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PRIMARY RESEARCH PAPER

Local consumers are the first line to control biological invasions: a case of study with the haemastoma (: )

A. Giacoletti . A. Rinaldi . M. Mercurio . S. Mirto . G. Sara`

Received: 30 October 2015 / Revised: 8 January 2016 / Accepted: 9 January 2016 Ó Springer International Publishing Switzerland 2016

Abstract The increasing spread of invasive higher handling time. Stramonita showed a greater in the determines several alter- preference for Brachidontes, that resulted as the prey ations in local food webs, changing the feeding habits with the higher energetic content, and the second most of native organisms. The whelk Stramonita haemas- profitable after Patella. This suggests that the higher toma is a widespread Mediterranean gastropod that energy gain is behind the change in the predator’s diet, consumes bivalves, and limpets. Previous with possible effects on its energy budget. studies showed a shift in its diet from the bivalve minimus to the invasive Brachidon- Keywords Invasive species Á Functional response Á tes pharaonis, presumably due to a higher energy gain. Gastropod Á Á Á Here we tested ’ preference among natives and a novel prey, calculating the profitability ratio, and integrating those results with biochemical analysis on prey tissues and the routine metabolism of the whelks. Further, we used the scaled functional response as a Introduction theoretical tool to describe whelk ability to obtain energy from their environment by using four different There are only so many ways in which local commu- prey species: B. pharaonis, Mytilus galloprovincialis, nities can control biological invasions. Autochthonous- M. minimus and Patella caerulea. Whelks evidenced a predators (sensu Schoener, 1986) are usually the first in Type II functional response for all prey, while line to influence the likelihood of invasion (Ricciardi Brachidontes displayed a lower attack rate and a et al., 2013). Invaders—often r-strategists—deploy their pronounced ability to compete for space—by exploiting local resources and promptly addressing Handling editor: Vasilis Valavanis local establishments. The importance of both predators and top-down control in structuring ecological com- & A. Giacoletti Á A. Rinaldi Á M. Mercurio Á G. Sara` ( ) munities has to date been widely discussed by ecolo- Dipartimento di Scienze della Terra e del Mare, University of Palermo - Local UO CoNISMa, Viale delle gists, and the loss of predators may provide an Scienze Ed. 16, 90128 Palermo, Italy important example of how they shape marine environ- e-mail: [email protected] ments (Terborgh, 2010). In particular, the common Indo-Pacific mussel Brachidontes pharaonis (Fischer A. Rinaldi Á S. Mirto IAMC-CNR, via Giovanni da Verrazzano 17, P., 1870), which entered the Mediterranean Sea after 90194 Castellammare del Golfo, Trapani, Italy the opening of the Suez Canal (1869), is slowly 123 Hydrobiologia colonising near all rocky upper subtidal and lower current literature, functional response experiments in intertidal substrates (Safriel & Sasson-Frosting, 1988) the natural field are quite rare. We thus decided to focus in the south-western Basin (Sara` et al., 2013), thereby on laboratory feeding trials at different prey densities. outcompeting the native bivalve Our specific aims were to investigate the prey choice (Poli, 1795) (Safriel & Sasson-Frosting, 1988). In sea process by studying prey profitability and assimilation locations, however, it seems that the initial Brachidon- efficiency, and comparing experimental results to their tes colonisation process is constrained by predators, respective energetic content derived from biochemical such as Muricid gastropods (Rilov et al., 2002), which analyses. We further estimated the routine metabolism are able to regulate the population dynamics of their of S. haemastoma by measuring the oxygen consump- prey (Safriel et al., 1980). In the Mediterranean Sea, the tion rate. At the same time, we derived the functional whelk Stramonita haemastoma (Linnaeus, 1767; Gas- response of S. haemastoma over B. pharaonis,com- tropoda: Muricidae) is a widely distributed intertidal paring it with some of their most common indigenous top consumer that adopts opportunistic strategies to prey, the model species used many times in companion consume a large number of species (Basedow, 1994). studies: mussels Mytilus galloprovincialis (Lamark, Before dense beds of the invading mussel B. pharaonis 1819) (Sara` et al., 2011; Montalto et al., 2014)andM. were established along the Israeli Mediterranean coast, minimus (Sara` & De Pirro, 2011), and the limpet indigenous barnacles, small native mussels, Vermeti- Patella caerulea (Linnaeus, 1758) (Prusina et al., 2014) dae gastropods and limpets were the main potential in order to analyse predator–prey trophic interactions in food items for S. haemastoma (Rilov et al., 2001). greater detail. We examined whether (i) whelks exhib- Previous laboratory experiments have shown that B. ited a Type II or Type III functional response; (ii) pharaonis was the food item preferred by S. haemas- functional responses differed between natives and a toma over the indigenous mussels and barnacles (Rilov, novel prey and (iii) the new prey determines changes in 1999), and that in sites where it was abundant (densities the whelk’s ecology by altering functional response [25 % of the available prey), it constituted the whelks’ parameters such as the attack rate (a) and prey handling main food, except when energetic limitations ‘‘forced’’ time (h), affecting the duration and the cost of the the predator to prey on the more abundant, but predatory event, with a probable different energy intake presumably less profitable prey (Rilov et al., 2002). from the new prey. Trophic interactions are generally well described by functional response models quantifying consumer per capita consumption