INTRODUCTION
Predation on parasites is a mechanism by which community composition can regulate the impact of parasitism. This may have important implications for diseases in general, including those of public health and commercial importance. Predation on parasites may occur in different ways. Here we address predation on parasite transmission stages in the environment. This can reduce the prevalence and abundance of such parasites in the target hosts. Predation on parasites may be commonplace. Lafferty et al. (Reference Lafferty, Dobson and Kuris2006) showed that 44% of food web links in a southern California estuary comprised predation on parasites. Hence, increasing predation on parasites may be a useful approach for disease management, incorporating top-down control on transmission dynamics. For example, predation on hosts has been investigated as a management strategy to control transmission of human infectious diseases. Mkoji et al. (Reference Mkoji, Hofkin, Kuris, Stewart-Oaten, Mungai, Kihara, Mungai, Yundu, Mbui, Rashid and Kariuki1999) and Sokolow et al. (Reference Sokolow, Huttinger, Jouanard, Hsieh, Lafferty, Kuris, Riveau, Senghor, Thiam, N'Diaye and Faye2015) found support for controlling schistosomiasis in human population by controlling transmission; in these cases, crayfish and prawns consumed snails (intermediate hosts), which shed transmission stages into the water where they come in contact with humans. In these studies, reducing snail densities could reduce the number of transmission stages in the water, reducing schistosomiasis in local human population. Thus, consumption of parasites may interrupt transmission dynamics, highlighting the potential importance of studying parasitism in context of community composition (Johnson et al. Reference Johnson, Dobson, Lafferty, Marcogliese, Memmott, Orlofske, Poulin and Thieltges2010).
Multiple laboratory studies document examples of predators of parasite transmission stages across a diverse group of animals. In southern California estuaries, predation on free-living transmission stages comprises 7% of food web links (Hechinger et al. Reference Hechinger, Lafferty, Dobson, Brown and Kuris2011). Sea anemones (Hopper et al. Reference Hopper, Poulin and Thieltges2008; Prinz et al. Reference Prinz, Kelly, O'Riordan and Culloty2009), barnacles (Prinz et al. Reference Prinz, Kelly, O'Riordan and Culloty2009), bivalves (Faust et al. Reference Faust, Stallknecht, Swayne and Brown2009), crabs (Thieltges et al. Reference Thieltges, Bordalo, Caballero Hernández, Prinz and Jensen2008), fishes (Kaplan et al. Reference Kaplan, Rebhal, Lafferty and Kuris2009; Orlofske et al. Reference Orlofske, Jadin, Preston and Johnson2012), newt larvae (Orlofske et al. Reference Orlofske, Jadin, Preston and Johnson2012), odonates (Orlofske et al. Reference Orlofske, Jadin, Preston and Johnson2012; Rohr et al. Reference Rohr, Civitello, Crumrine, Halstead, Miller, Schotthoefer, Stenoien, Johnson and Beasley2015) and shrimp (Thieltges et al. Reference Thieltges, Bordalo, Caballero Hernández, Prinz and Jensen2008; Orlofske et al. Reference Orlofske, Jadin, Preston and Johnson2012) can all be predators on parasite transmission stages. However, these studies measure predation with laboratory feeding trials and were conducted on trematode cercarial transmission (but see Faust et al. Reference Faust, Stallknecht, Swayne and Brown2009), limiting the generalizability of these patterns. Thus, while direct consumption of transmission stages of parasites has been theorized to reduce parasitism (Johnson et al. Reference Johnson, Dobson, Lafferty, Marcogliese, Memmott, Orlofske, Poulin and Thieltges2010), quantification remains limited in the field and across types of parasites. However, some studies have linked parasite predators to parasitism in the field (though only for cercariae) – Kaplan et al. (Reference Kaplan, Rebhal, Lafferty and Kuris2009) showed that cercariae were consumed by small fishes in the field. More indirectly, Rohr et al. (Reference Rohr, Civitello, Crumrine, Halstead, Miller, Schotthoefer, Stenoien, Johnson and Beasley2015) found increased predator diversity reduced parasitism in tadpoles and Mouritsen and Poulin (Reference Mouritsen and Poulin2003) reported increased anemone density reduced parasitism in cockles. Thus, there is some empirical evidence that natural community composition shapes predation on parasite transmission stages and drives spatial patterns of parasitism. However, the magnitude of this effect is needed to evaluate the role of predation on transmission dynamics.
