Effect of substratum drying on the survival and migrations of Ponto-Caspian and native gammarids (Crustacea: Amphipoda)

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1 Hydrobiologia (2013) 700:47 59 DOI /s PRIMARY RESEARCH PAPER Effect of substratum drying on the survival and migrations of Ponto-Caspian and native gammarids (Crustacea: Amphipoda) Małgorzata Poznańska Tomasz Kakareko Maciej Krzy_zyński Jarosław Kobak Received: 13 December 2011 / Revised: 23 May 2012 / Accepted: 3 June 2012 / Published online: 17 June 2012 Ó Springer Science+Business Media B.V Abstract Organisms inhabiting shallow near-shore waters are at risk of desiccation during water level fluctuations. Using laboratory experiments, we investigated the survival and behavioural defences of four freshwater amphipod species during substratum drying: three Ponto-Caspian invaders (Pontogammarus robustoides, Dikerogammarus haemobaphes and Dikerogammarus villosus) and the native Gammarus fossarum. We hypothesized that they would be able to survive air exposure events as well as to adjust their behaviour by following the decreasing water level and/or burying in the sediments. To test these hypotheses, we examined survival of each species on gradually drying sandy substratum as well as their horizontal and vertical migration behaviours. P. robustoides was most resistant to substratum drying and was the only species burying into the substratum. On the other hand, G. fossarum exhibited distinct horizontal migrations following the retreating waterline. Handling editor: John Havel M. Poznańska (&) M. Krzy_zyński J. Kobak Department of Invertebrate Zoology, Nicolaus Copernicus University, Institute of General and Molecular Biology, Gagarina 9, Toruń, Poland mpoznan@umk.pl T. Kakareko Department of Hydrobiology, Nicolaus Copernicus University, Institute of Ecology and Environment Protection, Gagarina 9, , Toruń, Poland These two species seem to be particularly well adapted to the drying environment. Defence mechanisms of D. haemobaphes and D. villosus were less efficient, though the former species also followed the retreating waterline to some extent. Our study demonstrates that exotic and native gammarids have several adaptations that enable them to invade and persist in habitats experiencing common water level fluctuations. Keywords Desiccation Drought tolerance Water level fluctuations Invasive species Gammarus Behaviour Survival Introduction Benthic fauna colonize all available habitat types containing valuable food resources, including the shallowest bottoms and land water interfaces (Otto, 1979; Extence, 1981; Pieczyńska, 1990; Guidicelli & Bournaud, 1997; de Szalay & Resh, 2000). These areas are strongly affected by water level fluctuations and temporary exposures of substratum to air (Pieczyńska, 1990; Richardson et al., 2002; Furey et al., 2006; Brauns et al., 2008; Poznańska et al., 2010). Invertebrates living in floodplains can use several strategies to survive air exposure: (1) forming desiccation-resistant stages; (2) emerging as adults (insects) or (3) living in a non-dormant state in exposed soil as long as the soil remains moist (Tronstad et al., 2005).

