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Specific damage recognised on land snail shells as a tool for studying predation intensity: differences related to habitat and predator types

In: Contributions to Zoology
Authors:
Tomáš Němec Department of Botany and Zoology, Masaryk University, Kotlářská 2, CZ-611 37 Brno, Czech Republic, neme.tomik@seznam.cz

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Michal Horsák Department of Botany and Zoology, Masaryk University, Kotlářská 2, CZ-611 37 Brno, Czech Republic, horsak@sci.muni.cz

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Shell formation is the main defensive strategy against predation for the majority of snails. Therefore, various predators have had to develop a variety of techniques how to overcome this barrier. As shells can persist in a calcium-rich environment for a long time, specific external or internal traces on shells left by predators indicate whether and who killed the snail. Based on litter samples collected at 30 sites of five different habitat types, the intensity and type of predation were assessed. The minimal predation rate varied between 0.0 and 21%, with an average of 8%. The highest rate was observed at limestone steppes, on average 15%. Beetles were found to be the most common predators of snails; however, predation by snails was more common in calcareous fens. Predation by some vertebrates and dipteran flies was also recognised. To test the role of mouth barriers as a means to reduce predation by carabid beetles that break the shell from an aperture, we analysed the predation rate separately on adult and juvenile shells using 24 populations of the steppe snail Granaria frumentum (Draparnaud, 1801). As expected, carabid beetles chiefly preferred juveniles compared to adult shells (Wilcoxon test, p < 0.001). On the contrary, the parasitoid fly Pherbellia limbata (Meigen, 1830) and Drilus beetles preferred adults. We found that predation by carabid beetles positively increased with prey abundance (R2 = 42.8%, p = 0.021), while no relation was observed for the parasitoid (p = 0.703), likely due to their feeding specialisation.

Introduction

Land snail communities of temperate and boreal zones are well known to be driven by several ecological gradients, mainly related to the calcium level (Wäreborn, 1970; Hylander et al., 2005; Juřičková et al., 2008), soil moisture and architecture (Wäreborn, 1992; Nekola, 2003; Hettenbergerová et al., 2013) and habitat history (Cameron et al., 1980; Kappes, 2006; Horsák et al., 2012). Additionally, the effect of macroclimate (Horsák et al., 2013a; Nicolai & Ansart, 2017; Horsák et al., 2018) and the existence of shelters to overwinter (Millar & Waite, 1999; Ansart et al., 2001; Horsák et al., 2015) have been found to be important. Although we know a good amount about the bottom-up limitation of snail communities, little is known about top-down mechanisms constituted by snail predators, pathogens, and other natural enemies (see Barker, 2004). A high importance of biotic interactions can be expected because land snails may provide suitable sources of water to many predators, especially in arid conditions (Yom-Tov, 1970; Shachak et al., 1981), as well as energy and nutrients, including calcium (Graveland & van Gijzen, 1994). Furthermore, snails can rasp parts of each other’s shells in calcium poor environments (Fournié & Chétail, 1984).

There is an extensive body of literature on natural enemies of land snails (see Barker, 2004 for the most recent review), among which several beetle families, particularly carabids, were identified as the most common predators of land snails (e.g., Konuma et al., 2011; Baalbergen et al., 2014). In the case of snail-killing carabid beetles, two types of feeding specialisation have evolved in two mutually exclusive directions, i.e., crawling into the shell (cychrisation) and breaking the shell apart (procerisation). Individual predators are morphologically adapted to one of these strategies (Thiele, 1977). Besides beetles, dipteran flies represent another known gastropod predators, often also as parasitoids. Species of the family Sciomyzidae are best known for their development associated with molluscs (Vala et al., 2000; Knutson & Vala, 2011). However, for most dipteran fly larvae it is difficult to determine their exact feeding relationship to molluscs, as they can fit to a saprophage, parasite, parasitoid or predator strategy. Importantly, some snails are also known to be carnivorous with clear preferences for other snails (Mordan, 1977; Griffiths et al., 1993; Barker & Efford, 2004; Myzyk, 2014). In addition, aggressive behaviour and cannibalism are well-known in predators among land snails (Baur, 1992). Several other invertebrate groups such as harvestmen (Nyffeler & Symondson, 2001) and terrestrial flatworms (Ogren & Sheldon, 1991; Sugiura et al., 2006), as well as many vertebrates such as small mammals (e.g., Rosin et al., 2011), birds (e.g., Shachak et al., 1981), and reptiles (e.g., Hoso et al., 2010) are among the common consumers of land snails. It is generally assumed that the number of malacophagous vertebrates is smaller in comparison with the number of vertebrates targeting other groups of animals (e.g., insects). Based on a large number of various malacophagous predators, it is clear that predation must have important regulatory effects on land snail communities, although there have been virtually no studies that have attempted to quantify this intensity using any method (but see Millar & Waite, 1999).