Spatial epidemiology – the description, quantification and explanation for spatial differences in diseases – is a burgeoning area of research; however, local predation on parasite transmission stages has received limited attention (but see Mouritsen and Poulin, Reference Mouritsen and Poulin2003; Rohr et al. Reference Rohr, Civitello, Crumrine, Halstead, Miller, Schotthoefer, Stenoien, Johnson and Beasley2015; Strauss et al. Reference Strauss, Shocket, Civitello, Hite, Penczykowski, Duffy, Cáceres and Hall2016). Recently, epidemiology models have begun to consider the effects of spatial patterns of transmission on parasitism. Regionally, transmission dynamics can be strongly influenced by abiotic factors such as climate (e.g. Brooker et al. Reference Brooker, Leslie, Kolaczinski, Mohsen, Mehboob, Saleheen, Khudonazarov, Freeman, Clements, Rowland and Kolaczinski2006; Brooker, Reference Brooker2007; Mordecai et al. Reference Mordecai, Paaijmans, Johnson, Balzer, Ben-Horin, Moor, McNally, Pawar, Ryan, Smith and Lafferty2013), which have been incorporated into models of spatial epidemiology. However, these have all been large-scale spatial investigations, unable to reveal the mechanisms whereby these patterns are regulated. Locally, the distribution of parasite predators may control spatial patterns in transmission and may provide a control for parasites. Further support for the role of proximity to parasite predators is offered by Mouritsen and Poulin (Reference Mouritsen and Poulin2003), who found increased density of anemones in tidal mudflats reduced larval trematode parasitism of bivalves.
The rocky intertidal zone is a classic ecological system with high biodiversity and a long history of study (e.g. Cranwell and Moore, Reference Cranwell and Moore1938; Connell, Reference Connell1961; Hopper et al. Reference Hopper, Poulin and Thieltges2008). This system is characterized by sessile occupiers of limited hard substrate space (Dayton, Reference Dayton1971). We suggest that this offers a powerful opportunity to study spatial epidemiology. In many environments, movement of the host can decouple risk of infection from the subsequently observed patterns of infection (e.g. Byers et al. Reference Byers, Malek, Quevillon, Altman and Keogh2015). The rocky intertidal zone, with its array of sessile and sedentary organisms, is an ideal system to evaluate drivers of spatial epidemiology. These relationships are durable due to the longevity of many of the space competitors. This will allow direct tests of the effects of these spatial associations on parasitism. Here, we initiate exploration of parasitism in this ecosystem and were able to correlate the spatial association of an anemone predator on parasitism of a barnacle. This parasite directly reduces host reproduction, and hence, the potential fitness of the barnacles.
Hemioniscus balani is an isopod parasite that infects at least 14 species of barnacles and Chthamalus fissus is the most frequently infected host on the California coast of the USA (Crisp, Reference Crisp1968; Goudeau, Reference Goudeau1970; Blower and Roughgarden, Reference Blower and Roughgarden1988). This parasite has generally been ignored in studies of barnacle ecology with the exception of Blower and Roughgarden (Reference Blower and Roughgarden1987, Reference Blower and Roughgarden1988, Reference Blower and Roughgarden1989a , Reference Blower and Roughgarden b ). The isopod enters the mantle cavity of the barnacle and attaches to its cuticle near the ovaries. The parasite then drains the ovarian fluid, rendering the barnacle unable to reproduce as a female (Goudeau, Reference Goudeau1972). After the parasite matures, it releases its offspring, and then dies. Hence, H. balani is an ephemeral, semelparous, parasitic castrator. In this study, we test whether association with a non-host species, Anthopleura elegantissima, protects the barnacle host from infection by consuming the cryptoniscus larva transmission stage of the isopod. Cryptonisci, are planktonic, when settled on the substrate they are highly motile. Whether swimming or benthic the cryptonisci may encounter and be consumed by sea anemones. This sea anemone reproduces clonally and can reach high densities in the rocky intertidal zone (54·4 ± 34 individuals m−2, Dayton, Reference Dayton1971). It is an abundant and generalist predator, largely of zooplankton (Zamer, Reference Zamer1986). Here we report the role of this sea anemone as a predator reducing parasitism of its barnacle host by a parasitic castrator.
