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Impact of short-term salinity stress on larval development of the marinegastropod Crepipatella fecunda (Calyptraeidae)

J.A. Montory a,c,⁎, O.R. Chaparro a, J.A. Pechenik b, C.M. Diederich b, V.M. Cubillos a

a Instituto de Ciencias Marinas y Limnológicas, Universidad Austral de Chile, Valdivia, Chileb Biology Department, Tufts University, Medford, MA 02155, USAc Programa de Doctorado en Ciencias Mención Ecología y Evolución, Universidad Austral de Chile, Valdivia, Chile

a b s t r a c ta r t i c l e i n f o

Article history:Received 24 January 2014Received in revised form 10 April 2014Accepted 5 May 2014Available online 24 May 2014

Keywords:Clearance rateLarvaeLarval growth rateOxygen consumption rateSalinity stress

Shallow-water coastal environments suffer frequent reductions in salinity due to heavy rains. This creates stressfulconditions for the organisms found there, particularly for the early stages of development, including pelagic larvae.Freshly hatched larvae of the gastropod Crepipatella fecunda were exposed to different levels of salinity stress (32(control), 25, 20 and15) for a single 6hperiod. Subsequently, all veligersweremaintained at normal control salinity(32) through metamorphosis. Periodic measurements were made of mortality, larval growth, and larval behavior.In particular, we measured changes in velar surface area, swimming velocity, clearance rate, oxygen consumptionrate, shell growth rate, larval mortality, time to metamorphosis, and size at metamorphosis. The short exposureto salinity stress decreased subsequent mean growth rates at normal salinity and mean size at metamorphosis,but increased the duration of the planktonic period and the extent of larval mortality. It also reduced the rate atwhich the velar surface area increased relative to shell growth, and reduced mean larval swimming velocity.Mean oxygen consumption rates and clearance rates were also significantly lower for larvae that had been stressedearly in larval life, compared with values obtained for control individuals. Exposure to low salinity for even a shorttime early in larval life can clearly have a substantial impact on the rest of larval development.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Organisms living in intertidal or shallowwater areas are periodicallyexposed to fluctuations in temperature, dissolved oxygen concentration,salinity, and food availability (Allen et al., 2012; Amado et al., 2011;Dahlhoff et al., 2002; Diederich and Pechenik, 2013; Diederich et al.,2011; Lohrer et al., 2000; Lowell, 1984; Richmond and Woodin, 1996;Sampath-Wiley et al., 2008; Underwood, 1979), among other stressors.

Salinity reductions canhave particularly strong effects on distribution,behavior, and survival in such environments (Berger and Kharazova,1997; Bodinier et al., 2009; Kinne, 1971; Sameoto and Metaxas, 2008;Spicer and Strömberg, 2003; Torres et al., 2006; Yen and Bart, 2008),due to the effects of changes in solute concentrations on the efficiencyof metabolic processes (Kinne, 1966). Such salinity declines are commonin shallow coastal waters, estuarine areas, tidepools, and areas of river orglacial input, or where heavy local rains during substantial low tides canstrongly reduce ambient salinity, sometimes to almost freshwater condi-tions (Stickle and Denoux, 1976; Toro and Winter, 1983). In extreme

situations, prolonged exposure to low salinitiesmay killmany individuals(Génio et al., 2008). However, shorter duration exposures may lead tosituations of physiological stress (Aarset and Aunaas, 1990; SameotoandMetaxas, 2008; Tedengren et al., 1988) that have negative but sub-lethal impacts (Pechenik, 1983; Roller and Stickle, 1985, 1989, 1993).Sublethal effects caused by low salinities can be especially pronouncedin free-living organisms, particularly during the early stages of develop-ment (e.g. pelagic larvae, Bas and Spivak, 2000). Larvae may find them-selves in tide pools (Moulin et al., 2011) or in other shallow watersduring periods of heavy rain, where they are potentially exposed toshort-term but extreme hyposaline events. Such salinity stress can pro-long larval swimming times and delay the process of metamorphosis(Diederich et al., 2011; Giménez, 2002; Zimmerman and Pechenik,1991), potentially leading to increased larval mortality (Giménez,2002). Moreover, sublethal stresses experienced in the early stages ofdevelopment often lead to later effects on individuals (Pechenik,2006), such as decreased growth rates, food clearance rates, and oxygenconsumption rates (e.g. Balanus amphitrite, Pechenik et al., 1993;Hydroides elegans, Qian and Pechenik, 1998; Haliotis iris, Roberts andLapworth, 2001; Crepidula fornicata, Pechenik et al., 2002; C. onyx,C. fornicata, C. fecunda, Diederich et al., 2011), either later in larvaldevelopment or even in more advanced stages after metamorphosis(Diederich et al., 2011; Marshall et al., 2003; Pechenik, 2006; Pechenik

Journal of Experimental Marine Biology and Ecology 458 (2014) 39–45

⁎ Corresponding author at: Instituto de Ciencias Marinas y Limnológicas, UniversidadAustral de Chile, Valdivia, Chile.

E-mail address: jaimemontory@gmail.com (J.A. Montory).

http://dx.doi.org/10.1016/j.jembe.2014.05.0040022-0981/© 2014 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Journal of Experimental Marine Biology and Ecology

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et al., 1993; Pechenik et al., 2002; Qian and Pechenik, 1998; Takamiet al., 2002; Wendt, 1996; Wendt and Johnson, 2006).

The gastropod Crepipatella fecunda (Gallardo, 1977, 1979) is espe-cially appropriate for studies on salinity impacts. The species is commonin intertidal and shallow subtidal areas along the coast of southernChile, where salinities can drop after heavy rains during the breedingseason. Females incubate their egg masses within the mantle cavitybeneath the shell for about 4 weeks before releasing feeding veligerlarvae into the plankton (Chaparro et al., 2005; Mardones et al., 2013).Although females may retain the veligers within the mantle cavity fora time before releasing them into the plankton, in this paper we willrefer to release into the plankton as “hatching”. Veliger larvae emergefrom the female at approximately 350 μm shell length (Gallardo,1979) and continue to feed and grow in the plankton for about another15 days before settlement and metamorphosis (Chaparro et al., 2005).During those several weeks, planktonic veligers of C. fecunda may beexposed to decreases in environmental salinity. Although the impactof prolonged larval exposures (12–48 h) to reduced salinities on larvalsurvival, larval growth, and juvenile survival and growth has beenreported for this species (Diederich et al., 2011), the possibility thateven short-term exposures to sublethal low salinities can affect subse-quent larval performance has not been previously considered, norwere any physiological effects considered. In this investigationwe stud-ied the after-effects of temporary low-salinity stress on larval survival,physiology, and swimming behavior after the veliger larvae were per-mitted to continue their development at normal salinity levels. BecauseKlinzing and Pechenik (2000) showed that velar lobe size varies withfood level and diet for larvae of the related species Crepidula fornicata,we also tested to see if velar lobe size in C. fecunda was affected byshort-term exposure to water of reduced salinity.