There are several commonly used approaches regarding how to detect predation such as analyses of (i) prey remains in a predator digestive system or its faeces (e.g., Griffiths et al., 1993; Hoso & Hori, 2006), (ii) stable isotopes of nitrogen and carbon to investigate dietary specialisation (e.g., Meyer & Yeung, 2011; Yanes et al., 2018), and (iii) DNA fragments in the predator intestine (Harper et al., 2005; Eskelson et al., 2011). However, using all these methods it is difficult to quantify the level of predation in natural communities. For land snails, there is a unique opportunity to retrospectively reconstruct a level of predation by the analysis of external and internal damage recognised on their empty shells. In a calcium rich environment, empty shells accumulating over several years (Říhová et al., 2018) provide an excellent record about the level of predation on land snail communities. As the types of shell damage made by various predators are well-described (e.g., Goodhart, 1958; Yom-Tov, 1970; Quensen & Woodruff, 1997; Chiba, 2007; Meyer & Shiels, 2009; Rosin et al., 2011; Baalbergen et al., 2014; Myzyk, 2014), we can easily and relatively accurately partition predation pressure among various groups of predators (see Millar & Waite, 2004). Although some predators do not damage shells, they do leave specific marks of their predation activity within the shell. These include for example larvae exuviae of predatory beetles of the genus Drilus (Baalbergen et al., 2014) and the puparia of parasitoid flies such as Pherbellia limbata (Nerudová-Horsáková et al., 2016). Additionally, the developmental stage of an individual victim can also be recognised based on the empty shell, which enables us to observe predator preferences for juveniles or adults of the prey species. Because some predators attack the snail through the shell mouth, leaving the victim’s shell undamaged, the predator pressure assessed by the above introduced approach needs to be viewed as minimal.

Despite a unique opportunity to assess predation pressure by using the analysis of specific damage in empty shells, this approach has been used very rarely. So far, there has been only a single study using damage recognised on empty shells and exploring predation made by various predators based on samples collected in a single forest site in England (Millar & Waite, 2004). To the best of our knowledge, there has been no study assessing predation intensity based on samples collected from a larger number of sites across a large area and comparing differences among various predators and habitat types.

Thus, the main goal of this study was to provide the first quantitative data on the intensity of predation on land snail communities sampled across many sites, stratified into five types of temperate habitat: talus forests, floodplain forests, wet meadows, calcareous fens, and limestone steppes. Secondly, we also aimed to investigate predation intensity in relationship to population densities and development stages of the steppe snail Granaria frumentum, a snail prey with a strongly armed aperture in the adult stage. We tested whether there were any differences in the minimal rate of predation and frequency of predator types among the studied habitat types. For populations of G. frumentum, we hypothesised a higher rate of predation by carabid beetles on juvenile stages in contrast to adult shells which are protected by a strong mouth incrustation. We also tested whether the predation rate is correlated to prey abundance for both true predators (i.e., carabid beetles) and parasitoid species (i.e., the fly P. limbata). We expected a positive association for true predators, but there was not a clear prediction made on a possible pattern for the parasitoid.