rate depending on local prey Materials and methods abundance (Holling, 1959). Several authors have measured the number of prey eaten by single predators Sampling and experimental design in small ‘‘arenas’’ (Hassel, 1978; Lang et al., 2012; Toscano et al., 2014), in order to eliminate the Specimens of S. haemastoma were collected alive at possibility of seeing anything other than prey depen- low tide during February 2014 from the intertidal dence (Abrams & Ginzburg, 2000). Using the func- shores near San Vito Lo Capo and the natural reserve tional response can explain and predict the impact of of Monte Cofano (Castelluzzo, Trapani). Whelks were predators on prey populations (Juliano, 2001;Dick brought back to the laboratory and placed in a 60-l et al., 2013). In particular, Twardochleb et al. (2012) aquarium at room temperature (18–20°C) and seawa- showed that the functional response might predict, ter salinity (37–38%) and they were allowed to along with simple population growth models, whether a acclimate for 1 week to reduce stress generated by predator will provide biotic resistance against non- manipulation and transport (Garton & Stickle, 1980). native preys at different prey densities. This prediction Specimens were then gradually transferred from room is made by determining the functional response shape temperature, at daily increase of *1to24°C, the and parameters (a, h). Predator functional responses optimal experimental temperature to elicit maximum interact with prey birth rate and abundance, and the feeding rates in the lab (Rilov, 1999; Brown & Stickle, magnitude of this interaction is reflected in the shape of 2002). Having reached this experimental temperature, the curve (Type II or III) and the intensity of the attack whelks were constantly acclimated there for 10 days, rate (a) (Twardochleb et al., 2012). According to the with no feeding (Garton & Stickle, 1980) prior to the 123 Hydrobiologia start of the experiment. We thereby standardised the Profitability of prey hunger level (Garton & Stickle, 1980). Aquaria used in the present study comprised n = 10-1l independent We also compared the biomass of the different prey by plastic compartments (15 9 8 cm base, 10 cm calculating their individual dry weights (oven 95°C for height), each containing a single whelk. We decided 24 h) and the ash content of their flesh (muffle furnace to use individual arenas so that feeding would not be at 450°C for 4 h) to estimate the organic matter as affected by scents from surrounding treatments, as Ash-Free Dry Weight (AFDW) to the nearest 0.001 g. gastropods use chemosensory cues to find food (Smith, Then, to assess the profitability of different prey, we 1983). Each aquarium was aerated, and kept under divided per capita dry tissue by handling times for constant light, while seawater was changed after each each prey species, according to Brown & Richardson trial. All the experiments were repeated twice for each (1987). treatment. Biochemical analysis of prey Prey selection A sample of n = 10 specimens for each prey (B. While previous studies have determined the prefer- pharaonis, M. minimus, P. caerulea) was analysed to ence of the Lessepsian species B. pharaonis over the determine the protein, carbohydrate and lipid content indigenous mussel M. minimus, the Balanus (Modica et al., 2006). The values for M. galloprovin- perforatus, and the limpet P. caerulea (Rilov et al., cialis were derived from the literature (Okumus et al., 2002), we conducted two further laboratory experi- 1998; Orban et al., 2002). Protein (PRT) determination ments, both aiming for greater detail of the S. was carried out according to Hartree (1972) and haemastoma prey preferences. In particular, we com- modified by Rice (1982), using bovine serum albumin pared the preference of S. haemastoma feeding on B. (BSA) as a standard. Carbohydrates (CHO) were pharaonis vs. the mussel M. galloprovincialis and determined according to Dubois et al. (1956) using repeated trials with the limpet P. caerulea, following D(?)glucose as a standard. Lipids (LIP) were the experimental design proposed by Underwood & extracted using the method of Bligh & Dyer (1959) Clarke (2005). Each experiment was replicated twice and measured according to Marsh & Wenstein (1966). for each treatment, with prey of standard shell length Tripalmitine was used as a standard. The caloric chosen as these corresponded to the most common content of mussel flesh was calculated using the prey upon which the whelks were observed feeding in equation: the field (author’s pers. obs.): B. pharaonis (25–35 mm), Kcal gÀ1 ¼ 0:041%CHO þ 0:055%PRT M. galloprovincialis (25–35 mm), and P. caerulea þ 0:095%LIP (20–25 mm) were offered to medium size (40–45 mm) whelks. To reduce the likelihood that where CHO, PRT, and LIP are, respectively, the further encounters with the same type of prey may percentage content of carbohydrate, protein, and lipid increase the predator experience by artificially affect- (Winberg, 1971). Caloric values expressed in Kcal g-1 ing the handling efficiency (Rovero et al., 1999), these were finally converted into J g-1 and related to the authors suggested simultaneously offering two differ- average tissue of each prey species. ent prey species (B. pharaonis and M. galloprovin- cialis, B. pharaonis and P. caerulea) to each Assimilation efficiency individual whelk. The trial ended as soon as whelks had made their choice of prey. Attacks were consid- As part of the manipulation, we also obtained a ered to start when we inserted prey in the arena and qualitative estimate of the whelk’s assimilation effi- ended when a feeding S. haemastoma crawled away ciency (AE). For every prey, AE was measured by the from its first prey. To meet the independence criterion ratio method of Conover (1966): (Underwood, 1997), each whelk was used in only one AE ¼ ðÞF À E =½ðÞ1 À E FŠ experimental trial and then killed by gentle freezing.

