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Thermal Preferences and Critical TemperatureRegimes of the Western North Atlantic InvasiveLionfish Complex (Pterois spp.)Benjamin BarkerNova Southeastern University, barker.ben294@gmail.com

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Recommended CitationBenjamin Barker. 2015. Thermal Preferences and Critical Temperature Regimes of the Western North Atlantic Invasive Lionfish Complex(Pterois spp.). Master's thesis. Nova Southeastern University. Retrieved from NSUWorks, Oceanographic Center. (385)http://nsuworks.nova.edu/occ_stuetd/385.

Nova Southeastern University

Halmos College of Natural Sciences

and Oceanography

Thermal preferences and critical temperature regimes of the western North Atlantic

invasive lionfish complex (Pterois spp.)

By

Benjamin D. Barker

Submitted to the Faculty of

Nova Southeastern University

Halmos College of Natural Sciences and Oceanography

in partial fulfillment for the requirements for

the degree of Master of Science with a specialty in:

Marine Biology

Nova Southeastern University

July 2015

Thesis of

Benjamin D. Barker

Submitted in Partial Fulfillment of the Requirements for the Degree of

Masters of Science:

Marine Biology

Nova Southeastern University

Halmos College of Natural Sciences and Oceanography

July 2015

Approved:

Thesis Committee

Major Professor: _______________________________

David W. Kerstetter, Ph.D.

Committee Member: ____________________________

Andrij Z. Horodysky, Ph.D.

Department of Marine Science and Environmental Science

Hampton University

Committee Member: ____________________________

James D. Thomas, Ph.D.

I

Acknowledgements

This research project was a collaboration between Nova Southeastern University’s

Halmos College of Natural Sciences and Oceanography and Hampton University. Thanks

to both of these institutions for providing the laboratory space and equipment to complete

this study. A President’s Faculty Research and Development Grant (PFRDG) provided

funding for this project. Thank you to Jesse Secord, Adam Nardelli, and the rest of the

NSU OC Fisheries Lab for assistance in setting up the laboratory, maintenance, and

monitoring of experiments. Thank you to everyone who offered assistance in collection

of fish subjects. A special thanks to Carlos and Allison Estapé and those who participated

in REEF Lionfish Derbies for help acquiring many of the lionfish subjects.

Thank you to my committee, Drs. David Kerstetter, Andrij Horodysky, and James

Thomas for their hard work and guidance with the project. It has been a pleasure to work

with such a supportive and knowledgeable group. The things I’ve learned over the past

few years will undoubtedly carry on with me for the rest of my professional career.

Thank you to all of the friends I’ve made at NSU for making these past three years as

enjoyable as possible. Finally, thank you to my family for all the support they’ve lent me

over the years. Completing this degree would not have been possible without all of you.

II

Abstract

Temperature preference, behavioral tolerance, and physiological tolerances were

determined for locally captured, invasive juvenile lionfish at four different acclimation

temperatures (13°C, 20°C, 25 °C and 32°C). Temperature preferences and avoidance

temperatures were evaluated using an automated shuttlebox system that presents subject-

driven temperature stimuli to subjects, who control the temperature with their movement

throughout the tank for 12 hours. Subjects are tracked by a computer system, with data

output approximately every second. Acute preference was calculated from the archived

data as the mean temperature that the fish occupied during the first two hours of dynamic

experimentation. Acute preference measurements were used to determine final

temperature preferendum and avoidance temperatures were used to determine behavioral

tolerance. Critical thermal methodology (CTM) determined the CTmin and CTmax of the

lionfish with loss of equilibrium (LOE) as the endpoint. It is assumed that beyond this

temperature, the fish would be unable to survive. Temperature was increased or

decreased by 0.33°C per minute until the end point was reached. Thermal tolerance

polygons provide a visual representation of the lower and upper thermal avoidance

temperatures, delineating the thermal range of the species. Their CTmin and CTmax

(acclimated to 25°C) were compared experimentally with two other Florida reef fish

species (Cephalopholis cruentata and Lutjanus apodus). Acute preferences of juvenile

invasive lionfish showed a final preferendum at 28.7 ± 1°C, but with no significant

difference between acclimation temperatures. The thermal tolerance polygon of invasive

lionfish shows a strong correlation between CTM and acclimation temperature, with the

highest CTmax at 39.5°C and the lowest CTmin at 9.5°C. The thermal polygon, preference,

and avoidance data describes the thermal niche of the lionfish. Lionfish CTM (24.61°C)

is narrower than those of C. cruentata (25.25°C) and L. apodus (26.87°C).

Keywords: Pterois volitans, lionfish, invasive, preference, thermal tolerance, shuttlebox,

CTM

III

Table of Contents

Acknowledgements ............................................................................................................ I

Abstract ............................................................................................................................. II

List of Figures .................................................................................................................. IV

List of Tables ................................................................................................................... IV

Introduction ....................................................................................................................... 1

Background ................................................................................................................... 1

Invasive Lionfish ......................................................................................................... 2

Temperature Preference ............................................................................................... 5

Behavioral Tolerance ................................................................................................... 7

Physiological Tolerance ............................................................................................... 9

Materials and Methods ................................................................................................... 13

Collection and Husbandry .......................................................................................... 13

Electronic Shuttlebox: Preference and Behavioral Tolerance ................................. 14

Critical Thermal Methodology (CTM): Physiological Tolerance ............................ 19

Results .............................................................................................................................. 23

Discussion......................................................................................................................... 30

Temperature Preference ............................................................................................. 30

Behavioral Tolerance ................................................................................................. 31

Physiological Tolerance ............................................................................................. 32

Thermal Niche ............................................................................................................ 38

Conclusion ....................................................................................................................... 40

References ........................................................................................................................ 41

IV

List Of Tables

Table 1. Lionfish collection log ...................................................................................... 15

Table 2. ANOVA summary for acclimation vs. temperature preferences ................ 24

Table 3. ANOVA summary for acclimation vs. upper avoidance .............................. 24

Table 4. ANOVA summary for acclimation vs. lower avoidance ............................... 24

Table 5. ANOVA summary for acclimation vs. CTmax ................................................ 24

Table 6. ANOVA summary for acclimation vs. CTmin ................................................ 24

Table 7. ANOVA summary for species vs. CTmax ........................................................ 25

Table 8. ANOVA summary for species vs. CTmin ........................................................ 25

Table 9. Comparisons of total polygonal area (ºC2)..................................................... 36

List of Figures

Figure 1. Zone of Final Preferendum .............................................................................. 8

Figure 2. Diagram of electronic shuttlebox system ...................................................... 16

Figure 3. Photo of electronic shuttlebox system ........................................................... 17

Figure 4. Zone of Final Preferendum ............................................................................ 20

Figure 5. Thermal Polygon for common goby.............................................................. 22

Figure 6. Lionfish Thermal Preference and Zone of Final Preferendum .................. 26

Figure 7. Lionfish Avoidance Temperature ................................................................. 27

Figure 8. Lionfish Thermal Polygon ............................................................................. 28

Figure 9. Species CTM Comparison ............................................................................. 29

Figure 10. Behavioral Tolerance Range........................................................................ 33

Figure 11. Thermal Tolerance Comparison ................................................................. 34

Figure 12. Lionfish Thermal Niche ............................................................................... 39

1

Introduction

Humans have been responsible for the introduction of many exotic species into

new areas. Many of these introductions will go unnoticed, but some have the potential to

impose direct impacts on the existing ecosystems, human health, or human activity.

Exotic species may thrive when introduced because their fitness is increased in new areas

compared with their native habitat, potentially due to reduced predation, parasitism,

competition, and/or increased food abundance (Kimbro et al. 2009; Mooney and Cleland

2001; Torchin et al. 2003). Unfortunately, exotic species can have an adverse affect on

the existing ecosystem by outcompeting native species. If an exotic species threatens the

native ecosystem, it is then considered invasive. Understanding invasive species is the

first step in minimizing undesirable impacts.

Invasive species can change how existing ecosystems are structured and often

negatively affect commercial, agricultural or recreational activities in the process

(Pimentel et al. 2005). One example is the introduction of lionfish Pterois spp. into

tropical Atlantic waters. Since their establishment, lionfish have changed the structure of

the tropical Atlantic reef ecosystem, impacted commercial and recreational fisheries, and

have received a great deal of attention from scientists, managers, and the general public

(Morris and Whitfield 2009). The invasive lionfish is quickly becoming one of the most

impactful marine invasive species to date and will continue to affect the tropical Atlantic

marine ecosystem (Morris and Whitfield 2009).