MATERIALS AND METHODS
To determine the frequency with which barnacles were associated with anemones, we surveyed the natural populations at Coal Oil Point, Santa Barbara County, California at a single tidal height, at which organisms are submerged an estimated 64% of the time (unpublished data). We ran five 1 m transects parallel to shore and sampled five random 5 × 5 cm quadrats per transect, to survey a total length of 5 m (n = 25). Chthalamus fissus barnacles were considered associated with anemones if they were ⩽1 cm from an anemone, which approximated the length of the anemone's tentacles. We counted the total number of barnacles in each quadrat associated with an anemone or not associated with an anemone. We then calculated the average density of barnacles and the percentage of barnacles associated with anemones and not associated with anemones within each quadrat.
To quantify the relationship between association with an anemone and parasitism, we haphazardly collected 125 barnacles associated (⩽1 cm away) and unassociated with anemones (>10 cm away, substantially farther than the tentacles of an anemone can extend to ensure no effect on distant barnacles) (total n = 250) (Fig. 1). All barnacles sampled were >2 mm in diameter, as smaller barnacles are immature and infrequently infected (Hines, Reference Hines1978; Fong et al. in preparation). Barnacles were placed into individual wells of a cell culture microplate in the field, returned to lab, and immediately processed. Each barnacle was measured to the nearest ¼ mm, assessed for the presence of H. balani, and scored as either reproductive as females (with ripe ovaries or with eggs in the mantle cavity), or not. Because H. balani blocks reproduction, we were able to categorize barnacles as infected, uninfected/reproductive, or uninfected/non-reproductive. Data met assumptions of normality and homogeneity, and we used a t-test to determine if barnacle size varied with association with anemones. To test whether prevalence and reproduction varied with respect to association with anemones, we analysed infection and reproduction data and whether double infections varied with association with anemones with Fisher's Exact Test (no triple infections were recorded in this study).
Photographs of barnacles and anemones at Coal Oil Point, Santa Barbara County, California at low tide. Anemones have small rocks, shells and debris attached to their column. A group of anemones are outlined while the two arrows point out examples of barnacles considered to be associated with anemones (i.e. those <1 cm away from an anemone).
By multiplying host density (survey data) and parasite prevalence (the percentage of infected hosts), we estimated the number of barnacles actively reproducing in each quadrat. Density was not considered as a covariate in this study because previous work has indicated there was no relationship of density with prevalence (Fong, Reference Fong2016). We then compared the estimated number of barnacles actively reproducing that were associated with anemones vs the number of such barnacles not associated with anemones using a t-test.
To determine whether anemones were capable of consuming parasites, we collected three anemones from the field and isolated them in individual finger bowls with 150 mL of seawater and allowed the anemones to acclimate for 30 min. Hemioniscus balani has a free-living, mobile transmission stage, allowing for collection in the field. We collected the cryptoniscus larva transmission stage of H. balani from the field by chiselling off sections of rock, transporting the rock to the laboratory, rinsing in seawater in the laboratory, and collecting free-living parasites from the water. After the anemone had acclimated for 30 min, we added ten cryptoniscus larvae in 25 mL of seawater to the finger bowl. As a control, we also placed ten cryptoniscus larvae in 25 mL of seawater to three finger bowls without anemones. After 30 min, we counted the number of cryptoniscus larvae remaining in the water and calculated a consumption rate (no./h). We then averaged individual anemone consumption rates for a mean larval consumption rate (n = 3). Because no larvae were lost in the controls, we performed a one sample t-test comparing our anemone predation rates to zero to determine if the anemones consumed significantly more than zero cryptonisci on average.
RESULTS
We surveyed a total of 1262 barnacles at Coal Oil Point. Their average density was 50·5 ± s.e. 10·4 barnacles per 25 cm2. Of all barnacles surveyed at the site, 29% were ⩽1 cm away from an anemone and thus associated with the anemone (370/1262). On average, 39·7 ± s.e. 7·8% of the barnacles within the 25 cm2 quadrat were ⩽1 cm away from an anemone. Barnacles collected for dissections were similar in size, irrespective of association with anemones (t-test, P = 0·25).