2. Material and methods

Adult specimens of the gastropod C. fecunda (35–45 mm shelllength) were collected from Pelluco beach, Puerto Montt (41°28′S;72°56′W) between September and December 2012 and transferred tothe laboratory. The individuals were kept in aquaria with circulatingunfiltered seawater (salinity of 32) at 14 °C with constant aeration.Feeding was supplemented with microalgae cultures of Isochrysisgalbana until females released veliger larvae into the surroundingseawater. Those larvae were then used in the experiments describedbelow.

2.1. Later effects of stress: mortality, duration of pelagic life, and size atmetamorphosis

Aquaria (200 mL capacity) were filled with filtered seawater(0.5 μm) at 4 different levels of salinity: 32 (control), 25, 20 and 15.Reduced salinities were obtained by adding distilled water to filteredseawater. We included 4 replicate aquaria for each salinity level, with200 newly hatched veligers (350 ± 40 um shell length) per replicate.Newly-hatched veligers were maintained at each salinity for 6 h. Theveligers from each salinity treatment were then transferred to separateaquaria with filtered seawater (0.5 μm) at a salinity of 32. Seawater waschanged daily, any dead larvae were removed, and remaining larvaewere fedwithmicroalgae (I. galbana, 50,000 cellsmL−1). Oncemetamor-phosis occurred, the number of settled juveniles was quantified; this in-formation was used to estimate the larval mortality rate during thestudy, by comparing the initial number of individuals in each aquariumwith the number of settled and metamorphosed individuals. For eachtreatment, the duration of pelagic life was estimated from the timeelapsed between the day of hatching and the day when at least 50% ofthe surviving individuals had metamorphosed in each aquarium. Meta-morphosis was identified through loss of the larval swimming organ,the velum (Pechenik, 1984).

Newly hatched larvae and metamorphosed juveniles were bothphotographed using an inverted microscope Olympus BX41 at 40×magnification. A calibrated slide was also photographed and used todetermine actual larval sizes. The images were later processed using animage analysis program (Scion image pro) that allowed us to estimatethe respective sizes.

2.2. Later effects of stress on velar surface area and larval swimming speeds

Veligers of C. fecunda were stressed as described above and thenmaintained at normal salinity (32). They were examined 5 and 11 daysafter the low-salinity stress treatment ended. From each experimentalaquarium, 6 veliger larvae were transferred to a small glass chamberwith filtered seawater (0.5 μm, salinity of 32). Larval swimming wasvideotaped at 35× magnification using a magnifier- equipped Leica EZ4with a video camera. The film obtained allowed us to determine thedistance traveled by the veligers and the time it took to travel that dis-tance. With this information, we were able to estimate the displacementvelocity (mm s−1) of each individual. The distance was measured in astraight line, but in short distances which reduced the problem of anynon-linear movements made by the veligers. Measurements weremade using a reference slide that was also videotaped at the samemagnification.

Larvae were videotaped at 100× magnification using an invertedmicroscope. Still pictures were obtained from the videos at timeswhen the larvae had the velum fully extended and frontally exposedto the observer. Using standard image processing software (Scionimage pro), the velar lobe surface area (mm2) was then determinedfor each of the veligers, by tracing the border of the extended velum(Chaparro et al., 2002b; Cubillos et al., 2007). An index that relates thelength of the shell (an indicator of larval biomass) and the surfacearea of the velum (which moves the larval biomass through thewater) was also estimated for each individual, again using still framesfrom the videos. This “propulsion index” was calculated for larvae ex-amined 5 and 11 days after they experienced the 6 h low-salinity stress.

2.3. Later effects of stress on larval physiology and growth rates

2.3.1. Oxygen consumption rate (OCR)Twenty-four aquaria with seawater of different salinities (450 mL

per aquarium) were prepared by mixing filtered seawater (0.5 μm)with appropriate amounts of distilled water. The final salinities inaquaria were 32 (control), 25, 20 and 15, with 6 replicates for eachsalinity. Newly-hatched veligers of C. fecunda (350 ± 40 μm shelllength, N = 500) were added to each aquarium and maintainedunder the conditions described for 6 h. The veligers from each treat-ment were then removed to new aquaria containing filtered seawater(0.5 μm) at a salinity of 32. Thus larvae were stressed for only 6 h, andthen reared under control conditions for the rest of the study, as before.Larvae were fed daily with pure cultures of the microalgae I. galbana at50,000 cells mL−1. After 5 days, 50 veliger larvae were collected atrandom from each aquarium and placed in 10 mL syringes, with eachsyringe containing larvae from a single treatment and also from a singleoriginal aquarium. Each syringe contained 3mLoffiltered seawater (sa-linity of 32, 0.5 μm filtered), which was previously saturated with ox-ygen by continuous bubbling. The syringes used were maintained in athermostated water bath at 14 °C. AMicrox TX oxygen sensor was intro-duced through the tip of each syringe; oxygen concentration was mea-sured at time 0 and after 2–4 h, to subsequently calculate mean rates ofoxygen consumption per individual for each syringe. We repeated thesemeasurements at 11 days post hatching, using the same procedure.

