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Do amphibious crabs have amphibious eggs? A case study of Armases miersii

Riccardo Simoni a, Stefano Cannicci a,⁎, Klaus Anger b, Hans-Otto Pörtner c, Folco Giomi c

a Department of Evolutionary Biology “Leo Pardi”, University of Florence, Via Romana 17, I-50125 Florence, Italyb Alfred Wegener Institute for Polar and Marine Research, Biologische Anstalt Helgoland, Postfach 180, D-27483 Helgoland, Germanyc Alfred Wegener Institute for Polar and Marine Research, Department Integrative Ecophysiology, Am Handelshafen 12, D-27570 Bremerhaven, Germany

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

Article history:Received 19 February 2011Received in revised form 14 August 2011Accepted 16 August 2011Available online 9 September 2011

Keywords:Air breathingCrab embryosSemi-terrestrial crabsTerrestrializationWater dependence

The efficiency of aerial respiration and the overall dependence on water availability during embryonic devel-opment have never been tested in semi-terrestrial or terrestrial crabs. In the present experimental study, weshow that respiration of the embryos of a semi-terrestrial intertidal crab, Armases miersii from Jamaica, is bi-modal. They are able to extract oxygen from both air and water, although a lower air respiration with approx-imately half O2 uptake than in water from the stage III onward was observed. Comparing the embryos ofovigerous females that were impeded for 18 h per day to submerge their egg clutches in seawater (n=8treatments), with those having unlimited access to water (n=7 controls), we observed no differences in em-bryonic respiration or development time, but the lack of water caused complete brood-loss in 5 females andmassive developmental problems in the remaining 3 broods, with an average hatching rate of only 28% anddeformations in most newly hatched larvae. Although the higher embryonic O2 uptake in water suggests areduced embryonic ability to extract oxygen from air, we propose as an alternative hypothesis an adaptivedown-regulation of the metabolic rate, which is not linked to oxygen extraction but to the water dependenceof vital metabolic pathways, such as the excretion of ammonia and CO2. Since bimodal embryonic respirationhas only been known from vertebrates, this is the first study demonstrating aerial respiration in brachyuranembryos. However, developmental deficiencies in water-limited egg clutches suggest that the embryos A.miersii still depend on water to avoid desiccation stress and, probably, an impediment of excretory processes.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The evolution of terrestrial forms and the consequent overcoming ofnon-aquatic constraints has been a recurrent process in crustacean radi-ation (Little, 1990). Considering the hypothesis that terrestrial invasionsare results of a gradual conquest via semi-aquatic (or semi-terrestrial)transitional zones, the comparison of physiological, morphological andbehavioral traits of intertidal, amphibious and terrestrial species aids tothe understanding of this evolutionary process (Little, 1989; Powersand Bliss, 1983).

Among the Crustacea, isopods and amphipods have been most suc-cessful in land colonization, and they include numerous taxa whose lifecycle is totally independent of aquatic environments (Little, 1983). Pa-leontological as well as biological evidence from extant forms corrobo-rates the hypothesis that also brachyuran crabs are at the dawn of landinvasion, thus constituting key models for investigations of the ongoingprocess of terrestrialization (Bliss and Mantel, 1968). Although manyextant taxa of semi-terrestrial and terrestrial brachyurans are, at least inthe juvenile and adult stages, effectively adapted to land environments,

the first fossil records of brachyurans with a semi-terrestrial life style goback no more than to the Cenozoic period (Greenaway, 1999; Naruseet al., 2003). Moreover, in the vast majority of land-adapted brachyurans,the early life-history stages are still strongly tied tomarine or other aquat-ic environments.

As in terrestrial crustaceans in general, the evolution of new mor-phological structures and the evolution of new respiratory and excreto-ry features have been crucial to overcome the physiological constraintsof terrestrial environments, with a particular emphasis on gas exchange,osmoregulation, and excretion (for reviews, see Bliss, 1968; Burggrenand McMahon, 1988; Burggren, 1992; Greenaway, 1999, 2003; Little,1983, 1990; Weihrauch et al., 2004). However, crabs do not have abrood pouch, which contributes in isopods and amphipods to successfulcolonization of terrestrial habitats, as it permits a protected embryonicdevelopment (Little, 1983). The vastmajority of crabs, despite a high de-gree of terrestrial adaptation in the adults, is therefore not completelyindependent fromwater, because an aquatic environment is still neededin the early (embryonic and larval) life-history stages (Anger, 1995a).

