Parasite Infection Directly Impacts Escape Response and Stress Levels in Fish Bridie J

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RESEARCH ARTICLE Parasite infection directly impacts escape response and stress levels in fish Bridie J. M. Allan1,2,3,*, Björn Illing2, Eric P. Fakan2,3, Pauline Narvaez2,3,4, Alexandra S. Grutter5, Paul C. Sikkel6,7, Eva C. McClure2,3,8, Jodie L. Rummer2 and Mark I. McCormick2,3

ABSTRACT behavioural and physiological phenotypes (for review, see McElroy Parasites can account for a substantial proportion of the biomass and de Buron, 2014). Unlike macropredators such as piscivores, in marine communities. As such, parasites play a significant micropredators (which we define broadly to include both parasites ecological role in ecosystem functioning via host interactions. Unlike and micropredators as defined more narrowly by Kuris and Lafferty, macropredators, such as large piscivores, micropredators, such as 2000, and Lafferty and Kuris, 2002) typically do not cause direct parasites, rarely cause direct mortality. Rather, micropredators impose mortality, but rather cause a constant drain on energetics, thus an energetic tax, thus significantly affecting host physiology and significantly affecting host physiology and behaviour (for review, behaviour via sublethal effects. Recent research suggests that see Barber, 2007). However, the magnitude of this change depends infection by gnathiid isopods (Crustacea) causes significant on the parasite type, parasitic loading and the size and ontogenetic physiological stress and increased mortality rates. However, it is stage of the host (Sun et al., 2012). For example, larval and juvenile unclear whether infection causes changes in the behaviours that fish are reported to be more vulnerable to the effects of infection underpin escape responses or changes in routine activity levels. than their adult counterparts, owing to low body reserves and high Moreover, it is poorly understood whether the cost of gnathiid infection metabolism (Strathmann et al., 2002; Grutter et al., 2011). manifests as an increase in cortisol. To investigate this, we examined Moreover, parasitic infection can also affect behaviours and the effect of experimental gnathiid infection on the swimming and physical attributes important for fleeing predators, such as escape performance of a newly settled coral reef fish and whether reducing visual acuity (Seppälä et al., 2005), limb malformation infection led to increased cortisol levels. We found that micropredation causing reductions in maximum jumping distance, burst swimming by a single gnathiid caused fast-start escape performance and speed and endurance (Goodman and Johnson, 2011), and reducing swimming behaviour to significantly decrease and cortisol levels to critical swimming speeds in adult and newly settled coral reef fishes double. Fast-start escape performance is an important predictor of (Binning et al., 2013; Grutter et al., 2011). recruit survival in the wild. As such, altered fitness-related traits and One of the most prevalent ectoparasites on coral reefs is gnathiid short-term stress, perhaps especially during early life stages, may isopods (Crustacea) (Grutter, 1994; Sikkel and Welicky, 2019). result in large scale changes in the number of fish that successfully Gnathiids, mobile temporary parasites of fish, feed using a trophic recruit to adult populations. strategy that might best be referred to as micropredation (Kuris and Lafferty, 2000; Lafferty and Kuris, 2002). Micropredators attack KEY WORDS: Escape performance, Fish behaviour, Micropredator, multiple prey (hosts), much like predators do, but an individual Predator–prey interactions micropredator’s effect on its prey tends to be small. Micropredators of vertebrate hosts, such as ticks, mosquitos and gnathiids, briefly feed INTRODUCTION on blood and are not transmitted trophically. Because micropredators Parasites can reach high biomass in marine communities (Kuris feed on several prey individuals, they also do not benefit from et al., 2008) and make up around 40% of the total biodiversity on minimising damage to prey (Barber et al., 2000) and can rapidly Earth, making them one of the most successful modes of life (Poulin abandon their prey if it is incapacitated (Murray, 1990; Lehmann, and Morand, 2000; Hatcher and Dunn, 2011). As such, parasites 1993). These reef-based parasites feed on a variety of coral reef fish play a significant role in ecosystem functioning as they exert sub- hosts from teleosts to elasmobranchs and on all host ontogenetic lethal effects on their host where they can modify and manipulate stages (Grutter and Poulin, 1998; Grutter et al., 2017). As such, they can cause significant physiological stress such as increased oxygen consumption (Grutter et al., 2011), reduced haematocrit (Jones and 1Department of Marine Science, University of Otāgo, Dunedin 9054, New Zealand. Grutter, 2005), increased cortisol load (Triki et al., 2016) and even 2ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD 4811, Australia. 3Department of Marine Biology and Aquaculture, mortality (Hayes et al., 2011). Previous work by Grutter et al. (2011) James Cook University, Townsville, QLD 4811, Australia. 4Centre for Sustainable estimated that a single gnathiid can consume up to 85% of the blood Tropical Fisheries and Aquaculture, James Cook University, Townsville, QLD 4811, volume of a late-stage larval damselfish, which has the potential to Australia. 5School of Biological Sciences, The University of Queensland, St Lucia, QLD 4072, Australia. 6Department of Biological Sciences, Arkansas State significantly affect behaviours that rely on aerobic activities, such as University, Jonesboro, AR 72467, USA. 7Water Research Group, Unit for swimming (Gallaugher et al., 1995; Grutter et al., 2011). Reduced Environmental Sciences and Management, North-West University, Potchefstroom swimming performance can affect the way in which a fish interacts 2520, South Africa. 8Australian Rivers Institute, Griffith University, Gold Coast, QLD 4215, Australia. with conspecifics and predators and whether it can settle successfully to the benthic environment (Allan et al., 2013; Grutter et al., 2011). *Author for correspondence ([email protected]) When coral reef fishes recruit to the benthic environment, it is B.J.M.A., 0000-0002-5991-9711; B.I., 0000-0002-5217-805X reported that predator-induced mortality can be absolute, but averages 60% within the first few days of settlement (Almany and

