The effects of temperature and oxygen availability on ...

155THE EFFECTS OF TEMPERATURE AND OXYGEN ON EMBRYO DEVELOPMENTRevista Chilena de Historia Natural79: 155-167, 2006

The effects of temperature and oxygen availability on intracapsulardevelopment of Acanthina monodon (Gastropoda: Muricidae)

El efecto de la temperatura y la disponibilidad de oxígeno sobre el desarrollo intracapsularde Acanthina monodon (Gastropoda: Muricidae)

MIRIAM FERNÁNDEZ1*, PAULA PAPPALARDO & KATHERINE JENO

Estación Costera de Investigaciones Marinas of Las Cruces and Center for Advanced Studies in Ecology and Biodiversity,Departamento de Ecología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D,

Santiago, 6513677, Chile1 International Associate Laboratory ‘Dispersal and Adaptation in Marine Species’ (Station Biologique de Roscoff and

Center for Advanced Studies in Ecology and Biodiversity);* e-mail for correspondence: [email protected]

ABSTRACT

Freshwater and marine organisms show similar models of parental care and are faced with similarconstraints to brood, which suggest that comparable environmental limits drive the evolution of parentalcare in aquatic systems. In fact, the low diffusion coefficient and solubility of oxygen in aquaticenvironments affect oxygen acquisition and therefore the capacity to aggregate embryos. The effect ofother critical environmental variables, such as temperature, is less clear. We assessed the effects oftemperature and oxygen availability on (1) the number of developed and undeveloped encapsulatedembryos, (2) the proportion of embryos reaching advanced stages during intracapsular development(counting not only developed and undeveloped embryos but also abnormal embryos), (3) asynchrony indevelopment (estimated only in capsules in which development occurred), and (4) final embryo size, as thefirst step toward identifying the main factors constraining parental care in the ocean. We used the gastropodAcanthina monodon as a model because it has an extended latitudinal range of distribution and exhibitsfeeding larvae during intracapsular development. The latter factor is relevant because previous studies havesuggested that sibling cannibalism could be triggered by intracapsular competition for oxygen. Freshly laidegg capsules were collected and incubated until embryos hatched under different experimental temperatures(7, 11, 15 and 19 °C) and oxygen conditions (hypoxia: 50-60 % air saturation; normoxia; and hyperoxia:150-160 %). More embryos remained in early stages at the end of the experimental period under hypoxiaand at the highest experimental temperature. The mean number of developed embryos was significantlylower under hypoxia conditions than under normoxia and hyperoxia, but was not influenced by temperature.However, temperature negatively affected embryo size of developed embryos and the level of asynchrony(number of different developmental stages per capsule). This suggests that even when a comparable numberof embryos develops at high temperature, subsequent survival may be affected, since developed embryosattained smaller sizes. The negative effect of high temperature on embryo aggregation has also beenreported for Brachyuran crabs, affecting female patterns of oxygen provision and brooding costs. Thisevidence suggests that aggregating embryos in the ocean, even under optimum oxygen conditions, may benegatively affected at high temperatures. Spatial patterns of distribution of brooding species in the oceantend to agree with this prediction. Our analysis is particularly relevant given the current increase intemperature and the proportion of anoxic areas in the world’s oceans.

Key words: brooding, intracapsular development, gastropods, oxygen, temperature.

RESUMEN

Los organismos marinos y dulceacuícolas muestran modelos similares de cuidado parental y estánconfrontados con similares restricciones para incubar, lo que sugiere que existen límites ambientalescomparables guiando la evolución del cuidado parental en sistemas acuáticos. El bajo coeficiente de difusióny la baja solubilidad del oxígeno en ambientes acuáticos afecta la adquisición de oxígeno y por lo tanto lacapacidad para agregar los embriones. El efecto de otras variables ambientales críticas, como la temperatura,es menos claro. Se evaluaron los efectos de la temperatura y la disponibilidad de oxígeno sobre (1) el númerode embriones desarrollados y sin desarrollar, (2) la proporción de embriones que alcanza estados avanzadosde desarrollo (contabilizando no solo embriones desarrollados y sin desarrollar sino también anormales), (3)

156 FERNÁNDEZ ET AL.

la asincronía en el desarrollo (estimada solo cuando ocurrió desarrollo embrionario), y (4) el tamaño final,con el objetivo final de identificar las limitaciones que estos factores imponen sobre el cuidado parental en elmar. La especie modelo fue el gastrópodo Acanthina monodon. Cápsulas recientemente depositadas fueroncolectadas e incubadas bajo diferentes condiciones experimentales de temperatura (7, 11, 15 y 19 °C) yoxígeno (hipoxia: 50-60 % saturación de aire; normoxia; e hiperoxia: 150-160 %). Más embrionespermanecieron en fases tempranas del desarrollo al final del experimento en hipoxia y a 19 °C. El númeropromedio de embriones desarrollados fue significativamente más bajo en hipoxia que bajo normoxia ehiperoxia, pero no fue influenciado por la temperatura. Sin embargo, la temperatura de incubación afectóotras variables de respuesta. Menores tamaños de los embriones y mayores niveles de asincronía al final deldesarrollo fueron observados en las más altas temperaturas experimentales, lo que podría tener consecuenciasnegativas sobre la sobrevivencia posasentamiento. Las altas temperaturas también afectan el comportamientoy los costos asociados a la provisión de oxígeno a los embriones en braquiuros. Estas evidencias sugieren queagregar embriones en el océano, aun en condiciones óptimas de oxígeno, podría ser desfavorable a altastemperaturas. Los patrones espaciales de distribución de especies incubadoras tienden a apoyar estapredicción. Nuestro análisis también cobra relevancia en el escenario actual de aumento de la temperaturamedia de los océanos y de la proporción de zonas anóxicas.