2 48 Hydrobiologia (2013) 700:47 59 Furthermore, mobile organisms can (4) escape from sites exposed by decreasing water level or (5) bury into the moist substratum. The organisms staying on the exposed bottom can survive for some time, but finally their mortality inevitably increases. Horizontal migrations following the retreating waterline were observed in the field for a variety of aquatic insects (chironomids, stratiomyids, ephemeropterans, trichopterans, coleopterans, plecopterans) as well as leeches and Acari (Extence, 1981; Richardson et al.,2002; Tronstad et al., 2005). Oligochaetes, trichopterans and sphaeriid clams have been observed to bury into the substratum when exposed to drying (McKee & Mackie, 1980; Imhof& Harrison, 1981; Richardson et al., 2002). On the other hand, potentially mobile organisms, such as amphipods and trichopteran larvae remained on the exposed substratum after the decrease of the water level (Extence, 1981; Richardson et al., 2002). Several taxa have been shown to be particularly resistant to air exposure, surviving for at least 7 days (chironomids) (Suemoto et al., 2005) to even 30 days (a trichopteran Diplectrona modesta) (Imhof & Harrison, 1981). Amphipoda (Crustacea) include several taxa particularly well adapted to life at a land water interface. For instance, sandhoppers (Talitridae) are semiaquatic beach dwellers using celestial and astronomical cues to locate the strandline on the beach, where they live (Ugolini, 2003; Ugolini et al., 2004). However, the number of studies on the impact of air exposure and drying on amphipods is limited (e.g. Preece, 1971; Holsinger & Dickson, 1977; Marsden, 1991). Gammarids are another group of amphipod crustaceans, often occurring in shallow water habitats and constituting an important part of near-shore freshwater communities. Some gammarid species live at extremely shallow depths, very close to the shoreline (Gruszka, 1999; _Zytkowicz et al., 2008). Thus, some adaptations of these organisms to potential air exposure events could also be expected, though probably of different nature than those exhibited by semi-aquatic species. Due to their high mobility, gammarids quickly spread in water bodies to which they are introduced, reach great densities and often dominate or subdominate the invertebrate assemblages of the littoral and near-shore zones of rivers and streams (Grabowski et al., 2007a; Czarnecka et al., 2009). In recent decades, several gammarids have proven to be efficient invaders: Pontogammarus robustoides (G.O.Sars),Dikerogammarus haemobaphes (Eichwald) and Dikerogammarus villosus (Sovinsky), with origins in the Ponto-Caspian region, have expanded their ranges in Europe (Grabowski et al., 2007b), where they compete with and feed on native taxa (Dick & Platvoet, 2000; bij de Vaate et al., 2002; MacNeil & Platvoet, 2005; Kley & Maier, 2006; van Riel et al., 2006; Grabowski et al., 2007a, b; Kinzler et al., 2009; Platvoet et al., 2009; van der Velde et al., 2009; Weis, 2010; MacNeil et al., 2010). Understanding the adaptations of invasive gammarids to survive in unsuitable environmental conditions could help predict their invasive potential and susceptibility of various habitats to their invasions. Evolving in the Black Sea limans, where they experienced great water level fluctuations due to the variable inflow of the river water (Rozengurt, 1971, 1974; Orlova et al., 2005) may preadapt these species to invading similar unstable environments, such as lowland reservoirs where air exposure of the substratum is common. Previous studies on the resistance and behavioural responses of gammarids to air exposure are limited and ambiguous. This group is not resistant to total drying, but quickly recolonises temporary stream sections by drift or migration from adjacent permanent sections (Meyer et al., 2004). Muskó et al. (2007), studying the effect of water level fluctuations in Balaton Lake, found that Dikerogammarus spp. (a mixture of D. haemobaphes and D. villosus) dominated during the dry period at most of the examined sites and gave way to another amphipod, Chelicorophium curvispinum, when the water level increased. Extence (1981) and Richardson et al. (2002) observed that Gammarus pulex, G. fasciatus and G. tigrinus stayed on the drying shore after the decrease of the water level. In the latter study (Richardson et al., 2002), gammarids were protected from the water loss by macrophyte cover, which could also prevent their escape towards the waterline. Thus, various gammarid species experience periods of air exposure during their life, but their defence mechanisms against this factor have not been well examined. We used experiments to test the survival and behaviour of three Ponto-Caspian gammarids: P. robustoides, D. haemobaphes and D. villosus during substratum drying. They are abundant in lowland reservoirs and large rivers of Central Europe, but clearly differ from one another in their habitat preferences, including the distance from the shore

