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Efficacy of Internal PIT Tagging of Small-bodied Crayfish for Ecological Study
Tyler R. Black, Shawna S. Herleth-King, and Hayden T. Mattingly

Southeastern Naturalist, Volume 9, Special Issue 3 (2010): 257–266

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Conservation, Biology, and Natural History of Crayfishes from the Southern US 2010 Southeastern Naturalist 9(Special Issue 3):257–266 Efficacy of Internal PIT Tagging of Small-bodied Crayfish for Ecological Study Tyler R. Black1,*, Shawna S. Herleth-King1, and Hayden T. Mattingly1 Abstract - Recent studies have demonstrated the feasibility of using passive integrated transponder (PIT) tags for tracking crayfish spatiotemporally in streams. PIT tags can be inserted internally for long-term tracking, assuming low tagging mortality, or attached externally for shorter-term tracking until the individual molts. To date, the practical use of internal cephalothorax tagging has been limited to individuals >30 mm carapace length (CL). The efficacy of internal tagging for small-bodied crayfish species or juveniles of large-bodied species remains poorly understood. We conducted studies with the small-bodied Orconectes compressus (Slender Crayfish) to assess whether internal placement of small PIT tags (8.5 mm long, 2.12 mm diameter) was a viable methodology for future ecological work. In the field, we tagged 63 crayfish and monitored them with a portable transceiver system for 1.5 weeks. In the laboratory, we tagged 21 crayfish and maintained 21 control crayfish for 12 weeks. Crayfish averaged 18 mm CL (n = 84). In the field, there was high initial (14 of 63) and delayed (16 of 63) mortality. We also observed initial (3 of 21) and delayed (11 of 21) mortality in the laboratory within the first 10 days. Smaller individuals had higher mortality rates in both studies. We constructed logistic regression models with field (P = 0.005) and laboratory data (P = 0.027) to show the likelihood of tagging mortality as a function of carapace length. Our results suggest that internal PIT tagging could induce undesirably high mortality in crayfish <22 mm CL for most ecological study objectives. Introduction The American Fisheries Society Endangered Species Committee recognizes 363 native crayfishes within the Unites States and Canada, of which 2 are listed as endangered - possibly extinct, 66 as endangered, 52 as threatened, 54 as special concern, and 189 as currently stable (Taylor et al. 2007). Crayfishes are affected by factors that also impact other aquatic organisms, such as freshwater fishes and mussels, including physical habitat alteration, introduced species, chemical alteration of habitat, hybridization, overharvesting, and limited distribution (Allan and Flecker 1993, Miller et al. 1989, Taylor et al. 1996, Warren and Burr 1994, Williams et al. 1993). Further exacerbating the imperilment of crayfishes within North America is the lack of attention given to crayfishes by biologists, regulatory agencies, policy makers, and the general public (Lodge et al. 2000). Currently, information regarding the biology of most crayfishes is incomplete or lacking (Taylor et al. 1996, 2007) due to the reasons 1Department of Biology, Box 5063, Tennessee Technological University, Cookeville, TN 38505. *Corresponding author - trblack21@tntech.edu. 258 Southeastern Naturalist Vol. 9, Special Issue 3 described above, but also due to technological barriers. For example, the inability to efficiently track individual crayfish throughout their lifespan has limited knowledge acquired from life-history and ecological studies. Molting of crayfish presents a major problem when attempting to repeatedly detect marked crayfish, because external tags and marks are typically lost or become indistinguishable after a few molts (Guan 1997). In addition, the small body size of many crayfish restricts the type and size of tag that can be attached and the substrate burrowing behavior of crayfish reduces efficient detection of marked individuals. Therefore, internal implantation of passive integrated transponder (PIT) tags has recently been explored as an approach to track individual crayfish (Bubb et al. 2002, Wiles and Guan 1993). A PIT tag is an electronic microchip encased in glass, which is activated by an electromagnetic field produced by a PIT tag reader. Upon activation, it transmits an alphanumeric code back to the PIT tag reader (Gibbons and Andrews 2004). The maximum PIT tag detection distance is up to 30 cm, and detection is relatively unimpeded by water and substrate, but orientation of the tag affects detection distance (Cucherousset et al. 2005). Wiles and Guan (1993) and Bubb et al. (2002) injected PIT tags into the cephalothorax of crayfish through a hypodermic needle incision at the base of the fifth pereiopod. Unfortunately, Wiles and Guan (1993) observed low survival (<33.3%) of small crayfish (<25 mm CL) when a 13-mm long x 2-mm diameter tag was implanted, but they did report 100% survival of larger crayfish (>36 mm CL). Smaller, untested PIT tags (8.5- mm long x 2.12-mm diameter) are currently manufactured by the Destron Fearing Corp. (South St. Paul, MN) and are available