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Survival and Growth of the Stygophilic Amphipod Gammarus troglophilus UnderLaboratory ConditionsAuthor(s): Daniel Nelson and Frank M. WilhelmSource: Journal of Crustacean Biology, 31(3):424-433. 2011.Published By: The Crustacean SocietyDOI: http://dx.doi.org/10.1651/10-3431.1URL: http://www.bioone.org/doi/full/10.1651/10-3431.1

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SURVIVAL AND GROWTH OF THE STYGOPHILIC AMPHIPOD GAMMARUS TROGLOPHILUS UNDER

LABORATORY CONDITIONS

Daniel Nelson and Frank M. Wilhelm

(DN) Department of Fish and Wildlife Resources, College of Natural Resources, University of Idaho, PO Box 441136, Moscow, ID

83844, U.S.A. Current address: Department of Biological Sciences, University of Alabama, Aquatic Biology Program, A126 Bevill

Building, 201 Seventh Ave, Box 870206, Tuscaloosa, Alabama 34587, U.S.A.;

(FMW, corresponding author, fwilhelm@uidaho.edu) Department of Fish and Wildlife Resources, College of Natural Resources,

University of Idaho, PO Box 441136, Moscow, Idaho 83844, U.S.A.

A B S T R A C T

Amphipods play important roles in the cycling of nutrients and energy in many aquatic systems where they display a wide range of

feeding modes ranging from detritivore to predator. Although the biology of many amphipod species has been examined, little is known

of hypogean amphipods inhabiting cave streams. Gammarus troglophilus is a stygophilic amphipod that co-occurs with the federally

endangered stygobiont G. acherondytes in cave streams of the Salem Plateau Karst Region of southwestern Illinois. With the goal to

establish a self-sustaining laboratory population of cave amphipods to obtain amphipods for lethality experiments, we tested hypotheses

relating the survival and growth rates of G. troglophilus collected from cave streams to different laboratory conditions of food and water

velocity. We used a series of microcosm experiments to test the hypotheses that survival and growth are not affected by type of water

(cave water vs. amended water), water velocity (static vs. dynamic/recirculating), or the type of available food (sediment vs. sediment,

leaf discs, and TetraMinH). We also tested if different food treatments affected the survival and/or growth of juvenile amphipods and

newly released neonates in static chambers. Our results indicate that cave water was important for survival because no amphipods

survived past 30 days in experiments with water amended to resemble cave water. The addition of food (leaf discs and TetraMinH) and

water velocity affected survival but not growth rates in microcosm experiments. Food treatment (leaf discs vs. TetraMinH) did not

significantly affect survival or growth rates of juvenile amphipods. However, leaf discs increased the survival and growth of neonate

amphipods. Overall, survival was low in all experiments and further research is needed to examine the effects of handling stress on

survival during experiments because amphipods left in stock tanks survived and grew well.

KEY WORDS: cave ecosystems, Gammarus troglophilus, growth, stygobionts, survival

DOI: 10.1651/10-3431.1

INTRODUCTION

Crustacean amphipods are common and widespread inaquatic ecosystems throughout the world (MacNeil et al.,1999) and are important in the cycling of matter and energy(Robinson and Mann, 1980; Howard, 1982; Karlson et al.,2007). In many freshwater ecosystems, gammarid amphi-pods are often the dominant macroinvertebrate in terms ofbiomass and/or abundance (Shaw, 1979; MacNeil et al.,1999). They display a range of feeding modes ranging fromdetritivores to grazers and predators/cannibals, while in turnthey are important prey for fish and other macroinvertebrates(MacNeil et al., 1997; MacNeil et al., 1999; Wilhelm andSchindler, 1999). Although much is known about crustaceanamphipods in surface waters, little is known about thebiology or ecology of amphipod species that live in caveecosystems, or groundwater – the hypogean environment.Because these systems are light, and thus energy-limited(Huppop, 2000; Graening and Brown, 2003), the role ofamphipods could be vastly different than in surfaceecosystems. To adequately protect and make informeddecisions about hypogean ecosystems requires that weunderstand the biology and ecology of species within them.

In southwestern Illinois, the Salem Plateau Karst Region(SPKR) extends from just north of East St. Louis south to theShawnee National Forest. It is characterized by a highsinkhole density, as high as 90 sinkholes/km2 (Panno et al.,1996), springs, and caves resulting from the dissolution ofcarbonate rocks (limestone). The soil cover is thin, andrecharge to the shallow groundwater is rapid and highlysusceptible to contamination from livestock waste, row cropagriculture, and private wastewater treatment systems(Panno et al., 1996; U.S. Fish and Wildlife Service, 2002).Farming and urbanization have been implicated as threats tothe groundwater fauna of the region (U.S. Fish and WildlifeService, 2002). Cave stream ecosystems in the SPKR areoften dominated by amphipods (Lewis, 2003; Lewis andLewis, 2007) including Gammarus troglophilus Hubrichtand Mackin, 1940, a common stygophilic (aquatic, faculta-tive cave organism) that co-occurs with the federallyendangered stygobiontic (aquatic, cave obligate) amphipod,Gammarus acherondytes Hubricht and Mackin, 1940,endemic to a 230 square kilometer area of the SPKR.