Palabras clave: cuidado parental, desarrollo intracapsular, gastrópodos, oxígeno, temperatura.

INTRODUCTION

Freshwater and marine organisms exhibit similarmodes of parental care and are faced withsimilar constraints in aggregating embryos. Forinstance, several taxa of aquatic organisms, frominvertebrates to vertebrates, show similar typesand shapes of embryo masses (Strathmann &Strathmann 1989, Seymour & Roberts 1991,Seymour et al. 1991, Crawford & Balon 1996,Crump 1996, Fernández & Brante 2003,Fernández et al. 2003, Green & McCormick2005). Moreover, these taxa are subjected tosimilar constraints on parental care at large bodysizes (Strathmann & Strathmann 1982, Crump1996), a restriction that has enormousecological, biogeographical and evolutionaryconsequences (Strathmann & Strathmann 1982,Jablonski & Lutz 1983). These similarities,exhibited by a wide range of taxa, suggest thatcomparable ecological conditions andenvironmental limits drive the evolution ofparental care in aquatic systems. However, themain factors determining embryo development,affecting embryo aggregations, triggering siblingconflicts, and therefore shaping parental care inaquatic systems are poorly understood.

The physical characteristics of aquaticenvironments (low solubility of oxygen, lowdiffusion coefficient) affect oxygen acquisitionin embryo aggregations (Strathmann &Strathmann 1989, Fernández & Brante 2003,Fernández et al. 2003, Green & McCormick2005). Over the last two decades several studieshave shown that oxygen availability is a limitingfactor in embryo aggregation in aquatic systems

(Strathmann & Chaffee 1984, Strathmann &Strathmann 1989, 1995, Cohen & Strathmann1996, Crump 1996, Fernández et al. 2003),directly affecting embryo developmental rates(Strathmann & Strathmann 1995, Cohen &Strathmann 1996, Cancino et al. 2003). Oxygenavailability also seems to affect shellcalcification in marine gastropods (Maeda-Martinez 1985, Cancino et al. 2003), which mayhave enormous consequences on subsequentsurvival. The problem of oxygen acquisitionimposed by aquatic systems can also help toexplain the positive relationship betweennumber of embryos and surface area of thecapsule in marine gastropods (genus Conus;Perron & Corpuz 1982). Moreover, oxygenavailability also appears to determine the ratiobetween developed embryos and nurse eggs(Lardies & Fernández 2002), suggesting thatstrong interactions (conflicts) may occurbetween siblings when an environmentalcondition such as oxygen availability becomeslimiting. This body of evidence suggests that thestrong oxygen limitation exhibited by embryomasses and capsules in aquatic systems may bean important evolutionary force, affecting thecapacity to aggregate the embryos and thereforethe spread of parental care in aquatic systems ingeneral (Strathmann & Strathmann 1982).

Embryo oxygen consumption determinesoxygen availability in embryo aggregations,since diffusion of oxygen into the center of theaggregations usually is not sufficient to satisfyoxygen demands (Cohen & Strathmann 1996,Baeza & Fernández 2002, Fernández et al.2003). As embryo oxygen demand increases

157THE EFFECTS OF TEMPERATURE AND OXYGEN ON EMBRYO DEVELOPMENT

throughout development, oxygen gradientsbetween the center and the periphery ofgelatinous embryo masses become stronger(Cohen & Strathmann 1996, Brante et al.2003). Actively brooding species, such asBrachyuran crabs, compensate for the higheroxygen demand of later embryonic stages byincreasing the frequency of brooding behaviorsthat help provide oxygen to the embryos (e.g.,abdominal flapping), thereby increasing thecosts of brooding (Baeza & Fernández 2002,Brante et al. 2003, Fernández et al. 2003).Temperature influences oxygen demand of theembryos (Wear 1974, Weathly 1981, Brante etal . 2003), which suggests that thisenvironmental factor can also indirectly affectoxygen availability in embryo aggregations andthus embryo development, sibling interactions,brooding behaviors and costs, and consequentlyparental care. In fact, a strong relationshipbetween temperature-dependent embryo oxygendemand and brooding behaviors associated tooxygen provision has already been reported inBrachyuran crabs (Brante et al . 2003).Moreover, the association between temperatureand patterns of oxygen supply to the embryomass increases the cost of brooding, suggestingthat parental care in the ocean may be favoredat low temperature (Brante et al. 2003). Thisprediction is supported by the high prevalenceof brooding species in the deep ocean and athigh latitudes, particularly in Antarctic waters(Thorson 1950, Gallardo & Penchaszadeh2001). The effect of temperature on otheraspects of parental care, such as embryodevelopmental success (Cancino et al. 2003)and sibling conflicts in embryo aggregationshas been less explored.