123 Hydrobiologia where F is the ratio between ash-free dry weight À1 RR ¼ ðÞÂCt0 À Ct1 Volr  60ðÞt1 À t0 (AFDW) and dry weight (DW) for food, and E is the same ratio for the faeces; this represents the efficiency where Ct0 is oxygen concentration at the beginning of with which organic material is absorbed from the the measurement, Ct1 is the oxygen concentration at ingested food material. Here, 15 specimens of S. the end of the measurement, and Volr is the volume of haemastoma feeding on each prey, acclimated as water in the respirometric chamber. After the mea- above, were used to estimate whelk AE in a simple surement, each was killed by gentle freezing feeding trial, where one prey was offered to a single and dissected, and shell was separated from body predator. According to experiments conducted on tissue, in order to calculate their individual dry other whelks, such as Rapana venosa (Valenciennes, weights and standardise respiration rates to body 1846), the emptying of 1 g fresh mussel started soon weights. Respiration rates were finally converted into after food was digested and lasted for about 8 h an energetic cost of routine activities, expressed in -1 -1 (Seyhan et al., 2003). In this study, we were not Jg day for a cost/benefit analysis. interested in estimating the evacuation time for S. haemastoma, so the whelks were given 24 h after the time predation event for the complete collection of faeces. Once collected by pipettes, faeces were washed with Predatory activity is usually described by two mutually 0.5 M ammonium formate (purest grade) to remove exclusive phases comprising the functional response of adventitious salts (Widdows & Staff, 2006), placed on Holling (1959): searching time and food handling. small aluminium disposable dishes, dried in the oven These components of feeding behaviour can be (95°C for 24 h), and then incinerated in a muffle integrated in a cost-benefit analysis, to determine the furnace (450°C for 4 h). After each step, the samples optimum maximising the net rate of energy intake were weighted using a precision balance (Sartorius BL (Pyke & Charnov, 1977). Four experiments were 120S ± 1 lg). For the calculation of AE, together conducted in order to investigate and measure the with the faeces collected from the whelks, 10 prey of foraging behaviour of S. haemastoma towards four each type were weighted, after removal of tissues, then different prey species: B. pharaonis (25–35 mm), M. dried and incinerated as above. minimus (*10 mm), M. galloprovincialis (20–30 mm) and P. caerulea (*20 mm). In each session, one Respiration rate specimen was offered to a single whelk acclimated as above, and the trial was repeated for each prey species, Rates of oxygen consumption by single whelks were in order to compare the predator’s responses. Whelk measured in a respirometric glass chamber (0.3 l) in a behaviour was recorded with a USB web camera placed temperature-controlled water bath. Air-saturated fil- in front of the experimental aquarium, connected to a tered seawater was added to each respirometric cham- laptop computer running video capture software (Debut ber and stirred with a magnetic stirrer bar beneath a video capture software, http://www.nchsoftware.com/ perforated glass plate (e.g. small Petri dish with holes) capture/). Searching and handling times were then supporting each individual (Widdows & Staff, 2006). recorded to the nearest second from the acquired videos The decline in oxygen concentration was measured by a through a digital chronometer. The response period calibrated oxygen fibreglass sensor connected to a data began when the siphon protruded through the siphonal canal, the foot was extended, and the shell lifted to logger (PyroScience Firesting O2), capable of four sensor connections. We used a total of n = 10 move towards the prey. Searching time ended once the for this experiment, acclimated as above, and then fed whelk was on top of its prey; at this point, the handling ad libitum for one week until the day before the time began. This then ended when the whelk crawled experiment. The decline was continuously recorded for away from the prey at the end of the predatory event. As at least 1 h, excluding the initial period (*10 min) we were only interested in studying the handling time of when there is a more rapid decline in oxygen caused by prey, searching times were analysed only to verify the a disturbance of the sensor’s temperature