Aquatic Invasions (2021) Volume 16, Issue 2: 186–207

CORRECTED PROOF

Research Article Assessing biotic interactions between a non-indigenous amphipod and its congener in a future climate change scenario

Paola Parretti1,2,3,*, Macarena Ros4, Ignacio Gestoso3,5, Patrício Ramalhosa3,6, Ana Cristina Costa1,2 and João Canning-Clode3,5 1CIBIO, Research Center in Biodiversity and Genetic Resources, InBIO Associate Laboratory and Faculty of Sciences and Technologies, University of the Azores, Portugal. Rua Mãe de Deus 13A, 9501-801 Ponta Delgada, São Miguel, Açores, Portugal 2Faculty of Sciences and Technology, University of the Azores, Rua Mãe de Deus 13A, 9501-801 Ponta Delgada, São Miguel, Açores Portugal 3MARE - Marine and Environmental Sciences Centre, Agência Regional para o Desenvolvimento da Investigação Tecnologia e Inovação (ARDITI). Edifício Madeira Tecnopolo, Caminho da Penteada, 9020-105 Funchal, Madeira, Portugal 4Departamento de Biología, Área de Ecología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, 11510, Puerto Real, Spain 5Smithsonian Environmental Research Center, 647 Contees Wharf Road, Edgewater, MD 21037, USA 6OOM- Oceanic Observatory of Madeira, Agência Regional para o Desenvolvimento da Investigação Tecnologia e Inovação, Edifício Madeira Tecnopolo, Piso 0, Caminho da Penteada, 9020-105 Funchal, Madeira, Portugal Author e-mails: [email protected] (PP), [email protected] (MR), [email protected] (IG), [email protected] (PR), [email protected] (ACC), [email protected] (JCC) *Corresponding author

Citation: Parretti P, Ros M, Gestoso I, Ramalhosa P, Costa AC, Canning-Clode J Abstract (2021) Assessing biotic interactions between a non-indigenous amphipod and To evaluate the impact of successful invasions of marine ecosystems by non- its congener in a future climate change indigenous (NIS) in a future climate change scenario, we analysed how an scenario. Aquatic Invasions 16(2): 186– increase in temperature may affect biotic interactions between resident species and 207, https://doi.org/10.3391/ai.2021.16.2.01 newcomers. In this context, we examined the effect of temperature on interference Received: 8 May 2020 competition (i.e. displacement) between two ecologically-similar caprellid species Accepted: 6 August 2020 that co-occur in the Portuguese Atlantic archipelago of Madeira: the NIS Published: 17 November 2020 scaura (Templeton, 1836) and its congener Caprella equilibra (Say, 1818). Mesocosm experiments were used to assess the interaction between the two species and the Handling editor: Alejandro Bortolus effects of warmer ocean waters on this interaction. Specifically, we investigated the Thematic editor: Joana Dias effect of an increase in temperature on: i) survivorship and intraspecific displacement Copyright: © Parretti et al. of the NIS C. scaura, ii) survivorship and interspecific displacement when this NIS This is an open access article distributed under terms and its congener C. equilibra coexisted at similar densities, and iii) survivorship of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). and interspecific displacement in the two species when the density of NIS is higher compared to its congener. Furthermore, we explored differences in the heart rate of OPEN ACCESS. the two species as proxy for physiological condition. Our results showed that in a future scenario of ocean warming for Madeira Island (29 °C), survivorship and intraspecific displacement of C. scaura most likely will not be affected. The survivorship of the congener C. equilibra will not be compromised, but its displacement will be affected by a combination of temperature and density of NIS. Overall, our results showed that warming exacerbates interspecific interactions between the two caprellid species suggesting that climate change can modify species distribution among habitats, potentially affecting community structure and diversity. The results of this study highlight the importance of biotic interactions and environmental context to predict and assess NIS success and potential impacts on resident species in the perspective of climate change.

Key words: non-indigenous species (NIS), Caprellids, ocean warming, displacement, competition, Madeira Island, North-east Atlantic

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 186 Caprella scaura drives Caprella equilibra displacement in a warmer sea

Introduction A significant increase in the number of invasive non-indigenous species (NIS) is recognised as a major threat to marine ecosystems worldwide, impacting their structure and function (Costello et al. 2010). Although only a small proportion of NIS are able to establish, spread, and impact an ecosystem (Williamson et al. 1986; Suarez et al. 2005; Blackburn et al. 2011), successful invasions can have a wide range of effects on native biota (Johnston et al. 2017). In some cases, the establishment of a new species can increase the density of the local population (Wright et al. 2014), while other NIS invasions can significantly reduce the growth and survival of the resident species (Shinen and Morgan 2009). In addition, habitat destruction by human activities (e.g., construction of recreational marinas and commercial ports), together with climate change, constitute a threat to worldwide biodiversity (Occhipinti-Ambrogi 2007). In fact, these factors can affect species distribution and resource dynamics in both terrestrial and aquatic ecosystems favourable for biological invasions (Dukes and Mooney 1999). Rising ocean temperature is one of the most relevant and serious aspects of climate change (Hoegh-Guldberg et al. 2014). It is expected that global warming will promote interspecific competition by species that can easily adapt to warmer water, replacing species that cannot adapt as well (Stachowicz et al. 2002). In aquatic environments, several studies were conducted to assess the response of NIS and native species to an increase in water temperature (Stachowicz et al. 2002; Sorte et al. 2010). For example, Nguyen et al. (2020) demonstrated, in a common garden simulated thermal stress experiment, that plants of the seagrass Halophila stipulacea (Forsskal) Ascherson, 1867 from the invasive population (in Limassol, Cyprus) performed better than plants from the native population (in Eilat, Israel). Recently, Suárez et al. (2019) showed that ocean warming significantly reduced survival and asexual reproduction rates in the native anemone Anthothoe chilensis (Lesson, 1830), but not in the NIS Anemonia alicemartinae Haussermann & Forsterra, 2011. These findings demonstrate that temperature is only one of the key factors driving the success of NIS (Früh et al. 2016). Consequently, temperature increase due to climate change and other anthropogenic pressures are expected to favour biological invasions (Lenz et al. 2011). With warmer temperatures, the pool of potential NIS is enlarged, the success of invaders is expected to increase, and the outcome of interspecific interactions are assumed to shift in favour of NIS (Rahel and Olden 2008). To fully understand the ecological implications of increasing ocean temperatures, it is important to understand individual responses of various species to environmental stressors, as well as the response of communities and ecosystems; species-specific responses add to the complexity of interspecific interactions and ecological processes like competition and predation (Gattuso et al. 2015; Lim and Harley 2018).