2.3.2. Clearance rate (CR)CR measurements were made at a salinity of 32, 5 and 11 days after

the 6 h salinity treatments. Into each of 24 syringes (6 syringes per treat-ment salinity), we added 5 veligers of C. fecunda. Each syringewas filled

40 J.A. Montory et al. / Journal of Experimental Marine Biology and Ecology 458 (2014) 39–45

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with 10mLof seawater at salinity of 32 previouslyfiltered to 0.5 μm. Theseawater in each syringe contained an initial concentration of50,000 cells mL−1 of the microalgae I. galbana. In parallel, 3 syringeswere prepared as described, but without veligers, to control for anysedimentation of algal cells or cell reproduction. Larvae were allowedto feed for 2–3 h, after which time algal cell divisions were determinedusing a particle counter (Beckman Coulter Z2). The mean CR per indi-vidual was estimated for the larvae in each syringe following themeth-odology described by Coughlan (1969).

2.3.3. Growth rate (GR)Larvaewere exposed to different levels of salinity for 6 h as previously

described. At the end of the 6 h stress period, 5 veligers were obtainedfrom each of the 24 experimental aquaria. The veligers were fixed in95% ethanol and stored for later determinations of average veliger shelllength immediately following the 6 h salinity stress. When veligerswere 13 days old and near the end of their pelagic development, 5 veli-gers from each aquarium were collected and fixed with ethanol (95%).We subsequently obtained images of both groups of veligers using an op-tical microscope (Olympus model BX41) coupled with a Micropublisher5.0 camera. The images were processed using standard image analysissoftware (Scion image pro), allowing us to determine mean larval shelllength and to then calculate mean daily growth rates.

2.4. Statistical analysis

When the normality and homogeneity of variance of the data wereverified, we used one-way ANOVA to determine the effects of exposureto different salinities on subsequent mortality, duration of planktoniclife before metamorphosis, swimming velocity, velar surface area, sizeat metamorphosis, and the “propulsion index” relating larval shelllength to velar surface area. The same analysis was used to identify dif-ferences in mean rates of oxygen consumption, particle clearance, andlarval growth. When significant differences between treatments wereidentified, a posteriori Tukey test were used to determine where thedifferences lay. In all analyses, we used a significance level of 0.05 todetermine whether differences were statistically significant or not(Underwood, 1997). Throughout this paper, variations about meanvalues are expressed as ±SD.

3. Results

3.1. Later effects of stress: mortality, duration of pelagic life, and size atmetamorphosis.

Control larvae (salinity of 32) of C. fecunda showed ameanmortalityduring the 15 days of development of 64% (±8). However, mortalitywas significantly higher (one-way ANOVA, F(3,16) = 20.01; P = 0.001)for larvae that had been exposed to reduced salinity early in their larvallives; the highest mortality occurred in larvae that had been exposed tosalinities of 15 and 20 (Fig. 1).

The mean time from hatching to metamorphosis was 16.3 (±1.2)days for control larvae. However, this period was significantly longer(one-way ANOVA, F(3,16) = 38.22; P = 0.0001) for veligers that hadbeen exposed to salinities of 15 and 20 early in their pelagic develop-ment, resulting in an average planktonic period of 19 (±1.5) and 18.5(±1.3) days, respectively (Fig. 2).

The shells of individuals at the time of metamorphosis were on aver-age smaller (one-wayANOVA, F(3,16)= 342.81; P= 0.0002) for juvenilesthat had been briefly exposed to salinity stresses of 15 and 20 as earlylarvae, with sizes at metamorphosis of 602 (±20) and 610 (±25) μmshell length, respectively. These sizes were about 7% smaller than thoseof control individuals (650 (±27) μm shell length) (Fig. 3).

3.2. Later effects of stress on velar surface area and on larval swimmingspeeds

After 5 days of pelagic life, the control veligers showed a meanvelar surface area of 0.0802 (±0.0192) mm2. For veligers exposedto low salinity early in their development (25, 20 or 15), only ex-posure to a salinity of 15 negatively impacted (one-way ANOVA,F(3,96) = 9.76; p = 0.001) velar surface area, reducing it by approxi-mately 30% (0.0568 ± 0.0192 mm2) relative to the velar dimensionsof control veligers (Fig. 4A). Even after 11 days of pelagic life, larvaethat had been exposed to the greatest stress (salinity of 15) still ex-hibited a significantly smaller velar surface area (one-way ANOVA,F(3,96) = 12.54; p = 0.0016), about 20% smaller than that of controllarvae (Fig. 4B).

Mean swimming velocity for 5-day old control veligers was 1.0106(±0.1006) mm s−1, statistically equivalent to that of veligers that hadbeen exposed to salinities of 25 early in their larval development(0.9152 ± 0.1012 mm s−1) and 20 (0.8501 ± 0.2018 mm s−1)

Level of salinity stress

Mo

rtal

ity

(%)

0

60

65

70

75

80

85

90

95

100

32 (control)

aa

a b

b

15 20 25

Fig. 1. Crepipatella fecunda. Average mortality (+SD) of veliger larvae during the pelagicpost-stress period. Larvae were exposed to the salinity levels indicated for 6 h and thentransferred to control seawater (salinity of 32) for the duration of the study. Different lettersabove the bars indicate significant differences (p b 0.05) between means. N total = 4 repli-cates per treatment with 200 larvae per replicate.

Salinity

Tim

e u

nti

l met

amo

rph

osi

s (d

)

10

12

14

16

18

20

22

32 (control)

aa

b b

15 20 25

Fig. 2. Crepipatella fecunda. Average time (+SD) of pelagic life of the veliger larvae fromthemoment of hatching to metamorphosis. Larvae were exposed to the salinity levels in-dicated for 6 h and then transferred to control seawater (salinity of 32) for the duration ofthe study. Different letters above the bars indicate significant differences (p b 0.05) be-tween means. N total = 4 replicates per treatment with 200 larvae per replicate.

41J.A. Montory et al. / Journal of Experimental Marine Biology and Ecology 458 (2014) 39–45

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(Fig. 5A). However, 5-day old veligers that had been exposed to thelowest salinity (15) showed a significantly lower (by approximately17%: 0.8402 ± 0.1008 mm s−1) mean swimming speed than that ofthe control individuals (one-way ANOVA, F(3,96) = 25.63; p = 0.0003)(Fig. 5A). Similarly, the propulsion index relating shell length to velar

surface area was only significantly different when control larvae werecompared with those that had been previously exposed to a salinity of15 (one-way ANOVA, F(3,16) = 20.33; p= 0.0024) (Fig. 5A).