equilibration. efficiency of the arena in standardising the searching -1 component, as long as it requires a different study Respiration rate (RR, lmol O2 h ) was calculated according to Widdows & Staff (2006): approach and probably field observations. During 123 Hydrobiologia experimental trials, in order to simulate a natural where Ne is the number of prey eaten, N0 is the initial predatory event, the prey was introduced first, and left prey density, a and h are the attack rate and handling acclimating for *1 h, before introducing the predator time, respectively, and T is the total time available. and starting the recording session. Coefficients a, h used in (1) were derived from individual predation experiments for each prey (de- Functional response scribed in the Predation time section); functional response models were fitted using the maximum- Four experiments were conducted in order to measure likelihood estimation (bbmle:: mle2, version 1.0.52, the functional response of S. haemastoma on different Bolker and R Development Core Team, 2014). prey species. Whelks involved in these experiments were divided into four groups, and each group was Statistical analysis tested for a different kind of prey: the mussels B. pharaonis, M. galloprovincialis and M. minimus, and In order to test for significant differences in handling the limpet P. caerulea. Each experiment was repli- times between prey species and AE, a Permutational cated twice: an increasing number of B. pharaonis Univariate Analysis of Variance (PERMANOVA) (25–35 mm; n = from 1 to 6) or M. galloprovincialis was used due to different sample sizes. Analyses were (25–35 mm; n = from 1 to 7), or M. minimus performed considering the prey species as a fixed (*10 mm; n = from 1 to 11), or P. caerulea (20– factor (4 levels), and variables were ln (y ? 1) 25 mm; n = from 1 to 6) were offered to 40–45 mm transformed to retain information on relative concen- predators, and the consumed items were counted after trations but reduce differences in scale among the a period of 24 h, without replenishing them. Data from variables. The Euclidean similarity measure was used, these experiments were useful to investigate if whelks and all P-values were calculated using 9999 permu- displayed Type II or Type III functional response, and tations of the residuals under a reduced model to do this a phenomenological analysis was used (Anderson, 2001). Data from prey choice experiments according to Juliano’s (2001) and Paterson’s (2015) were analysed using t-tests. ANOVA was performed methods. All statistical analyses were performed using on biochemical data, considering the caloric content as R v. 3.1.3 (R Core Team, 2015). For each prey, logistic a fixed factor (4 levels). When significant differences regressions of proportion of amount killed against were detected, the Student–Newman–Keuls (SNK) density were performed using a function (frair:: post hoc pair wise comparison of means was used frair_test) provided in an integrated package for (Underwood, 1997). Cochran’s test was used prior to functional response analysis in R (frair, Pritchard, ANOVA to test the assumption of homogeneity of 2014). Type II functional responses were indicated by variance (Underwood, 1997). a significant negative first-order term, whereas Type III responses were indicated by a significant positive Results first-order term followed by significant negative second-order term (Juliano, 2001; Pritchard, 2014). Prey selection The dynamics of predator–prey systems are described by several equations assuming either a constant prey Stramonita haemastoma showed a larger significant density (i.e. a ‘‘replacement’’ experimental design) or preference (4 times more) for the invasive mussel B. a diminishing one (i.e. a ‘‘non-replacement’’ experi- pharaonis with respect to the blue mussel of similar mental design). While Hollings original Type II size (t test, P \ 0.05), while no significant preference functional responses assume a constant prey density, was exhibited by whelks between B. pharaonis and in the case of a reduction in prey density due to limpets (t test, P [ 0.05; Fig. 1). consumption, Roger’s (1972) ‘‘random predator’’ equation is more appropriate (Pritchard, 2014). This Profitability of prey equation accounts for prey depletion and their non- replacement over time: The Brachidontes pharaonis dry weight (0.15 ± Ne ¼ N0ðÞð1 À expðÞaNðÞeh À T 1Þ 0.01 g) was similar to that of P. caerulea (0.13 ±