from Biomark, Inc. (Boise, ID). This new development in PIT tag technology warrants experimentation to determine if these smaller tags can be safely implanted in small-bodied crayfish. Thus, this study examines the likelihood of tagging mortality as a function of carapace length. Methods Efficacy of internal PIT-tagging for small-bodied crayfish was determined by field and laboratory studies conducted during July 2007 and July–October 2007, respectively. Orconectes compressus (Faxon) (Slender Crayfish) was selected for these studies because it is locally abundant in Tennessee, widely distributed, and has a relatively small body size (body length <51 mm). Field study Two 50-m study reaches were selected on Little Trace Creek in Clay County, TN, upstream from the Highway 52 bridge crossing. The two reaches were established such that the upstream margin was at the upstream end of a riffle, and the reaches were separated by approximately 80 m. Mean wetted-channel width was 5.7 m for the upstream reach and 3.6 m for the 2010 T.R. Black, S.S. Herleth-King, and H.T. Mattingly 259 downstream reach, and mean thalweg depths were 15 and 21 cm, respectively. The water level remained relatively constant throughout the study, and the mean water temperature was 20.5 ºC. Slender Crayfish were collected within each reach using a 1.5-m kick seine (4-mm delta mesh) and aquarium nets. Twenty-five crayfish with carapace lengths (CL, measured from the anterior tip of the rostrum to the posterior boundary of the cephalothorax) ranging from 16.1 to 22.7 mm were captured in each reach. Captured individuals were internally tagged with a Destron Fearing TXP1485B PIT tag (8.5-mm long x 2.12-mm diameter, weight = 0.067 g). To insert the tag, crayfish were held by the cephalothorax with the ventral surface facing up. A 12-gauge hypodermic needle was used to make an incision at the base of the fifth pair of pereiopods. The tag was then inserted through the incision and pushed anteriorly until it rested within the cephalothorax cavity and underneath the digestive gland (Bubb et al. 2002). Once tagged, individuals were held in a 51-L cooler containing freshwater and sections of 2.54-cm PVC pipe for shelter for a minimum of one hour before being released throughout the study reach. Individuals that expired prior to release were replaced with newly tagged crayfish. Carapace length, gender, and individual tag identification number were recorded for each tagged crayfish. Tagged crayfish were searched for the day after tagging and subsequently every other day thereafter for 1.5 weeks (9–18 July 2007). A 304.8-mm triangular waterproof multi-directional antenna (Biomark Inc.) mounted on a 3.05-m adjustable pole was used to scan each reach for tagged crayfish. Each scanning pass began at the downstream periphery of the reach and was conducted by traveling upstream in a zig-zag pattern to cover available habitats. The antenna was connected to a portable transceiver system (FS2001F-ISO, Destron Fearing Corp. ), which displays an alphanumeric identification code when a tag is detected. Once a tagged crayfish was detected, a 0.25-m2 quadrat sampler was placed over that area. The sampler was constructed of 2.54-cm PVC pipe covered with 6-mm square mesh that extended approximately 40 cm below the bottom so that the frame could be sealed to the substrate (Walton 2008). A bag was built into the back of the sampler’s frame and a 6-cm fringe of rubber shower liner was added to the bottom perimeter. The area within the sampler was searched thoroughly for the tagged individual by gently removing substrate until the tagged individual or tag was recovered. Once recovered, the individual’s status (alive or dead) and tag number were recorded. If an individual was alive upon capture, it was returned to its capture location. This process was repeated for each tagged crayfish encountered within the two 50-m sites. Laboratory study Slender Crayfish 16.6–22.1 mm CL were collected with a 1.5-m kick seine (4-mm delta mesh) from Little Trace Creek on 18 July 2007 and 260 Southeastern Naturalist Vol. 9, Special Issue 3 transported to a laboratory at Tennessee Technological University. Crayfish were held overnight in aquariums to allow acclimation to laboratory conditions. Forty-two crayfish were used in the study: 21 were tagged (13 females and 8 males) and 21 served as controls (12 females and 9 males). Crayfish were divided into groups based on the following CL size classes: 16.0–16.9 mm, 17.0–17.9 mm, 18.0–18.9 mm, and ≥19.0 mm. Six crayfish were placed in each of 7 aquariums, set up as described below. Three crayfish from each aquarium were then tagged, while the remaining three individuals were used as controls. All tagged crayfish were paired with control crayfish of similar CL and gender, except for one female and male pair that was used in the ≥19 mm size class. Tagging procedures followed the methodology described above for the field study. Once tagged, the crayfish were placed back into their assigned aquariums. Each 19-L aquarium was set up with a sponge filter for waste treatment, pea-sized gravel substrate 3.5 cm deep, and six sections of 2.54-cm PVC pipe for shelter, and filled with dechlorinated water. A