Received 12 June 2020; Accepted 26 June 2020 Webster, 2006). Predator avoidance and evasion are key ecological Journal of Experimental Biology

1 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb230904. doi:10.1242/jeb.230904 traits that are directly related to growth and survival. When a predator ambon damselfish, Pomacentrus amboinensis Bleeker 1868 attacks, prey are faced with a series of decisions, such as how fast to (Pomacentridae) [standard length range 9–12 mm, mean±s.d. 10.3 respond, which direction to turn, and how fast and how far to escape ±0.05 mm] were collected using light traps (Meekan et al., 2001) in in an overall whole-organism behaviour called a fast start (for review, the waters off Lizard Island (14°40′S, 145°28′E) in the northern see Domenici and Blake, 1997). Fast-start escape behaviour can Great Barrier Reef, Australia. This species is a common component significantly increase the probability of prey escape (Walker et al., of the benthic fish fauna of Indo-Pacific reefs, and adults inhabit 2005; Allan et al., 2013, 2015, 2017). The effectiveness of fast-start sandy areas of lagoons and inshore reefs. Pomacentrus amboinensis escape behaviour is a consequence of body morphology, muscle naturally settle on patch reef environments near the continuous reef. mass and muscle cell physiology and energy reserves (Langerhans, In this habitat, juveniles are exposed to reef-associated gnathiids 2009). Fast starts are characterised by rapid acceleration, which is and macropredators that use a variety of feeding modes from driven by the rapid anaerobically powered contraction of large ambush (lizardfish Synodus dermatogenys and the small grouper myotomal blocks of fast glycolytic muscle (Rome et al., 1988; Cephalopholis microprion) to pursuit (dottybacks Pseudochromis Josephson, 1993). Although anaerobically powered, fast starts are a fuscus and wrasse Thalassoma lunare). These fishes can be strenuous form of activity in which the active muscles require more observed to prey on juveniles that venture too far from shelter oxygen than can be supplied during the period of activity. Therefore, (McCormick, 2012), including the species used in this study, an oxygen debt is accrued that needs to be repaid via aerobic P. amboinensis. After capture, P. amboinensis were transferred from metabolism (Scarabello et al., 1991). light traps to aquaria (65×35×30 cm) with aeration and water flow To date, few studies have addressed the effects of parasitic load for a minimum of 48 h before use in trials. Coral reef fish recruits, on fast-start escape behaviours. Blake et al. (2006) examined the when captured using light traps, habituate to life in aquaria effects of parasite load on the C-start performance of the three- extremely quickly and will feed within several hours following spined stickleback, Gasterosteus aculeatus, and found negative removal from light traps. effects on escape kinematics (Blake et al., 2006). By contrast, Binning et al. (2014) tested the escape performance of the monocle Gnathiid exposure bream, Scolopsis bilineata, following infection by the large In the evening, prior to behavioural trials (17:00 h), well-fed ectoparasitic cymothoid isopod Anilocra nemipteri, and observed P. amboinensis were individually transferred to randomly assigned no change in the escape performance of parasitised fish, suggesting 700 ml black aquaria filled with filtered seawater. Fish were left to that infection may not compromise escape performance. However, habituate for 1 h, after which a single, unfed, stage three gnathiid these studies used adult fish (overall range in body length of 4 to (∼1.5 mm long, harvested from a well-established gnathiid culture 13 cm) to measure the effects of parasite infection on escape tank at the Lizard Island Research Station; Grutter et al., 2010) was performance, and it seems likely, given the physiological cost of carefully transferred to each aquarium via a pipette. Control fish parasitic infection (Grutter et al., 2011; Sun et al., 2012), that the were treated in the same way and transferred into 700 ml black escape performance of coral reef fish recruits would be negatively aquaria filled with filtered seawater. However, instead of a gnathiid, affected. Therefore, the main goal of the current study was to filtered seawater was added via a pipette. After