Gammarus acherondytes is the focus of a U.S. Fish andWildlife Service recovery plan aimed to delist it by the year

JOURNAL OF CRUSTACEAN BIOLOGY, 31(3): 424-433, 2011

424

2030 (U.S. Fish and Wildlife Service, 2002). However, thiswill require tough management decisions including poten-tially setting minimum acceptable concentrations ofconstituents such as dissolved oxygen to ensure a suitablehabitat for G. acherondytes. Because the species’ lifehistory, ecology, and role in its subterranean habitat havenot been well-studied, setting such limits is difficult.Furthermore, because many populations of G. acherondytesare at a low density or declining, harvesting wildindividuals for dose-response-type lethality experiments isnot possible. Instead, a self-sustaining laboratory popula-tion from which individuals could be harvested to discoverwhich environmental parameters and their bounds are crucialfor its survival would be ideal. Continuous cultures of G.acherondytes also could provide individuals for potentialreintroduction into habitats from which it is currentlyextirpated. Jenio (1980), Taylor et al. (2003), and Wilhelmet al. (2006) have demonstrated that G. acherondytes and G.troglophilus can be maintained in the laboratory forsufficient periods of time to conduct short-term experiments.Based on life history information obtained for G. acher-ondytes, Venarsky et al. (2007) suggested that a successfullaboratory population of G. acherondytes could be estab-lished in approximately 1.5 years.

Because G. troglophilus is not endangered, but is verysimilar to G. acherondytes in terms of habitat occupied(Taylor and Webb, 2000), apparent food selection andtrophic position (Nelson, 2010), it has been suggested as asurrogate species to develop protocols for the laboratoryculture of G. acherondytes (U.S. Fish and Wildlife Service,2002).

Our objectives were to experimentally test hypothesesrelating the survival and growth rates of G. troglophiluscollected from cave streams to different laboratory condi-tions of water type, food, and flow to determine optimalconditions under which G. acherondytes should survive in alaboratory setting. Specifically, to test the hypothesis thatsurvival and growth were not affected by type of water,water velocity, or the type of available food, we used a seriesof small microcosm experiments filled with natural cavewater or amended water (amended to resemble cave water inregards to certain chemical constituents) under static orcontinuous flow with different food treatments (cavesediment vs. cave sediment, leaf discs, and TetraMinH fishflakes). Amended water was tested because of the potentialdesire to rear and perform experiments with cave amphipodsaway from a source of cave water. In addition, a rearing/maintenance protocol may offer the chance to temporarilyharbor fauna should a waterway in a cave be impacted, e.g.,surface spill that could threaten cave fauna. In addition tothese microcosm experiments, we also tested if differentfood treatments affected the survival and/or growth ofjuvenile amphipods (approximately 4 mm total length) andnewly released neonates (approximately 2.5 mm totallength). Few studies have compared growth rates ofamphipods given different diets, especially those recentlyreleased from the brood pouch. Thus, our results willincrease the understanding of amphipods inhabiting thegroundwater of the SPKR. Furthermore, our outcomes willprovide conservation personnel with strategies to rear the

endangered G. acherondytes as well as shape recommenda-tions to better manage karst habitat.

MATERIALS AND METHODS

Collection Sites

Gammarus troglophilus were obtained from Reverse Stream Cave inMarch 2009 and Spider Cave in June 2009 and February 2010 in the SPKRof southwestern Illinois. Reverse Stream Cave is a small cave withapproximately 50 m of humanly enterable passage and a maximum ceilingheight of 1 m. Technically, it is a karst window with access to thegroundwater system that is part of a much larger system (Aley et al., 2000;Aley and Moss, 2001). Stream width varied from 1 to 3 m, while depthvaried from 0.05 to 0.5 m. The substrate was composed of large (3-10 cmdiameter) gravel in the riffles and fine silt and sediment in the pools. Thecave stream is fed from a small rise pool adjacent and upstream of the caveentrance. Due to the small size of the cave, the entire accessible portion ofthe groundwater system expressed in this cave is in the twilight zone.

Spider Cave is accessed via a 3.5 m deep pit entrance at the base ofwhich is a wide, standing height stream passage. The ceiling lowers in bothdirections to approximately 1 m, 50 m up- and downstream from theentrance. The stream width varied from 1 to 2 m, while depth varied from0.025 m to 0.5 m. The substrate varied from large breakdown boulders, tocobbles, gravel, and fine cave sediment. Collections were madeapproximately 20 m upstream of the pit entrance.

The stream fauna of both caves are similar to the fauna of other caves inthe region. Based on census data (Lewis, 2003; Lewis and Lewis, 2007),both caves contain G. acherondytes, G. troglophilus, and the isopodsCaecidotea brevicauda (Forbes, 1876) and C. packardi Mackin andHubricht, 1940. Spider Cave also contains the flatworm Sphalloplanahubrichti Hyman, 1945.

Collection of Amphipods

To collect G. troglophilus from the cave streams, a small aquarium net wasplaced vertically in the stream and the substrate just upstream of the netwas gently disturbed. Amphipods disturbed from the substrate were carriedinto the net by the current. In addition, amphipods were collectedopportunistically if encountered. The contents of the net were inspectedand carefully placed into plastic holding containers filled with cave water.Amphipods identifiable as G. acherondytes and other invertebrates(isopods C. brevicauda and C. packardi; flatworm S. hubrichti) werecarefully removed from the nets and immediately returned to the stream.Remaining amphipods were identified on site to species with the aid of adissecting microscope and only G. troglophilus were collected, while G.acherondytes were returned to the stream. Retained amphipods wereseparated by eye into small, medium, and large body size and placed into5 L plastic containers filled with cave water in a cooler for shipping viaovernight courier to the University of Idaho in Moscow, Idaho.

