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Do invaders always perform better? Comparing the response of native and invasive shrimps to
temperature and salinity gradients in south-west Spain
Lejeusne C.a*, Latchere O.a, Petit N.a, Rico C.a, Green A. J.a
a
Doñana Biological Station-CSIC, EBD-CSIC, Wetland Ecology Department, Avenida Américo Vespucio
s/n, 41092 Sevilla, Spain
* Corresponding author:
Christophe Lejeusne
Doñana Biological Station-CSIC, EBD-CSIC, Wetland Ecology Department, Avenida Américo Vespucio
s/n, 41092 Sevilla, Spain
lejeusne@ebd.csic.es
Ph.: +34 954 232 340
Abstract
Invasive species are often thought to benefit from climate change, outcompeting native species as
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temperatures increase. However, the physiological tolerance has been little explored as a potential
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mechanism explaining biological invasion success. In this study, we used empirical data from both
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invasive and native estuarine species as a case study to address the hypotheses that (1) invasive species
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show a better resistance to acute thermal stress, (2) invasive species present lower oxygen consumption
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rates owing to greater resistance to environmental stressors, and (3) native species have lower survival
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rates under chronic temperature and salinity stress. We conducted various comparative experiments on
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three sympatric and syntopic closely related shrimp species (one invasive Palaemon macrodactylus, and
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two natives Palaemon longirostris and Palaemonetes varians). We evaluated their critical temperature
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maxima, their oxygen consumption rates under different salinities and temperatures, and their survival
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rates under chronic salinity and temperature. We found that the invasive species was the most tolerant to
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rapid increase in temperature, and consistently consumed less oxygen over a broad range of temperatures
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and salinities. P. macrodactylus also had lower mortality rates at high temperatures than P. longirostris.
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These results support previously reported differences in physiological tolerance between native and
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invasive species, with the invasive species always performing better. The consistently higher tolerance of
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the non-indigenous species to temperature variation suggests that climate change will increase the success
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of invaders.
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Keywords: Introduced species, Estuarine organisms, Environmental factors, Biological Stress, Palaemon
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macrodactylus
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Regional index terms: Europe, Spain, Andalusia, Guadalquivir River
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1. Introduction
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Invasive species often have tremendous ecological impacts on invaded ecosystems and native species
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(Nentwig, 2007; Richardson and Pysek, 2008). They also have huge economic impacts estimated at more
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than five per cent of the global economy (Burgiel and Muir, 2010). Together with climate change, they
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constitute a “deadly duo” threatening worldwide biodiversity (Halpern et al., 2008; Burgiel and Muir,
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2010; Barnosky et al., 2012). Both factors can act individually on species abundances, distributions and
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biotic interactions, inducing local and regional extinctions (Grosholz, 2002; Parmesan, 2006; Lejeusne et
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al., 2010; Durrieu de Madron et al., 2011), but they also can act synergistically (Dukes and Mooney,
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1999; Stachowicz et al., 2002; Hellmann et al., 2008)
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To become established then invasive, a non-indigenous species (NIS) has to successfully pass through a
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series of biotic and abiotic filters acting as barriers between the different steps of the invasion process (see
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Blackburn et al., 2011 for synthesis). However, the mechanisms leading to a successful invasion are
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poorly understood in most cases. The numerous non-exclusive hypotheses proposed to explain invasion
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mechanisms, include evolutionary hypotheses (e.g. hybridisation) and ecological hypotheses (e.g. enemy
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release) (Hufbauer and Torchin, 2007; Sax et al., 2007; Catford et al., 2009). Another potential
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mechanism, the physiological tolerance hypothesis, is as yet relatively unexplored (Zerebecki and Sorte,
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2011). This hypothesis predicts that invasive species have a greater and/or broader physiological tolerance
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than native species occupying the same habitat. Predictions of this hypothesis have been verified in a
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large taxonomical panel of species and stress factors (e.g. Lenz et al., 2011). However, owing to the
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importance of climate change, most of the studies dealing with this hypothesis have focused on
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temperature effects and eurythermality of invasives compared to a more stenothermal tolerance of natives
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(Dukes and Mooney, 1999; McMahon, 2002; Rahel et al., 2008; Zerebecki and Sorte, 2011). In the
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present study, we address tolerance to two major environmental factors (salinity and temperature) as
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potential contributors to the success of an invasive estuarine species.
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Estuaries are very productive ecosystems providing nursery habitats to many marine and commercial
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species. These marine-freshwater ecotones show strong fluctuations of physical and chemical parameters
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at both spatial and temporal scales (e.g. tidal-based salinity fluctuations with a decreasing spatial gradient
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from the inner mouth). Estuaries are particularly impacted by climate change but are also especially
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susceptible to biological invasions (Ruiz et al., 1997; Cohen and Carlton, 1998; Wasson et al., 2001;
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Grosholz, 2002). In the San Francisco estuary, one new NIS is recorded every 14 weeks, and in Europe
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one fifth of estuarine species are NIS (Cohen and Carlton, 1998; Reise et al., 2006). One key question is
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whether NIS have more resistance to environmental stressors than native estuarine species, being better
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adapted to strong fluctuations in temperature and salinity.
