2008 SOUTHEASTERN NATURALIST 7(1):159–164 Visible Implant Elastomer as a Tool for Marking Etheostomine Darters (Actinopterygii: Percidae) Michael R. Weston1 and Ronald L. Johnson1,* Abstract - Mark-recapture studies for investigating migration patterns and population dynamics of large numbers of individuals require an effective, non-lethal marking technique. Our goal in the present study was to determine the suitability of visible implant elastomer (VIE) for a species in nominal decline, Etheostoma moorei (Yellowcheek Darter). Mortality and mark retention were initially studied in a laboratory setting using a surrogate species, Etheostoma caeruleum (Rainbow Darter) due to Yellowcheek Darter’s conservation status (candidate species for listing as endangered). There was 100% survival and mark retention over the 58-day period for the fourteen Rainbow Darters injected with VIE marks, and there were no observable lesions or scars resulting from marking. Adult Yellowcheek Darters ≥30 mm (n = 385) were individually marked with VIE, and their soft dorsal fins were clipped. They were then returned to their original riffl e sites on the Middle and South forks of the Little Red River, AR. There were a total of 26 recapture events, with a mark retention rate of 88% (12% of recaptures had evidence of fin clips, yet no VIE marks). The interval between initial capture and last recapture ranged from 13–401 days. We identified no relationship between position of injection, color marked, or darter size at injection and mark retention. The duration of the present study (13 months) appears suitable for VIE mark retention in Yellowcheek Darter. Adults are both slow-growing and short-lived, characteristics which enhance the suitability of VIE for mark-recapture use. Introduction Biologists have been marking fish for centuries to estimate population sizes and their migration dynamics (McFarlane et al. 1990). Marking techniques include external, internal, natural, genetic, and chemical marks, in addition to biotelemetry. Primary assumptions for external, internal, and chemical marks are that they do not alter the subject’s growth, survival, or behavior. Assumptions for genetic marks are that they must be inherited in Mendelian fashion, and the frequency of the alleles studied is stable within the population (for review, see Nielsen 1992). Selecting the correct mark depends on the goals and limitations specific to each study. Fish size, conservation status, habitat, budget, and duration of the study may direct mark selection. The method of marking will also differ depending upon whether the study of individual behaviors or larger population dynamics is the desired goal. Our goal in the present study was to identify a mark that would later enable us to estimate population sizes and movement patterns of individual Etheostoma moorei Raney and Suttkus (Yellowcheek Darter). Yellowcheek Darters are endemic to four headwater streams of the Little Red River in Arkansas (Raney and Suttkus 1964), with an estimated nominal decline of 80% in population size over two decades (Wine et al. 2001). The Yellowcheek Darter is a 1Department of Biological Sciences, Arkansas State University, State University, AR 72467. *Corresponding author – rlj@astate.edu. 160 Southeastern Naturalist Vol.7, No. 1 candidate species for listing under the Endangered Species Act (C. Davidson, US Fish and Wildlife Service, Conway, AR, pers. comm.). Due to these characteristics, it is critical for the marking process to not be lethal. Adult Yellowcheek Darters range in size from 30 to 70 mm total length (TL). The marking of small fish in studies requiring individually identifiable marks can be challenging. Several techniques were considered for the present study, but not used for various reasons. Set-up costs for using passive integrated transponder (PIT) tags are quite expensive. Coded wire tags must physically be recovered from the fish; for a small fish, recovery of these tags can cause extensive trauma and/or fatality. Visible implant alpha numeric tags are less expensive, yet must be injected into transparent tissue where they are externally readable. Yellowcheek Darters are heavily pigmented, and the option of injecting these tags into the eye region was not available to us. We decided to test the suitability of visible implant elastomer (VIE; Northwest Marine Technology, Shaw Island, WA), a mixture of color elastomer and curing agent, for Yellowcheek Darters. This technique has a relatively low cost and has been used to mark a wide variety of fishes (e.g., Oncorhynchus mykiss Walbaum [Rainbow Trout], Close 2000; several marine rockfishes, Griffiths 2002; and Gadus morhua L. [Atlantic Cod] , Olsen et al. 2004). However, mark retention for VIE has been tested in only two species of darters (Percina roanoka Jordan and Jenkins [Roanoke Darter] and Etheostoma podostemone Jordan and Jenkins [Riverweed Darter]), and this research was done in a laboratory setting (Roberts and Angermeier 2004). Additionally, Yellowcheek Darters are darkly pigmented relative to those darter species. We were interested in studying both mortality and mark retention using VIE on darters. Mortality and mark retention were initially studied in a laboratory setting using a surrogate species due to the conservation status of the Yellowcheek Darter. Etheostoma caeruleum Storer (Rainbow Darter) is a common riffl e inhabitant in the Little Red River drainage and is sympatric with Yellowcheek Darter. A primary limitation of a laboratory study is the inability to mimic field conditions, particularly in regards to infection rates. Once the VIE injection was demonstrated to be safe for Rainbow Darter, we performed a field test of mark retention of VIE for Yellowcheek Darters in the Middle and South forks of the Little Red