Carpinteria Salt Marsh Reserve

31 Jan, 11 PM - 31 Dec, 01 PM

Introduction and study system In many host-parasite systems, parasitologists and ecologists note that certain individuals accumulate parasites much more quickly than others in the same population. In parasites that can be passed between individuals, measures of social interactions frequently explain differences in parasite load (e.g., Godfrey et al. 2009; Grear et al. 2009; Otterstatter and Thomsom 2007; Natoli et al. 2005). Both behavior and the underlying hormones influencing these behaviors are important in transmitting parasites between individuals. Despite the ecological importance of trophically transmitted parasites (e.g., Lafferty 2008), we know little about the mechanisms that underlie parasite load differences between individuals. As these parasites frequently have heavy fitness impacts on their hosts, differences in encounter and infections rates could have important ecological implications. An ideal study system for interactions between behavior, hormones, and parasites is California killifish (Fundulus parvipinnis) and their trematode parasite Euhaplorchis californiensis. Euhaplorchis californiensis (EUHA) has a multi-host life cycle, infecting California horn snails (Cerithidea californica), California killifish and piscivorous birds (Martin 1950). EUHA reproduces asexually in California horn snails and, when mature, emerge as free-swimming larvae known as cercariae, which are capable of infecting California killifish. All California killifish over a certain size are infected by the parasite (Shaw 2007), but the magnitude of infection for similarly sized and aged individuals can vary by an order of magnitude (Kuris, personal communication). In order for EUHA be successfully transmitted to its definitive host, piscivorous birds, its current killifish host must be depredated. Work done by Lafferty and Morris (1996) suggests that EUHA increases the likelihood that piscivorous birds consume killifish by manipulating their behavior. This study found that infected killifish exhibit more ?conspicuous? behaviors (including quick dashes to the surface of the water) and found that infected killifish were 10-30 times more likely to be depredated by piscivorous birds than uninfected individuals (Lafferty and Morris 1996). Encounters rates with EUHA by California killifish may vary for individuals as a function of their differences in activity patterns, shoaling tendency, and/or willingness to engage in risky situations. Particular hormones (e.g., testosterone and cortisol) may also affect the magnitude of the behaviors listed above and impact the immune system. Individual variations in behavioral type and hormone profile are frequently observed and may help to explain why particular individuals accumulate parasites more rapidly than others. This project has 2 main objectives: 1) To better understand the ecology of Euhaplorchis californiensis (EUHA) in order to predict how killifish behavior should influence encounter rates with this parasite, and 2) To quantify how the behavioral type and hormone profile of California killifish influences encounter and infection rates with EUHA. Proposed Experiments This research will be conducted in the joint lab of Armand Kuris and Kevin Lafferty at the University of California Santa Barbara. Objective 1: Explore the ecology of Euhaplorchis californiensis. In order to understand how California killifish behavior should influence encounter rates with EUHA, we must first understand EUHA?s distribution in the reserve and how EUHA responds to the presence of a potential host. Experiment 1: Is EUHA?s distribution across Carpinteria Salt Marsh Reserve homogeneous or patchy? The distribution of EUHA across Carpinteria Salt Marsh will determine the extent to which California killifish can avoid parasitism risk altogether. If EUHA?s distribution is found to be homogenous, then the killifish will be unable to avoid the parasite and may instead rely on behaviors such as schooling and reduced activity to reduce risk. Methods: Field surveys of Carpinteria Salt Marsh Reserve will be conducted, and water samples taken at 150 random locations in the reserve. At each location, 3 water samples will be collected (one on either side and one in the middle of the slough). Cylindrical collecting devices will capture water from the entire water column and water samples will be examined for the presence and abundance of EUHA. Samples will be collected around dusk, as this is when the largest number of cercariae emerge (Fingerut et al. 2003). Experiment 2: Where in the water column do EUHA typically reside? A reduction in activity in response to the presence of trematode cercariae has been observed in fishes and amphibians (Thiemann and Wassersug 2000, James et al. 2008). This is presumably an adaptive response to decrease parasite encounter rates. These studies, however, did not measure the response to naturally