After 11 days of pelagic life, the control veligers exhibited a meanswimming velocity of 1.2011 (±0.2530) mm s−1, which was not signifi-cantly different (one-way ANOVA, F(3,96)= 1.28; p= 0.067) from that ofveligers that had been exposed to salinities of 15, 20 and 25 earlier in de-velopment. The swimming speeds of the latter were 1.003 (±0.1504),1.0910 (±0.1200) and 1.1009 (±0.1038) mm s−1, respectively(Fig. 5B). Also by this time, there were no longer any significant dif-ferences in the propulsion index relating shell length to velar surfacearea for larvae from the various salinity treatments (one-way ANOVA,F(3,16) = 3.46; p = 0.079, Fig. 5B).

3.3. Later effects of stress on larval physiology and growth rates

3.3.1. Oxygen consumption rate (OCR)By 5 days after hatching, control veligers exhibited a mean OCR

(oxygen consumption rate) of 0.0056 (±0.00037) mg O2 h−1 ind−1,while individuals that had been previously exposed to a salinity of 15exhibited a significantly lower mean OCR of 0.0045 (±0.00057) mgO2 h−1 ind−1 (one-way ANOVA, F(3,24) = 19.97; p = 0.0034)(Fig. 6A). By 11 days after hatching, themean OCRwas still significantlylower for individuals that had experienced a salinity of 15 earlier inlarval development (one-way ANOVA, F(3,24) = 53.26; p = 0.0017).No other significant differences in OCR were seen (Fig. 6B).

3.3.2. Clearance rate (CR)The CR (clearance rate) for control veligers at 5 days post-hatching

was 0.096 (±0.0107) mL h−1 ind−1, approximately 40% higher (one-

Salinity

Met

amo

rph

osi

s si

ze (

µm

)

500

550

600

650

700

750

32 (control)15 20 25

aa

abb

Fig. 3. Crepipatella fecunda. Influence of short-term salinity stress on size (+SD) at meta-morphosis. Larvae were exposed to the salinity levels indicated for 6 h and transferred tocontrol seawater (salinity of 32) for the duration of the study. Different letters above thebars indicate significant differences (p b 0.05) between means. N total = 4 replicatesper treatment with 200 larvae per replicate.

Salinity

0.00

0.02

0.04

0.06

0.08

0.10

0.12

32 (Control)15 20 25

a

ab

ab bA 5 days

Salinity

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

32 (Control)15 20 25

B 11 days

a

ab ab

b

Vel

um

su

rfac

e ar

ea (

mm

2 )V

elu

m s

urf

ace

area

(m

m2 )

Fig. 4. Crepipatella fecunda. Influence of short-term salinity stress on average velar surfacearea (+SD) at day 5 after the stress (A) and day 11 (B). Larvaewere exposed to the salinitylevels indicated for 6 h and transferred to control seawater (salinity of 32) for the durationof the study. Different letters above the bars indicate significant differences (p b 0.05)between means. N total = 4 replicates per treatment with 200 larvae per replicate.

Salinity

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4Swimming velocity

32 (Control)15 20 25

a

ab abb

A 5 Days

Pro

pu

lsio

n in

dex

(S

hel

l siz

e (µ

m)

/ Vel

um

are

a (µ

m2 )

)

0.005

0.006

0.007

0.008

0.009

0.010

0.011

0.012Propulsion index

a

ab ab

b

Salinity

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75 Swimming velocity

32 (Control)15 20 25

B 11 Days

a a a

a

Pro

pu

lsio

n in

dex

(Sh

ell s

ize

(µm

) / V

elu

m a

rea

(µm

2 ))

0.002

0.004

0.006

0.008

0.010

0.012Propulsion index

aa a

aS

wim

min

g v

elo

city

(m

m s

-1)

Sw

imm

ing

vel

oci

ty (

mm

s-1

)

Fig. 5. Crepipatella fecunda. Influence of short-term salinity stress on average swimmingvelocity (+SD) of displacement of veliger larvae (bars) and average propulsion index(+SD) relating shell length versus velar area at day 5 after the stress (A) and day 11 (B).Larvae were exposed to the salinity levels indicated for 6 h and transferred to control sea-water (salinity of 32) for the duration of the study. Different letters above the bars indicatesignificant differences (p b 0.05) between means. N total = 4 replicates per treatmentwith 200 larvae per replicate.

42 J.A. Montory et al. / Journal of Experimental Marine Biology and Ecology 458 (2014) 39–45

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wayANOVA, F(3,24)= 31.59; p= 0.0002) than that for veligers that hadpreviously experienced salinities of 20 and 15, with average values ofonly 0.0636 (±0.0163) and 0.0633 (±0.0077) mL h− ind−1, respec-tively (Fig. 7A). For 11-day old veligers, significant differences in CRwere again found (one-way ANOVA, F(3,24) = 53.26; p = 0.0017) forcontrol larvae in comparison with those that had been exposedto salinities of 15 and 20 earlier in larval life. The CR averages were:controls, 0.6871 ± 0.0407, salinities of 20, 0.5931 ± 0.0387 and 15,0.5803 ± 0.0560 mL h−1 ind−1 (Fig. 7B).

3.3.3. Growth rate (GR)Larvae from the different treatments grew at significantly different

rates (one-way ANOVA, F(3,90) = 51.08; p = 0.001). Control larvaeshowed an average GR of 18 (±1.2) μm day−1 ind−1, while veligersthat had been exposed to the two lowest salinities (20 and 15) showedgrowth rates that were about 12.2% lower, only 15.8 (±1.2) and 15.5(±1.4) μm day−1 ind−1, respectively (Fig. 8).

