation through local environments likely occurs primarily through use of visual mapping and chemoreception (Braithwaite 1998; Atema et al. 2002; Myrberg and Fuiman 2002).
Ocean acidification, caused by the uptake of additional CO2 at the ocean surface, is recognised as a significant threat to marine species (Orr et al. 2005; Royal Society 2005; Hoegh-Guldberg et al. 2007; Fabry et al. 2008). The extent to which it disrupts the chemical environment and therefore critical behavioural and physiological processes is still being evaluated. Recent studies simulating ocean acidification have revealed profound effects of elevated CO2 on the chemosensory behaviour of larval reef fishes. Larvae exposed to elevated CO2 are incapable of distinguishing between chemical cues likely used for navigation and settlement site selection at the end of their larval phase (Munday et al. 2009; Dixson et al. 2010). Furthermore, CO2-treated larvae are more active and exhibit riskier behaviour after settlement than fish exposed to present-day conditions, leading to higher mortality rates from predation (Munday et al. 2010). Based on current emission trajectories, CO2 concentrations causing alterations in larval behaviour and survival rates (~700 ppm CO2) will be observed in the oceans before 2100 (Meehl et al. 2007; Raupach et al. 2007). However, whether elevated CO2 could have similar effects on olfactory-mediated behaviours and homing in adult fish is unknown.
Testing the potential ecological effects of ocean acidification on adult homing is challenging, as homing often takes place in the open ocean and can involve migrations of thousands of kilometres. However, the localised homing behaviour of coral reef fish (Sale 1971; Meyer et al. 2000; Willis et al. 2001) provides a manageable arena for testing the effects of elevated CO2 on olfactory-mediated behaviours in a natural setting. Some of the best examples of homing are from the family Apogonidae (cardinalfish) (Marnane 2000; Døving et al. 2006; Gardiner and Jones 2010), a diverse and abundant group of fish found in both temperate and tropical regions (Randall et al. 1997). Cardinalfish maintain daytime resting sites, often forming large, multispecific aggregations primarily within branching corals (Greenfield and Johnson 1990; Gardiner and Jones 2010). By night, cardinalfish depart home resting sites to forage over sand and reef habitat (Chave 1978; Marnane and Bellwood 2002). Apogonids display strong site fidelity, returning to the same resting sites each day, with some species maintaining site persistence for over 12 months (Marnane 2000). Previous studies have demonstrated successful homing of individuals following displacement 2 km away from home sites (Marnane 2000) and spatial memory in migratory cardinalfish allowing return to within 30 cm of previously occupied areas after 6 months away from breeding territories (Fukumori et al. 2010). Homing likely occurs through navigation based on visual landmarks and chemical cues, with preference for odours of home site conspecifics and substrate marked by conspecifics at home resting sites having been experimentally demonstrated (Døving et al. 2006). Cardinalfish therefore provide an ideal group in which to test the potential impacts of ocean acidification on olfactory-mediated behaviour and homing ability in marine fishes.
We used a combination of laboratory and field-based experiments to investigate how near-future levels of CO2 (550, 700, and 950 ppm) could affect olfactory discrimination and homing ability in adult cardinalfish. First, we used pair-wise choice experiments in the laboratory to test if elevated CO2 affected the ability of cardinalfish to distinguish between olfactory cues of home site conspecifics versus conspecifics from foreign reef sites. Tagged cardinalfish exposed to control or elevated CO2 levels were then released back on the reef, either 0 or 200 m from their home resting site, with presence of released individuals at home sites monitored over a 3-day survey period. Finally, observations were performed to determine if normal daytime behaviour at home sites was affected by CO2 exposure.
Materials and methods
Study site and fish collection
This study was conducted at Lizard Island in the Great Barrier Reef, Australia (145°27′E, 14°41′S) during March and October 2010. Adult five-lined cardinalfish, Cheilodipterus quinquelineatus, were collected from various sites throughout the lagoon and returned to the research station where they were placed in one of four CO2 treatments (control, 550, 700 or 950 ppm). Previous experiments have demonstrated that the behavioural effects of elevated CO2 manifest within 4 days of CO2 exposure (Munday et al. 2010), and therefore cardinalfish were maintained in CO2 treatments for four consecutive days prior to testing. Approximately 30 individuals (mean standard length 8.5 cm) were removed from each of 18 sites with aggregations of >50 individuals per aggregation. Cardinalfish were fed commercial fish pellets (Spectrum Aquaculture) twice daily.