Background

The phylogeny of the invasive lionfish complex in the Atlantic and Caribbean

remains uncertain. Most literature describes the invasive lionfish complex in the Atlantic

and Caribbean to be composed of two species, Pterois volitans [Linnaeus 1758; “red

lionfish”] and Pterois miles [Bennett 1828; “common lionfish”] (Hamner et al. 2007;

Kochzius et al. 2003). However, recent studies have shown that P. volitans may instead

be a hybrid of other Pterois species (specifically, P. miles and P. lunulata/russelii), rather

than a distinct species (Wilcox 2014). Species of Pterois are members of the family

Scorpaenidae, which includes other species of lionfish, stonefish, and scorpionfish. The

2

maximum size of the invasive lionfish within the literature is 48-49 cm; however,

individuals reach sexual maturity at approximately 10 cm (Darling et al. 2011; Jud and

Layman 2012). Invasive lionfish in the western North Atlantic are also much larger

compared to individuals from their native ranges (Darling et al. 2011), which is known to

also occur in other invasive species (e.g. invasive plants; Blossey and Notzold 1995).

Invasive lionfish coloration is typically alternating white and reddish vertical bands on

the body with spots on their soft fins. They have 13 dorsal spines, 2-4 pelvic spines, and

2-3 anal spines, all containing an acetycholine-based venom. Lionfish also display a

number of soft fin rays, including their fan-like pectoral fins. Their heads are usually

covered with soft tentacles (cirri), which change shape and size throughout their life.

Invasive lionfish are highly adaptable, since they inhabit many different habitats,

utilize different feeding strategies, and prey upon a wide range of fish and invertebrates.

Invasive lionfish have been documented not just on coral reefs, but also in nearly all

habitats that provide structure. This list includes artificial habitats, deep habitats,

mangroves, sea grasses, and estuaries (Barbour et al. 2010; Jud et al. 2011). Lionfish are

known to use multiple feeding strategies. They can ambush, stalk, and corner their prey

with their large pectoral fins, or they can stir up sand and capture fleeting invertebrates

(Morris and Akins 2009). Although lionfish are mainly piscivorous as adults, they do

consume more crustaceans as juveniles, suggesting an ontogenetic diet shift occurring

over their life span (Dahl and Patterson 2014; Jasper 2013). Feeding usually occurs

during the crepuscular times when they are typically more active, but they have been

shown to eat mid-day as well (Morris and Akins 2009). Predation upon lionfish is mostly

unknown in both their native and invasive range. However, there are reports of the

Pacific cornetfish Fistularia commersoni feeding on lionfish in their native habitat

(Robins 2014). Lionfish have also been found in the stomachs of grouper species in their

invasive range (Robins 2014).

Invasive Lionfish

Confirming the mechanism for introduction of invasive lionfish into North

Atlantic waters is nearly impossible. However, deliberate aquarium release is widely

considered the most likely mode due to the similarity between invasive specimens and

3

lionfish from Indonesia and the Philippines (Hare and Whitfield 2003), a region from

which roughly 85% of marine aquarium fishes imported into the United States are

collected (Whitfield et al. 2002). The first sighting of lionfish in the wild within the

western North Atlantic occurred in 1985, when one individual was captured in a lobster

trap off of Dania Beach, Florida (Schofield 2009). Subsequent reported lionfish sightings

were relatively scarce until 2000, when lionfish spread up the U.S. Atlantic coast and to

Bermuda. In 2004, lionfish reached the Bahamas (Freshwater et al. 2009). Lionfish have

now spread to the rest of the Caribbean islands (Schofield 2009), the coasts of Central

America (USGS-NAS 2015), Florida Keys (Ruttenberg et al. 2012), and the Gulf of

Mexico (Aguilar-Perera and Tuz-Sulub 2010). More recently, Ferriera et al. (2015)

reported a confirmed lionfish capture in Brazil, south of the Amazon River outflow.

Nucleotide and haplotype diversity for lionfish throughout the invasive range are

moderate to low, suggesting that the now-extensive lionfish invasion originated from a

very small founder population (Butterfield et al. 2015; Hamner 2007).

The proliferation of invasive species is typically aided by concepts within the

enemy release hypothesis (ERH), which states that introduced species flourish in new

surroundings due to the reduction of “enemies” (Keane and Crawley 2002). In the case of

the invasive lionfish, this includes release from both predators and parasites. Lionfish are

equipped with 18 venomous spines that deter predators (Morris 2009). The acetycholine-

based venom causes sufficient pain and localized swelling, but is non-lethal to humans if

treated (Cohen and Olek 1989). Venomous spines, unfamiliarity to native predators, and

aposematic coloration and form are the most probable reasons that lionfish have no

known regular predators in their invaded range (Morris 2009). Grouper, sharks, and eels

are considered the most likely candidates to become consistent predators of lionfish, but

none of these species groups have been documented as regularly feeding upon them.

Only on rare occurrences have there been reports of predation on uninjured lionfish;

however, some groups believe that feeding native predators injured or dead lionfish will

help them “acquire a taste” (Diller et al. 2014; Pimiento et al. 2012). In addition to this

being dangerous for lionfish cullers, there is no indication that this method has been

successful, yet most dive operators now condone the practice. Another competitive

advantage that invasive species have over native reef fishes is the scarcity of parasites.

4

Invasive lionfish have fewer parasites in the western North Atlantic as compared to

native fishes in the same range (Sikkel et al. 2014; Simmons 2014).

Invasive lionfish have rapidly spread throughout the tropical Western Atlantic

because of their reproductive successes. They are gonochoristic batch spawners, in which

the female releases two buoyant egg masses, which are externally fertilized by the male

(Morris 2009; Morris et al. 2011). This reproductive strategy allows lionfish to reproduce

throughout the year, as long as conditions are suitable. They are estimated to spawn every

four days throughout the year, possibly assisting their rapid spread throughout the

Caribbean region (Morris 2009). Studies examining lionfish fecundity show averages

from about 24,000 to 75,000 eggs per female (Morris 2009; Priyadharsini et al. 2013),

which is also dependent on individual size. Lionfish are also known mature very quickly:

males can be sexually mature at just 100 mm TL, while females can be mature at 175 mm

TL (Morris 2009).

Furthermore, invasive lionfish have been able to out-compete native fish for both

food and space (Albins 2013, Albins and Hixon 2008; Green et al. 2012). In their invaded

range, lionfish are known to exist in higher densities than in their native range (Darling et

al. 2011). In Kenya and the Red Sea, for example, densities of 25 and 80 lionfish per

hectare were reported, respectively (Darling et al. 2011; Fishelson 1997). In contrast,

natural reef sites from North Carolina, Bahamas, and northern Gulf of Mexico have

shown densities of 150, 393, and 49 lionfish per hectare, respectively (Morris and

Whitfield 2009; Green and Côté 2009; Dahl and Patterson 2011). While densities were

lower on natural Gulf of Mexico sites, they also reported densities of 1470 lionfish per

hectare on their artificial sites (Dahl and Patterson 2011). According to recent studies in

the Bahamas, lionfish have become 40% of the predatory fish biomass on their reef

ecosystems; in turn, they have also reduced their prey fish biomass by an average of 65%

(Green and Côté 2009; Green et al. 2012). Another study comparing predatory effects of

lionfish versus the native coney Cephalopholis fulva on native prey fishes shows that the

presence of lionfish negatively affects fish biomass, while the presence of coney did not

have nearly the same affect (Albins 2013).

Lionfish are known to be voracious consumers in their invasive range, where their

diet mainly consists of small reef fish and occasionally small invertebrates. They are

5

generalist predators, and consume about 40-70 different teleost species (Morris and Akins

2009; Muñoz et al. 2011). The primary teleost prey items depend largely on the local

abundance because lionfish exhibit high site fidelity and generalist nature, and thus

changes from area to area (Jasper 2013; Layman and Allgeier 2012; Morris and Akins

2009; Muñoz et al. 2011). The small-bodied species consumed by lionfish are also typical

prey for the economically important competitors, such as large-bodied serranids and

lutjanids (Morris and Akins 2009). This competition for food can create over-lapping

niches and potentially decrease prey availability for native predators. Invasive lionfish are

likely out-competing these native large-bodied predators (Albins 2013; Green et al.