3 Hydrobiologia (2013) 700: ( _Zytkowicz et al., 2008). Particularly P. robustoides, living in sandy substratum at a very shallow depth (Gruszka, 1999; _Zytkowicz et al., 2008), may be exposed to drying during water level fluctuations. This species was previously found buried in sand above the waterline by Poznańska et al. (2010). The other species usually live in deeper waters, among stones and mussel shells ( _Zytkowicz et al., 2008), but may also occur near the shore at shallow depths (Muskó et al., 2007). For comparison, we also examined a native species, Gammarus fossarum Koch, which is common in smaller rivers and streams with fast water flow, good oxygen conditions and alkaline, nutrientpoor water (Ja_zd_zewski, 1975; Peeters & Gardeniers, 1998; Wijnhoven et al., 2003), in which the water level may fluctuate due to variable precipitation. Because of their natural occurrence in shallow habitats, we hypothesized that P. robustoides and G. fossarum would be most resistant to substratum drying and would exhibit the most efficient defence mechanisms. Furthermore, because of their occurrence in the deeper part of reservoir, we hypothesized that D. villosus and D. haemobaphes would be less resistant to substratum drying. To check these hypotheses, we exposed gammarids on gradually drying sandy substrata, with or without the possibility of their vertical and/or horizontal migrations to more humid locations. Materials and methods Animals We collected the individuals of D. haemobaphes (mean body length: 11.3 mm, range: 9 13 mm) together with zebra mussel clusters and submerged hard substrata from the Smukalski Reservoir, a reservoir on the lower River Brda (the Vistula River catchment) near the town of Bydgoszcz, central Poland. We obtained the individuals of D. villosus (12.2, mm) from the Włocławek Reservoir (a reservoir on the lower River Vistula, central Poland) using plastic Christmas tree branches as substrata for colonization (Czarnecka et al., 2010). We captured the individuals of P. robustoides (12.0, 9 14 mm) from the near-shore sandy bottom of the Włocławek Reservoir, at a depth of ca m, using a 1-mm mesh sieve. The native species, G. fossarum (10.6, 8 13 mm), occurred in the River Ruda, a small left tributary of the Włocławek Reservoir (Gierszewski, 2000). We captured the individuals of this species using a 1-mm mesh sieve from the sandy bottom at a depth of ca m. In the laboratory, each species was maintained separately in 50-l tanks with sandy substratum (with plant remnants in the case of G. fossarum), and stony shelters, in aerated tap water (left for at least 24 h before use) at room temperature. We fed them frozen chironomids and used them in the experiments within 1 week after collection. Following rough sorting for experiments, we identified ethanol-preserved gammarids at magnification with the keys by Ja_zd_zewski (1975) and Konopacka (2004). Experimental conditions We collected sandy substratum and water for the experiments from the locations where particular species were also sampled. Before the tests, we dried the substratum at 60 C for 6 h to get rid of any living invertebrates and eliminate the potential effects of their uneven distribution and/or movements on the responses of gammarids. The substratum was then cooled at room temperature and conditioned in aquarium water for 24 h before using it in the experiments. Water was aerated for 24 h before the tests to prevent potential oxygen deficits, but we did not apply aeration during the experiments to avoid the influence of air bubbles on gammarid behaviour. We carried out the experiments at room temperature controlled with an air conditioner. Air temperature and humidity, measured daily with a thermo-hygrometer (EMR812HGN, Oregon Scientific, UK), were 22.5 C (21 24 C) and 38% (35 45%), respectively. We monitored water quality at the beginning, in the middle and at the end of the tests using a multimeter Multi340i (WTW GmbH, Weilheim, Germany), except the cases when the water level was too low to insert the probes. Most of the environmental parameters did not differ among the tests with various species. Water oxygen concentration was always high (range mg l -1 ) and ph varied little (range: ). Only conductivity was lower in the water from the River Ruda, used for G. fossarum (mean: 424 ls cm -1 ), than in the reservoir water, in which the other gammarids were tested (mean: 762 ls cm -1 ).