Cardinalfish from each site were divided across CO2 treatment levels and placed in replicate 35-L aquaria with approximately 6–8 individuals per group. All elevated CO2 treatments were maintained with a pH–stat system. Seawater was pumped directly from the ocean into 4 × 60-L sumps and diffused with ambient air (control) or CO2 to achieve a pHNBS of approximately 8.14 (control), 8.06, 8.00 or 7.86. A pH-controller (Tunze Aquarientechnik, Germany) was attached to each CO2-treated sump to maintain pH at the desired level, with a solenoid injecting a slow stream of CO2 into a powerhead in each sump whenever seawater pH rose above the set point. Equilibrated seawater from each sump was supplied at a rate of ~500 mL min−1 to four replicate 35-L aquariums, each housing a group of fish. To maintain oxygen levels and the required pCO2 levels, aquariums were individually aerated with air (control ~390 ppm) or CO2-enriched air (~550, 700, or 950 ppm). The concentration of CO2-enriched air was measured continuously with an infrared CO2 probe (Vaisala GMP222). Temperature and pHNBS of each aquarium was measured each morning and afternoon using a HQ40d pH meter (Hach, CO, USA). Dissolved CO2 in the aquariums was measured at regular intervals using a submerged CO2-permeable membrane connected to an infrared CO2 probe (Vaisala GMP222) in a closed loop (Hari et al. 2008). Average pCO2 for the treatments were: 450 ± 6.84 (SE) ppm (control), 563 ± 15.69, 678 ± 46.65, and 961 ± 7.14 ppm, respectively.
The effect of CO2 on the ability of adult cardinalfish to distinguish non-visual cues of their home resting sites was tested using a pair-wise choice experiment. Individuals were tested in outdoor, cylindrical 300-L aquaria containing different stimuli on opposite sides of the arena. Each stimulus consisted of a porous opaque 3.5-L container housing either a native conspecific, collected at the same home site as the test individual, or a foreign conspecific collected at a reef site approximately 400 m away. We assumed that the stimulus was primarily olfactory, as previous studies have demonstrated that cardinalfish respond to odours of home site conspecifics and substrate marked by home conspecifics (Døving et al. 2006), although we cannot exclude the possibility that auditory cues might have been present. Test subjects were released in the centre of the aquarium and allowed to acclimate to the arena for 30 min. Following this acclimation period, the position of the test individual in relation to the two containers was recorded at 5-min intervals for 1 h, with preference for a stimulus source indicated by the presence of the test subject in an aquarium half for >50% of the test period. Test water was changed between trials, with no water flow during the test period. Prior to each trial, test water was maintained still for 5 min to allow concentration gradients to form before introduction of the test subject. Binomial distribution tests were used to analyse preferences based on the probability (0.5) of test subjects spending >50% of the test period within the home site conspecific cue for each trial.
A displacement experiment was used to determine if exposure to elevated CO2 impairs the ability of adult cardinalfish to navigate to their home resting sites. To resight individuals after release all fish were uniquely tagged with coloured elastomer (Northwest Technologies) injected into the dorsal musculature. Following 4 days in CO2 treatments, fish were measured, transported to the reef and released at 0 or 200 m from their respective home site. Fish were released in the late afternoon between 1500 and 1700 hours. For individuals released away from home resting sites, the release point in relation to the home site was determined through random rotation of cardinal compass direction. Each home resting site was surveyed between 0800 and 1000 hours on the following three mornings for the presence or absence of tagged individuals. Additional releases (n = 15–16 individuals per treatment group) followed by night dives were conducted at three sites to determine if normal nocturnal feeding migration occurred following release of fish at home sites. Previous research with damselfish has found that the behavioural effects of elevated CO2 are retained for at least 24 h after fish are returned to current-day conditions, but dissipate over a period of 48 h, and full sensory ability is restored within 72 h (Munday et al. 2010). Therefore, the 3-day survey period is sufficient to capture any effects caused by exposure to elevated CO2. Chi-square tests for independence were used to compare return success (for fish displaced 200 m) and site persistence (for fish released back on home sites) among treatments based on the number of individuals observed at home sites throughout the 3-day test period.
Behavioural observations of tagged individuals from each CO2 treatment at their home sites were conducted the morning after release. The maximum distance (cm) ventured from habitat structure, maximum cumulative distance (cm) moved throughout the habitat, and activity level was recorded during 3-min observation periods. Activity level was scored on a scale from 1 to 3 where 1 is remaining predominately stationary with movement no more than 5 cm, 2 consisting of periodic movements not remaining still for longer than 30 s with frequent advances out of habitat structure and possible conspecific interactions, and 3 is nearly constant movement throughout home site with frequent interactions with conspecifics initiated by the test individual. ANOVAs were used to compare maximum distance ventured and cumulative distance moved among treatments, with means from each CO2 treatment compared with control fish in post-hoc tests. Categorical data from activity level assessments were analysed using Chi-square tests. In this analysis the number of fish scored in each activity level in CO2 treatment groups was compared with the observed distribution of the controls.