2012). The effect of lionfish presence on economically important species is increasingly

important from a management perspective, though lionfish rarely prey directly on these

species (Morris and Akins 2009).

Temperature Preference

Cellular processes that occur within an organism are influenced by temperature

(Evans and Claiborne 2006). Since lionfish are poikilothermic, their bodily processes are

dependent on the surrounding water temperature. If temperatures change, the individual

fish must either make adjustments internally (“acclimation”) or seek out more suitable

conditions externally (“behavioral thermoregulation”; see Reynolds and Casterlin 1980).

Internally, the fish will involuntarily adjust biochemical processes at a cellular

level to adapt to temperature changes via mechanisms such as homeoviscous adaptation

(HVA) and homeophasic adaptation (HPA) (Evans and Claiborne 2006; Hazel 1995).

These cellular-level responses are key to maintaining organismal equilibrium and

functionality. Another means of responding to temperature changes is behavioral

thermoregulation. Poikilothermic animals accomplish thermoregulation behaviorally by

physically moving within an available temperature gradient to the most ideal temperature

available (Reynolds and Casterlin 1979). In nature, individual fish utilize a combination

of acclimation and behavioral thermoregulation to adjust to the changing environment.

Closely related to behavioral thermoregulation is the temperature preference of

fish. Reynolds and Casterlin (1979) described temperature preference, “When presented a

choice of ambient temperatures, as in a thermal gradient, motile organisms tend to

6

congregate in, or spend the most time in, a relatively narrow range of temperatures.”

There are two different concepts of temperature preference, final preferendum and acute

preferendum. Final preferendum describes the temperatures at which the fish will

aggregate, regardless of prior acclimation. This final preferendum temperature is thought

to be species-specific and ideal to their physiological performance (Fry 1947; Reynolds

and Casterlin 1979). Alternatively, acute preference is simply the range of temperatures

at which fish congregate while under the influence of their prior acclimation temperature.

Behavioral thermoregulation has been measured in fishes with several methods.

The oldest experimental designs used gradients to measure temperature preference. There

are many variations of gradient tanks, from chambered devices to continuous vertical or

horizontal gradients (McCauley 1977; Shelford and Allee 1913; Sullivan and Fisher

1953). In these experiments, individuals are presented with either chambers of different

water temperatures or a gradient of water temperatures in a single tank. The fish are

allowed to gravitate to their preferred temperature (McCauley 1977). Gradient methods

often encountered problems with supersaturation of gases, maintenance of temperature

gradients and other design flaws that influenced fish behavior (McCauley 1977). Another

design is the use of shuttlebox methods. These designs have conditioned fish to perform a

specific task to regulate their own temperature (McCauley 1977). Early designs required

fish to press against a lever to control water temperature (Rozin and Mayer 1961). Neill

(1972) introduced an apparatus called the “electronic shuttlebox”, which “allows fishes,

by their spatial movement, to regulate the temperature in experimental tanks.” Since its

first application, it has been used in a number of studies to measure preferences in

temperature, salinity, and water turbidity (among other environmental variables; Meager

and Utne-Palm 2008; Serrano and Serafy 2010).

Thermal preference is measured because it gives insight into the physiology and

ecology of an organism. Regarding lionfish, acute preference measurements could

correlate to field observations of distributions. Lionfish may be more likely to congregate

in areas where water temperatures are within their Zone of Final Preferendum (Figure 1).

This has been demonstrated in freshwater fishes and could be relevant to marine species

as well (Cincotta and Stauffer 1984). Also, final preferendum has been linked to the

temperature for optimal metabolic scope and optimal growth in fishes (Jobling 1981;

7

Kelsch and Neill 1990). Metabolic scope, or the difference of oxygen uptake between a

resting and active individual, can be maximized at a specific temperature (Kelsch and

Neill 1990). In other words, an individual resting at this temperature is most efficiently

utilizing their energy for metabolic demand, allowing for the surplus of energy to be

applied to growth and reproduction. Jobling et al. (1981) supports this by showing that

final preferendum in fishes is also correlated to the temperature for optimal growth in

fishes, defined as the temperature at which the growth rate is highest under conditions of

maximum feeding. In addition to metabolic scope, these temperatures are optimal for

other biochemical and physiological processes in ectotherms, such as digestion, enzyme

activity, or swimming speed (Hutchinson and Maness 1979; Kelsch and Neill 1990).

Behavioral Tolerance

The avoidance temperatures of an individual determine their behavioral tolerance

range. Avoidance temperatures are measured because they quantify when a fish will

likely move in the wild as a result of temperature. When temperatures become too warm

or too cold for an individual, they instinctively seek out more favorable conditions or

endure in their current location. Past conventional tagging studies have shown that

lionfish demonstrate high site fidelity, but there is still evidence that lionfish may move

substantial distances (Akins et al. 2014; Jud and Layman 2012). The reasons for lionfish

movement in the wild are still unclear. Temperature may be a contributing factor to

lionfish movement in certain habitats or regions. For example, lionfish avoidance

temperatures would be increasingly relevant in areas of frequent and drastic temperature

fluctuations, such as estuaries. Also, lionfish living near the edge of their thermal range

would encounter extreme temperatures seasonally. Lionfish living in these regions, such

as in North Carolina waters, may need to evade live threatening temperatures during

winter months.

8

Figure 1. Diagram of thermal niche. Line “AP” is the regression line for the acute thermal

preference, while line “LE” is the line of equality. The Zone of Final Preferendum is

where these two lines intersect. Additional measurements in Jobling (1981) are critical

thermal maximum as “CTM”, upper incipient lethal temperature as “UILT”, ultimate

upper incipient lethal temperature as “UUILT”, and lower incipient lethal temperature as

“LILT” (Figure from Jobling et al. 1981).

9

Physiological Tolerance

Fish not only have optimal temperatures for bodily processes, but also thermal

limits at which those processes degrade or cease to function at all. Fish struggle at these

extreme temperatures because of the effect temperature has on cellular proteins and

enzymes. As temperature changes within a given range, proteins and enzymes needed for

metabolism and normal cell function change shape or function. When temperatures near

extremes, proteins and enzymes have difficulty performing their metabolic function,

limiting the ability of the fish to survive. Heat shock proteins are generated within the

cells in an attempt to maintain the regularity of cellular proteins and enzymes, so the

individual fish may continue to function or survive until conditions return to a livable

state (Roberts et al. 2010). At this point, the fish is exceeding its thermal limit and may

not be able to maintain equilibrium or may experience muscle spasms (Lutterschmidt and

Hutchinson 1997).

Critical thermal methodology (CTM) has been used for a number of decades as a

method for measuring thermal tolerance in fishes. In a CTM experiment, temperature is

increased or decreased from the acclimation temperature at a constant rate until a

predetermined endpoint (Becker and Genoway 1979; Cox et al. 1974; Hutchinson 1961).

This chosen endpoint is considered to be the physiological state where the fish would not

be able to avoid death, should the temperature remain stable. At these extreme

temperatures, their locomotory movements should be so disorganized that they would not

be able to behaviorally thermoregulate or move to a more suitable environment (Cox et

al. 1974). The most commonly used endpoints are loss of equilibrium (LOE) or the onset

of muscle spasms (OS) (Beitinger et al. 2000; Lutterschmidt and Hutchinson 1997).

The one aspect of critical thermal methodology that receives the most scrutiny

among fish physiologists would be the rate at which the temperature changes. The rates

range from 1°C per minute to 1°C per hour, with the former being most common in the

literature (Becker and Genoway 1979). Although there has been much debate over the

proper rate, the ideal CTM rate would be one that is slow enough that the fish’s body

temperature does not lag behind the water temperature, but is fast enough that the fish is

not able to acclimate to the temperature changes (Beitinger et al. 2000). Becker and

Genoway (1979) suggested the use of a rate of 0.3 °C per minute, based on a literature

10

review of past CTM studies and their own rate comparison study. They found that faster

rates of temperature change, such as 1°C per minute, could result in heat shock or a lag of

the fish’s body temperature behind the water temperature (Becker and Genoway 1979).