transfer, the understand whether gnathiid infection would compromise the fast- gnathiids were observed to be swimming freely in the aquaria. Fish start escape kinematics of newly settled, coral reef fish recruits. were exposed to the gnathiids during the night, as gnathiids tend to Furthermore, we evaluated whether experimental exposure to be nocturnally active when their fish hosts are less active (Grutter gnathiids induced changes in cortisol levels. The physiological and Hendrikz, 1999; Sikkel et al., 2009). Fish were left undisturbed processes by which fish respond to a stressor can be grouped into for 2 h and were subsequently checked at 2 h intervals (using a red primary, secondary and tertiary responses (Barton and Iwama, light to minimise disturbance) throughout the night, and the status of 1991). Initially, catecholamines from chromaffin tissue are released, the gnathiid (fed, unfed or gnathiid missing – presumably eaten by thus stimulating the hypothalamic–pituitary–interrenal (HPI) axis, the fish) recorded. The next day, the fish were tested for swimming which causes the release of corticosteroid hormones. This is behaviour and fast-start responses in the order in which they followed by a secondary response, which involves haematological had been parasitised, meaning that they were tested no more than preparations to increase the efficiency of metabolic and immune 10 h after the gnathiid was observed to be attached. To control responses (for review, see Barton, 2002). Finally, tertiary responses for a temporal effect, control fish and non-parasitised fish (i.e. the manifest as changes in whole-animal performance, such as changes gnathiid remained unfed at the end of infection exposure) were in condition and behaviour. Increased cortisol following infection also tested throughout the day. For sample sizes per treatment, see by haematophagous parasites has been observed across multiple Fig. 1 legend. taxa, including birds (Quillfeldt et al., 2010), rodents (St Juliana et al., 2014) and fishes (Triki et al., 2016). These variables were Routine swimming and fast-start protocol selected as they are key metrics of individual performance and are Routine swimming and fast starts were examined using individual predictors of fish survival in the wild (McCormick et al., 2018). fish in a transparent circular acrylic arena (diameter 200 mm, Newly settled fish were chosen as prey because the life-history shift height 70 mm) within a large opaque-sided plastic tank between pelagic larvae and settled juveniles represents an important (585×420×330 mm; 60 l) with a transparent Perspex bottom to bottleneck where mortality is intense and selective. allow responses to be filmed from below using the fish’s silhouette. The water level was maintained at a height of 60 mm to reduce MATERIALS AND METHODS movements in the vertical plane, and the water in the arena was Study species emptied and refilled with fresh seawater after approximately every All work carried herein was in accordance with the James Cook 20 min to maintain water quality and temperature. The arena was University Animal Ethics guidelines (JCU Animal Ethics approvals illuminated by an LED light strip wrapped around the outside of the A2080, Great Barrier Reef Marine Park Authority collection permit holding tank with light penetrating with even illumination through

G12/35117.1.). During December 2016, newly metamorphosed the white plastic sides. At the end of the 5 min habituation period, Journal of Experimental Biology

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Canonical 2 Fig. 1. Comparison of the effect of gnathiid infection (9.6%) on swimming and escape kinematics in the ambon 1 damselfish Pomacentrus amboinensis. A canonical discriminant analysis comparing the behavioural Max. speed changes in swimming and escape behaviour after Control exposure to a gnathiid, showing parasitized fish, those 0.5 Distance that managed to avoid parasitism (non-parasitised) and Speed control fish. Vectors represent the direction and Parasitised intensity of trends in prey performance: latency, maximum speed, distance, speed, routine swimming 0 Canonical 1 (90.4%) (RS) speed and RS distance. The circles represent –2 –1.5 –1 –0.5 0 0.5 1 1.5 95% confidence intervals. n=34 controls, n=34 non- RS distance parasitised and n=27 parasitised. RS speed

