Embryonic water uptake during pregnancy is stage- and fecundity-dependent in the snake Vipera aspis

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Abstract

Water is a crucial resource that can profoundly impact the biology of terrestrial organisms. Early life stages are particularly sensitive to hydric constraints because water uptake is an important component of embryonic development. While amniotic eggs constitute a key innovation to terrestrial life, many vertebrates are viviparous wherein the mother must be the source of water for her developing embryos. Since most viviparous squamates are lecithotrophic (i.e., energy is supplied to the offspring as yolk deposited into pre-ovulated follicles), water is the predominant resource allocated from the mother to the offspring during development. Contrary to energy that can be stored (e.g., as fat reserves), water typically cannot be acquired in advance. Therefore, the embryos' need for water can impose significant constraints on the pregnant female. We detailed water flux during pregnancy in a viviparous snake, the aspic viper (Vipera aspis). We found that embryonic water uptake occurred mostly during the second half of pregnancy—a period dominated by somatic growth. We also found that, somewhat unexpectedly, changes in female plasma osmolality were negatively related to fecundity. This latter result suggests that water consumption by the female is especially important for large litter sizes, and thus may suggest an important sensitivity of reproductive females to environmental water availability.

Introduction

Variation in environmental resources can have profound influences on physiological traits, tradeoffs, and life history traits (O’Connor et al., 2006). Water is a critical resource that can exhibit important seasonal fluctuations and is known to affect terrestrial organisms' physiology (Bradshaw, 1997), growth (Lorenzon et al., 2001), and survival (Shine and Brown, 2008, McKechnie and Wolf, 2010). Embryonic life is particularly sensitive to water availability, and eggs are sensitive to dehydration (Du, 2004, Stein and Badyaev, 2011). For instance, water supply is critical for the conversion of stored energy reserves (yolk) to embryonic mass (Vleck, 1991, Thompson and Speake, 2003, Belinsky et al., 2004). Therefore, water limitation can profoundly alter embryonic development and affect offspring quality or result in embryonic death (Brown and Shine, 2005, Lourdais et al., 2007). Such negative impacts have favored the emergence of multiple adaptations, including the selection of an appropriate nesting site and/or parental care of the eggs to minimize water loss (Shine, 2004a, Shine and Brown, 2008, Stahlschmidt and DeNardo, 2010). The eggshell's structure can also be modified either to minimize water loss (hard-shell eggs are laid in a desiccating atmosphere) or, conversely, favor water uptake (parchment shell eggs laid in a humid environment or substrate) (Deeming and Ferguson, 1991, Shine and Thompson, 2006).

While most terrestrial organisms are oviparous, viviparity has emerged on multiple occasions in amphibians, non-avian reptiles, and mammals (Blackburn, 2000, Shine, 2004b). These repeated transitions are associated with a diversity of embryonic nutrition strategies that have attracted considerable interest (Blackburn, 2006, 1999. Despite the focus on energy allocation, a viviparous female must also be the source of water for embryonic development (Thompson, 2007), and this facet of maternal resource provisioning has multiple implications to consider (Oftedal, 2002). For instance, contrary to energy that can be stored (as body fat) to prepare for energy investment during reproduction (Lourdais et al., 2002b), water typically cannot be accumulated in advance. Therefore, water availability in the environment must match the timing of the reproductive requirement for water. In many environments, water may well be more constraining than energy during reproduction because gestation often occurs during dry summer months (Lourdais et al., 2004a, Le Galliard et al., 2012). Water limitation may alter reproductive success either by inducing embryonic mortality or by affecting offspring quality (Dauphin-Villemant and Xavier, 1986, Ross and Desai, 2005). Importantly, water demand during reproduction should be directly related to reproductive effort since an increase in the number of developing embryos increases water demand. Therefore, increased fecundity should either increase female water acquisition from the environment or cause greater hydric deficit in the female.

Despite the likely importance of water in influencing female behavior, physiology, and fecundity, maternal water flux remains largely overlooked. Squamate reptiles are unique among vertebrates for the vast number of times that viviparity has independently evolved (> 100, Blackburn, 2000, Stewart and Thompson, 2000). While some species show complex placentotrophy, most lizards and snakes are lecithotrophic where yolk reserves support the energy demands during embryonic requirement (Stewart and Thompson, 2000, Thompson and Speake, 2003, Van Dyke et al., 2014). Therefore, maternal resource demand during pregnancy is primarily focused on water. This situation offers a simple context to address the implication of water allocation independent of nutritional aspects. A previous study in the common lizard, Zootoca vivipara, suggested that water uptake is greatest during late embryonic stages when most somatic growth occurs (Dauphin-Villemant and Xavier, 1986). The same study also revealed an important impact of experimental water deprivation on reproductive success. To improve our understanding of water allocation, it is also critical to consider the impact of pregnancy and fecundity on female water balance.

We used a viviparous snake, the aspic viper, Vipera aspis, to describe water flux during pregnancy. We used high-resolution ultrasonography to monitor embryonic volume and estimate water intake during development. We also monitored relevant maternal traits including body mass and plasma osmolality over pregnancy. Our main hypothesis was that embryonic water requirements should influence maternal water intake and water balance.

We tested the following predictions:

  • (1) Water uptake by the embryos should mainly occur during the second half of gestation when embryonic somatic growth occurs.

  • (2) Maternal mass change during pregnancy should reflect water intake and be closely related to the number of developing embryos.

Section snippets

Study species and maintenance

The aspic viper, V. aspis, is a small viviparous snake of the western Paleartic region. This species is a typical capital breeder and thus accumulates energy needed for reproduction over an extended period before engaging in a reproductive effort (Bonnet et al., 2002, Lourdais et al., 2002b). Pregnancy is a long process, lasting up to three months, and it is associated with an increase in thermal preference and precision, which necessitates an increase in thermoregulatory activities (Saint

Embryonic volume

We detected significant variation in embryonic volume over time (F2, 80 = 151.68, p < 0.001) with a clear increase over the three consecutive periods (mean embryonic volume 2.71 ± 0.10, 4.90 ± 0.25, and 8.42 ± 2.6 cm3 for early, mid, and late pregnancy measures, respectively). All three stages were significantly different from each other (Tukey's post hoc tests, all p values < 0.001). Total volume change represented, on average, an increase of 210% of initial volume. When considering time from ovulation

Discussion

Our study on the aspic viper provides significant insight into the dynamics of water transfer between mother and offspring, but also into the potential impact of embryonic water demands on maternal physiology and drinking behavior. We found that water uptake occurred mostly during the second half of pregnancy and was closely related to the number of developing embryos. We discuss our results in details below.

High-resolution ultrasonography has been previously used to assess reproductive output

Acknowledgments

We thank François Brischoux for providing valuable comments on earlier drafts of the manuscript. This research was made possible by financial support from the “Programme opérationnel plurirégional Loire FEDER” (# PRESAGE 30810), the “Etablissement Public Loire”, the FYSSEN foundation, and the Centre National de la Recherche Scientifique.

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