Maintenance of Amphipods

Individuals of G. troglophilus were kept in 5 L glass aquaria filled withapproximately 4.5 L of cave water and stocked with a bottom layerapproximately 0.5 cm deep of small (5 mm to 10 mm diameter) aragonite(CaribSea, Inc.), 8 to 12 cave rocks of various sizes, and small amounts ofcave sediment (approximately 15 g wet weight). Aquaria were equipped withsingle air stones set to release bubbles at a slow rate (20-50 bubbles/min) andfood (TetraMinH fish flakes) was added ad libitum. Aquaria were housed in atemperature controlled room at 15uC under total darkness. Amphipods werekept in these aquaria for several days before the experiments began. Surfacearea to volume ratio in this set up was 10. We did not measure dissolvedoxygen (DO) concentrations of the water in the stock tanks, but assumed thatthe air stone supplied sufficient oxygen replenishment. This assumption wasmade based on DO measurements made by Wilhelm et al. (2006) who used asimilar set up to hold cave amphipods.

Experimental Design

Microcosm Experiments.—Experimental microcosms were made fromsections of plastic gutter material sealed with end caps and equipped with

NELSON AND WILHELM: SURVIVAL AND GROWTH OF GAMMARUS TROGLOPHILUS 425

standpipes to remove water. Standpipes functioned only in dynamic/recirculating microcosms. For experiments ASN and ADN (see Table 1 forexplanation), microcosm dimensions (101.6 cm L 3 11.5 cm W 3 6.5 cmD) were slightly larger than all other experiments (61.0 cm L 3 11.5 cm W3 6.5 cm D). After experiments ASN and ADN, microcosms wereshortened to make visual observations of amphipods easier and to shortenthe time of disturbance when searching and locating individuals. Of theoverall length, a 10 cm section of each microcosm was screened off with500 mm-mesh Nitex to allow installation of the standpipe to control waterlevel and prevent amphipods from exiting. To maintain total darkness andhigh humidity (high humidity to reduce evaporative loss) similar to caveconditions, microcosms were kept under a frame covered with 10 mil thickblack construction plastic. All microcosms were housed on a bench in awalk-in temperature controlled room set at 15uC. Each microcosm wasstocked with 12 to 15 rocks (10 to 200 mm in diameter, longest axis), asmall handful of pebbles (, 10 mm in diameter, longest axis), andsediment (approximately 15 g wet weight) collected from the cave streamsat the same time of amphipod collections. Sediment was first mixed withsome cave water and filtered through a 1 mm-mesh sieve to removeparticles . 1 mm.

We used static and dynamic water velocities to mimic pool and rifflesections of cave streams. In its native cave streams, G. troglophilusoccupies both riffles and pools with fast and slow moving water,respectively (Taylor and Webb, 2000; Lewis and Lewis, 2007). Staticmicrocosms were initially filled with 3 L of water and aerated using asingle 2.5 cm air stone. The surface area to volume ratio in this set up was25. Dynamic microcosms consisted of the experimental flume directlyabove a reservoir and were initially filled with 5 L of prepared water.Water was re-circulated from the reservoir to the experimental flume witha small aquarium pump (Micro-Jet 320, Aquarium Systems, Inc.) at a rateof approximately 5.28 L/min. The surface area to volume ratio in this setup was 50. Approximately 1.5 L of cave water was maintained in eachexperimental flume that contained amphipods, while the remaining waterwas in the reservoir containing the pump. Half of the entire water volumein each microcosm was replaced biweekly to prevent build up ofnitrogenous waste, replace any water lost to evaporation, and prevent largefluctuations in chemical concentrations.

We tested different treatment combinations of water source (native cavewater vs. amended cave water), food addition (conditioned leaf discs andTetraMinH vs. no food), and water velocity (static system vs. dynamic/recirculating system) in triplicate (Table 1). Alkalinity in each experi-mental microcosm was measured approximately weekly using standardtitration methods (Eaton et al., 2005) with a digital titrator (Hach Model16900).

To test the hypothesis that water type (cave water or amended water)does not affect the survival or growth of G. troglophilus, we used nativecave water collected at the same time as amphipods and returned to the labin bulk, or ground water from Moscow, Idaho that was amended with ReefBuilder (SeaChem Laboratories, Inc.) to increase alkalinity concentrationssimilar to those reported by Panno et al. (1996) and Taylor and Webb(2000) for caves of the SKPR. The Moscow ground water came from aUniversity of Idaho well and had been tested at the University of IdahoAnalytical Sciences Laboratory in Moscow, Idaho for alkalinity, hardness,and other trace elements prior to the start of the experiments.

To test the hypothesis that the survival and growth of G. troglophilus isnot affected by type of food, we provided different food sources. Theseincluded cave sediment only (No food) or the addition of a mixtureincluding red maple (Acer rubrum) leaf discs (7 mm diameter) and

TetraMinH fish flakes (in addition to the cave sediment and rocks). Redmaple was used because it is commonly found in forests of the SPKR.Also, skeletonized maple leaves (Acer spp.) are often found in cavestreams of the Salem Plateau (Nelson, personal observation). Leaf discswere conditioned for at least two weeks prior to the beginning of eachexperiment. Several studies have found that gammarid amphipods preferand often survive better on leaves which have a viable fungal flora(Kostalos and Seymour, 1976; Barlocher and Kendrick, 1975). In addition,TetraMinH fish food has been used to rear amphipods successfully in thelaboratory (DeMarch, 1981). Ten leaf discs (approximately 0.100 g wetweight) and 0.075 g of TetraMinH fish flakes were added to themicrocosms every 4-5 days. Uneaten food and skeletonized leaf discsfrom previous additions were removed when new food was added.

At the beginning of each experiment and at 30 day intervals thereafter,surviving amphipods from each experiment were photographed using aCanon A630 digital camera mounted on a Wild M3 dissecting microscopewith trinocular head. We measured the head capsule length (HD) of eachamphipod on digital images using ImageJ (Rasband, 1997). Total lengthwas estimated from the relationship:

TL~8:33 HDð Þ{0:018

Where: HD - head capsule length (mm), and TL - total length (mm)(Wilhelm et al., 2002). Attempts were made to check survival morefrequently (weekly), but this proved to be difficult without disassemblingthe microcosm and thus compromising the experiments.

