RESEARCH ARTICLE

Mechanical properties of the venomous spines of Pterois volitansand morphology among lionfish speciesKatherine A. Galloway* and Marianne E. Porter

ABSTRACTThe red lionfish, Pterois volitans, an invasive species, has 18venomous spines: 13 dorsal, three anal and one on each pelvic fin.Fish spines can have several purposes, such as defense, intimidationand anchoring into crevices. Instead of being hollow, lionfish spineshave a tri-lobed cross-sectional shape with grooves that deliver thevenom, tapering towards the tip. We aimed to quantify the impacts ofshape (second moment of area) and tapering on the mechanicalproperties of the spine. We performed two-point bending at severalpositions along the spines of P. volitans to determine mechanicalproperties (Young’s modulus, elastic energy storage and flexuralstiffness). The short and recurved anal and pelvic spines are stifferand resist bending more effectively than the long dorsal spines. Inaddition, mechanical properties differ along the length of the spines,most likely because they are tapered. We hypothesize that the highlybendable dorsal spines are used for intimidation, making the fish looklarger. The stiffer and energy-absorbing anal and pelvic spines aresmaller and less numerous, but they may be used for protection asthey are located near important internal structures such as the swimbladder. Lastly, spine second moment of area varies across thePterois genus. These data suggest there may be morphological andmechanical trade-offs among defense, protection and intimidation forlionfish spines. Overall, the red lionfish venomous spine shape andmechanics may offer protection and intimidate potential predators,significantly contributing to their invasion success.

KEY WORDS: Lionfish, Biomechanics, Stiffness, Elastic energy,Flexural stiffness

INTRODUCTIONSpines are multi-functional biological materials found in nature thatcan greatly benefit organisms in terms of gripping, injection,damage and defense (Anderson, 2018). For example, cacti use spinymodified leaves that prevent water loss in their dry desert habitat andprotect against herbivores (Koch et al., 2009). Hedgehogs use theirquills for protection against predators and the quills absorb energyduring impact from high falls (Vincent and Owers, 1986). Stonefishhave a lachrymal saber that is an elongation of an anterior spine,which they are able to rotate into a locked lateral position possiblyfor defense (Smith et al., 2018). In addition, triggerfish have amodified anterior dorsal fin spine that has several purposesincluding self-defense, anchoring into crevices in the coral reef

when sleeping and providing protection against a strong ocean surgeor waves (Cleveland and Lavalli, 2010).

Similar to differences in anatomy, spine material varies, andaffects the overall mechanics. Both lionfish and stingray spines aremade of mineralized collagen, a combination of hydroxyapatite andcollagen (Halstead and Modglin, 1950; Halstead et al., 1955).However, the mechanical properties of lionfish and stingray spinesremain unknown. Spines in porcupines, hedgehogs and echidnas aremade of keratin (Vincent and Owers, 1986) and have Young’smoduli (E) ranging from 5.56 GPa in porcupines to 11.56 GPa inhedgehogs (McKittrick et al., 2012; Vincent and Owers, 1986).Biomechanical properties have only been examined for stingers(bees, wasps and scorpions), where venom is delivered through themiddle of the spine (Zhao et al., 2015; Zhao et al., 2016). Lionfishspines, similar to those of stingrays, have venom glands and groovesthat line the sides of the spine, whereas in bees, wasps andscorpions, venom flows through the middle (Halstead and Modglin,1950; Halstead et al., 1955). Venom delivery morphologies incombination with material composition may affect the properties ofthe spine under various loading regimes.

In several organisms, mechanical properties vary along the lengthof the structure. In wasp stingers, the elastic modulus and hardnessdecrease along the length from the base to the tip (Das et al., 2018).In contrast, Young’s modulus increases towards the tip of owlfeather shafts (Bachmann et al., 2012). The tapered morphology ofporcupine fish spines changes the location of maximum stress to thedistal end (tip) of the spine, but does not change spine stiffness ortoughness (Su et al., 2017). By focusing spine damage toward thedistal ends, porcupine fish may conserve the energy required forregrowth.