Encapsulated embryos offer a good modelto investigate the effects of temperature andoxygen avai labi l i ty on embryonicdevelopment in aggregated masses, and theconsequences on parental care, for two mainreasons. First, species with encapsulatedembryos can show wide ranges of latitudinaldistribution (and therefore wide ranges ofthermal conditions). Second, some speciesdepend on siblings or nurse eggs as nutritionsources (e.g., Gallardo 1979, Gallardo &Garrido 1987), which offers the opportunity toanalyze the interactions between siblingstriggered by environmental factors (Lardies &Fernández 2002). Our goal was to assess the

effects of temperature and oxygen availabilityon several indicators of embryonicdevelopment , to contr ibute to theunderstanding of the main factors constrainingparental care in the ocean. We used thegastropod Acanthina monodon (Pallas 1774)as a model because it exhibits a feeding larvaduring intracapsular development and anextended latitudinal range of distribution. Weanalyzed four response variables: final numberof developed and undeveloped encapsulatedembryos, developmental success measured asthe proportion of embryos reaching advancedstages during intracapsular development(counting not only developed and undevelopedembryos but also abnormal embryos) ,asynchrony in development (estimated only incapsules in which development occurred), andfinal embryo size. We also analyzedintracapsular oxygen availability to assess theeffectiveness of the experiment. We discussthe effects of temperature and oxygenavailability on embryo aggregations as well asthe consequences on parental care and on thedistribution of brooding species in the ocean.

MATERIAL AND METHODS

Sample collections

Acanthina monodon is a muricid gastropod thatinhabits low intertidal rocky shores of centraland southern Chile from 28 to 55º S (Gallardo1979, Valdovinos et al. 1999). Capsules ofAcanthina monodon exhibit fairly transparentwalls which are yellowish shortly afterdeposition and turn grey as embryos develop(Gallardo 1979). Following this criteria, freshlylaid egg capsules were collected from one sitein central Chile (Consistorial, 33º29’ S), wherehigh densities of adult and capsules are found,and transported to the laboratory at theEstación Costera de Investigaciones Marinas(Las Cruces, central Chile) approximately 2 kmaway. Mean surface temperature in the studysite is 13 °C during the reproductive months(April-September), ranging between 11,5 and15,1 °C. Females deposit capsules inaggregations, attached to rocky platforms, inareas of high wave exposure year round(Gallardo 1979). However, in our study areacapsules are present only in autumn and winter.


Experimental design

In order to assess the effects of temperature andoxygen availability on the response variableslisted above, laboratory experiments wereconducted. Capsules from different clutcheswere collected and randomly assigned to fourexperimental temperatures and three oxygenlevels, until hatching. The experimentaltemperatures were 7, 11, 15 and 19 °C. Thisrange of temperatures was selected because itincludes the extreme temperatures within thelatitudinal range of distribution of the species.Each temperature treatment was run at differenttimes due to logistic constraints. Oxygentreatments were (1) Hypoxia, (2) Normoxia, and(3) Hyperoxia. Normoxia was established with aconstant flow of air in the aquaria to maintain100 % air saturation. Hyperoxic conditions werecreated by bubbling O2 and CO2 to maintain150-160 % air saturation. Hypoxic conditionswere created by bubbling nitrogen and CO2 tomaintain 50-60 % air saturation. We bubbledCO2 to minimize the changes in the pH whichwould have occurred if only nitrogen or oxygenhad been used for the hypoxia and hyperoxiatreatments, respectively. This level of airsaturation was selected because oxygen levelsbelow 50 % air saturation have been shown toaffect oxygen consumption of gastropods(Cancino et al. 2003). Temperature and oxygenconditions were monitored twice daily for alltreatments. The effectiveness of theexperimental oxygen conditions was assessed bymonitoring extra and intracapsular oxygenavailability (see Fig. 3).

