www.elsevier.com/locate/jembe

Journal of Experimental Marine Biolog

Dietary selectivity for the toxic cyanobacterium Lyngbya majuscula

and resultant growth rates in two species of opisthobranch mollusc

Angela Capper a,*, Ian R. Tibbetts a, Judith M. O’Neil a,b, Glendon R. Shaw c

a Centre for Marine Studies, School of Life Sciences, The University of Queensland, Brisbane, Queensland 4072, Australiab University of Maryland, Centre for Environmental Science, Cambridge, MD 21673, USA

c National Centre for Environmental Toxicology (EnTox), The University of Queensland, Kessels Road,

Coopers Plains, Queensland 4108, Australia

Received 29 November 2004; received in revised form 18 July 2005; accepted 16 October 2005

Abstract

Trophodynamics of blooms of the toxic marine cyanobacterium Lyngbya majuscula were investigated to determine dietary

specificity in two putative grazers: the opisthobranch molluscs, Stylocheilus striatus and Bursatella leachii. S. striatus is

associated with L. majuscula blooms and is known to sequester L. majuscula metabolites. The dietary specificity and

toxicodynamics of B. leachii in relation to L. majuscula is less well documented. In this study we found diet history had no

significant effect upon dietary selectivity of S. striatus when offered a range of plant species. However, L. majuscula chemotype

may alter S. striatus’ selectivity for this cyanobacterium. Daily biomass increases between small and large size groups of both

species were recorded in no-choice consumption trials using L. majuscula. Both S. striatus and B. leachii preferentially

consumed L. majuscula containing lyngbyatoxin-a. Increase in mass over a 10-day period in B. leachii (915%) was significantly

greater than S. striatus (150%), yet S. striatus consumed greater quantities of L. majuscula (g day�1) and thus had a lower

conversion efficiency (0.038) than B. leachii (0.081) based on sea hare weight per gram of L. majuscula consumed day�1. Our

findings suggest that growth rates and conversion efficiencies may be influenced by sea hare maximum growth potential,

acquisition of secondary metabolites or diet type.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Bursatella leachii; Feeding preference and deterrence; Lyngbya majuscula toxins; Sea hare; Stylocheilus striatus (formerly longicauda)

1. Introduction

Opisthobranch molluscs are known to sequester die-

tary-derived secondary metabolites from chemically

defended foods (Faulkner, 1984; Hay and Fenical,

1988; Avila, 1995; Rogers et al., 1995; Faulkner, 1997;

Yamada and Kigoshi, 1997; Pennings et al., 2001,

0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jembe.2005.10.009

* Corresponding author. Smithsonian Marine Station, 701 Seaway

Drive, Fort Pierce, FL 34949, USA. Tel.: +1 772 465 6630x106; fax:

+1 772 461 8154.

E-mail address: capper@sms.si.edu (A. Capper).

Masuda et al., 2002). Sequestered compounds can accu-

mulate in the skin or mantle (Ginsburg and Paul, 2001),

be incorporated as exudants (such as ink, opaline secre-

tions and mucous) (Paul and Pennings, 1991; Rogers et

al., 2000) or stored in the digestive gland (Winkler, 1969;

Watson, 1973; Pennings et al., 1993, Pennings and Paul,

1993a; Pennings and Carefoot, 1995; Pennings et al.,

1999). Retention of these compounds is thought to en-

hance defense strategies (Paul and Pennings, 1991;

Rogers et al., 1995, 2000; Ginsburg and Paul, 2001),

however the location of storage sites are not optimally

situated for defensive purposes (Pennings and Paul,

y and Ecology 331 (2006) 133–144

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144134

1993a,b) and may be present as the result of either a

passive or active mechanism of degradation (Avila,

1995) or detoxification (Pennings et al., 1996, 1999).

Whilst there is some debate regarding the function of

dietary sequestration, association with a chemically

defended host can at least provide the oligophagous

consumer with a source of food and a refuge from

predation (Hay, 1992; Rogers et al., 2000; Ginsburg

and Paul, 2001; Cruz-Rivera and Paul, 2002).

Stylocheilus striatus Quoy and Gaimard, 1832 (for-

merly Stylocheilus longicauda Quoy and Gaimard,

1824 (Rudman, 1999)) is a herbivorous grazer that

often exhibits a narrow diet breadth (Paul and Pennings,

1991) and sequesters secondary metabolites from its

algal host (Pennings and Paul, 1993a,b). Blooms of

the toxic cyanobacterium Lyngbya majuscula often

yield dense aggregations of S. striatus (Paul and Pen-

nings, 1991; Pennings and Paul, 1993b) although they

were also observed in large numbers in areas devoid of

L. majuscula growth where other food sources probably

supported their growth (personal observation; Turtle-

Trax, 2000).

Bursatella leachii de Blainville 1817 is a generalist

grazer of benthic cyanobacteria (Ramos et al., 1995)

and macroalgae (Paige, 1988), and may periodically

dominate marine communities. Little is known of se-

questration and secondary metabolite uptake mechan-

isms in B. leachii. Like S. striatus, B. leachii uses

chemical cues from L. majuscula to induce settlement

and rapid growth of larvae (Switzer-Dunlap and Had-

field, 1977; Paige, 1988). A high temporal and spatial

variability in such cues has been observed (Cardellina

et al., 1979; Hay and Fenical, 1988; Paul and Pennings,

1991; Nagle and Paul, 1999; Thacker and Paul, 2001).