distributed cercariae. Trematode cercariae of many species have a tendency to aggregate near the top of the water column (Haas 1994), indicating that not just the amount of activity, but also the location of activity may be important. Methods: This experiment will be conducted under semifield conditions employing methods outlined in Haas et al. 2008. Briefly, a Plexiglas cylinder will be submerged in water at Carpinteria Salt Marsh and EUHA cercariae will be added to the cylinder. The cercariae will be collected from 50 California horn snails hand collected around the marsh. These snails will be encouraged to shed the cercariae in the lab and EUHA cercariae will be brought back out to the marsh. At regular intervals, 100 mL water samples will be drawn from the Plexiglas cylinder at 4 different depths. Water samples will be analyzed and the number of EUHA at each of the 4 depths will be determined. Haas et al. 2008 found that the behavior of cercariae in the Plexiglas cylinder approximated the behavior of cercariae naturally distributed in a pond. Experiment 3: How do EUHA increase contact rates with California killifish? Water turbulence, chemical cues, touch, and shadows are cues commonly used to alert trematode cercariae to the presence of a potential host. Cercariae seeking fish hosts typically do not use chemo-orientation as these cues are not reliable for highly mobile hosts such as fish (Haas 2003). Fish-invading cercariae have been shown to alter their swimming behaviors in response to touch, water turbulence, and dark shadows (such as that cast by a fish swimming overhead) (Haas 2003). The effect of these three stimuli on EUHA behaviors will be explored. How fish behaviors such as activity patterns influence the strength and frequency of information received from the fish by EUHA will be considered. Methods: The methods employed in Haas et al. 1990 will be used to explore changes in EUHA behavior in response to tactile stimuli, water turbulence, and shadows. Fifty Califonia horn snails will be hand collected from CSM and brought back to the lab for this purpose. Cercariae will be placed in a Plexiglas cuvette, and their behaviors in response to each of these cues will be observed using a horizontal dissection microscope. Model: Data collected from these three experiments will be used to create a model to predict how California killifish behavior influences encounter rates with EUHA. Objective 2: Quantify how the behavioral type and hormone profile of California killifish influence encounter and infection rates with EUHA. To achieve this objective, I will quantify behavioral type and hormone profile in 90 uninfected California killifish before exposing the fish to EUHA in field mesocosm enclosures. The California killifish will be collected by beach seines and will be brought back to UCSB. Uninfected fish will be obtained by collecting adults from Carpinteria Salt Marsh Reserve which will be bed in the lab. Methods: AT UCSB: Marking: All fish will be marked with elastomer tags (Northwest Marine Technologies) for individual identification. Behavioral assays: To quantify behavioral type, each fish will be run through behavioral assays to measure its shoaling tendency, activity level, boldness, and exploratory tendency. The data collected from these assays will be run through a principal components analysis to determine if behaviors are correlated, indicating the presence of consistent behavioral types. I have previous experience running these assays in mosquitofish (Gambusia affinis) (Cote et al., submitted) and smallmouth bass (Micropterus dolomieui) (Smith et al. 2009). Activity: If EUHA uses water turbulence, touch, or shadows to find their host, then active fish may provide these cues to EUHA more frequently than less active fish. Activity may therefore be an important factor determining encounter rate. Fish will be housed individually prior to behavioral assays. A camera turned on remotely will record undisturbed fish behavior in their home tank for 10 minutes. The video will be analyzed in Image J to determine how much time was spent moving, where the movements occurred (e.g., top of the tank or near the bottom), how much distance was covered, and the fishes? average speed. Shoaling tendency: Depending on EUHA?s distribution in the water, shoaling could decrease parasitism rates by spreading the risk amongst members of the shoal. Individual fish differ in the degree to which they associate with conspecifics in a shoal (Cote et al., submitted) and may therefore differ in the degree to which they are at risk of parasitism. Shoaling tendency will be measured as an individual?s time spent near a shoal of conspecifics. A 38 liter tank will be split by glass partitions into 3 compartments: an outer compartment which holds an experimental shoal, a center compartment holding the focal individual, and an empty outer control compartment. An observer behind a black curtain will