Capsules were collected by detaching themfrom aggregations deposited by differentfemales in the field. The experiment wasstarted two days after the collection of capsules(initial time), allowing the capsules 48-hacclimation to the experimental conditionsbefore the first measurements were made. Five1-L containers were used for each combinationof temperature and oxygen conditions. Tencapsules were randomly assigned to eachcontainer in order to have enough capsules tomonitor embryo development throughout theexperiment. However, only five replicates (onecapsule from each container) were analyzed atthe initial and final time for all the variables ofinterest. Therefore, the same number ofreplicates were used for the analysis of final

number of embryos, proportion of developedembryos, asynchrony and embryo size. In eachcontainer, the capsules were allowed to floatseparately in order to avoid potential restrictionin oxygen diffusion. At the initial time, onecapsule from each treatment combination wasrandomly selected, and the following variableswere measured: (a) intracapsular % airsaturation, (b) capsule size (maximum lengthand width), (c) number of embryos, and (d)stage of development of the embryos. Sincetemperature treatments were run at differenttimes using different sets of capsules, a oneway ANOVA was used to compare the initialnumber of embryos among temperatures. At theinitial time, all embryos were at early stages ofdevelopment (earlier than post-ingestiontrochophore). No differences in the initialnumber of embryos per unit of capsule areaamong temperature treatments were detected(F3,17 = 1.327, P = 0.298), which was anecessary condition to test for differences inembryo number at the end of the experiments.Thereafter, embryo development wasmonitored regularly until the advanced stagesof veliger and pediveliger were reached in oneof the oxygen treatments, at which time theexperiment was ended. This was considered thefinal time, and then one capsule from eachtreatment combination was again randomlyselected and the variables indicated abovemeasured.

Number of embryos

The effects of experimental treatments on thenumber of embryos at the end of theexperiment were evaluated. In some capsules,embryo development was severely retarded andearly stage embryos were observed in somecapsules while hatching was attained in theremaining contemporal treatments. Therefore,total embryo count at the end of the experimentdid not necessarily reflect the effects ofexperimental treatments unless embryo stagewas accounted for. Thus, the statistical analysesto assess the effects of experimental treatmentson final number of embryos were conductedseparately for developed and undevelopedembryos. Embryos were counted and the stagedetermined using a NIKON-SZ binocularmicroscope. Embryo stages were categorizedfollowing Gallardo (1979). The five categories


used were: early embryos, post-ingestiontrochophore, veliger, pediveliger, prejuveniles.We classified as developed embryos thosereaching veliger to prejuvenile stages at the endof the experiment and as undeveloped embryosthose attaining only the trochophore stage(Gallardo 1979). The number of embryos percapsule differed enormously between capsulescontaining undeveloped and developedembryos, because embryo number decreases 90% throughout development in Acanthinamonodon, as developing embryos depend onsiblings as a source of food (e.g., Gallardo1979, Gallardo & Garrido 1987, Lardies &Fernández 2002). Thus, two differentapproaches were used to estimate embryonumber depending on developmental stage. Incapsules containing undeveloped embryos atthe end of the experiment, a subsample of theembryos was counted. Embryos were spreadevenly on a dish divided in 113 equally sizedsquares and all the embryos were counted in 16randomly selected squares. Based on this count,total egg number was estimated. All embryoswere counted in capsules containing late stageembryos. When more than one developmentalstage was found within a capsule, all embryoswere counted and the stage determined. Thesize of the capsules ranged from 7.75 to 13.00mm (mean ± SD = 10.41 mm ± 1.15). Sinceembryo number was positively correlated withcapsule size (length: P < 0.0001, width: P <0.0001), the number of embryos wasstandardized per unit of surface area of thecapsule (mm2) and used as a response variable.We selected capsule area because nocorrelation was found between number ofembryos per unit of capsule area and capsulelength (P = 0.745) and because capsule areacould be an indicator of oxygen exchangesurface (Perron & Corpuz 1982). Capsule areawas estimated assuming the shape of acylinder. Two-way ANOVAs were conductedto evaluate the effects of temperature andoxygen conditions on mean final number ofundeveloped and developed stage embryos perunit of capsule area. Square-root transformationwas used for undeveloped embryos to meet theassumptions of the model; arcsin for developedembryos. Tukey HSD tests were used forcomparison among treatments.

In some capsules, a few developing butabnormal embryos were found. Since those

embryos were neither classified as developed,nor as retarded, but could affect the number ofembryos hatched, the proportion of developedembryos was estimated at the end of theexperiment (number of developed embryos /total number of embryos) and was used as anindicator of developmental success. Totalembryos resulted from the sum of abnormal,developed and retarded embryos. Two-wayANOVAs were used to assess the effects oftemperature and oxygen condition (% airsaturation) on the mean proportion of embryosthat developed. In the oxygen treatments inwhich embryo development occurred(excluding hypoxia, see below), a second two-way ANOVA was run to comparedevelopmental success as well as asynchronyacross treatments. Asynchrony was estimatedas the sum of different embryonic stageswithin a capsule. Square-root transformationwas used for the proportion of developedembryos to meet the assumptions of theANOVA model. Tukey HSD tests were usedfor a-posteriori comparison.

Embryo size

In order to assess the effects of theexperimental conditions on embryo size, thelength of between 10 and 15 embryos percapsule were measured. The most advancedstages within a capsule were measured using anocular micrometer using a Nikon-SZ binocularmicroscope. Then, the mean size of the mostadvanced embryos in each capsule wasestimated. We focused on the most advancedstages in this case because otherwise the resultswere strongly biased by the number ofundeveloped or slowly developed embryos.Other variables analyzed considered differencesin embryo development among treatments.Mean embryo size was compared amongtemperatures and oxygen conditions using atwo-way ANOVA. A Tukey HSD test was usedfor comparison among treatments.