Nagle et al., (1998) suggested that different chemotypes

of L. majuscula (which differ in composition or con-

centration of secondary metabolites) should differ dra-

matically in acceptability among potential grazers. L.

majuscula blooms in Moreton Bay, Australia often

differ in toxic metabolite expression (SEQRWQMS,

2001): Deception Bay predominantly produces debro-

moaplsyiatoxin (DAT); Eastern Banks produces lyng-

byatoxin-a (LTA); and Adams Beach produces DAT

and LTA in the early stages of bloom growth, followed

by LTA only during later growth phases (N.T. Osborne,

personal communication; Capper et al., unpublished

data). These blooms are genetically similar (T. Salmon,

personal communication) and differences in secondary

metabolite production may have a physico-environmen-

tal origin.

Large aggregations of both S. striatus and B. leachii

were observed residing in L. majuscula at the Adams

Beach site over a period of 4 months (personal obser-

vation). Both LTA and DAT were isolated from S.

striatus (Kato and Scheuer, 1975a; Mynderse et al.,

1977; Cardellina et al., 1979; Capper et al., 2005),

however the dynamics of algal toxin sequestration by

B. leachii has yet to be examined.

In this study we evaluate the dietary selectivity of S.

striatus with regard to chemotype using L. majuscula

collected from three study sites. To ascertain whether

diet history affected feeding choice, the feeding prefer-

ence of S. striatus previously exposed to L. majuscula

were compared to dnaı̈veT S. striatus, which had been

reared on a microalgal diet in a site devoid of L.

majuscula or macroalgal growth. To compare interspe-

cific variation in response to different diets we exam-

ined the feeding preferences of both S. striatus and B.

leachii using L. majuscula from the Adams Beach site

which contained two secondary metabolites (LTA and

DAT) in preliminary trials. Conversion of L. majuscula

to actual body mass in these two species was also

investigated.

2. Materials and methods

2.1. Study sites

Two areas were chosen as persistent dnuisanceTbloom sites (Fig. 1): Eastern Banks (EB) situated in

Eastern Moreton Bay (27826VS, 153822VE) in clear

oceanic waters; and Deception Bay (DB), situated

near the Western shore of Moreton Bay (27805VS,153809VE) where it often receives pulse run-off events

from terrestrial and riverine sources (Albert et al.,

2005). Adams Beach (AB) was chosen as the third

site (Fig. 1). It is situated in a sheltered bay (27851VS,153841VE) and is subjected to localised run-off during

storm events from North Stradbroke Island (personal

observation) and turbid run-off from Southern More-

ton Bay during periods of elevated rainfall. Blooms at

all sites produce either LTA or DAT or a combination

of both. All sites have extensive seagrass beds with

macroalgae locally dominating in places (Abal et al.,

2001).

2.2. Study organisms

S. striatus were collected from blooms (dbloomTStylocheilus) at the aforementioned sites throughout

the year. A second group of S. striatus was obtained

from a prawn pond at Bribie Island Aquaculture Re-

search Centre (BIARC). Sea hares had recruited in the

pond as larvae and as there was no apparent L. majus-

Fig. 1. Map of Lyngbya majuscula bloom sites in Moreton Bay with

blooms superimposed. Circle size provides approximate estimation of

bloom size between sites.

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144 135

cula growth, it is unlikely they were exposed to

L. majuscula (dnon-bloomT Stylocheilus). In the ab-

sence of L. majuscula or macroalgae it is assumed

this group of S. striatus was feeding on microalgae.

In the laboratory the dnon-bloomT group of S. striatus

was maintained on a diet of blanched lettuce prior to

feeding assays. B. leachii were collected from a bloom

at Adams Beach. Sea hares were maintained, and

experiments conducted, in separate 50-l tanks in a

closed circuit aquarium system at Moreton Bay Re-

search Station (MBRS), located in close proximity to

the Adams Beach site. Salinities were kept between 34

and 36 ppt and temperature consistent with ambient (24

8C) with 12:12 h light and dark cycles.

L. majuscula was collected when available at the

three different locations and maintained in aquaria at

MBRS. Macroalgae and seagrasses were collected lo-

cally from the shore at Dunwich, North Stradbroke

Island. A representative range of locally abundant rho-

dophytes, phaeophytes and chlorophytes were collected

on a daily basis to ensure only healthy robust samples

were used in feeding trials. Algal availability differed

seasonally leading to different choices being presented

in some experiments. Plant species tested included the

seagrasses Zostera capricorni and Halophila ovalis;

red alga Acanthophora spicifera; green alga Caulerpa

taxifolia; brown algae Dictyota dichotoma, Sargassum

flavicans and Lobophora variegata; and the cyanobac-

terium L. majuscula.

