Australian Journat of Eeotogy (1998) 23, 129-137

Evaluation of the toxicity of eggs, hatchlings and tadpolesof the introduced toad Bufo marinus (Anura: Bufonidae) tonative Australian aquatic predators

MICHAEL R, CROSSLAND AND ROSS A, ALFORD*Department of Zoology and Tropical Ecology, School of Biological SciencesJames Cook University, Townsville Qld 4811, Australia

Abstract The early life history stages of anurans in the Family Bufonidae often possesschemicals that are noxious or toxic to predators. Predators with no evolutionary history ofexposure to bufonids may be particularly susceptible to these toxins. We conducted a seriesof laboratory experiments to investigate the toxic effects of eggs, hatchlings and tadpolesofthe introduced toad, Bufo marinus (Linnaeus), on native Australian aquatic predators.There was considerable interspecific and intraspecific variation in these effects. Bufo marinuswere highly toxic to some predator species, but were readily consumed by other specieswithout apparent ill effect. Interspecific variation in toxic effects was not related to preda-tor feeding mode or the number of B. marinus ingested by predators, and there was no clearpattern of distribution of vulnerability among species within higher taxa. Intraspecific vari-ation in responses to toxins may result from individual variation in the resistance of predatorsto B. marinus toxins, or from individual variation in toxicity among B. marinus. Some nativespecies adversely affected by B. marinus appeared unable to detect and avoid B. marinustoxins. This may result from a general inability to assess the toxicity of food items or froma lack of evolutionary exposure to B. marinus toxins.

Key words: exotic species, impact, mortality, native species.

INTRODUCTION

Numerous plant and animal species have noxious ortoxic chemicals in their tissues (e.g. Fraenkel 1959;Mosher et at. 1964; Pawlik et at. 1986; Harvell et at.1988). However, these chemical defences may notalways be effective deterrents (Brodie 1968; Licht &Low 1968; Pawlik et at. 1986; Robineau et at. 1991).One factor that is likely to influence a species' behavi-oural and physiological responses to such chemicals isits evolutionary history of exposure to them. Speciesthat regularly incorporate toxic food items in their dietmay evolve detoxification mechanisms (Ehrlich &Raven 1964; Krieger et at. 1971; Whittaker & Feeny1971; Blau et at. 1978; Scriber 1978). In contrast,species that are naive to such chemicals may be unableto detect (Speiser et at. 1992) or detoxify them (Blauetal. 1978; Ryan & Byrne 1988; Gilbert 1994).

In the present study we investigated the toxicity ofthe eggs, hatchlings and tadpoles ofthe introduced toadBufo marinus (Anura: Bufonidae) to native Australian

*Corresponding author.Accepted for publication June 1997.

aquatic predators. Bufo marinus is native to Centraland tropical South America (Zug & Zug 1979) but wasintroduced to Australia in 1935 (Mungomery 1935),Many species of Bufo are unpalatable and/or toxic topredators during their early life history stages (e.g, Voris& Bacon 1966; Licht 1967, 1968; Wassersug 1971;Kruse & Stone 1984). However, little is known ofthetoxicity of B. marinus eggs, hatchlings and larvae tonative Australian aquatic predators. Chemical analyseshave verified the presence of bufodienolides in B. mar-inus eggs and larvae (Flier et at. 1980; Akizawa et at.1994), and previous studies indicate that these earlylife history stages are toxic to some native Australianpredators but not to others (Covacevich & Archer1975; Hutchings 1979; Hamley & Georges 1985;Hearnden 1991). As no species of Bufo are nativeto Australia, and no Australian frog is known topossess chemical defences based on steroidal bufo-genins and bufotoxins (Tyler 1987), native aquaticpredators may be particularly susceptible to B. marinustoxins.

Bufo marinus breeds in both temporary and perman-ent water bodies in northern Queensland, Australia, somany predators are exposed to their eggs, hatchlingsand larvae. The aim of this study was to assess the

130 M. R. C:ROSSI.AND AND R. A. AI .FORD

to.xicity of B. iiiariiius eggs, hatchlings and tadpoles tonative aquatic predators that are likely to encounterB. marinus in nature.

