Bull Mar Sci. 90(2):611–621. 2014 research paper http://dx.doi.org/10.5343/bms.2013.1060
Ecological effects of the invasive parasite Loxothylacus panopaei on the flatback mud crabEurypanopeus depressus with implications for estuarine communities
1 School of Coastal and Marine Kathryn A O’Shaughnessy 1 * Systems Sciences, Coastal Juliana M Harding 2 Carolina University, PO Box 2 261954, Conway, South Carolina Erin J Burge 29528-6054. 2 Department of Marine Science, Coastal Carolina University, PO Box 261954, Conway, South Abstract.—The rhizocephalan barnacle Loxothylacus Carolina 29528-6054. panopaei (Gissler, 1884) is a parasitic castrator that infects xanthid crabs and is invasive on the US Atlantic coast. It was * Corresponding author email: introduced with infected crabs to Chesapeake Bay in the mid-
The Rhizocephala includes parasitic barnacles that castrate decapod crustaceans, including xanthid crabs. Parasitic anecdysis of the crab host results from infection (O’Brien and Van Wyk 1985), while endocrine and central nervous systems sustain damage from the parasitic internal rootlet system (Høeg 1995). This internal system ramifies throughout the host hemolymph, absorbs nutrients, and emerges from the crab abdomen as a reproductive sac called the externa (O’Brien and Van Wyk 1985). The rhizocephalan barnacle, Loxothylacus panopaei (Gissler, 1884), infects mud crabs and is native to coastal estuarine habitats from Cape Canaveral, Florida, south into the Gulf of Mexico and Caribbean waters as far east as Venezuela (Hines et al. 1997, Kruse and Hare 2007, Kruse et al. 2012). Infection with L. panopaei halts the molt- ing process, thereby inhibiting host growth (O’Brien and Van Wyk 1985). Parasitic castration removes the infected individual from the genetic pool, reducing its eco- logical success, and potentially lowering the effective population size (Van Engel et al. 1966, Daugherty 1969). Additionally, host feeding behavior may be compromised
Bulletin of Marine Science 611 © 2014 Rosenstiel School of Marine & Atmospheric Science of the University of Miami 612 Bulletin of Marine Science. Vol 90, No 2. 2014 by internal damage to host organs from the parasitic rootlet system (Høeg 1995) and the presence of the parasitic externa (Bishop and Cannon 1979). Loxothylacus panopaei is invasive along the US Atlantic coast where prevalence reports have ranged from 10% to 93% in the flatback mud crab, Eurypanopeus de- pressus (Smith, 1869) (e.g., Daugherty 1969, Kruse and Hare 2007, Freeman et al. 2013). Crabs infected with L. panopaei were transplanted from the Gulf of Mexico to Chesapeake Bay with oysters during the mid-1960s (Van Engel et al. 1966). Since then, L. panopaei has invaded western Atlantic habitats from Long Island Sound, New York, to just north of Cape Canaveral, Florida (Kruse and Hare 2007, Kruse et al. 2012, Freeman et al. 2013). Mud crabs exert top-down control within temperate intertidal oyster reefs (Silliman et al. 2004) because they are voracious consumers of bivalves including the eastern oyster, Crassostrea virginica (Gmelin, 1791) (e.g., McDermott 1960, Bisker and Castagna 1987) and the Atlantic ribbed mussel, Geukensia demissa (Dillwyn, 1817) (e.g., Seed 1980). In intertidal US Atlantic oyster reef habitats, E. depressus feeds on small bivalves (McDermott 1960, Kulp et al. 2011) and macroalgae in oyster cultch interstices (Meyer 1994). Kulp et al. (2011) found that E. depressus of 15.8 mm mean carapace width consumed 22.7 (SD 2.9) C. virginica (5.9 mm shell length) 96 hrs−1 crab−1 at 25 °C. McDermott (1960) observed E. depressus (16.4–22.9 mm cara- pace width) consumption of C. virginica (3–30 mm) at a rate of 1.6 C. virginica hrs−1 crab−1 at 23 °C. Community-level changes in intertidal oyster reef trophic structure may occur when parasites are prevalent (Mouritsen and Poulin 2002). Because L. panopaei is an invasive parasite, the effects on native host populations and related trophic structure are unknown but they potentially decrease native species abundance (Van Engel et al. 1966, Ruiz et al. 1997). The presence of L. panopaei on an oyster reef may impact predator (E. depressus) demographics, population size and, thus, the relative impor- tance of prey (bivalve) species. Investigations of L. panopaei in South Carolina waters are lacking. There has been only a single parasite prevalence study in South Carolina, which reported an ab- sence of L. panopaei in the 1980s (Hines et al. 1997). Recent parasite prevalence studies have been restricted mainly to Florida (Tolley et al. 2006, Kruse and Hare 2007, Kruse et al. 2012), North Carolina (Reisser and Forward 1991, Hines et al. 1997), Georgia (Hines et al. 1997, Kruse and Hare 2007, Kruse et al. 2012), Maryland, Virginia (Hines et al. 1997, Kruse and Hare 2007, Kruse et al. 2012), and New York (Freeman et al. 2013). While mud crab feeding rates have been studied (Seed 1980, Milke and Kennedy 2001, Kulp et al. 2011), feeding behavior in mud crabs parasitized by Loxothylacus species has not been examined, and little is known about the overall effects of theR hizocephala on intertidal oyster reef food webs. The present study de- scribed parasite prevalence from E. depressus populations at Clambank Creek, North Inlet, South Carolina, and examined prey consumption in E. depressus infected with L. panopaei.
