An evolutionary solution of terrestrial isopods to cope with low atmospheric oxygen levels

Atmospheric oxygen concentration is a driving force for many major events in the evolution of life (reviewed in Berner et al., 2007). The origin of metazoan life (Canfield et al., 2007), land invasion by animals (Ward et al., 2006), animal radiation and diversification (Saltzman et al., 2011; Sperling et al., 2013), evolution of modern phytoplankton (Falkowski et al., 2004), placental reproduction (Falkowski et al., 2005), gigantism (Dudley, 1998; Payne et al., 2012) and mass extinction (Huey and Ward, 2005) all represent key evolutionary events closely tied to high, low or rapidly changing oxygen levels on Earth. Many evolutionary innovations such as brood care (Baeza and Fernandez, 2002), metamorphosis (Callier et al., 2013), sexual reproduction (Nevalainen et al., 2011), gas exchange patterns in insects (Lighton and Berrigan, 1995), arrested embryonic development in reptiles (Rafferty and Reina, 2012) and dormancy (Katajisto, 2004) have been put into context with environmental oxygen availability and/or organismal oxygen requirements. These evolutionary strategies may thus either represent adaptations that evolved independently multiple times, in direct response to fluctuating levels of atmospheric oxygen, or alternatively represent secondary adaptations to meet changing oxygen requirements in organisms subject to environmental change.

Here, we show that catch-up growth is probably a further example of such an evolutionary innovation. Switching from aquatic to air conditions during development within the motherly brood pouch of the terrestrial isopod Porcellio scaber relaxes oxygen limitations and facilitates accelerated growth under motherly protection. Our findings provide important insights into the role of oxygen in brood care in present-day terrestrial crustaceans.

Early development in terrestrial isopods occurs sequentially in aquatic conditions followed by aerial conditions in the motherly brood pouch (Fig. S1; Hoese and Janssen, 1989). Boxes containing gravid females of the terrestrial isopod Porcellio scaber Latreille 1804 were maintained in plexi-chambers (40×50×55 cm, YETI- Advertisement Agency, Kryspinów, Poland) with regulated normal (22%) and low oxygen (10%) levels (ROXY-4, Sable Systems Europe GmbH, Berlin, Germany). Plexi-chambers with normoxic and hypoxic conditions were maintained in two climatic chambers with constant temperature of either 15 or 22°C (POL-EKO APARATURA, Sp.j., Wodzisław Śląski, Poland), providing a two-factorial experimental design (15°C and 22% oxygen, 15°C and 10% oxygen, 22°C and 22% oxygen, and 22°C and 10% oxygen). A uniform circulation of air through the closed plexi-chambers was provided by a fan. Relative humidity was regulated to 75% by a separate dew point generator for each of the four plexi-chambers (DG-4, Sable Systems Europe GmbH). The temperature difference of 7°C in our experiment resulted in a twofold increment in oxygen consumption rate (Q₁₀≈2.8) of adult isopods (T.H., unpublished data). Gravid females were examined under a stereomicroscope for the presence/absence of liquid inside the marsupium every 2 days to define the length of the aqueous and gaseous phase (see Fig. S1 for a description of marsupial phases). The current approach is an improvement on our previous study (Horvathova et al., 2015) as it includes a higher time resolution of sampling, determination of the length of both the aqueous and gaseous phases separately, instead of only the total developmental time, and represents a more conservative approach for the determination of the onset of marsupial development. Intramarsupial development is characterized by 20 discrete stages (Milatovič et al., 2010). Because we could not precisely define the first day of gravidity, the length of the aqueous phase was calculated as the time between the appearance of stage 13 (Milatovič et al., 2010) and the day of liquid absorption (see Table S1 for the duration of the first 12 stages). Stage 13 can be easily defined by the presence of hepatopancreatic glands that enclose a conspicuous yolk sac (fig. 1f in Milatovič et al., 2010). The length of the gaseous phase was calculated as the time between the absorption of marsupial liquid and the day of hatching. Females were removed and weighed to the nearest 0.01 mg (Mettler Toledo XP26, Greifensee, Switzerland) after hatching. We determined the clutch size and body mass of 10 hatchlings (to the nearest 0.001 mg) for a random subsample of clutches in each condition (see Table 1). Mean hatchling mass was calculated by dividing the total mass by the number of weighed offspring. Mortality of the brood was checked every 2 days and dead females or aborted broods were removed.