partial water change was conducted approximately every 3–6 d to maintain water quality, and a temperature similar to that of the collection site was maintained throughout the study. Crayfish were exposed to a 12 h light:12 h dark cycle and were fed shrimp pellets biweekly. On the initial day of tagging, crayfish status (alive or dead) was checked two hours after implantation of the PIT tag. Crayfish status was then checked once every 1–2 d for the duration of the 3-month study (19 July–19 October 2007). Statistical analyses Logistic regression models were constructed to show the probability of tagging mortality as a function of gender and carapace length (LOGISTIC procedure; SAS Institute 2001). For the field study, initial mortality was defined as all individuals that expired prior to release, and delayed mortality was defined as all individuals that expired post-release or when a tag was found without its associated crayfish. For the laboratory study, initial mortality was defined as all individuals that expired within two hours after tagging, and delayed mortality was defined as all individuals that expired after the first two hours. Only the first 10 days of laboratory data were used in model construction. Models followed the format of Hosmer and Lemeshow (2000): π(Mortality) = ef / (1 + ef ), where π(Mortality) equals the probability of tagging mortality and f equals β0 + β1x1 + β2x2, where β0 equals the model intercept (constant), βi equals the parameter estimate, and xi equals the gender or carapace length of an individual. The likelihood ratio test statistic (G) was used to assess model significance (Hosmer and Lemeshow 2000). Models were constructed separately for field and laboratory data. 2010 T.R. Black, S.S. Herleth-King, and H.T. Mattingly 261 Results Field study Sixty-three crayfish (38 females and 25 males) with a mean CL of 18.2 mm (SD ± 1.4 mm) were tagged during the field study (Fig. 1a). Initial Figure 1. Length-frequency histogram for Orconectes compressus (Slender Crayfish) used during field (a) and laboratory (b) studies in 1-mm intervals beginning with 16.0–16.9 mm carapace length (CL) and ending with 22.0–22.9 mm CL. Control crayfish were not tagged. 262 Southeastern Naturalist Vol. 9, Special Issue 3 mortality was high, with 14 of 63 individuals expiring within an hour of tagging (and being replaced by tagging additional individuals). Delayed mortality was high as well, with another 16 individuals expiring within the 1.5-week study (Fig. 2a). Throughout the duration of the study, 14 crayfish were located only once, 11 crayfish were located more than once, 8 crayfish were never located and were therefore modeled as alive, 6 crayfish were found dead (presumably from tag implementation or infection at the injection site), and 10 PIT tags were found without crayfish (5 from each reach). Thirty-three crayfish were considered alive at the completion of the study (22 females and 11 males). The mean carapace length for surviving crayfish was 18.6 mm (SD ± 1.6 mm). The gender of an individual was not a significant predictor of mortality, but carapace length was a significant predictor of mortality when includ- Figure 2. Length-frequency histogram for initial and delayed tagging mortality during the first 10 days for internally PIT tagged Orconectes compressus (Slender Crayfish) during field (a) and laboratory (b) studies in 1-mm intervals beginning with 16.0–16.9 mm carapace length (CL) and ending with 22.0–22.9 mm CL. Control crayfish survival was 100% for the first 10 days of the 3-month study. 2010 T.R. Black, S.S. Herleth-King, and H.T. Mattingly 263 ing initial mortalities with delayed mortalities (Wald χ2 = 6.15, P = 0.005; Table 1). The linear function within the logistic regression model developed to predict the probability of mortality as a function of carapace length is given by f = 10.6785 - 0.5952 (CL). When plotted, the inflection point (i.e., 50:50 probability of mortality) is at approximately 18 mm CL, and the probability of mortality decreased from approximately 20% at 20 mm CL to near zero by 24 mm CL (Fig. 3). Laboratory study Twenty-one crayfish (13 females and 8 males) with a mean CL of 18.0 mm (SD ± 1.2 mm) were tagged for the laboratory study (Fig. 1b). Three of 21 individuals expired within two hours after tagging. Delayed mortality primarily occurred within the first 10 days of the study, when 11 individuals died (7 females and 4 males; Fig. 2b). Three additional males died by the end of the study giving a total of 17 tagged crayfish expiring; conversely, only 6 control crayfish (4 females and 2 males) expired during the duration of the study. All surviving tagged individuals were females (mean CL of 19.1 mm; SD ± 2.1 mm). The gender of an individual was not a significant predictor of mortality, but carapace length was marginally significant when including initial mortalities with delayed mortalities occurring within the first 10 days (Wald χ2 = 2.95, P = 0.027; Table 1). The linear function within the logistic regression model developed to predict mortality as a factor of carapace length is given by f = 19.9963 - 1.0674 (CL). When plotted, the inflection point is at approximately 19 mm CL, and the probability