4 50 Hydrobiologia (2013) 700:47 59 This reflects the distribution of alien and native gammarids in waters of different conductivity levels in Europe (Grabowski et al., 2009). We also determined substratum water content (WC) at the start of each test and during them, from the difference in the weight of a 14 g sediment sample before and after drying for 24 h at 100 C. We conducted the experiments described below with five replicate containers for each gammarid species. We also ran five control replicates with constant water level in each experiment. We did not feed the gammarids during the tests, because it was impossible when the water level dropped below the substratum surface. Van der Velde et al. (2009) observed very high survival rates of G. fossarum and D. villosus after 10 days of starvation. As the experiments in the present study lasted for not more than 8 days, it seems unlikely that the lack of food in our experiments could considerably affect the mortality of gammarids. Experiment 1: survival We conducted the survival experiment in ceramic trays with their bottoms covered by a 2-cm layer of sandy substratum (Fig. 1A). We filled the trays with aerated water to the level of 1.5 cm above the substratum surface (volume: 456 ml). After 24 h, we introduced five gammarids to each tray. In the test trays, water was left to dry, so that its level gradually dropped and then the substratum began to dry out. In the control trays, the water level was kept constant. Every day, we determined the mortality of gammarids and the substratum WC. Dead animals were easily visible on the substratum surface. We conducted the experiment until reaching 100% mortality of the gammarids in the test trays (6 7 days). We compared the survival of the tested species using a one-way ANOVA followed by a Tukey HSD post hoc test. Because the drying rate differed somewhat among the trays, we used the sediment WC on the day when 80% mortality (4 out of 5 individuals in a replicate) occurs as a dependent variable in this analysis. We applied a probit regression with Schneider- Orelli correction for the control mortality (Finney, 1971) using MASS 7.2 package (Venables & Ripley, 2002) for R statistical computing environment (R Development Core Team, 2008) to calculate the lethal sediment WCs at which 50 and 90% mortality (LC 50 and LC 90, respectively, and their 95% confidence intervals) took place, after pooling the results from all replicates in each treatment. We considered the LC values for two particular species as statistically different when their 95% confidence intervals did not overlap. Experiment 2: horizontal migrations We conducted this experiment in glass tanks with a 1.5-cm layer of sand on the bottom (Fig. 1B). We filled the tanks with aerated water to the level of 3 cm above the substratum (864 ml). After 24 h, we divided the tank into three equal zones using glass barriers and added 10 gammarids to one of the side zones of the tank. After the next 24 h, we raised the shorter tank wall adjacent to the gammarid zone so that it was elevated 4.5 cm above the table surface. This allowed us to imitate the shore inclination. As a result, the water depth at the raised tank side (in the zone of gammarid introduction) was lowest, while near the other side depth was highest. Then we removed the barriers and let the animals migrate freely all over the tank. In the experimental tanks, the water level dropped gradually exposing larger and larger part of the substratum surface (Fig. 1B). In the control tanks, the water level was kept at a constant level of 2 cm in the shallowest place. To hasten the process of drying, we removed daily 100 ml of water from each drying tank with a syringe. When water in the tank dropped to the final level indicated in Fig. 1B (after aprox. 6 8 days), we finished the experiment. At this moment, the substratum consisted of three equal zones: (1) the air-exposed zone with humid sand, (2) the air-exposed zone with infiltration water below the substratum surface and (3) the submerged zone. At the end of the experiment, we put the glass partitions between the zones to prevent further migrations of animals and determined the number of gammarids, their mortality and final sediment WC in each zone. We applied the same procedure to the control tanks, checked at the same time as the drying ones. Had the gammarids not responded to the decreasing water level at all, they should have been randomly distributed, with 1/3 of individuals in each tank zone. We checked the departures of their actual distributions from this random pattern with one-sample t tests,