123 Hydrobiologia

100 compared to native prey, that resulted, respectively, 90 in: 580 ± 28 J/ind. for M. minimus (*10 mm), and 80 12,870 ± 2,188 J/ind. for P. caerulea (20–25 mm), 70 while the energetic content of M. galloprovincialis 60 derived from the literature was equal to 10,052 ± 50 669 J/ind. (ANOVA P \ 0.001; Table 1; Fig. 2d). 40

% Prey choice 30 Assimilation efficiency 20 10 AE showed no differences between B. pharaonis 0 (0.70 ± 0.02), M. galloprovincialis (0.66 ± 0.01) and B. pharaonis P. caerulea B. pharaonis M. galloprovincialis P. caerulea (0.77 ± 0.03) (PERMANOVA P [ 0.05; Prey species Table 3; Fig. 2c), while M. minimus (0.57 ± 0.04) Fig. 1 Results of prey choice experiment on different prey resulted in a significantly lower AE (P \ 0.05). P. items in laboratory caerulea further resulted in an higher AE compared to M. galloprovincialis (P \ 0.001) and M. minimus 0.02 g) and they both resulted higher than M. gallo- (P \ 0.01), while the two native mytilid species provincialis (0.05 ± 0.01 g) and M. minimus showed a non-significantly different AE (P [ 0.05). (0.0049 ± 0.0005 g) (ANOVA P \ 0.001; Table 1). When dry masses of prey were divided by handling Respiration rate times, S. haemastoma showed greater profitability ratios for Patella (PERMANOVA P \ 0.01; Table 2; The average energetic cost of routine metabolism Fig. 2a) then in order for Brachidontes (P \ 0.001), derived from the measurement of oxygen consumption Mytilus (P \ 0.01) and Mytilaster (P \ 0.001). resulted equal to 194.29 ± 27 J g-1 day-1.

Biochemical analysis Predation time

Brachidontes pharaonis resulted in a significantly Searching times within experimental arenas resulted higher energetic content (20,983 ± 1,375 J/ind.) not different among different prey (PERMANOVA

Table 1 ANOVA table of results (dry weights and energetic content) of different prey species consumed by S. haemastoma (* p \ 0.05; ** p \ 0.01; *** p \ 0.001; ns not significant) Dry weight Energetic content Source df MS F P Source df MS F P

DW 3 0.049 30.79 *** Joule 3 26.131 861.38 *** Res 36 0.0016 Res 36 0.0303

Table 2 PERMANOVA table of results (profitability) and group analysis of different prey species consumed by S. haemastoma (* p \ 0.05; ** p \ 0.01; *** p \ 0.001; ns not significant) Source df MS Pseudo-F P(perm) Groups (Ht) t P(perm) Unique perms

Sp 3 0.000000607 23.881 *** Bp, Pc 3.408 ** 2867 Res 21 0.000000025 Bp, Mg 3.72 ** 2887 Bp, Mm 9.765 *** 2884 Pc, Mg 3.785 * 126 Pc, Mm 5.697 ** 126 Mg, Mm 7.238 ** 126 Bp = B. pharaonis,Pc=P. caerulea,Mg=M. galloprovincialis,Mm=M. minimus 123 Hydrobiologia

360 0.0012 SNK: Patella > Brachidontes > Mytilus > Mytilaster a b 340 320 0.0010 300 280 260 0.0008 240 220 200 0.0006 180 160 140 0.0004 120 100 80