The most neglected aspect in the study of land colonization bybrachyurans is the biology of their embryonic stages (Cannicci et al.,2011). It is well known that female crabs brood their eggs, which arefixed through filaments to the pleopods under the abdomen (Fernándezet al., 2002), throughout the embryonic period. This strategy allows anactive protection of the brood, but at the same time incurs massive costs

Journal of Experimental Marine Biology and Ecology 409 (2011) 107–113

⁎ Corresponding author. Tel.: +39 0552288210.E-mail address: stefano.cannicci@unifi.it (S. Cannicci).

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due to enhanced energy expenditure by the mother (Fernández et al.,2000; Fernández and Brante, 2003; Ruiz-Tagle et al., 2002).

In the great majority of semi-terrestrial and terrestrial crabs, be-longing to the Ocypodidae and Gecarcinidae, the females incubatetheir egg masses in burrows that are excavated close to the sea andcharacterized, compared to the adult habitats, by a stable microhabi-tat with higher humidity, and reduced temperature and UV radiation(for review see: Crane, 1975; Hartnoll, 2010; Wolcott, 1988). One ex-ample is the terrestrial speciesGecarcoidea natalis, which performs dur-ing each reproductive season impressive migrations over 4–5 km frominland forests to the coast for mating and egg extrusion (Adamczewskaand Morris, 2001). As another example, ovigerous fiddler crabs (Ucaspp.) spend most of their time in burrows, where the temperature is10–15 °C lower than on the bare sandy surface, where temperaturescan reach 35–40 °C and where the adults are normally active (Edney,1961).

Nevertheless we still do not know the extent to which the eggs ofterrestrial and semi-terrestrial species may rely on aquatic or aerialgas exchange and excretion. The 30-fold higher oxygen content inair than in water, a lower viscosity, and faster oxygen diffusion inair could theoretically facilitate embryonic respiration. On the otherhand, if air can easily diffuse through the egg mass, problems of os-moregulation and an accumulation of metabolic waste productsmay more likely occur in air than in water.

In the present study, we tested if an aerial oxygen uptake occurs inthe eggs of a semi-terrestrial brachyuran crab, representing a possibleinitial evolutionary step towards a conquest of land (Anger, 1995a).In respirometric laboratory experiments, we investigated the depen-dence of developing eggs on water availability. As an experimentalmodel, we used the neotropical sesarmid species Armases miersii(Rathbun 1897), a semi-terrestrial crab that is distributed along theCaribbean islands, Florida and the Bahamas, inhabiting the intertidalzone of rocky shores and coastal limestone caves with highly variablesalinities (Abele, 1992). Although it lives in a coastal marine environ-ment, hiding during daytime in crevices of immersed limestone rocks,it also shows highly terrestrial behavior. During night time, it regu-larly performs foraging migrations for extended periods up to sev-eral hours per day to habitats well above the water line, wherethe ovigerous females are regularly observed to feed and releasetheir larvae using small supratidal rock pools filled with seawaterfrom wave splash, often mixed with highly variable amounts ofrainwater (Anger, 1995b; Schuh and Diesel, 1995). Compared tocongeners, this species passes through an abbreviated and partiallyfood-independent larval development (Anger, 1995a; Cuesta et al.,1999; Schuh and Diesel, 1995). An early ontogenetic appearanceof physiological adaptations, namely a strong capability of osmoreg-ulation expressed already at hatching, allows for successful larvaldevelopment in a land-locked habitat with highly unpredictable sa-linity conditions depending on occasional seawater intrusions, rain-fall, and strong evaporation (Anger et al., 2008). These unusualcharacteristics indicate a high degree of independence from the ma-rine environment in A. miersii, suggesting that special adaptationsto non-marine conditions may be expected also in the embryonicphase of its life cycle, which so far has remained largely unknown.