2.3. Chemical extraction

To determine concentrations of L. majuscula sec-

ondary metabolites, voucher samples from all assays

were frozen at �20 8C and then freeze-dried. Plant

material was then extracted three times in acetone,

sonicated and filtered under vacuum. Three replicates

were pooled and dried by rotary evaporation. Concen-

trations of secondary metabolites (mg kg�1) were de-

termined by HPLC-MS/MS using a PE/Sciex API 300

mass spectrometer equipped with a high flow electro-

spray interface (TurboIonspray) coupled to a Perkin

Elmer series 200 HPLC system. Separation was

achieved using a 150�4.6 mm Altima C18 column

(Alltech) run at 35 8C, with a mobile phase consisting

of 80:20 acetonitrile/hi-pure water containing 0.1%

formic acid and 2 mM ammonium formate at a flow

rate of 0.8 ml min�1. The flow was split post column

such that the flow to the mass spectrometer interface

was 250 Al min�1. Under these conditions, the reten-

tion times were 11.72 min for LTA and 9.3 min for

DAT. The mass spectrometer was operated in the pos-

itive ion, multiple ion monitoring mode. Ions monitored

with dwell times of 300 ms were 410.3 and 438.3

(M+H)+ for LTA and 543.3 for DAT. Quantification

was achieved by comparison to standards of DAT

(kindly provided by Dr. R.E. Moore, Department of

Chemistry, University of Hawaii at Manoa) and LTA

(Calbiochem) run under the same conditions. Using a

20-Al injection the detection limit for both toxins is

typically 0.01 mg l�1 in the extracted solution.

2.4. Multiple choice feeding assays (intra- and inter-

specific dietary selectivity)

Dietary preferences of S. striatus was determined by

conducting multiple choice feeding assays using

L. majuscula from the three different locations in Mor-

eton Bay, i.e. Adams Beach, Eastern Banks and Decep-

tion Bay (Table 1).

Sea hares were acclimatized for 48 h in either four

or five replicate (depending upon availability of sea

hares at time of testing) 50 l aquaria that were masked

to eliminate behavioural interactions. Sea hares were

Table 1

Sea hare multiple choice feeding assays using seasonally available plant species in Moreton Bay over a 24-h period

Sea hare species

and statusaLyngbya majuscula

locationbPlant speciesc No. of treatment

replicatesdNo. of control

replicates

Mean animal

weighte (gwwt)

dBloomT S. striatus DB (LTA and DAT) Lm, Dd, Zc, As, Lv, Sf, Ho 4 (5) 4 1.62 (F0.06)

dNon-bloomT S. striatus DB (LTA and DAT) Lm, Dd, Zc, As, Lv, Sf, Ho 4 (5) 4 1.48 (F0.04)

dBloomT S. striatus EB (LTA) Lm, Dd, Zc, As, Ho, Ct 5 (4) 5 1.80 (F0.25)

dBloomT S. striatus AB (LTA) Lm, Dd, Zc, As, Lv, Ct 5 (4) 5 3.10 (F0.30)

dBloomT B. leachii AB (LTA) Lm, Dd, Zc, As, Lv, Ct 5 (4) 5 2.20 (F0.25)

a dBloomT refers to Stylocheilus striatus collected from three Lyngbya bloom sites; dnon-bloomT refers to S. striatus collected from prawn ponds at

BIARC.b DB refers to Deception Bay; EB refers to Eastern Banks; AB refers to Adams Beach. Letters in parentheses refer to type of secondary metabolite

detected in L. majuscula sample post-exposure: LTA refers to lyngbyatoxin-a; and DAT to debromoaplysiatoxin.c Plant species used in experimental feeding trials according to seasonal availability. Lm, Lyngbya majuscula; Dd, Dictyota dichotoma; Zc,

Zostera capricorni; As, Acanthophora spicifera; Lv, Lobophora variegata; Sf, Sargassum flavicans; Ho, Halophila ovalis; Ct, Caulerpa taxifolia.d Number in parentheses are number of animals per treatment replicate.e Number in parentheses are standard error.

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144136

weighed (blotted wet weight, gwwt) and allocated to

groups with a similar mean wet weight per replicate

(Table 1). Variability in starting size of sea hares is due

to availability of animals at time of testing. Macroalgae

and seagrass samples were rinsed with clean seawater

(all visible epiphytes and particulate matter were re-

moved by hand) and blotted dry. The number of blots

was standardised for each algal species depending

upon its water retention capacity. The plant material

was then weighed (F0.5 g) and tied into a bundle

using monofilament fishing line (0.05 mm). Six or

seven simultaneous choices of plant material (depend-

ing upon seasonal availability at time of testing) were

randomly assigned to predetermined positions, secured

to a weight and placed on the tank base. To commence

the experiment sea hares were placed on the mid-floor

of the aquarium. Control tanks (those without sea

hares) with plant material were interspersed between

treatment tanks (those with sea hares) to determine

autogenic changes in the food mass (Peterson and

Renaud, 1989). A fixed-time design (Lockwood,

1998) of 24 h was allocated to each experiment, after

which the test was terminated and food items were

removed, re-blotted and re-weighed.

To determine the influence of diet history upon

dietary preference we compared wild S. striatus reared

on a diet of L. majuscula (dbloomT Stylocheilus) to

dnaı̈veT S. striatus reared on a suspected microalgal

diet (dnon-bloomT Stylocheilus) (Table 1). This multiple

feeding choice assay followed the protocol outlined

above for plant material preparation and removal.

