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Species (common name) Domain I Domain II Domain III Domain IVR. norvegicus (rat) RL MT QDF WEN RV LC GEWI ET VA TF KGWMDI I T TS A GWDGLT. pardalis (puffer fish) RL MT QDNWES RI LC GEWI ET I A TF KGWMDI I T TS A GWDT LD. melanogaster (fly) RL MT QDF WED RV LC GEWI ES VA TF KGWI QI MS TS A GWDGVL. opalescens (squid) RL MT QDYWEN RV LC GEWI ES VA TF KGWI NI MS TS A GWDGVA. californica (sea hare) RL MT QDF WEW RV LC GEWI ES VA TY KGWI DI MCTS A GWSDVB. candida (flatworm) RL MT QDF WED RV LC GEY I ES VA TF KGWT DI I S TT GGWHS VC. capillata (jellyfish) QV CT L DYWES RI LC GKWI EP T A TL EGWF EE I S TA A GWNGVM. arenaria sensitive RL MT QDYWEN RV LC GEWI ES VA TY KGWI DI MCTS A GWDGVM. arenaria resistant RL MT QDYWEN RV LC GEWI DS VA TY KGWI DI MCTS A GWDGV

Locomotory Cost of Resistance to Algal Toxins in Relation to Nutritional State in the Softshell Clam, Mya arenaria

Jamie Dolan1, Kendra Waters2, and Monica Bricelj2

1Univ. of North Carolina Wilmington, Wilmington, NC 2Inst. of Mar.and Coastal Sci., Rutgers Univ., New Brunswick, NJ

Background

Objectives• Determine if there is a locomotory cost to PST resistance under non-toxic conditions by

comparing burrowing times (or rates) of juvenile clams of known genotype

• Determine if the clams’ (RR and SS) burrowing performance is affected by their nutritional state (exposure to a non-toxic high vs. low food regime)

• Determine if starvation differentially affects burrowing of RR and SS clams, and establish the relationship between burrowing performance and the loss of reserves during starvation.

Methods

Juvenile clams (~ 20 mm shell length, SL) were F1 progeny obtained by controlled breeding of adults of known genotype (SS or RR) collected from LE and BF respectively (Fig. 2). Juveniles were used as at these sizes they are most vulnerable to predation and environmental stressors if exposed in the intertidal zone. They were kept in 6 cm-deep washed, coarse sand and acclimated for 15 d in seawater at 15˚C, 33 salinity.

For burrowing time observations (Fig. 3), clams were placed on the sediment surface in an observation tank containing 2 trays with fresh sediment, and assayed sequentially. SL and total body wet weight were determined at the end of the experiment to calculate the Burrowing Rate Index (BRI) which corrects for size-related differences in burrowing time [5].

Results

Conclusions

References:

Acknowledgements:

[1] Bricelj et al. 2005 Nature 434: 763-766, [2] Geffeny et al. 2005 Nature 434: 759-763, [3] Bricelj & Shumway 1998 Rev. Fish. Sci. 6(4): 315-383, [4] Brodie & Brodie 1999 Evolution 53: 626-631, [5] Alexander et al. 1993 Palaios 8(3): 289-303

Burrowing rate is sensitive to the effect of nutritional condition (low food supply or starvation) suggesting that burrowing is a metabolically demanding process

Our results suggest that the PST resistant mutation is associated with a locomotory cost, reflected in an increase in burrowing times in RR clams. The observed small, but highly significant difference in burrowing times may have ecological significance by increasing predation risk, although this remains to be assessed.

A confounding effect in our study is that the two genotypes originated from two different source populations (LE and BF). Future studies will compare genotypes derived from a single source population to confirm these results.

The locomotory advantage of SS clams under non-toxic or low-toxicity conditions may help to explain the persistence of the S allele in nature despite the continued selection of resistant clams during recurrent toxic blooms.

This study provides yet another parallel between M. arenaria and the garter snake, suggesting that this model of adaptation of populations to neurotoxins may be widespread across phyla.

Toxic algal blooms (red tides) of Alexandrium spp. produce paralytic shellfish toxins (PSTs). Similarly newts, T. granulose, produce tetrodotoxin (TTX). These neurotoxins bind to voltage-gated sodium (Na+) channels in excitable muscle and nerve cells, resulting in block of the action potential causing paralysis and even death.

Results (cont.)

Table 1. Results of two-way ANOVA showing that both variables, food and genotype, had a highly significant effect on burrowing rates, but that there was no significant interaction.

STX binding site

TTX binding site

BA

• SS clams consistently burrowed faster than RR clams in both diets

• The mean burrowing time ranged from 18.4 min (High Food/SS) to 26.9 min (Low Food/RR)

• A 34% reduction in reserves (tissue organic weight)

resulted in a 19 to 27% reduction in BRI in RR and SS clams respectively

• Starvation differentially affected the two genotypes; it only adversely affected SS clams

Softshell clam (Mya arenaria)

Dinoflagellate (Alexandrium spp.)