2. Material and methods

2.1. Maintenance of experimental animals

The experimentswere performed at the BiologischeAnstalt Helgoland(BAH) of the Alfred Wegener Institute (AWI, Germany), using A. miersiioriginating from Jamaica, but born and raised in the laboratory (formore details of origin and long-term cultivation, see Anger et al., 2008).The rearing and all experiments were carried out in a temperature-controlled room at 24 °C, with a relative humidity close to saturation,under a 12h:12h light:dark cycle. Fifteen ovigerous females were

transferred one day after egg laying from large communal rearing tanksto smaller plastic aquaria (50×20×30 cm). Up to four females wereplaced in each aquarium and provided with a water layer (10 cmhigh) and 1–2 vertically placed bricks to allow the females to choosestaying inside or outside the water. The experimental salinity (25‰)was obtained mixing seawater from the North Sea (32‰) with tapwater. The animals were fed daily with mixed diet (isopods, carrots).

To assess the water dependence of embryonic development, 15females were allocated to a control and a treated group. The controlcomprised 7 females with permanent access to water, while thetreatment consisted of 8 females with limited access to water (only6 h per day). This was achieved by fixing a rigid plastic net (10 mmmesh size) 5 cm above the bottom of the tank and by periodicallydraining the water through a hole in the base. The 18-hour periodof exclusion from water sources was chosen on the basis of field ob-servations that showed how adult A. miersii in Jamaica undergo reg-ular and extended natural period of emersion, during which theymigrate, forage and release larvae. They actively return to seawateronly during the hours characterized by strong sunshine (Anger,pers. obs.).

The tanks were equipped with a plastic lid with two holes, one ofwhich held the aerator tubing, while the other one was used for waterrenewal. In this way, animal disturbance during the experimental pe-riod was minimized.

2.2. Embryonic development and hatching success

The developmental stage of embryos and egg size were assessedby microscopic examination after each respirometric measurement(microscope: Olympus SZX16; camera: Olympus Dp 77; lens: Olym-pus SDF PLAPO1×PF; software: Cell D). Egg volume (V)was calculatedfrom measurements of the minor and major axis of the eggs, using theformula for oblate spheroids: V=1/6(п·d2·D), where d=smaller di-ameter, D=larger diameter.

The hatching success of eggs, expressed as the percentage of nothatched embryos versus the newly hatched larvae was evaluated forboth control and treated females.

2.3. Larval quality

In order to estimate offspring fitness at hatching, zoeae were mi-croscopically inspected and assigned to three categories: I) activelyswimming larvae; II) larvae that hatched but were unable to swim,moving or lying on the bottom; III) dead larvae. Each category wasphotographically documented for 10–12 randomly chosen larvaetaken from the treated and control groups, respectively, and usedfor morphometric analyses. The following measures were recorded:total larval length from the base of the rostrum to the end of the tel-son spine (TL); dorsal spine length (DSL); eye length (EL), and dorsalspine length as distance from the base of the rostrum to the base ofthe dorsal spine (RSL). Image-J software was adopted for imageprocessing.

2.4. Embryonic respiration

Embryonic respiration in water and air was measured with in eggscarefully separated from the pleopods of ovigerous females (bothcontrol and experimental group), and placed in a closed respirometrysystem. Hamilton microliter precision syringes (Volume: 250 μl;Hamilton Bonaduz AG, Bonaduz, Switzerland), filled with seawateror ambient air, were used as chambers to measure the aquatic and ae-rial respiration, respectively.

Oxygen saturation was recorded with an oxygen micro-optodesensor (needle-type, fiber-optic microsensor, flat broken tip, diame-ter: 140 μm) introduced through the top of a Hamilton syringe by ahole in the low bleed high temperature septa. The optode was placed

108 R. Simoni et al. / Journal of Experimental Marine Biology and Ecology 409 (2011) 107–113

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at a distance of about 5 mm from the experimental egg clutch andconnected to a Microx TX2 (PreSens GmbH, Regensburg, Germany).Prior to insertion, optodes were calibrated with air-equilibrated sea-water at 25‰ (100%) and with oxygen-free water (0%) obtained bydissolving 1% of sodium sulfite (Na2SO3) in seawater at 25‰. Oxygenconsumption was determined by measuring the decline in oxygensaturation in known volumes of water and/or air over time. Eachtrial ended before oxygen saturation dropped below 70%, in bothmedia, in order to avoid to exposure of the embryos to hypoxicconditions.