4. Discussion

Organisms living in intertidal zones, tide pools, or shallow coastalareas are often exposed to fluctuations in environmental salinity(Amado et al., 2011; Toro and Winter, 1983). These stressful eventsmay be particularly shocking for animals in the early stages of ontogeny.Pelagic larvae should be especially vulnerable because they have neither

maternal physical protection nor the capacity to effectively protectthemselves from exposure to poor environmental conditions (Anger,1996; Torres et al., 2007). The results of this investigation indicate thatfor veligers of the gastropod C. fecunda, even short-term exposure tolow salinities can impair development in various ways during the rest

Salinity

0

1

2

3

4

5

6

7

8

32 (Control)15 20 25

aab

abb

A 5 day

Salinity

0

1

2

3

4

5

6

7

8

9B 11 days

32 (control)15 20 25

a ab

abb

Oxy

gen

co

nsu

mp

tio

n (

x10-3

mg

O2

h-1

ind

-1)

Oxy

gen

co

nsu

mp

tio

n (

x10-3

mg

O2

h-1

ind

-1)

Fig. 6. Crepipatella fecunda. Influence of short-term salinity stress on rate of oxygen con-sumption (+SD) of veliger larvae at day 5 after the stress (A) and day 11 (B). Larvaewere exposed to the salinity levels indicated for 6 h and transferred to control seawater(salinity of 32) for the duration of the study. Different letters above the bars indicate sig-nificant differences (p b 0.05) between means. N total = 6 replicates per treatment with50 larvae per replicate.

Salinity

0.030

0.045

0.060

0.075

0.090

0.105

0.120

32 (Control)15 20 25

a

aab

b

A 5 days

Salinity

0.50

0.55

0.60

0.65

0.70

0.75

0.80

32 (Control)15 20 25

B 11 days

a a

ab

b

Cle

aran

ce r

ate

(mL

h-1

ind

-1)

Cle

aran

ce r

ate

(mL

h-1

ind

-1)

Fig. 7.Crepipatella fecunda. Influence of short-term salinity stress on average clearance rate(+SD) for veliger larvae at day 5 after the stress (A) and day 11 (B). Larvae were exposed tothe salinity levels indicated for 6 h and transferred to control seawater (salinity of 32) for theduration of the study. Different letters above the bars indicate significant differences(p b 0.05) between means. N total = 6 replicates per treatment with 5 larvae perreplicate.

Salinity

12

13

14

15

16

17

18

19

20

32 (Control)15 20 25

aa

b

b

Gro

wth

rat

e (µ

m d

-1 in

d-1

)

Fig. 8. Crepipatella fecunda. Influence of short-term salinity stress on average daily growthrate (+SD) of veliger larvae from themoment of hatching tometamorphosis. Larvaewereexposed to the salinity levels indicated for 6 h and transferred to control seawater (salinityof 32) for the duration of the study. Different letters above the bars indicate significantdifferences (p b 0.05) between means. N total = 6 replicates per treatment with 5 larvaeper replicate.

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of larval life. Hyposaline events (6 h exposures to salinities of 20 and 15)increased subsequent larval mortality by 15 to 20%, with respect to thatseen in control veligers that were not exposed to low ambient salinities.This elevated larval mortality corroborates previous reports indicatingthat the veligers of C. fecunda have a particularly poor tolerance ofhyposaline events, compared with larvae of other calyptraeid species(e. g. Crepidula fornicata and Crepidula onyx, Diederich et al., 2011).These interspecific differences in low salinity tolerance probably reflectdifferent capacities for regulating cell volume in the face of reducedsolute concentration, thus preventing permanent cell damage (Bradley,2009; Diederich et al., 2011). Short-term exposure to low salinity earlyin larval life prolonged the planktonic period for C. fecunda in our study.Such a phenomenon has also been identified in larvae of other inverte-brate species (Strongylocentrus droebachiensis, S. pallidus, S. purpuratus,Pisaster ochraceus, Roller and Stickle, 1985; Stramonita haemastomacanaliculata, Roller and Stickle, 1989; Balanus amphitrite, Pechenik et al.,1993; Lytechinus variegates, Roller and Stickle, 1993; Haliotis discushannai, Takami et al., 2002; Diplosoma listerianum, Marshall et al., 2003;C. onyx, C. fornicata, C. fecunda, Diederich et al., 2011). The larvae ofC. fecunda have been reported to spend approximately 15 days develop-ing in the plankton before metamorphosing (Chaparro et al., 2005), avalue that is consistent with the results recorded for the control larvaein our study. However, short-term exposure to either of the two lowestsalinities used in our study (20 and 15) prolonged the planktonic periodof C. fecunda by approximately 20%, increasing the time to metamorpho-sis by about 3 days. This increase in planktonic duration may favor in-creased larval dispersal and gene flow (Jablonski, 1986; Pechenik et al.,1993); however, it should also increase vulnerability to planktonic larvalpredators (Pechenik, 1999).

Larvae that had suffered the most extreme hyposaline exposure(salinity of 15) also showed the lowest feeding rates and the smallestvelar lobe surface areas, whichmay be responsible for the lower shellgrowth rates recorded in this study. Veligers of C. onyx, C. fornicata andC. fecunda showed a significant decrease in growth when exposed tohypoosmotic stress situations (Diederich et al., 2011), but this is thefirst study to show that even short-term exposure to reduced salinityearly in larval life can have prolonged effects on subsequent larvalperformance.

The mean size of unstressed control individuals at metamorphosiswas approximately 650 μm shell length, which is similar what was pre-viously reported by Chaparro et al. (2005). However, in this investiga-tion, individuals that had been stressed soon after hatching presentedsmaller average sizes at metamorphosis, compared with metamorphicsizes of control individuals. The smaller size of the stressed individualsat metamorphosis may reduce their subsequent fitness as juveniles(Rivest, 1983; Spight, 1976), as smaller individuals have a lower growthrate, less ability to feed, increased vulnerability to predators, and in-creased post-metamorphic mortality (Gebauer et al., 1999; Rivest,1983; Strathmann and Leise, 1979; Wendt, 1996).

In the present investigation, veligers that had been exposed to asalinity of 15 had a smaller average velar surface area at both 5 and11 days post-stress. Previous studies have shown a close relationshipbetween larval shell length, the size or area of the food-collectingvelum, and clearance rates (Chaparro et al., 2002a, b; Klinzing andPechenik, 2000). Since one of the main functions of the velum ofplanktotrophic veliger larvae is particle capture (Chaparro et al., 1999,2002a, b; Fretter and Graham, 1994; Hadfield and Iaea, 1989;Strathmann and Leise, 1979;Widdows, 1991), the smaller velar surfacearea that we identified in this investigation could well explain the re-duced clearance rates and lower growth rates of veligers that hadbeen stressed early in larval development.