Materials and Methods
Parasite Prevalence.—Monthly (January–August 2012) xanthid crab (E. depressus and Panopeus herbstii H. Milne-Edwards, 1834) collections were made by hand (Hines et al. 1997, Kruse et al. 2012) from a natural fringing oyster reef in O’Shaughnessy et al.: L. panopaei reduces feeding in E. depressus 613
Clambank Creek (33°20.07´N, 79°11.52´W). Collection consisted of excavating all surface oyster cultch, buried shell, and aerobic sediment from a 0.25 m2 area. The excavated material was placed into a bin and the cultch was broken apart to ensure crabs of all sizes were captured. This process was repeated until approximately 100 crabs were collected, except between December and March when the target collection was set to 50 crabs because mud crabs have been found to be less dense intertidally (Dame and Vernburg 1982). All xanthid crabs present were collected (Hines et al. 1997) to avoid misidentification of E. depressus in the field and to confirm that L. panopaei infects only E. depressus in Clambank Creek. Crabs were returned to the laboratory where they were frozen for later examination. Water temperature (°C) and salinity were recorded monthly with a YSI 30 wa- ter temperature and salinity meter. Continuous water temperatures and salinities (January–August 2012) in Clambank Creek were also obtained from NOAA (http:// cdmo.baruch.sc.edu) to provide additional context for field temperature and salinity measurements. Crabs were identified to species and sexed based on external morphology in the laboratory (Williams 1984). Each crab abdomen was separated from the body and examined for an externa using a dissecting microscope. Crabs were classified as parasitized if an externa of any size (mature or virgin) was present. Maximum crab carapace width (CW) was measured to the nearest 0.1 mm using digital calipers. Feeding Experiments.—Laboratory feeding experiments were conducted in May and June 2012 in flow-through seawater tanks at the University ofS outh Carolina Baruch Marine Field Laboratory, located in the North Inlet-Winyah Bay National Estuarine Research Reserve near Georgetown, South Carolina. Experimental E. de- pressus [range: 8–13 mm CW; median: 11.1 mm CW; mean: 11.0 (SD 1.5) mm CW] were collected by hand from oyster cultch in Clambank Creek, North Inlet. Mussels (G. demissa and Brachidontes exustus Linneaus, 1758, 5–9 mm shell length) were collected from pilings and among oyster cultch in Clambank Creek. Eurypanopeus depressus used in experiments were given at least 72 hrs to accli- mate to laboratory conditions and were maintained on natural, living oyster cultch in a flow-through seawater tank adjacent to and receiving the identical flow rate (19 L min−1) as the experimental tanks. Mussels were collected 1 wk before experimental runs, acclimated to laboratory conditions, and allowed to attach via byssal threads to matte ceramic tiles (95 × 95 mm) before placement in experimental containers. Source water was pumped from Oyster Landing in Crab Haul Creek, North Inlet (33°20.96´N, 79°11.35´W). Crab sex and species were assessed in the field and con- firmed with a dissecting microscope post experiments to minimize handling stress prior to experiments. Two treatments were used: unparasitized (externa-lacking) and parasitized (exter- na-bearing) E. depressus. The unparasitized treatment consisted of male E. depressus mud crabs (8–13 mm CW). Male E. depressus were used to avoid accidental inclusion of gravid females that feed at lower frequencies than nongravid crabs (Mantelatto and Christofoletti 2001). Externa-bearing E. depressus (8–13 mm CW) of both sexes were used as the parasitized treatment because parasitic castration effects made sex difficult to discern in the field (Daugherty 1969). Only infected E. depressus with mature externae were used. Crabs 8–13 mm CW were used because concurrent field 614 Bulletin of Marine Science. Vol 90, No 2. 