There was no significant difference among CO2 treatments with respect to the ability of tagged individuals to persist at home sites when released 0 m from their home resting habitat (p > 0.21). Site persistence was 64% for control fish and 51, 73, and 66% for 550, 700, and 950 ppm CO2 treatments, respectively (Fig. 2). Of cardinalfish present at home sites after release, 64–84% persisted for 2 or more days throughout the survey period in both control and CO2 treatments. Night dives indicated that fewer CO2 exposed fish might be departing each night to feed compared with control fish, with 2 of 15 tagged individuals from each elevated CO2 treatment sighted at home sites whereas all control fish departed at night to feed, although this trend was not statistically significant.
Behavioural traits of fish from control, 550, 700 and 950 ppm CO2 treatments released at home resting sites
Max distance ventured (cm)
Cumulative distance moved (cm)
Activity level (1 to 3)
3.16 ± 0.87
14.72 ± 3.54
1.42 ± 0.14
4.10 ± 0.83
13.89 ± 3.35
1.55 ± 0.17
7.15 ± 1.87
26.00 ± 4.55
1.55 ± 0.15
7.95 ± 1.44
26.84 ± 5.62
1.89 ± 0.17
Since navigation and homing in fishes likely involves a suite of sensory cues, including detection of chemical cues, ocean acidification has the potential to interfere with critical migratory behaviours. Recent studies have shown that larval behaviour is severely disrupted by increasing CO2 (Munday et al. 2009, 2010; Dixson et al. 2010). Given that olfaction is also a critical sense used by adult fish, there is a strong potential for acidification to impact fish at multiple life history stages. Our results demonstrate that continued uptake of CO2 by the ocean could affect navigational capabilities and homing behaviour of adult cardinalfish. Exposure to elevated CO2 appeared to impair olfactory discrimination in adult cardinalfish and significantly affected their ability to home to resting sites after displacement. However, CO2-treated fish returned to their home sites were still able to navigate from nocturnal excursions, likely following unaffected individuals or using other sensory cues, demonstrating the importance of testing the impacts of sensory impairment in an appropriate ecological setting.
Cardinalfish exposed to elevated CO2 exhibited impaired sensory and homing ability at concentrations as low as 550 ppm. Current IPCC emission scenarios predict that atmospheric CO2 will exceed this level during the second half of the century, with present-day CO2 emissions already surpassing the stabilisation trajectory for 650 ppm, and could exceed 800 ppm by the end of the century (Meehl et al. 2007; Raupach et al. 2007). While Munday et al. (2010) found no significant difference in larval damselfish behaviour following exposure to low CO2 concentrations (550 ppm), it appears that members of the family Apogonidae may be more sensitive to alterations in ocean chemistry and could therefore be one of the first groups of reef fish to be affected by ocean acidification. Apogonids form an important dietary component for many reef-dwelling piscivores (Chrystal et al. 1985; Beukers-Stewart and Jones 2004) and likely play an important role in energy transfer and provisioning for reef ecosystems (Marnane and Bellwood 2002), hence factors affecting their fitness and survival could have important implications for other reef species.
Chemical cues are important for navigation and site selection at a variety of life history stages in aquatic animals (Hara 1993; Døving and Stabell 2003). Although the potential for auditory disruption was present in this study (Simpson et al. 2011), olfaction is believed to be the dominant sensory mechanism involved in cardinalfish homing. Cardinalfish are known to be attracted to the chemical cues from shelters previously marked by home site conspecifics (Døving et al. 2006), suggesting they use olfactory cues to help locate home territories after nocturnal foraging. Adult cardinalfish exposed to elevated CO2 were no longer able to distinguish between conspecifics at their home site versus conspecifics from a foreign social group, and consequently would have impaired ability to locate their home resting site. Consistent with this hypothesis, CO2-treated individuals were less able to locate their home site when displaced 200 m for their resting site. Furthermore, tagged individuals from 700 and 950 ppm treatments released both at 0 and 200 m were sighted on multiple occasions associating with foreign conspecific groups at least 400 m from the nearest collection site, likely due to their inability to recognize home site odours. The decrease in homing success following displacement, which coincided with impaired olfactory discrimination behaviour at all elevated CO2 levels tested, suggests that adult cardinalfish use chemical cues associated with home sites to aid in navigation to and identification of their diurnal resting sites. If ocean acidification interferes with homing and home site recognition, individuals may be forced to seek refuge in foreign territories with unfamiliar social groups following nocturnal excursions to foraging sites. It is unknown how easily individuals may be incorporated into foreign social groups as resident adults often defend established territories from foreign intruders (Turner 1994; Chellappa et al. 1999).
