Transcript Jamie Dolan

Locomotory Cost of Resistance to Algal Toxins in Relation to Nutritional
State in the Softshell Clam, Mya arenaria
Jamie
1
Dolan ,
Kendra
2
Waters ,
and Monica
2
Bricelj
1Univ. of North Carolina Wilmington, Wilmington, NC
2Inst. of Mar.and Coastal Sci., Rutgers Univ., New Brunswick, NJ
Background
Methods
Results (cont.)
Garter snake (Thamnophis sirtalis)
Softshell clam (Mya arenaria)
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.
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.
A
TTX
binding
site
B
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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.
A
0.10
B
C
0.06
0.04
0.02
1
Low Food
Fig. 6. Burrowing Rate Index (mean ± SE; n = 22 to 28).
Different letters indicate statistically significant differences
(p < 0.05).
umbo
Tv
BRI = 100 x
Tu
Ts
[Total Body Wet Weight (g)]
Two - way ANOVA
1/3
Burrowing Time (Ts-Tv) (sec)
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.
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.
33% Reduction
**
p = .002
BC
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High Food
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18% Reduction
NS
High, Sensitive
High, Resistant
Low, Sensitive
Low, Resistant
0.00
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).
S
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BRI
Newt (Taricha granulosa)
Dinoflagellate (Alexandrium spp.)
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].
Experiment 2
Experiment 1
Variable
df
F
Significance
Food
1
20.53
p =<.001 ***
Genotype
1
11.59
p = .001
***
Interaction
1
1.69
p = .197
NS
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.
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)
•
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
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.
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.
Conclusions
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].
Results
Experiment 1
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.
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].
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.
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.
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.
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.
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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).
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.
.
• 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
References:
[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
Acknowledgements:
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.