METHODS

Experimental protocol

We conducted a series of laboratory experimentsat James Cook University, Townsville (19''20'S,146°46'E) and at Heathlands Reserve, Cape YorkPeninsula (1 l''45'S, 142°35'E) between January 1992and June 1994. Experiments were conducted in either440 mL plastic containers (containing 350 mL water)or 10 L buckets (containmg 6 L water). The size of thecontainer depended on the size of the predator beingtested. The predator species tested are listed in Tables1-3. Containers in the fish, dytiscid, belostomatid,nepid, snail and crustacean predation experiments werecovered with lids to prevent predator escape.Containers in the crustacean, snail, leech and fishpredation experiments were also constantly aeratedfrom a common source to ensure a sufficient supply ofoxygen to the predators.

For each experiment, containers were positioned ona benchtop in a 3 x j ; array (;; = up to 10 replicates)and filled to equal levels from a common water source.Each experiment was a randomized block design andconsisted of three treatments: (1) one predator percontainer (predator control); (2) one predator percontainer plus B. marinus eggs, hatchlings, or tadpoles(predator exposure); and (3) B. marinus eggs, hatch-lings, or tadpoles with no predator (B. marmus control).Treatments were randomly assigned to containerswithin blocks, with the constraint that no adjacentcontainers had the same treatment.

Predators exposed to B. marinus (treatment 2) wereoffered either 50 B. marinus eggs, 10 B. marinus hatch-lings (3.2-4.4 mm snout-vent length; Gosner (1960)stages 21-24) or 10 B. marinus tadpoles (4.0-8.0 mmsnout-vent length; Gosner (1960) stages 25-28).Predators and B. marmus were allocated to containerswithin treatments by using a random number table.All predators and prey were used only once duringexperiments.

All predators (except crayfish Cherax quadricarinatusvon Martens) used in the experiments were collectedfrom local temporary and permanent water bodiesusing dip-nets and baited traps. Cherax quadricarinatuswere obtained from breedmg stock maintained at theJames Cook University aquaculture facility. The fertil-ized B. marinus eggs and the tadpoles used in experi-ments were collected from local temporary andpermanent water bodies. The B. marinus hatchlingsused in experiments were initially collected as eggs fromlocal water bodies and were reared to hatchling stage

in the laboratory. We starved potential predators ofhatchlings and tadpoles for 24 h prior to experiments.We did not starve potential egg predators because it wasdifficult to predict when we would obtain eggs, andbecause of the short duration of the egg stage. Allpredators were measured using vernier callipers priorto each experiment. Predator sizes are given as totallengths, except for tadpoles (snout-vent length), crabs(carapace width) and leeches (wet weight). Prior toeach hatchling and tadpole predation experiment, onecontainer in the predator exposure treatment waschosen at random and all B. marmus hatchlings ortadpoles in it were measured using vernier callipers(snout-vent length) and staged (Gosner 1960). Becauseanimals were randomly allocated to containers, thisconstituted a random subsample of the availableB. marinus.

Predator condition and the number of B. marmuspresent in each experimental container were monitoredat 12-hour intervals. Egg and hatchling experimentscontinued for the duration of these early life historystages: eggs were exposed to predators until hatchingcommenced (36-48 h), while hatchlings were exposedto predators until they reached Gosner (1960) stage 25(24-36 h). Tadpoles were exposed to predators for 72 h.Predators that consumed eggs, hatchlings or tadpoleswithout apparent ill effect were kept for 3 days after theexperiment was completed to monitor their condition.

Statistical analyses

We analysed our data in several stages. We initiallytested the overall hypothesis that exposure to the eggs,hatchlings, or tadpoles of B. marmus affected predatorsurvival using three Chi-squared contingency tests.Each of these tests examined the numbers of individualpredators, summed across all predator species, in a2 X 2 table. The tables classified predators by exposureto B. marinus (not exposed/exposed) and predatorsurvival (survived/did not survive for 72 h following endof experiment). Testing across all predator speciesensured maintenance ofthe type I statistical error ratefor the general hypotheses that exposure to B. marinusof each ofthe three stages affected predator survival.