In addition, it was difficult to accurately measure LOE and death at this rate. Becker and

Genoway (1979) also demonstrated that rates of temperature change slower than 0.3°C

per minute allowed fish to acclimate throughout the experiment. The 0.3 °C per minute

rate has been frequently used for CTM experimentations in the subsequent literature, but

other rates are still being employed as well. As examples, 0.3°C per minute was used in

Currie et al. (1998) and Debnath et al. (2006), 1°C per minute was used in Rajaguru

(2002), and 1°C per hour was used in Mora and Ospina (2001).

Critical thermal methodology offers insights into the ecological role of a species.

Primarily, CTM describes the temperature at which fish of a certain species would not be

able to survive. Therefore, when water reaches these high or low temperatures, it can be

assumed that any fish exposed to this water would soon die or must move to more

suitable temperatures (Hutchinson 1961). Temperature-related fish kills normally involve

a closed environment, such as a river or lake, where the fish is unable to escape.

However, in marine waters, fish kills related to temperature changes are almost

exclusively caused by rapid onset cold-water events (Beitinger et al. 2000). This could be

caused by either their ability to adjust to warmer temperatures more quickly or because

cold water causes lethargic behavior, making it more difficult to move to warmer

temperatures or increasing susceptibility to predation (Beitinger et al. 2000).

In addition to addressing ecological role questions for a single species, CTM is

also frequently used as a tool for comparisons of thermal tolerance between species.

Thermal tolerance is displayed using a thermal tolerance polygon, which encompasses

the entire inhabitable thermal range of the species (Eme and Bennett 2009). It not only

displays the CTmin and CTmax at different acclimation temperatures, but also quantifies the

eurythermic potential of a species by calculating the area within the polygon (Eme and

Bennett 2009). The dimensions of the thermal polygons are constructed by connecting

the CLmin and CLmax of the species with the CTmin and CTmax regression (Eme and

Bennett 2009).

11

However, the ecological relevance of CTM data has been debated since its

introduction. Some critics note that conditions simulated by CTM experiments are rarely

experienced by wild organisms and thus unproven (for example, see discussion in

Lutterschmidt and Hutchinson 1997). This may be particularly true for organisms that

generally are not physically “confined” to an area, such as marine fishes. However, for

species that are able to live near their thermal extremes, there are indeed encounters with

these life-threatening temperatures. The lionfish is a prime example of this type of

species, as studies have shown that individuals in the invasive range live extremely close

to their thermal limit (Kimball et al. 2004; Whitfield et al. 2014).

A trend among many tropical species (aquatic, marine, and terrestrial) is that they

generally live closer to their thermal maximum and have a narrow thermal niche in

comparison to temperate species (Tewksbury et al. 2008). Both of these characteristics

stem from tropical climates being relatively stable in temperature year-round, or

aseasonal (Madeira et al. 2012; Tewksbury et al. 2008). The species adapted to an

aseasonal climate will be less suited for changing temperatures because their bodies are

not internally prepared to adjust to them, compared to temperate species. Temperate

species experience large changes of temperature every year, and thus have a wider

thermal niche (Tewksbury et al. 2008). Aseasonal climates also allow tropical species to

live closer to their thermal maximum, while rarely encountering temperatures above it.

Thus, there is no need to be able to adapt to them (Tewksbury et al. 2008).

Few studies have examined the ecological effect of temperature on lionfish. In a

study, conducted off North Carolina, researchers examined the transition of fish

assemblages and lionfish abundance with depth (Whitfield et al. 2014). They found

lionfish abundance increased with depth (most abundant at 38-46 m), as these depths

maintained a winter mean above 15.3 °C and the shallower depths are too cold to

overwinter. This study also suggested that increased ocean temperatures due to climate

change might allow lionfish to overwinter in shallower water. The potential effects of

climate change were also examined by Côté and Green (2012), who concluded that the

lionfish larval duration would shorten, leading to an increase in the local retention of the

individuals. Côté and Green (2012) also determined that lionfish predation rates on native

fishes would increase in response to an increased metabolic rate.

12

In the western Atlantic, lionfish are currently established year-round from Cape

Hatteras, NC through the southern Caribbean. North of Cape Hatteras, juvenile

individuals are found seasonally in coastal habitats, which presumably die off during the

winter months (Kimball et al. 2004). Since lionfish are a tropical/sub-tropical species,

they probably only encounter temperatures approaching their thermal maximum in rare

circumstances. Kimball et al. (2004) was the first to examine the effects of temperature

tolerance on the invasive range of the lionfish. It investigated their chronic lethal

minimum (CLmin) to discuss the possible limits of their invasive range, reporting a CLmin

of 10.7 °C and cessation of feeding at 15.3 °C. Kimball et al. (2004) hypothesized that

lionfish were limited by their inability to overwinter at temperatures colder than 12 °C.

With this threshold, lionfish are able to overwinter as far North as Cape Hatteras, but

only in deeper water. However, throughout Florida and the Gulf of Mexico, lionfish are

able to overwinter at shallower depths. Kimball et al. (2004) also tested the effects of

acclimation temperature and the rate of temperature change on their CLmin, showing no

difference between rates of temperature change (1 °C per day, 2 °C per day, and 3 °C per

day) or acclimation temperatures (15 °C, 20 °C, and 25°C).

Understanding the relationships between physiology, behavior, and ecology

provide additional insights into ecological problems, and provide the background for

implementing the correct management techniques. This study aims to investigate the

physiology and behavior of the invasive lionfish by describing their thermal niche. It

defines three important biological parameters; temperature preference, behavioral

tolerance, and physiological tolerance. Temperature preference data is used to determine

final preferendum, which describes the temperature for optimal metabolic scope.

Avoidance temperatures determine behavioral tolerance. These temperatures represent

when an individual will move to seek out better conditions as a result of warming or

cooling temperatures. CTM determines physiological tolerance, which are the

temperatures that result in the death of the fish. The results serve as a platform for future

research investigating the effect of temperature on this invasive species.

Acute preferences of lionfish were measured at different acclimation temperatures

(20 °C, 25 °C and 32 °C). These acute preference measurements were used to graphically

determine their final preferendum (Figure 1). In addition, differences in temperature

13

preference at different acclimation temperatures were tested. For poikilotherm fishes,

acute preference is measured during the first two hours after being presented with a

thermal gradient. During this time, their preference is under the influence of their

acclimation temperature (Reynolds and Casterlin 1979). Therefore, we would expect to

see differences in preference between lionfish acclimated to different temperatures.

Avoidance temperatures were measured at three different acclimation temperatures (20

°C, 25 °C and 32 °C). Regression lines were developed from these measurements, and

between these lines will be their behavioral tolerance range. Differences between

avoidance temperatures at different acclimation temperatures were also tested. Lionfish

were expected to display higher avoidance temperatures, as acclimation increases

(Reynolds and Casterlin 1979). Expanding upon Kimball et al. (2004), CTmin and CTmax

temperatures were recorded at different acclimation temperatures (13 °C, 20 °C, 25 °C

and 32 °C) to establish the complete physiological tolerance range of the invasive

lionfish. Differences in physiological tolerance in response to acclimation temperature

were tested. According to Kimball et al. 2004, there were no significant differences in

CLmin between different acclimation temperatures. The same is to be expected for CTmin

and CTmax. To investigate how lionfish thermal tolerance compares to other tropical

species, this study also measured the CTmin and CTmax for two other co-occurring tropical

predatory fishes: schoolmaster snapper Lutjanus apodus and graysby grouper

Cephalopholis cruentata. Eme and Bennett (2009) show similar temperature tolerance

between reef-associated marine tropical fish species. Since the three species are primarily

tropical, there should be no significant difference between their CTmin and CTmax.

Materials and Methods

Collection and Husbandry

Forty-two juvenile lionfish (87 mm-121 mm total length) were collected by

SCUBA throughout southeast Florida and the Florida Keys. Hand nets were used for

capture, and the individual lionfish are then transferred to a dry bag for the remainder of

the dive. Fish were then taken back to the holding tanks at the Nova Southeastern

University (NSU) Guy Harvey Oceanographic Center as soon as possible. About half

14

were collected by a team of Oceanographic Center graduate students and REEF (Reef

Environmental Education Foundation) volunteers, diving underneath bridge pilings in the

Florida Keys. Participants in REEF Lionfish Derbies located in Ft. Lauderdale, West

Palm Beach, and Key Largo collected the other individual lionfish. A small number (n=4)

were also purchased from “Carib Fish & More, Inc.” in Pompano Beach (these were

caught by commercial collectors off the Broward County reef tract). A list of the

individuals collected by location, size, and date is included as Table 1.