All data were tested for normality of distribution and homogeneity of variance prior to analyses. The total number of females used in the analysis was 219. Differences in the total length of brood development, length of the aqueous and gaseous phases, duration of the gaseous phase as a percentage of total development time, clutch size and hatchling body mass were analysed by two-factorial ANCOVA with oxygen and temperature as fixed factors and mother mass as covariate, and all possible interactions were stepwise removed. The interaction term between temperature and oxygen was always retained in the minimum model. Because isopod progeny growth occurs during both the aqueous and gaseous phases (Helden and Hassall, 1998), we analysed this pattern in more detail and tested whether the length of the gaseous phase depends on the length of the aqueous phase. For this analysis, we included the length of the gaseous phase as a dependent variable, temperature and oxygen as fixed factors and the length of the aqueous phase as a covariate, with all possible interactions. The GLIMMIX procedure was used to analyse differences in the survival rates during the aqueous and gaseous phases of brood pouch development. The model included survival as a binary response variable (died/survived during development in the aqueous or gaseous phase), temperature and oxygen as fixed factors and the interaction between these two factors. We did not differentiate between mortality of the brood and mortality of gravid females because females that aborted clutches died within a few days. All statistical analyses were performed with SAS 9.4 statistical software (SAS Institute Inc., Cary, NC, USA).

Our manipulation of oxygen did not affect total development time within the brood pouch (sum of aqueous and gaseous phases; Fig. S1), but total development lasted approximately twice as long in the cold as in the warm (Fig. 1A, Table 1; Fig. S2). Differences in total developmental time did not translate into differences in hatchling body mass; offspring body mass at hatching did not differ significantly between experimental conditions (Table 1). This suggests that development within the marsupium lasts until a predetermined body mass is reached and that ambient temperature – an important driver of growth rate in crustaceans (Hartnoll, 2001; Horvathova et al., 2015) – only determines the time required to reach the predetermined body mass. Because both mortality during development within the brood pouch and mortality during the juvenile stage can reach up to 60–80% in terrestrial isopods (see Table S2; Dangerfield, 1997; Hassall et al., 2005; Zimmer, 2002), females may increase fitness more by increasing hatchling size, rather than increasing fecundity, as increased hatchling body mass will enhance growth and survivorship as a juvenile. This may result in relative constancy of body size, but varying brood size.

With respect to the two constituent phases of marsupial development of present-day terrestrial isopods, we found that oxygen limitations occur during development of the progeny in the aqueous phase but not the gaseous phase within the motherly brood pouch. Under hypoxia, longer development in the initial aqueous conditions in the cold (Fig. 1B, Table 1) was followed by more rapid development in the subsequent gaseous phase at both temperatures (Fig. 1C, Table 1; Fig. S2). Given that oxygen limitation is expected to be greater under warm conditions, this result is of special interest in view of the capacity of brooding P. scaber females to supply oxygen to embryos under varying temperature conditions. Female isopods actively provide oxygen to their progeny only during the aqueous phase, through special structures called cotyledons within the marsupial cavity (Hoese and Janssen, 1989) that have evolved as a novel adaptation to terrestrial habitats (Csonka et al., 2013; Hornung, 2011). Females under conditions of low environmental oxygen and temperature may be limited in the supply of oxygen as a result of their slow metabolic rate and increased oxygen demand of the brood, and this might explain why progeny spent a proportionally longer time in the aqueous phase. Longer development in the aqueous phase may be necessary to achieve a state of development in preparation for subsequent development in gaseous conditions. This result suggests that the limits to oxygen supply imposed by the environmental conditions may restrict the mother's capacity to provide adequate oxygen to the brood.

The total developmental time of the progeny was not affected by oxygen levels; however, the relative distribution of time spent in the aqueous and air phases differed significantly (Fig. 1D, Table 1; Fig. S2). Progeny developing under 22% oxygen and thus less constrained by oxygen availability during the initial aqueous phase spent a proportionally longer time developing in the subsequent gaseous phase compared with progeny developing in 10% oxygen (Fig. 1D). Because the same hatchling size was attained, we infer that processes that govern completion of intramarsupial development might be differentially affected by oxygen availability during the two marsupial phases; any effect during the aquatic phase may be compensated for during the gaseous phase. At present, we can only speculate about potential constraints leading to the ratio between the two phases, such as growth and development of the digestive system, cuticular protuberances or the tubular heart (Milatovič et al., 2010; Mrak et al., 2014; Štrus et al., 2008; Wolff, 2009).