Juvenile Amphipod Experiment.—To compare growth rate and survival ofjuvenile amphipods reared on different foods, 17 juvenile amphipods ofsimilar size (approximately 4 mm total length) from a single amphipodstock tank were placed into individual 150 ml wide-mouthed glass flasksfilled with 90 mL of cave water and randomly assigned to either leaf disc(n 5 8) or TetraMinH fish flake (n 5 9) food. The surface area to volumeratio in this set up was 40. The amphipods used in this experiment musthave been released by a female (or multiple females) while in the stocktank because we did not collect amphipods of this size from cave streams.Amphipods in the leaf disc treatment were given one conditioned leaf disc(7 mm diameter, approximately 0.01 g wet weight) once a week, whilethose receiving TetraMinH were given 0.01 g of TetraMinH once a week.Old food was removed when new food was added and water was changedon a weekly basis. All jars contained one small rock (approximately 10 mmL 3 10 mm W 3 10 mm H) as shelter and the entire experiment wasmaintained in the dark at 15uC. To examine survival, each individual waschecked weekly by carefully examining each amphipod for movement ofpleopods. As a result of the weekly check, survival was recorded as thenumber of weeks surviving. At the beginning of the experiment and everytwo weeks thereafter, all surviving amphipods were photographedindividually and measured as described above.

Neonate Amphipod Experiment.—To determine the growth rates andsurvival of newly hatched amphipods on different foods, 24 neonates werecollected and separated immediately after release from a single gravidfemale amphipod from an amphipod stock tank. Neonates were placed inindividual 100 mL wide-mouthed plastic sample jars filled with 80 mL ofcave water. The surface area to volume ratio in this set up was 14. Each jarwas randomly assigned to one of four food treatments: Control (C) (n 5 6) -no food or cave sediment. In the Disc (D) treatment (n 5 6), amphipodsreceived one red maple leaf disc (7 mm diameter) once a week. Amphipods

Table 1. Table showing water source (A - amended, C - cave), water velocity (S - static, D - dynamic flow at 88 ml/s), and food (N - no food, F - foodpresent) treatments in replicated (33) microcosm experiments in small stream channels. Dates refer to the starting and ending date of each experiment andvary because of limited space to run each experiment as well as the availability of amphipods.

Experiment ID H2O source H2O velocity Food treatment Source cave Date

ASN Amended Static No Food Reverse Stream 8 April 2009-7 May 2009ADN Amended Dynamic No Food Reverse Stream 8 April 2009-7 May 2009CSF Cave Static Food Spider Cave 26 June 2009-25 August 2009CDF Cave Dynamic Food Spider Cave 26 June 2009-25 August 2009ASF Amended Static Food Spider Cave 22 February 2010-23 March 2010ADF Amended Dynamic Food Spider Cave 22 February 2010-23 March 2010CSN Cave Static No Food Spider Cave 22 February 2010-23 March 2010CDN Cave Dynamic No Food Spider Cave 22 February 2010-23 March 2010

426 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 31, NO. 3, 2011

in the Sediment (S) treatment (n 5 6) received 0.005 g (wet weight) ofsediment as food, while amphipods in the Disc + Sediment (DS) treatment (n5 6) received 0.005 g of sediment and one leaf disc once a week. Water,sediment, and food were changed weekly. All neonates were given onesmall (approximately 10 mm L 3 10 mm W 3 10 mm H) rock for shelter.Amphipods were kept at 15uC in total darkness. Survival was checkedweekly. At the beginning of the experiment and every two weeks after, eachsurviving neonate was measured as described above.

Statistical Analyses

Microcosm Experiments.—Because amphipods only survived past day 30in the CSF and CDF experiments, they are the only experimentsconsidered for full analysis. To compare the survival of amphipods inthese experiments, we used a repeated-measures ANOVA with onebetween-subject factor (water velocity - static or dynamic) and one within-subject factor (time - T1 5 Day 30 and T2 5 Day 60) (von Ende, 1993).The treatment (water velocity) 3 time interaction was also considered.Percent survival data were arcsine square-root transformed to meetassumptions of normality and homogeneity of variance and forundertaking parametric tests with percent data.

Growth rates were calculated using the following linear growth rateformula:

G~ S2{S1ð Þ= t2{t1ð Þ

Where G is growth in mm day21; S2 is the mean TL in mm at time periodt2; S1 is the mean TL in mm at time period t1. We used a linear growth rateformula because growth over the short period of time (60 days) was bestrepresented by a linear compared to a specific (exponential or logarithmic)growth model. To compare growth rates over time between CSF and CDF,we used a repeated measures ANOVA with one between-subject factor(water velocity - static or dynamic) and one within-subject factor (time -T1 5 Day 1 to Day 30 and T2 5 Day 30 to Day 60).

Juvenile Amphipod Experiment.—To compare survival curves of juvenileamphipods between food treatments (leaf disc vs. TetraMinH), we used thenon-parametric log rank test (Pyke and Thompson, 1986; Bewick et al.,2004). The log rank tests the null hypothesis of no difference betweensurvival curves (Bewick et al., 2004). The test was performed in SASH 9.2using the procedure ‘‘PROC LIFEEST’’ (SAS Institute, 2009).

Growth rates for individual juvenile amphipods were calculated usingthe linear growth rate formula because growth over the short duration ofthe experiment was linear rather than exponential. To compare growthrates of amphipods between food treatments, we used a repeated measuresANOVA with one between-subject factor (food) and one within-subjectfactor (time). We also considered the treatment (food) 3 time interaction.