Intracapsular oxygen availability

Intracapsular oxygen availability at the initialand final times was measured using a fiberoptic sensor (Microx I, Presens) with a tipdiameter ranging between 20 and 50 µm. Thesensor was calibrated in a solution saturated


with Na2O3S (0 % air saturation) and in aeratedwater (100 % air saturation). The calibrationand measurements were conducted at constanttemperature, regulated by a thermostat. Aftercalibration, oxygen availabili ty (% airsaturation) was measured outside and theninside the capsules. The tip of the fiber opticwas inserted in the center of the capsule and thecapsule was then immersed in the experimentaltank for the duration of the measurements.Each measurement was conducted for 15 min,recording oxygen availability (% air saturation)every 5 s. The first and the last 2.5 min werediscarded to avoid possible disturbances whenthe fiber optic was inserted or removed. Thus,% air saturation was calculated as the averageof the remaining 10 min of measurements.Intracapsular % air saturation was comparedamong temperatures and oxygen conditionsusing a two-way ANOVA, for eachexperimental time (initial and final). For initialoxygen conditions, cosine transformation wasused. A-posteriori Tukey’s tests (HSD) wereconducted to determine differences amonglevels.

RESULTS

Developmental time

The trocophore was the most advanced stageobserved at the end of the experiment in allhypoxia treatments. In contrast, in the othertwo oxygen treatments the veliger orpediveliger stages were attained. At normoxia,developmental t ime (days) increasedsubstantially as temperature decreased beyond11 ºC. Hatching occurred 112 days after theexperiment started at 7 °C, after 70 days at 11°C, 50 days at 15 °C, and 47 days at 19 °C. Thecapsules were freshly laid and the embryoswere all at the same developmental stage whenthe experiment started.

Number of embryos

The significant interaction between the twomain factors precluded us from testing theeffects of temperature and oxygen conditionson mean final number of undeveloped embryos(Table 1, Fig. 1A). The interaction was due tothe lack of change in the mean number of

undeveloped embryos from low to hightemperatures under hypoxia (P > 0.05), whichcontrasted with the increase in the number ofundeveloped embryos with temperature undernormoxia and hyperoxia (Fig. 1A). Moreembryos remained in early stages at the end ofthe experimental period under hypoxia and atthe highest experimental temperature undernormoxia and hyperoxia (P < 0.05). Theremaining treatments formed a homogenousgroup lacking statistical distinction (P > 0.05).The mean number of embryos attaining latestage (developed embryos) was significantlyaffected by oxygen conditions, but not bytemperature (Table 1, Fig. 1B). The meannumber of developed embryos was significantlylower under hypoxia conditions than in theother two oxygen treatments (P < 0.05). Nodifferences were found between normoxia andhyperoxia (P > 0.05). The interaction term wasnot significant (Table 1).

Due to differences in the final number ofundeveloped and developed embryos acrosstreatments, and the presence of abnormalembryos, the mean proportion of developedembryos was compared. The interaction termwas significant because again different patternsof developmental success occurred acrosstemperatures in hypoxia than under the othertwo oxygen conditions (Table 2, Fig. 2A). Thelowest proportion of developed embryos at theend of the experimental treatment was observedunder hypoxia and at the highest temperature (19°C both under normoxia and hyperoxia; P <0.05, Fig. 2A). The remaining treatments form asecond homogenous group lacking statisticaldistinction (P > 0.05). When the hypoxiatreatment was excluded, since embryos neverreached advanced developmental stages, asignificant effect of temperature was detected(F3,29 = 6.03, P = 0.0025). The mean proportionof advanced embryos at the end of developmentwas lowest at 19 °C (P < 0.05). Asynchrony wassignificantly higher at the highest temperature(F3,29 = 13.96, P < 0.0001, Fig. 2A). Nodifferences were found among the remainingtemperature treatments for asynchrony ordevelopmental success (P > 0.05) and betweennormoxia and hyperoxia (developmentalsuccess: F1,29 = 0.99, P = 0.33; asynchrony: F 1,29

= 1.19, P = 0.28). The interaction terms were notsignificant (developmental success: F3,29 = 1.19,P = 0.33; asynchrony: F3,29 = 2.58, P = 0.07).


TABLE 1

Results of the two-way ANOVA conducted to compare mean number of embryos in capsules ofAcanthina monodon at the end of the developmental period among oxygen conditions (hypoxia,

normoxia, hyperoxia) and temperatures (7, 11, 15 and 19 ºC). The response variables were number ofundeveloped and developed embryos per unit of capsule area. P-values < 0.05 are indicated in bold

Resultados de los ANDEVA de dos vías realizados para comparar el número promedio de embriones en cápsulas deAcanthina monodon al final del período experimental bajo los diferentes niveles de los tratamientos de oxígeno (hiperoxia,

normoxia e hipoxia) y temperatura (7, 11, 15, y 19 ºC). Las variables de respuesta fueron número de embriones desarrollados(tardíos) y sin desarrollar (tempranos) por unidad de área de cápsula. En negritas se indican los valores de significancia < 0,05