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 187 Caprella scaura drives Caprella equilibra displacement in a warmer sea

Competition among and between species plays an important role in community structure (Gurvevitch et al. 1992) and invasion dynamics (Levine and D’Antonio 1999; Stachowicz et al. 2002; Levine et al. 2004). Interference competition is the dominant form of competition in marine ecosystems (Branch 1984; Branch and Barkai 1988) and occurs when different species, usually congeners, interact directly (Shucksmith et al. 2009 and references therein). In the case of species competing for space, interference competition often results in displacement, defined as localised exclusion, of a weaker competitor from habitats occupied by a superior one (Branch 1984; Branch and Barkai 1988; Amarasekare 2002). For example, the non-indigenous seagrass H. stipulacea has been shown to displace local seagrass species, both in the Caribbean (Willette and Ambrose 2012) and in the Mediterranean Sea (Sghaier et al. 2014). In New England and Nova Scotia, the non-indigenous European green crab Carcinus maenas (Linnaeus, 1758) contributed to the displacement of endemic such as the American lobster Homarus americanus H. Milne Edwards, 1837 (Williams et al. 2006). In northern California (USA), Shinen and Morgan (2009) tested the performance of the invasive Mytilus galloprovincialis Lamarck, 1819 and two other native mussels (Mytilus trossulus Gould, 1850 and Mytilus californianus Conrad, 1837), and the results suggest that M. galloprovincialis likely contributed to the displacement of M. trossulus. Moreover, Shucksmith et al. (2009) showed that the non-native Schurin, 1935 displaced two ecologically-similar native caprellids: Caprella linearis (Linnaeus, 1767) and Pseudoprotela phasma (Montagu, 1804), even when the density of C. mutica was much lower than the density of native caprellids. In some cases, competition between NIS and native species may be stable until a disturbance takes place that benefits one species over the other (Desmet 2011). Therefore, to evaluate the impact of a successful invasion in a future climate change scenario, it is important to analyse how temperature increase may affect biotic interactions between NIS and native species (Olabarria et al. 2016). Caprellids are small crustaceans that form an important trophic link between primary producers and higher trophic levels in marine ecosystems (Woods 2009). These organisms are epibionts of algae and invertebrates (e.g., sponges, hydroids, sea anemones, gorgonians, mollusks, and other crustaceans), but they can reach high densities on artificial structures such as fish cages, netting buoys, pontoons, and oil platforms (Buschbaum and Gutow 2005; Greene and Grizzle 2007; Page et al. 2007; Rensel and Forster 2007; Shucksmith 2007). Caprellids have limited dispersal abilities other than rafting (e.g. Keith 1971; Caine 1979; Aoki 1988). However, some species have attained widespread distribution aided by human transport vectors. An example of this is C. scaura Templeton, 1836, a known invader in the Mediterranean Sea (Ulman et al. 2019) and in the Atlantic and Indian oceans (Martínez and Adarraga 2008 and references therein).

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 188 Caprella scaura drives Caprella equilibra displacement in a warmer sea

In Europe, C. scaura was recorded for the first time in 1989 in Mar Piccolo di Taranto, Italy (Scipione 2015). According to Cabezas et al. (2014), this species spread to the western Mediterranean and subsequently to the eastern Atlantic (see also Guerra-García et al. 2011 and Ros et al. 2014a). In 2015 it was detected for the first time in the Portuguese Atlantic islands of Madeira (Ramalhosa and Canning-Clode 2015) where it has been classified as NIS. On the other hand, its congener C. equilibra Say, 1818 is a resident species on Madeira island since 1954, with a sample deposited at the Natural History Museum of Funchal (Sample reference: MMF442823). Caprella equilibra also has worldwide distribution and its true origin is unclear. In Europe, it is traditionally considered a native species (Guerra- García et al. 2011), while in other parts of the world—South Africa (Griffiths et al. 2011), New Zealand and Australia (Ahyong and Wilkens 2011), and Japan (Doi et al. 2011)—C. equilibra is classified as cryptogenic (i.e., species of unknown origin). After detection of C. scaura at different locations, several studies (Guerra-García et al. 2011; Fernandez-Gonzalez and Sanchez-Jerez 2013) reported a decrease in the abundance of the congener C. equilibra when C. scaura was present. In particular, a field study conducted by Ros et al. (2015) along the Iberian Peninsula highlighted the critical role of temperature and salinity in modulating the interaction between the two species. Therefore, C. scaura presents a proxy to study the synergistic effects of NIS in a climate change scenario. The present study examined the effect of temperature on interference competition (i.e., displacement) between two ecologically-similar caprellid species that co-occur in the Portuguese Atlantic archipelago of Madeira – the NIS C. scaura and its congener C. equilibra. Mesocosm experiments were performed to assess the interaction between the two species and potential climate change effects on the outcome of interactions (interference competition) between species. Specifically, we investigated the effect of an increase in temperature on i) survivorship and intraspecific displacement of the NIS C. scaura, ii) survivorship and interspecific displacement when this NIS and its congener C. equilibra coexist at similar densities, and iii) survivorship and interspecific displacement when the density of NIS is higher than the one of its congener. Furthermore, we explored differences in the heart rate between the two species. We hypothesised, based on the aggressive behaviour of C. scaura and previous research, that the combined effect of increased temperature and the dominance of C. scaura would reduce survivorship and trigger displacement of C. equilibra. Finally, considering that NIS are physiologically prepared to adjust to new habitat because they are more tolerant of environmental stressors (Braby and Somero 2006), and that the measurement of heart rate in crustaceans is a physiological tool that reflects stress levels in (Lim and Harly 2018; McGaw et al. 2018; Zainal and Noorani

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 189 Caprella scaura drives Caprella equilibra displacement in a warmer sea

2019), we hypothesised that the heart rate of C. scaura will be less affected by changes in temperature and biotic interactions between species than the heart rate of C. equilibra.