Neonate Amphipod Experiment.—To compare survival curves of neonateamphipods among treatments, analyses followed the methods described forjuveniles above. In addition, the number of weeks each individual survivedwas compared among treatments using an ANOVA followed by a Fisher’sLeast Significant Difference (LSD) multiple comparison test. Growth ratesof individual neonate amphipods were calculated using the linear growthrate formula. To compare growth rates during the first two weeks of lifeamong all four food treatments, we used an ANOVA followed by aFisher’s LSD multiple comparison. A test on the homogeneity of variancesindicated that variances were unequal. Therefore, we log transformed thedata before the ANOVA. Only one amphipod survived 28 days afterrelease in each of the S and DS treatments. Therefore, we used a repeatedmeasures ANOVA as above to compare growth rates of neonates betweentreatment C and Treatment D from day 1 to day 42 and omitted treatmentsS and DS from analysis. All statistical analyses were performed usingSASH 9.2 (SAS Institute, 2009).

RESULTS

Microcosm Experiments

Survival of amphipods in experiments with different watervelocity (static vs. dynamic), food additions, and watertypes was surprisingly low, with amphipods only surviving

past Day 30 in the CSF and CDF experiments (Table 2).Analysis of the CSF and CDF experiments showed thatsurvival declined over time (Table 3) and was related towater velocity (RMANOVA, P , 0.001), with highersurvival in the static vs. dynamic microcosms (Fig. 1).

The mean amphipod lengths of 10.1 mm 6 0.3 (SE) and10.3 mm 6 0.3 in experiments CSF and CDF, respectively,at the start of the experimental period were similar (t test,d.f. 5 45, t 5 0.49, P 5 0.730). After the 60 days, the meansize had increased to 12.3 mm 6 0.5, and 11.9 mm 6 0.2 inthe CSF and CDF experiments, respectively. Although theincrease in mean size of 2.2 mm in the CSF experiment waslarger than the 1.6 mm increase in the CDF experiment(Fig. 2), water velocity did not have a significant effect ongrowth (RMANOVA, P 5 0.276, Table 4). Althoughalkalinity varied between experiments, concentrationsusing cave water were within the annual range recordedfor caves in the Salem Plateau (Table 5).

Juvenile Amphipod Experiment

Results of the log rank test indicated no difference insurvival curves of juvenile amphipods between foodtreatments (d.f. 5 1, x2 5 0.73, P 5 0.396). The averagenumber of weeks amphipods survived was higher in theleaf disc treatment compared to the TetraMinH treatment,however, it was not significant (t test, d.f. 5 15, t 5 0.79, P5 0.442) (Fig. 3). In addition, the longest an amphipodsurvived in the TetraMinH treatment was 11 weeks whileone amphipod survived 14 weeks in the leaf disc treatment.

The length of juvenile amphipods at the beginning of theexperiment did not differ significantly between treatments(t test, d.f. 5 15, t 5 20.32, P 5 0.754) and ranged from3.21 to 4.37 mm. Average individual linear growth rates for

Table 2. Mean (6 SE) proportion of Gammarus troglophilus survivingafter 30 and 60 days of each experiment (CSF and CDF). The size range ofGammarus troglophilus used in each experiment is given; while Nindicates the number of individuals in each microscosm.

Experiment ID

Proportion surviving (mean 6 SE)

Size range (mm) N30 days 60 days

ASN 0.00 6 0.00 0.00 6 0.00 6.1-10.1 5ADN 0.00 6 0.00 0.00 6 0.00 5.3-9.1 5CSF 0.75 6 0.07 0.71 6 0.10 5.0-13.1 8CDF 0.90 6 0.08 0.35 6 0.18 7.6-14.8 8ASF 0.00 6 0.00 0.00 6 0.00 5.4-10.1 5ADF 0.00 6 0.00 0.00 6 0.00 6.1-10.6 5CSN 0.00 6 0.00 0.00 6 0.00 4.6-10.6 5CDN 0.00 6 0.00 0.00 6 0.00 6.2-10.9 5

Table 3. Results of repeated measures ANOVA comparing survivalbetween microcosm experiments CSF and CDF. Percent survival data werearcsine square-root transformed prior to analysis.

Effect d.f. F P

Between subjects

H2O velocity 1 128.74 , 0.001

Within subjects

Time 1 1.46 0.294H2O velocity 3 Time 1 0.62 0.477

NELSON AND WILHELM: SURVIVAL AND GROWTH OF GAMMARUS TROGLOPHILUS 427

the duration of the experiment or until time of death rangedfrom 0.001 mm d21 to 0.047 mm d21. In the leaf disctreatment, average individual growth rates ranged from0.008 mm d21 to 0.047 mm d21, while in the TetraMinHtreatment they ranged from 0.001 mm d21 to 0.037 mm d21.A repeated measures ANOVA indicated no time, treatment,or interaction effects (time 3 treatment) on juvenile growthrates (Table 6).

Neonate Amphipod Experiment

The log rank test indicated differences in neonate survivalamong the four food treatments (d.f. 5 3, x2 5 9.31, P 50.03). ANOVA comparisons showed differences in thenumber of weeks that amphipods survived in eachtreatment (F3, 20 5 4.56, P 5 0.014) with amphipods intreatment D surviving the longest (Fig. 4). At the beginningof the experiment, total lengths of neonate amphipodsranged from 2.31 mm to 2.68 mm but did not differbetween treatment (ANOVA, F3, 20 5 0.73, P 5 0.549).Average individual growth rates ranged from 0.003 mm d21

to 0.033 mm d21 (Fig. 5) and were higher in treatments

containing leaf discs (D and DS) than in other foodtreatments (ANOVA F3, 20 5 5.02, P 5 0.009; Fig. 5).Over the first 42 days after release from the brood pouch,growth rates were significantly higher in treatment D(0.023 6 0.002 mm day21) than in treatment C (0.009 60.002 mm day21) (Table 7).