When one of the overall tests showed significanteffects of exposure to B. marmus on predator survival,we proceeded to a second stage in which we performedseparate contingency table analyses for each predatorspecies. These analyses tested the hypothesis that eachspecies was affected by B. marinus at a significance levelof a = 0.05. We did not adjust these tests for multiplecomparisons because they were conducted to separatelytest the hypothesis that each predator species was sus-ceptible to B. marinus with a comparisonwise error rateof 0.05, and followed an overall test ofthe significanceof the general hypothesis that some predators areaffected (Hochberg & Tamhane 1987). The statistical

TOXICITY OF BUI'U TO AQUATIC PREDATORS 1 31

analysis for each predator species used one of threetechniques. If predators did not experience anymortality following exposure to B. niarinus, we did notstatistically compare xhe mortality rates of predators incontrol and B. niarinus treatments. When all individualsof a predator species consumed B. ntarinus and therewas predator mortality, we compared the numbers ofliving and dead predators in control and B. niariinistreatments by using 2 X 2 Fisher's Exact tests. Whenonly some individuals of a predator species consumedB. ntarinus and there was predator mortality, we com-pared the numbers of living and dead predators in thefollowing categories: (i) control animals, (ii) animalsexposed to B. niannus that consumed at least oneB. ntarinus, and (iii) animals exposed to B. marimis thatdid not consume any B. marimts, by using 2 x 3Fisher's Exact tests.

In addition, we tested whether invertebrate andvertebrate predators differed in susceptibility to B. iiiar-inus toxins by comparing the numbers of invertebrateand vertebrate species that experienced significant mor-tality after feeding on B. inarinus with the numbers of

invertebrate and vertebrate species that consumedB. iitarimis without ill effect, by using a 2 x 2 Fisher'sExact test. We also tested the effect of predator feedingtechnique on the susceptibility of predators to B. ntar-inus toxins. In this 2 X 3 Fisher's Exact test, predatorswere categorized by their response to B. marintis(significant mortality/no ill effect) and their feedingtechnique (bite then chew; pierce then suck; swallowwhole). Classification of predator feeding techniquefollowed Britton (1979), Woodward et al. (1979),Williams (1980) and our personal observations.

All analyses were performed using SAS PROCFREQ (SAS Institute Inc. 1987).

RESULTS

Results from the B. ntarinus egg, hatchling and tadpolepredation experiments are presented in Tables 1-3, res-pectively. Mortality of B. marintis in control treatmentsduring all experiments was minimal. Egg mortality incontrols was apparently due to fungal infection. The

Table 1. Predators tested with Bufo marinus eggs

Predator

NepidaeRanarra sp."Laccotrephes sp."

DytiscidaeCv6iSttrsp.(L)"Hydaticus sp.(L)"Cybisier godeffroyi "Hydaticus vittatus^Sandracottus bakezvelli "

BelostomatidaeLethocerus insulamts

OdonataTrapezosrignia sp.(L)'̂

CrustaceaMacrobrachium sp."Holrhuisana sp.^Cherax quadricarinams^

GastropodaAustropeplea lessoni^

AnuraLitoria bicolor^Litoria rubella^Litoria infrafrenata^Litoria nigrofrenara "Litoria albogutiata^Limnodynastes ornatus^

PiseesCraterocephalus stercusmuscaritn

Feedingmode

PSPS

PSPSBCBCBC

PS

BC

BCBCBC

BC

BCBCBCBCBCBC

SW

10(0)1(1)

10(6)5(0)4(4)10(10)4(4)

10(10)

10(0)

5(4)10(10)10(10)

10(10)

10(10)10(3)10(10)10(6)10(10)10(9)

10(1)