Materials and methods collection All specimens used in this study were collected at the end of July 2019 from two marinas located on Madeira Island off the Atlantic coast of Portugal: marina Funchal (32.64°N; 16.91°W) and marina Machico (32.71°N; 16.75°W). Specimens of C. scaura and C. equilibra were collected from their bryozoan substrate of Amathia verticillata (delle Chiaje, 1822), Bugula neritina (Linnaeus, 1758), and Cradoscrupocellaria bertholletii (Audouin, 1826). No species-specific substrate-epifauna relationship was observed in the field. Colonies of bryozoans attached to floating pontoons, buoys, and ropes inside the marinas were picked by hand and preliminary sorting by species and sex was conducted in situ. To reduce stress, the caprellid samples were isolated in buckets filled with water collected in situ and aerated, along with some colonies of the original substrate, and transported to the laboratory. At each marina, water quality parameters (i.e. temperature, salinity, pH) were measured with a handheld multiparameter meter (YSI Professional Plus, Supplementary material Table S1). Caprellids were kept isolated in eight 10-L aquaria per species and per sex until the start of the experiment. Acclimatisation was performed for at least 24 h in aquaria supplied with filtered seawater (1 µm) in a continuous water flow system (1.2 L/h; volume per aquarium: 10 L) at collection site temperature (23 °C) and salinity (35.55 ± 0.22, mean ± SD; n = 3). Additionally, in each aquarium, constant aeration systems and artificial light (four eco+ LED bars REEF 11000K, LEDaquaristik.de, Schierbusch, Germany) were provided for a photoperiod of 12/12 h (light/dark). Some of the original host bryozoans were added into each of the acclimatisation aquaria to provide substrate for attachment.

Experimental design and set up Three simultaneous mesocosm experiments were conducted to explore the effect of temperature on the biotic interaction between the NIS C. scaura and the congener C. equilibra. Specifically, experiment 1 examines survivorship and intraspecific displacement of C. scaura while experiment 2 assesses survivorship and interspecific displacement when NIS and its congener C. equilibra coexist at similar densities. Finally, experiment 3 investigates survivorship and interspecific displacement in the two species when the density of NIS is higher than the congener C. equilibra. Three levels of temperature were selected: the first level (23 °C) is the temperature at the collection site, the second level (26 °C) corresponds to the average

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 190 Caprella scaura drives Caprella equilibra displacement in a warmer sea

maximum value of temperature registered in marinas of Madeira for the last six years (MARE-Madeira temperature data set), and the third level (29 °C) represents the predicted summer maximum temperature for the A2 SRES (Special Report on Emission Scenarios, Nakicenovic et al. 2000) for the period 2070–2099 in Madeira (Santos et al. 2004). A preliminary experiment revealed that neither species survived at 32 °C, so no further investigation was conducted above a temperature of 29 °C. Differences in heart rates between the two species of caprellids in experiments 2 and 3 were measured as proxies to evaluate stress levels and quantify physiological responses to environmental and biotic variables (Lim and Harley 2018; McGaw et al. 2018). Different specimen densities were chosen in order to test if displacement, survivorship, or heart rate could be influenced by density or presence of the NIS under limited space conditions. The experiments were conducted in August 2019 at the mesocosm system and laboratory facilities of MARE (Marine and Environmental Research Centre) located at Quinta do Lorde Marina, Madeira (Portugal). In all experiments, a constant temperature was maintained with water baths provided with titanium heaters (Schego 600W, Schemel & Goetz GmbH and Co KG) and chillers (Aqua Medic Titan 2000), both connected to a powerbar type 5.1-D-PAB and ProfiLux 4 aquarium controller (GHL International, Advanced Aquarium Technology, Germany) and a continuous water flow system of 1 µm filtered seawater (1.2 L/h) heated to the desired temperature. Water quality parameters (i.e., temperature, oxygen, salinity, pH) were checked in experimental unit (EU) without caprellids at the beginning and end of each experiment using the same multiparameter meter previously mentioned (see also Table S2). Additionally, two water pumps (Aqua Medic EcoDrift 4.1) were placed inside each water bath to ensure temperature homogeneity. Artificial light was provided as in the acclimatisation aquaria. Specimens were isolated, according to the experimental design, into EU of 10.5 cm × 5 cm, (Figure 1 and Figures S1a–c). Each EU was designed with two perforated holes on opposite sides of the tank to maintain a constant water volume of 160 mL. These holes were covered with two vertical 1 mm plastic mesh (7 cm × 2.5 cm) to avoid loss of caprellids during the experiment. Inside each EU, the two plastic meshes were placed 5–7 cm apart to provide a substrate for caprellid attachment and to test microhabitat displacement of the specimens (see Figure 1). At the beginning of each experiment, all caprellids were carefully deposited on the mesh on the left side of the EU. Displacement from the mesh on the left to the mesh on the right side of the EU was used as a proxy for interference competition for space. The temperature was increased only after all caprellids were allocated into their respective EU and the water reached the designated temperature (26 °C was reached after 50 min and 29 °C after 100 min).

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 191 Caprella scaura drives Caprella equilibra displacement in a warmer sea

Figure 1. Locations of new records of Callinectes sapidus. Previous records in the Iberian captures in this study are shown with white circles. For details see Supplementary material.

Survivorship and displacement were assessed after 48 h from the start of the experiment. The displacement that occurred within each EU was measured as the number of live individuals on the right mesh at the end of the experiment. No food was supplied during the experiments.