Dissolved Oxygen Concentration in Experiments

Although we did not directly measure DO in the abovesuite of experiments, the surface area to volume ration in allexperimental set ups was between 1.4 to 5 fold higher thanin the stock tank in which amphipods survived well andfemales even released young. These young also grew andsurvived well. Thus, it is unlikely that low DO concentra-tions occurred in the experiments or influenced results. Inaddition, Wilhelm et al. (2006) found that stock tanks setup in a similar manner maintained DO at saturation.

DISCUSSION

Amphipods have been successfully maintained in contin-uous culture for long periods of time for their use intoxicity tests (DeMarch, 1981; U.S. Environmental Protec-tion Agency, 2000; McCahon and Pascoe, 1988; Nelsonand Brunson, 1995; Othman and Pascoe, 2001). However,our results suggest that establishing a continuous culture ofG. troglophilus and perhaps other cave amphipods may bedifficult. Of our microcosm experiments, only those inwhich we supplied native cave water and food to

Fig. 2. Average 6 SE linear growth rates over 60 days for amphipods inexperiments CSF and CDF.

Table 4. Results of a repeated measures ANOVA comparing growthrates between experiments CSF and CDF.

Effect d.f. F P

Between subjects

H20 velocity 1 1.76 0.276

Within subjects

Time 1 1.72 0.281H20 Velocity 3 Time 1 1.75 0.277

Table 5. Alkalinity (mg/L) of water used in microcosm experiments andcave stream water from caves in the Salem Plateau of southwestern Illinois.Ranges are reported when available. For our microcosm experiments, wereport means 6 standard error. *from Panno et al. (1996), **from Taylorand Webb (2000).

Experiments Alkalinity (as mg/L CaCO3)

ASN 285 6 14-513 6 32ADN 314 6 12-626 6 36CSF 199 6 2-212 6 3CDF 186 6 3-244 6 6ASF 281 6 4-318 6 6ADF 290 6 6-326 6 8CSN 218 6 10-238 6 3CDN 217 6 12-228 6 6

Cave streams

Illinois Caverns * 195-248 (N 5 15)Illinois Caverns ** 200 (N 5 12)Foglepole Cave ** 220 (N 5 12)Krueger-Dry Run Cave ** 243 (N 5 12)Stemler Cave ** 244 (N 5 12)

Fig. 1. Survival as a function of time in microcosm experiments withcave water, food (leaf discs and TetraMinH flakes), and static (solidcircles) or dynamic (open circles) water flow regimes. Data points areslightly offset from days 30 and 60 to clearly show error bars (6 SE).

428 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 31, NO. 3, 2011

amphipods (CSF and CDF) did amphipods survive after30 days. This does not support our hypothesis that survivalof G. troglophilus is not affected by type of water or thetype of food.

The food we provided to amphipods (leaf discs andTetraMinH flakes) in the microcosm experiments shouldhave been sufficient and not the cause of mortalities. AdultG. troglophilus were observed to readily feed on condi-tioned maple leaves and TetraMinH (Nelson personalobservation, this study). Similarly, Jenio (1980) found leafmaterial in 89% (n 5 76) of the G. troglophilus guts heexamined, while in selection experiments he found thatamphipods preferred elm over oak leaves. In addition, oneof us (FWM unpublished data) has previously kept G.troglophilus from Illinois Caverns in the laboratory for over1.5 yrs on a diet of pre-conditioned maple leaves. AlthoughG. troglophilus may consume leaf material and TetraMinHin the laboratory, additional food sources that may bepresent in a cave environment or a neglected, i.e., notregularly cleaned culture tank, could be required to achievemaximum growth and survival.

Although gammarid amphipods are typically regarded asshredders under the functional feeding group (FFG)classification, they can exhibit a wide range of feedingstrategies including cannibalism and predation (MacNeil etal., 1997; Kelly et al., 2002). Stable isotope analysis of thefood web in Spider Cave, Monroe County, Illinois suggeststhat G. troglophilus is an omnivore, consuming mostlyFPOM and the isopod Caecidotea brevicauda (Nelson,

2010). This suggests that animal protein may be an importantfood in the diet of G. troglophilus. Hence, we expected thatTetraMinH, which includes animal protein, to provide acomplete diet. Our results suggest that additional research isneeded to investigate if the direct addition of animal proteinin the form of another animal such as the isopod C.brevicauda to the diet of G. troglophilus would change itsgrowth and survival in the laboratory. Alternatively, constantremoval of accumulated unused food materials and con-comitant removal of amphipod fecal pellets may haveresulted in a microcosm that was too ‘clean’. This presents adichotomy for research in developing a rearing protocol.Leaving a tank(s) with amphipods undisturbed for longperiods of time would not provide insights on trajectories ofsurvival upon which to recommend optimum conditions.

Fig. 4. Average 6 SE number of weeks that neonate amphipods survivedunder different treatments. C 5 control group (no food, no sediment); D 5

leaf disc treatment; S 5 sediment treatment; DS 5 leaf disc + sedimenttreatment. Letters indicate similar means identified using Fisher’s leastsignificant difference (LSD).

Fig. 5. Average 6 SE growth rates of neonate amphipods during the first14 days after release from the brood pouch given four different foodtreatments. C 5 control group (no food, no sediment); D 5 leaf disctreatment; S 5 sediment treatment; DS 5 leaf disc + sediment treatment.Means were log transformed for analysis (ANOVA) due to unequalvariances and back transformed to calculate standard error. Letters indicatesimilar means.

Fig. 3. Average 6 SE number of weeks that juvenile amphipods survivedin the leaf disc and the TetraMinH treatments. Letters indicate similarmeans.