Latency –0.5 Non-parasitised

–1 routine activity (used to determine routine swimming) was recorded back to James Cook University, Townsville, QLD, Australia, as a silhouette from below, at 30 frames s−1 for 2 min (Casio where samples were analysed for cortisol (n=14 controls, n=13 EX-ZR1000). Routine swimming was analysed on these 2 min non-parasitised, n=14 parasitised). video sequences and measured by tracking the distance (in metres) covered by the fish every second, resulting in 120 data points per fish. Cortisol extraction and ELISA From this distance measure, average speed was also calculated (m s−1). Individual fish were freeze-dried (0.2 mbar, >16 h, Alpha 1-2 A fast start was then stimulated by the release of a conical weight LDplus, Martin Christ, Osterode am Harz Germany) and weighed with a tapered end into the testing arena and was recorded at (Mettler Toledo UMX2 Ultra-Microbalance, 0.1 µg readability) then 480 frames s−1 (Casio EX-ZR1000). This was only carried out when homogenised in 2 ml Eppendorf vials, using a glass bead, 0.5 ml 1× fish had moved to the middle portion of the tank, allowing an phosphate-buffered saline (PBS, pH 7.4) and a shaking mill (3 min, individual to move an equal distance in any direction and FastPrep24, MP Biomedicals, Santa Ana, CA, USA). Homogenised standardising for fish position relative to the stimulus. The weight tissue was transferred to a 10 ml glass vial and rinsed with 0.4 ml was released from an electromagnet and was governed by a piece of PBS. Ethyl acetate (Ajax Finechem, Thermo Fisher Scientific) was fishing line that was long enough such that the tapered tip of the added (1:9 ratio), and samples were vortexed (1 min, Vortex Mixer, weight only just touched the surface of the water. To avoid a Ratek, Boronia, VIC, Australia) and centrifuged (3500 rpm, 5 min, premature fast-start response associated with visual stimulation 4°C, Eppendorf 5810 R). Ethyl acetate has been shown to be an occurring, the weight was released through a 550 mm piece of effective organic solvent for extracting whole-body cortisol from 48.5 mm diameter PVC pipe with the bottom edge at a distance of early life stages of fish (Yeh et al., 2013). The supernatant was 10 mm above the water level. To ensure a standardised protocol, fast- collected and transferred to a 28.5 ml glass vial, and this extraction start variables were only measured when fish performed a C-start step was performed 4 times with all collected supernatants being (commencement of fast-start that results in the individual forming a pooled. The ethyl acetate was dried off in glass reaction tubes using a C-shape, sensu Domenici and Blake, 1997). A minimum of 27 centrifugal vacuum concentrator (43°C, Savant SpeedVac SC110A, replicates (individual fish) per treatment group were startled to ensure Thermo Fisher Scientific). The samples were reconstituted on the statistical robustness (n=34 controls, n=34 non-parasitised and n=27 same day with 1 ml assay buffer and processed following the parasitised). Trials were conducted between 08:00 h and 16:00 h. enzyme-linked immunosorbent assay (ELISA) protocol provided by Kinematic variables associated with the fast-start response were Cayman Chemical (Cortisol ELISA Kit 500360, Cayman Chemical). analysed using ImageJ with a manual tracking plug-in. The centre of The samples were analysed in triplicate with a spectrophotometer mass of each fish was tracked for the duration of the response. The (SpectraMax Plus 384 Microplate Reader, Molecular Devices; following kinematic variables were measured. (1) Response latency average absorbance calculated from readings at 405 to 420 nm). (s), measured as the time interval between the stimulus onset and the first detectable movement leading to escape of the animal. Cortisol ELISA validation (2) Response distance (m), a measure of the total distance covered by Several assay validation steps were performed to test for parallelism, the fish during the first two flips of the tail [the first two axial bends, accuracy and precision of the cortisol ELISA kit, following i.e. stages 1 and 2 defined based on Domenici and Blake (1997), recommendations by Metcalfe et al. (2018). Parallelism was which is the period considered crucial for avoiding ambush predator confirmed by comparing dose–response curves of diluted samples attacks (Webb, 1976)]. (3) Response speed (m s−1), measured as the against a standard curve (ANCOVA, P>0.05, n=3). Briefly, distance covered within a fixed time (25 ms). This fixed duration was reconstituted samples (n=3) were diluted (1:4, 1:8, 1:12, 1:16, 1:20 based on the average duration (22.8 ms) of stage 1 and 2 (as defined and 1:24) and compared against the cortisol standard curve (Cayman above). (4) Maximum response speed (m s−1), measured as the Chemical ELISA kit, 6.6–4000 pg ml−1 range). An optimal dilution maximum speed achieved at any time during stage 1 and stage 2. for the samples (20×) was observed at 50% relative maximum After fish had been assessed for their routine swimming and binding, and sample dilutions falling within 20–80% B/B0 relative fast-start responses, they were killed by cold shock, blotted maximum binding were considered as acceptable (Metcalfe et al., dry, immediately frozen in liquid nitrogen, and then transferred 2018). The accuracy of the method (i.e. the recovery of a known Journal of Experimental Biology