The red lionfish, Pterois volitans, has 13 dorsal fin spines, threeanal fin spines and one spine on each pelvic fin (Fig. 1A). In cross-section,P. volitans spines are solid and have a tri-lobedmorphology,thought to be exclusive to lionfish (Halstead et al., 1955; Fig. 2A).This tri-lobed shape is formed by a pair of lateral grooves along theouter two-thirds of the length and these grooves contain glandulartissue that houses venom (Fig. 2B). Both the spines and theglandular tissue are covered by a thin membrane, which ruptureswhen the spines penetrate an object, releasing the venom (Halsteadet al., 1955). The length of the refractory period between venomdelivery events and whether the presence of venom in the lateralgrooves affects the mechanical properties remain unclear.

The tri-lobed cross-section of the lionfish spine is reminiscent ofI-beams used in building design and construction. Engineeringbeam theory demonstrates that the I-beam shape is able to carry bothbending and shearing loads because most of the material isdistributed away from the neutral axis. As a result, I-beams have ahigh second moment of area and span-to-depth ratio, meaning thatthis shape effectively resists bending (Vogel, 2013). The lionfishspine tri-lobed cross-section also has a large portion of the materiallocated away from the center of the structure (Fig. 2A).Received 7 December 2018; Accepted 22 February 2019

Department of Biological Sciences, Florida Atlantic University, Boca Raton,FL 33431, USA.

*Author for correspondence (kgalloway2016@fau.edu)

K.A.G., 0000-0003-0711-6893

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The goal of this study was to investigate the mechanical (bending)properties of the venomous spines of the red lionfish, P. volitans.Our specific goals were to: (1) determine Young’s modulus(material stiffness; E), elastic energy storage (ability to absorbenergy and restore the sample to its original shape), second momentof area (shape characterization; I ) and flexural stiffness (bendingresistance; EI) of dorsal, pelvic and anal spines; (2) determinewhether mechanical properties differ along the length of the spines;and (3) compare second moment of area among seven lionfishspecies to determine spine structural variation. First, wehypothesized that mechanical properties differ among dorsal,pelvic and anal spines as a result of differences in morphology(Fig. 1B). Specifically, we hypothesized that pelvic and anal spineswill be stiffer and can absorb more energy than the long, thin dorsalspines. Second, we hypothesized that mechanical properties differalong the length of the lionfish spines because of the taperingmorphology, which results in venom grooves being absent at thespine tips. We predicted that the base of the spines will be stiffer andbend less, reducing the likelihood of damage at this location andpreventing the energetic cost of regrowing an entire spine. Lastly,we hypothesized that the second moment of area will differ amongPterois species, indicating a difference in cross-sectional shape thatmay be due to varying ecological constraints of native habitats.

MATERIALS AND METHODSSpine preparation and mechanical testingAdult Pterois volitans (Linnaeus 1758) specimens (n=6 females, 4males) were obtained dead from local fishermen and lionfishtournaments on the Western Atlantic coast of Florida, USA.

Lionfish are invasive and no permit is required for fishing thisspecies in the state of Florida (Florida Wildlife Commission). Foreach specimen (total length, TL=188–370 mm; N=30 spines), thefourth dorsal, left pelvic and third anal spines had respective spinelengths in the range 45–90 mm, 24–39 mm and 16.5–37 mm.Spines were extracted from their proximal attachments on the body.The fourth dorsal spine is one of the longest, and was always intactin specimens collected and used in this study. The third anal spine isalso the longest on that fin, and there is only one pelvic spine oneach fin.

The proximal base of freshly dissected spines was potted inapproximately 5 mm of marine epoxy, which was determined to bethe smallest amount of epoxy necessary to secure the spines(Loctite, Westlake, OH, USA). We standardized the preparationprocess by measuring spine length before and after potting in epoxy.Point loads were applied and measurements were obtained at 60%and 0% (tip) of the unpotted spine length. Previous work has shownthat embedding stiff materials in epoxy does not significantly alterthe Young’s modulus (Hoffler et al., 2005; Zysset, 2009). Pottedspines were left to set in epoxy and cure for 48 h. To assess theimpact of curing time on spine mass and shape, we conducted a pilotexperiment on a separate subset of dorsal spines. We found that wetmass did not change for 14 days (Table S1). After 14 days, spineelastic energy storage remained the same, but stiffness increased.We also found therewas no significant difference in secondmomentof area after 14 days (Table S2). As mass and shape remained thesame for the first 13 days (Tables S1 and S2), we assumed that thespine mechanical properties presented here were not impacted bythe 48 h curing time.