Experiment 1: Survival and intraspecific displacement The main objective of experiment 1 was to examine the effect of temperature on intraspecific displacement and survivorship of C. scaura, considering the effect of density and the composition of the community (i.e. male and female alone versus male and female together). The experimental design comprised single-sex treatments at different densities and mixed-sex treatments at different densities. A total of 576 individuals of C. scaura were used (288 of each sex) to explore four factors: 1) temperature at three levels: 23 °C (ambient), 26 °C (summertime), and 29 °C (climate change scenario); 2) density at two levels: D1(4 adult individuals) and D2 (12 adult individuals) per experimental unit; 3) sex at two levels: male and female; and 4) population at two levels: P1 (one sex only (male or female) and P2 (both sexes in the same experimental unit (mixed sex)). Each treatment was replicated four times (for details, see Figure S1a). Measurements of survivorship and displacement from the left to the right mesh were taken after 48 h. This experiment could not be performed with C. equilibra due to low number of specimens available.

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 192 Caprella scaura drives Caprella equilibra displacement in a warmer sea

Experiment 2: Survival and interspecific displacement The main objective of experiment 2 was to explore the effect of temperature on survivorship and interspecific displacement of the sympatric C. equilibra by C. scaura, considering the effect of total density but keeping the same proportion of each species. This experiment was performed simultaneously with experiment 1. In experiment 2, a total of 192 individuals of C. equilibra (96 per sex) and 192 individuals of C. scaura (96 per sex) were used to explore three factors: 1) temperature at two levels: 23 °C (ambient) and 29 °C (climate change scenario), 2) density at two levels: D1 (4 individuals as 2 couples) and D2 (12 individuals as 6 couples), and 3) composition at two levels: couples of C. equilibra alone and when couples of C. equilibra co-exist with couples of C. scaura in the same EU. Each treatment was replicated four times (for details, see Figure S1b). Data on survivorship and displacement were collected after 48 h.

Experiment 3: Density of NIS The main objective of experiment 3 was to examine the effect of temperature on survivorship and interspecific displacement of the sympatric C. equilibra by C. scaura considering the effect of a different proportion of NIS compared to the congener. This experiment was also performed at the same time of the previous ones. In experiment 3, a total of 152 individuals of C. equilibra (76 per sex) and 184 of C. scaura (92 per sex) were used to explore two factors: 1) temperature at two levels 23 °C (ambient) and 29 °C (climate change scenario) and 2) density of NIS at three levels: absent (6 couples of C. equilibra), equal (3 couples of C. scaura co-existing with 3 couples of C. equilibra), and high (4 couple of C. scaura co-existing with 2 couple of C. equilibra). The initial total density in each EU was of 12 adult individuals. Each treatment was replicated four times (for details, see Figure S1c). Measurements of survivorship and displacement from the left mesh to the right mesh were collected after 48 h.

Assessment of heart rate and body size Heart rates were measured on caprellid specimens from the previous experiments after data on survivorship and displacement were assessed. Caprellid heart rates were measured in 4 individuals per EU (2 males and 2 females if enough individuals survived) using an adapted methodology (Lim and Harley 2018). Caprellids were transferred from the EU to a Petri dish with a drop of original experimental water and a small piece of 1 mm mesh. Caprellid heart rates, visible in the second pereonite, were recorded under a stereo microscope (Leica S8APO) for a minimum of 20 seconds. Videos were edited in iMovie (Apple version 10.1.14) and then played at half speed to estimate the heart rate. For each video, the number of heart beats was counted three times and the average count was used to estimate the average number of heart beats per minute.

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 193 Caprella scaura drives Caprella equilibra displacement in a warmer sea

To measure caprellid body size, one frame of the video clip was saved as an image and the length from the first pereonite to the fourth pereonite was measured with Image J software. This length was chosen because it was not possible to record the heart rate simultaneous with an image of the entire body due to magnification limits under stereo microscope. Although it does not represent total body size, this measurement allows size comparisons between individuals using linear regression between the second pereonite and total body length, as demonstrated by Campbell (1979) in C. californica, a morphologically similar species. We also included measurements of first, third, and fourth pereonites, which have a lower elongation than the second pereonite during growth (Hosono 2011) to increase accuracy of the measurements.

Statistical analysis Data on survivorship and displacement were transformed into percentages. Dead individuals were discarded from the analysis of displacement, and the average and standard errors were used for descriptive statistics. In experiment 1, variations in survivorship and displacement among treatments were compared using analysis of variance (ANOVA) with three fixed orthogonal factors: 1) temperature (T at three levels: 23 °C, 26 °C, and 29 °C); (2) density (D at two levels: D1 with 4 adults and D2 with 12 adults); and 3) population (P at two levels: P1 (only one sex: male or female) and P2 (males and females in the same EU). Because sex was not an independent variable in all cases (i.e. in P2), ANOVAs were conducted to assess variations in survivorship and displacement for males and for females separately. In experiment 2, variations in survivorship and displacement among treatments were compared using ANOVA with three fixed orthogonal factors: 1) temperature (T at two levels: 23 °C and 29 °C); (2) density (D at two levels: D1 (4 adults) and D2 (12 adults), and (3) composition (C at two levels: C. equilibra alone and C. equilibra coexisting with C. scaura). In experiment 3, variations in survivorship and displacement among treatments were compared using ANOVA with two fixed orthogonal factors: 1) temperature (T at two levels: 23 °C and 29 °C) and 2) density of NIS

(DNIS at three levels: absent, equal, and higher than the density of C. equilibra). Prior to all ANOVA routines, homogeneity of variance was tested with Cochran’s C-test. A p value < 0.05 was considered statistically significant. When ANOVA indicated a significant difference for a given factor, the source of difference was identified using the Student-Newman-Keuls (SNK) test. Univariate analysis were conducted using GMAV-5 (Underwood et al. 2002). Variations in heart rate among treatments were compared using univariate PERMANOVA because the data were not balanced. Analyses were based on a Euclidean distance matrix generated using untransformed data and 9999 permutations. To assess intraspecific differences in heart rate

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 194 Caprella scaura drives Caprella equilibra displacement in a warmer sea

Table 1. Results of three-way ANOVA for C. scaura survivorship and displacement. Temperature (23, 26, and 29 °C), density (4 individuals; 12 individuals) and population (single sex; mixed sex) treatment levels. Significant p-values are highlighted in bold.