3 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb230904. doi:10.1242/jeb.230904 amount of added cortisol) was assessed by spiking three samples subsequently analysed using a Kruskal–Wallis test with Dunn’s test with 800 pg cortisol ml−1, more than half of the samples’ cortisol and Holm–Šidák adjustment as post hoc tests. All statistical concentration and within the detection limit of the ELISA kit (see analyses were performed in R, version 3.5.1. Guest et al., 2016). For each of the three samples, two fish were homogenised, pooled and the homogenate split into halves, with RESULTS one half receiving the spike and the other the assay buffer. Both Kinematic results parts were then processed in the same way as all other samples. The Exposure to a single gnathiid affected nearly all measured kinematic spike’s recovery (percentage) was expressed as spiked sample traits (Figs 1 and 2, Table 1). The MANOVA revealed a significant result−unspiked sample result×100/known spike (800 pg ml−1), and difference in the overall change in behaviour in response to gnathiid the mean recovery (94.3%, n=3) was used as a correction factor for exposure (Pillai’s trace 0.414, F12,164=3.568, P<0.0001). A CDA calculating the samples’ cortisol concentration. Intra-assay precision displayed the nature of the differences found among treatment of triplicate samples was determined using the coefficient of variation centroids and shows a clear separation of the three treatments (CV), and found to be 5.5±4.9 (mean±s.d., n=41). into two distinct groups with respect to the six behavioural measurements, with the parasitised treatment being separate from Statistical analyses the non-parasitised and control treatments (Fig. 1). Control fish and Kinematic analysis non-parasitised fish were differentiated from parasitised fish along A preliminary analysis of covariance (ANCOVA) found that latency the first canonical axis, which accounted for 90.4% of the difference to respond to the startle was positively related to distance to the among treatments. This axis was principally driven by trends in fast- stimulus, and the slope of the relationship did not differ between the start kinematics, which indicated that control fish and non- two treatments (i.e. homogeneous slopes; F2,84=1.77, P=0.177). To parasitised fish travelled further, had higher average speeds and remove the influence of distance to the stimulus on latency exhibited a more rapid response to the drop stimulus (i.e. lower (F1,84=11.29, P=0.001), the residuals of the relationship were used response latency) than parasitised fish. This suggestion was for subsequent analyses. No other variable was affected by distance statistically confirmed by the results of the one-way ANOVA, of the fish to the startle stimulus. A multivariate analysis of variance with the parasitised group exhibiting reductions in performance in (MANOVA) was undertaken to determine whether there was a nearly all measured traits (Fig. 2). For example, parasitised fish were difference in the routine swimming or fast-start kinematics of slower to respond to the stimulus, with increased latency in this P. amboinensis after exposure to a single gnathiid. Dependent group (F2,86=11.425, P=0.001). The distance achieved during variables included were: the fast-start variables distance, speed, stage 1 and 2 and the speed achieved during this same period maximum speed and latency (residuals), and the routine swimming were significantly reduced (F2,88=3.871, P=0.0025; F2,88=3.987, variables distance and speed. The nature of significant differences P=0.0022) in fish that had been parasitised. In addition, the distance found by MANOVA in relation to the original variables values was and speed over a 2 min period were significantly reduced, with then compared between treatments using canonical discriminant parasitised fish covering half the distance covered by the control analyses (CDAs) to determine how escape and swimming and the non-parasitised groups (F2,91=9.929, P=0.001; speed kinematics differed between treatments. Trends in the behavioural F2,91=9.997, P=0.001). There was, however, no significant variables were represented as vectors, which were plotted on the first difference among treatments in the maximum speed achieved two canonical axes, together with treatment centroids and their 95% during an escape (F2,87=1.818, P=0.160). The repeated measures confidence clouds (Seber, 1984). The strength or importance of MANOVA revealed a significant effect of treatment (Wilks 0.337, each of the original variables in discriminating among groups was F8,28=2.523, P=0.033). However, the order in which the trial displayed graphically as the length and direction of these vectors. To occurred was insignificant (Wilks 0.814, F4,14=0.7861, P=0.547). further explore the differences between treatments, one-way There was also an insignificant interaction between order of trial and ANOVA were used to identify significant differences within treatment (Wilks 0.646, F8,28=0.851, P=0.566). These results individual behaviours of interest. When significant, differences suggest that despite being tested in the same water as a previous were further examined using Tukey’s HSD means comparison tests. trial, there was no effect of this on routine swimming or fast-start Pairs of fish were successively tested in the same water; however, in escape behaviour. doing this, it is possible that the behaviour of the second fish may have been influenced by chemical signals excreted from the first Cortisol analysis fish. To remove this potential risk, we suggest using clean water for Cortisol concentrations were significantly different among each trial. To account for this possible bias, we undertook a treatments (Kruskal–Wallis test, P<0.001) but highest in ambon repeated-measures approach to test the potential effect of trial order damselfish that were parasitised by gnathiids (Dunn’s post hoc test, influencing the behaviour of the fish, while still allowing us to P<0.001; see Fig. 3, Table 2). Non-parasitised ambon damselfish determine whether there was an effect of gnathiid exposure. Here, a showed comparable cortisol levels to fish maintained under control two-way repeated-measures MANOVA was undertaken on a subset conditions (Dunn’s post hoc test, P=0.244). of pairs of fish to test the effect of trial order (1st or 2nd trial) and treatment (n=8 control pairs, n=7 non-parasitized pairs, n=5 DISCUSSION gnathiid pairs) on the routine swimming and fast-start kinematics Predation is a central tenet in ecology – predators capture, kill of P. amboinensis. All assumptions of normality and homogeneity and consume their prey (Lima and Dill, 1990). By contrast, of variance were visually inspected and found to have been met. micropredators attack multiple hosts, may briefly feed on blood, and Analyses were carried out in Statistica version 13. can influence the mortality schedules of fish through changes in physiology, morphology and behaviour (Grutter et al., 2011, 2017; Cortisol analysis Binning et al., 2013, 2014; Artim et al., 2015; Triki et al., 2016; The cortisol results were tested for homogeneity of variance, which Sellers et al., 2019). Here, we demonstrate that experimental infection was found to be violated (Bartlett’s test, P<0.001). Data were by a single gnathiid has a marked influence on the fast-start escape Journal of Experimental Biology