A B

Dorsal AnalAnal: 3 spines Pelvic: 2 spines

Dorsal: 13 spines

Pelvic

Fig. 1. Venomous spines of the red lionfish.(A) Pterois volitans has 18 venomous spinesassociated with the dorsal, pelvic and anal fins.(B) Comparison of spines from each region.

Anterior

Posterior

Lateral point load

1 mmC

A B

60% 0%

Fig. 2. Lionfish spine morphology and testing set up. (A) Pteroisvolitans tri-lobed dorsal spine anteroposterior cross-sectionalshape, and point load direction for mechanical testing. This pointload would imitate a side attack from a predator. (B) Lateral view ofthe spine and the tapered distal end. (C) Clamp holding the epoxypot at a 90 deg angle to the stand. Point loads were applied at60% and 0% of dorsal spine total length.

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A clamp on an adjustable steel stand secured potted spines at a90 deg angle to the stand. Point loads were applied at 60% and 0% ofthe spine length (Fig. 2C) to examine the effects of tapering onmechanical properties (Young’s modulus, elastic energy storage,second moment of area and flexural stiffness). A tapered cantileverbending test was modified for an irregular cross-sectional shapeon an Instron E1000 with a 250 N load cell (Su et al., 2017).A dissection pin was used to apply the point load along the spine at adisplacement rate of 0.3 mm s−1, and all spines were deflected to10% of their total unpotted length. A lateral point load (Fig. 2A) waschosen for the following two reasons: (1) we were able to place thedissection pin in a single position within the lateral groove and (2)we could assess spine loading simulating a predator attacking fromthe side. Common displacement speeds for hard biomaterials rangedramatically from 0.003 to 400 mm s−1 (Halstead et al., 1955;Galloway et al., 2016; Su et al., 2017; Summarell et al., 2015;Whitenack and Motta, 2010). We decided to load the mineralizedcollagenous lionfish spines at a slower speed within this rangebecause there are no previous studies on their mechanical behavior,and testing at faster speeds may have resulted in fracture before wewere able to test at other point load locations. A previous study onthe compressive modulus of keratinous horse hooves comparedspeeds from 0.167 to 1.667 mm s−1 and found no significant effectbetween displacement rate and compressive properties (Landeauet al., 1983).Instron BlueHill Software was used to collect force (N) and

displacement (mm) data, which were converted into stress (σ, GPa)and strain (ε, %) using tapered beam equations (Eqns 1 and 2;Su et al., 2017):

smax ¼ 128PL

27pdb3b2ð1� bÞ ; ð1Þ

1max ¼ 2vmaxdb9bð1� bÞL2 : ð2Þ

To account for the tri-lobed morphology, the denominator of themaximum tensile stress equation (Eqn 1) was multiplied by 0.75,because the lionfish spine is approximately 75% of the cross-sectional area of a circle (Eqn 1). Stress is a measure that is sizeindependent and these equations take into account the length (total,unpotted length) and diameter changes seen in the tapered lionfishspines. A ratio of beam diameter, β, is calculated by da/db, where db(mm) is the larger diameter of the tapered beam at the proximal baseand da (mm) is the smaller diameter at the tip. Beam length ismeasured as L (mm), the point load is denoted as P (N) and vmax ismaximum deflection of the beam. All constants are derived in Suet al. (2017).Elastic energy storage is the material’s ability to absorb energy

and to restore the sample to its original shape when the load isremoved, and this property can be calculated as the area under thestress–strain curve (Vogel, 2013; Fig. S1). Elastic energy storage inthese data refers to the energy absorption before deformation,because we did not test to yield (permanent deformation). Young’smodulus (E, GPa), a size-independent measure of material stiffness,is calculated as the slope of the linear portion from the stress–straincurve (Fig. S1). A tapered beam equation was used to calculateYoung’s modulus (Eqn 3; Su et al., 2017):

E ¼ 64PL3

3pbd 4b vmax

: ð3Þ

All spines were tested after curing in the epoxy pot for 48 h, andonce mechanical tests were completed, spines were cross-sectioned

at the point load locations (60% and 0% spine length; unpotted) andphotographed in the anteroposterior orientation. We used BoneJ(ImageJ 1.x) software to calculate anteroposterior second momentof area, I (mm4) at point load locations for all spines. The secondmoment of area can be calculated by integrating the areas of manysmall pieces (dA) of an object’s cross-section, and then multipliedby the distance from the neutral axis squared (y2): I=∫y2dA. Flexuralstiffness (EI, N mm2), or the ability to resist bending, was calculatedby multiplying Young’s modulus by the second moment of area.