We interpret our results for the two-factorial experimental design with low and normal oxygen levels and high and low ambient temperature levels to provide an example of catch-up growth (Metcalfe and Monaghan, 2001). Our analysis on differences in developmental timing within individual broods showed that offspring that were subjected to a period of constrained development in the aqueous phase under 10% oxygen shortened development time in the gaseous phase (aqueous phase×oxygen: F_5,103=4.48, P=0.037; Fig. 2) and attained a final size not different from that of non-constrained individuals under 22% oxygen (Table 1). These results are in agreement with the physical properties of oxygen in water and air, with oxygen being 20- to 40-fold more abundant and diffusing 10,000 times more rapidly in air than in water (Denny, 1993). Constrained growth in water may be attributed to the brooding costs of oxygen provisioning by the mother (Fernández et al., 2000), which can be later offset by increased oxygen availability in air. Growing in sheltered conditions under motherly protection but completing intramarsupial development under relaxed oxygen constraints – through the incorporation of a gaseous phase – may thus represent an important evolutionary strategy for life under transient hypoxic environments.

Catch-up growth represents an evolutionary strategy that compensates for a poor start in an animal's life, such that: ‘animals that have experienced a period of retarded growth […] then enter a phase of growth acceleration when conditions improve’ (Metcalfe and Monaghan, 2001). Catch-up growth is a widely used strategy among vertebrates and invertebrates and it commonly occurs under conditions of nutritional deficit (Dmitriew and Rowe, 2007; Hector and Nakagawa, 2012; Malzahn and Boersma, 2012; Morgan and Metcalfe, 2001), but accelerated growth is also seen in response to environmental cues (De Block and Stoks, 2008; Orizaola et al., 2014). Here, we provide evidence for catch-up growth during early development within the motherly brood pouch of terrestrial isopods, which we believe to be the first report regarding catch-up growth in conjunction with oxygen availability. In the present case, low environmental oxygen concentration initially increased development duration and was subsequently compensated through catch-up growth after the oxygen limitation was relaxed.

Colonization of land appeared in two major waves, each corresponding with high atmospheric oxygen content: the first invasion occurred between 425 and 360 million years ago (Mya) and was followed by a second one beginning approximately 345 Mya (Berner et al., 2007; Ward et al., 2006). Isopods – the most successful terrestrial colonizers among crustaceans (>3600 species; Schmalfuss, 2003) – invaded land during the second major wave (Broly et al., 2013, 2015), when atmospheric oxygen content reached its maximum value (Fig. 3), indicating relaxed conditions for processes with high oxygen demands. A brood pouch as a brooding strategy of crustaceans evolved long before, as indicated by fossil records of marsupia (Fig. 3; Broly et al., 2017; Siveter et al., 2014). It has been suggested that animals that evolved under such high oxygen levels and then survived the mass extinction during the subsequent period of low oxygen content (Fig. 3) underwent major modifications of their body plans (Huey and Ward, 2005; Payne et al., 2013; Ward, 2006). We hypothesize that oxygen may have played an important role in the evolution of brood care in isopods (Fig. 3). While in a marine environment, a brood pouch may have primarily protected eggs from mechanical damage (Steele, 1991), it may have progressively evolved into a more complex structure with a supply of water (Carefoot and Taylor, 1995) and oxygen (Hoese and Janssen, 1989) and the inclusion of a gaseous phase at a later stage of development (this study). Concluding development within the motherly brood pouch in air by implementing gaseous conditions may have relaxed the constraints imposed by the female's limited capacity to supply oxygen and may also have enabled progeny to respond to hypoxic conditions via catch-up growth as a key innovation for life on land. We hypothesize that spatial and temporal variation in oxygen availability may possibly result in strong selective forces promoting the evolution of novel traits.

We thank Edyta Sadowska, Maciej Pabijan, Aleksandra Walczyńska and Manfred Bauchinger for critical comments. We thank the two anonymous reviewers for their valuable comments on the manuscript.