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The oriental shrimp (also known as migrant prawn, or grass shrimp) Palaemon macrodactylus is an
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estuarine caridean shrimp native to China, Japan and Korea. It was initially introduced to San Francisco
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Bay, CA in the 1960s, before spreading northward along the US coast. Since 1992, it has reached Europe,
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Argentina and the north-eastern USA coast (Newman, 1963; Cuesta et al., 2004; Spivak et al., 2006;
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Warkentine and Rachlin, 2010). In European estuaries, the species has spread rapidly and extensively
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since its first introduction. It is now present from SW Spain to Germany and England, and in the western
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Black Sea. On the Atlantic coast, the species can interact with two other commercially exploited native
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species: the Atlantic ditch shrimp Palaemonetes varians (a brackish water species found mainly in non-
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tidal ponds, marshes and canals with hydrological connections to estuaries) and the delta prawn Palaemon
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longirostris (an estuarine species). Despite its relatively small size, P. varians is often captured for human
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consumption, use as fishing bait, or use as live diet for aquaculture (Palma et al., 2008), while traditional
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fishing of P. longirostris has local economic importance (Holthuis, 1980; Béguer et al., 2012). Both
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native Palaemonidae can be very abundant and they occupy a central position in the estuarine trophic
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network (Salgado et al., 2004), being prey of many European native and commercial fishes (e.g. the
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European sea bass Dicentrarchus labrax for P. longirostris) (Salgado et al., 2004; Dauvin and Desroy,
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2005).
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Competitive interactions between the NIS P. macrodactylus and the native P. longirostris may be strong,
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especially for space and food. Both species are estuarine with strong overlap in habitat and trophic
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preferences (González-Ortegón et al., 2010; Béguer et al., 2011a). In the Guadalquivir estuary (SW
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Spain), this habitat overlap is maximal in autumn during low abundance of their shared mysid prey
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Mesopodopsis slabberi (González-Ortegón et al., 2006; González-Ortegón et al., 2010). Since the NIS
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was first recorded, an increase in P. macrodactylus densities recorded in some European estuaries has
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coincided with a decrease in density of the native P. longirostris (González-Ortegón et al., 2010; Béguer
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et al., 2011a). A previous study comparing the osmoregulatory capacities of P. macrodactylus with the
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two natives P. longirostris and P. varians indicates that the three species have similar osmoregulatory
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capacities (González-Ortegón et al., 2006). However, oxygen consumption rates measured under different
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salinities and dissolved oxygen concentrations suggested that the NIS has a more efficient metabolism
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and higher tolerance to hypoxic conditions (González-Ortegón et al., 2010). However, despite field
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surveys showing the salinity-related and spatial distribution patterns of these estuarine species (González-
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Ortegón et al., 2006; Béguer et al., 2011a), little is known of the ecophysiology of the NIS P.
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macrodactylus compared to the natives P. longirostris and P. varians, in particular regarding the
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influence of temperature variations. Taking into account the climate change expected in the Euro-
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Mediterranean area, the interaction between temperature and salinity might be central to the success of
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NIS and to changes in status of native species (see Coccia et al., 2013 for an example from the
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Guadalquivir delta).
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Studying the relative performance of NIS and natives under a range of environmental conditions allows
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evaluation of the likely mechanisms of a successful invasion, and testing of the physiological tolerance
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hypothesis. We therefore conducted a series of three experiments to test if P. macrodactylus performs
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better under extreme conditions of temperature and salinity, the two main abiotic stress factors found in
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estuaries. We evaluated behavioural activity and the critical temperature maxima of different shrimp
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species under an acute short-term thermal stress. We hypothesized that the NIS would show greater
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resistance to acute thermal stress, reflected in a higher critical thermal maximum. We also measured
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oxygen consumption under different conditions of temperature and salinity to test whether the NIS
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species present lower consumption rates owing to greater resistance to environmental stressors. Finally,
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we quantified survival under different chronic thermal and salinity stress to test whether the native species
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had lower survival rates.
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2. Material and methods
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2.1.
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The oriental shrimp P. macrodactylus and the delta prawn P. longirostris were collected in the
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Guadalquivir estuary, SW Spain (see Figure 1) at three distinct, tidal sites S1-S3, with P. macrodactylus
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was only found at site S2 (environmental parameters at each site are described in Appendix A). The
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Atlantic ditch shrimp P. varians was sampled in Veta La Palma (S4 and S5), a complex of fish ponds
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connected to and supplied with water from the Guadalquivir estuary (Figure 1 and Appendix A) and
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protected within Doñana Natural Park, where it is abundant and harvested commercially (see Rodriguez-
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Perez and Green, 2012 for details of the study site). Living individuals were collected in 2011 using
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shrimp keep-nets (mesh size 4mm) placed at low tide for S1-S3 and recuperated 24h later. Size of the
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shrimps was estimated by measuring the carapace length from the orbital edge of the eye to the edge of
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the cephalothorax under a stereomicroscope SteREO Discovery V8 (Zeiss) using the AxioVision Rel
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4.8.2 (Zeiss) software. In order to reduce catching and manipulation stress, living shrimps were
Shrimp collection and laboratory acclimation
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acclimated during at least 48h before any experiment in aerated aquariums with artificial saltwater at
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20°C and a salinity of 5, obtained by dissolving dry sea-salt Instant Ocean (Aquarium Systems, Mentor,
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Ohio) in distilled water. Salinity was measured using the Practical Salinity Scale. Aquariums were placed
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in a climatic chamber (Fitoclima 10000EHHF, Aralab) on a 12h:12h dark:light photoperiod. Shrimps
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were fed daily ad libitum with commercial aquarium food (gammarids) before and during all the
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experiments. In order to reduce stress and injury associated with its determination, sex was characterized
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after the experiments by looking for the presence or absence of the masculine appendix on the endopodite
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of the second pleopod (Siegfried, 1980). A summary of size and sex ratio of the specimens used in each
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experiment is given in Table 1.
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2.2.