Comparative secondmoment of area among lionfish speciesWe obtained specimens from The Smithsonian (Washington, DC,USA) to collect comparative spine second moment of area in theanteroposterior orientation (I, mm4) from six additional lionfishspecies [Pterois andover G. R. Allen and Erdmann 2008, Pteroisantennata (Bloch 1787), Pterois lunulata Temminck and Schlegel1843, Pterois radiataG. Cuvier 1829, Pterois russelii E. T. Bennett1831 and Pterois sphex D. S. Jordan and Evermann 1903]. Thesesix species, combined with P. volitans, allowed for the investigationof seven out of 12 recognized species in the lionfish genus(FishBase; USNM Fish Catalog). At University of Washington’sFriday Harbor Laboratories, fish were wrapped in cheesecloth andcling wrap, and scanned in a Bruker Skyscanner 1173 (Kontich,Belgium) at 70 kVp (kilovoltage potential), 114 mA (x-rayintensity), 35.7 μm slice resolution and 2K resolution for largerspecies and a lower resolution for smaller species. Multiple fishwere scanned in a single canister, producing a DICOM file with datafor several species. Individual fish were then digitally dissected(segmented) into separate files and reconstructed using BrukerDataViewer. We used Horos software (Horosproject.org; sponsoredby Nimble Co LLC d/b/a Purview in Annapolis, MD, USA) todigitally segment spines and each spine was saved as a DICOM file.Dorsal spines ranging from the fourth to sixth spine (dependent onwhich spines were not damaged from museum specimens) weredigitally dissected to obtain cross-sections in the anteroposteriororientation, and I was measured using BoneJ at 60% and 30% ofspine length. Wewere unable to obtain the cross-section of spines at0%. Lionfish spines narrow at the distal tip and it was not possible toobtain a cross-section at that location using Horos software.

StatisticsWe evaluated morphological relationships among spine length, fishtotal length and spine region using a two-way ANOVA, where fishtotal length and spine region were main effects. To evaluate data forgoals 1 and 2, we used two-way ANOVA to examine mechanicalproperties: Young’s modulus (E), elastic energy storage, secondmoment of area (I ) and flexural stiffness (EI). Spine region (dorsal,anal and pelvic) and testing location (proximal and distal) weremain effects, and we examined their interaction term. Fish totallength was also included in the models as a covariate. If a maineffect was significant, we used pairwise Tukey tests to evaluatethose differences. If the interaction term was significant for amechanical property, we present those data in the respective figure.If the interaction term was not significant, we present data for onlysignificant main effects. All statistics were analyzed using JMP(SAS Institute Inc., Cary, NC, USA).

Using a separate subset of spines in a pilot study, we found thatsecond moment of area (I ) did not differ between hydrated anddehydrated spines (Table S2). Data for dehydrated spines arepresented here because obtaining the cross-sections for secondmoment of area calculations was destructive and needed to becompleted after all mechanical testing. Based on these results, we

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present I as a hydrated spine property for goals 1 and 2 above. Inaddition, we treated flexural stiffness (EI) as a hydrated spineproperty when we multiplied hydrated Young’s modulus (E) withdehydrated second moment of area (I ).