2.5. Chronic exposure and biomass assays

To determine increase in biomass of sea hares fed an

exclusive diet of L. majuscula 10 sea hares of both

species were individually housed in 1 l jars over a 10-

day period (treatment), with seawater changed every

other day. Replicate jars containing L. majuscula and

no sea hares were randomly interspersed between treat-

ment jars to ascertain autogenic changes in the food

mass (control) (Peterson and Renaud, 1989). L. majus-

cula samples were obtained from Adams Beach. Sam-

ples were blotted and weighed (as per multiple feeding

choice assay) and placed into the treatment and control

containers. Plant material was removed, weighed and

replaced with fresh L. majuscula on days 2, 4, 6 and

8 during the 10-day exposure period.

A concurrent test was carried out to ascertain a

conversion rate of wet weight to dry weight biomass

using 10 S. striatus and 10 B. leachii. These animals

were split into two groups: Group 1 was fed L. majus-

cula on days 0–4 and then sacrificed 24 h later (allow-

ing time for gut evacuation) on day 5; Group 2 were fed

from days 0 to 9, with no food between days 4 and 5,

and sacrificed on day 10. Individuals were dried in an

oven (60 8C) for 5 h and re-weighed. The difference

between the final wet weight and the subsequent dry

weight for Group 1 on day 5 were used to calculate a

theoretical day 5 dry weight for Group 2.

2.6. Conversion efficiency

Conversion of algal mass (from a monospecific diet

of L. majuscula) to body mass (Econv) was extracted

from each species using data from the chronic exposure

and biomass assays as total change of sea hare mass

(Dshm). This was subjected to a conversion efficiency

calculation (Eq. (1)) that consisted of the total change

of sea hare mass divided by the quantity of L. majus-

cula consumed (control adjusted for autogenic changes

in mass, Lcons), the sum of which was divided by the

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144 137

experimental duration (t =10 days) (Rogers et al.,

1995).

Econv ¼Dshm=Lconsð Þ

t: ð1Þ

The effect of sea hare size was also assessed. Smal-

ler animals have a rapid growth rate and thus conver-

sion efficiency is expected to be greater. Mean initial

size of species were: (1) large B. leachii 1.077 gwwt(F0.080 SE); (2) small B. leachii 0.251 gwwt (F0.024

SE); (3) large S. striatus 3.246 gwwt (F0.058 SE); (4)

small S. striatus 0.112 gwwt (F0.005 SE).

2.7. Statistical analysis

Data were analysed using StatisticaR software.

Results of multiple choice feeding assays were analysed

using paired-sample t-tests on individual plants. t-tests

were chosen as they are very robust to departures from

normality. If significant differences occurred between

means for replicate treatment arenas and control arenas

then differential consumption and a preference was

Fig. 2. Stylocheilus striatus and Bursatella leachii multiple choice feeding a

(A) dbloomT (7) vs. dnon-bloomT(X) S. striatus from Deception Bay (cont

control (5), n =5; (C) S. striatus from Adams Beach (n) vs. control (5), n

Paired sample t-tests were used to analyse data. Asterisk (*) denotes a signific

data sets using Levene’s test. Lyngbyatoxin-a (LTA) and debromoaplysiatox

bars. n =number of replicates.

interpreted (Peterson and Renaud, 1989). Whilst their

use may be inappropriate for checking differences be-

tween feeding preferences within the same aquaria, the

technique is not inappropriate for testing for differences

in the same food between aquaria, since observations

from different tanks are independent (W. Venables,

personal communication). Levene’s homogeneity of

variance was used to ascertain normality of data.

Prior to analyses, data that deviated from normal dis-

tribution were transformed using either square root or

logarithmic transformations. To assess variance be-

tween L. majuscula chemotypes, factorial ANOVA

was used on individual (and thus independent) plant

species. To determine the influence of diet history

(dbloomT vs. dnon-bloomT) upon dietary preference t-

tests were used on control adjusted proportions of

individual plants. Biomass changes and consumption

of L. majuscula results were subjected to repeated

measures ANOVA and corresponding post-hoc analy-

sis, which allow comparisons among multiple non-in-

dependent treatments based on ranks (Conover, 1980).

Fisher’s LSD pair-wise comparison tests were used to

ssays. Data are mean proportion of plants consumed (blotted gwwt) by:

rol adjustedFS.E, n =4); (B) S. striatus from Eastern Banks (n) vs.

=5; and (D) B. leachii from Adams Beach (n) vs. control (5), n =5.

ant difference ( p b0.05). Homogeneity of variance was verified for all

in (DAT) concentration in L. majuscula is shown in mg kg�1 above

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144138

compare the means. A comparison of the gain in dry

weight between the two species from days 5 and 10 was

carried out using single factor ANOVA. Conversion

efficiency data was square-root transformed to meet

parametric criterion and subjected to factorial ANOVA.

3. Results

3.1. Multiple choice feeding assays

Two populations of S. striatus were examined and

compared: those collected from a L. majuscula bloom

(dbloomT Stylocheilus) and those unlikely to have been

exposed to L. majuscula (dnon-bloomT Stylocheilus).