In C. fecunda, velar surface area increases in proportion to the size ofveligers (Chaparro et al., 2002a). However, in the present investigation,a brief exposure of young larvae to low salinity (15) changed the allo-metric relationship between the size of the velum and the size of thelarval shell. That is, the increase of velar surface areawas proportionally

slower than shell growth at low salinity (i.e., the shell length-velum sur-face area propulsion index was increased), which may explain thelower swimming velocity of previously-stressed veligers recorded inthis investigation. Differences in growth allometry of these two structures(velum and shell) have also been described for the encapsulated veligersof Crepipatella dilatata (Chaparro et al., 2002a). This species has directintracapsular development (Brante et al., 2012; Chaparro et al., 2012;Gallardo, 1979), and although it includes a veliger stage, the veligermeta-morphoses before hatching.Whenmanually excapsulated, the veligers ofC. dilatata were not able to swim in the water column (Chaparro et al.,2002a), suggesting that swimming requires a particular threshold valuein the relationship between what needs to be moved (larval biomass)and what moves that biomass (e.g., length of the velum and cilia charac-teristics and beat frequency).

The effect of low-salinity stress (salinity of 15) on the relationshipbetween velum growth, shell growth, and larval swimming velocitywas reversible in C. fecunda; by 11 days post-stress, this relationshipwas not significantly different from that seen in control individuals,and mean swimming velocity was the same for larvae from all treat-ments, including controls. The food clearance capability of pelagic larvaeis directly related to the particle-capturing area of the velum (Chaparroet al., 2002a, b; Klinzing and Pechenik, 2000), which in turn reflects thevelum perimeter along which food particles are captured by the longvelar cilia of the opposing-band particle capture system (Chaparroet al., 2002b). Both velar surface area andmean clearance rate values re-ported in this study for control C. fecunda veligers coincide with thoserecorded by Chaparro et al. (2002b). Note that the larval velum doesnot function solely in food collection and locomotion; because thevelum is also an important gas-exchange organ for veliger larvae(Fretter and Graham, 1994), the reduced velar surface area seen inlarvae that had been briefly stressed earlier in development could alsobe affecting the rate of oxygen consumption. Indeed, we found thatlarvae that were stressed at the lowest salinity we tested (15) had re-duced oxygen consumption rates.

In summary, when newly-hatched veligers of C. fecunda were ex-posed to hypoosmotic stress for only 6 h, there were surprisinglyprolonged effects on various physiological variables, growth rates, andbehavior for much of subsequent larval development. Future studiesin this species should seek to identify potential latent effects beyondeffects on growth rate (Pechenik, 2006), because the larval characteris-tics that were affected in this study (e.g. morphological, physiologicaland behavioral traits) could influence the future fitness of the juveniles(Crean et al., 2011). In addition, it is interesting that in our study thestressed larvae took longer to metamorphose and metamorphosed ata smaller size. This suggests that the short-term salinity stress alteredthe subsequent relationship between rates of growth (as determinedby increases in shell length) and rates of differentiation (i.e., rates ofprogress towards whatever it is in development that causes metamor-phosis) (Zimmerman and Pechenik, 1991), something that should belooked at in future studies.

Acknowledgment

The authors thank CONICYT by the Doctoral fellowship 24121345given to JAM and to Fondecyt-Chile by the financial support throughthe Fondecyt grant 1100335 to ORC. [SS]

References

Aarset, A.V., Aunaas, T., 1990. Effects of osmotic stress on oxygen consumption andammonia excretion of the Arctic sympagic amphipod Gammarus wilkitzkii. Mar.Ecol. Prog. Ser. 58, 217–224.

Allen, B.J., Rodgers, B., Tuan, Y., Levinton, J.S., 2012. Size-dependent temperature and des-iccation constraints on performance capacity: implications for sexual selection in afiddler crab. J. Exp. Mar. Biol. Ecol. 438, 93–99.

Amado, E.M., Vidolin, D., Freire, C.A., Souza, M.M., 2011. Distinct patterns of water andosmolyte control between intertidal (Bunodosoma caissarum) and subtidal(Anemonia sargassensis) sea anemones. Comp. Biochem. Physiol. A 158, 542–551.

44 J.A. Montory et al. / Journal of Experimental Marine Biology and Ecology 458 (2014) 39–45

Author's personal copy

Anger, K., 1996. Salinity tolerance of the larvae and first juveniles of a semiterrestrialgrapsid crab, Armases miersii (Rathbun). J. Exp. Mar. Biol. Ecol. 202, 205–223.

Bas, C.C., Spivak, E.D., 2000. Effect of salinity on embryos of two southwestern Atlanticestuarine grapsid crab species cultured in vitro. J. Crustac. Biol. 20, 647–656.

Berger, V.J., Kharazova, A.D., 1997.Mechanisms of salinity adaptations inmarinemolluscs.Hydrobiologia 355, 115–126.

Bodinier, C., Boulo, V., Lorin-Nebel, C., Charmantier, G., 2009. Influence of salinity on thelocalization and expression of the CFTR chloride channel in the ionocytes ofDicentrarchus labrax during ontogeny. J. Anat. 214, 318–329.

Bradley, T.J., 2009. Animal Osmoregulation. , Oxford University Press, Oxford pp. 59–71.Brante, A., Fernández, M., Viard, F., 2012. Phylogeography and biogeography concordance

in the marine gastropod Crepipatella dilatata (Calyptraeidae) along the southeasternpacific coast. J. Hered. 103, 630–637.

Chaparro, O.R., Thompson, R.J., Emerson, C.J., 1999. The velar ciliature in the brooded larvaof the Chilean oyster Ostrea chilensis (Philippi, 1845). Biol. Bull. 197, 104–111.

Chaparro, O.R., Charpentier, J.L., Collin, R., 2002a. Embryonic velar structure and functionof two sibling species of Crepidula with different modes of development. Biol. Bull.203, 80–86.

Chaparro, O.R., Soto, A.E., Bertran, C.E., 2002b. Velar characteristics and feeding capacity ofencapsulated and pelagic larvae of Crepidula fecunda Gallardo, 1979 (Gastropoda,Calyptraeidae). J. Shellfish Res. 21, 233–237.

Chaparro, O.R., Saldivia, C.L., Pereda, S.V., Segura, C.J., Montiel, Y.A., Collin, R., 2005. The re-productive cycle and development of Crepidula fecunda (Gastropoda: Calyptraeidae)from southern Chile. J. Mar. Biol. Assoc. U. K. 85, 157–161.