Mean predator Gosner Numbersize (mm) ±SD stage eggs eaten

44.0 ± 16.166.1

28.0 ± 8.120.8 ±4.531.8 ± 5.414.1 ±0.611.5 ± 2.4

66.3 ± 3.2

23.5 ± 3.0

33.1 ± 11.628.2 ± 10.785.1 ± 3.9

23.8 ±2.6

10.5 ±0.75.7 ± 0.6

12.5 ± 1.218.2 ± 1.519.0 ± 1.911.2 ± 1.4

40.4 ± 5.0

31-402526-3126-3525-3230-38

07

0-2503-26

23-5016-25

1-16

0-410-5048-50

1-2

0.5-20-21-30-21-70-4

0-1

Predatormortality {%) P

000

100

100301006010090

——

—

< 0.0001

< 0.0001

0.0009

< 0.0001

< 0.0001

< 0.0001

< 0.0001

10 0.1000

Overall x '= 65.84, d.f. = 1, P< 0.0001. H, Heathlands; T, Townsville. BC, bite then chew; PS, pierce then suck;SW, swallow whole, n = number of replicates; numbers in parentheses indicate the number of predators that ate B. mariimseggs. P values are results of Fisher's Exact tests as explained in the text. Only one predator (C. siercusnniscarum) died in controltreatments during the experiments. All aquatic insects tested were adults except where indicated: L, larvae.

132 M. R. CROSSLAND AND R. A. ALHORD

causes of hatchling and tadpole mortality in controltreatments are unknown. Only one predator control (afish, Crateroceptiaius stcrcusmustarum Giinther) diedduring the experiments.

Overall, exposure to B. marinus eggs (Yates' correctedX" = 65.84, P < 0.0001), hatchlings (Yates' correctedX" = 7.42, P= 0.0064) and tadpoles (Yates' correctedX" = 7.30, P= 0.0069) significantly affected the sur-vival of native aquatic predators; predators experiencedhigher mortality- when exposed to ii. marinus than incontrol treatments. Mortality of predators exposed toB. marinus always occurred within 24 h ofthe consump-tion of B. marinus. None of the predators that con-sumed B. marinus without ill effect during theexperiments died during the 3 day monitoring periodfollowing the completion of the experiments.

In the results that follow, we distinguish two typesof consequences of predator exposure to B. marinus:increased risk of mortality for individual predators('toxic effects'), and increased mortality at the popu-lation level ('significant mortality'). Population-levelincreases were tested for significance as outlined in thestatistical methods above. Because our sample sizes ofpredators were relatively small, it is possible that in-creased risk at the individual level might not always leadto significant changes at the level of the population.To determine whether individual mortality risk was

affected by exposure to B. marinus, we calculated theoverall probability of predator mortality in control treat-ments by combining mortality data for all controlpredators in all experiments ( P = 0.00222). We thencalculated the cumulative binomial probability of oneor more predators dying by chance in B. marinus ex-posure treatments for experiments with the greatestnumber of replicates (and therefore the highest prob-ability of predator mortality by chance; n = 10 repli-cates; P = 0.022). This low probability suggests thatmost instances of predator mortality in B. marinusexposure treatments are likely to have been caused bythe toxic effects of B. marinus.

Toxic effects

Many predators readily consumed B. marinus withoutapparent ill effect, while others (e.g. belostomatids)killed B. marinus but either did not consume them oronly consumed a small portion of them (Tables 1-3).However, B. marinus eggs, hatchlings and/or tadpoleswere toxic to native tadpoles, snails, fish, dytiscid larvae,notonectids and leeches (i.e. individuals of these taxadied affer consuming B. marinus; Tables 1-3). Con-sumption of B. marinus was always lethal to tadpoles,snails and fish. However, dytiscid larvae, notonectidsand leeches experienced differential mortality after con-

Table 2, Predators tested with Bufo marinus hatchlings

Predator

NepidaeRanatra sp."Laccotrepties sp."

DytiscidaeCj'ftisttr sp.(L)"Cybisier godeffroyi"Hydaticits vitlatus^Saudracottiis batzewetti

OdonataTrapezostigma sp.(L)"Hemianax papiiensis (L)"

NotonectidaeAnisops sp."