RESULTSMorphology and mechanical properties of P. volitansSpine length increased with fish total length (P<0.0001; n=10 fish;N=30 spines, 10 spines from each region); as fish got larger thespines got longer. Based on P. volitans used in this study, as fishincreased from 188 to 370 mm, dorsal spine length doubled. Overthe same range of fish sizes, anal spines increased in length 66% andpelvic spines increased 40%. Dorsal spines were significantlylonger than pelvic and anal spines (P<0.0001).A two-way ANOVA showed that elastic energy storage varied

significantly (F6,59=13.85, P<0.0001) among spine regions(P<0.0001) and testing locations (P=0.012), and increased withincreasing fish total length (P<0.0001). The interaction betweenspine region and testing location was not significant (F1,54=0.6591,P=0.52). The anal and pelvic spines can store about 85% moreenergy than the dorsal spines (Fig. 3A). The proximal end of thespine (base) can absorb about 42% more energy than the distal (tip)end (Fig. 3B). These data support our hypothesis that mechanicalproperties vary along the length of the tapered lionfish spines.A two-way ANOVA showed that Young’s modulus (E) varied

significantly (F6,59=78.56, P<0.0001) among spine regions andtesting locations (P<0.0001), and increased with increasing fishtotal length (P<0.0001). The interaction between spine region andtesting location was significant (P=0.0057). The anal and pelvicspines at the proximal end (base) were stiffer than the pelvic andanal spines at the distal end (tip) (Fig. 4A). The dorsal spines weresignificantly less stiff at both testing locations in comparison to thepelvic and anal spines (Fig. 4A). These data align with ourhypothesis predicting that the base of the spines will be stiffer,which may reduce the likelihood of damage towards the basepreventing the energetic cost of regrowing an entire new spine.A two-way ANOVA showed that second moment of area (I )

varied significantly (F6,59=176.58, P<0.0001) among spine regionsand testing locations (P<0.0001), and second moment of areaincreased with increasing fish total length (P<0.0001). Theinteraction between spine region and testing location wassignificant (P=0.0003). The distal ends, or tips, of lionfish spineshad significantly lower I compared with the base of all spines(Fig. 4B). This aligns with our hypothesis that the spines are taperedand there are no venom grooves at the tip of the spines, andI becomes smaller towards the tip. The anal spines at the proximalregion (base) had the highest I (Fig. 4B), and the anal spine at theproximal region is the stiffest (Fig. 4A).A two-way ANOVA showed that flexural stiffness (EI) varied

significantly among spine regions and testing locations

(F6,59=49.50, P<0.0001), and increased with increasing fish totallength (P<0.0001). The interaction between spine region and testinglocation was significant (P<0.0001). The anal and pelvic spines atthe proximal end (base) had a higher EI (Fig. 4C), which indicatesthey are more resistant to bending than the anal and pelvic distalends, and the dorsal spines. These data align with our hypothesisthat the base of the anal and pelvic spines can better resist bending,and will incur less damage than the tips of the spines. Overall, thedorsal spines do not resist bending as well, do not absorb a highamount of elastic energy and are not as stiff (Fig. 4A–C).

Second moment of area (I) among seven Pterois speciesSecond moment of area (I ) of dorsal spines ranged from 0.0029 to0.9267 mm4 (Table 1). There are no statistics for these comparativedata (n=1 from each species). Pterois russelii had the highest I incomparison to the other species, suggesting that P. russelii dorsalspines are stiffer and more resistant to bending. Pterois radiata hadthe lowest I in comparison to the other species, suggesting thatP. radiata dorsal spines are the least stiff and are less resistant tobending. Interestingly, P. russelii and P. radiata overlap in somenative ranges, but P. radiata exhibits a broader native habitat range.Pterois sphex had a relatively high secondmoment of area comparedwith the other species examined here, and is only native to theHawaiian Islands (Table 1).

DISCUSSIONMechanical properties of P. volitans spines differed among spineregions and testing locations. Pelvic and anal spines were stiffer,could store more elastic energy and could resist bending more thandorsal spines (Figs 3A, 4A,C). Dorsal spines did not have a highresistance to bending and were not stiff structures, and we wouldexpect them to be able to bend substantially under large lateral loads(Fig. 4A,C). Proximal regions of all spines were stiffer, could storemore elastic energy and could resist bending more effectively thanthe distal ends (Figs 3B, 4A,C). Second moment of area (I ) differedamong lionfish species, and there may be relationships among spinemorphology, second moment of area and native versus invasivelocations (Fig. 4B and Table 1). For example, in P. volitans, thepelvic and anal spines had similar lengths, but their shape (asmeasured by second moment of area) was significantly different inthe proximal region. Overall, spine length and mechanicalproperties increased with fish total length.