Chaparro, O.R., Lincoqueo, L.A., Schmidt, A.J., Veliz, D., Pechenik, J.A., 2012. Comparing bio-chemical changes and energetic costs in gastropods with different developmentalmodes: Crepipatella dilatata and C. fecunda. Mar. Biol. 159, 45–56.

Coughlan, J., 1969. The estimation of filtering rate from the clearance suspensions. Mar.Biol. 2, 356–358.

Crean, A.J., Monro, K., Marshall, D.J., 2011. Fitness consequences of larval traits persistacross the metamorphic boundary. Evolution 65, 3079–3089.

Cubillos, V.M., Chaparro, O.R., Montiel, Y.A., Véliz, D., 2007. Unusual source of food: impactof dead siblings on encapsulated embryo development of Crepipatella fecunda(Gastropoda: Calyptraeidae). Mar. Freshw. Res. 58, 1152–1161.

Dahlhoff, E.P., Stillman, J.H., Menge, B.A., 2002. Physiological community ecology: varia-tion in metabolic activity of ecologically important rocky intertidal invertebratesalong environmental gradients. Integr. Comp. Biol. 42, 862–871.

Diederich, C.M., Pechenik, J.A., 2013. Thermal tolerance of Crepidula fornicata(Gastropoda) life history stages from intertidal and subtidal subpopulations. Mar.Ecol. Prog. Ser. 486, 173–187.

Diederich, C.M., Jarrett, J.N., Chaparro, O.R., Segura, C.J., Arellano, S.M., Pechenik, J.A., 2011.Low salinity stress experienced by larvae does not affect post-metamorphic growthor survival in three calyptraeid gastropods. J. Exp. Mar. Biol. Ecol. 397, 94–105.

Fretter, V., Graham, A., 1994. British prosobranch molluscs: their functional anatomy andecology. The Ray Society (820 pp.).

Gallardo, C.S., 1977. Two modes of development in the morphospecies Crepidula dilatata(Gastropoda: Calyptraeidae) from Southern Chile. Mar. Biol. 39, 241–251.

Gallardo, C.S., 1979. Especies gemelas del género Crepidula (Gastropoda, Calyptraeidae) en lacosta de Chile; una redescripción de C. dilatata Lamarck y descripción de C. fecunda n. sp.Stud. Neotropical Fauna Environ. 14, 215–226.

Gebauer, P., Paschke, K., Anger, K., 1999. Costs of delayed metamorphosis: reducedgrowth and survival in early juveniles of an estuarine grapsid crab, Chasmagnathusgranulata. J. Exp. Mar. Biol. Ecol. 238, 271–281.

Génio, L., Sousa, A., Vaz, N., Dias, J.M., Barroso, C., 2008. Effect of low salinity on the survivalof recently hatched veliger of Nassarius reticulatus (L.) in estuarine habitats: a casestudy of Ria de Aveiro. J. Sea Res. 59, 133–143.

Giménez, L., 2002. Effects of prehatching salinity and initial larval biomass on survival andduration of development in the zoea 1 of the estuarine crab, Chasmagnathusgranulata, under nutritional stress. J. Exp. Mar. Biol. Ecol. 270, 93–110.

Hadfield, M.G., Iaea, D.K., 1989. Velum of encapsulated veligers of Petaloconchus(Gastropoda), and the problem of re-evolution of planktotrophic larvae. Bull. Mar.Sci. 45 (2), 377–386.

Jablonski, D., 1986. Larval ecology and macroevolution in marine invertebrates. Bull. Mar.Sci. 39, 565–587.

Kinne, O., 1966. Physiology of estuarine organisms with special reference to salinity andtemperature: General aspects. Proceedings of the conference on estuaries, JekyllIsland, Georgia, 1964.

Kinne, O., 1971. Salinity. In: Kinne, O. (Ed.), Marine Ecology, vol. 1, Part 2. J. Wiley andSons, London, pp. 821–995.

Klinzing, M.S.E., Pechenik, J.A., 2000. Evaluating whether velar lobe size indicates foodlimitation among larvae of the marine gastropod Crepidula fornicata. J. Exp. Mar.Biol. Ecol. 252, 255–279.

Lohrer, A.M., Fukui, Y., Wada, K., Whitlatch, R.B., 2000. Structural complexity and verticalzonation of intertidal crabs, with focus on habitat requirements of the invasive Asianshore crab, Hemigrapsus sanguineus (de Haan). J. Exp. Mar. Biol. Ecol. 244, 203–217.

Lowell, R.B., 1984. Desiccation of intertidal limpets: effects of shell size, fit to substratum,and shape. J. Exp. Mar. Biol. Ecol. 77, 197–207.

Mardones, M.L., Chaparro, O.R., Pechenik, J.A., Segura, C.J., Osores, S.J.A., 2013. Embryonicbrooding in Crepipatella fecunda: implications for processes related to maternal feeding.J. Exp. Mar. Biol. Ecol. 443, 141–146.

Marshall, D.J., Pechenik, J.A., Keough, M.J., 2003. Larval activity and delayed metamorphosisaffect post-larval performance in the colonial ascidian Diplosoma listerianum. Mar. Ecol.Prog. Ser. 246, 153–162.

Moulin, L., Catarino, A.I., Claessens, T., Dubois, P., 2011. Effects of seawater acidification onearly development of the intertidal sea urchin Paracentrotus lividus (Lamarck 1816).Mar. Pollut. Bull. 62, 48–54.

Pechenik, J.A., 1983. Egg capsules of Nucella lapillus (L.) protect against low-salinity stress.J. Exp. Mar. Biol. Ecol. 71, 165–179.

Pechenik, J.A., 1984. The relationship between temperature, growth rate, and duration ofplanktonic life for larvae of the gastropod Crepidula fornicata (L.). J. Exp. Mar. Biol.Ecol. 74, 241–257.

Pechenik, J.A., 1999. On the advantages and disadvantages of larval stages in benthicmarine invertebrate life cycles. Mar. Ecol. Prog. Ser. 177, 269–297.

Pechenik, J.A., 2006. Larval experience and latent effects-metamorphosis is not a new be-ginning. Integr. Comp. Biol. 46, 323–333.