Barnacles associated with anemones were significantly less likely to be infected (Fisher's Exact Test, P < 0·0001). Whereas 69·6% (87/125) of the barnacles not associated with anemones were infected, only 28·0% (35/125) of barnacles associated with anemones were infected. Thus, barnacles not associated with anemones were 2·5 times more likely to be infected (Fig. 2). Additionally, double infections differed with association with anemones (Fisher's Exact Test, P = 0·0393). Near anemones, 2·9% of barnacles were infected by more than one parasite, compared with 22·5% of barnacles distant from anemones (n = 1, n = 16, respectively). Thus, multiple infections increased 7·7 times away from anemones, and double infections increased with increased prevalence.
Number of barnacles infected (I), uninfected but not actively reproducing (UI/NR) and uninfected and actively reproducing (UI/R) when associated (+A) or not associated (−A) with an anemone.
When associated with anemones, barnacles were more likely to be actively reproducing (Fisher's Exact Test, P = 0·0007). The 23·2% of barnacles associated with anemones were actively reproducing (n = 29) compared with only 7·2% of barnacles not associated with anemones (n = 9). Thus, barnacles near anemones were 3·2 times more likely to be actively reproducing (Fig. 2). However, among uninfected barnacles, the same fraction of individuals were actively reproducing, when adjacent or away from anemones (Fisher's Exact Test, P = 0·4005).
The density of actively reproducing barnacles that were associated with anemones was not significantly different from barnacles that were not associated with anemones (t-test, P = 0·46). Hence, while only 30% of the barnacles were associated with an anemone in the 25 cm2 quadrats, the number of reproductive barnacles reproducing was equivalent between the two groups.
In laboratory trials, anemones consumed parasite transmission stages. On average, under these conditions, anemones consumed cryptoniscus larvae at a rate of 8·7 ± s.e. 1·3 per hour (n = 3), while in the control treatment, no cryptonisci were lost (one sample t-test, P = 0·0136).
DISCUSSION
Barnacles associated with anemones had reduced parasitism and this resulted in higher reproduction, presumably due to predation on parasite transmission stages. In a community context this factor reduces parasitism at a local scale. Thus, anemones provide a spatial refuge for barnacles from parasites. It is possible that other factors result in the spatial patterns of parasitism we documented in the field. For example, it is possible that anemones settle in microhabitats inaccessible to parasite transmission stages. Similarly, it is possible that parasite transmission stages actively avoid areas colonized by anemones. However, parasites are delivered to hosts by water movement and we collected all barnacles from a narrow tidal height in the rocky intertidal zone with no obvious microhabitats. Further, because parasitism was reduced and not eliminated near anemones, at least some parasites do reach barnacles near anemones. It is also possible that parasitism was reduced near anemones because barnacles near anemones were of lower quality. For example, if barnacles adjacent to anemones were less likely to be actively reproducing as females, possibly due to competition with the anemone for food, they would be inappropriate hosts for a parasite that consumes ovarian fluid. However, we found increased reproduction in barnacles adjacent to anemones; thus, barnacles adjacent to anemones were compatible hosts, making reduced host quality an unlikely explanation. Further, barnacles next to anemones were similar in size to barnacles away from anemones, making size-based differences in host quality or compatibility unlikely. While genetic differences can drive patterns of resistance (e.g. Little and Ebert, Reference Little and Ebert2000), we find it unlikely that barnacles collected from a single site and a single tidal height are genetically distinct. Finally, barnacle density could be lower adjacent to anemones, reducing any signalling cue required by the parasite to find the host. However, parasitism does not increase with density or aggregation of this barnacle host in the field (Fong, Reference Fong2016). Thus, we find predation by anemones to be the simplest explanation for the spatial pattern of reduced parasitism near sea anemones.