0.0002 Handling time (min. ± s.e.) 60 40 20 Profitability Index (DW / HT of prey) 0.0000 0 P. caerulea B. pharaonis M. galloprovincialis M. minimus B. pharaonis M. galloprovincialis M. minimus P. caerulea 1.0 25500 c 24000 SNK: Brachidontes > Patella > Mytilus > Mytilaster d 0.9 22500 21000 0.8 19500 0.7 18000 16500 0.6 15000 13500 0.5 12000 0.4 10500 9000 0.3 7500 6000 0.2 4500 0.1 3000 Energetic content (J/prey ± s.e.) 1500 0.0 0

Assimilation efficiency (AE, adimensional) P. caerulea B. pharaonis M. galloprovincialis M. minimus B. pharaonis P. caerulea M. galloprovincialis M. minimus Prey species

Fig. 2 a Profitability Index of prey (dry tissue consumed per (AE) calculated for the different preys; d energetic content (J/ prey/handling time); b handling times recorded from video prey) calculated from LPG analysis of tissues from the different analysis for different prey species; c assimilation efficiency species

Table 3 PERMANOVA table of results and group analysis for assimilation efficiency (AE) of different prey species consumed by S. haemastoma (* p \ 0.05; ** p \ 0.01; *** p \ 0.001; ns not significant) Source df MS Pseudo-F P(perm) Groups T P(perm) Unique perms

Sp (species) 3 5.8615E-2 7.014 *** Bp, Pc 1.708 ns 9843 Res 59 8.3567E-3 Bp, Mg 1.787 ns 9824 Bp, Mm 2.676 * 4181 Pc, Mg 3.667 *** 9818 Pc, Mm 3.187 ** Mg, Mm 1.976 ns Bp = B. pharaonis,Pc=P. caerulea,Mg=M. galloprovincialis,Mm=M. minimus P [ 0.05) (32.1 ± 9.9 min. for B. pharaonis, 14.0 ± (301.2 ± 35.8 min. for B. pharaonis, 151.5 ± 11.1 6.4 min. for M. galloprovincialis,61.8± 14.8 for M. min. for M. galloprovincialis, 134.2 ± 40 for M. minimus and 44.4 ± 21.7 min. for P. caerulea). By minimus, and 131 ± 32.24 min. for P. caerulea). In contrast, the main component of feeding measured in particular, handling times with B. pharaonis were this study, handling time, showed significant differ- greater than those exhibited by whelks feeding on the ences (PERMANOVA P \ 0.001; Table 4; Fig. 2b) other prey species. Whelks did not show significantly

123 Hydrobiologia different handling times among native prey species tissue content, the highest energy gain from consump- (P [ 0.05). tion and generated the best assimilation efficiency in whelks, thereby justifying the switch to the alien Functional response species as showed by Rilov et al. (2002). This change in preference may also be part of some ecological Phenomenological analysis showed that S. haemas- processes controlling the abundance and biogeograph- toma met the Type II functional response for each prey ical range size of an invasive species, and even its species, as revealed by the significant negative first- impacts within a community (Maron & Marler, 2008; order term (Table 5; Fig. 3a–d). Brachidontes resulted Ricciardi et al., 2013). From surveys conducted during in a significant lower attack rate (0.103 h; Table 5) the same period along the intertidal zone of Castel- respect to M. minimus (0.267 h), while it resulted in luzzo (Trapani), not affected by noticeable anthro- the higher handling time (5.8 h) followed by M. pogenic activities as trampling (Milazzo et al., 2004) galloprovincialis (5.696 h), P. caerulea (4.438 h) and or whelk harvesting, and Capo Gallo - Isola delle M. minimus (2.799 h). Rogers’ analysis further con- Femmine, where trampling and harvesting are usually firmed that the whelks spent significantly more time much more intense due to the proximity to the city of handling B. pharaonis than the other native prey Palermo, S. haemastoma densities, recorded along (Table 6). 100 9 1 m transects, resulted *0.56 ind./m2 and *0.03 ind./m2, respectively (A. Giacoletti, pers. obs.). The presence of P. caerulea was recorded in both sites, Discussion while B. pharaonis, occasional and rare in Castel- luzzo, reached more than 9000 individuals ± 4033 Foraging for food in the rocky intertidal zone involves ind. per m2 in the second site (Isola). M. minimus a number of potential dangers, such as exposure to reached densities of *19,753.3 ± 9,445 ind./m2 (G. predators and the increased likelihood of being Sara`, unpublished data) in Castelluzzo, while very low dislodged or affected by thermal stress (Burrows & Hughes, 1989; Denny et al., 2009). Longer handling Table 5 Phenomenological analysis of the functional times imply greater risks to the predator that must be responses for each prey species (* p \ 0.05; ** p \ 0.01; compensated at least by a higher energy gain, in order *** p \ 0.001; ns not significant) to justify the choice and fulfil energetic requirements Prey species Type II for routine metabolism, as measured in the present study. Before the dense beds of the invading mussel B. N0 P pharaonis were established along the Mediterranean B. pharaonis -0.2937 * coast, the indigenous mussels, limpets and barnacles M. galloprovincialis -0.4788 *** constituted the main food items of S. haemastoma. M. minimus -0.4036 *** Here, according to our results, the invasive species P. caerulea -1.1440 * showed, in the following order, the highest edible