The fish were acclimated to four different temperatures before undergoing

experimentation. Three large plastic water tanks were utilized for acclimation, each

containing around 60 gallons of salt water, allowing for around 10 fish per tank at a time.

Salinity was maintained between 32-36 ppt via occasional water changes and vigorous

aeration ensured near saturation oxygen levels. UV sterilizers and biofiltration sustained

optimal water quality. The acclimation temperatures were 13ºC, 20ºC, 25ºC, and 32ºC,

chosen to represent typical water temperatures in New England, North Carolina, Florida,

and the southern Caribbean region, respectively. Ideally, these temperatures were chosen

for acclimating lionfish near the boundaries of their thermal range, but still are reasonably

found in nature. The fish were acclimated to their respective temperatures 2-4 weeks

before experimentation (Chung 1995; Segnini et al. 1993). They were fed ad libitum the

same diet of feeder guppies Poecilia reticulate, goldfish Carassius auratus auratus, or

frozen silversides from local pet stores over the course of their holding and acclimation.

Electronic Shuttlebox: Preference and Behavioral Tolerance

The experimental shuttlebox tank (Figures 2 and 3) included two circular

chambers with a passage between them, allowing the fish to move freely between the two

chambers. Infrared (IR) lights shined from directly underneath the tank, which was

detected by an IR-sensitive camera above the tank. The fish creates a “shadow,” blocking

the infrared light and allowing the computer to monitor the position of the fish via the

contrast difference between dark shadow and the well-lit background of the tank.

Temperature fluctuation was controlled by a computerized system, which pumps cold or

warm water into the tank, depending on the position of the fish.

15

Fish ID # Collection location Collection Date Acclimation temp TL (mm)

LF100 Fort Lauderdale 7/27/13 25 132

LF99 Fort Lauderdale 7/27/13 25 104

LF97 Islamorada 7/27/13 25 138

LF96 Islamorada 7/27/13 25 129

LF95 Islamorada 8/13/13 20 162

LF94 Islamorada 8/13/13 20 154

LF93 West Palm Beach 8/17/13 25 132

LF92 West Palm Beach 8/17/13 25 89

LF91 Islamorada 8/13/13 25 106

LF90 Islamorada 8/13/13 25 109

LF89 Islamorada 8/13/13 25 116

LF88 Islamorada 8/13/13 20 117

LF87 Islamorada 8/13/13 20 120

LF86 Islamorada 8/13/13 20 104

LF85 West Palm Beach 8/17/13 20 123

LF84 Key Largo 9/14/13 20 122

LF83 Key Largo 9/14/13 20 151

LF82 Key Largo 9/14/13 20 156

LF81 Key Largo 9/14/13 20 87

LF79 Islamorada 9/17/13 13 97

LF78 Key Largo 9/14/13 13 137

LF77 Key Largo 9/14/13 13 169

LF76 Key Largo 9/14/13 13 128

LF75 Key Largo 9/14/13 32 152

LF74 Key Largo 9/14/13 32 148

LF73 Key Largo 9/14/13 32 131

LF72 Key Largo 9/14/13 32 104

LF70 Islamorada 9/17/13 32 141

LF69 Islamorada 9/17/13 32 163

LF68 West Palm Beach 11/23/13 32 137

LF67 West Palm Beach 11/23/13 32 117

LF66 Ft. Lauderdale

25 183

LF65 Ft. Lauderdale

20 201

LF64 Ft. Lauderdale

32 143

LF63 Ft. Lauderdale

20 161

LF60 Ft. Lauderdale

32 129

LF59 Ft. Lauderdale

32 114

LF58 Ft. Lauderdale

25 170

LF57 Ft. Lauderdale

25 153

LF56 Ft. Lauderdale

25 212

LF55 Ft. Lauderdale

25 137

LF54 Ft. Lauderdale

25 152

Table 1. List of 42 lionfish identification numbers, collection site, date of collection, and

acclimation temperature. Fish size is measured after completion of the experiments or

death.

16

Figure 2. A diagram of the electronic shuttlebox (Horodysky 2013). The experimental

“dumbbell-shaped tank” contains the specimen. The fish can move freely between the

“hot” or “cold” side of the experimental tank. The mixing towers allow for the dynamic

changes in temperature on each side of the experimental tank.

17

Figure 3. Photo of the shuttlebox as assembled at the NSU Oceanographic Center, circa

2013. Image includes the “dumbbell-shaped tank” and the mixing towers behind. Not

pictured are the infrared lights underneath the tank and the sensors above, the

chiller/heater systems, or the computer and control switches for the mixing tower flows

18

Lionfish were individually removed from their acclimation tanks for

experimentation. Each lionfish underwent two 12-hour phases of experimentation: the

first was the acclimation phase, then followed secondly by the dynamic phase. The

acclimation portion occurred over night and maintained two invariant temperatures,

separated by a temperature difference of Δ3 ºC between the two tanks. One side of the

tank was designated the “hot” side and the other is the “cold” side. During the

acclimation stage, the temperature of each side of the tank was set around the acclimation

temperature of the fish. Lionfish acclimated to 20 ºC were set to 18.5 and 21.5 ºC (Δ3

ºC), and those acclimated at 25 ºC were set to 23.5 and 26.5 ºC. Meanwhile, due to how

close the acclimation temperatures were to their thermal limits, lionfish acclimated to 13

ºC had tank temperatures set to 13 to 16 ºC (Δ3 ºC), and those acclimated to 32 ºC were

set to 29 to 32 ºC. This phase allowed the fish to sense and learn that there was a “hot”

and “cold” side of the tank, and that it could move freely between them. After 12 hours,

the acclimation phase ended, and the dynamic portion began.

The dynamic phase involved temperature changes in both sides of the tank in

response to the movement of the fish. When the fish was on the “hot” side of the tank,

temperature in each chamber continually increases, while still maintaining Δ3 ºC. While

the fish is on the “cold” side of the tank, temperatures continually decreased in the same

fashion. System-wide minimum and maximum temperatures were set at 10.7 ºC and 36.7

ºC, respectively. The lionfish undergo this experimentation for 12 hours or until the fish

lost equilibrium, although only the first two hours of dynamic data were used for the

acute preference measurement. Following experimentation in the shuttlebox, each

lionfish was tagged using small, plastic T-bar tags (Hallprint, Ltd.; Australia), allowing

for identification of individual fish for the remainder of experimentation.

.

19

For measurement of acute preferences, only the first two hours of dynamic data

were used from each lionfish shuttlebox experiment. According to Reynolds and

Casterlin (1979), preference measurements taken during this time frame are strongly

influenced by prior acclimation temperatures. Temperature was recorded roughly every

second based on where the lionfish was located within the experimental tank. Avoidance

temperatures were determined as the temperature when the subject switched sides. These

temperatures were taken during the same two-hour dynamic phase.

Analysis of the acute preference data was conducted by measuring the mean of all

the temperature values that each lionfish occupied during the experiment to get a single

value. A nonlinear second polynomial (quadratic) line was fit to these data. Using the

graphical process employed by both Reynolds and Casterlin (1979) and Jobling (1981),

the zone of final preferendum was determined as the point along the acute preference line

that intersects with the line of equality (LE; Figure 4). The line of equality represents the

point where acclimation temperature is equal to the acute preferendum (Cincotta and

Stauffer 1984; Jobling 1981). Linear regression lines were also fit for the upper and lower

avoidance temperatures. These data were tested using one-way ANOVAs to determine

whether lionfish preference and avoidance were significantly different at each

acclimation temperatures.

Critical Thermal Methodology (CTM): Physiological Tolerance

Juvenile lionfish were collected in the same manner as previously mentioned.