Experiment 1: critical Thermal maximum (CTmax) experiment
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In order to compare thermal stress resistance between the shrimp species, Critical Thermal maximum
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(CTmax) experiments were conducted in May and August 2011. Carapace lengths were measured before
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the experiment. The experiment was not started until at least 24h after measurements of length.
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Acclimated shrimps were placed individually in a beaker filled with 200 ml of artificial water (salinity 5
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and initial temperature 20 ºC) and capped with a transparent lid to allow observation throughout the
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experiment. The beaker was placed in a water bath with a magnetic stirrer allowing rapid homogenisation
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of surrounding water. Temperature was monitored every minute with an electronic thermometer (model
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SA880SSX, Oregon Scientific) and a temperature ramp of 1°C.min-1 was applied as in Ravaux et al.
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(2012).
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During the experiment, behavioral activity of each individual shrimp was continuously monitored over 30
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s periods until reaching the end-point when the shrimp lay on its side or its posterior face for more than 30
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s. We subdivided behaviour into four categories based on previous literature (Ravaux et al., 2003; Shillito
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et al., 2006; Oliphant et al., 2011): ‘Movement’: any kind of motion of the animal except for active
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movement (see below): pereopods or pleopods movements, antennal lateral sweeping on the dorsal side,
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cleaning of mouth parts by rubbing them together; ‘Active movement’: when shrimps moved a distance
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(either by walking or swimming) exceeding their own length in less than 30 seconds; ‘Loss Of
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Equilibrium’ (LOE): shrimp on the bottom of the beaker in either an ‘upside-down' or a 'sideways'
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position for more than 2 seconds; ‘Spasmodic motions or spasms’: vibrations of the pleopods and/or
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sudden contraction of the abdomen.
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The CTmax was determined as the temperature at which coordinated movements were lost, using LOE as
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the reference parameter. The CTmax was calculated for a total number of 20 individuals per species for P.
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macrodactylus and P. varians, and 18 individuals for P. longirostris.
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2.3.
Experiment 2: oxygen consumption rate
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In order to compare oxygen consumption under different temperatures and salinities, we performed two
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series of treatments with varying temperatures (20°C, 25°C and 30°C at a constant salinity of 5) and
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salinities (salinity of 5, 15, 25, 35 and 45, at constant 20°C) respectively in May 2011 (9-10 shrimps per
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treatment for P. longirostris and P. varians, and 5 shrimps per treatment for P. macrodactylus). Each
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shrimp was weighted 24h before experiment with a Voyager analytical balance (Ohaus) after removal of
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excess water using blotting paper. In order to avoid any heat shock when moving shrimps from their
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original aquarium (20°C at salinity 5) to aquariums with higher temperatures, they were acclimated
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overnight prior to the experiments by gradually increasing temperature (2°C.h-1) or salinity (10 salinity
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units.h-1) depending on the treatment. Temperature within each treatment was maintained within ± 0.2°C
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using a heater (Jäger 300; Eheim) controlled by a Biotherm Pro (Hobby) temperature regulator.
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To measure oxygen consumption rate (OCR), shrimps were put into cylindrical flasks (12.3 mL) and the
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flow rate of water circulating in each flask was measured. The difference between oxygen concentrations
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in water at the entrance and exit of the flasks was recorded using a 10-channel OXY-10 mini (PreSens)
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fiber optic oxygen transmitter connected to a computer with the OXY10v3_33 software. OCR was
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calculated according to the formula: OCR = F × ([O2]in – [O2] out)/BW, where OCR is oxygen consumption
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rate (mg O2.gwwt-1.h-1), F is water flow rate (L.h-1) circulating in each flask, [O2]in is oxygen content of
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the water inflow (mg O2.L-1), [O2]out is oxygen content of the water outflow (mg O2.L-1), and BW is wet
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mass (g).
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2.4.
Experiment 3: comparative survival of P. longirostris and P. macrodactylus under chronic stress
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Shrimps were placed individually in small, closed plastic aquaria (0.35L) with a 1mm mesh sieve at the
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bottom and placed within 91L experimental aquaria.
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Acclimatised shrimps were reared in different 91L aquaria at three different temperatures (20°C, 24°C
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and 28°C, with a constant salinity of 5) and three different salinities (5, 25, and 45, with a constant
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temperature of 20°C) during 28 days. As environmental parameters are constantly varying in an estuary,
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especially salinity that has tide-based regime, submitting estuarine organisms to constant salinity or
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temperature as here represents thus a chronic stress. Shrimp size and weight were measured twice a week
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and survival was checked daily.
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Experiments were conducted in October 2011 when both species were caught at the same time and place.
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The experiment was repeated twice (15 day interval between sampling) on both species (8 individuals per
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treatment for both P. longirostris and P. macrodactylus and per sampling time).
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2.5.
Statistical analysis
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All statistical analyses were performed using R 2.15.2 (R Core Team, 2012). The CTmax data were not
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normally distributed, even after transformations (Shapiro-Wilk’s test, p<0.001) and had unequal variances
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(Bartlett test, p<0.001). These data were therefore analysed using the non-parametric Kruskal-Wallis
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ANOVA and Wilcoxon-Mann-Whitney tests. However, for oxygen consumption data, a parametric
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ANOVA was performed, after data transformation when necessary. The post-hoc Tukey HSD test was
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used to compare treatments. Survival analysis were performed with the Survival package in R (Therneau
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and Lumley, 2009) using Kaplan-Maier estimates and log-rank tests.
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3. Results
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3.1. Experiment 1: behavioral analysis and Critical Thermal maximum (CTmax) experiment
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P. macrodactylus was collected at only one site (S2) in the estuary (Fig. 1). In contrast, P. longirostris
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was collected at the three sampling sites in the Guadalquivir River (Fig. 1), representing a decreasing
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salinity gradient from S1 to S3.