2014 collections in Clambank Creek (see Parasite Prevalence) found that 90% of parasit- ized crabs were within this size range (52/58). Crabs were arbitrarily assigned to identical flow through containers (150 × 150 × 90 mm) and separated into treatment-specific tanks to prevent the spread of parasite larvae to the unparasitized treatment. Containers held 0.75 L of seawater and had ap- proximately 60 holes 3 mm in diameter throughout to promote seawater exchange. Crabs were starved for 12 hrs before each experiment to standardize hunger levels (Bisker and Castagna 1987). Experiments were conducted in two flow-through sea- water tanks (100 cm diameter and 40 cm height). Tank 1 held 19–21 containers per experiment, while Tank 2 held 17–23 containers per experiment, depending on how many crabs were available for each treatment after collection and acclimation period. Treatments were switched between tanks at the start of each experiment to mini- mize potential tank effects. The experiment was conducted four times, with each experiment containing 19–23 parasitized and 17–21 unparasitized crabs housed in- dividually in containers. A single experiment lasted 72 hrs and consisted of identical containers, each containing one crab of either treatment and 15 live mussels attached to a tile. Flow-through seawater temperature [23.9 °C (SD 2.0)] and salinity [32.8 (SD 2.7)] were measured twice daily during the experiments. Daily mean water temperature and salinity in Clambank Creek when experimental crabs and mussels were collect- ed (2 May–2 June, 2012) was 23.7 °C (SD 1.2) and 34.6 (SD 2.3), respectively (NOAA, http://cdmo.baruch.sc.edu). The target temperature and salinity conditions for this study were 20–25 °C and 24–35, respectively (Whetstone and Eversole 1981, Bisker and Castagna 1987). Any mussels remaining in containers at the end of the 72-hr period were count- ed and consumption rate was estimated as the number of mussels eaten crab−1 72 hrs−1. Normal feeding behavior in crabs is disrupted immediately prior to ecdysis (O’Halloran and O’Dor 1988), so containers were examined after each experiment for molted exoskeletons and/or “soft” crabs. All E. depressus were frozen at the con- clusion of each experiment for subsequent examination of sex and measurement of CW. Because the time from parasite settlement on the host to inoculation takes 48– 72 hrs, and from infection to externa emergence (internal phase) is 25–42 d (Walker et al. 1992, Glenner 2001), some E. depressus in the unparasitized treatment may have been infected but had not yet expressed an externa. Crabs in the unparasitized treatment were therefore dissected, microscopically examined, and all tissue was ex- tracted for DNA using DNeasy Blood and Tissue Kit (Qiagen, Inc., Valencia, CA), following the protocol for animal tissue. Externa-bearing E. depressus were used as the positive control (Sherman et al. 2008). Extracted DNA was amplified (PCR) using L. panopaei-specific primers to identify the presence of the interna stage S( herman et al. 2008). The PCR conditions used to amplify the parasite and host 18S rDNA were: 5.0 µl 5× MyTaq Red Reaction Buffer, 0.25 µl MyTaq HS DNA polymerase, 1 µl DNA template and 10 µM of each primer in a 25-µl reaction using the standard MyTaq PCR cycling conditions, but with an initial denaturation time of 2:45 and a 45 °C annealing temperature. Crabs that molted (n = 3), died (n = 2), possessed a double externa (n = 3), were be- low the target size threshold (n = 1), lost a chela (n = 1), did not consume mussels (n = 60), or whose sex was misidentified in the fieldn ( = 17) were excluded from analyses after post-experiment examination. Non-feeding E. depressus were excluded from O’Shaughnessy et al.: L. panopaei reduces feeding in E. depressus 615 analysis here because captivity effects can cause a loss of appetite S( eed 1980), and so some crabs were not capable of foraging (they exhibited an abnormal feeding be- havior). The number of non-feeding E. depressus removed from analysis was equally distributed between the parasitized and the unparasitized treatments (45% and 46%, respectively). No crabs died during the acclimation period. Statistical analyses were conducted using data from 43 parasitized and 29 unparasitized E. depressus. Data Analyses.