Paired t-tests were used to determine differences in

individual plant consumption (control adjusted) be-

tween populations. Diet history had no significant effect

on dietary selectivity (t-test, p N0.01 for all alga, Fig.

2A). L. majuscula used in this assay contained both

LTA (0.24 mg kg�1) and DAT (0.02 mg kg�1).

In S. striatus and B. leachii multiple choice feeding

assays, L. majuscula was the most preferred food (t-

test, p b0.0001, Fig. 2B, C, D) compared to all other

6

5

4

3

2

1

0

7S. striatus weight(A)

Mea

n bl

otte

d w

et w

eigh

t (g)

L. majuscula consumed by S. striatus (B)

DayDay 0 Day 2 Day 4

6

5

4

3

2

1

0

7

n=10

*14.32

0.69

p=0.1

p=0.00

Fig. 3. Small Stylocheilus striatus and Bursatella leachii chronic exposure

striatus (0.11F0.01 SE) and B. leachii (0.25F0.02 SE) over a period of 10

by S. striatus and B. leachii over a period of 10 days (control adjustedFSE)

indicate a significant difference ( p b0.05) using Fisher’s LSD pair-wise co

toxin-a in mg kg�1 (n/d=not detected).

plants tested at Eastern Banks and Adams Beach.

Whilst S. striatus from Eastern Banks also consumed

small quantities of C. taxifolia (t-test p =0.035; Fig.

2B) and H. ovalis (t-test, p=0.036, Fig. 2B), both S.

striatus and B. leachii from Adams Beach consumed

only L. majuscula ( p b0.0001, Fig. 2C, D) suggesting

there were no interspecific differences in choice of

preferred food.

Voucher samples of L. majuscula used in S. striatus

multiple choice feeding assays yielded different concen-

trations of LTA for each location: Deception Bay (0.24

mg kg�1, Fig. 2A); Eastern Banks (56.98 mg kg�1, Fig.

2B); and Adams Beach (28.37 mg kg�1, Fig. 2C,D).

DAT was also detected in Deception Bay L. majuscula

(0.02 mg kg�1, Fig. 2A). Intraspecific differences were

observed in L. majuscula consumption from the three

locations (Deception Bay*Eastern Banks*Adams Beach

ANOVA F73.08, df2, p b0.0001, Fig. 2A, B, C).

3.2. Chronic exposure and biomass assays

All sea hares increased in biomass during laboratory

trials between days 0 and 10 (Fig. 3A and Fig. 4A).

B. leachii weight

*

L. majuscula consumed by B. leachii

of testDay 6 Day 8 Day 10

*

n=9

*n/d

0.33

0.16

19

02

and biomass assays. (A) Mean blotted wet weight (gwwt) of small S.

days. (B) Mean blotted wet weight (gwwt) of L. majuscula consumed

. Repeated measures ANOVAwere used to analyse data. Asterisks (*)

mparisons. Numbers above bars represent concentrations of lyngbya-

Day of test

Mea

n bl

otte

d w

et w

eigh

t (g)

Day 0 Day 2 Day 4 Day 6 Day 8 Day 10

10

6543210

789

S. striatus weight B. leachii weight(A)

L. majuscula consumed by S. striatus L. majuscula consumed by B. leachii

10

6543210

789

(B)

*

**

*

*

*

*n=10n=9

n/d

14.32

0.69

0.33

0.16

p<0.001

p<0.001

Fig. 4. Large Stylocheilus striatus and Bursatella leachii chronic exposure and biomass assays. (A) Mean blotted wet weight (gwwt) of large S.

striatus (3.25F0.06 g SE) and B. leachii (1.08F0.08 g SE) over a period of 10 days. (B) Mean blotted wet weight (gwwt) of L. majuscula

consumed by S. striatus and B. leachii over a period of 10 days (control adjustedFSE). Repeated measures ANOVA was used to analyse data.

Filled symbols represent significant differences ( p b0.05) between species increase in biomass using Fisher’s LSD pair-wise comparisons. Asterisks

(*) indicate a significant difference ( p b0.05) between rate of daily biomass increase between species using Fisher’s LSD pair-wise comparisons.

Numbers above bars represent concentrations of lyngbyatoxin-a in mg kg�1 (n/d=not detected).

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144 139

Whilst small B. leachii (0.25F0.02 g SE) and S.

striatus (0.11F0.005 g SE) differed in initial wet

weights at the start of the experiment (ANOVA,

F28.33, df1, p b0.0001), their final weights were not sig-

nificantly different (ANOVA, F3.703, df1, p =0.0713).

Likewise, the daily rate of mass change� dx�ymass

t

�was not

significantly different between species over the trial

period (ANOVA, F2.69, df1, p =0.119) (Fig. 3A). There-

fore, we can say that the average rate of growth between

the species in this size range was approximately the

same over the trial period. However, a significant dif-

ference in this variable was noted between days 8 and

10 using Fisher’s LSD post-hoc analysis (Fig. 3A).

Whilst daily increases in mass were not dissimilar,

small S. striatus actually consumed significantly larger

amounts of L. majuscula throughout the trial period

(ANOVA F6.23, df4, p =0.0002) (Fig. 3B). No correla-

tion was observed between consumption rates and LTA

concentrations for small S. striatus (r2=0.067,

p =0.674) or small B. leachii (r2=0.200, p =0.450).