5 Hydrobiologia (2013) 700: Fig. 1 Experimental vessels used in Experiment 1 (A), Experiment 2 (B) and Experiment 3 (C). The dimensions are given in mm comparing the mean percentage of animals in a given zone with a theoretical value of 33.3%. As 100% of G. fossarum individuals in the drying tanks moved to the submerged zone (see Results section), the lack of the within group variability made it impossible to analyse the data with a parametric test. Therefore, to find differences among the distributions of various species, we used separate Kruskal Wallis tests for each tank zone and treatment (drying or control tanks). In the case of significant results, we applied by a post hoc procedure described by Sokal & Rohlf (1995). We also used Mann Whitney U tests to compare the percentages of each gammarid species between the drying and control tanks (separately for

6 52 Hydrobiologia (2013) 700:47 59 each zone). We applied the same methods to analyse the survival of gammarids at the end of the experiment. We checked the differences in the final sediment WC among the particular zones of the drying tanks with a two-way ANOVA followed by a Tukey post hoc test, with species as a between-groups factor and tank zone as a repeated measures factor. Experiment 3: vertical migrations We ran this experiment in glass tanks filled with 15-cm layer of sand and a 3-cm layer (432 ml) of aerated water above the substratum surface (Fig. 1C). After 24 h, we introduced five gammarids to each tank. The water level dropped gradually in the test tanks and was kept constant in the control tanks. To hasten drying, we removed 100 ml of water each day from each drying tank with a syringe. As soon as the substratum in the drying tanks became exposed to air, we divided it into six 2.5-cm vertical layers and determined the number of gammarids and final WC in each layer. We applied the same procedure to the control tanks, checked at the same time as the drying ones. The experiment lasted for 6 8 days. Despite our expectations, based on the earlier field observations (Poznańska et al., 2010), gammarids did not migrate deeply into the substratum, but only buried just below the substratum surface. Thus, we simply analysed the percentage of buried animals. As some species never buried, the use of a parametric test was problematic due to the lack of data variability. Therefore, we applied two Kruskal Wallis tests, run separately for the data from the control and drying tanks and followed by a post hoc procedure (Sokal & Rohlf, 1995). We also ran Mann Whitney U tests to compare the burying activity of gammarids between the control and drying tanks. Moreover, we checked the differences in the final WC in the surface layer of sediments among the drying tanks with different gammarid species using a one-way ANOVA. Results Experiment 1: survival The mean substratum WC at which 60% mortality took place differed significantly among the studied Fig. 2 Substratum water content (WC) causing 50 and 90% mortality of gammarids (Experiment 1). LC 50 and LC 90 values are corrected for the control mortality and shown with 95% confidence intervals. Groups labelled with the same letter indicate species that did not differ significantly from one another (as indicated by non-overlapping confidence intervals) species (ANOVA: F 3, 16 = 11.82, P \ 0.001), with P. robustoides (mean WC ± SE: 3.6 ± 1.8%) being significantly more resistant than the other three species (11.5 ± 0.6%) (Tukey test). Pontogammarus robustoides could survive very low WC (LC 50 = 4.6% WC, LC 90 = 1.8% WC, Fig. 2). The LC 90 WC corresponds to sand, that nearly dry and with loose grains. The other species did not differ from one another with respect to their resistance to air exposure (LC 50 = , LC 90 = %, Fig. 2). Experiment 2: horizontal migrations All G. fossarum individuals followed the decreasing water level and moved to the submerged zone of the drying tanks (Fig. 3A). Therefore, their distribution could not be formally analysed with one-sample t tests, but certainly it was not random. G. fossarum was the only species that also exhibited a non-random distribution in the control tanks, with ca. 70% of individuals found in their deepest zone. P. robustoides was always randomly distributed among the zones of both control and drying tanks, though the percentage of animals found in the air-exposed zone was higher than elsewhere (Fig. 3B). D. haemobaphes gathered in the submerged zone of the drying tanks (Fig. 3C). D. villosus grouped in the middle zone of the drying tanks, containing infiltration water below the substratum surface (Fig. 3D), whereas it avoided the submerged zone.