Between 30 and 60 eggs were used for each respiratory assay inwater. Eggs in early and late embryonic stages were immersed for20 or 40 min in 100 or 200 μl of seawater, respectively, (expecting dif-ferent magnitudes of respiration rate). During each experiment, thesyringe was gently inverted, so that the continuously moving eggs en-sured that the water was thoroughly mixed. Due to the higher concen-tration of O2 in air, masses of 60 to 100 eggs, a volume of 60 μl, and alonger measurement time (1 h) were used for respiration assays in airto obtain a significant decrease in oxygen saturation. Recordings wereinitiated only after an initial exposure to ambient air for 60 min. This ap-proach standardized the degree of dryness of the eggmasses. During re-spiratory assays in air no attempt was made to stir the volume insidethe syringe, which was immersed in water to avoid an ingress of ambi-ent air.

Repeated measurements were performed without eggs in both airand water in order to correct for the influence of microbial respira-tion. All respiration assays were performed at an average temperatureof 24°±0.5 °C.

2.5. Statistics

Permutational analysis of variance (PERMANOVA, Anderson,2001), based on at least 9800 permutations per term, was used totest the null hypothesis that there were no differences in volumeacross embryonic stages in treatment and control groups, both factorsbeing treated as fixed and orthogonal. Since in the embryonic respira-tion tests an unbalanced set of replicate groups of eggs from each ex-perimental female was used, a full factorial 4-way PERMANOVA wasalso utilized to test differences in oxygen uptake, with factors “em-bryonic stage”, “respiratory medium” and “treatment vs. control”,fixed and orthogonal and factor “female” random and nested in“treatment vs. control”. In both cases post-hoc tests were performedwhen appropriate. The Principal Component Analysis (PCA) wasused to visualize patterns in the morphometric larval characteristicsamong the different categories, while a 1-way multivariate PERMA-NOVA was performed to test for statistical differences across the cat-egories. All data are expressed as means±SE and the analyses wereperformed using PRIMER v. 6.1 and the PERMANOVA+for PRIMERroutines (Anderson et al., 2008).

3. Results

3.1. Embryonic development and hatching success

Among the seven control females, fiveweremonitored from embry-onic stage I, the remaining two only from stage III. All eight femalesmaintained under experimental treatment conditions were monitoredfrom stage I.

We could microscopically distinguish five different stages of em-bryonic development (I–V; Fig. 1). In stage I, the yolk represented al-most 100% of the total volume, while the undifferentiated embryodeveloped into a blastula (Fig. 1A). Stage II is characterized by gastru-lation (Fig. 1B), stage III by the appearance of the first cromatophoresand eye spots, with yolk occupying about 50% of egg volume (Fig. 1C).Stage IV was characterized by the appearance of a regular heart-beat,further developed eyes, and star-shaped chromatophores (Fig. 1D). At

this stage, the yolk filled only about 25% of total egg volume. In thefinal stage V, the embryo filled almost the entire egg volume withminor traces of yolk (Fig. 1E).

Six out of 7 control females produced successfully hatching larvae,while only one female lost the eggs at stage III. In the treatmentgroup, by contrast, only 3 females produced successfully hatching lar-vae, while the remaining 5 females lost their eggs before hatching(one at stage I, two at II, one at III, and one at V).

500 µm

A B

C D

F

H

E

G

Fig. 1. Armases miersii embryonic development and larvae. Stage I: Blastula, 100% of yolk21–12 days to hatch(A). Stage II: Gastrula 12–7 days to hatch (B). Stage III: first appear-ance of chromatophores and eye spots, 7–4 days to hatch (C). Stage IV: well developedeyes, regular heart beats, star-like chromatophores, 4–2 days to hatch (D). Stage V: welldeveloped embryo, yolk almost completely consumed, 2–0 days to hatch (E). Larvae:healthy larvae (F); alive larvae unable to swim and only moving and lying on the bottom(G); dead larvae (H).