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ABFig. 2. Effect of gnathiid infection on 0.24 0.8 swimming and escape kinematics of a,b a,b P. amboinensis. (A) Response distance, 0.6 (B) response speed, (C) maximum speed, 0.16 ) –1 (D) response latency, (E) routine swimming 0.4 distance (over 2 min) and (F) routine swimming 0.08 (m s speed (over 2 min). Means±s.e.m. Letters above 0.2 bars represent Tukey’s HSD groupings of means. Response speed n=34 controls, n=34 non-parasitised and n=27 0 0 Response distance (m) parasitised.

1.6 C 0.8 D ) –1 1.2 0.6

0.8 0.4

0.4 0.2 Max. speed (m s 0 Response latency (s) 0 ) EF–1 5 1.2 4 3 0.8

2 0.4 1 0 0 Control Non-parasitised Parasitised Control Non-parasitised Parasitised Routine swimming distance (m) Routine swimming speed (m s kinematics and the routine swimming behaviour of settlement-stage metabolic performance measured as oxygen uptake, and found ambon damselfish. For example, latency to respond when startled infected fish had reduced performance, probably driven by blood increased following gnathiid infection, and high latencies have been loss. Consequently, fish infected with strongly debilitating parasites associated with lower survival (McCormick et al., 2018). In addition may exhibit markedly reduced activity levels to conserve energy; to latency, all locomotory behaviours, with the exception of this may explain the observed decrease in fast-start behaviour in the maximum speed, were found to be reduced when compared against current study. Infected fish may have substantially decreased energy the control and non-parasitised groups, indicating that there was a reserves (via blood loss), thus reducing the ability to recover after kinematic cost associated with infection. eliciting an energetically costly escape. In addition, we also Fast-start escape behaviour is a measure of whole-organism observed a 50% decrease in routine swimming and average speed performance and is influenced by intrinsic (i.e. physiological and following infection by a single gnathiid. biochemical) and extrinsic processes (i.e. habitat degradation, Our results contrast those of Binning et al. (2014), who found that predation stress, temperature and oxygen) (McCormick et al., 2017; the escape performance of S. bilineata was unaffected following Allan et al., 2015; Domenici et al., 2019). It is the interaction infection by the cymothoid isopod A. nemipteri, with little between these processes that can trigger and modify how an escape difference in escape kinematics between non-infected and is undertaken (Breed and Sanchez, 2010). Any factor that disrupts infected fish. However, these contrasting results may be driven by these processes can lead to increased mortality rates (Allan et al., ontogeny. For example, Binning et al. (2014) used infected adult 2013). Grutter et al. (2011) quantified the cost of infection by a