Materials and methods

Study sites and data collection

Samples were collected between 2015 and 2018. The study sites were selected based on an extensive previous survey of snail assemblages by the senior author in various habitat types across southern Moravia (Czechia) and Slovakia (fig. 1). To explore the predation rate and its relation to habitat types, six site replicates were sampled for each of the five habitat types studied (i.e., talus forest, floodplain forest, wet meadow, calcareous fen, and limestone steppe). At each site, an approx. 10-litre sample of leaf litter and the upper soil layer was collected from a representative plot of 20 × 20 m. Additionally, the other 18 limestone steppe sites within the same area were sampled specifically for the steppe snail Granaria frumentum populations (see below). Predation by carabid beetles was observed at each of them, but there was no predation by the parasitoid fly Pherbellia limbata observed at three sites, indicating the absence of this predator. Therefore, these were excluded from the analyses related to this predator. Each sample was sieved in the field and then placed into a plastic bag. Samples were left to dry out in the laboratory and then processed by a standard protocol (Ložek, 1956). Snail shells were extracted from the processed samples using a soft forceps, determined to the species level under a dissection microscope based on Horsák et al. (2013b), and counted. All shells were examined for any sign of damage left by predators. Undamaged shells were crushed manually to search for the presence of predator exuviae within the shell or dry tissue of the snail body. Examined shells were then categorised as: (i) live individuals at the time of collection, (ii) undamaged empty shells, and (iii) empty shells showing any evidence of predation. The shells of the former type were excluded from the analyses as these represented snail individuals unaffected by predation. We were able to unambiguously recognise several types of unique evidence of predation including both external shell damage left by a predator or remains of the predator (exuviae) left within the shell (fig. 2). These include: (1) characteristic damage by gradual spiral biting that begins at the shell mouth and continues towards the apex of the shell, most likely caused by carabid beetles (Millar & Waite, 2004); (2) the presence of Drilus beetle larval exuviae, which was found in the shell after breaking it manually (Schilthuizen et al., 1994; Baalbergen et al., 2016); (3) damage caused by gastropod predation represented by a specifically rasped hole in the shell wall with a typical gradual transition at the edges of the opening; (4) a large part of shell sharply chipped off, usually from a side or spire, most likely by a bird; (5) the presence of the puparium of a predatory dipteran taxa hidden inside the shell; and (6) holes in the shell wall caused by unidentified predators, presumably by small mammals, classified as ‘non-specific’.

Figure 1
Figure 1

Location of 30 study sites categorised into five habitat types.

Citation: Contributions to Zoology 88, 3 (2019) ; 10.1163/18759866-20191402

Figure 2
Figure 2

Examples of the recorded evidence of predation caused by various predators. a) shell of Cochlicopa lubrica predated on by a snail; b) characteristic damage caused by a carabid beetle, here on a shell of Discus rotundatus; c) puparium of the parasitoid dipteran fly Pherbellia limbata within a Granaria frumentum shell (the shell was crushed manually); d) external damage to a Xerolenta obvia shell caused by bird predation; e) Drilus beetle larval exuviae within an Alinda biplicata shell (the shell was crushed manually).

Citation: Contributions to Zoology 88, 3 (2019) ; 10.1163/18759866-20191402

Data analyses

We calculated the proportion of predated individuals for each site to analyse the relationship between predation rate and habitat types. It represented the number of all shells that clearly showed any of the predation types described above and divided by the all recorded empty shells in the sample. Differences in the predation rate among habitat types were tested using the F-statistic in the generalised linear regression model (GLM). Differences among individual predator intensities related to habitat types were tested using the Kruskal-Wallis test. The Pearson correlation coefficient was used to test whether predation by snails is correlated with the occurrence of specific predatory snail species. To visualise changes in predation type composition among habitat types, multidimensional scaling (MDS) on the Bray-Curtis dissimilarity matrix was performed to find the main directions of variability in predation types among the habitats.

Predator specific preferences for different G. frumentum life stages were tested using the pairwise Wilcoxon test. Because a clear preference for different life stages of G. frumentum was detected for individual predators, i.e., chiefly carabid beetles and the parasitoid fly P. limbata, we analysed the predation rate separately for non-adult and adult shells (i.e., with fully developed mouth barriers; fig. 3a). The relationship between the predation rate on G. frumentum and its population densities was modelled using a linear regression model (lm) with significance tested by the F-statistic. Counts were transformed using the natural logarithm (ln) to normalise their distribution. All analyses and graphics were performed in R program, version 3.4.4 (www.r-project.org).

Figure 3
Figure 3

Shells of the steppe snail Granaria frumentum, a species with a thickened mouth lip in adults: a) shell of an adult individual from the front and lateral view; b) non-adult shell damaged by a carabid beetle as a result of predation.