7 Hydrobiologia (2013) 700: Fig. 3 Horizontal migrations of gammarids following the decreasing water level (Experiment 2). The black parts of the bars indicate dead individuals. The error bars show standard errors of means (SE). The black arrows indicate significant departures of gammarid percentages in a particular tank zones from a theoretical value of 33.3%, indicating a random distribution (one-sample t test: 2 arrows \ P \ 0.01, 1 arrow 0.01 \ P \ 0.05). Upward and downward arrows denote a preference for or an avoidance of a given zone, respectively. The asterisks show significant differences in gammarid percentages of drying tanks from the control tanks (Mann Whitney tests: ***P \ 0.001, **0.001 \ P \ 0.01, *0.01 \ P \ 0.05). Significant differences among species percentages (post hoc procedure following a Kruskal Wallis test) are indicated by the letter symbols: Gf G. fossarum, Pr P. robustoides, Dh D. haemobaphes, Dv D. villosus. The species labelled with the same letter in a square frame did not differ from one another with respect to their mortality in the drying and control tanks (post hoc procedure following a Kruskal Wallis test) Significant differences among the distributions of various species occurred in the drying tanks only (Table 1). The percentage of G. fossarum in the submerged zone was significantly higher, and that in the dry zone significantly lower compared to the other species. Moreover, D. villosus was more common in the middle zone and less common in the submerged zone than D. haemobaphes and G. fossarum. The differences in gammarid percentages between the corresponding zones of the drying and control

8 54 Hydrobiologia (2013) 700:47 59 Table 1 Kruskal Wallis tests of the differences in distribution and survival among the studied species in Experiment 2 (horizontal migrations) v 2 (df = 3) P Distribution in the drying tanks Dry zone * Intermediate zone * Submerged zone * Distribution in the control tanks Shallowest zone Intermediate zone Deepest zone Survival in the drying tanks All zones * Survival in the control tanks All zones Asterisks indicate statistically significant results tanks were significant for all species but P. robustoides (Fig. 3), indicating that they responded to the decreasing water level. We only observed significant differences among gammarid mortalities in the drying tanks (Table 1), with the survival of D. haemobaphes being lowest (68%) and that of G. fossarum highest among the studied species (Fig. 3). Most of the D. haemobaphes individuals found on the dried substratum were dead (Fig. 3C). In the control treatment, the survival of all species was high (85 100%). The substratum WC differed significantly among the zones of the drying tanks (ANOVA: F 2,32 = 1.57, P \ 0.001), but not among the tanks with various species (F 3, 16 = 2.76, P = 0.076), indicating that the test conditions for all species were similar. The WC in the submerged zone (mean ± SD: 20.2 ± 1.7%) differed significantly (Tukey test) from those found in the two other zones (17.7 ± 0.8%), which did not differ significantly from each other. Experiment 3: vertical migrations The gammarids never migrated deeply into the sediments. They only buried themselves just below the substratum surface, usually with their backs visible from the top. We found significant differences in burying activity among species both in the control and drying tanks (Kruskal Wallis tests: v 2 = 26.1, P \ and v 2 = 10.4, P = 0.015, respectively). Fig. 4 Vertical migrations of gammarids into the substratum (Experiment 3). The error bars show standard errors of means (SE). The species labelled with the same letter did not differ significantly from one another with respect to their burying activity (post hoc procedure following a Kruskal Wallis test) Only P. robustoides often buried in sand (Fig. 4). We found 75% individuals of this species (the experimental and control tanks pooled) under the sand surface. The behaviour of P. robustoides differed significantly from that of the other species (post hoc test, P \ 0.05). Three other gammarids did not differ significantly from one another with respect to their burying activity, though we found a few individuals of G. fossarum immersed in sand (10% on average), whereas both Dikerogammarus species always stayed on the substratum surface (Fig. 4). We observed no differences in gammarid behaviour between the control and drying tanks for any species (Mann Whitney U tests, P [ 0.05). The mortality ranged from 20 to 30% in the drying tanks and 4 to 24% in the control tanks. The final substratum WC in the surface zone of the substratum, where the burying animals were present, was 15.5 ± 3.5% (mean ± SD) and did not differ among the tanks with various gammarid species (ANOVA: F 3, 16 = 0.95, P = 0.441). Discussion Our study has demonstrated that various gammarids exhibit different strategies allowing them to survive air exposure events during water level fluctuations. The variability of these strategies likely reflects differences among habitats preferred by particular species and adaptations to their specific living conditions in the field.