C. scaura survivorship C. scaura displacement Source of variation df Female Male Female Male MS F p MS F p MS F p MS F p Temperature (T) 2 12,905.093 2.42 0.104 21,976.273 4.97 0.012 848.557 1.38 0.265 12,677.826 1.76 0.187 Density (D) 1 5.787 0.01 0.918 19,806.134 4.48 0.041 0.393 0 0.98 6,429.493 0.89 0.351 Population (P) 1 5.787 0.01 0.918 26,750.579 6.05 0.019 3,760.033 0.61 0.439 8,529.581 1.18 0.284 T × D 2 752.315 0.14 0.869 192.419 0.44 0.650 545.211 0.89 0.421 526.16 0.73 0.489 T × P 2 5,266.204 0.99 0.383 4,094.329 0.93 0.405 1,852.257 0.3 0.742 9,060.928 1.26 0.297 D × P 1 1,446.759 0.27 0.606 7,653.356 1.73 0.197 89.808 0.15 0.705 265.141 0.04 0.849 T × D × P 2 3,530.093 0.66 0.523 8,695.023 1.97 0.155 18,796.702 3.05 0.059 14,071.951 1.95 0.157 Residual 36 5,343.364 4,422.261 6,159.303 7,211.849

Cochran’s C-test C = 0.219 C = 0.314 C = 0.310 C = 0.241

among treatments, two PERMANOVA designs were used. In both cases, caprellid body size was used as a co-variable. PERMANOVA design 1 followed the design of experiment 2 with three fixed factors: temperature at two fixed levels, density at two fixed levels, and composition at two fixed levels. PERMANOVA design 2 followed the design of experiment 3 with two fixed factors: temperature at two levels and density of NIS at three levels. Univariate PERMANOVA analyses were conducted with PRIMER v. 6+ PERMANOVA package (Anderson et al. 2008). To assess differences in heart rate between the two species of caprellids and between sexes, we used a Wilcoxon signed rank test given the non-independence of the observation (e.g., individuals were in the same EU in mixed-sex treatments). The Wilcoxon signed rank test was conducted using SPSS (IBM Statistic version 26).

Results Experiment 1: Survival and intraspecific displacement Overall, an increase in temperature slightly reduced, but not significantly, survivorship and displacement of C. scaura. Specifically, after 48 h at ambient temperature (23 °C), C. scaura showed 86 ± 3.1% (mean ± SE) survivorship; at 26 °C, caprellids had 81 ± 3.8% survivorship; and in the climate change scenario, 66 ± 4.9% of caprellids survived. No significant differences were found in survivorship or displacement of females for the variables considered (Table 1, Figure 2a–f). In the case of males, survivorship dropped significantly with an increase in temperature (Table 1, Figure 2a). Moreover, survivorship of male C. scaura was inversely related to density (Table 1, Figure 2b) and directly related to the presence of females: in mixed sex treatments, male survivorship was significantly higher (Table 1, Figure 2c). No significant variations in displacement of males was found (Table 1, Figure 2d–f). At 23 °C, 35 ± 5.2% of C. scaura individuals moved from the left mesh to the right mesh of the EU. At 26 °C, movement decreased to 30.4 ± 5.3% and at 29 °C, the displacement of the caprellids was 20.2 ± 3.9%. Males consistently showed higher displacement rates than females (Figure 2d–f).

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 195 Caprella scaura drives Caprella equilibra displacement in a warmer sea

Figure 2. Results of experiment 1 on survivorship (left side) and displacement (right side) of C. scaura (average ± SE). Green correspond to males and yellow to females. The symbol * represents significant SNK test results. Pairwise comparisons for ANOVA results with p < 0.05 (Table 1). a) Survivorship at different water temperatures, b) survivorship between differing densities regardless of temperature, c) survivorship of populations regardless of temperature (Single sex = males or females alone; Mixed sex = males in same EU as females), d) displacement at different water temperatures, e) displacement at different densities regardless of temperature, and f) displacement of populations regardless of temperature (Single sex = males or females alone; Mixed sex = males in same EU as females).

Moreover, results from ANOVA assessed displacement of females as marginally significant (p = 0.0597) due to interaction among the factors of temperature, density, and population. The SNK post hoc test showed a significant reduction in displacement of females when the temperature increased from 26 °C to 29 °C and in the mixed-sex treatment at low density (2 couples).

Experiment 2: Survival and interspecific displacement In general, temperature did not have a significant influence on the survival or the intraspecific displacement of C. equilibra. However, the presence of C. scaura increased the displacement of C. equilibra. At ambient temperature (23 °C), survivorship for C. equilibra was 86 ± 4.8%, and for C. scaura, it was 81.7 ± 4.8%. Survival rates in both species decreased at higher temperature (29 °C) with 78.1 ±5.5% for C. equilibra and 63.5 ± 6.2% for C. scaura. Three-way ANOVA results showed no significant differences in C. equilibra survivorship among the variables considered (Table 2).