Citation: Contributions to Zoology 88, 3 (2019) ; 10.1163/18759866-20191402

Results

Predation in relation to various habitats

In total, there were 11,242 empty shells represented by 90 land snail species recorded at 30 study sites of five different habitat types. The number of empty shells per site varied from 84 to 1,877 (table 1). The highest numbers of empty shells were recorded in limestone steppes (688 shells on average) and the lowest in floodplain forests (201 shells on average). Out of all empty shells, 1,038 showed clear signs of external traces of predation or the predator’s remains left within the shells. The total numbers of shells collected for each habitat type, along with the numbers of shells showing particular predation types are given in table 1.

T000001

The minimal rate of predation per site ranged between 0 and 21% (8% on average). Using a generalised linear model, a significant difference in the minimal rate of predation was found among the habitat types (fig. 4). The highest values of minimal predation rate, significantly differing from all the other habitat types, were recorded at the steppe sites, showing on average 16% of shells with any traces of predation. Floodplain forest sites were, except for limestone steppe sites, also significantly different from calcareous fens, i.e., the habitat type showing the lowest minimal rate of predation. There were no differences between the predation rate detected in wet meadows, talus forests, and calcareous fens, which all showed the low rates of predation.

Figure 4
Figure 4

Variation in the minimal rate of predation recorded in six samples collected in each of five habitat types. Different letters refer to significant differences (p < 0.05) among habitat types, based on a generalised linear model. The central line of each box refers to the median value, box height to the interquartile range, whiskers to the non-outlier range (i.e., 1.5 times the interquartile range at each side), and small circles to outliers.

Citation: Contributions to Zoology 88, 3 (2019) ; 10.1163/18759866-20191402

Beetles were found to be the most common predators of terrestrial snails in our samples. Shells indicating predation by beetles in proportion to all collected shells ranged from 0 to 15% (5% on average), and this type of predator was recorded at all study sites (except in one calcareous fen). With the exception of calcareous fens, about 55% to 76% of all predation events were addressed exactly to carabid beetle predation (fig. 5). However, predation by carnivorous snails was more common in calcareous fens, occurring at five out of six studied sites and causing on average 65% of all predation events in the fens. Predation by some vertebrates and dipteran flies was observed at a low rate as well. Thereafter, we tested pairwise comparisons of the most common predators (carabid beetles and snails) among individual habitat types. Although, carabid beetle predation was clearly lowest at fen sites in contrast to the remaining habitat types, there were no significant differences found (Kruskal-Wallis test, p > 0.06). In contrast, predation intensity by snails was the highest in calcareous fens and significantly differed from all the other habitat types (Kruskal-Wallis test, p < 0.05).

Figure 5
Figure 5

Relative representation of individual predation types recognised in six samples collected at each of five habitat types. The height of the column represents the average value, while the lines indicate the standard deviation.

Citation: Contributions to Zoology 88, 3 (2019) ; 10.1163/18759866-20191402

By using proportional representation of individual predation types as variables in the multivariate analysis, the main change in the predation scheme was significantly shaped by differences in carabid beetle and snail predation rates (p < 0.001, in 1999 permutations). Furthermore, differences in the dipteran fly predation rate significantly loaded onto the second ordination axis (p = 0.019). In the ordination diagram, a cluster of calcareous fens was found to express a unique nature, mainly due to the low rate of predation by carabid beetles (fig. 6). Likewise, limestone steppes did not overlap with the other sites, as they were characterised by a high rate of carabid predation and a null rate of snail predation. In contrast, talus forests and wet meadows almost entirely overlapped, and they also partially overlapped with floodplain forest sites.

Figure 6
Figure 6

Non-metric multidimensional scaling (NMDS) of predation type composition at five study habitat types based on the Bray-Curtis distance. Parameters with a significant relationship to the first two ordination axes are shown in the bottom left corner.

Citation: Contributions to Zoology 88, 3 (2019) ; 10.1163/18759866-20191402

Predation in relation to Granaria frumentum life stages and population densities

At 24 limestone steppe sites, 6,466 juvenile and 3,178 adult empty shells were collected, among which 1,571 shells showed any kinds of predation evidence (table 2). Predation on juveniles occurred in 918 cases caused by four different types of predators, and in 653 cases on adults, represented by three different predator types.

T000002