109R. Simoni et al. / Journal of Experimental Marine Biology and Ecology 409 (2011) 107–113

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The durations of these stages were roughly the same in controland treated embryos, with 9 days in stage I, 5 days in II, 3 days inIII, 2 days each in stages IV and V.

Egg size increased during development, but the increment dif-fered between the control and experimental groups. The final volumeof the control eggs was 42% larger than at hatching (0.110 vs0.078 mm3), while the treatment eggs showed an increase by 57%(from 0.076 to 0.120 mm3; Table 1). However, the egg volume atstage IV was significantly smaller in treatment broods compared tocontrol groups, whereas the opposite was true for stage V eggs(Fig. 2, Table 2). Hence, the increase in egg volume was in the treatedbroods gradual throughout development, while control eggs de-creased in size between the final stages IV and V (Fig. 2, Table 2).

3.2. Larval quality

In the treatment broods, nearly all newly hatched larvae died shortlyafter hatching or were lying on the bottom of the tank, being unable toswim. An exact counting of all hatched larvae in one treatment and onecorresponding control group showed that 20% of the control larvaefailed to hatch, remaining within the egg membrane, while 63% of thelarvae were actively swimming (Fig. 1F); 10% hatched alive but werethen unable to swim (category II; Fig. 1G), and 7% died shortly afterhatching (category III; Fig. 1H). In the treatment group, by comparison,72% of larvae were unable to hatch, only 2% were healthy (category I),3% belonged to category II, and 23% died.

The abnormal development of embryos in categories II and III waseasy tonote (Fig. 1G andH). PCA confirmeda strongmorphometric differ-ence between these categories, with larvae from category II being charac-terized by proportionally shorter body length than in the other categories(Fig. 4). Dead larvae (category III) were characterized by smaller eyes andshorter RSL compared to healthy larvae. PERMANOVA comparison amongthe groups showed significant differences among all categories (pseudo-F=19.224; df=3; pb0.0001), and between healthy swimming larvaefrom control and treatment broods.

3.3. Embryonic respiration

Respirometric measurements were performed in a total of 133 eggclutches (including both treatment and control embryos at differentstages; Table 1). No treatment effect on embryonic respiration ratewas recorded, but there was a significant difference in respirationrates among egg masses collected from different females (Table 3).Notwithstanding these differences among clutches, we found cleardifferences in oxygen uptake (MO2) among developmental stagesand between respiratory media (Table 3). Although MO2 rose in bothmedia exponentially during the course of embryonic development(Fig. 3), oxygen uptake in water increased by a factor of 17 (from 223±25 to 3907±275 pmol·egg−1·h−1). In air, by comparison, it increasedby a factor of 10 (from 234±41 to 2269±240 pmol·egg−1 h−1).

Furthermore, the early developmental stages (I–III) showed nosignificant difference in respiration rates between the two media,

while a significantly higher respiration rate in water than in air wasobserved in the final phase of development (Fig. 3; Tables 1 and 5).

4. Discussion

The present study demonstrated that the embryos of the amphib-ious brachyuran A. miersii shows bimodal respiration, being able toextract oxygen from both air and water and that embryonic metabo-lism in air appeared to be down-regulated from stage III onward. Toour knowledge, this is the first time that aerial respiration was inves-tigated in brachyuran embryos. This new finding may change ourview of evolutionary trends in terrestrial crab reproduction.

While no data are available for decapod crustaceans to compareembryonic respiration rates in air, our results on aquatic oxygen up-take are in good agreement with results obtained in previous studies.Studies on the intertidal species Cancer pagurus, Heterozius rotundifronsand Cyclograpsus lavauxi have shown an exponential increase of meta-bolic rates during embryonic development (Naylor et al., 1997, 1999;Taylor and Leelapiyanart, 2001; Wheatly, 1981). Similar to A. miersii,the respiration rate increases nearly linear through the first stages, butthe rise becomes exponential at the onset of cardiac activity. Furthermore,in all species for which data are available, it is worth-while mentioningthat the embryonic metabolic rate correlates positively with egg size,but not necessarily with the duration of development. However, differ-ences in the ecology and biology of the species previously studied donot allow any further comparison with A. miersii in terms of respirationrate and developmental time. In fact, only H. rotundifrons showed a com-parable respiration rate, but its developmental time spans to 194 daysagainst 21 days in A. miersii, whose eggs also are three times larger.