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Due to density variations at each sampling site, the experiment was conducted on 9, 7 and 2 individuals of
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P. longirostris. sampled the same day at sites S1, S2 and S3 respectively. We pooled the different sites
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into one group for further comparative analysis as no statistical differences were found among sites
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(Kruskal-Wallis test, p=0.37 and p=0.82 for the CTmax and temperature at first spasmodic motion
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respectively).
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In the same manner, P. varians was sampled at two sites (S4 and S5; Fig. 1) differing in salinity. The
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experiment was conducted on 10 individuals from each site. No statistical differences were found for the
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CTmax and for the temperature at first spasmodic motion between the two sites (Wilcoxon-Mann-Whitney
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test, p=0.15 and p=0.17 respectively). Hence, we pooled the two sites for further analysis.
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Despite identical pre-experimental acclimation conditions, at the start of the experiment, no P.
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longirostris individual was presenting an active motion, whereas 60% and 20% of P. varians and P.
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macrodactylus, respectively were presenting an active motion (Fig. 2). The temperatures at which 50% of
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individuals were actively moving were reached earlier for P. varians (20.0 ºC) and P. macrodactylus
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(22.0 ºC) than for P. longirostris (24.6 ºC). The maximal number of individuals presenting active
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movements was reached as early as ca. 28 ºC for P. longirostris, compared to ca. 33 ºC and ca. 32.0 ºC
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for P. varians and P. macrodactylus respectively. Moreover, the active moving curves closely preceded
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the loss of equilibrium (LOE) curves (Fig. 2). In the case of P. varians and P. macrodactylus, the LOE
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and spasms curves were closer together. As a consequence, LOE was observed at much lower
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temperature for P. longirostris (mean CTmax ± SE = 27.24 ºC ± 2.16) compared to P. varians (mean CTmax
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± SE = 31.71 ºC ± 2.21) and P. macrodactylus (mean CTmax ± SE = 33.0 ºC ± 1.11)(Fig. 2 and 3). The
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CTmax values were significantly different between species (Kruskal-Wallis test, H=33.3276, df=2,
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p<0.001), with all pairwise comparisons between the three species being significantly different
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(Wilcoxon-Mann-Whitney test, p<0.001).
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For individuals used in the above experiments, information about size and sex ratio of the different
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samples have been gathered into Table 1. When considering each species separately, there was no effect
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of size (Spearman rank correlation, p>0.15) or sex on LOE values (Kruskal-Wallis test, p>0.05).
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3.2. Experiment 2: oxygen consumption rate
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In the salinity experiment, OCR values were significantly different between species and salinity
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treatments (four-way ANOVA, F8,109=15.58, p<0.001) without effects of sex or size (see detailed value of
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these parameters in Table 1). The significant differences between species were due to lower OCRs of P.
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macrodactylus compared with the two native species (post hoc Tukey HSD test, p<0.001), which did not
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differ between them (post hoc Tukey HSD test, p=0.075). Whatever the salinity treatment, P. varians had
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the highest OCR values and P. macrodactylus the lowest (Fig. 4).
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The significant effect of salinity treatment reflected an increase in OCR with salinity (Fig. 4), for all
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species (134%, 186% and 236% increase for Palaemonetes varians, P. longirostris, and P. macrodactylus
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respectively, between the lowest and highest treatments). In the case of P. longirostris, which presented
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intermediate OCR values, OCR was significantly lower at the lowest salinity treatment (salinity 5) than at
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all others. Low salinity effects were more gradual for the other two species (Fig. 4).
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In the temperature experiment, OCRs were significantly different between species and temperature
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treatments (four-way ANOVA, F6,65=21.30, p<0.001) without effects of sex or size (see Table 1 for
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details on values of these parameters). All pairwise species differences were significant (post hoc Tukey
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HSD tests, p<0.015). As for the salinity experiment, P. macrodactylus had the lowest OCR, and P.
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varians the highest. OCR increased significantly with increasing temperature for all species (Fig. 4;
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183%, 257% and 200% increase for Palaemonetes varians, P. longirostris, and P. macrodactylus
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respectively, between the lowest and highest treatments).
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3.3. Experiment 3: comparative survival of P. longirostris and P. macrodactylus under chronic stress
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We did not find any significant effect of sampling date for P. macrodactylus (log-rank test, χ2=0, df=1,
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p=0.923) or P. longirostris (log-rank test, χ2=0, df=1, p=0.989) on survival rate at different salinities.
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Likewise, no significant effect of the date of sampling was found in the temperature experiment (P.
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macrodactylus, log-rank test, χ2=0, df=1, p=0.946; P. longirostris, log-rank test, χ2=0, df=1, p=0.993).
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Thus, samples from different dates were pooled for further analyses. In the treatment 20 ºC-salinity 5 that
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provided the least stressful conditions, only one individual (P. macrodactylus) showed a premature death
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(two weeks before the end of the experiment). No significant size differences was found between the two
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shrimp species in each of the treatment (see Table 1).
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Survival curves of both species according to salinity are shown in Fig. 5. There was a significant effect of
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salinity treatment on the survival of P. macrodactylus (log-rank test, χ2=54.0, df=2, p<0.001) and of P.
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longirostris (log-rank test, χ2=41.3, df=2, p<0.001). No significant difference was found between the two
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species when comparing their general survival trend according to salinity (log-rank test, χ2=1, df=1,
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p=0.308): the higher the salinity, the lower the survival (Fig. 5). However, when comparing survival of
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both species in detail at each salinity treatment, significant interspecific differences were noted only at the
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high salinity of 45 (log-rank test, χ2=8.6, df=1, p=0.00338).