Large B. leachii increased more rapidly in biomass

on a daily basis when fed a mono-specific diet of L.

majuscula compared to large S. striatus (ANOVA

F14.29, df4, p b0.0001) (Fig. 4A). However, large S.

striatus consumed significantly more L. majuscula

than large B. leachii (ANOVA F79.87, df4, p b0.0001)

until day 8 when biomass increase reached a steady

state, apparently due to cessation of feeding (Fig. 4B).

The final weights of both species were not significantly

different (ANOVA, F0.29, df1, p =0.596). A single mor-

tality occurred in both small B. leachii and large S.

striatus during experimental trials; however, it is not

clear if this can be attributed to this particular diet or to

the levels of secondary metabolites contained within L.

majuscula. No correlation between consumption rates

and LTA concentrations were observed for large S.

striatus (r2=0.008, p =0.890) or large B. leachii

(r2=0.485, p =0.192).

A concurrent trial to show conversion to actual body

mass (wet weight to dry weight, gdwt) of sea hares fed

L. majuscula from the Adams Beach site was carried

out (Fig. 5). S. striatus and B. leacheii were sacrificed

at day 10. Similar amounts of L. majuscula were con-

sumed between species on days 0–4 (ANOVA, F2.17,

0.5

0.4

0.3

0.2

0.1

0

12

10

8

6

4

2

0

Day 5 Day 10

L. majuscula consumed by S. striatus

L. majuscula consumed by B. leachii

Dry

wei

ght o

f sea

har

e (g

dwt)

Blo

tted

wet

wei

ght o

f L. m

ajus

cula

(g w

wt)

Day of test

S. striatus dwt biomass

B. leachii dwt biomass

*

Day 0-4 Day 5-9

4.35

3.35

Fig. 5. Bursatella leachii and Stylocheilus striatus conversion of wet weight to dry weight biomass (n =5). Consumption rates of L. majuscula

(mean blotted wet weight gwwtFSE) and conversion to dry weight body mass (gdwt). Day 5 dry weight of B. leachii and S. striatus is based upon

dry weights obtained from a concurrent 5-day termination experiment. Dry weight of sea hares on the primary vertical axis and blotted wet weight

of L. majuscula on secondary vertical axis are compared using single factor ANOVA with asterisk (*) indicating significantly different values

( p b0.001). Numbers above bars represent concentrations of lyngbyatoxin-a in mg kg�1.

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144140

df1, p =0.179) compared to consumption rates in the

same animals between days 5 and 9 (ANOVA, F27.45,

df1, p =0.0008). However, there was no significant

difference between dry weight mass of the two species

at the termination of the trial (ANOVA, F0.19, df1,

p =0.673).

3.3. Conversion efficiency

Conversion efficiencies were significantly higher in

B. leachii than S. striatus (ANOVA, F244.96, df1,

p b0.0001) with significant differences also observed

between sea hare size (ANOVA, F12.99, df1, p =0.001)

(Fig. 6).

Fig. 6. Bursatella leachii and Stylocheilus striatus conversion of algal mass

mono-specific diet of L. majuscula. Data are given as mean proportional

consumed/day (FSE) (Rogers et al., 1995). Data have been transformed

significant differences in pair-wise comparisons using Fisher’s LSD and Tu

4. Discussion

4.1. Multiple choice feeding assays

Consumption experiments demonstrated a narrow

diet breadth for both S. striatus and B. leachii amongst

the range of plant species tested. While some brown,

green and red algae were consumed, both species pri-

marily consumed L. majuscula. Suggestions that B.

leachii is a generalist feeder with a wider dietary rep-

ertoire (including Lyngbya, Chrysophyta, Rhodophyta,

Tracheophyta) (Paige, 1988) were not borne out in this

study. Whilst, S. striatus is a purported L. majuscula

specialist (Pennings et al., 1996; Nagle and Paul, 1999),

to body mass (n-10). Conversion efficiency in two size groups fed a

change in S. striatus and B. leachii wet weight/(gwwt) L. majuscula

to meet parametric criterion for factorial ANOVA. Letters refer to

key’s HSD.

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144 141

it can occasionally be found on other plant species once

its host supply is depleted (Pennings and Paul, 1993a).

The ability to actively sequester secondary metabolites

from L. majuscula and other host plant species (Pen-

nings and Paul, 1993a,b) may confer some ecological

advantages to S. striatus. Whilst some L. majuscula

secondary metabolites can actively stimulate feeding

(Nagle et al., 1998; Cruz-Rivera and Paul, 2002), at

higher concentrations these compounds may deter feed-

ing (Pennings and Paul, 1993b). An ability to detect

changes in secondary metabolite concentrations could

potentially reduce negative post-ingestive conse-

quences for the consumer (Freeland and Janzen, 1974).