Expt. 1. Thalassiosira weissflogii was continuously delivered via a peristaltic pump from a stock culture at a rate sufficient to match consumption, at two food levels: high (a concentration that supports clam growth) and low (a food-limiting concentration) (Fig. 4) in 2 tanks per diet, each containing both SS and RR clams.

Expt. 2. Clams acclimated to common food conditions for 11 d were held in 1 m-filtered seawater. The BRI of a subsample was determined on Day 0 and after 1 wk, 2 wks and 4 wks of starvation, and tissues frozen to determine protein and carbohydrate reserves.

Fig. 2. Mendelian inheritance results in 3 clam genotypes: homozygote resistant (RR), homozygote sensitive (SS) and heterozygote (RS). RR clams feed readily on toxic algae, accumulate higher toxin levels, and have a higher fitness (growth and survival) than SS clams during toxic blooms. PSTs also impair burrowing of adult SS, but not RR. Coastline affected by toxic Alexandrium blooms is indicated in red. A higher proportion of RR clams is found in BF, which has a recurrent history of PSTs, than in LE, with no history of red tides [1].

In marine softshell clams, Mya arenaria, that consume toxic algae, and in terrestrial garter snakes, T. sirtalis, that prey preferentially on T. granulosa, individual differences in nerve toxin resistance were attributed to a mutation in the Na+ channel gene that reduces the binding affinity of PSTs [1] and TTX respectively [2], to the pore region of the Na+ channel protein (Fig. 1).

A tradeoff between locomotory capacity and resistance to PSTs may thus also occur in M. arenaria, which are often scoured out of sediment in their high-energy intertidal habitat, and which rely on rapid burrowing to escape predators.

We thank C. Noji (IMCS) for her technical assistance, and L. Connell (Univ. of ME) for providing the genotyped clams. This research was supported by an award from NOAA-ECOLAB; J. Dolan was supported by an NSF RIOS internship.

Garter snake (Thamnophis sirtalis)

Newt (Taricha granulosa)

The proportion of resistant individuals in M. arenaria and T. sirtalis populations correlates with their history of toxin exposure (Fig. 2) [3]. Thus PSTs and TTX may act as a strong natural selection agent in nature, leading to spread of toxin resistance in softshell clam and garter snake populations.

Resistance in garter snakes is associated with a locomotory cost, resulting in lower maximum crawling speeds under non-toxic conditions, and thus greater vulnerability to their own top predators [4].

BRI = 100 x[Total Body Wet Weight (g)] 1/3

Burrowing Time (Ts-Tv) (sec)

Tv Tu Ts

umbo

• An 86% reduction in food supply from the high to the food regime resulted in a 34% reduction in organic weight of soft tissues at the end of 15 d for a standard-sized juvenile clam (SL = 23

mm)

• Thus this difference in food supply was effective in establishing a marked contrast in nutritional condition of clams

Fig. 1. A. 3D model of the pore region of the Na+ channel protein showing binding sites of saxitoxin (STX), the most potent PST, and TTX. The pore is formed by folding of the 4 domains shown in B. B. 2D structure of the Na+ channel protein (ɑ-subunit). This is highly conserved across taxa, but a mutation resulting in a single amino acid substitution (see arrow) in resistant M. arenaria causes a conformational change that decreases toxin binding affinity. Amino acids crucial to STX binding in yellow; those in the pore region in red.

Fig. 3 Stages of clam burrowing used in determining the BRI. Tv = time to attain a vertical position, Tu = time for the umbo to be buried below the sediment surface, and Ts = when only siphons are visible and the shell has disappeared below the sediment surface. Clams were individually numbered and color-coded by genotype.

Fig. 4. Daily averages of T. weissflogii cell concentrations in the high and low food treatments (mean of 2 tanks), as determined using the Coulter Counter over the 15 d acclimation period. Dashed lines indicate overall means ± SE.

.

Fig. 5. Relationship between M. arenaria shell length (SL) and ash-free dry weight (ADFW) or organic weight of soft tissues in the 2 diet treatments (pooled genotypes) (n = 20 per food level).

Experiment 1

Fig. 6. Burrowing Rate Index (mean ± SE; n = 22 to 28). Different letters indicate statistically significant differences (p < 0.05).

Fig. 7. Preliminary results of the effects of starvation on the BRI (at 1 wk) on the two clam genotypes Values = mean ± SE; (n = 24 to 28)

Experiment 1 Experiment 2

33% Reduction**

p = .002

18% ReductionNS

Low, Resistant

High, ResistantHigh, Sensitive

Low, Sensitive

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

1

BR

I

Low Food

C

A

BBC

High Food

Two- way ANOVA

Variable df F Significance

Food 1 20.53 p =<.001 ***

Genotype 1 11.59 p = .001 ***

Interaction 1 1.69 p = .197 NS