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Table 2. Results of three-way ANOVA for C. equilibra survivorship and displacement. No transformations were necessary for either survivorship or displacement values. Temperature had two levels (23 °C and 29 °C), density had two levels (4 individuals; 12 individuals), and composition had two levels (C. equilibra alone and C. equilibra co-existing with C. scaura). Significant p-values are highlighted in bold. C. equilibra survivorship C. equilibra displacement Source of variation df MS F p MS F p Temperature (T) 1 976.562 1.13 0.292 850.694 0.61 0.437 Density (D) 1 351.562 0.41 0.526 1,029.340 0.74 0.393 Composition (C) 1 3,164.062 3.66 0.061 1,029.340 0.74 0.393 T × D 1 39.062 0.05 0.832 2,100.694 1.51 0.224 T × C 1 4.340 0.01 0.944 434.027 0.31 0.578 D × C 1 351.562 0.41 0.526 8,633.507 6.21 0.016 T × D × C 1 733.507 0.85 0.361 17.361 0.01 0.911 Residuals 56 864.955 1389.670 Cochran’s C-test C = 0.309 C = 0.257

Figure 3. Results of experiment 2. Displacement (mean ± SE) of C. equilibra at different levels of density and composition (blue = EU with C. equilibra only; orange = C. equilibra co-exists with C. scaura in same EU). Different letters represent significant differences at different compositions within the same level of density (lowercase letters for low density, uppercase letters for high density). The asterisk (*) represents significant differences between density.

Globally, C. equilibra and C. scaura showed similar displacement at ambient temperature (23 °C): 33 ± 6.6% and 29.3 ± 6.1%, respectively. At 29 °C, 40.2 ± 7% of C. equilibra and 15.8 ± 4.3% of C. scaura moved to the opposite side of the experimental unit. Three-way ANOVA results showed significant differences in displacement of caprellids for the interaction between variables of density and composition (Table 2). The SNK post hoc test showed that C. equilibra was displaced more at low density (D1) and co-existing with C. scaura (Figure 3).

Experiment 3: Density of NIS In general, the survival of C. equilibra was not affected by an increase in temperature or an increase in the density of C. scaura. Meanwhile, temperature and density of NIS factors significantly affected the displacement of C. equilibra. At ambient temperature, C. equilibra showed a survivorship of 79.1 ± 6.5% and C. scaura of 75.7 ± 5.1%. At higher temperature survival of

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 197 Caprella scaura drives Caprella equilibra displacement in a warmer sea

Table 3. Results of two-way ANOVA for C. equilibra survivorship and displacement. No transformations were necessary for either survivorship or displacement values. Temperature had two levels (23 °C and 29 °C). Density of NIS had three levels: absent (no C. scaura); equal (6 couples of C. scaura and 6 couples of C. equilibra), and high (4 couples of C. scaura and 2 couples of C. equilibra). Significant p-values are highlighted in bold. C. equilibra survivorship C. equilibra displacement Source of variation df MS F p MS F p Temperature (T) 1 468.75 0.5 0.482 1,672.454 1.76 0.192 Density of NIS 2 989.583 1.06 0.354 1268.287 1.33 0.275 (DNIS) T × DNIS 2 329.861 0.35 0.703 3,130.787 3.29 0.047 Residuals 42 930.060 952.596

Cochran’s C-test C = 0.376 C = 0.2465

Figure 4. Results of experiment 3. Displacement of C. equilibra at different temperatures and densities of the NIS (blue = absent; orange = equal; red = high) represented as the mean ± SE. Different letters represent significant differences at different density of NIS at same temperature (lowercase letters represent 23 °C and uppercase letters represent 29 °C). The asterisk (*) represents significant differences between temperatures.

both species decreased 73 ± 6.2% for C. equilibra and 59.7 ± 6.4% for C. scaura. Analysis of two-way ANOVA showed no significant differences in C. equilibra survivorship among the variables considered (Table 3). Overall, C. equilibra and C. scaura showed similar displacement at 23 °C: 29.4 ± 7% and 28 ± 6.3%, respectively. At 29 °C, different trends were observed, with an increase in displacement for C. equilibra of 41.2 ± 6.3% and a decrease in displacement of 14.9 ± 4.2% for C. scaura. Two-way ANOVA results showed significant differences in displacement of caprellids for the interaction between the variables temperature and density of the NIS (Table 3). The SNK post hoc test suggest that in a future climate change scenario of warmer oceans, C. equilibra experienced greater displacement

when the density of C. scaura was higher (i.e. DNIS = high) compared to the congener (Figure 4).

Heart rate Of the 122 caprellids used, the heart rate of C. equilibra was always higher than that of C. scaura. At 23 °C, the heart rate of C. equilibra (n = 32) averaged 208.7 ± 53.3 beats/min and the heart rate of C. scaura (n = 28)

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Table 4. Results of PERMANOVA conducted to assess differences in heart beats per min for C. scaura and C. equilibra. Based on Euclidean distances of untransformed data. Significant p-values were obtained by computing 9999 permutations. Significant p-values are highlighted in bold. C. equilibra C. scaura Source of variation df p (perm) p (perm) MS Pseudo-F MS Pseudo-F Unique permutations Unique permutations Size (co-variable) 31.735 0.017 0.893 (9828) 6920.7 49.998 0.029 (9839) Temperature (T) 1 47903 26.455 0.000 (9832) 22646 16.361 0.000 (9818) Density (D) 1 3245.5 17.923 0.192 (9832) 210.8 0.152 0.710 (9835) Composition (C) 1 87.163 0.005 0.946 (9850) 4080.9 29.482 0.096 (9832) T × D 1 2061.2 11.383 0.284 (9847) 115.62 0.083 0.772 (9846) T × C 1 119.32 0.066 0.802 (9836) 581.01 0.420 0.517 (9844) D × C 1 540.42 0.298 0.592 (9850) 2889.8 20.878 0.158 (9830) T × D × C 1 3344.3 18.469 0.185 (9859) 3785 27.345 0.106 (9838) Residuals 44 1810.7 1384.2