Intraspecific differences were observed in feeding

behaviour from S. striatus collected at sites Adams

Beach, Deception Bay and Eastern Banks in relation

to their L. majuscula chemotypes (i.e. those containing

different secondary metabolites). L. majuscula from

Eastern Banks was consumed in higher quantities

than either Deception Bay or Adams Beach, yet it

contained the highest concentrations of LTA, leading

us to infer LTA did not act as a feeding deterrent to S.

striatus collected from this site. L. majuscula from

Deception Bay, which contained the lowest concentra-

tions of LTA, but also contained DAT, was consumed in

much lower quantities during the 24-h test period and

ranked second in preference to the brown algae D.

dichotoma by both dbloomT and dnon-bloomT S. stria-tus. The cumulative effect of the two secondary meta-

bolites or the presence of DAT itself (or other

compounds not tested for) may have deterred feeding

in S. striatus. However, this seems unlikely as Nagle et

al. (1998) found that DAT had no deterrent effect upon

S. striatus at natural concentrations. S. striatus is capa-

ble of detoxifying both DAT and LTA into less harmful

acetates (Kato and Scheuer, 1975a,b; Pennings et al.,

1996). The capability to store or detoxify compounds

allows it to continually consume L. majuscula with no

apparent deleterious effects allowing this species to

capitalise on an ephemerally abundant food source.

Interestingly, both dbloomT and dnon-bloomT S. striatusfollowed the same feeding pattern, suggesting recent

diet history does not affect preference. High consump-

tion levels of D. dichotoma highlight the ability of this

herbivore to switch to other host species if its preferred

host becomes unavailable or unpalatable due to intol-

erable concentrations of active compounds or synergis-

tic effects between multiple secondary metabolites.

Host switching could also be a strategy to maximize

nutritional intake. Whilst the broader dietary spectrum

reported for B. leachii may result in a more nutritionally

rich diet, it may also be a means of preventing harmful

additive accumulations of secondary metabolites. When

fed a monospecific diet of L. majuscula, B. leachii

transfers LTA from body tissue to body secretions,

such as ink and faecal matter (Capper et al., 2005).

Excretion rather than storage of secondary metabolites

may help prevent autotoxicity in this species.

4.2. Chronic consumption, biomass increase and

conversion efficiency

The nutritional value of L. majuscula to S. striatus

and B. leachii was measured using growth (wet

weight, gwwt; and dry weight, gdwt) and conversion

efficiencies as indicators of performance. It has been

suggested that S. striatus is a specialist feeder of L.

majuscula (Pennings et al., 1996; Nagle and Paul,

1999). If this is the case, one might then predict that

S. striatus would exhibit better growth rates and con-

version efficiencies than a grazer with a wider dietary

repertoire, e.g. B. leachii (Paige, 1988). Differences in

growth rates were indeed observed between size

groups (small vs. large) of S. striatus and B. leachii.

Whilst small animals of both species maintained an

equivalent growth rate, consumption of L. majuscula

by S. striatus was much greater. Large S. striatus also

consumed significantly greater amounts of L. majus-

cula on days 0–8 compared to large B. leachii, yet B.

leachii increased more rapidly on a daily basis in wet

weight mass (gwwt) between days 0 and 8. Therefore, it

appears that S. striatus needs to consume a greater

biomass of L. majuscula to gain an equivalent mass

increase to B. leachii. The reason that no L. majuscula

was consumed by large S. striatus on days 8–10 is

unknown. A satiation point may have been reached or

body mass may have already increased to maximum

size for this organism. A corresponding decrease in

biomass was observed between days 8 and 10 con-

firming that high levels of L. majuscula consumption

are required to maintain overall body mass in S. stria-

tus. There was no correlation between this lack of

consumption and LTA concentrations. A new batch

of L. majuscula was collected for this trial on day

8 from the same site and whilst it contained equivalent

concentrations of LTA to previous samples, it is pos-

sible that it may have contained other unidentified

secondary metabolites, to which S. striatus may have

been sensitive. Malyngamides produced by L. majus-

cula in Guam have resulted in suppressed growth rates

in S. longicauda (Pennings and Paul, 1993b). Whilst

this study was limited to identifying the two predom-

inant toxins produced by L. majuscula in Moreton

Bay, other compounds may have been present which

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144142

deterred feeding in S. striatus (with a resultant de-

crease in mass), but not B. leachii.

In a test to determine conversion of L. majuscula to

actual body tissue (i.e. dry mass, gdwt) we found no

significant difference in dry weights between species

on day 5 or day 10 even though a significant variation

was observed between levels of L. majuscula consump-

tion in the later stages of the trial. Large B. leachii

increased in blotted wet weight (gwwt) body mass by

915% between days 0 and 9 (mean startingwwt1.09F0.07 g SE; mean finalwwt 9.98F0.02 g SE,

whereas large S. striatus only showed a 150% increase

(mean startingwwt 2.61F0.13 g SE; mean finalwwt3.92F0.87 g SE). The actual dry mass of both animals

at the end of 10 days (i.e., conversion of known quantity

of L. majuscula to actual tissue mass) was 9.5% of the

final wet weight for S. striatus (meandwt 0.372F0.03 g

SE) and 3.13% for B. leachii (meandwt 0.312F0.07 g

SE). Thus, we infer that whilst B. leachii consumed a

greater quantity of L. majuscula, this was not converted

to tissue mass and might be associated with increased

tissue fluid levels, mucous production or egg production.

Using the conversion efficiency equation suggested

by Rogers et al. (1995) Econv ¼ Dshm=Lconsð Þt

, all sea hares

had positive conversion efficiencies in the laboratory.