River, AR. Methods Laboratory evaluation A laboratory experiment was conducted to investigate short-term markinginduced mortality, mark retention, and mark recognition in Rainbow Darters. Fourteen individuals were collected in 2004 from the Beech Fork of the Little Red River, AR and transported to Arkansas State University. Darters were anesthetized with tricaine methanesulfonate (MS-222; Finquel) and given two green VIE marks, one immediately anterior to the spiny dorsal fin and one in the upper caudal peduncle. Both marks were injected left of the mid-dorsal line with a nearly vertical orientation. After recovery from the anesthetic, darters were released into a 947.5-L Living Stream® with a cobble and gravel substrate. Two submersible pumps with a 2839 liter/hour capacity were also used to 2008 M.R. Weston and R.L. Johnson 161 provide a current to simulate a shallow riffl e. Darters were fed frozen bloodworms and brine shrimp daily. After 58 days, surviving darters were counted and examined for mark retention. These data were used to calculate percent survival, and percent mark retention (i.e., number of recognizable marks retained divided by the number of darters surviving). Field evaluation Yellowcheek Darters were collected from August 29, 2003 through September 9, 2004 at three sites on the Middle Fork and two sites on the South Fork of the Little Red River, AR. The Middle Fork ranges from a third-order stream at the uppermost sample site to a fifth-order stream at the lowest sample site, whereas the South Fork ranged from a second- to third-order stream at the sample sites. Kick seining proved to be the most effective nonlethal method of collecting Yellowcheek Darters Captured adults ≥30 mm were anesthetized with MS-222, measured to the nearest mm TL, and individually marked with VIE using varying combinations of four fl uorescent colors (red, orange, green, and yellow) and four injection locations (left and right of the mid-dorsal line anterior to the spiny dorsal fin and left and right sides in the upper caudal peduncle). Markings were for determining individual fish movement as part of a later study. One to three subcutaneous injections using a 28-gauge needle marked each darter. The VIE was mixed immediately preceding anesthesia and was kept on ice between fish injections. We were able use the VIE mixture for up to two hours and mark up to 25 individuals with excellent results and no elastomer hardening or fish mortality. The soft dorsal fin of each Yellowcheek Darter was also completely removed to provide information for VIE mark retention and for later genetic analysis. After recovery from anesthesia, darters were released back to their point of capture. Sampling using kick-seining was continued over a 13-month period. Captured Yellowcheek Darters were examined both for soft dorsal fin clips and VIE marks. We compared fish identified as recaptures by fin clips to those observed with VIE marks. Darters possessing either mark were considered recaptures. Percent mark retention for both methods was calculated by the proportion possessing that particular mark (fin clip or VIE) versus the total number of recaptures. Only darters possessing documented injection patterns were considered as retaining the mark. The possibility exists that some darters may have regenerated their soft dorsal fin and lost the VIE mark, thus infl ating mark-retention estimates. A second field limitation was the inability to determine marking-induced mortality. We performed chi square analysis on captured fish to compare mark retention relative to the following variables: mark placement (dorsal fin or caudal peduncle), color injected (green, orange, red, and yellow) and darter size (35–44 mm, 45–54 mm and 55–64 mm) (see Henry et al. 2005). Results and Discussion There was 100% survival and mark retention for the 14 Rainbow Darters studied over the 58-day period in the laboratory. The blue light and amber shaded glasses provided by the manufacturer were not needed to recognize the VIE marks. Additionally, there were no observable lesions or scars resulting from 162 Southeastern Naturalist Vol.7, No. 1 VIE marking. The lack of Rainbow Darter mortality in the laboratory due to VIE injection is consistent with several other laboratory studies of mark-induced mortality (Dewey and Zigler 1996, Close 2000, Olsen and Vøllestad 2001). Results from field testing were more variable. We marked and released 385 Yellowcheek Darters at five study-site riffl es on the Middle and South forks of the Little Red River, AR. Mean TL of Yellowcheek Darters captured was 46.0 mm (± 0.38 SE, range = 30–70 mm). Twenty-four darters were recaptured, with two recaptured twice, for a total of 26 recapture events over a 13-month period. The interval between initial capture and last recapture with distinguishable VIE marks ranged from 13–401 days (mean = 134.3 days, ± 30.09 SE). One recaptured darter, identified by dorsal fin clip, had no VIE marks, whereas two darters had VIE marks that did not correspond to any combinations of VIE marks applied and recorded during this study (21/24 = mark retention of 88%). These unidentified darters indicated that at least one color of a multiple color mark had been lost or more probably that we had made a mistake in the recording of injection patterns. There was no evidence of color fading; color loss was complete for individuals lacking marks. None of the darters recaptured had complete fin regeneration, yet two individuals had greater than 75% regeneration within 12 months. A conservative estimate of the soft dorsal fin mark retention is 92%. Mark-retention studies using VIE have variable results based upon study duration, position of VIE marking, developmental stage and growth characteristics of the species studied, and colors used