Patterns of Multi-Symbiont Community Interactions in California Freshwater Snails

Virtually all organisms function as hosts for a variety of symbionts that can interact and form complex communities. Recently, research has begun to highlight the influence that symbiont communities can have on human and animal health through multi-symbiont interactions. Here, we examine symbiont community patterns both by host species and specific symbiont interactions in California freshwater snails. Specifically we explore two questions. First, what are the broad patterns observed among five commonly occurring snail species (Helisoma trivolvis, Physa spp., Gyraulus spp., Lymnaea columella, and Radix auricularia)? Second, what are the dynamics between the symbiont annelid Chaetogaster limnaei limnaei and larval trematode infections? We sampled and necropsied 12,713 snail hosts from Contra Costa, Alameda, and Santa Clara counties in California and found that among wetlands, symbiont communities varied significantly between snail species. The prevalence of symbionts and the richness of symbiont communities were both positively correlated to the abundance of each snail host species across the landscape. Within individual snail hosts, larval trematode infection and C. l. limnaei abundance correlated positively, with ~30% more C. l. limnaei in trematode infected hosts as compared to uninfected snails. This relationship, however, was variable among trematode species, suggesting that the underlying mechanism of interaction may be a combination of preferred predation by the annelid worm and other less direct interactions. This study presents evidence that links patterns of host availability to symbiont community richness and prevalence as well as potential interactions among symbionts, stressing the importance of considering multi-host and multi-symbiont communities when studying community interactions. Introduction Despite a rich history of research and recent exposure by world leaders and the media, global biodiversity decline remains a major concern for the scientific community (Balvanera et al. 2006, Carpenter et al. 2009). Changes in biodiversity affect a variety of ecosystem processes, such as nutrient cycling and disease transmission which, in turn, influence social issues like food security and human and animal health (Butchart et al. 2010, Cardinale et al. 2012). As humancatalyzed biodiversity loss continues to alter ecosystems, several biases within our knowledge of biodiversity have emerged (Hortal et al. 2008). The Millennium Ecosystem Assessment (2005) underlined that, historically, most efforts to catalogue the variety of life on earth have been focused heavily on plants, mammals, birds, and reptiles. The richness and diversity of other organisms like fungi, bacteria, insects, and flatworms (helminthes), remains largely unknown. One possible reason for this lack of clarity is that many of these organisms live as symbionts: organisms that utilize another organism as a host. This use of a host causes symbionts to be more difficult to observe than free-living species. The task of classifying symbiont biodiversity is daunting; by some predictions there are 300,000 species of vertebrate parasites (a type of symbiont) alone (Dobson et al. 2008) and others estimate that about 50% of all species are parasitic (Toft, 1986). Not only is symbiont diversity poorly understood, we are just beginning to understand the importance of symbiont interactions within hosts. The consequences of symbiont biodiversity loss will largely depend on what ecological interactions are also lost when a symbiont species goes extinct. Symbionts are well known for interacting with their hosts, but can also interact with other symbionts and free-living organisms in an ecosystem (Bush and Holmes 1986, Fernandez at al. 1991, Ibrahim 2007, Mieog et al. 2009). Recently, there has been a growing appreciation for the importance of symbiont interactions within an individual host and how these interactions form unique symbiont communities (Dale and Moran 2006). These communities tend to be complex because multiple symbionts often inhabit a single host, proximity and shared reliance on that host is a potential catalyst for multi-symbiont interactions. Associations within a symbiont community can have important effects on host disease patterns, like virulence (severity) and transmission (communicability) (Graham 2008, Johnson et al. 2012). For example, Rodgers et al. (2005) experimentally demonstrated a decrease in the prevalence (commonness) of Schistosoma mansoni in aquatic snails (Biomphalaria glabrata) when the latter were co-infected with the commensal annelid Chaetogaster limnaei limnaei. This small-scale interaction has potential for real-world consequences on human health because S. mansoni infects more than 80 million people worldwide (Crompton 1999), causing intestinal schistosomiasis, which is known for its symptoms of anemia, malnutrition, and learning disabilities (King 2005). Thus far, most studies focus on interactions between two or, at most, three specific symbionts, and rarely have complete inventory of symbiont organisms living within a studies’ host species. It is inherently difficult to draw conclusions about symbiont communities because there are a number of variables that structure community interactions (Johnson and Buller 2011). Symbionts determined to be correlated through observation could be interacting in three ways. First, directly within a host though predation or mechanical facilitation (Bandilla et al. 2006). Second, indirectly