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In the temperature experiment (Fig. 5), no significant effect of temperature was found on survival of P.
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macrodactylus (log-rank test, χ2=1.2, df=2, p=0.555), despite a slight increase of mortality with increasing
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temperature (Fig. 5). However for P. longirostris, temperature had a significant effect (log-rank test,
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χ2=19.4, df=2, p<0.001), the highest temperature (28 ºC) being the only one to induce mortality (50% of
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individuals dead by the end of the experiment; Fig. 5). The general trend for survival with temperature
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was not significantly different between the two species (log-rank test, χ2=0.3, df=1, p=0.570). However, a
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marginally significant difference in survival could be noted between the two species at 28 ºC (log-rank
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test, χ2=3.8, df=1, p=0.051).
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4. Discussion
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The primary objective of this work was to compare the resistance to two major environmental stressors
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(temperature and salinity) between native and NIS palaemonid shrimps, through the comprehensive study
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of their critical temperature maximal, oxygen consumption and long-term survival. Such a series of
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experiments has rarely been conducted at the same time on NIS and native species using both sympatric
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and phylogenetically close taxa (but see González-Ortegón et al., 2006; González-Ortegón et al., 2010).
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In the case of P. macrodactylus and P. longirostris, they were even syntopic and congeneric.
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4.1.
CTmax
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We evaluated simultaneously the upper thermal tolerance of the two native shrimps P. varians and P.
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longirostris, and of the NIS P. macrodactylus. Such an approach comparing CTmax of both sympatric and
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related, native species and NIS under identical experimental conditions has rarely been adopted (but see
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Coccia et al., 2013). There were clear significant differences in the CTmax values found between the three
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species (Fig. 3). The species P. longirostris presents by far the lowest CTmax value (27.24 ºC ± 2.16),
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while P. varians and P. macrodactylus present closer though significantly different CTmax values (31.71
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ºC ± 2.21 and 33.0 ºC ± 1.11 respectively).
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The high interspecific difference found in CTmax values between the two European native species is likely
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to be associated with their habitat preferences. The Atlantic ditch shrimp P. varians is a ubiquitous shrimp
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inhabiting shallow waters (mainly ponds and canals) in and around NE Atlantic estuaries. It typically
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tolerates salinity ranges from 1-2 to >45 (though it has a preference for brackish waters), associated with
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seasonal fluctuations of water temperature ranging from 0 to 33 °C, and summer daily variations >10 °C
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(Nielsen and Hagerman, 1998; Gelin and Souty-Grosset, 2006; González-Ortegón and Cuesta, 2006;
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Ravaux et al., 2012). The delta prawn P. longirostris is strictly estuarine, present in all the NE Atlantic
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(Bah et al., 2006; Béguer et al., 2010). The species is known to be euryhaline though being more
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abundant in brackish waters at the outer estuarine zone and in intermediate salinities, with spatial sexual
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segregation and within-estuary reproductive migrations (Campbell and Jones, 1989; González-Ortegón
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and Cuesta, 2006; Béguer et al., 2011b).
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Temperature ranges experienced by this species are therefore those of the estuary itself. In the
18
Guadalquivir river where P. longirostris and P. macrodactylus were sampled, temperature is quite
19
homogenous along the estuary and presents a consistent seasonal pattern oscillating in the 10-30ºC range,
20
with summer daily variations of <5ºC (Baldó et al., 2005; González-Ortegón et al., 2006; Navarro et al.,
21
2011; García-Lafuente et al., 2012). Major differences in habitat preferences between the two native
22
species thus imply differences in water depth and temperatures: maximum water temperatures in ponds
23
and canals frequented by P. varians are much higher with strong daily and seasonal temperature
24
fluctuations (especially in summer), while in the core of the river estuary used by P. longirostris such
14
1
variations are highly buffered. Adaptation of each species to its environment is reflected in their different
2
CTmax values and ecophysiology may partly explain the niche partitioning observed between these two
3
sympatric species.
4
As for the NIS P. macrodactylus, we report in this study the first CTmax value (33.0 ºC ± 1.11) for this
5
worldwide invader, which is much higher than for the two European natives (27.24 ºC ± 2.16 and 31.71
6
ºC ± 2.21 for Palaemonetes varians and P. longirostris respectively). Compared to values found in the
7
literature for other Palaemonid adults, these values are low. The Mississippi grass shrimp Palaemonetes
8
kadakiensis or shrimps of the widespread genus Macrobrachium present much higher values (e.g. M.
9
acanthurus 34.2 ºC ± 0.48; Díaz et al.). However, those species are tropical freshwater species living in
10
waters that never cool down to 20ºC, and as such their acclimation was conducted at higher temperatures
11
than in the present study (Oliphant et al. (2011). More interestingly, the CTmax of P. macrodactylus is
12
closer to that of its Euro-Mediterranean euryoecious temperate congener P. serratus (31-37ºC for
13
acclimation temperatures in the natural range 14-25ºC; details in Richard, 1978; Oliphant et al., 2011). In
14
any case, the present study clearly demonstrates that the NIS P. macrodactylus presents a higher upper
15
thermal tolerance than the native P. varians and P. longirostris when submitted to an acute thermal stress.