Source of variation Degrees of freedom F-value P-value Degrees of freedom F-value P-value

Embryos in early stage (undeveloped) Embryos in late stage (developed)

Temperature 3 7.03 0.0006 3 1.73 0.17

Oxygen 2 74.85 < 0.001 2 64.11 < 0.0001

Interaction 6 3.02 0.015 6 2.09 0.072

Error 44 44

Fig. 1: Mean number of (A) undeveloped (early stages) and (B) developed (late stages) embryos atthe end of the experimental period under different levels of oxygen (hyperoxia, normoxia andhypoxia) and temperature (7, 11, 15, and 19 ºC). Vertical bars indicate one standard deviation.Número promedio de embriones (A) sin desarrollar (estadíos tempranos) y (B) desarrollados (estadíos tardíos) al final delperíodo experimental bajo los diferentes tratamientos de oxígeno (hiperoxia, normoxia e hipoxia) y temperatura (7, 11, 15,y 19 ºC). Las barras verticales indican una desviación estándar.


TABLE 2

Results of the two-way ANOVA conducted to compare the mean proportion of developed embryos(developmental success) among oxygen (hypoxia, normoxia, hyperoxia) and temperature (7, 11, 15

and 19 ºC) treatments in capsules of Acanthina monodon. P-values < 0.05 are indicated in bold

Resultados del ANDEVA de dos vías realizado para comparar el éxito del desarrollo embrionario, medido como laproporción de embriones desarrollados al final del experimento, entre los diferentes niveles de los tratamientos de oxígeno

(hiperoxia, normoxia e hipoxia) y temperatura (7, 11, 15, y 19 ºC) en cápsulas de Acanthina monodon. En negritas seindican los valores de significancia < 0,05

Source of variation Degrees of freedom F-value P-value

Temperature 3 609 0.0015

Oxygen 2 42.54 <0.0001

Interaction 6 2.42 0.041

Error 44

Fig. 2: Mean (A) proportion of developed embryos and (B) embryo size under different levels ofoxygen (hyperoxia, normoxia and hypoxia) and temperature (7, 11, 15, and 19 ºC). Vertical barsindicate one standard deviation.Proporción promedio de embriones desarrollados (A) y tamaño promedio de los embriones (B) al final del período experi-mental bajo los diferentes tratamientos de oxígeno (hiperoxia, normoxia e hipoxia) y temperatura (7, 11, 15, y 19 ºC). Lasbarras verticales indican una desviación estándar.


Embryo size

A significant interaction between the mainfactors precluded us from testing the effects oftemperature and oxygen condition on meanembryo size (Table 3). Hypoxia conditionsaffected embryo size of the most advancedstages within the capsule, as the smallestembryos were found at the end of theexperimental period under hypoxia, regardlessof temperature (Fig. 2B). Since developmentonly reached the trocophora stage under hypoxiaconditions, a second ANOVA including only theoxygen treatments in which more advancedembryo stages (between veliger and prejuvenile)were attained was conducted. The interactionterm was also significant (Table 3) due to thelack of consistent patterns in embryo sizebetween normoxia and hyperoxia acrosstemperatures. It is worth pointing out that thestatistically homogeneous group that exhibitedthe largest embryos included the lowesttemperatures (hyperoxia incubated at the twolowest temperatures and the three lowesttemperature of the normoxia treatment; Fig. 2B).

Intracapsular oxygen conditions

Extracapsular oxygen conditions reflected theexperimental treatments (Fig. 3). A significantinteraction in the ANOVA prevented us fromtesting for the effects of experimental oxygen

condition and temperature on intracapsular % airsaturation at the beginning of the experiment(Table 4, Fig. 3). The interaction was due to thelack of difference between hyperoxia andnormoxia at 7 °C in contrast with the higher %air saturation found under hyperoxia in theremaining temperatures (Fig. 3A). Threehomogeneous group were identified, whichreflected oxygen treatments (hyperoxia >normoxia > hypoxia; P < 0.05). At the end of theexperiment, temperature treatments did notaffect final intracapsular oxygen conditions, butoxygen treatments had a significant effect (Table4, Fig. 3B). Capsules incubated under hyperoxiashowed higher % air saturation than the othertwo treatments (P < 0.05), which did not showsignificant differences (normoxia and hypoxia; P> 0.05, Fig. 3).