CrustaceaCtierax quadricarinatus^Hoithuisana sp.^

AnuraLitoria rubella^Litoria lufrafrenataLitoria nigrofrenata^Liloria atboguttatcJLimnodynastes ornatus^

Feedingmode

PSPS

PSBCBCBC

BCBC

PS

BCBC

BCBCBCBCBC

K

10(0)6(6)

5(5)2(2)

10(10)2(2)

5(5)5(4)

5(4)

10(10)8(8)

10(0)5(4)5(0)

10(2)10(1)

Meanpredator size(mm) ±SD

44.3 ± 15.946.6 ± 3.5

34.2 ± 13.336.1 ± 3.914.2 ±0.412.9 ± 2.4

20.5 ± 3.542.4 ± 13.4

7.9 ± 1.1

88.2 ± 8.723.9 ± 4.3

12.3 ± 1.013.9 ± 0.917.4 ±0.622.7 ± 2,49.4 ±0.8

Gosnerstage

33-3729-3226-3126-3728-32

Numberhatchlings

eaten

04-10

1-36-8

1010

8-100-10

0-8

103-10

00-100-10-1

Predatormortality (%)

00

20000

00

20

00

080

02010

P

——

1.0000———

——

0.500

_—

—0.0048

0.00530.0500

Overall x' = 7.42, d.f. = 1, P= 0.0064. H, Heathlands; T, Townsville. BC, bite then chew; PS, pierce then suck, n =number of replicates; numbers in parentheses indicate the number of predators that ate B. marinus hatchlings. P values areresults of Fisher's Exact tests as explained in the text. No predators died in control treatments during the experiments. Allaquatic insects tested were adults except where indicated: L, larvae.

TOXICITY OF BUFO TO AQUATIC PREDATORS 133

suming B. marinus. Overall, this intraspecific variationwas not related to the number of B. marinus eaten; therewas no significant difference between the numbers ofB. marinns consumed by dytiscid larvae, notonectidsand leeches that lived and the numbers consumed bythose predators that died (Mann-Whitney P = 0 1889-Fig. 1).

Significant mortality

Native tadpoles and snails experienced significantlyreduced population survival rates when exposed toB. marinus eggs, and native tadpoles also experiencedsignificantly reduced population survival rates when ex-

posed to B. marinus hatchlings (Tables 1, 2). Leecheswere the only predator to experience significantlyreduced population survival rates when exposed toB. marinus tadpoles (Table 3). The reduction in popu-lation survival rates of the remaining susceptible taxawas not statistically significant because either fewindividuals of these taxa ate B. marinus (e.g. fish) or fewof the individuals that did eat B. marinus died (e.g.dytiscid larvae, notonectids; Tables 1-3).

General patterns

There was a significant difference between the suscepti-bilities of invertebrate and vertebrate predators to

Table 3. Predators tested with Bufo marinus tadpoles

Predator

NepidaeRanatra sp.**Laccotrephes sp.^

DytisddaeHydaticus sp.(L)"Cybister godeffroyi ̂Hydaticus vittams^Sandracottus bakewelli^

BelostomatidaeLethocerus itisulanus^

V.̂ U \Jl Xu Lu

Trapezostignia sp.(L)"Hemianax papuensis (L)^

NotonectidaeAnisops sp."

CrustaceaCherax quadricarinatus^Hollhuisana sp.^

HirudineaGoddardobdella elegens^

AnuraLitoria rubella^Litoria infrafrenata ̂Litoria nigrofrenata^Litoria albogunata^Limnodynastes ornatus'^

PiscesHypseleotris compressa^Neosilurus hyrtlii ^Ambassis agrammus^Craterocephalus siercusmuscariim^Melanotaenia splendida australis^

ChelidaeElseya latisternum^Emydura krefftii^

Feedingmode

PSPS

PSBCBCBC

PS

BCBC

PS

BCBC

PS

BCBCBCBCBC

SWSWSWSWSW

SWSW

n

10(0)4(4)

5(5)5(5)

10(10)4(4)

10(10)

5(4)5(4)

5(0)

10(10)2(0)

10(9)

10(0)10(0)10(0)10(0)10(0)