16
Intraspecific comparisons for our specimens of P. varians sampled in SW Spain can be made with French
17
specimens stressed with a similar temperature ramp (0.9 ºC.min-1) but acclimated at 10 ºC and 20 ºC
18
(Oliphant et al., 2011; Ravaux et al., 2012). The CTmax value we observed for P. varians (31.71 ºC ± 2.21)
19
is more similar to the previous CTmax value reported for the 10 ºC-acclimated P. varians than for the 20
20
ºC-acclimated shrimps (respectively 30.9ºC ± 1.0 and 35.9ºC ± 0.6; Ravaux et al., 2012). Such a
21
discrepancy might be explained by either the differences in acclimation duration (4 months vs. 48h in our
22
study) or salinity of water used (salinity 35 vs. 5 in our study), or both.
23
In the case of P. longirostris, Madeira et al. (2012) obtained a much higher CTmax value of 34.4 ºC
24
(Portuguese shrimps acclimated for 2 weeks at 24 ºC and salinity 35 and stressed with a temperature ramp
15
1
of 1 ºC.h-1) than in the present study (27.24 ºC ± 2.16). Unlike for P. varians, the very different heating
2
rates used in the two studies are the most likely explanation for this difference. Indeed, an exhaustive
3
literature review demonstrated that commonly-used temperature ramps (in the 0.5–1.5°C.min–1 range, as
4
in the present study) allow avoidance of either thermal acclimation (so called heat-hardening) or
5
mismatching of body and environmental temperatures, due to slow or excessive heating rates respectively
6
(Lutterschmidt and Hutchison, 1997). Moreover, non-significant differences have been shown between
7
environmental and body temperatures for animals of <150g submitted to a heating rate of 1 ºC.min-1
8
(Shillito et al., 2006). As a consequence, a match between experimental and shrimp body temperatures
9
without heat-hardening can be assumed in the present study.
10
In any case, the examples of P. varians and P. longirostris illustrate that caution must be taken when
11
comparing CTmax values between studies, even within a single species, as they clearly depend on both
12
acclimation temperature and experimental procedures and can also differ markedly between seasons
13
(Hopkin et al., 2006; Ravaux et al., 2012; Somero, 2012). Adequate interspecific comparisons can
14
however be made through experiments that use the same behavioral parameters and similar heating rates
15
(Shillito et al., 2006) as in Madeira et al. (2012) or in the present study.
16
17
4.2.
Oxygen consumption
18
In the present study, under the different stressful conditions of temperature and salinity tested, the NIS
19
consistently showed better respiratory performances, with lower OCRs than its native counterparts (Fig.
20
4). Such a pattern of better NIS performance had previously been observed between the NIS P.
21
macrodactylus and the native P. longirostris, for various combinations of salinity and dissolved oxygen
22
concentrations (but not of temperature), although such interspecific differences were not always
23
statistically significant and acclimation conditions were much less drastic (González-Ortegón et al.,
24
2010). P. macrodactylus was shown to be much more tolerant than P. longirostris under stressful hypoxic
16
1
conditions which, associated with the present eutrophication of estuaries in Europe, might help the NIS to
2
outcompete its native congener (González-Ortegón et al., 2010). Our results for both salinity and
3
temperature agree with and support this scenario, and are in accordance with other studies dealing with
4
NIS from a wide range of phyla such as tunicates, other crustaceans, and fishes (Lenz et al., 2011;
5
Maazouzi et al., 2011; Morosawa, 2011). Overall, our results reinforce previous findings that a successful
6
NIS is often associated with a much better respiratory performance and more efficient metabolism
7
compared to native congeners.
8
We recorded a similar trend for increase in OCRs with salinity and temperature for all species (Fig. 4).
9
Because of the major role of these two major abiotic variables in physiological responses of most
10
invertebrates (Angilletta, 2009), such increases are quite common and were previously recorded for
11
congeners such as Palaemonetes pugio, Palaemon peringueyi or Palaemon pacificus (Allan et al., 2006;
12
Oliphant et al., 2011; Purcell et al., 2013; Vilibić et al., 2013). However, direct comparisons of values
13
from the literature are difficult, due to differences in methodology and study aims. However, trends can
14
be compared, and a similar increase of OCR with dissolved oxygen concentration and salinity (but not for
15
temperature) was previously found for the two Palaemon species from the Guadalquivir river (González-
16
Ortegón et al., 2010). A similar pattern was also found previously for P. varians (Lofts, 1956), and is
17
quite common for decapod shrimps and other invertebrates (see for example Allan et al., 2006; Lenz et
18
al., 2011; Oliphant et al., 2011). However, unlike the present study, OCRs have rarely been compared
19
between sympatric NIS and native species reared under identical conditions of both temperature and
20
salinity.
21
22
4.3.
Comparative survival of P. longirostris and P. macrodactylus under chronic stress
23
In the present study, we deliberately used normal to acute parameters of chronic stress compared to those
24
found in the wild in order to evaluate the comparative survival abilities of the native species and the NIS.
17
1
Chronic exposure to a stress factor increases physiological stress, sometimes compromising fitness and
2
even survival, while in other cases it might induce a better resistance of the organism to future, more
3
acute stress through an increase of the tolerance threshold (Boonstra and Fox, 2013). Furthermore, the
4
duration, frequency and intensity of stress factors are fundamental in conditioning the organismal
5
response to chronic stress.