Similarly, research has linked predator abundance to decreased trematode parasitism in the field. Mouritsen and Poulin (Reference Mouritsen and Poulin2003) showed that an anemone attached to bivalves in New Zealand tidal flats reduced infection of those bivalves and Rohr et al. (Reference Rohr, Civitello, Crumrine, Halstead, Miller, Schotthoefer, Stenoien, Johnson and Beasley2015) reported that increased odonate diversity depressed parasitism, presumably due to predation on cercaria transmission stages. Laboratory trials (Hopper et al. Reference Hopper, Poulin and Thieltges2008; Thieltges et al. Reference Thieltges, Bordalo, Caballero Hernández, Prinz and Jensen2008; Faust et al. Reference Faust, Stallknecht, Swayne and Brown2009; Kaplan et al. Reference Kaplan, Rebhal, Lafferty and Kuris2009; Prinz et al. Reference Prinz, Kelly, O'Riordan and Culloty2009; Orlofske et al. Reference Orlofske, Jadin, Preston and Johnson2012; Rohr et al. Reference Rohr, Civitello, Crumrine, Halstead, Miller, Schotthoefer, Stenoien, Johnson and Beasley2015) and one field study (Kaplan et al. Reference Kaplan, Rebhal, Lafferty and Kuris2009) indicate that a variety of animals consume parasite transmission stages, supporting the potential for predation on parasites to control parasite transmission. Kaplan et al. (Reference Kaplan, Rebhal, Lafferty and Kuris2009) found predation on trematode cercariae in the laboratory and detected this consumption in the field. Further support of predator-mediated transmission comes from mesocosm experiments. Thieltges et al. (Reference Thieltges, Bordalo, Caballero Hernández, Prinz and Jensen2008) determined the presence of a crab and shrimp species reduced parasitism in a bivalve host, and attributed this effect to the directly observed predation. Additionally, Rohr et al. (Reference Rohr, Civitello, Crumrine, Halstead, Miller, Schotthoefer, Stenoien, Johnson and Beasley2015) found odonate predators of cercariae reduced transmission to tadpole hosts in mesocosm experiments. Finally, Faust et al. (Reference Faust, Stallknecht, Swayne and Brown2009) found that in mesocosms, bivalves were able to filter avian influenza virions and reduce transmission of the virus to ducks. Thus, while there was evidence predators can consume parasite infectious stages, we provide the first evidence that predation may translate to fine-scale spatial patterns of parasitism in nature. Predation on other free-living parasite transmission stages warrants investigation.
Multiple infections were substantially higher away from anemones, suggesting anemones may be quite effective at locally reducing parasite abundance and providing protection to the associated barnacles. A single castrator consumes all of the reproductive energy of the host; thus, multiple infections necessarily result in competition between the parasites (for review see Lafferty and Kuris, Reference Lafferty and Kuris2009). As for parasitoids and predators, parasitic castrators face severe resource limitation with increasing prevalence and can approach saturation of a host population as uninfected hosts become unavailable. Kuris et al. (Reference Kuris, Poinar and Hess1980) found that multiple infections by an entoniscid isopod, a parasitic castrator of crabs, only became common at sites where >70% of the hosts were infected. The high incidence of multiple infections in the barnacles not associated with anemones suggests that the parasite may approach saturation of the available susceptible hosts at this particular site.
Barnacle reproduction varies substantially in space, and many studies have sought explanations for this spatial variation (e.g. Hines, Reference Hines1978; Leslie et al. Reference Leslie, Breck, Chan, Lubchenco and Menge2005; Berger, Reference Berger2009; Freuchet et al. Reference Freuchet, Tremblay and Flores2015). Despite their lower abundance near anemones, those barnacles appear to contribute reproductive productivity equivalent to the more numerous barnacles in the sampled quadrats that were relatively distant from anemones. Food availability (Hines, Reference Hines1978; Leslie et al. Reference Leslie, Breck, Chan, Lubchenco and Menge2005), salinity gradient (Berger, Reference Berger2009), and temperature stress (Freuchet et al. Reference Freuchet, Tremblay and Flores2015) all influence barnacle reproduction, both at regional and local scales. We show that parasitism can contribute strongly to these spatial patterns since A. elegantissima and C. fissus are both widespread, abundant and often co-occur at a fine scale in the rocky intertidal zone along the Pacific coast of North America. Thus, we suggest future studies examining factors influencing barnacle reproduction should consider parasitism. Additional factors that potentially influence the success of barnacles include the possible reduction in plankton available to barnacles near anemones, reducing growth or reproduction, and the possible differential predation on barnacle nauplii released from barnacles adjacent to or more distant from the anemones.
ACKNOWLEDGMENTS
We would like to thank the Parasite Ecology working group at UCSB, particularly Ryan Hechinger and Kevin Lafferty for insights on this paper.
FINANCIAL SUPPORT
Funding was provided by NSF EEID grant no. 1115965 awarded to A.M.K.