DISCUSSION

This study provides further support for theimportance of oxygen conditions onencapsulated embryo development in marineinvertebrates (Strathmann & Strathmann 1989,1995, Cohen & Strathmann 1996, Cancino et al.2003), assessing also the effects of temperatureon intracapsular development (Wear 1974,Weathly 1981, Brante et al. 2003). Theexperiments carried out showed a clear effect ofhypoxia and high seawater temperature on

TABLE 3

Results of the two-way ANOVA conducted to test for differences in mean embryo size at the end ofthe experimental period in capsules of Acanthina monodon between oxygen (hypoxia, normoxia,hyperoxia) and temperature (7, 11, 15 and 19 ºC) treatments. The response variable was the mean

size of the most advanced stages reached in each capsule. P-values < 0.05 are indicated in bold

Resultados de los ANDEVAs de dos vías realizados para comparar el tamaño medio de los embriones al final delexperimento en cápsulas de Acanthina monodon entre los diferentes niveles de los tratamientos de oxígeno (hiperoxia,

normoxia e hipoxia) y temperatura (7, 11, 15, y 19 ºC). La variable de respuesta fue el tamaño medio de los estadíos másavanzados por cápsula. En negritas se indican los valores de significancia < 0,05

Source of variation Degrees of freedom F-value P-value Degrees of freedom F-value P-value

All treatments Excluding hypoxia treatments

Temperature 3 0.52 0.67 3 0.98 0.4

Oxygen 2 172.03 <0.0001 1 19.64 0.0001

Interaction 6 4.95 0.0006 3 9.46 0.0001

Error 44 30


TABLE 4

Results of the two-way ANOVA conducted to compare intracapsular oxygen conditions (% airsaturation) under oxygen (hypoxia, normoxia, hyperoxia) and temperature (7, 11, 15 and 19 ºC)treatments in capsules of Acanthina monodon, at the initial and the final experimental times. P-

values < 0.05 are indicated in bold

Resultados de los ANDEVAs de dos vías realizados para comparar las condiciones de oxígeno intracapsular (% desaturación de oxígeno) bajo los diferentes tratamientos de oxígeno (hiperoxia, normoxia e hipoxia) y temperatura (7, 11,15, y 19 ºC) en cápsulas de Acanthina monodon, en los tiempos experimentales inicial y final. En negritas se indican los

valores de significancia < 0,05

Source of variation Degrees of freedom F-value P-value Degrees of freedom F-value P-valueInitial experimental time Final experimental time

Temperature 3 0.76 <0.0001 3 0.64 0.59Oxygen 2 120.27 0.52 2 28.26 < 0.0001Interaction 6 2.30 0.04 6 0.24 0.96Error 44 44

Fig. 3: Mean intracapsular oxygen condition at (A) initial and (B) final experimental times underdifferent levels of oxygen (hyperoxia, normoxia and hypoxia) and temperature (7, 11, 15, and 19ºC). The numbers above indicate the average extracapsular oxygen conditions (and standard de-viation, between parentheses) in the hypoxia, normoxia and hyperoxia treatment throughout theexperiment for each temperature. Vertical bars indicate one standard deviation.Condición de oxígeno intracapsular en los tiempos (A) inicial y (B) final bajo diferentes condiciones de oxígeno (hipe-roxia, normoxia e hipoxia) y temperatura (7, 11, 15, y 19 ºC). Los números mostrados arriba del gráfico superior indican lacondición promedio de oxígeno extracapsular (y entre paréntesis la desviación estándar) bajo hipoxia, normoxia e hipe-roxia. Las barras verticales indican una desviación estándar.


embryo developmental success, size, andasynchrony in development. Both environmentalvariables exhibit strong spatial (Roy et al. 1998)and temporal (Smith et al. 1999, Gille 2002,Helly & Levin 2004) variations in the ocean.

Embryo development was strongly affectedby our experimental oxygen conditions, whichis in line with other results suggesting thecrucial effect of oxygen availability on embryodevelopment (Booth 1995, Strathmann &Strathmann 1989, Cohen & Strathmann 1996,Lee & Strathmann 1998, Woods 1999, Lardies& Fernández 2002, Fernández et al. 2003),particularly during early development (Cancinoet al. 2003). Our results show that underpermanent low oxygen conditions (50-60 %extracapsular air saturation) development wasstrongly hindered; hatching was not achievedand a large number of embryos remained inearly stage at the end of the experiment. In ourstudy, developmental success reached only 0.02% under hypoxia in comparison with the 79 %of developed embryos found under normoxia.Since intracapsular oxygen conditions duringlate development did not differ betweenhypoxia and normoxia, the significantdifferences in intracapsular oxygen conditionsamong oxygen treatments during earlydevelopment seemed to have determinedembryo development in our study. Afteraltering oxygen conditions between hypoxiaand normoxia throughout development,Cancino et al. (2003) showed similar effects ofhypoxia and also concluded that embryosensitivity to low oxygen conditions is strongerduring early development than in latedevelopment in another gastropod species. Wesuspect that hypoxic conditions are not rare inour study area, even in the intertidal zone, asadults and capsules can be exposed to extendedperiods (several weeks) of burial in sand.