Comparative mechanical propertiesLionfish spines, composed of mineralized collagen, ranged inYoung’s modulus from 0.11 to 10.88 GPa (Fig. 4A; Halstead et al.,1955). Lionfish spine stiffness falls within the range of other bonyfish structures such as porcupine fish spines (6.8–20.5 GPa), whichare made of nanocrystalline hydroxyapatite, collagen and water (Suet al., 2017). Teleost rib material stiffness (tilapia: 4.1–11.0 GPa,

25A B

a

bb

a

b20

15

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Ela

stic

ene

rgy

stor

age

(MP

a)

Pelvic

Spine region

Anal Proximal Distal

Testing location

20

15

10

5

0

Fig. 3. Elastic energy storage of P. volitans spinesvaries significantly among spine regions andbetween testing locations. (A) Elastic energy storagewas more than three times greater in the pelvic andanal spines than in the dorsal spines. (B) Proximalspines point loads result in greater elastic energystorage than distal point loads. Data are means±s.e.m.(n=10 individuals; N=30 spines, 10 from each region).Bars sharing the same letter are statistically similar.

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and carp: 3.6–14.5 GPa) is also similar to that of lionfish spines(Cohen et al., 2012; Horton and Summers, 2009). As lionfish spinestiffness is comparable to bone stiffness, we hypothesize thatthey are also heavily mineralized. Flexural stiffness of lionfishspines ranged from 2.28 to 8257 N mm2 (Fig. 4C), which ismuch higher than the mineralized cartilage of batoid propterygia(33.74–180.16 N mm2; Macesic and Summers, 2012). These datasuggest that the mineralized collagen spines of lionfish resistbending better than skeletal elements in cartilaginous fishes and arefunctioning similar to bony fish skeletal elements. In addition tobeing able to resist bending effectively (EI), lionfish spines can storea high amount of energy (particularly the pelvic and anal spines)(Fig. 3). We hypothesize that the combination of high bendingresistance and elastic energy storage (especially in the pelvic andanal spines) is important for puncturing predators and prey, andreducing damage, which has potential energetic savings of avoidingspine regrowth. Future studies could examine puncture mechanics,the amount of mineralization in lionfish spines and detailedhistology to empirically determine tissue type.It has been suggested that the lionfish dorsal spines are important

for defense in terms of predator avoidance and prey capture, becauseof their location on the dorsal surface, their length and the number(13) of spines. The dorsal spines are significantly longer and

probably contain more venomous glandular tissue than the pelvicand anal spines. Dorsal spines are less stiff and can absorb lessenergy than the pelvic and anal spines (Figs 3 and 4A), whichsuggests that they may be less effective at puncturing and prone tobreaking. A broken dorsal spine may not be detrimental to the fish,as they would have 12 other spines in the same location. Based onour results, we hypothesize that the dorsal spines primarily serve asan intimidation strategy, making the fish look larger to predators,rather than being strictly a defense apparatus.

Lionfish morphology and biologyThe many native habitats of Pterois species, in combination withdiffering spine morphology, suggest a potential variation ineco-morphological pressures. Our data from seven lionfish speciesshow that second moment of area (I ) of dorsal spines does not relateto the body size range. For example, P. sphex was the smallestspecies examined here (22 cm TL; Randall, 1985) but had thesecond highest I (Table 1). In contrast, P. lunulata can reach up to35 cm TL (Kuiter and Tonozuka, 2001) and had a low I (Table 1).The maximum size of P. volitans is documented at 38 cm and it hadan intermediate I (Table 1). However, these comparative datasuggest that spine morphology may vary with habitat. Pteroisradiata inhabits shallower rocky crevices and reefs and has a low

10A B

C

a a

a

b

a

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b

a

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b

d

b b

cc

c

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mom

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f are

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m4 )