3(0)10(l)t10(l)t5(0)5(0)

1(1)4(4)

Meanpredator size(mm) ±SD

32.4 ± 2.342.3 ±2.9

24.2 ±29.8 i14.0 i13.5 1

60.3 ±

21.2 ±41.1 1

7.1 ±

87.4 ±46.9 ±

1.2 ±

11.9 ±13.1 ±17.1 ±23.9 ±10.5 ±

66.3 ±77.7 ±45.1 ±50.4 ±56.0 ±

100.2132 ±

: 9.8:4.2:0.5: 2.5

:4.5

3.4: 5.7

; 1.8

6.8:4.5

:6.7*

0.90.51.12.50.7

3.69.92.05.12.2

32.2

Gosnerstage

33-3928-3127-3428-3728-34

Numbertadpoles

eaten

08-10

5-105-10

1010

5-10

0-70-8

0

7-100

0-^

00000

00-10-100

83-10

Predatormortality (%) P

0 —0 —

60 0.16670 —0 —0 —

0 —

0 —0 —

0 —

0 —0 —

60 0.0022

0 —0 —0 —0 —0 —

0 —0 —0 —0 —0 —

0 —0 —

Overall x" = 7-30, d.f. ^ 1, P -0 .0069 . H, Heathlands; T, Townsville. BC, bite then chew; PS, pierce then suck;SW, swallow whole, n = number of replicates; numbers in parentheses indicate the number of predators that ate B. marinustadpoles. P values are results of Fisher's Exact tests as explained in the text. *Weight (grains); ^consumption of tadpoles notcertain. No predators died in control treatments during the experiments. All aquatic insects tested were adults except whereindicated: L, larvae.

I 34 M. R. CROSSl.AND AND R. A. ALFORD

1 0 •

9 '

8 '

7 •

6

5

4

3

1

0

V T

DDD

V D

oa

DO

DD

Ahve Dead

Predator condition

Fig. 1. Number otBi(fo eaten by predators that experienceddifferential mortality after consuming Bufo ( • , Cybisier sp.;V, .4niiops sp.; • . Hydaticiis sp.; and D G. elt\i;aus).

Table 4. Effect of feeding mode on susceptibility of predatorsto Bufo niaruius toxins

Feedingmode

Bite then chewPierce then suckSwallow whole

No. speciesnot

experiencingmortality

89

9

No. speciesexperiencing

significantmortality

710

Mortalit\'(%)

46.733.3

0

B. marinus toxins (Fisher's Exact test P = 0.0194;Tables 1-3). A higher proportion of vertebrate predatorsexperienced significant mortality after consuming B.marintis than did invertebrate predators. Unfortunately,this result is not corrected for, and is almost certainlyconfounded with, phylogenetic patterns. Feeding modedid not significantly affect the susceptibility of predatorsto B. marinus toxins (Fisher's Fxact test P = 0.762;Table 4), although the sample sizes for two ofthe feedingmodes were very small, making the lack of significancedifficult to interpret.

DISCUSSION

Native aquatic predators exhibited considerable inter-specific variation in their susceptibility to the toxins inB. ntarinus eggs, hatchlings and tadpoles. Bufo ntarinuswere highly toxic to some native species, while otherspecies either consumed them without ill effect oravoided consuming them at all. None ofthe predators

that died after consuming B. ntarinus were adverselyaffected by the consumption of the equivalent life his-tory stages of native anurans (Eimnodynastes ornatusGray; MRC unpubl. data). The ability of many preda-tors to consume B. marinus without ill effect was sur-prising as it has generally been presumed that few nativeAustralian predators can consume B. ntarinus eggs andlarvae due to their high toxicity (Covacevich & Archer1975). In fact, some native aquatic predators (dytiscidbeetles, crustaceans) can be maintained on a diet of B.marintis eggs and tadpoles for at least 4 weeks withoutany apparent ill effect (MRC unpubl. data). The con-sumption of large numbers of i?. martnus by predatorssuch as dytiscid beetles and crustaceans (Tables 1-3)suggests that they may be potential predators of B. mar-iitus eggs, hatchlings and tadpoles in nature.