Table 4 PERMANOVA table of results (handling time) and group analysis of different prey species consumed by S. haemastoma (* p \ 0.05; ** p \ 0.01; *** p \ 0.001; ns not significant) Source df MS Pseudo-F P(perm) Groups (Ht) t P(perm) Unique perms

Sp 3 1.48 6.7366 ** Bp, Mg 3.532 ** 2887 Res 22 0.21 Bp, Pc 3.763 ** 5623 Bp, Mm 3.505 ** 2890 Mg, Pc 1.001 ns 461 Mg, Mm 0.928 ns 462 Pc, Mm 0.019 ns 461 Bp = B. pharaonis,Pc=P. caerulea,Mg=M. galloprovincialis,Mm=M. minimus. Ht handling time 123 Hydrobiologia

6 a 6 b

5 5 eaten (±SE) 4 4

3 3

2 2 B. pharaonis eaten (±SE)

1 M. galloprovincialis 1 Mean

0 Mean 0 01234567 012345678

8 c 6 d 7 5 6 4 5

4 3

3 2 P. caerulea eaten (±SE) M. minimus eaten (±SE) 2 1

1 Mean Mean

0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 Prey density, n

Fig. 3 a–d Functional response: relationship between prey eaten (items consumed in 24 h) and prey density

Table 6 Phenomenological analysis of the functional responses: attack and handling parameters estimate for each species (* p \ 0.05; ** p \ 0.01; *** p \ 0.001; ns not significant) Prey Parameter Estimate SE z value P (z)