Schoolmaster and graysby were captured live via hook and line from Broward county

reefs and estuaries. Air bladders of captured lionfish were vented, if necessary, to ensure

their survival. Lionfish were acclimated to four different temperatures (13ºC, 20ºC, 25ºC,

and 32ºC) before undergoing the experiment, while the other species were only

acclimated to 25ºC. The fish were acclimated to their respective temperatures 2-4 weeks

before experimentation, allowing for proper time to acclimate to their respective

temperatures (Chung 1995; Segnini et al. 1993). They were fed ad libitum a diet of live

feeder guppies, live goldfish, or frozen silversides Menidia menidia from local pet stores

over the course of their holding and acclimation.

20

Figure 4. The Zone of Final Preferendum was determined by constructing the diagram

above. It shows the relationship between acclimation temperature and preference

temperature for white sucker Catostomus commersonii. The curved line is the quadratic

model used to fit the plotted points and the straight line is the line of equality. They

intersect at 27.10 ºC. (Figure from Cincotta and Stauffer 1984).

21

Each fish was randomly selected for testing in either the acute CTmin or CTmax

trials. Half of the fish in each acclimation temperature underwent the CTmin trial, while

the other half underwent the CTmax trial. Each trial occurred in thermostatically controlled

plastic containers holding clean seawater. Water was continuously aerated and circulated

to maintain homogenous oxygen and temperature levels throughout the container.

Temperature began at the fish’s acclimation temperature and increased or decreased by

0.3 °C per minute, as suggested by Beitinger et al. (2000). Since the acute critical thermal

method (CTM) was being employed, the endpoint of the experiment was non-lethal. The

endpoint was defined as when the fish achieved a loss of equilibrium (hereafter, “LOE”;

i.e., it cannot maintain dorso-ventral orientation for 1 minute, per Beitinger et al. 2000).

Upper and lower thermal tolerance for each replicate treatment group were

calculated as the mean of the CTmin or CTmax trials per Eme and Bennett (2009);

similarly, the grand mean of the collective replicate endpoints are the CTmin or CTmax for

the temperature acclimation regime (Cox 1974). Thermal tolerance polygons were

constructed for lionfish by connecting the CTmin and CTmax with CTM regressions to

produce a quadrilateral figure (Figure 5). Similarly, these polygons demonstrated an

intrinsic tolerance zone (i.e., thermal tolerance independent of previous thermal

acclimation) and acquired tolerance zones (i.e., thermal tolerance gained through

acclimation) by dividing polygons with horizontal lines from extrapolated CTmin and

CTmax values at CTM limits. An additional thermal tolerance polygon was constructed

(per the methods of Eme and Bennett [2009]) for the purpose of comparing polygonal

area (ºC2). This additional polygon was formed by connecting CLmin (from Kimball

[2004]) and CLmax (estimated to be 35 °C) with the CTM regressions.

Data analysis was conducted using R software (version 3.1.2; R Development

Core Team 2014). Two separate ANOVAs were utilized to detect differences in mean

CTmin and CTmax values between lionfish acclimated to different temperatures. Normality

of residuals was tested using the Shapiro-Wilk test and homogeneity of variances was

tested using Bartlett’s test. ANOVAs were conducted to detect differences in mean CTmin

and CTmax values between the different fish species.

22

Figure 5. Example of a thermal polygon (figure from Eme and Bennett 2009). The top

and bottom lines represent the CTmax and CTmin regression lines for the common goby

Bathygobius fuscus. Black circles represent actual CTM measurements. White circles

represent the predicted CTM measurements at their highest and lowest theoretical

acclimation temperature, determined by the CLmin and CLmax.

23

Results

The second order polynomial model of the lionfish acute preference data is

displayed by the following equation: y = -0.0729x2 + 3.9981x - 26.007. The acute

preference quadratic line and the line of equality intersect at 28.7 °C, estimating 27.7-

29.7 °C (or 28.7 ± 1 °C) to be the zone of final preferendum for lionfish (Figure 6).

Statistical analysis with the ANOVA found there to be no significant difference in

temperature preference with acclimation temperature (p>0.05; Table 2).

Linear regressions for upper and lower avoidance temperatures of lionfish were fit

to the data (Figure 7). Upper avoidance data yielded an R-square value of 0.11 (y=

0.1745x + 25.21), while lower avoidance showed an R-square value of 0.14 (y= 0.1938x

+ 18.42). The ANOVAs for both upper and lower avoidance showed significant

differences between avoidance temperatures at different acclimation temperatures

(p<0.05; Table 3 and Table 4).

Lionfish displayed an absolute CTmax of 40 ºC and an absolute CTmin of 9.5 ºC.

CTmax and CTmin were different for lionfish acclimated to different temperatures

(ANOVA, p<0.05; Table 5 and Table 6). CTmax for lionfish ranged from 40 ºC to 30.5 ºC

and the linear regression had an R-square value of 0.97 (y = 0.501x + 23.975). CTmin

ranged from 16.5ºC to 9.5 ºC and the linear regression displayed an R-square value of

0.92 (y = 0.4273x +2.0228). The thermal polygon showed a total estimated polygonal

area of 573 ºC2

(Figure 8). Comparisons of thermal limits between species show a

difference in CTmax at 25 ºC (ANOVA, p<0.05; Table 7), but no difference in CTmin

(ANOVA, p>0.05; Table 8). The schoolmaster CTmax (39.9 ± 0.67 ºC) was 1.4 ºC higher

than graysby (38.5 ± 0.53 ºC) and 2.8 ºC higher than lionfish (37.1 ± 0.25 ºC). However,

the lionfish CTmin (12.7 ± 0.52 ºC) is 0.58 ºC lower than graysby (13.3 ± 0.46 ºC) and

0.40 ºC lower than schoolmaster (13.1 ± 0.42 ºC) (Figure 9). The entire inhabitable range

at this acclimation temperature for lionfish, graysby, and schoolmaster is 24.5 ºC, 25.3

ºC, and 26.9 ºC respectively.

24

DF SS MS F p

Acclimation Temperature

1 13.86 13.86 0.909 0.353

Residuals 18 274.38 15.24

Table 2. ANOVA summary for the effect of acclimation temperature on temperature preference. DF SS MS F p

Upper Avoidance

Temperature

1 219.6 219.59 31.66 4.85e-08*

Residuals 254 1762.0 6.94

Table 3. ANOVA summary for the effect of acclimation temperature on upper avoidance. DF SS MS F p

Lower Avoidance

Temperature

1 271.8 271.76 40.38 9.6e-10*

Residuals 254 1709.3 6.73

Table 4. ANOVA summary for the effect of acclimation temperature on lower avoidance.

DF SS MS F p

CTmax 1 119.7 119.73 444.8 1.94e-11*

Residuals 13 3.5 0.27

Table 5. ANOVA summary for the effect of acclimation temperature on CTmax.

DF SS MS F p

CTmin 1 87.35 87.35 163.2 1.83e-09*

Residuals 15 8.03 0.54

Table 6. ANOVA summary for the effect of acclimation temperature on CTmin.

25

DF SS MS F p

Acclimation Temperature

2 20.782 10.391 33.92 1.76e-06*

Residuals 16 4.902 0.306

Table 7. ANOVA summary for the effect of species on CTmax. DF SS MS F p

Acclimation Temperature

2 1.187 0.5933 2.782 0.0872

Residuals 19 4.052 0.2133

Table 8. ANOVA summary for the effect of species on CTmin.

26

Figure 6. Thermal preference graph for lionfish, including the Zone of Final Preferendum

(ZFP) at the intersection of the acute thermal preference line (AP) and line of equality

(LE). Zone of Final Preferendum was determined to be 28.7 ± 1 °C.

15 20 25 30 35 4015

20

25

30

35

40

Acclimation Temp (°C)

Mean A

cute

Pre

fere

nce (

°C)

ZFP (28.7 ± 1 °C)

LE

AP

27

Figure 7. Behavioral tolerance range of lionfish, featuring the upper avoidance regression

line (UA) and the lower avoidance line (LA). Triangles represent upper avoidance data

points, while circles represent lower avoidance data points.

10 20 30 4010

20

30

40

Acclimation Temperature (°C)

Avoid

ance T

em

pera

ture

(°C

)

UA

LA

28

Figure 8. Invasive lionfish thermal polygon, showing the CTmax and CTmin regression

lines. Total estimated polygonal area is 573 ºC2.