in the marking process. The duration of the present study (13 months) appears suitable for both VIE and fin-clip mark retention in Yellowcheek Darters. The 92% retention rate of the soft dorsal fin clip is indicative that this was an effective check system for VIE mark retention. Studies of shorter duration have similar VIE percent retention rates to those of Yellowcheek Darter. Several studies have demonstrated retention rates >90% for short-term marking of fishes (1 month up to 8 months; Close 2000, Dewey and Zigler 1996, Hale and Gray 1998, Olsen and Vøllestad 2001, Roberts and Angermeier 2004). However, several studies have demonstrated long-term declines in retention rates (e.g., FitzGerald et al. 2004, Haines et al. 1998), with retention rates dropping as low as 29% for Rainbow Trout 35 months post-injection (Close and Jones 2002). There was no difference in recapture rates of individuals having VIE injection sites anterior to the spiny dorsal fin versus the upper caudal peduncle (χ2 = 0.0044; p = 0.950). Likewise, Hale and Gray (1998) found no positional difference in the retention of VIE marks in either the eye adipose tissue or the anal fin of Salmo trutta L. (Brown Trout), nor did Roberts and Angermeier (2004) find retention differences for injections in the midventral, middorsal, and upper and lower caudal peduncle of Roanoke Darters and Riverweed Darters. However, Haines and Modde (1996) found that ventral marks had higher retention than dorsal marks for Ptychocheilus lucius Girard (Colorado Squawfish). Also, anal fin marks had the best mark retention for Lepomis macrochirus Rafinesque (Bluegill; Catalano et al 2001) and for Xyrauchen texanus Abbott (Razorback Suckers; Haines et al. 1998). FitzGerald et al. (2004) found a greater long-term decline in mark retention in the jaw than in adipose eye tissue for Salmo salar L. (Atlantic Salmon). 2008 M.R. Weston and R.L. Johnson 163 VIE mark retention has been demonstrated to be variable based upon the size of fish at time of injection and growth characteristics for individual species. However, we identified no relationship between darter TL at injection and mark retention (χ2 = 0.4076; p = 0.997), yet all individuals injected were adults. VIE injection in juvenile fishes has often resulted in poor retention (Dewey and Zigler 1996, Hale and Gray 1998). Close (2000) found that percent detection of VIE marks increased with greater O. mykiss size at the time of marking; retention rates were inversely related to post-marking growth. Adult darters such as Yellowcheek Darters are both slow-growing and short-lived. For example, the maximum TL of Yellowcheek Darters observed was 70 mm. Mean growth rate of recaptured individuals was 1.29 mm per month (± 0.22 SE). Additionally, with a maximum lifespan of only 4 years (McDaniel 1984), the period of studying individual fish is limited, as is the potential for VIE mark loss. There was no relationship of color injected and recapture rates of marked Yellowcheek Darters (χ2 = 4.4599; p = 0.225). The blue light and amber shaded glasses supplied by the manufacturer were required for field identification, however. Visible implant elastomer mark retention as a function of color is inconsistent among other species and may be tied to species-specific pigment patterns. For example, Roberts and Angermeier (2004) studying Roanoke Darters and Riverweed Darters and Catalano et al. (2001) studying Bluegill found that green marks had better retention than yellow marks. However, Close (2000) identified that yellow marks were detectable at a greater rate than were green marks after 6.5 months in Rainbow Trout. Red and orange VIE marks were retained more than green and blue for Colorado Squawfish and Razorback Suckers (Haines et al. 1998), whereas red and blue marks were more visible than orange in Rainbow Trout (Hale and Gray 1998). Mark-recapture studies for investigating migration patterns and population dynamics of large numbers of individuals require an effective, non-lethal marking technique. This information can assist researchers in producing information important to fisheries conservation and restoration; in the present study, we were able to use mark-recapture data for a candidate species for listing as an endangered species. Visible implant elastomer proved to be a non-lethal method for marking Rainbow Darters in the laboratory and has high mark retention for Yellowcheek Darters in a field setting. Visible implant elastomer can be applied to small fish that have previously been difficult to individually mark. The minimum TL we felt would not harm fish was 30 mm. For this study, adult Yellowcheek Darters are both slow-growing and short-lived, characteristics which enhance the suitability of VIE for mark-recapture use. As each species has unique morphometries, growth dynamics, and pigmentation which can affect mark retention, we recommend laboratory study to optimize injection sites and colors used for each study species prior to field implementation. Acknowledgments This research was funded by the United States Fish and Wildlife Service (USFWS). We are thank J. Fullington, K. Gillespie, M. Johnson, A. Peck, T. Sanders, M. Trevino, and C. Weston for assistance with collection of darters, and M. Wine of the USFWS for 164 Southeastern Naturalist Vol.7, No. 1 technical assistance. Helpful criticism in manuscript preparation was provided by M. Huss and anonymous reviewers, to whom we are most grateful. Literature Cited Catalano, M.J., S.R. Chipps, M.A. Bouchard, and D.H. Wahl. 2001. Evaluation of injectable fl uorescent tags for marking centrarchid fishes: Retention rate and effects on vulnerability to predation. North American Journal of Fisheries Management 21:911–917. Close, T.L. 2000. 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