Pechenik, J.A., Rittschof, D., Schmidt, A.R., 1993. Influence of delayed metamorphosis onsurvival and growth of juvenile barnacles Balanus amphitrite. Mar. Biol. 115, 287–294.

Pechenik, J.A., Jarrett, J.N., Rooney, J., 2002. Relationships between larval nutritional expe-rience, larval growth rates, juvenile growth rates, and juvenile feeding rates in theprosobranch gastropod Crepidula fornicata. J. Exp. Mar. Biol. Ecol. 280, 63–78.

Qian, P.-Y., Pechenik, J.A., 1998. Effects of larval starvation and delayed metamorphosis onjuvenile survival and growth of the tube-dwelling polychaeta Hydroides elegans(Haswell). J. Exp. Mar. Biol. Ecol. 227, 169–185.

Richmond, C.E., Woodin, S.A., 1996. Short-term fluctuations in salinity: effects on plank-tonic invertebrate larvae. Mar. Ecol. Prog. Ser. 133, 167–177.

Rivest, B.R., 1983. Development and the influence of nurse egg allotment on hatching sizein Searlesia dira (Reeve, 1846) (Prosobranchia: Buccinidae). J. Exp. Mar. Biol. Ecol. 69,217–241.

Roberts, R.D., Lapworth, C., 2001. Effect of delayed metamorphosis on larval competence,and post-larval survival and growth, in the abalone Haliotis iris Gmelin. J. Exp. Mar.Biol. Ecol. 258, 1–13.

Roller, R.A., Stickle, W.B., 1985. Effects of salinity on larval tolerance and early develop-ment rates of four species of echinoderms. Can. J. Zool. 63, 1531–1538.

Roller, R.A., Stickle, W.B., 1989. Temperature and salinity effects on the intracapsular de-velopment, metabolic rates, and survival to hatching of Thais Haemastomacanaliculata (Gray) (Prosobranchia: Muricidae) under laboratory conditions. J. Exp.Mar. Biol. Ecol. 125, 235–251.

Roller, R.A., Stickle,W.B., 1993. Effects of temperatura and salinity acclimation of adults onlarval survival, physiology, and early development of Lytechinus variegates(Echinodermata: Echinoidea). Mar. Biol. 116, 583–591.

Sameoto, J.A., Metaxas, A., 2008. Can salinity-induced mortality explain larval vertical dis-tribution with respect to a halocline? Biol. Bull. 214, 329–338.

Sampath-Wiley, P., Neefus, C.D., Jahnke, L.S., 2008. Seasonal effects of sun exposure andemersion on intertidal seaweed physiology: fluctuations in antioxidant contents,photosynthetic pigments and photosynthetic efficiency in the red alga Porphyraumbilicalis Kützing (Rhodophyta, Bangiales). J. Exp. Mar. Biol. Ecol. 361, 83–94.

Spicer, J.I., Strömberg, J.-O., 2003. Metabolic responses to low salinity of the shipworm Teredonavalis (L.). Sarsia 88, 302–305.

Spight, T.M., 1976. Ecology of hatching size for marine snails. Oecologia (Berl.) 24,283–294.

Stickle, W.B., Denoux, G.J., 1976. Effects of in situ tidal salinity fluctuations on osmotic andionic composition of body fluid in southeastern Alaska rocky intertidal fauna. Mar.Biol. 37, 125–135.

Strathmann, R.R., Leise, E., 1979. On feedingmechanisms and clearance rates of molluscanvelígeras. Biol. Bull. 157, 524–535.

Takami, H., Kawamura, T., Yamashita, Y., 2002. Effects of delayedmetamorphosis on larvalcompetence, and postlarval survival and growth of abalone Haliotis discus hannai.Aquaculture 213, 311–322.

Tedengren, M., Arnér, M., Kautsky, N., 1988. Ecophysiology and stress responses of marineand brackish water Gammarus species (Crustacea, Amphipoda) to changes in salinityand exposure to cadmium and diesel-oil. Mar. Ecol. Prog. Ser. 47, 107–116.

Toro, J.E., Winter, J.E., 1983. Estudios en la ostricultura Quempillén, un estuario del sur deChile. Parte I. La determinación de los factores abióticos y la cuantificación del sestoncomo oferta alimenticia y su utilización por Ostrea chilensis. Mems. Asoc. Latinoam.Acuic. 5, 129–144.

Torres, G., Anger, K., Giménez, L., 2006. Effects of reduced salinities onmetamorphosis of afreshwater-tolerant sesarmid crab, Armases roberti: is upstream migration in themegalopa stage constrained by increasing osmotic stress? J. Exp. Mar. Biol. Ecol.338, 134–139.

Torres, G., Giménez, L., Anger, K., 2007. Effects of osmotic stress on crustacean larvalgrowth and protein and lipid levels are related to life-histories: the genus Armasesas a model. Comp. Biochem. Physiol. B 148, 209–224.

Underwood, A.J., 1979. The ecology of intertidal gastropods. Adv. Mar. Biol. 16, 111–210.Underwood, A.J., 1997. Experiments in Ecology. Their Logical Design and Interpretation

Using Analysis of Variance, 1st ed. , Cambridge University Press, Sydney.Wendt, D.E., 1996. Effect of larval swimming duration on success of metamorphosis and size

of the ancestrular Lophophore in Bugula neritina (Bryozoa). Biol. Bull. 191, 224–233.Wendt, D.E., Johnson, C.H., 2006. Using latent effects to determine the ecological impor-

tance of dissolved organic matter to marine invertebrates. Integr. Comp. Biol. 46,634–642.

Widdows, J., 1991. Physiological ecology of mussel larvae. Aquaculture 94, 147–163.Yen, P.T., Bart, A.N., 2008. Salinity effects on reproduction of giant freshwater prawn

Macrobrachium rosenbergii (de Man). Aquaculture 280, 124–128.Zimmerman, K.M., Pechenik, J.A., 1991. How do temperature and salinity affect relative

rates of growth, morphological differentiation, and time to metamorphic competencein larvae of the marine gastropod Crepidula plana? Biol. Bull. 180, 372–386.

45J.A. Montory et al. / Journal of Experimental Marine Biology and Ecology 458 (2014) 39–45