Despite clear disruption of homing ability in fish displaced from their home resting sites, there was no effect of elevated CO2 on site fidelity for fish returned directly to their resting sites. Night observations suggest that a smaller proportion of CO2-treated individuals might be departing home sites each night to feed, whereas all control fish appeared to resume nocturnal foraging behaviour. While cardinalfish maintain a high degree of spatial separation between species groups by night (Marnane and Bellwood 2002), many species form small intraspecific aggregations while foraging, suggesting that CO2-treated fish may also be able to return to their daytime resting sites by simply following other individuals. Likewise, interpretation of visual cues appears to be unaffected by exposure to the CO2 levels used in this study, therefore feeding nearby within visual reference to home sites may help CO2-treated individuals return. Thus, failure to vacate resting sites, or reliance on small feeding aggregations and visual cues for direction, could account for the high persistence rates we observed in the elevated CO2 treatments.
Variation in behaviour observed in CO2-treated fish at daytime resting sites could potentially decrease survival rates by increasing susceptibility to predation. Individuals from 700 and 950 ppm CO2 treatments appeared less cautious, spending more time further from shelter and displaying an increase in activity throughout the habitat. Behavioural changes such as these have been found to cause higher mortality rates from predation in newly settled damselfish larvae (Munday et al. 2010), and would likely increase mortality of adult cardinalfish at home sites.
The prospects for adaptation of sensory systems to maintain performance in high CO2 environments remain unknown. Although the acute exposure to elevated CO2 used in this study does not allow observation of long-term effects, the consistency of behavioural responses observed at CO2 concentrations likely to occur within the next 30 years (550 ppm) indicates there is limited time for adaptation to occur before impacts are present in wild populations. While Munday et al. (2010) observed substantial variation among damselfish larvae exposed to 700 ppm CO2, variation between treatment levels was not observed in this study. The short generation time observed in most apogonid species might contribute to development of adaptations over time; however, the lack of variability in behavioural responses observed here suggests there may be limited opportunity for selection to occur. The plasticity of the physiological mechanisms responsible for impairment of sensory systems will be important in predicting the potential for adaptation; however, these mechanisms are currently unknown and are an important area for further research. Homing itself may be critical for local adaptation and any impairment of homing ability may significantly impact the ability of species to evolve in rapidly changing environments.
Alterations in homing ability, olfactory discrimination, and behaviour caused by elevated CO2 suggest possible impairment of general cognitive function. Cardinalfish held in elevated CO2 were no longer able to distinguish cues associated with home sites, and consequently displayed an inability to successfully return to home sites following displacement, suggesting failure both to recognize familiar home site chemical cues and to retain spatial memory of the surrounding reef environment. Attaining the benefits provided by maintenance of home ranges is dependent on mental retention of familiar aspects, such as locality of food, shelter and conspecifics throughout the territory. Without the capacity to identify previously learned resources, many of the benefits gained from maintaining home territories may be lost. Therefore, interruption of pathways necessary for sensory cue recognition and subsequent elicitation of appropriate behavioural responses could have significant implications for individual fitness.
This study is the first to demonstrate effects of ocean acidification on ecologically important behaviour of adult fish in the field and at CO2 concentrations that could occur in the oceans within 40 years (Meehl et al. 2007). While the apogonids tested here appear to be more sensitive to rising CO2 than the damselfish species tested previously (Munday et al. 2009, 2010; Dixson et al. 2010), potential effects on other species are unknown. If other species are affected at similar CO2 levels, there could be significant implications for homing and site attachment in fish from a range of different ecosystems. Further research is needed to explore the sensitivity of other fish species to elevated CO2 in order to understand the mechanisms responsible for impairment of sensory cue recognition and the ecological impacts likely to be experienced in natural ecosystems.
Special thanks to Danielle Dixson and Ingrid Cripps for assistance with field work and the Australian Museum Lizard Island Research Station for providing excellent logistical support and facilities. Funding to P.L.M. from the ARC Centre of Excellence for Coral Reef Studies and to B.M.D. from the Great Barrier Reef Marine Park Authority supported the project. Research was conducted in accordance with JCU ethics approval A1468.
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