Comparisons between aerial and water respiration rates were car-ried out in a frog, Pseudophryne bibroni (Bradford and Seymour, 1985)and in two intertidal fish, Fundulus heteroclitus (DiMichele and Powers,1984) and Leuresthes tenuis (Darken et al., 1998). The embryos of these

Table 1Armases miersii embryonic respiration (MO2) and egg volume recorded during the five developmental stages in control (water constantly available) and treatment (water availablefor 6 h per day) experiments. Values are means±SE.

Control Treatment

Stage Air MO2

(pmol·egg−1·h−1)n Water MO2

(pmol·egg−1·h−1)n Volume

(mm3)n Air MO2

(pmol·egg−1·h−1)n Water MO2

(pmol·egg−1·h−1)n Volume

(mm3)n

I 200±43 8 194±19 8 0.078±0.001 63 289±80 5 274±59 6 0.076±0.001 88II 414±85 9 484±52 8 0.092±0.001 77 253 ±54 7 453±79 6 0.092±0.001 76III 794±174 7 1248±133 7 0.104±0.001 117 871±222 5 1117±177 6 0.107±0.002 33IV 1217±208 8 2332±129 7 0.117±0.002 40 1467±321 3 2566±251 3 0.111±0.002 27V 2249±264 10 4041±336 9 0.110±0.001 103 2310±416 5 3705±495 6 0.120±0.002 47

n: number of measurements.

II III IV V

Embryonic stageI

Eg

g v

olu

me

(mm

3 )

0.07

0.08

0.09

0.10

0.11

0.12

0.13

ControlTreatment

Fig. 2. Armases miersii embryonic development. Average egg volume (±SE) of eggs intreated (open circles) and control experiments (filled circles) at each developmentalstage. *pb0.05; **pb0.01.

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three vertebrates are able to carry out a complete development in moistmicro-environments with no apparent metabolic failures due to desicca-tion and, in consequence, disturbance of excretion or osmoregulation.Their larvae hatch in water, being highly synchronized with periodicfloods or spring tides, in frogs and fishes, respectively. However, it is dif-ficult to compare the respiration physiology of the embryos of these ver-tebrateswith the one shownbyA.miersii, since the former lay eggmassesadherent to the substratum, thus showing strongdifferences in the clutchecology and morphology. P. bibroni lays about 100 eggs with 2–3 mm indiameter, each surrounded by a thick-walled mucopolysaccharide cap-sule (Woodruff, 1977), while F. heteroclitus has small clutches, with afew tens to a few hundred eggs, and L. tenuis lays between several hun-dreds and several thousands of eggs. The two fish species have similaregg size of about 2 mm (Able and Castagna, 1975; Marteinsdottir andAble, 1988; Smyder and Martin, 2002). The oxygen uptake and devel-opmental time of embryos were similar in the twomedia throughoutthe developmental period, suggesting that these embryos are trulyamphibian. In A. miersii embryos, by contrast, the aerial/aquaticMO2 ratio dropped from about 1.0 at the first developmental stageto about 0.5 in the last two developmental stages.

The two-fold higher respiration rate of A. miersii embryos in watercompared to air could be explained with a reduced ability of embryosto extract oxygen from air. Combined effects of surface tension andgravity may produce a collapse of the interstices within the eggmass, preventing oxygen diffusion and generating an anoxic microen-vironment, as observed in gelatinous egg masses (Seymour, 1999;Strathmann and Chaffee, 1984; Strathmann and Hess, 1999). This

would imply a bimodal respiration of embryos, but a weaker capabil-ity of aerial oxygen extraction compared to the adults. Consequently,embryos could undergo metabolic depression as a response to oxygenlack at the tissue level, causing e.g. bradycardia as observed in embry-os of marine crustaceans experiencing anoxic conditions in water(Reiber, 1997; Spicer, 2001).