The initial intracapsular % air saturationclearly mimicked the experimental oxygenconditions, which is reflected in the increase inintracapsular oxygen availability from hypoxiato normoxia to hyperoxia. Those differencestended to disappear as embryos developed,showing the impact of embryo oxygenconsumption on intracapsular oxygenavailability, especially in treatments in whichembryo development was successful (normoxiaand hyperoxia). Although only the trocophorastage was reached in the hypoxia treatment,

oxygen consumption seems to have occurredthroughout the experimental period sinceintracapsular oxygen availability averaged 46% air saturation at the end of the experimentwhile mean extracapsular % air saturation was62 %. This suggests that (1) oxygenconsumption still occurred under the lowoxygen conditions of the hypoxia treatment atthe end of the experiment, although oxygenconsumption was comparatively low, and (2)embryos may arrest development but do notnecessarily die even after extended periods ofhypoxia (Strathmann & Strathmannm 1989,1995). The 25 % decrease between extra andintracapsular % air saturation in the hypoxiatreatment contrasts with the 49 and 42 % airsaturation differences between intracapsularand extracapsular oxygen availability in thenormoxia and hyperoxia treatments. Thisimplies that embryo oxygen consumption (1)was higher under normoxia and hyperoxia thanin hypoxia, and (2) affected intracapsularoxygen conditions (Fig. 3). The effect ofembryo oxygen consumption was more evidentunder normoxia (Fig. 3). The lack of effect oftemperature on intracapsular oxygen conditionsdriven by temperature-dependent oxygenconsumption of the embryos remainsunexplained.

Although our results in general coincidewith evidence in the literature, showing theeffect of hypoxia on embryo development, thelack of differences in the number of developedembryos at the end of the experimental periodbetween normoxia and hyperoxia contrasts withprevious findings showing a clear effect ofhyperoxia on the final number of developedembryos (Lardies & Fernández 2002). Whenthe response variable used by Lardies &Fernández (2002) is compared with our results,the differences are notorious. We found that themean number of developed embryos per mm ofcapsule was 5 % higher (although nosignificant) in hyperoxia than in normoxia, incontrast with the 32 % significant increase inthe number of developed embryos underhyperoxia reported by Lardies & Fernández(2002). Our study clearly shows that hyperoxiadoes not positively affect the mean number ofdeveloped embryos, regardless of temperature.Therefore, the conclusion that embryodevelopment is determined by intracapsularoxygen conditions rather than by the


assignation of nurse egg by the mother needs tobe reconsidered (Lardies & Fernández 2002). Itis also interesting that slightly higherextracapsular oxygen conditions than ourhypoxia treatment (75 % air saturation) did notaffect developmental success of Acanthinamonodon embryos when compared to normoxicconditions (Lardies & Fernández 2002).

Although in general the effects oftemperature were less clear than the effects ofhypoxia, some variables were affected at thehighest experimental temperature. The lowestdevelopmental success was observed at 19 °C,mostly driven by the higher number ofundeveloped embryos found at the end of theexperimental period. Developmental successwas not affected by the number of abnormalembryos as similar patterns in the number ofdeveloped embryos and proportion ofdeveloped embryos were found acrosstreatments. The mean number of developedembryos was not affected by temperature;however, the level of asynchrony and the meansize of the embryos were negatively affected athigh temperatures. This suggests that evenwhen a comparable number of embryos maydevelop at high temperature, their subsequentsurvival may be affected since developedembryos attained smaller sizes (see alsoStrathmann & Strathmann 1995), which couldaffect rates of predation after hatching. Thesmall size of the embryos may be associatedwith the lower consumption of nurse eggs (orsiblings) since an important fraction of theundeveloped embryos remained at the end ofthe experiment (Fig. 2A). The negative effectof temperature on embryo aggregation has alsobeen reported for Brachyuran crabs, affectingfemale patterns of oxygen provision andbrooding costs (Brante et al. 2003). Moreover,a trade-off between investment in eggs andbrooding at different temperatures (latitudes)has been documented (Brante et al. 2003). Ourstudy, showing the effect of temperature onembryo development in aggregated masses, andprevious studies on Brachyuran crabs suggestthat aggregating embryos in the ocean, evenunder optimum oxygen conditions, may benegatively affected at high temperatures. It isremarkable that the frequency of broodingspecies seems to be higher in areas exhibitinglow temperatures (Thorson 1950). Clear spatialpatterns of distribution of brooding species

have also been found among amphibians,supporting the generality of our analysis ofcausal factors affecting embryo aggregationsand brooding in aquatic systems. This type ofanalysis becomes relevant considering thecurrent increase in both the proportion ofanoxic areas (Helly & Levin 2004) andtemperature in the world oceans (Smith et al.1999, Gille 2002)

ACKNOWLEDGMENTS

We are grateful to A. Brante, R. Calderon, C.Gonzalez, M. Cifuentes, C. Inostroza, R. Sotoand F. Veliz for their help collecting capsulesand in the laboratory. We also thank R. Finke,J. Holl, S. Kimberlin, and two anonymousreviewers for their suggestions on themanuscript. This study was fully funded byFONDECYT 1020860 (to MF). We alsoacknowledge the Humboldt Foundation. MFacknowledges the FONDAP-Fondecyt grant1501-0001 to the Center for Advanced Studiesin Ecology & Biodiversity and the Pew FellowsProgram in Marine Conservation.

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Associate Editor: Jorge NavarroReceived December 10, 2005; accepted March 20, 2006

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