Both small and large B. leachii have similar conver-

sion efficiency levels (prior to statistical transforma-

tion) of 0.081 and 0.080, respectively, whereas small

S. striatus and large S. striatus were much lower at

0.038 and 0.022, respectively. Therefore, it appears

that whilst large and small S. striatus may consume

greater quantities of L. majuscula in general, they

have lower growth rates and conversion efficiencies

than B. leachii when wet weight mass is used. This

may then lead us to question why S. striatus is the

dspecialistT in this case. This may be related to the size

of the sea hare. Whilst body size was similar for small

S. striatus and B. leachii during the test period, S.

striatus may have reached its maximum size during

the test period. B. leachii on the other hand do not

reach sexual maturity until 2–3 months post-hatching

(Paige, 1986, 1988) and may still have been in a

dgrowing phaseT, resulting in higher conversion effi-

ciencies to sustain maximum growth potential. Mea-

suring conversion efficiencies for both species until a

growth plateau is reached may assist in the interpre-

tation of these results.

The acquisition of secondary metabolites from a host

plant may come at a cost. Storage and/or detoxification

mechanisms require a high energy expenditure that can

greatly reduce net energy gained from nutrition (Porter

and Orcutt, 1980) and potentially limit S. striatus’

ability to grow (Pennings and Carefoot, 1995). It is

widely known that S. striatus is able to actively seques-

ter many secondary metabolites from L. majuscula

(Paul and Pennings, 1991; Pennings and Paul,

1993a,b; Nagle et al., 1998; see reviews by Faulkner,

1984; Avila, 1995; Nagle and Paul, 1999) including

DAT and LTA (Kato and Scheuer, 1975a,b; Capper et

al., 2005), which are often compartmentalized in the

digestive gland (Watson, 1973; Pennings et al.,

1993a,b; Pennings and Carefoot, 1995; Pennings et

al., 1999). The cost/benefit ratios of different accumu-

lated compounds are likely to vary significantly be-

tween species (Rogers et al., 1995) and it may be less

energetically expensive for B. leachii to excrete or egest

sequestered metabolites than to detoxify them (Pen-

nings and Paul, 1993a; Capper et al., 2005). A further

compounding factor may be food type. Whilst S. stria-

tus exhibited low conversion efficiencies for L. majus-

cula, it may be even lower for other plant species.

Conversion efficiencies for the range of plants tested

both species for is not known.

Whilst the high degree of variability in LTA and

DAT recorded during these experiments may have af-

fected the results presented, we regarded it as important

to expose both species of sea hares to levels that are

reflected in the environment, i.e. secondary metabolites

can vary dramatically both temporally and spatially in

L. majuscula blooms in South East Queensland, Aus-

tralia (personal observation; N.T. Osborne, personal

communication). Therefore, the findings of this study

have allowed us to gauge the impacts of daily changes

in the two secondary metabolites investigated (LTA and

DAT) upon sea hare feeding patterns. Whilst S. striatus

did show significant intraspecific differences between

L. majuscula chemotypes, no correlation was found

between LTA concentration and consumption patterns

in either species during testing.

In summary, this study demonstrates that while other

macrophytes may be consumed, L. majuscula is a

preferred food choice for the two species of sea hare

tested. Both species preferentially consumed and grew

well on an exclusive diet of L. majuscula. Whilst, B.

leachii exhibited a greater wet weight daily mass in-

crease, this may be associated with either fluid retention

or mucous production as tissue dry weights were lower

than S. striatus. Growth rates and conversion efficien-

cies may be influenced by sea hare age and maximum

growth potential, acquisition of secondary metabolites

resulting in higher energetic costs or be related to

specific food type and its nutritional content. Further

studies of purported dspecialistT and dgeneralT L. majus-cula grazers are required that incorporate not only

A. Capper et al. / Journal of Experimental Marine Biology and Ecology 331 (2006) 133–144 143

dietary preference and secondary metabolite acquisi-

tion, but also growth rates at different life stages on

monospecific and alternative diets to fully understand

grazer bloom dynamics.

Acknowledgements

The authors wish to thank W. Knibb and D. Willett

for supply dnaı̈veT Stylocheilus striatus from prawn

ponds at Bribie Island Aquaculture Research Centre

(BIARC); K. and K. Townsend, S. Litherland for help

at Moreton Bay Research Station (MBRS); G. Eagle-

sham and N. Osborne for help with toxicology work at

National Research Centre for Environmental Toxicol-

ogy (EnTox); J. Phillips (CMM, University of Queens-

land) for identifying marine plant species used in trials;

and N. Khan (University of Queensland) and W. Ven-

ables (CSIRO, Queensland) for help and guidance with

statistics. This project has been supported by: Centre

for Marine Studies Moreton Bay Research Station

Scholarship; the South East Queensland Regional

Water Quality Management Strategy (SEQRWQMS)

award of $25,000 for biotic interactions work; research

grants and International Post-Graduate Scholarships

from the Department of Zoology and Entomology

and the Centre for Marine Studies, School of Life

Sciences, University of Queensland, Brisbane, Austra-

lia; and R. Ritson-Williams for feedback and valuable

critique of the manuscript. [SS]

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