The reasons why certain native aquatic taxa aresusceptible to B. martnus toxins, while others remainunaffected, are unknown. Although more vertebratepredators experienced significant mortality afterconsuming B. marinus than did invertebrate predators,all of these vertebrate species were anuran larvae(Tables 1-3) and therefore are phylogenetically non-independent. Thus, although anuran larvae were sus-ceptible to B. ntarinus toxins, there is no strong evidenceto suggest that vertebrate predators in general are moresusceptible to B. inarinus toxins than are invertebratepredators. Interspecific variation in the toxic effects ofB. marinus is also unlikely to be related to the numbersof B. marinus consumed by different predator species.For example, anuran larvae died after eating few(0.5-7) B. inarinus eggs while some crustaceans ate upto 50 eggs without ill effect (Table 1). Similarly,differences in predator feeding mode did not accountfor interspecific variation in the toxicity of B. marinusto aquatic predators (Table 4), although small samplesizes make this lack of difference inconclusive. Themechanisms that allow some native aquatic predatorsto consume B. marinus without ill effect remain to bedetermined.

There are few published data regarding the effectsof i?. marinus eggs, hatchlings and tadpoles on aquaticpredators in native B. marinus habitat. In Costa Rica,Heyei etal. (1975) found that B. )Harm»s hatchlings andtadpoles were non-toxic to larval anurans and odonates.During our experiments, B. ntarinus tadpoles were non-toxic to native odonate larvae but were always toxic tonative tadpoles. The ability of native odonate larvae toconsume B. marinus tadpoles without ill effect mayindicate that they have adapted to detoxify B. marimistoxins, or that they have always been unaffected bythese toxins. Differences between the responses ofanuran larvae in Australia and Costa Rica to B. marinustoxins may result from differences in their evolutionaryhistories of exposure to these toxins (e.g. Ehrlich &Raven 1964; Krieger et al. 1971; Whittaker & Feeny1971; Blau et al. 1978; Scriber 1978), or may reflect

TOXICITY OF BUFO TO AQUATIC PRHDATORS 1 35

variation in the toxicity of different populations ofB. marinus (e.g. Daly ct at. 1986).

Although the consumption of B. marinus was alwaysfatal to some native species (fish, tadpoles, snails), otherspecies (notonectids, dytiscid larvae, leeches) exhibitedintraspecific variation in susceptibility to B. marinus.There was no strong evidence for any taxon that thissusceptibility depended on the number of B. marinusconsumed (Fig. 1). This may indicate that there is intra-specific variation in the ability of these predators to copewith B. marinus toxins, or that there is intraspecific vari-ation in the toxicity of B. marinus hatchlings and tad-poles. Although intraspecific variation in the palatabilityof anuran larvae has been investigated (Brodie &Formanowicz 1987; Peterson & Blaustein 1992), weknow of no published data regarding intraspecificvariation in the toxicity of their early developmentalstages.

Despite the high toxicity of B. marinus to severalnative aquatic taxa, not all of these taxa experiencedsignificantly reduced survival when exposed to B. mar-inus. It seems likely that this lack of statistical signifi-cance is due, in most cases, to small sample sizes. Noexperiment produced results suggesting that predatormortality occurred at random. However, the results doindicate that variation in the propensity to consume andbe poisoned by B. marmus exists among susceptibletaxa. This variation may refiect differences in the abili-ties of susceptible native taxa to detect and avoidB. marinus toxins. For example, B. marinus eggs weretoxic to native fish. The only fish that died during thee.xperiments was the individual that ate B. marmus eggs.However, native fish did not experience significantlyreduced survival when exposed to B. marmus eggsbecause few fish ate them (Table 1). Fishes probablyavoid B. marinus eggs because they can detect their nox-iousness (Licht 1968, 1969; Wells 1979). In contrast,other susceptible native taxa (anuran larvae, gastro-pods, leeches) did not avoid eating B. marinus and con-sequently experienced significantly reduced survivalwhen exposed to B. marinus (Tables 1-3). This lack ofavoidance suggests that these species have limited abil-ity to detect B. marinus toxins, although the absence ofalternative food may have forced some predators toattempt to feed on unpalatable B. marmus. The be-havioural responses of native tadpoles and snails toB. marinus eggs, however, suggest that these species areunable to detect the toxicity of B. marinus eggs. Nativetadpoles and snails showed no avoidance responsewhen they came into contact with B. marinus eggs.Rather, they persisted in grazing on egg strings untilthey had penetrated the gelatinous string and con-sumed the fertilized eggs within, after which theyalways died.