9 Hydrobiologia (2013) 700: Pontogammarus robustoides, which turned out to be most resistant to the loss of the water content (WC) from the substratum (Fig. 2), tends to inhabit sandy bottoms at very shallow depths (Gruszka, 1999; _Zytkowicz et al., 2008). In the current study, some individuals of this species were still alive in sand that was nearly dry (1.8% WC), with loose grains and no visible traces of moisture. P. robustoides did not migrate horizontally following the decreasing water level, but rather remained on the exposed bottom (Fig 3B). This behaviour is consistent with the earlier field findings of P. robustoides individuals buried in sand above the waterline after a temporary water level decrease in a reservoir (Poznańska et al., 2010). In our present study, this species also buried into the substratum, though only just below the surface. However, as the gammarids in the control tanks buried similarly to those exposed to drying, this behaviour cannot be regarded as a response to air exposure, rather being a usual behaviour of this species. Nevertheless, staying below the sand surface can considerably facilitate the survival of gammarids during the periods of air exposure. P. robustoides seems to be particularly predisposed to burying due to its short antennae I and II (Karaman & Barnard, 1979, M. Grabowski, pers. inf.). Two other invasive gammarids: D. haemobaphes and D. villosus turned out to be much less resistant to substratum drying (Fig. 2), probably because that they did not bury into the sediments when the surface became dry (Experiment 3). Dikerogammarus haemobaphes responded to air exposure by migrating horizontally to the deeper, submerged zone, though this response was not as pronounced as that shown by G. fossarum. Most of the individuals that remained on the exposed bottom died before the end of the experiment. There are two possible explanations of this result: (1) these specimens died because of drying or (2) they had died of some other reasons or had been in a poor physiological condition and therefore did not migrate as the others. The mortality of this species occurred in the drying tanks only, which would support the first option. On the other hand, the WC in the air-exposed substratum of the experimental tanks (17.7%) was well above the 50% mortality level of D. haemobaphes found in Experiment 1 (9.7%), showing that the mortality of gammarids was unlikely to result from drying only. Dikerogammarus villosus also migrated horizontally in response to the decreasing water level, but, surprisingly, instead of moving to the submerged tank zone, it grouped in the central zone, with no surface water, though containing infiltration water under the substratum surface. This behaviour is rather difficult to explain. D. villosus usually inhabits hard substrata of large grain diameter (Devin et al., 2003; Muskó et al., 2007; MacNeil et al., 2010). Thus, it is likely that it would try to search for a shelter under experimental conditions. However, such behaviour would have rather lead to the occupation of the tank corners, also in the control treatment, which did not happen in our experiment. Low oxygen concentration might have also affected gammarid movements, making them leave the hypoxic water zone. Upward vertical migrations up to the air water interface in response to oxygen deficiency were observed in another amphipod, Gammarus roeseli (Henry & Danielpol, 1999). However, in our experiments, oxygen conditions in water were always good (C6.8 mg l -1 ), which excludes such a possibility. Perhaps, when we finished our experiment, the gammarids just started to migrate and were on their way to the submerged zone. However, as shown by Experiment 1 (Fig. 2), this species was least resistant to substratum drying and therefore should have responded faster than the other gammarids to have a chance to survive. Thus, its location in the infiltration water zone of the drying tanks remains unexplained. In the field, both Dikerogammarus species usually live at greater distances from the shore than P. robustoides and G. fossarum, typically inhabiting deeper sites covered with stones, zebra mussel colonies and macrophytes (Muskó, 1993; Devin et al., 2003; Kobak & _Zytkowicz, 2007; MacNeil et al., 2008; _Zytkowicz et al., 2008; Platvoet et al., 2009; MacNeil et al., 2010), which are less exposed to drying. Such distribution could explain the fact that their defence mechanisms turned out to be weaker than those of the other tested species. Furthermore, both Dikerogammarus species may need shelters formed by habitat structure, absent in our experiments, to survive in adverse environmental conditions. This hypothesis is supported by Martens & Grabow (2008), who observed that D. villosus had survived for at least 6 days out of water in a dense colony of zebra mussels fouling an outboard motor exposed to air. In the field,