B. pharaonis a 0.103 0.051 2.008 * h 5.807 2.074 2.800 ** M. minimus a 0.267 0.064 4.179 *** h 2.799 0.277 10.096 *** M. galloprovincialis a 0.322 0.191 1.687 ns h 5.696 0.945 6.027 *** P. caerulea a 0.504 0.315 1.602 ns h 4.438 0.758 5.854 *** densities were evident in site 2 (Isola), probably due to pharaonis between the two sites. Under natural the competition with the invasive mussel. The lack of conditions, whelks tend to select prey of optimal size, an important predator may be one of the factors, such as those chosen for this study, thereby exhibiting together with other abiotic characteristics of the site an active choice of an easily accessible or energeti- not considered in this study, that explain this remark- cally rich species of prey over others (Garton, 1986; able difference in density and abundance of B. Brown & Richardson, 1987; Brown, 1997), gaining 123 Hydrobiologia the most energy with the least handling effort. Our effect of a predator on prey at low prey density. The profitability data confirmed this statement, with our pattern of food consumption in relation to food density whelks actively selecting prey characterised by the in our whelks met the Type II Holling functional higher tissue content such as P. caerulea and B. response for both invasive and indigenous prey. While pharaonis.Palmer(1983, 1984) demonstrated that prey in the Type II function, as in the Type III, the number selection in three thaidid species could be explained by of killed prey approaches the asymptote following a the food value of prey provided in an enclosed arena. sigmoid function, the first part of the Type II curve is Food value integrates several complex factors deter- exponential. Such a fact is crucial as it describes a mining prey choice: energy content, abundance and consumption rate that increases exponentially for low vulnerability (handling time) of a prey species. Accord- densities of prey even though it gradually slows down ing to this theory, and as demonstrated by our results, to an asymptote. This type of function is usually the change in feeding preferences is driven by the defined as destabilising at low densities (Dick et al., higher profitability (energy gain relative to energy 2013). The main consequence is that handling time expenditure, Garton, 1986) of the invasive mussel can be the limiting factor in the feeding process. compared to the native prey, and suggests the whelk’s Ecological repercussions will occur if local recipient ability to assess the prey’s value, regardless of their communities have predators displaying Type II feeding history, as hypothesised by Rilov et al. (2002). Holling functional response at their top, but the Accordingly, the present S. haemastoma significantly colonisation of new invaders can result in different preferred the invasive species to their regular food item, fates if they have Type II or Type III predators as, in such as barnacles and mussels (Rilov, 1999)withthe the second case, they are not able to exclude invasive exception of, in our study, the limpet P. caerulea. This species when they are at low density. This is an suggests that the prey’s energetic content was important important ecological aspect that has only recently been in the choice of species singularly because whelks, with considered in the biological invasion literature (Dick the invasive species, could attain foraging optimisation et al., 2013, 2014; Dodd et al., 2014; Paterson et al., (Krebs & Davies, 1993). The ability of S. haemastoma 2015). Indeed, according to a common scheme (Drake to assess food value has also been shown by other & Lodge, 2006), an invasive species reaching new authors (Richardson & Brown, 1990;Brown,1997), but recipient sites with habitat-suitable conditions for its the current study has finally demonstrated that the establishment (Peterson & Vieglas, 2001; Sutherst, content of edible tissue (biomass) plays a more 2003) and with a sufficient propagule pressure (Drake important role than handling time in determining & Lodge, 2006), starts to increase the population profitability. Our study supported this hypothesis, i.e. density, intercepting local resources, as shown by that whelks actively preferred mussels with a greater Leung et al. (2004) and Mack et al. (2000). In most amount of edible tissue like B. pharaonis to M. invaders, the first period of colonisation is delicate: the galloprovincialis and M. minimus, despite an appar- initial small nucleus of an invasive species should ently longer handling time being necessary. Our trials, contrast with the suppressing effects of environmental however, revealed no such preference between B. stochasticity and with the biotic resistance exerted, for pharaonis and the limpet P. caerulea,incontrastto instance, by the local presence of predators (e.g. all what has been previously demonstrated (Rilov et al., components of the Allee effect). Thus, if local 2002), probably because of the similar edible biomass predators possess Type II or Type III functional of the two species, that led to an equal choice. Rilov response characteristics, this becomes crucial in et al. (2002) recorded handling times between 3 and addressing the new establishment of the introduced 16 h on the same type and size of prey (B. pharaonis species (sensu Drake & Lodge, 2006). From recent 25–35 mm), while handling times from our experi- surveys conducted at site 1 (Castelluzzo), whelks seem mental trials ranged between 2 and 9 h. Such handling able to control densities of possible invaders such as B. times varied widely for different kinds of prey: 2–3 h pharaonis, and may have sufficient plasticity to for M. galloprovincialis, and 1–4 h for M. minimus and modify feeding patterns in order to accommodate this P. caerulea (Fig. 2b). new species in their diet. Predation rates derived from One of the most important ecological extrapola- functional response modelling seem somewhat tions from our results on functional response is the reduced on Brachidontes (lower attack rates and 123 Hydrobiologia higher handling times) when compared to native prey, functional response may be considered a simple, cost- but this may be attributed to either longer manipula- effective and accurate tool to predict ecological tion time or satiety, eventually leading to an higher impacts of invasive species on aquatic systems. energy gain. There is currently limited knowledge about the absorption efficiency of marine predatory Acknowledgments PRIN TETRIS 2010 Grant (n. gastropods, so our results can only be compared to a 2010PBMAXP_003) funded to Gianluca Sara` by the Italian Minister of Research and University (MIUR) supported this few others. These do report similar absorption effi- research. We thank Dr. Francesca Ape for her help in the ciency for predatory gastropods of the same class-size biochemical analysis of prey and Anna Lossmann for the (e.g. Huebner & Edwards, 1981). Other comparisons English fine tuning. The authors declare no competing financial have also been made via experiments performed on interests. scavenger or herbivorous gastropods (Dozey-Stenton Author contributions: AG and GS conceived the idea and led et al., 1995; Huebner & Edwards, 1981), producing the writing, AG and MM carried out all experiments in meso- results that do not differ greatly from our experiments. cosms, AG & AR performed modelling work and analysed Significant differences in the absorption efficiency output data, SM provided grant funds for AG while GS provided lab facilities at DISTEM and research funds. (AE) resulted between B. pharaonis and the native M. minimus, while such differences were not detected between Brachidontes and Patella or Brachidontes and Mytilus. 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