However, our findings also can be explained with an alternativehypothesis assuming that the metabolic rate is not limited by reducedoxygen extraction from air. Instead, a down-regulation of the respira-tion rate in air is possible as a metabolic response of the embryos tocope with the water-dependence of other metabolic processes suchas ammonia excretion and acid–base balance. If the egg mass canuse the thin layer of water on the egg surface to extract oxygen, itshould be well oxygenated, and the oxygen uptake may even be

Table 2Armases miersii embryonic development. PERMANOVA test on differences in egg vol-ume across developmental stage and experimental conditions, both factors treated asfixed and orthogonal. Pairwise post hoc tests are also shown for the significant interac-tion term between the two factors.

Source df MS Pseudo-F p

Stage – St 4 0.0328 253.17 0.0001Experiment – Exp 1 0.0001 0.79 0.38St×Exp 4 0.0009 7.28 0.0001Res 661 0.0001Total 670

Pairwise tests t p

Control vs. treatment within stage I 0.970 0.326Control vs. treatment within stage II 0.257 0.799Control vs. treatment within stage III 1.029 0.309Control vs. treatment within stage IV 2.512 0.014Control vs. treatment within stage V 4.440 0.0001

df: degree of freedom, MS: variance, P: probability level of Pseudo-F statistic value.

Embryonic stageI II III IV V

pm

ol *

eg

g-1

* h

ou

r-1

0

1000

2000

3000

4000

5000

AirWater

Fig. 3. Armases miersii embryonic respiration. Embryonic oxygen uptake rates (±SE) inwater (open circles) and air (filled circles). The data from treatment and control conditionswere pooled according to the results of the PERMANOVA test (see Table 3). *pb0.05;**pb0.01.

Healthy Larvae (Control)

Healthy Larvae (Treatment)

Dead Larvae (Treatment)

Inactive Larvae (Treatment)

-4 -2 0 2 4

PC1

-4

-2

0

2

PC

2

r.s.l.

d.s.l.

e.l.

t.l.

Fig. 4. Armases miersii embryonic development. Two-dimensional scatter plot of thefirst and second principal components (a total of 79% of variation among samplesexplained) of the three morphological larval categories after the normalization ofdata. Vectors of the linear correlations between individual variables are superimposedon the graph. s. l. = dorsal spine length, e. l. = eye length, r. s. l. = rostrum-dorsalspine length, t. l. = total length.

Table 3Armases miersii embryonic respiration. PERMANOVA test on differences in embryos ox-ygen uptake (MO2) across developmental stage, respiratory medium and experimentalconditions, all factors treated as fixed and orthogonal. Pairwise post hoc tests are alsoshown for the significant interaction between the factors “stage” and “respiratorymedium”.

Source df MS Pseudo-F p

Stage – St 1 7,875,400.0 48.90 0.001Medium – Me 4 27,981,000.0 74.57 0.000Experiment – Exp 1 59,922.0 0.09 0.813female(Exp) – Fe(Exp) 7 742,440.0 3.28 0.017Me×St 4 2,114,100.0 5.21 0.009Me×Exp 1 178,980.0 1.11 0.342St×Exp 4 15,655.0 0.04 0.997Me×Fe(Exp) 7 151,400.0 0.67 0.631St×Fe(Exp) 15 378,090.0 1.67 0.125Me×St×Exp 4 118,050.0 0.29 0.878Me×St×Fe(Exp) 15 409,370.0 1.81 0.105Res 69 226,260.0Total 132

Pairwise test t p

Water vs. Air MO2 within stage I 0.319 0.789Water vs. Air MO2 within stage II 2.933 0.0309Water vs. Air MO2 within stage III 2.358 0.0615Water vs. Air MO2 within stage IV 8.084 0.0068Water vs. Air MO2 within stage V 3.882 0.0151

df: degree of freedom, MS: variance, P: probability level of Pseudo-F statistic value.

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enhanced in air due to higher concentration and diffusiveness. How-ever, all other metabolic pathways would still need a constant supplyof water. In this case, the embryos might down-regulate their meta-bolic rate when exposed to air, although they still can be consideredbimodal in relation to oxygen uptake. Water evaporation from theegg mass can have two effects: 1) an increasing osmotic concentra-tion due to dehydration; 2) a disappearance of the water layer onthe egg surface, which may result in a failure of ammonia excretionand a disturbance of the acid–base balance. In conclusion, the distur-bance of principal metabolic processes could be mitigated by reducedrespiration while waiting for the next immersion in water.