of mortality rapidly decreased at 20 mm CL in a similar fashion to the field model (Fig. 3). Discussion Advancements in PIT tag technology resulting in the development of smaller tags (8.5 mm long, 2.12 mm diameter) have allowed small crayfish to be successfully internally tagged. However, our results suggest that their practical use is limited to crayfishes with a carapace length >22 mm. The death of several crayfish shortly after tagging indicates that Table 1. Logistic regression models to predict the probability of tagging mortality as a function of Slender Crayfish carapace length (CL, in mm). The Wald chi-square statistic tests the significance of each coefficient. The odds ratio is the multiplicative factor by which the odds of mortality increase when CL decreases by 1 mm. Odds Model term Coefficient SE Wald χ2 P > χ2 ratio G P Model constructed with field data 7.8 0.005 Intercept 10.6785 4.33 6.09 0.014 CL -0.5952 0.24 6.15 0.013 0.551 Model constructed with laboratory data 4.9 0.027 Intercept 19.9963 11.25 3.16 0.076 CL -1.0674 0.62 2.95 0.086 0.344 264 Southeastern Naturalist Vol. 9, Special Issue 3 tagging mortality was caused by insertion of the PIT tag. Wiles and Guan (1993) reported that crayfish <25 mm did not have adequate space within the cephalothorax to hold a 13-mm PIT tag, and Bubb et al. (2002) further suggested that tagging mortality may result from inserting a tag too close to the ventral nerve cord. The high initial mortality rates found in our study could have been caused by one or both of these tagging problems. Furthermore, 13-mm, 12-mm, and 8.5-mm PIT tags all have a diameter that is approximately 2 mm, suggesting that the diameter of a tag may also be a critical factor in tagging mortality. A reduction of diameter for 8.5-mm PIT tags is likely to greatly reduce the likelihood of damaging the ventral nerve cord during tag insertion, and thereby could reduce initial tagging mortality in small-bodied crayfishes. Tagging experiments conducted by Wiles and Guan (1993) show that there was a higher survival rate for males than females in the 21–25-mm CL size class; however, the reverse was true for both the 26–30-mm and 31–35-mm CL size classes. The majority of surviving individuals in our study were female, although gender of an individual was not a statistically significant predictor of tagging mortality. The tagging procedure required that an incision and tag insertion be made at the base of the fifth pair of pereiopods, which proved to be more difficult in males because of the presence of gonopods and steeper tag entry angle. Therefore, we suggest that additional care should be used when inserting a PIT tag into male small-bodied crayfish. Figure 3. Field and laboratory probabilities of tagging mortality as a function of Orconectes compressus (Slender Crayfish) carapace length as predicted by logistic regression models given in Table 1. 2010 T.R. Black, S.S. Herleth-King, and H.T. Mattingly 265 Our results demonstrate that small-bodied crayfish can be successfully internally PIT tagged and detected with a portable PIT tag detector (89% of the tagged individuals were detected at least once in the field study). Even though high mortality was experienced, internal PIT tagging provides a novel technique for tracking crayfish through time. Bubb et al. (2002) highlight the benefits of internal PIT tagging, such as repetitive non-destructive sampling, indefinite tag life span, negligible tagging mortality of crayfish >33.7 mm CL, and no apparent effects on growth and survival of tagged individuals. However, a high level of tagging mortality can be expected for crayfish <22 mm CL, and researchers should use caution when tagging smaller individuals. Therefore, for crayfish <22 mm CL, it would be more cost-effective to externally attach the PIT tag to the cephalothorax exoskeleton to obtain short-term data until molting occurs. Bubb et al. (2002) reported that there was no significant difference between the growth of internally PIT tagged and control crayfish >33 mm CL, but further research is needed to determine if there is a reduction in growth and long-term survival for internally PIT tagged crayfish in the 22–33-mm CL size range. Acknowledgments We thank the Tennessee Technological University Department of Biology for the use of PIT equipment and laboratory space. We would also like to thank the anonymous reviewers whose valuable editorial comments improved this manuscript, and Stuart Welsh for publication support. The publication of this manuscript was supported, in part, by the US Geological Survey Cooperative Research Unit Program, including the West Virginia Cooperative Fish and Wildlife Research Unit. Literature Cited Allan, J.D., and A.S. Flecker. 1993. Biodiversity conservation in running waters. Bioscience 43:32–43. Bubb, D.H., M.C. Lucas, T.J. Thom, and P. Rycroft. 2002. The potential use of PIT telemetry for identifying and tracking crayfish in their natural environment. Hydrobiologia 483:225–230. Cucherousset, J., J.-M. Roussel, R. Keeler, R.A. Cunjak, and R. Stump. 2005. The use of two new portable 12-mm PIT tag detectors to track small fish in shallow streams. 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