—Monthly parasite prevalence was calculated as the number of infected E. depressus divided by the total number of E. depressus collected during that month and expressed as a percentage. A Pearson correlation was used to eval- uate the relationship between mean monthly water temperatures calculated from continuous data collected by the NERRS monitoring program (NOAA, http://cdmo. baruch.sc.edu) and monthly parasite prevalence. The cumulative demographic of un- parasitized E. depressus was compared with that of parasitized individuals. Nonparametric Kruskal-Wallis tests were used to assess biotic (consumption rates among parasitized crabs, consumption rates among unparasitized crabs, CW among parasitized crabs, CW among unparasitized crabs, and CW between parasitized and unparasitized crabs) and abiotic (water temperature and salinity) parameters across all experiments to determine if they could be analyzed together. Consumption rates between parasitized and unparasitized E. depressus were compared using a Kruskal- Wallis test because the data were not normally distributed and could not be normal- ized by routine transformations. Data analyses were performed using the statistical software PAST (PAlaeontological STatistics; Hammer et al. 2001) with an a priori alpha set at 0.05.
Results
Parasite Prevalence.—In total, 546 xanthid crabs were collected and exam- ined (384 E. depressus and 162 P. herbstii), and only E. depressus crabs were found with L. panopaei infection. A total of 66 out of 384 E. depressus were parasitized (overall prevalence 17.2%) with a mean monthly prevalence of 18.2% ± 6.2 (mean ± 95% CI), which ranged from 9.4% to 30.3% (January and June, respectively). Sample sizes of E. depressus collected per month were as follows: January, n = 64; February, n = 43; March, n = 55; April, n = 44; May, n = 53; June, n = 33; July, n = 32; August, n = 60. There was a significant positive correlation between mean monthly water temperatures and monthly parasite prevalence (Pearson correlation: r = 0.72, P = 0.04). Collections from 26 January–29 June indicated that prevalence increased with increasing water temperature until it reached 30.3% and then declined through 31 July (21.9%) and 28 August (13.3%) as water temperatures remained stable (26.2–30.3 °C; Fig. 1). Size Characteristics of Collection.—The mean CW of all E. depressus col- lected (parasitized and unparasitized, n = 384) was 8.2 mm (SD 2.7; median 8.4 mm). The size range for unparasitized crabs n( = 318) was 2.3–17.0 mm CW with a mean CW of 8.0 mm (SD 2.8), while the size range for parasitized crabs (n = 66) was nar- rower at 5.8–14.0 mm CW with a mean CW of 9.7 mm (SD 1.4; Fig. 2). The total population sex ratio was 1.7:1 (F:M), with sex ratios of 1.5:1 and 2.2:1 in the unpara- sitized and parasitized collections, respectively. 616 Bulletin of Marine Science. Vol 90, No 2. 2014
Figure 1. Percent prevalence of Loxothylacus panopaei and associated daily mean water temper- ature (°C) in Clambank Creek, North Inlet, South Carolina (January–August 2012). Error bars represent standard deviation. At least 32 Eurypanopeus depressus were collected each month (January, n = 64; February, n = 43; March, n = 55; April, n = 44; May, n = 53; June, n = 33; July, n = 32; August, n = 60).
Feeding Experiment.—The four experiments were treated as replicates and all data were pooled after Kruskal-Wallis tests determined there were no significant differences within biotic and abiotic parameters across all experiments. Parasitized E. depressus consumed significantly fewer mussels than unparasitized E. depressus (Kruskal-Wallis: H = 5.94, df = 1, P = 0.02). The median number of mussels consumed by unparasitized E. depressus was 4 per 72 hrs [mean: 4.9 (SD 3.7)], while parasitized E. depressus consumed a median of only 2 mussels [2.7 (SD 2.0)]. When the crabs in the unparasitized treatment were dissected and examined, no crabs showed visual signs of the internal L. panopaei stage. DNA analyses of the control and unparasit- ized crab tissues resulted in no or weak PCR products, and so these analyses could not be used to verify parasite absence in the unparasitized treatment. Therefore, we defined parasitized E. depressus as “externa-bearing” and unparasitized E. depressus as “externa-lacking.”