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1000

100

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1

Spine region and testing location

P D P D P DAnal Pelvic Dorsal

P D P D P DAnal Pelvic Dorsal P D P D P D

Anal Pelvic Dorsal

Fig. 4. Mechanical properties of P. volitans spines vary significantly among spine regions and between testing locations. (A) The interaction termbetween spine region and testing location was significant for Young’s modulus (E; P=0.0057). Dorsal spines in general are less stiff than pelvic and anal spines.The base (proximal, P) of anal and pelvic spines is stiffer than the tip (distal, D). (B) The interaction term between spine region and testing location was significantfor secondmoment of area (I;P=0.0003). All spines at the distal (tip) regions have a smaller secondmoment of area than the proximal (base) regions. At the base,dorsal spine secondmoment of area is smallest compared with the pelvic and anal spines. (C) The interaction term between spine region and testing location wassignificant for flexural stiffness (EI; P<0.0001). The base of the anal and pelvic spines can resist bending more than the dorsal spines and the tips of the anal andpelvic spines. In general, the tips of all spines have low flexural stiffness values and do not resist bending. Data are means±s.e.m. (n=10 individuals;N=30 spines,10 from each region). Bars sharing the same letter are statistically similar.

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spine second moment of area, while P. russelii dwells on muddy/brackish substrates in shallow and offshore depths and has a highspine second moment of area (Kuiter and Tonozuka, 2001). Furtherrelationships investigating eco-morphology are needed on lionfishin their native habitats – information that is currently limited.Lionfish, specifically P. volitans, have become a successful

invasive predator for various reasons. First, lionfish can survivewithout food for extended periods of time, although they have beenshown to have a very healthy appetite and are generalist feeders(Albins and Hixon, 2008). In a starvation study, lionfish lost only5–16% of their body mass over a period of 3 months without food(Fishelson, 1997). In addition, lionfish have specialized bilateralswim bladder muscles that provide pitch control for prey strikebehavior (Hornstra et al., 2003). This novel buoyancy adaptationmay aid in stability in the water column as well. Lionfish also havean extremely high reproductive rate of two million eggs per year andan early maturity size of 4–6 inches (Morris and Whitfield, 2009).Finally, they have bacterial communities in skin mucus that providedisease resistance, a novel trait that may have facilitated theirsuccessful invasion in the Western Atlantic and Caribbean (Stevenset al., 2016). These characteristics combined with 18 venomousspines make lionfish an aggressive and intimidating marine invader,although we show here that the numerous dorsal spines are not stiff,energy-absorbing structures. As a result, not all of the venomousspines of P. volitans may be effective at defense.All lionfish spines that we examined lacked grooves at the tips

and had tapered structures; second moment of area (I ) decreasedtowards the tips of the spines for all species, similar to P. volitans

(Table 1). As we suspect that the spines of all lionfish species arecomposed of the same material (mineralized collagen), the materialstiffness or Young’s modulus (E) should remain constant, andI would be the critical variable determining the spine bendingproperties among species. We predict that flexural stiffness (EI,bending resistance) decreases towards the tips of the spines for allspecies of lionfish, as it does in P. volitans investigated in this study.

Table 1. Second moment of area (I ) of dorsal spines from seven Pterois species and corresponding anteroposterior cross-sectional shape at 60%and 30% of total spine length

Species and museum ID Micro-habitat

60% spine length 30% spine length

I (mm4)Anteroposteriorcross-sectional shape I (mm4)

Anteroposteriorcross-sectional shape

P. andover Spine length 31.6 mm(USNM 390776)

Benthopelagic, tropical,depth 3–70 m

0.0117

0.0112

P. antennata Spine length 57.1 mm(USNM 420972)

Reef associated, tropical,depth 2–86 m

0.0087

0.0083

P. lunulata Spine length 32.9 mm(USNM 99680)

Reef associated, tropical,depth 132–172m

0.01930.0172

P. volitans Spine length 41.8 mm(OSF, Scan All Fishes)

Reef associated, tropical,depth 2–55 m

0.41860.3955

P. radiata Spine length 41.4 mm(USNM 385903)

Reef associated, tropical,depth 1–30 m

0.00290.0019

P. russelii Spine length 39.4 mm(USNM 168219)

Reef associated, brackishwater, tropical, depth 15–60 m

0.92670.9255

P. sphex Spine length 28.2 mm(USNM 50650)

Reef associated, tropical,non-migratory, depth 3–122 m

0.82730.8209

Citations for micro-habitat data are found in the Discussion.