single gnathiid on newly recruited ambon damselfish, using 500

) –1 400

Table 1. Results of ANOVA on fast-start and routine swimming variables mg comparing Pomacentrus amboinensis parasitised by a gnathiid, those –1 300 that managed to avoid parasitism and control fish 200 Variable FPη2 Latency 11.60 <0.0001 0.21 100

Distance 3.96 0.023 0.080 ml (pg Cortisol Speed 4.06 0.02 0.086 0 Parasitised Maximum speed 1.60 0.21 0.035 Control Non-parasitised Distance travelled* 9.31 0.0002 0.18 Fig. 3. Effect of gnathiid infection on cortisol concentration of Speed* 9.36 0.0002 0.18 P. amboinensis. Cortisol levels of non-parasitised fish were significantly lower Asterisks denote routine swimming variables. η2 values are given as a than those of fish that were parasitised by a gnathiid. Means±s.e. n=14 measure of effect size. d.f.=2,87 (86 for routine swimming variables). controls, n=13 non-parasitised and n=14 parasitised. Journal of Experimental Biology

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Table 2. Cortisol content of P. amboinensis exposed to gnathiids and Alarcon-Chaidez, 2001; Fulford and Harbuz, 2005). Regardless Cortisol (pg ml−1 mg−1 dry mass) of the mechanism(s), our results suggest that short-term exposure to a gnathiid ectoparasite causes the release of cortisol. Whether the Treatment n Mean s.d. s.e.m. release of cortisol following attachment has long-term effects is Control 14 150.55 41.36 11.05 unknown. However, this seems unlikely, given that cortisol rises Non-parasitised 13 175.55 62.30 17.28 quickly, within the first 4–10 min of an experienced stress, and lasts Gnathiid 14 365.98 179.35 47.85 for only a few hours (Foo and Lam, 1993; Sumpter, 1997). We found that experimentally exposing coral reef fish recruits to fish (∼130 mm body length) that may have a higher physiological gnathiids negatively affected their fast-start escape performance. We tolerance to infection than the newly recruited fish (∼15 mm body also observed increased cortisol levels following infection. A loss of length) used in the current study. By examining adult fish, the fitness can decrease survival during metamorphosis as fish transition results may have been biased toward those individuals that could from the pelagic to the benthic environment where they face myriad cope with infection. Those that could not cope with infection may predators (Hoey and McCormick, 2004). Therefore, any external have been removed from the population, thus underestimating the stressor (i.e. parasitism) that reduces condition, affects behaviour and/ cost of infection. Moreover, the life history strategies of the parasites or alters physiology may indirectly increase mortality rates. For used in both studies are markedly different. Anilocra nemipteri example, Grutter et al. (2017) examined the effect of gnathiid remain on their host for between 12 and 16 months and may not exert infection on 14 species of pre-settlement coral reef fish and found that, a major cost to the host, owing to their dependence on host survival. for small fish (<12 mm), there was significant mortality following By contrast, gnathiids have a larval phase consisting of three stages infection by a single gnathiid. This suggests that micropredators may and associated moults during which they feed on the blood of their contribute to size-selective mortality during settlement. Moreover, host before releasing from their host (Tanaka, 2007). Therefore, the parasites can interact with other ecological drivers such as habitat fitness cost exerted on their host is much greater (i.e. 85% blood loss, degradation (Sikkel et al., 2019), resulting in an increase in infection sensu Grutter et al., 2011) and depends on the size of the juvenile host rate with potentially detrimental effects on biodiversity and ecosystem (Grutter et al., 2017). Given an individual parasite is large, relative to health. The early life-history stages of marine fishes are critical for the its small hosts (a 1:10 ratio of gnathiid to a newly recruited ambon replenishment and abundance of keystone species to marine damselfish), it is not surprising that we observed a significant ecosystems (Almany et al., 2007). As such, any changes at this reduction in the effectiveness of fast-start escape behaviour in the stage can compromise the integrity of adult populations. ambon damselfish as a result of infection. Aside from gnathiid