Table 5. Results of PERMANOVA conducted to assess differences in heart rate within C. scaura and C. equilibra due to differences in C. scaura abundance. Based on Euclidean distances of untransformed data. Significant p-values were obtained by computing 9999 permutations. Significant p-values are highlighted in bold. C. equilibra C. scaura Source of variation df p (perm) p (perm) MS Pseudo-F MS Pseudo-F Unique permutations Unique permutations Size (co-variable) 1 1713.9 0.837 0.3581 (9810) 675.42 0.442 0.5185 (9852) Temperature (T) 1 53919 26.327 0.0002 (9852) 12867 84.111 0.0102 (9827) Density of NIS (DNIS) 2 812.32 0.397 0.6698 (9952) 3777.5 24.694 0.1071 (9946) T × DNIS 2 1485.5 0.726 0.4918 (9953) 1863.7 12.183 0.3079 (9948) Residuals 29 2048.1 1529.7

averaged 183.3 ± 39.5 beats/min. At 29 °C, the heart rate of C. equilibra (n = 35) averaged 151.8 ± 36.4 beats/min and the heart rate of C. scaura (n = 27) averaged 143.3 ± 33.4 beats/min. Thus, the heart rate of C. equilibra decreased at 29 °C by 27.26%, and by 21.82% in C. scaura. Wilcoxon signed ranks test showed no differences in heart rate between C. equilibra and C. scaura and no differences in heart rate between males and females of the same species. The results of PERMANOVA analyses showed that the heart rate of both species decreased significantly with an increase in temperature, while body size only affected the heart rate in C. scaura (Table 4; for information on body size, see Table S3). There were no significant differences in heart rate due to the presence of one species on the other. The results of PERMANOVA used to assess differences in heart rates of C. scaura and C. equilibra due to differences in density of NIS showed a significant reduction in the heart rate of both species when the temperature increased (Table 5).

Discussion Thermal sensitivity is a fundamental physiological attribute and a primary reason for climate-induced changes in natural communities (Pörtner and Farrell 2008). Moreover, interspecific interactions under warming conditions can modify the magnitude of a species response to climate change (Harley et al. 2006; Milazzo et al. 2013). Because each species reacts differently to climate stressors, linking interspecific interactions between local and NIS

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with environmental conditions as potential factors in the success of invasive species is important to predict invasions and subsequent impact on local assemblages (Früh et al. 2016). In general, the results of our three short-term experiments suggest that, in a scenario of ocean warming on Madeira Island (29 °C), survivorship of the NIS C. scaura and its congener C. equilibra will not be compromised. However, further studies are needed to assess long-term effects on both species. Moreover, due to the lower number of C. equilibra found in the field, it was not possible to conduct a deeper assessment of the thermal response of this species. Future research should assess the intraspecific displacement of C. equilibra in a climate change scenario. Finally, this study revealed that a combination of high temperature and high density of NIS will increase displacement of C. equilibra. One hypothesis in invasion science theory states that NIS are physiologically prepared to adjust to a new habitat if the species can tolerate the environmental stressor (Braby and Somero 2006). In fact, results from experiment 1 showed that temperature increase did not affect the displacement of C. scaura, but may affect survivorship of the male sex. If males are more sensitive than females to increases in temperature, the population structure may become strongly impaired. Nevertheless, a female-biased sex ratio is common in amphipods (Lewbel 1978; Prato et al. 2013; Velazquez et al. 2017) and could prove advantageous for the invasive species because a high number of females enhance the population’s reproductive potential (Devin et al. 2004; Cabiddu et al. 2013). However, further studies are needed, because an increase in temperature might decrease the reproductive activity of C. scaura (Sconfietti and Lupari 1995; Prato et al. 2013). In addition, survivorship of male C. scaura appeared to be inversely related to density and directly related to the presence of females (Figure 2b–c). This response is likely due to the aggressive behaviour of C. scaura males (Schulz and Alexander 2001; Guerra-García et al. 2011) that in our experiment seems to be reduced in the presence of females. This result contrast to the observations of Lewbel (1978) and Lim and Alexander (1986) on mating-related male mortality. Both studies, conducted with C. gorgonia Laubitz & Lewbel, 1974 and C. scaura, respectively, reported a greater number of male survivors in a “males only” condition than in “males with receptive females” and no significant difference between the number of male survivors in the “male only” EU and males with ovigerous females. In our experiment, we selected adult females without taking into consideration their moult cycle or ovarian maturation; therefore, our results could be explained by heterogeneity in female sexual maturity. High temperatures can trigger anger and hostility in some crustacea (e.g., crayfishes and crabs) and increase their willingness to fight (Belair and Miron 2009; Gherardi et al. 2013). In fact, for the swimming crab Portunus trituberculatus (Miers, 1876), an increase in intensity, duration, and

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 200 Caprella scaura drives Caprella equilibra displacement in a warmer sea

frequency of fighting was observed under higher temperature conditions (Su et al. 2020). Finally, results from experiment 1 showed that males of C. scaura displaced more females despite temperature, density, or the sex ratio of the population, and none of these factors affected the general displacement of the species. Higher mobility in males was observed in the experiment and seems to be a typical characteristic of caprellids (Caine 1991) and other amphipods (Borowsky 1983). In fact, both authors observed that males actively searched for receptive females. Moreover, the lower propensity to move in C. scaura females as compared to males could be explained by the maternal care behaviour in this species (Aoki 1997). Results from experiment 2 showed that survivorship of C. equilibra was not influenced by an increase in temperature nor by the presence of C. scaura or a combination of these factors. However, the displacement of C. equilibra was dependent on a synergy between the total density of specimens and the presence of the conspecific C. scaura. The displacement of C. equilibra due to the presence of C. scaura when the total population was low density (i.e. D1) as observed in our mesocosm experiment agrees with previous observations on the potential impact of C. scaura on its congener (e.g., Guerra-García et al. 2011; Fernandez-Gonzalez and Sanchez-Jerez 2013; Ros et al. 2015). Interestingly, under high density conditions (i.e. D2), there were no significant differences between the “only C. equilibra” and the “co-existence of species” scenarios, both at ambient and high temperature. This result seems to contradict the output of experiment 1. In fact, we were expecting an increase in aggressive behaviour, especially from C. scaura males, and a subsequent decrease in C. equilibra survivorship and/or an increase in C. equilibra displacement. Possibly, in experiment 2, the population reached equilibrium between total specimen density and availability of space. In addition, considering that survival was not affected, the competition for space was not worth a fight. Future testing could examine whether other aspects of competition (i.e. changes in feeding strategies, reproductive behaviour, etc.) emerge. In fact, both species exhibited trophic plasticity: C. scaura is mainly a detritivore but it is able to feed on other resources (Ros et al. 2014b), while C. equilibra can consume a mixed diet consisting mainly of detritus and small crustaceans and polychaetes (Guerra-García and Tierno de Figueroa 2009). In experiment 3 no effects from climate stress were observed in C. equilibra when C. scaura was below a certain density (i.e. absent or equal). The advantage of our experimental design is that a control experiment for density dependence was performed for C. scaura (NIS) and C. equilibra separately; therefore, it is not possible to attribute the displacement seen at the highest densities of C. scaura to an overall increase in caprellid density. This result is consistent with that of Milazzo et al. (2013) for two species of coastal wrasses. The rainbow wrasse Coris julis (Linnaeus, 1758) that tolerates colder water and the ornate wrasse Thalassoma pavo (Linnaeus, 1758)