record the time the focal fish spends within 3 cm of the experimental shoal. Boldness and exploratory behavior: Boldness is frequently a measure of how an individual responds to a simulated predator attack, while exploratory behavior measures its movements in an unfamiliar environment. Both assays measure how an individual responds to risk. Wild caught California killifish appear anxious in the presence of EUHA (Lafferty, personal communication), indicating that EUHA is perceived as risky. Aversion to EUHA likely arises from pain the fish feels when the parasite burrows into its skin upon contact. The experimental arena for this assay will be a large, unfamiliar white tank (Figure 1). The fish will be placed in a chamber and allowed access to the experimental arena after a 10 min acclimation period. The first measure of boldness will be the latency to exit the chamber and begin exploring the novel environment. The activity of the fish in the novel environment will be videotaped for 5 min after it exits the chamber for later quantification of exploratory behavior. Following the 5 min of activity, blood worms will be added to the food dish on one end of the experimental arena. When the fish begins eating, a replica heron?s beak will be released, simulating a strike from a predatory bird. The duration of continuous movement following attack (referred to as duration of active evasion), the location where the fish ceases movement following active evasion (i.e., in the open or in a refuge), and the time it takes for the fish to resume normal activity will be recorded as measures of boldness. Figure 1: Experimental arena for boldness/exploratory behavior trial. Hormone Assays: Testosterone, 11-ketotestosterone, estrogen, and cortisol are steroid hormones that influence behavior and immunity. So, hormones are likely important in determining both encounter and infection rates. A new technique has recently been developed, which takes advantage of the fact that free steroids, such as the hormones mentioned above, pass through the gills of fishes and into the surrounding environment, where they can be extracted (Vermeirssen and Scott 1996, Ellis et al. 2005, Scott and Ellis 2007). Hormones are released into the water at a rate that mirrors plasma concentrations in the blood (Sebire et al. 2007, Wong et al. 2008). I will measure waterborne hormone concentrations using the techniques described in Ellis et al. 2004. During Spring 2010 I will validate the use of this technique in California killifish and establish concentration curves with the help of Dr. Ryan Earley (University of Alabama). I have already received funding to conduct this validation procedure and have prior experience executing this technique in mosquitofish. Infection Rates: This experiment will help to distinguish between the effects of hormones on behaviors that influence encounter rates as opposed to the effects of hormones on immune function. 30 uninfected fish that have had their behavioral type and hormonal profile assayed will be anesthetized with MS-222 and exposed to 100 EUHA cercariae in a petri dish. These cercariae will be obtained by hand collecting 30 California horn snails at CSM. This procedure will standardize encounter rates and allows for quantification of the effects of hormones on infection rates. These fish will then be placed in a tank for a month, allowing time for successful parasites to encyst. Fish will be euthanized and the number of parasites that successfully encysted in their brains will be determined. Mesocosm Experiments: Mesh enclosures measuring 2 meters by 2 meters will be placed in the subtidal channels. They will be anchored into place using tent stakes and will remain in place for a period of thirty days. Previous field surveys of EUHA distribution will inform where to set up the enclosures. The enclosures will be large enough to allow shoaling and natural activity patterns and will exclude predatory birds. Fish will be left in the field enclosures for a month, at which point they will be euthanized and the number of metacercariae established in their brains will be determined. Statistical analysis: A path analysis will determine if hormone levels explain behavioral type, which could then explain parasite load. This analysis will also determine if the hormonal metrics have a direct effect on parasite load above and beyond their indirect effects on behavioral type.

Visit #19497 @Carpinteria Salt Marsh Reserve

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Under Project # 20721 | Research

Effects of California killifish behavior and hormone profile on encounter and infection rates with the trematode parasite Euhaplorchis californiensis

research_scientist - University of California, Davis

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Kelly Smith Jan 31, 2010 - Dec 31, 2011 (700 days)

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Day Use Only 1 Jan 31, 2010 - Dec 31, 2011