From an evolutionary viewpomt, feeding deterrenceis not necessarily expected for novel compounds(Speiser et at. 1992). Although some gastropods

(Speiser t-t at. N92) and leeches (Licht 1964; Pough\'^7\) are able to detect and avoid noxious food items,Australian species of these taxa may have limited abilityto detect and avoid B. marinus toxins because they havehad no evolutionary experience with them. Tadpolesalso may have limited ability to taste their food (Heyeret at. 1975). If this is true, native tadpoles may con-tinue to be highly susceptible to B. marinus toxinsbecause selection to avoid their taste cannot lead to theevolution of aversion if native tadpoles cannot detectthat taste.

Several native predators were tested with B. marmuseggs, hatchlings and tadpoles, allowing us also to com-ment on possible ontogenetic changes in the toxicityof the early life history stages of B. marinus. Some ofthese predators consumed more than one B. marinuslife history stage. When this occurred, there was noapparent change in the toxicity of B. marinus. Bufoiitariiius hatchlings and tadpoles were always non-toxicto odonate larvae, while B. marinus eggs and hatchlingswere always toxic to anuran larvae. In addition, cold-killed B. marmus tadpoles (Gosner 1960, stage 25) arealways toxic to native tadpoles that feed on theircarcasses (MRC unpubl. data). In contrast, nepids,dytiscid beetles and crustaceans consumed B. iiiariiiusegg, hatchling and tadpole stages without ill effects.Thus, there is no evidence of ontogenetic changes inthe toxicity of B. marinus eggs, hatchlings and earlydevelopmental stage tadpoles; these stages remain toxicto some species but non-toxic to others.

The extent to which susceptible species are affectedby B. marmus in natural water bodies may depend ontheir ability to detect and avoid B. marinus toxins andon which stage of B. marmus they prey upon. Nativepredators that can detect and avoid B. marinus toxinsare likely to be unaffected by B. marinus, while speciesthat are unable to detect and avoid B. marinus toxinsmay be significantly affected. As the tadpole stage ofB. marinus lasts longer than the egg and hatchlingstages, species that prey on B. marinus tadpoles (e.g.dytiscid larvae, leeches) probably are exposed to riskfor longer periods and may be more adversely affectedthan species that prey upon B. marinus eggs or hatch-lings (e.g. anuran larvae, snails). Predators may playan important role in structuring freshwater communi-ties (Hurlbert et at. 1972; Macan 1977; Zaret 1980).Therefore, any changes in native aquatic predatorassemblages that result from predation on B. marinusare likely to cause alterations in species compositionand interactions at other trophic levels. We are currentlyinvestigating this possibility.

ACKNOWLEDGEMENTS

We thank the McLeod and Lyon families (QDEH) fortheir assistance and hospitality while staying at

136 M. R. CROSSLAND AND R. A. ALFORD

Heathlands, and the Department of Defence (Army)for allowing access to the Mt Stuart Training Area toundertake field collections. Kay Bradfield, StephenRichards, Bruce and Sara Russell, Lin Schwarzkopf andJohn Stewart provided valuable assistance in the field.Professor N. Milward provided the crayfish. Dytiscidswere identified by Dr C. Watts, while dragonfly larvaewere identified by Dr R. Rowe. Mr Alan Webbidentified the glass perch. This research was funded byCSIRO grants to RAA and MRC, and a Peter RankmTrust Fund Award to MRC. Earlier drafts of thismanuscript were improved by comments from KayBradfield, Stephen Richards and Lin Schwarzkopf.

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