and cymothoid isopods, other isopods are known to feed on blood or Acknowledgements fluids of marine fishes, including cirolanid, coralanid and aegeid We thank all the staff at the Lizard Island Research Station, and all the students and volunteers who helped with the light traps and sorting of fish and with the isopods (Poore and Bruce, 2012; Smit et al., 2019). maintenance of the gnathiid culture. To date, few studies have explored how short-term infections with gnathiids affect coral reef fish host stress physiology (Grutter and Competing interests Pankhurst, 2000; Grutter et al., 2011; Binning et al., 2014; Triki The authors declare no competing or financial interests. et al., 2016). We quantified total body cortisol levels following exposure to a parasite and found that infection led to nearly a 2-fold Author contributions Conceptualization: B.J.M.A., A.S.G., P.S.; Methodology: B.J.M.A., B.I., E.P.F., P.N., increase in cortisol levels. The effects of elevated cortisol on E.C.M.; Formal analysis: B.I., M.I.M.; Writing - original draft: B.J.M.A.; Writing - behaviour in fish have been well documented (Barton and Iwama, review & editing: B.I., E.P.F., P.N., A.S.G., E.C.M., J.L.R., M.M.; Funding acquisition: 1991). However, to the best of our knowledge, this is the first study A.S.G., P.S., M.M. to investigate the relationship between elevated cortisol and fast- start escape performance in fish. Increased glucocorticoids prime Funding Funding was provided by an Australian Research Council Centre of Excellence for animals for a number of activities, including reproduction, Coral Reef Studies grant (EI140100117). This work was supported by the Australian competition and avoiding predation. Therefore, it seems likely Research Council (A00105175, A19937078, ARCFEL010G, DP0557058, that glucocorticoids would play an important role in fast-start escape DP120102415) and the US National Science Foundation (OCE-724 1536794). B.I. behaviour. However, if the stressor is severe, the ability of the fish to was supported by a postdoctoral research fellowship from the German Research Foundation (Deutsche Forschungsgemeinschaft, IL-220/2-1) and the Australian cope may be reduced, and the overall effect of stress may become Research Council Centre of Excellence for Coral Reef Studies. maladaptive (Barton and Iwama, 1991). Increased cortisol may be due to either the physiological cost of Data availability infection or the discomfort caused by attachment of the parasite. For Data are available from the figshare repository: 10.6084/m9.figshare.12730847 example, gnathiids were observed to be attached around the anterior region of the fish, which is often dense with nociceptors that, when References stimulated, lead to quantifiable changes in neurological activity Allan, B. J. M., Domenici, P., McCormick, M. I., Watson, S.-A. and Munday, P. L. (Sneddon et al., 2014) indicative of pain. To date, the effect of (2013). Elevated CO2 affects predator-prey interactions through altered parasite attachment on nociception has not been examined. performance. PLoS ONE 8, e58520. doi:10.1371/journal.pone.0058520 Allan, B. J. M., Domenici, P., Munday, P. L. and McCormick, M. I. (2015). Feeling However, it is possible that attachment could cause the release of the heat: the effect of acute temperature changes on predator–prey interactions in cortisol via nociceptive system hormones (for review, see Galhardo coral reef fish. Conserv. Physiol. 3, cov011. doi:10.1093/conphys/cov011 and Oliveira, 2009). By contrast, it is possible that attachment could Allan, B. J. M., Domenici, P., Watson, S. A., Munday, P. L. and McCormick, M. I. – trigger an immune response with a resulting increase in cortisol. The (2017). Warming has a greater effect than elevated CO2 on predator prey immune system and the release of glucocorticoids are tightly interactions in coral reef fish. Proc. R. Soc. B Biol. Sci. 284, 20170784. doi:10. 1098/rspb.2017.0784 coupled. Glucocorticoids have a strong anti-inflammatory effect Almany, G. R. and Webster, M. S. (2006). The predation gauntlet: early post- and can induce relevant changes in immune cells as well as settlement mortality in reef fishes. Coral Reefs 25, 19-22. doi:10.1007/s00338- cytokines having the power to stimulate cortisol production (Wikel 005-0044-y Journal of Experimental Biology

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