10 56 Hydrobiologia (2013) 700:47 59 the ability to find a deeper water microhabitat during an incoming air exposure event may help them survive in a complex environment with submerged refugia remaining among stones, shells and pieces of debris. Dikerogammarus villosus is regarded as the most efficient gammarid invader in Europe, being the strongest competitor and predator of native fauna, causing considerable changes in the local environments (Dick & Platvoet, 2000; MacNeil & Platvoet, 2005; MacNeil et al., 2010). Nevertheless, its susceptibility to air exposure events may limit its invasion success, and areas susceptible to drying will probably be avoided by this species, where it could be replaced by more resistant taxa, such as P. robustoides. The native species G. fossarum turned out to be quite susceptible to substratum drying (Fig. 2). Its defence mechanism against air exposure was the horizontal migration following the retreating waterline. All individuals of this species moved to the submerged zone of the drying tanks and survived. It should be noted that the migratory responses of G. fossarum seem to be a more efficient defence mechanism than the drying resistance exhibited by P. robustoides, as shown by the higher survival of the former species in Experiment 2, when the escape from the drying area was possible. G. fossarum may be well adapted to the adverse effects of drought in small, shallow, fast-flowing streams, where water level changes rapidly with local precipitation. The species often occurs very close to the stream banks, at sites overgrown by macrophytes (pers. observations). Several gammarid species have been found to be capable of fine adjustment of their microhabitat with respect to various environmental cues, such as predation pressure, intra- and interspecific competition and substratum quality (Devin et al., 2003; Van Overdijk et al., 2003; Kobak & _Zytkowicz, 2007; MacNeil et al., 2008; Kobak et al., 2009; Platvoet et al., 2009; Czarnecka et al., 2010). These adaptations certainly contribute to their survival in variable environments in which they occur. Here we show that gammarids can also respond to the decreasing water level, using behaviour to survive air exposure and drying. Their different adaptations may help explain their distribution in the field. For instance, Gammarus pulex is unable to survive the total drying of a stream bed, though it is a typical inhabitant of temporary water bodies (Meyer et al., 2004). Probably the same applies to G. fossarum, which is ecologically and morphologically similar to G. pulex and both species may coexist in the same area (Ja_zd_zewski, 1975). Meyer & Meyer (2000) observed G. fossarum at an air-exposed site in a stream, though the duration of this exposure is unknown. Another species, Echinogammarus berilloni, invasive in Western Europe and tolerant to a wide range of environmental factors (Meyer et al., 2004 after Pinkster, 1993), avoids river sections with a low water flow and susceptible to drying (Meyer et al., 2004), as well as river headwaters (Josens et al., 2005). On the other hand, P. robustoides, highly tolerant to the water loss from the substratum, often colonizes shallow, sandy areas very close to the shoreline (Gruszka, 1999; _Zytkowicz et al., 2008). Other invasive gammarids more often inhabit deeper parts of water bodies ( _Zytkowicz et al., 2008), which is reflected by their weaker adaptations to air exposure events. The evolution of Ponto-Caspian gammarids in estuaries and near-sea limans, where they often face considerable changes of the water level may facilitate their invasions, particularly in anthropogenic water bodies, such as reservoirs, where water level fluctuations occur at a much greater scale than in natural waters (Richardson et al., 2002; Brauns et al., 2008; Poznańska et al., 2009) to which native species have been adapted. Acknowledgments This research was supported by Nicolaus Copernicus University (NCU grant no. 308-B) as well as Polish National Science Centre (NSC grant no. N N ). We wish to thank Marcin Budrewicz, a SCUBA diver, who helped us collect Dikerogammarus haemobaphes. We are very grateful to Dr. Alicja Konopacka (University of Łódź, Poland) for confirming the taxonomic identity of the studied gammarid species. We also deeply acknowledge the help of Dr. Mikhail Son (Odessa Branch Institute of Biology of Southern Seas, Ukraine) who provided us with the literature on the environmental conditions in the native range of Ponto-Caspian gammarids. Finally, we would like to thank Professor John E. Havel (Missouri State University) for his valuable editorial comments that helped us improve our MS. References bij de Vaate, A., K. Ja_zd_zewski, H. A. M. Ketelaars, S. Gollasch & G. van der Velde, Geographical patterns in range extension of Ponto-Caspian macroinvertebrate species in Europe. Canadian Journal of Fisheries and Aquatic Sciences 59:

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