0 10 20 30 400

10

20

30

40

50

Acclimation Temperature (°C)

Tem

pera

ture

of LO

E (

°C)

41.7

29.4

16.9

6.6

Total= 573 °C2

CTmax

CTmin

29

Figure 9. CTmax and CTmin comparisons of lionfish compared to two other species. All

subjects were acclimated to 25 ºC. Significant differences were found between the CTmax

values of the three species. CTmin values were not significantly different between species.

Lion

fish

Gra

ysby

Sch

oolm

aste

r0

10

20

30

40

50

CT

M T

em

pera

ture

(ºC

)

12.6413.0613.25

37.2538.5

39.93

24.6

1 26.8

7

25.2

5

CTmax

CTmin

30

Discussion

Temperature Preference

The zone of final preferendum for lionfish defined by this study was estimated to

be 27.7-29.7 ºC (81.9-85.5 ºF), which generally describes either a warm sub-tropical

summer or a tropical winter in the western north Atlantic. Other tropical marine species

have displayed similar temperature preferences. Measurements of juvenile pebbled

butterflyfish (Chaetodon multicinctus), banded damselfish, (Abudefduf abdominalis)

and convict tang (Acanthurus triostegus sandvicensis) are 27.0 ºC, 30.2 ºC, and 29.3

ºC, respectively (Medvick and Miller 1979). Some tropical marine fish have shown a

lower final preferendum, such as the juvenile humpback grouper (Epinephelus altivelis)

and adult yellow tang (Zebrasoma flavescens), which exhibited 24.5 ºC and 21 ºC,

respectively (Reynolds and Casterlin 1980). These studies utilized similar shuttlebox

procedures, but measured final preferendum directly instead of determining final

preferendum from acute preferences.

Final preferendum correlates well with optimal temperature for metabolic scope,

growth, cardiac performance, swimming activity, and digestion (Brett 1971; Jobling et al.

1981; Kelsch and Neill 1990). A previous study also investigated the bioenergetics of

lionfish, relating temperature to their feeding rate (Cerino et al. 2013). This study

determined that the 29.1 ºC was the optimal temperature of feeding for lionfish, which is

within our estimated final preferendum. It appears that the invasive lionfish are at their

physiological optimum when living near this temperature range.

Acute preference data showed significant amounts of variation. Acclimation

temperature explained about 15% of the lionfish’s movements in the shuttlebox, while

85% of the variation remains unexplained. In comparison, Cincotta and Stauffer (1984)

reported R-squared values from 0.40 to 0.78, meaning acclimation temperature accounted

for 40%-78% of their fish movement. However, the Cincotta and Stauffer (1984) results

were from gradient preference measurements, instead of shuttlebox measurements.

Shuttlebox studies have reported differing amounts variation in shuttlebox behavior. For

example, Mortensen et al. (2007) reports an R-squared value of 0.48 when analyzing the

relationship between temperature preference and specific growth rate. Other studies

31

report large amounts of temperature preference variation between individuals, mentioning

individual behavior and exploratory movement as possible explanations (Killen 2014;

Konecki et al. 1995). Measuring fish behavior is extremely difficult because their actions

are often unpredictable, even when controlling for as many variables as possible. For

statistical testing, there was no significant difference of acute preference between

acclimation temperatures. The test results were likely due to a low sample size and high

variation between the points, since a power test revealed only 27% probability of

detecting a significant difference.

The electronic shuttlebox measured thermal preferences and avoidance

temperatures for 20 lionfish. A total of 34 fish were tested in the shuttlebox, with 20 of

those successfully demonstrating behavioral thermoregulation (59%). There are a couple

of possible explanations for the 14 lionfish that did not thermoregulate. First, lionfish

have the tendency to demonstrate high site-fidelity, meaning they prefer to stay still

rather than explore (Jud and Layman 2012). Reduced exploratory movement could hinder

their ability to learn the shuttlebox system, as they would have less of an opportunity to

discover the difference in temperatures between the tanks. Second, due to the design of

the shuttlebox, small water current is created within each side of the tank. In currents,

lionfish tend to lie close to the substrate and avoid movement (James Morris, NOAA,

pers. comm. 2014). This small current could discourage lionfish from exploring the tank.

Some of lionfish spent long lengths of time at very high temperatures due to the

rate of change slowing within the shuttlebox chambers. When the shuttlebox reached

temperatures nearing 37 ºC, the rate of temperature change slowed down, due to the fact

that the “hot bath” was only a few degrees higher than the actual temperature. A few

subjects could withstand these temperatures and would not move to the other tank,

despite showing the capability of doing so earlier in the experiment.

Behavioral Tolerance

Mean upper avoidance temperatures ranged from 28.1-31.0 ºC, while mean lower

avoidance temperatures ranged from 22.1-24.5 ºC. Significant differences existed

between avoidance temperatures at different acclimation temperatures, demonstrating

that lionfish behavior is affected by surrounding water temperatures. The regression lines

32

for upper and lower avoidance were used to demonstrate the lionfish behavioral tolerance

range (Figure 10). Using the regression lines as a predictor, the maximum and minimum

avoidance temperatures are 32.2 ºC and 20.5 ºC, respectively. This range is much

narrower, in comparison to the physiological tolerance range of the invasive lionfish. In

total polygonal area, behavioral tolerance is actually just 26% of the total physiological

tolerance. This means that lionfish, when given the opportunity, will stay well away from

their thermal limits. When inside of this behavioral tolerance zone, it is likely that

lionfish are physiologically unaffected by the surrounding water temperature. Outside of

the behavioral tolerance zone is the resistance zone, utilized by Jobling (1981; Figure 11).

At these temperatures lionfish can survive, but their physiological performance may be

limited, with potential deleterious effects for fitness (Kimball et al. 2004).

Physiological Tolerance

Lionfish in the present experimental trials displayed differences in CTmin and

CTmax in response to changing acclimation temperatures. This is contrary to the CLmin

results reported by Kimball et al. (2004). One possible reason for this could be the rate of

temperature change. This study used a rate of 0.3 ºC per minute, while Kimball et al.

(2004) used 1 ºC per day (0.0167 ºC per minute). The slower rate used in the present

study presumably allows the lionfish to acclimate to the changing temperatures

throughout the experiment, masking any effect of prior acclimation. Kimball et al. (2004)

also reports a mean CLmin of 10.7 ºC, in comparison to a mean CTmin of 12.67 ºC in this

study (prior acclimation at 25 ºC). The difference in these results is most likely due to a

combination of slower temperature rate of change and the endpoint. Kimball et al. (2004)

used death as the endpoint, while this study used LOE. This is not surprising, as LOE

always comes before death, allowing for time to pass (and temperature to decrease) in

between. Although Kimball et al. (2004) did not state specific temperatures of LOE; they

did report that temporary and permanent LOE occurred just prior to death. It is likely that

their LOE temperatures would be lower than the CTmin reported by the current study.

33

Figure 10. Behavioral tolerance range (shaded area) shown in relation to physiological

tolerance (dashed lines). Upper avoidance (UA) and lower avoidance (LA) regression

lines form the upper and lower boundary of the behavioral tolerance range.

10 20 30 40

10

20

30

40

Acclimation Temperature (°C)

Avoid

ance T

em

pera

ture

(°C

)

UA

LA

31.3

20.5

27.1

25.2153.1 °C2

34

Figure 11. Tolerance zone comparison, showing the resistance zone (shaded gray

region), behavioral tolerance zone (striped region), and the zone of final preferendum

(black region).

10 20 30 40

10

20

30

40

Acclimation Temperature (°C)

Response T

em

pera

ture

(°C

)

35

Lionfish responses were consistent with other CTM studies of tropical fishes.

Eme and Bennett (2009) portrayed thermal tolerance polygons of five tropical mangrove-

associated and reef-associated fish species. Similar to the lionfish, these five species also

displayed differences in CTmin and CTmax with acclimation temperature. Additionally,

Eme and Bennett reported overall polygon area (reported as ºC2) as a comparative

measure for eurythermy between species. The mangrove-associated species showed

higher polygonal area than the reef-associated species. The increased eurythermy

demonstrated by these mangrove species is likely due to their exposure to frequently

changing temperatures from tidal movement.

Using the CLmin from Kimball et al. (2004) and an estimated CLmax, overall

polygon area was determined for lionfish (Table 9). A polygon area of 573 ºC

2 for

lionfish is smaller than the tropical mangrove-associated species (~820 ºC2), but higher

than the reef-associated species (~430 ºC2) (Table 9). This result suggests that lionfish are

not necessarily more eurythermal than all tropical species, but may be slightly more

eurythermal than Atlantic reef-associated species.