Fig. 5. Dorsal spine of P. volitans showing potential area of damage andregrowth. During dissections of lionfish used in this study, we observed thatapproximately 10% of dorsal spines showed potential regrowth. In thesespines, damage and regrowth always occurred between 30% and 60% ofthe spine total length, which may indicate a region susceptible to fracture.

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The shape of lionfish spines among species is also informational forgroove size and possibly glandular tissue and venom quantities. Forstructures with venom or poison, it is important to take into accountthe biological structure as a whole (the material, shape, and venomor poison). To better understand the relationships betweenmechanical properties and spine shape, future studies could focuson these properties over a range of fish and spine sizes.

Spine regrowthSeveral aquaria have observed that lionfish can regrow their spines,particularly damaged dorsal spines. There are currently no datadiscussing regrowth rate and whether spine regrowth always occurs.Regrowth of mineralized collagen spines may be influenced bywater pH or temperature, which can vary among aquaria. Fromdissections for this study, we documented instances where regrowthmay have occurred in approximately 10% of dorsal spines (Fig. 5).In these cases, regrowth always occurred between 30% and 60% oftotal spine length, suggesting this is the area of the spine wherefracture is likely to occur. These predictions are supported by ourdata, which found that testing location was a significant effectimpacting mechanical properties. We argue that the location ofspine loading is important because of possible tapering or change inshape of biological materials.

Applications and bioinspirationBiomimetic designs draw inspiration from nature to engineerdevices for human use. Fish scales and shark skin have gainedattention in recent years for applications such as impact-resistantarmor and self-cleaning materials (Nishimoto and Bhushan, 2013;Naleway et al., 2016; Zhu et al., 2012). Here, we presentexperimental data on lionfish spines and quantify their variationsin shape, length and mechanical properties. Many researchersstudying spine mechanics have argued that spines can be used forbiomedical inspiration for devices such as hypodermic needles (Baiet al., 2015; Cho et al., 2012; Sahlabadi et al., 2017). Lionfish spinesare not hollow, serrated or barbed, and they instead deliver venomfrom grooves along the sides (Fig. 2B). The lionfish spine designmay be useful in creating reusable syringe needles and plungers thatcan be sterilized, which would decrease biomedical waste andsharps disposal costs. We hypothesize that the second moment ofarea and tapering of lionfish spines will reduce puncture forces.Further collaborations among biologists, medical doctors andengineers would be beneficial for such bioinspired applications.

AcknowledgementsWe thank REEF and FWC for lionfish specimens. We thank Kent Wallace forInstron support. We thank Dr Justin Grubich (Field Museum) for lionfish expertise,commentary on experimental design, and thoughtful conversation. We thank theFlorida Atlantic Biomechanics lab members for support and comments. We thankDr Adam Summers and the Friday Harbor Laboratories at University of Washingtonfor access to themicro-CT scanner.We thank Danielle Ingle for scanning specimensand the Smithsonian National Museum of Natural History for various lionfishspecies. We thank Breanna Nelson for help with data collection. We thank theJEB reviewers and editors for their thoughtful critiques of our manuscript.

Author contributionsConceptualization: K.A.G., M.E.P.; Methodology: K.A.G., M.E.P.; Software: K.A.G.;Validation: K.A.G.; Formal analysis: K.A.G.; Resources: M.E.P.; Data curation:K.A.G.; Writing - original draft: K.A.G.; Writing - review & editing: K.A.G., M.E.P.;Supervision: M.E.P.; Project administration: M.E.P.; Funding acquisition: K.A.G.,M.E.P.

FundingThe Marine Technology Society Graduate Scholarship (K.A.G.) and the Walterand Lalita Janke Innovations in Sustainability Science Research Fund (M.E.P.

and K.A.G.) provided funding for this research. Fish fins that would otherwise bediscarded are repurposed for lionfish jewelry to promote research and aid in researchfunding (Fishgirl Fashion).

Data availabilityRaw data are available upon request from the corresponding author. Summary data,in .xls format, are available from the Dataverse repository: https://doi.org/10.7910/DVN/HGALDO.

Supplementary informationSupplementary information available online athttp://jeb.biologists.org/lookup/doi/10.1242/jeb.197905.supplemental

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