within a host by competing for limited host resources (Ishii et al. 2002, Hardin 1960), or by altering host immune responses (Su et al. 2005). Third, indirectly through site level co-existence by altering host behavior (Daly and Johnson 2010). Some of the most thoroughly explored symbiont-interaction studies focus on parasite antagonism in where the presence of one parasite reduces the success of another. In the same system described above, Sandland et al. (2007) demonstrated that co-infection of S. mansoni-positive snails with Echinostoma caproni (a complex-lifecycle trematode) reduced pathology and prevalence within the snail host as compared to snails solely infected with S. mansoni. However, two parasite-in-host interactions may be an oversimplification of what occurs at the symbiont community level because it’s likely that one or more such interactions are co-occurring within any given symbiont community (Pedersen and Fenton 2007). Looking at symbiont interactions from this more broad perspective have led to interesting insights on how symbionts can influence host disease. One major hypothesis linking biodiversity to disease is the ‘dilution effect’ which proposes that an increase in parasite richness (number of different parasites species) will increase cross-parasitic species competition. This, in turn, reduces the success of the most virulent parasites. Johnson and Hoverman (2012), using lab and field data, demonstrated an intra-host dilution effect by showing that an increase in parasite richness was correlated with a decrease in overall infection success, including infection by the most virulent parasites. For these reason it is crucial to include all organisms inhabiting a studies’ host when studying symbiont communities. Freshwater pond snails are an excellent system for studying symbiont interactions at both individual and community levels. Aquatic gastropods serve as obligatory first hosts for a range of trematode parasites and are considered a keystone species to freshwater ecosystems (Esch, Curtis, and Barger 2001). The trematode lifecycle begins as the parasite egg enters water and a miracidium hatches to find a mollusk host. Upon infection, trematode rediae or sporocysts develop in the snail host’s gonad, causing pathology that can lead to castration and even death of the host (Sousa 1983). The trematode then produces motile cercariae, which swim through the water column looking for a second intermediate host that can be, depending on the trematode, a variety of fishes, amphibians, or invertebrates. The final stage of the trematode lifecycle occurs when the second intermediate hosts is consumed by a vertebrate definitive host, where the adult parasite develops and reproduces. Because trematodes have long been a focus in parasitology, extensive identification manuals are readily available (see Yamaguti 1971 or Schell 1985). Additionally, lentic ecosystems are well delineated from one another, facilitating sampling of multiple replicate ponds across a landscape. The snails featured in this study have long been known to host a variety of other symbiotic organisms, one example is C. l. limnaei. These annelid worms, which live under the mantle, can then feed on various small organisms like rotifers, algae, and trematode cercariae. With these feeding habits in mind, C. l. limnaei offers an interesting case study for symbiont interactions because of its high probability of interacting directly with other symbionts. Other known snail symbionts include insect larvae (Chironomidae), the parasitic nematode Daubaylia potomaca, and the leech Helobdella punctato-lineata (Hugh 1971, Prat et al. 2004) .One study by Zimmermen et al. (2011b) examined the relationship between C. l. limnaei, trematode infection, and D. potomaca. From their data, the authors postulated that C. l. limnaei indirectly increases nematode presence by down-regulating the presence of trematode infections, which were negatively associated with the nematode through apparent competition. While this study highlights interesting patterns derived from snails in a single pond, other organisms or interactions may be at play in the mechanisms underlying these relationships. Given the need to better understand both symbiont community diversity and potential interactions among symbionts, we analyzed data collected through comprehensive necropsies of field-caught host specimens. This study asks two specific questions. First (Aim 1), how do symbiont communities differ among freshwater snail species? We address this question by testing whether symbiont community richness and total symbiont prevalence varied among snail species which, based on past research, might differ by host body size, local host abundance or life history characteristic (Blower and Roughgarden 1988). Second (Aim 2), what patterns of cooccurrence are observable between trematode infections and populations of the symbiont annelid C. l. limnaei in Helisoma trivolvis snail hosts? We choose to examine this specific example because trematodes and C. l. limnaei were the most common symbionts observed in H. trivolvis, which was the most common snail host at our field sites. Additionally, C. l. limnaei predation upon trematode cercariae has been suggested as a potential device against human and animal disease (Michelson 1964, Fried et al. 2008, and Ibrahim 2006, 2007). However, these studies have been limited to single pond systems and rarely look at their relationship at a regional level or assessed the patterns of co-occurrence among multiple species of trematodes. Materials and Methods