The limited access to water in our experiments resulted in lowhatching success, with high incidence of unhatched embryos andunviable or dead larvae. Larvae that were not capable to swim dif-fered also morphologically from healthy larvae hatching under con-trol conditions. Even those larvae being able to swim showed in thetreatment experiments different rostrum-spine length proportions,suggesting subtle developmental deficiencies that may become cru-cial drawbacks in later larval life. These results suggest that the exper-imental time of exposure to air was probably near the limit ofphysiological tolerance of A. miersii embryos. This may be tested in fu-ture studies with a wider set of different exposure conditions thatshould be intermediate between our treatment and control conditions.

Delayed hatching ormetabolic failure due to stressful conditions dur-ing embryonic developmentwas also demonstrated in several other taxa(Booth, 1995; Crisp, 1959; Giorgi and Congleton, 1984; Rankine et al.,1990; Seymour, 99; Strathmann and Strathmann, 1995; Strathmannand Hess, 1999). Thus, in the treated embryos we would expect notonly deficiencies in hatching success and larval quality but also differentoxygen uptake in bothmedia aswell as delays in the hatching time com-pared to controls. In A. meirsii, we did not observe delays in hatchingtime. This could be due to a very strictly, genetically controlled em-bryonic developmental time, not leaving suffering embryos an op-tion to delay the embryonic development and hatching time.

In our opinion, we did not observe differences in oxygen uptakebetween the two treatment groups because the respiration tests,both in treatment and control experiment, were performed after the6 h of water availability, when the embryos could already have recov-ered the transient metabolic stress suffered during the 18 h of dry-ness. We suggest that the lack of difference in respiration ratesobserved between treatment and control groups was caused by ourexperimental procedure. In fact, all respiration tests were performedimmediately after the 6 h of water availability, when the embryoscould already have recovered the transient metabolic stress sufferedduring the 18 h of dryness. However, even if an extraction of similarquantities of oxygen by treatment and control eggs at each stagewas recorded, the 18 h spent in air were enough to cause deficienciesin development.

Five females in experimental treatment conditions and one in acontrol lost their entire egg masses long before hatching. These lossesmay be explained as a form of brood-desertion behavior, an adaptivestrategy that saves energy which is otherwise required for parentalcare of a compromised clutch. This has also been reported for a vari-ety of other brooding animal taxa (Székely et al., 1996; Webb et al.,2002). This implies that female A. miersiimay be able to perceive em-bryonic deficiencies and that they can evaluate the trade-off betweenthe costs and benefits of continued investment in their current breed-ing attempt.

The suggested disturbance of osmoregulation and excretion inembryos exposed to air finds support in data on developmental vari-ation of egg volume. Embryos carried by control females displayed adecreasing volume between stages IV and V, as reported also for thesemiterrestrial crab Neohelice (=Chasmagnathus) granulata (Basand Spivak, 2000). Embryos with limited access to water, by contrast,showed a constant increase in egg volume, possibly due to a continu-ous accumulation of catabolic end products.

In conclusion, our data show for the first time in a crab species, A.miersii, that embryos can breathe in both air and water, although theyare not truly amphibious. Both reduced oxygen uptake in air and thefact that there was a high rate of mortality and developmental failuresin embryos experimentally deprived of water, suggest that they ac-tively slow-down their metabolic rate when exposed to air. Althoughthe adult crabs show conspicuous terrestrial adaptations, their em-bryos depend still strongly on water availability. Further studies areneeded to understand how embryonic respiration and excretion canlimit the conquest of land by crabs.

Acknowledgments

This study was funded by Italian MIUR funds (ex 60%) and by an ESFShort Visit Grant (no. 3300) within the ESF activity entitled ‘Thermal ad-aptation in ectotherms: linking life history, physiology, behavior and ge-netics’, granted to SC. FG is grateful to the Hanse Institute for AdvancedStudies (Hanse Wissenschaftskolleg) in Delmenhorst, Germany for thesubstantial support during the junior fellowship. We thank R. Kikofor his extremely helpful comments on the text. The present workconforms to the legal requirements of the country in which it wascarried out. [SS]

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