Table 6. Results of a repeated measures ANOVA comparing juvenileamphipod growth rates between food treatments (leaf discs vs. TetraMinH).

Effect d.f. F P

Between subjects

Treatment 1 0.61 0.44

Within subjects

Time 4 0.13 0.97Treatment 3 Time 4 2.11 0.10

NELSON AND WILHELM: SURVIVAL AND GROWTH OF GAMMARUS TROGLOPHILUS 429

However, if disturbance and cleaning are detrimental to theculture of cave amphipods in the laboratory, such anapproach may be necessary.

The lack of survival in amended water suggests that forthese amphipods, natural cave water or some chemicalcompound in cave water is necessary. Other researchers, e.g.,Fong (personal communication, American University), havesuccessfully cultured G. minus using commercially availablespring water with dolomite added to increase alkalinity andhardness. However, Fong indicated in conversations that die-offs happen for inexplicable reasons. It is unclear if the highmortality was natural mortality, mortality related to handling,or mortality related to the amended water. Had handling beenthe sources of our mortality, we would have expected theCSD and CDF experiments to have had the same mortalityrates as the other experiments. They did not, suggestinghandling was not the sole source of mortality. Our initialanalyses of Moscow ground water showed that onlyalkalinity and hardness differed significantly from cavewater (Nelson and Wilhelm, unpublished data). We attempt-ed to rectify this with the addition of ReefBuilder to increasealkalinity. However, it was difficult to maintain concentra-tions within the range reported for Illinois caves. Also, anelement not analyzed may have been critical to survival. Thisdeserves further investigation as it would be highly beneficialto make ‘‘amended cave water’’ in which cave amphipodscould be reared.

In cave streams, G. troglophilus is found in riffles andpools with fast and slow moving water, respectively. In ourexperiments, survival of amphipods was significantlyhigher in static microcosms (CSF) than in dynamicmicrocosms (CDF). When collecting amphipods, wetypically found more amphipods in pools compared toriffles (Nelson personal observations). In addition, al-lochthonous detritus, composed of fine and coarse partic-ulate organic matter (FPOM and CPOM, respectively)often accumulates in pools rather than riffles because of thelower velocity. Thus, pools may provide essential habitatfor G. troglophilus and possibly other cave amphipods,including G. acherondytes. Not only may detrital materialcontribute to the diet of G. troglophilus and G. acher-ondytes, but it could also provide shelter. Our results withwater velocity suggest that future rearing trials should becarried out under static conditions. The use of staticmicrocosms also simplifies the experimental setup andreduces the probability of failure associated with relying onelectrical power for recirculating pumps.

Our growth rate estimates for G. troglophilus inexperiments CSF and CDF were similar to those found

for other gammarid amphipods. For example, Venarsky etal. (2007) reported a growth rate of young G. acherondytesof 0.003 mm day21 to 0.065 mm day21. Similarly,Marchant and Hynes (1981) reported a constant growthrate of 0.038 mm day21 for Gammarus pseudolimnaeusBousfield, 1958. However, Welton (1979) calculatedgrowth rates ranging from 0.3 mm day21 to 0.8 mm day21

for the amphipod Gammarus pulex Linnaeus, 1758 in anEnglish chalk stream, while Fredette and Diaz (1986)reported laboratory growth rates of 0.6 mm day21 at 14uCfor Gammarus mucronatus Say, 1818. This range is notunexpected given the wide range of species and possibledifferences in their feeding habits, food sources andmetabolic rates.

Growth rates of juvenile G. troglophilus varied consid-erably but were within the range calculated for G.acherondytes by Venarsky et al. (2007). Because therewere no significant differences in juvenile growth rates orsurvival between food treatments and leaf discs, andTetraMinH flakes were readily consumed, it is not possibleto recommend one over the other for culture of juvenile G.troglophilus. Because the growth rate of neonates duringthe first two weeks after release was highest in the leaf disctreatment compared to the other food treatments suggeststhat leaves are an important food source. Although cavemud is a rich nutritional source for other cave amphipods(Dickson, 1979; Culver, 1985), it is apparent that cavesediment alone is not a good growth medium for G.troglophilus in the laboratory.

It is well known that many bottom-feeding invertebratesuse sediments from lakes, rivers, and streams as a foodsource (Hargrave, 1970). Some troglobitic amphipod specieshave been observed feeding on cave sediments (Dickson,1979). Cave mud plays an important role in the diet of theamphipod Niphargus virei Chevreux, 1896. In the laborato-ry, young N. virei almost always die before maturity unlessclayey mud is present (Dickson, 1979; Culver, 1985).However, the same clayey mud does not supply sufficientnutrition for adults (Culver, 1985). Adults of the speciesappear to need allochthonous organic material as well. Incontrast to studies on the amphipod N. virei, Dickson (1979)found the survival of juvenile Crangonyx antennatus Copeand Packard, 1881 in the laboratory to be independent of thepresence of cave sediments. Although C. antennatus readilyconsumed cave sediment in its natural environment, it was apoor growth medium in the laboratory (Dickson, 1979). Ingrowth experiments, Dickson (1979) did not find differencesin growth rates between trials of leaves versus cavesediment, but final body lengths were greater for amphipodsreceiving leaves. As well, in food preference experiments, C.antennatus selected leaves over cave sediment. Similarly, inlaboratory feeding experiments, Kostalos and Seymour(1976) reported that the diet of Gammarus minus Say,1818 was based on detritus and the fungi associated with thedetritus. In preference experiments with 10 diets differing incomposition of detritus-microflora complexes, G. minuspreferred leaves with a viable flora of fungi. These alsoprovided the highest survivorship. Thus, fungi comprise animportant nutritive component in the diet of G. minus andmay well be important to G. troglophilus as well.