6
The chronic exposure to temperature gave contrasting results between the two syntopic, congeneric
7
species as the survival of the NIS P. macrodactylus was not affected by temperature, while the native
8
species P. longirostris was negatively affected by high temperature (28ºC). Differences between the two
9
species at this high temperature were significant. These results are of particular interest in the present
10
context of climate change, which often results in an increase of both air and water temperatures (Walther
11
et al., 2009). The chronic exposure at a temperature of 28ºC is quite realistic in estuarine waters in
12
southern Europe. Such a temperature is presently often reached during summer periods (e.g. in the
13
Guadalquivir river) and might be more frequently encountered in the wild with the observed and
14
predicted increasing temperature in the Euro-Mediterranean zone, and especially in SW Spain (Lejeusne
15
et al., 2010; del Río et al., 2011; Philippart et al., 2011; Albouy et al., 2013). This double stress of climate
16
change and competition with NIS is becoming a very common pattern of threats for native species in
17
general. Indeed, many of them are presently close to their upper thermal tolerance, with dispersal abilities
18
being much lower than NIS and insufficient to cope with the pace of climate change (Stachowicz et al.,
19
2002; Walther et al., 2009; Burrows et al., 2011; Maclean and Wilson, 2011; Zerebecki and Sorte, 2011;
20
Madeira et al., 2012).
21
Chronic exposure to high salinity had a significant effect on the survival of both P. longirostris and P.
22
macrodactylus. As long as salinity remained in a range similar to that found in the estuary (5-25), no
23
difference in survival could be noted between the native and NIS. However, at higher salinity (45),
24
survival of both species was severely affected, with the NIS presenting a significantly higher mortality
18
1
than the native species (Fig. 5). Such a discrepancy in the survival under acute chronic salinity stress
2
mirrors the different habitat preferences found between the two species. Indeed, despite similar
3
osmoregulatory capacities in the 3-35 salinity range, P. longirostris and P. macrodactylus present
4
different, though largely overlapping, salinity preferences (González-Ortegón et al., 2006; González-
5
Ortegón et al., 2010; Béguer et al., 2011a). Those differences seem to have been accentuated since the
6
introduction of P. macrodactylus that displaced the midpoint of distribution of the native P. longirostris
7
in invaded estuaries (González-Ortegón et al., 2010; Béguer et al., 2011a). Presently the midpoint of P.
8
macrodactylus is more commonly found in the inner part of European estuaries with greater abundance at
9
lower salinities, while P. longirostris is more common in the outer estuarine zone and at intermediate
10
salinities (González-Ortegón and Cuesta, 2006; González-Ortegón et al., 2010; Béguer et al., 2011a).
11
The NIS preference for lower salinity in the Guadalquivir River associated with different survival at
12
experimental long-term high salinity might thus reflect a higher sensitivity of the NIS to salinity.
13
However this result should be treated with caution, as the experimental salinity at which differences
14
between species was found is much higher than the natural salinities both species experience in the wild.
15
Moreover, P. macrodactylus can be found in polyhaline to mixoeuhaline waters in other invaded areas
16
(e.g. Argentina or the United Kingdom), and in the Gironde estuary, France, the species prefers
17
mesohaline to polyhaline waters while the native species is found in lower salinity waters, unlike in SW
18
Spain (Béguer et al., 2011a). In any case, as expected for estuarine species, there were no significant
19
differences between species in survival when exposed to chronic salinity stress within the range of natural
20
estuarine fluctuations.
21
.
22
23
4.4.
Concluding remarks
24
We found that the NIS was more tolerant to rapid increase in temperature than the two native species, and
19
1
consistently consumed less oxygen than native species, over a broad range of temperatures and salinities.
2
P. macrodactylus also had lower mortality rates at high temperatures than P. longirostris. Overall, using a
3
rare combination of comparative experiments on sympatric and congeneric species, this work further
4
substantiates the broader physiological tolerance hypothesis of NIS as evoked by Zerebecki and Sorte
5
(2011) through the greater eurythermality of invasive species. Indeed, it is often assumed that NIS tend to
6
inhabit locations with broader ranges of stress factors (e.g. temperature) and higher maxima than native
7
species. Range width has often been evoked as a general trait of invasive success, with propagule pressure
8
and broad physiological tolerance as main explicative variables acting jointly or separately (Zerebecki
9
and Sorte, 2011). Propagule pressure can play a fundamental, but not exclusive, role providing new
10
individuals and genotypes due to the close proximity of the primary sites of introduction (e.g. ballast
11
water release from shipping traffic) with the surrounding habitats (Roman and Darling, 2007; Simberloff,
12
2009). However, during pre- and post-establishment, NIS have to pass through a series of abiotic filters to
13
become introduced, then invasive (see Blackburn et al., 2011). Species presenting broader physiological
14
tolerances may thus be more able to survive and establish (Zerebecki and Sorte, 2011). Without excluding
15
the role of propagule pressure in the success of P. macrodactylus, this work provides empirical evidence
16
that the NIS P. macrodactylus has a broader tolerance to abiotic stress (especially temperature) and better
17
physiological performance than closely related native species, particularly the European syntopic P.
18
longirostris. This is especially true in the present context of climate change where increased temperatures
19
associated with more frequent and severe thermal events are expected (Lejeusne et al., 2010; Durrieu de
20
Madron et al., 2011). Tolerance to abiotic factors and especially to temperature may play a fundamental
21
role and might help NIS to spread faster than expected (e.g. in the Mediterranean Sea) and out-compete
22
natives (see Lejeusne et al., 2010; Burrows et al., 2011; Zerebecki and Sorte, 2011). Future research
23
should take more into account the physiological tolerance hypothesis and explore whether the effect of
24
eurytolerance (not only eurythermality) might be a general pattern in invasion success, and occur during
20
1
the pre- or post-establishment of NIS. For the shrimps under study here, it would be useful to compare
2
experimentally the physiological tolerance at the egg and larval stages for each species, as well as to use
3
an ecogenomic approach to compare gene expression under stress, and genetic adaptation to
4
environmental extremes.