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which is expanding northwards as the Mediterranean warms. The authors were able to assess the displacement of the cool-water wrasse species from preferred habitat due to a combination of two factors: i) increase in temperature and ii) higher relative dominance of the warm-water fish. As in our experiments, no displacement effect was recorded in the present-day scenario (i.e. ambient temperature). The results of experiment 3 at ambient temperature corresponded to the output of experiment 2 and confirms our theory that the population inside EUs reached equilibrium, considering that the availability of substrate was a limiting factor. Future studies should investigate the predicted displacement of C. equilibra in the field. Given the important role of caprellids as a vital trophic link between primary production and successively higher trophic levels, a cascade effect may occur in the food chain. Finally, we observed a decline in heart rate related to an increase in temperature in both species. These results could be interpreted as “counter current” because several studies on crustaceans have shown a positive relationship between an increase in temperature and heart rate (Kushinsky et al. 2019; Zainal and Noorani 2019). On the other hand, it is possible that we did not observe such a positive relationship because heart rate tends to increase exponentially with increasing temperature until a species-specific break point is reached, beyond which heart rate drops (Eymann et al. 2019; Levinton et al. 2019). In such cases, our observed decrease in heart rate may be due to the fact that at 29 °C the break point of both species of caprellids was exceeded. The 100% mortality found at 32 °C in the preliminary experiment together with the decrease in survival rate of both species when temperature rises may support this second possibility. In this case, a decrease in heart rate may reflect stress in both species when temperature increases. Unfortunately, it was not possible to observe whether caprellids would recover if relocated to lower temperature water. Further studies should investigate the relationship between heart rate and temperature for these species in a more refined circumstance (i.e. different temperature gradients, longer exposure times, and addition of a recovery phase). In our case, it was not possible to conduct a deeper assessment of heart rate variation without compromising the results on displacement. A suitable description of NIS traits is fundamental to development of good management policy aiming to prevent or contain the arrival and subsequent dispersion of NIS in marine ecosystems (Cardeccia et al. 2016). Future studies should focus on long term effects of warming oceans together with an increase in density of the invading species. In fact, testing the displacement along different instar stage and successive generations of C. scaura and C. equilibra was not the aim of this study, but it might be fruitful. Finally, due to the habitat selection preferences shown by caprellids (Lacerda and Masunari 2011; Awal et al. 2016; Martineź -Laiz et al. 2018), the consequences of warming oceans related to climate change in the area

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or quality of the substrate must be addressed. In this context, future studies may incorporate the substrate in which these species interact, such as the bryozoans A. verticillata or B. neritina, and assess changes in the biotic interaction.

Acknowledgements We would like to acknowledge the managers of Funchal and Machico marinas for facilitating access and sampling procedure. Special thanks to João Silva, from Natural History Museum of Funchal (MMF) Madeira, for providing information on the oldest record of C. equilibra on the Madeira Island. Finally, we acknowledge three anonymous reviewers for their helpful suggestions that greatly improved the manuscript.

Funding Declaration The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PP was funded by a PhD grant ref. M3.1.a/F/065/2015 by Fundo Regional de Ciência e Tecnologia (FRCT) and the program AÇORES 2020. MR was funded by a Juan de la Cierva Postodoctoral Fellowship (IJCI-2017-34656). IG was financially supported by a post-doctoral grant in the framework of the 2015 ARDITI Grant Programme Madeira 14-20 (Project M1420-09-5369-FSE-000001). PR was partially funded by the Project Observatório Oceânico da Madeira-OOM (M1420-01-0145-FEDER-000001), co-financed by the Madeira Regional Operational Programme (Madeira 14-20), under the Portugal 2020 strategy, through the European Regional Development Fund (ERDF). JCC is funded by national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Scientific Employment Stimulus – Institutional Call – [CEECINST/00098/2018]. This work was partially funded by projects MIMAR (MAC/4.6.d/066), MIMAR+ (MAC2/4.6d/249), PLASMAR (MAC/1.1a/030) and PLASMAR+ (MAC2/1.1a/347), in the framework of INTERREG MAC 2014-2020 Programme. Finally, this study also had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project [UIDB/04292/2020] granted to MARE UI&I and also by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE and by National Funds through FCT – Foundation for Science and Technology under the UID/BIA/50027/2019 and POCI-01-0145-FEDER-006821.

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Supplementary material The following supplementary material is available for this article: Table S1. Water quality parameters in marinas collected with a YSI Professional Plus handheld multiparameter meter. Table S2. Water quality parameters in EU without caprellids. Table S3. C. scaura and C. equilibra body size per sex. Figure S1. Detailed design of the three experiments conducted in this study. This material is available as part of online article from: http://www.reabic.net/aquaticinvasions/2021/Supplements/AI_2021_Parretti_etal_SupplementaryMaterial.pdf

Parretti et al. (2021), Aquatic Invasions 16(2): 186–207, https://doi.org/10.3391/ai.2021.16.2.01 207