When discussing these CTM data and their potential ecological significance on

the boundaries of lionfish, it may be most effective to use the constructed thermal

polygon as a predictor. Using the results of this study in this way can improve our

understanding of the invasive lionfish’s thermal limits and the potential effect of global

climate change on their range.

The current overwintering limit in the northern hemisphere is Cape Hatteras,

North Carolina (Kimball et al. 2004), and this point typically serves as the northern-most

boundary for other reef-fishes as well. The average water temperature during 2014 off

Beaufort, NC was 17.6 ºC (NOAA National Oceanographic Data Center). Using the

CTmin regression line and 17.6 ºC as the acclimated temperature, 9.5 ºC is the predicted

CTmin for lionfish in this region. Even with ocean temperatures expected to increase by at

least 2-3 ºC in the next 50-100 years (2.7ºC according to IPCC in 2014, Pörtner et al.

2014), it will still not be warm enough north of this boundary to allow lionfish to

overwinter absent a biochemical change within the species to facilitate low-temperature

survival (Hoegh-Guldberg et al. 2007; Rummer et al. 2014). According to NOAA NODC

data, Virginia and New Jersey have average coastal winter water temperatures (January-

36

Species Primary

Habitat

Total

Polygonal

Area (°C2 )

Source

Common goby

Bathygobius fuscus

mangrove/tidepool 828 Eme and Bennett

(2009)

Square-tail mullet

Liza viagiensis

mangrove/tidepool 823 Eme and Bennett

(2009)

Sandflat goby

Bathygobius sp.

mangrove/tidepool 639 Eme and Bennett

(2009)

Lionfish

Pterois spp.

reef 573 Present study

White-tailed humbug

Dascylus aruanus

reef/seagrass 442 Eme and Bennett

(2009)

Nine-banded cardinalfish

Ostorhinchus

novemfasciatus

reef/seagrass 408 Eme and Bennett

(2009)

Table 9. Comparisons of total polygonal area (ºC2) between lionfish and other tropical

species measured by Eme and Bennett (2009). The mangrove and tidepool-associated

species show higher total polygonal area than reef-associated species. The total polygonal

area of lionfish is higher than reef-associated species, but lower than mangrove/tidepool-

associated species.

37

March) of 4.6 and 3.7 ºC, respectively. This shows that these water temperatures would

have to increase by at least 5 ºC for lionfish to overwinter in these areas, which is higher

than most predicted water temperature changes (Hoegh-Guldberg et al. 2007; Pörtner et

al. 2014). However, increasing ocean temperatures could allow lionfish abundance to rise

closer to the North Carolina shore, especially during the colder winter months (Whitfield

et al. 2014).

The southern boundary of the invasive lionfish range as of 2014 was off the coast

of South America, east through Venezuela to Trinidad and Tobago (USGS-NAS 2015).

Some have questioned whether lionfish will expand their invasive range down the East

coast of Brazil due both the Amazon-Orinco Plume and the North Brazil Current (Luiz et

al. 2013). However, the first lionfish was recorded off the coast of Brazil at Arraial do

Cabo in May of 2014 (Ferreira et al. 2015). According to the measured CTmin,

lionfish would likely expand their overwintering range to the coast of Uruguay, similar to

the predictions of Morris and Whitfield (2009) and Miloslavich et al. (2011).

The threat of increased ocean temperatures from global climate change has

prompted studies and discussions of the upper thermal tolerance of reef-fish and other

reef-inhabiting organisms (Booth et al. 2011; Figueira and Booth 2010; Hoegh-Guldberg

et al. 2007; Mora and Ospina 2001; Ospina and Mora 2004; Rummer et al. 2014). There

is some disagreement as to whether reef-associated fishes will be affected by an

estimated increase in ocean temperatures of 2-3 ºC over the next 100 years (Mora and

Ospina 2001; Rummer et al. 2014). When examining CTmax data, it appears unlikely that

the abundance of many species will be significantly affected by a temperature change of

2-3 ºC. Most species have CTmax values about 8 ºC above the average Caribbean sea

surface temperature (SST) (Mora and Ospina 2001). In the case of the invasive lionfish,

they will likely be unaffected by increasing ocean temperatures at this latitude, since their

CTmax in this area is around 36-40 ºC. However, indirect effects of increasing

temperatures could still influence the populations of reef-associated fishes in these areas

(including lionfish) through such problems as habitat degradation, coral bleaching, and

ocean acidification (Hoegh-Guldberg et al. 2007).

38

This study also examined the differences in CTM at 25 ºC between three different

reef-associated species, lionfish, graysby, and schoolmaster. Lionfish displayed the

smallest tolerance range (degrees from CTmin to CTmax) of the three species at 24.5 ºC,

while the others were only larger by 1-2 ºC. This is likely because lionfish do not have

larger thermal tolerance ranges, but actually very similar ranges compared to other

tropical reef species (Eme and Bennett 2009). Based on the described ranges within the

literature, all three tested species can each live year-round from North Carolina to South

America; therefore the similarity of tolerance range should not be surprising (Allen 1985;

Kimball 2004; Rocha et al. 2008).

Past studies have also used CTM on tropical fishes to examine thermal tolerance.

Mora and Ospina (2001) tested the CTmax of 15 difference reef fishes with a rate of 1 ºC

per hour at an acclimation of 26.5 ± 0.5 ºC. This study described CTmax that ranged from

34.7ºC to 40.8 ºC, with estuarine and intertidal species having higher CTmax values.

Another study investigated thermal maxima in tropical estuarine fishes, showing CTmax

between 39.5-44.5 ºC (Rajaguru 2002). Although it is unclear why CTmax varies from

species to species, it may be because of their primary habitat. Estuarine species are

subjected to more fluctuating temperatures while reef species inhabit a more stable

environment. The fluctuating temperatures of an estuary system could explain the

difference in thermal limits, either because these fish are more accustomed to changing

temperatures or they actually experience a wider range.

Thermal Niche

This study defines the entire thermal niche of the invasive lionfish (Figure 12).

This allows for a better understanding of the invasive lionfish and their interaction with

temperature. Figure 12 describes how a lionfish would be affected by temperature under

certain circumstances. For example, an invasive lionfish in warm tropical temperatures

(30 ºC) would have a temperature preference around 27.5 ºC, CTmin around 14.75 ºC, and

CTmax around 39 ºC. Figure 12 also shows the relationship between the different

39

Figure 12. Lionfish thermal niche incorporates the linear regression lines of CTmax, CTmin,

upper avoidance (UA) and lower avoidance (LA). The nonlinear quadratic line for acute

thermal preference (AP) is also included. Also shown is the line of equality (LE) and

Zone of Final Preferendum (ZFP), which intersect at 28.7 ºC.

0 10 20 30 40 500

10

20

30

40

50

Acclimation Temperature (°C)

Response T

em

pera

ture

(°C

)

CTmax

CTminLE

LA

UA

AP

ZFP (28.7 ± 1 °C)

40

measurements collected during this study. It appears that lionfish prefer temperatures that

are closer to their thermal maximum than their thermal minimum.

Additionally, this study demonstrates the differences between measuring

physiological parameters versus behavioral parameters. The difference of slopes between

the thermal limits (physiological) and preference/avoidance temperatures (behavioral)

indicate that lionfish thermal limits were more affected by the acclimation temperature.

Also, the thermal limits measured showed much less variation than the preferences and

avoidances. Behavioral studies traditionally have high amounts of variation because of

the unpredictability of a live animal. There are many unknown factors that contribute to

the actions of an individual and it is very difficult to understand the driving factors

behind their behavior.

Conclusion

For the invasive lionfish, temperature can define geographic range, impact

physiological processes, and influence behavior within their range. This study further

elucidates the relationships between the invasive lionfish and temperature. The

temperatures at which lionfish are at their physiological optimum, their behaviorally

selected tolerance range, and their physiological limits are now established for juveniles.

Investigating the underlying physiology and behavior behind the lionfish is important to

understanding the possible ecological effects of this invasion. Our thermal information

will be key to predicting and analyzing future geographic range changes for the invasive

lionfish and investigating their behavioral response to temperatures within their habitats.

41

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