Table 7. Results of a repeated measures ANOVA comparing neonateamphipod growth rates between treatments C (control) and D (leaf disc).

Effect d.f. F P

Within subjects

Treatment 1 26.76 , 0.001

Between subjects

Time 1 10.05 0.010Treatment 3 Time 1 0.49 0.499

430 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 31, NO. 3, 2011

Work on the diet of the amphipod G. pseudolimnaeus byBarlocher and Kendrick (1975) also supports the idea of theimportance of fungi. Through assimilation efficiencyexperiments, Barlocher and Kendrick (1975) showed thatG. pseudolimnaeus assimilated up to 90% of the fungalprotein while only 14-18% of the leaf protein wasassimilated. In addition, preliminary experiments byHargrave (1970) showed that sediment held in thelaboratory for longer than several days was not ingestedas rapidly as freshly collected sediment. The sediment usedin our experiments had been collected several days orweeks prior to the beginning of the experiments. This mayhave decreased the desirability of the sediment to theamphipods and should be examined in future studies.

We were somewhat surprised by the lack of survival inany of the neonate experiments with different foods.However, other studies have reported high mortality amongneonate amphipods. For example, Jenio (1980) reportedapproximately 65% mortality in G. troglophilus before thefirst molt and only 15 of 100 immature G. troglophilussurvived to a size of 10.1 mm in length, which he estimatedas adult size. In our study, dead neonate amphipods wereoften found half in and out of the exoskeleton, suggestingthat problems during ecdysis were a major source ofmortality. Sample jars containing neonate amphipods wereonly provided with one small rock. However, we havenoted that amphipods may need a rocky substrate with avariety of rock sizes to molt properly as this has beenobserved in other amphipod species as well. Given the highmortality of young, the high number of young produced perfemale (2 to 22 per brood, Jenio, 1980), and the scarcity ofadults in cave streams, high mortality in G. troglophilusmay be common. Alternatively, because neonates in ourexperiment came from a single female, survival may havebeen related to the small genetic pool represented versusthat available in a natural cave ecosystem. However, thelack of abundant gravid females in caves of the SPKRmeans that newly released neonates are relatively scarce. Inaddition, it would be nearly impossible to identify neonatescollected from the wild to species. Thus growth experi-ments focused on neonates are limited to those released inthe laboratory from gravid females. Complete mortality inthe neonate experiment was not observed in the controltreatment (C) until 8 weeks after release from the female’sbrood pouch. However, no neonates survived longer than7 weeks in our food addition treatments. Survival of thecontrol group that was not provided any food may indicatea possible microbial diet or coprophagy by neonates similarto that reported for older individuals feeding on leafmicroflora (see above). Coprophagous activity has beenreported in young of the troglobitic amphipod Crangonyxantennatus (Dickson, 1979) and other crustaceans (Fran-kenberg and Smith, 1967; Frankenberg et al., 1967). Thus,success with leaf discs and TetraMinH may not be related tothe ‘food’ per se, but rather the ‘food’ may serve as asubstrate for microbes that are the focus of the amphipoddiet. This is akin to the cracker and peanut butter analogy -where what researchers think of as food, in this case theleaf disc, in reality only serves as a vehicle (cracker) for thetransfer of microbes (the peanut butter) that are the source

of the energy and focus of the animal’s diet. Thus, furtherexamination of the microbes associated with cave streamdetritus and food preference experiments using G. troglo-philus are needed to fully understand their dietary needs.

CONCLUSIONS

Further examination of diet is required to determine if G.troglophilus can be cultured in a laboratory setting. Overall,the survival of neonate and juvenile amphipods was low. Wewere unable to rear neonates and juveniles to a length of10.1 mm, the minimum length considered for maturity(Jenio, 1980). Jenio (1980) found that it took 30 weeks forcultured immature amphipods to reach this size. Sevenweeks was the longest any neonate survived in ourexperiments. However, the juveniles we found in the stocktank and used in the leaf disc vs. TetraMinH experiment wereapproximately 4 mm when we first discovered them. Basedon neonate and juvenile growth rate estimates, it could havetaken anywhere from 6 to 16 weeks after release from thebrood pouch to reach that size, with somewhere in themiddle being more likely. During that early growth period,the amphipods had access to small aragonite, cave sediment,and TetraMinH fish flakes. It is impossible to know if thosejuveniles would have survived longer had they been leftalone rather than handled for experimental purposes.Therefore, it is still unclear if, or to what degree handlingaffects the survival of young G. troglophilus. Regardless, wemay be missing an important component in the diet of youngamphipods. Results from the stable isotope analysis (Nelson,2010) suggest that amphipods in caves are omnivorous. Thegeneral lack of food in cave ecosystems dictates omnivory inmany species (Gibert and Deharveng, 2002). Although theTetraMinH diet should have been complete, as it is designedto provide a full complement of dietary needs (carbohy-drates, proteins and lipids), it may be possible that caveamphipods have more specialized needs or require certainfoodstuffs in different quantities than available in TetraMinHflakes. This deserves further investigation.

ACKNOWLEDGEMENTS

We are grateful to the cave owners for granting us access to theirproperties and their caves. Philip Moss assisted in the field and providedaccommodations during field collections. We also thank James ‘‘Ding’’Johnson and Kevin Simon for providing comments on this manuscript.This research was supported with funding from the United States Fish andWildlife Service and the Department of Fish and Wildlife Resources andthe College of Natural Resources at the University of Idaho. We alsoacknowledge the assistance and cooperation of the Illinois Department ofNatural Resources and the United States Fish and Wildlife Service withpermitting and collections.

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RECEIVED: 13 November 2010.ACCEPTED: 20 January 2011.

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