5
6
Acknowledgements
7
We are indebted to Raquel López-Luque, Cristina Pérez-González, Cristina Coccia, Carmen Diaz, Alice
8
Saunier, J. Miguel Medialdea and to Pesquerías Isla Mayor, S.A. for their assistance during fieldwork.
9
Technical assistance was kindly provided by Francisco M. Miranda-Castro and by the staff of the
10
Laboratory of Ecophysiology and of the Laboratory of Aquatic Ecology at the Doñana Biological Station-
11
CSIC. We are also grateful to Doñana Natural Space for sampling authorization. We thank the three
12
anonymous reviewers for the useful comments. This work was funded by the Spanish Ministry of
13
Economy and Competitiveness (program CGL2010-16028).
21
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Table 1: Summary of treatments applied in the different experiments with initial experimental conditions and size and sex ratio of the
shrimps. T: temperature; S: salinity; PL: Palaemon longirostris (native); PV: Palaemonetes varians (native); PM: Palaemon
macrodactylus (non indigenous); F: female; M: male; SD: standard deviation; SE: standard error. Different uppercase letters indicate
statistical significant differences (Wilcoxon-Mann-Whitney test with p<0.05), while an asterisk indicates divergence from an equal sexratio (Chi-squared test with p<0.05).
Experiment
(number)
Initial
conditions
CTmax (1)
T = 20ºC
S=5
Oxygen
consumption
rate (2)
T = 20ºC
S=5
Comparative
survival under
chronic stress
(3)
Specimen carapace length
Treatments
-1
+1ºC.min until spasms
 1h at T = 20, 25, or 30ºC
 1h at S=5, 15, 25, 35, 45
 4 weeks at 20, 24, 28ºC
Mean
Size range
PL
PV
PM
PL
PV
PM
PL
PV
PM
12.44a mm (± 1.37 SD)
5.82b mm (± 1.10, SD)
5.49b mm (± 1.00, SD)
6.19c mm (± 0.12, SE)
5.69d mm (± 0.20, SE)
6.32cd mm (± 0.33, SE)
6.08e mm (± 0.09, SE)
6.01e mm (± 0.20, SE)
5.88e mm (± 0.28, SE)
8.98-14.26 mm
4.50-8.22 mm
4.05-8.35 mm
5.06-7.86 mm
3.76-8.08 mm
4.44-8.86 mm
4.47-7.34 mm
3.76-8.87 mm
3.90-8.68 mm
PL
PM
6.76f mm (± 1.73, SD)
6.57f mm (±1.70, SD)
4.32-12.06 mm
4.51-12.36 mm
T = 20ºC
S=5
Specimen
sex ratio
(F/M)
3.5*
1.22
1.86
3.67*
1.64
1.50
4.44*
3.70*
1.44
NA
 4 weeks at S=5, 25, 45
PL
PM
26
g
6.69 mm (± 1.82, SD)
6.30g mm (±1.36, SD)
3.70-12.06 mm
4.51-10.19 mm
27
Figure 1: Sampling locations in the Guadalquivir estuary, south-west Spain. Sampling points are indicated
by black dots.
Fig. 2: Distribution of behavioral categories (lower figures) of the three palaemonid shrimps Palaemon
longirostris, Palaemonetes varians and Palaemon macrodactylus according to temperature increase
(upper). Moving: empty circles; active moving: solid black circles; loss of equilibrium (LOE): solid black
triangles; spasms: solid black squares. Note the change of scale for the horizontal and vertical axes.
Figure 3: Boxplot of the critical thermal maximum (CTmax) values for the three palaemonid shrimps
Palaemon longirostris, Palaemonetes varians and Palaemon macrodactylus. For each box, the first and
third quartiles delimitate the box, the bold line represents the median value, the dashed line the mean of
the CTmax, the whiskers represent the minimum and maximum values, and the empty circle represents an
outlier. Values of mean CTmax with different letters are significantly different.
Figure 4: Oxygen consumption rates according to salinity (left) and temperature (right) for Palaemon
macrodactylus (circles), Palaemon longirostris (squares) and Palaemonetes varians (triangles). For each
species, values with different letters are significantly different.
Figure 5: Kaplan–Meier survival estimates (filled lines) with 95% confidence bounds (dashed lines) for
Palaemon longirostris (PL) and Palaemon macrodactylus (PM) under different conditions of temperature
and salinity.
re gene expression under stress, and genetic adaptation to environmental extremes.
28
Figure 1
Sampling locations in the Guadalquivir estuary, south-west Spain. Sampling points are indicated by black dots.
29
Fig. 2
Distribution of behavioral categories of the three palaemonid shrimps (lower figures) according to temperature increase (upper). Moving: empty circles;
active moving: black filled circles; loss of equilibrium (LOE): black filled triangles; spasms: black filled squares. Note the change of scale for the
horizontal and vertical axes.
30
Figure 3
Boxplot of the critical thermal maximum (CTmax) values for the three palaemonid shrimps. For each box, the first
and third quartiles delimitate the box, the bold line represents the median value, the dashed line the mean of the
CTmax, the whiskers represent the minimum and maximum values, and the empty circle represents an outlier.
Values of mean CTmax with different letters are significantly different.
31
Figure 4
Oxygen consumption rates according to salinity (left) and temperature (right) for P. macrodactylus (circles), P. longirostris (squares) and P. varians
(triangles). For each species, values with different letters are significantly different.
32
Figure 5
Kaplan–Meier survival estimates (filled lines) with 95% confidence bounds (dashed lines) for Palaemon
macrodactylus (PM) and Palaemon longirostris (PL) under different conditions of temperature and salinity.
33
Appendix A: Temperature, salinity, and pH monitored at the five sampling sites (S1-S5) during one year
in 2011-2012.
34