Discussion
Field observations describe a trend in increasing parasite prevalence with increas- ing water temperatures from 26 January to 29 June, with observed population preva- lence as high as 30.3% in late June 2012. The mean monthly prevalence of L. panopaei was 18.2% ± 6.2 (mean ± 95% CI). Eurypanopeus depressus composed 70% of the North Inlet collections and were the only crabs found with infection in Clambank Creek, while Panopeus herbstii made up 30% of the collections and were never infected. The present study is the first to investigate the effects of the invasive rhizocephalan parasite L. panopaei on E. depressus consumption of bivalves. Infection by L. pano- paei reduced E. depressus mussel consumption by a factor of 2. Future descriptions O’Shaughnessy et al.: L. panopaei reduces feeding in E. depressus 617
Figure 2. Carapace width frequency distribution of parasitized and unparasitized Eurypanopeus depressus collected January–August 2012 in Clambank Creek, North Inlet, South Carolina. Total crab sample size (parasitized and unparasitized) is the sum of the filled and unfilled bars. Numbers above bars represent carapace width range (mm) within each bar. of mud crab-bivalve dynamics in regions with L. panopaei should consider the indi- rect effects of parasite-mud crab interactions on oyster reef trophic dynamics. Sampling from Clambank Creek indicates that an average of almost one in five E. depressus is unable to reproduce due to infection with L. panopaei. Parasitic cas- tration causes a reduction in the effective E. depressus population size (Van Engel et al. 1966) and may decrease genetic diversity, although it is difficult to determine to what extent (Daugherty 1969). More females than males were infected in North Inlet (2.2:1, F:M), similar to findings from Chesapeake Bay (1.7:1, Daughtery 1969). Infection may reduce overall population fecundity by removing females at a higher rate than they are represented in the total population. These consumption data in conjunction with previous rhizocephalan–host behav- ioral studies (Bishop and Cannon 1979, Wardle and Tirpak 1991) suggest that in- fected E. depressus may be energetically compromised, reducing overall fitness and mobility. Decreased mobility of an infected individual may reduce foraging as well as burrowing behavior (Bishop and Cannon 1979, Wardle and Tirpak 1991). Parasitized Callinectes sapidus Rathbun, 1896 were less aggressive when feeding, and rarely bur- rowed below the sediment (Wardle and Tirpak 1991). Sand crabs Portunus pelagi- cus (Linnaeus, 1758) infected by the rhizocephalan Sacculina granifera Boschma, 1973 buried slower [72 s (SD 56)] than ovigerous female sand crabs [39 s (SD 31)], and significantly slower than non-ovigerous sand crabs [10 s S( D 7); Bishop and Cannon 1979]. Normal mud crab feeding behavior is likely interrupted in an infected individu- al because of mechanical and physiological hindrances. The crevices within oyster shell cultch provide protection for E. depressus (Meyer 1994) and a place for them to forage (McDermott 1960), but the externa on infected individuals likely compli- cates crab maneuverability in oyster reef interstices. Rhizocephalan-infected P. pe- lagicus exhibit a different stance because the externa pushes the exterior portion 618 Bulletin of Marine Science. Vol 90, No 2. 2014 of the cephalothorax higher so that it is almost horizontal (Bishop and Cannon 1979), which may alter normal feeding behavior. Isaeva et al. (2001) found that the rhizocephalan Sacculina polygenea Lützen and Takahashi, 1997 internal structure surrounds digestive and reproductive organs of its brachyuran host Hemigrapsus sanguineus (De Haan, 1835). It is possible that internal organ stress may interfere with a host’s digestive efficiency, indirectly altering foraging ability. The L. panopaei invasion may cause trophic changes in intertidal oyster reef eco- systems because the parasite reduces predation intensity in the mud crab-mussel predator-prey relationship. But the presence of this invasive rhizocephalan changes the natural relationships between predators and prey because the parasite arrests growth of the host at inoculation. O’Brien and Van Wyk (1985) suggested that rhi- zocephalan parasites may skew the mud crab population toward smaller individuals that typically consume smaller prey (McDermott 1960, Seed 1980). In our study, the mean CW of infected crabs [9.7 mm (SD 1.4)] was larger than the CW of uninfected crabs [8.0 mm (SD 2.8)]. Eurypanopeus depressus collections by Hines et al. (1997) found infection in crabs 6–18 mm CW, and thus support for a smaller-skewed in- fected population is lacking. Regardless of how the parasite skews the infected popu- lation, mussels consumed by crabs of the infected size range may never be released from predation by E. depressus under the infection scenario, creating a bottleneck whereby few mussels survive to reach larger sizes. We compared feeding rates of unparasitized male E. depressus with parasitized male and female E. depressus. Elner (1980) and Sumpton and Smith (1990) showed that feeding rates between male and non-gravid female crabs were not significantly different, thus the results reported here for unparasitized males are likely unbiased. Future feeding rate studies should accommodate potential sex-biased behavioral dif- ferences to further support the differences between parasitized and unparasitized crab foraging behavior. We amplified host and parasite 18S rDNA post experimentation to detect the par- asite’s cryptic internal stage during the feeding experiments, but DNA analysis re- sulted in no or weak PCR products in the control and unparasitized crabs. Sherman et al. (2008) were successful in amplifying Loxothylacus texanus Boschma, 1933 18S rDNA in a background of host (C. sapidus) DNA using L. texanus-specific primers. They preserved crab and parasite DNA by injecting 95% ethanol into the crab oral cavity, and then stored all crabs in 95% ethanol, whereas we froze all crabs for pres- ervation. In our study, amplification of L. panopaei DNA was possibly problematic because the parasitic DNA was degraded from the freeze-thaw process (Lahiri and Schnabel 1993). Knowledge of L. panopaei populations in South Carolina estuaries has, to date, been limited. Hines et al. (1997) did not find parasitizedE. depressus at their South Carolina collection sites (North Inlet and Charleston; 1983, 1986), despite finding L. panopaei in Maryland, Virginia, and North Carolina. Mud crab reports of collec- tions made in North Inlet, South Carolina, in the 1970s and 1980s make no mention of infected individuals (Dame and Vernberg 1982, McDonald 1982). The absence of L. panopaei in North Inlet reported by Hines et al. (1997) may relate to the number of E. depressus examined (n = 20). Our collections in June and July 2012 (n = 33 and n = 32 E. depressus, respectively) reported L. panopaei prevalence at 30.3% and 21.9%, respectively. The relatively high prevalence reports from these sample sizes here sug- gest that the parasite was absent in North Inlet E. depressus in 1986. Hines et al. O’Shaughnessy et al.: L. panopaei reduces feeding in E. depressus 619
(1997) showed there was a significant temporal fluctuation in E. depressus parasite prevalences collected in the summers of 1983 and 1986. They reported 47.4% para- site prevalence in Bogue Sound, North Carolina, in 1983, but 0% infection in 1986. Similarly, they found 0% prevalence in Chincoteague, Virginia, in 1983, but 16.7% in 1986. It is possible that L. panopaei was present in North Inlet in 1986, but in such low densities that a sample of 20 crabs might have been too small to detect parasite presence due to likely prevalence fluctuations over time. As far as we are aware, the present study is the first report of L. panopaei at Clambank Creek, North Inlet, South Carolina. Here we demonstrated that L. pano- paei has a significant negative effect on E. depressus consumption of prey with clear implications for individual growth and fitness. Additionally, these data suggest that L. panopaei invasion may have wider effects on the role of mud crabs in oyster reefs ecosystems.
Acknowledgments
The authors extend thanks to K Godwin C( oastal Carolina University, Department of Biology) for guidance in statistical analyses. D Allen and P Kenny (Baruch Marine Field Laboratory, Belle W Baruch Institute for Marine and Coastal Sciences, University of South Carolina) provided wet lab and facilities. Access to Clambank Landing was provided by the Belle W Baruch Foundation–Hobcaw Barony. This work was completed in partial fulfillment of the requirements for an MS thesis (K O’Shaughnessy) from Coastal Carolina University. The research was partially funded by grants to K O’Shaughnessy from the Coastal Carolina University Research Incentive Grant program, the MK Pentecost Foundation, and the Slocum-Lunz Foundation.
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