Evaluation of Visual Implant Alphanumeric Tagging on Rio Grande Silvery Minnow Free

Fisheries management requires methods to determine unbiased population sizes, survival, growth, mortality, habitat use, and movement (Pine et al. 2003). Mark-recapture studies with tagged fish are used to estimate fish abundance. Mark-recapture efforts are especially important in estimating population size of imperiled fish because these data influence protected status and inform recovery efforts. Studies using uniquely marked animals allow for more robust estimates of demographic parameters than batch-marking (Skalski et al. 2009). However, studies on tag effects on small-bodied fishes (e.g., < 100 mm standard length) are relatively uncommon compared to tagging studies in larger fish, as larger fish experience lower mortality, less stress, and have higher tag retention rates than small fish (Sandford et al. 2020). Field studies of small-bodied fishes generally use batch marks (Ficke and Myrick 2009; Archdeacon et al. 2022; Steffensmeier et al. 2022), but other tagging methods may allow for uniquely tagging fishes in studies examining more than 10 individuals. Examination of the effects of different types of tags on small fish and applicability to field studies will inform experimental designs.

Several alternatives to batch-mark tags are available that allow fish to be uniquely identifiable. Passive integrated transponder tags (PIT) are one commonly preferred option. Passive integrated transponder tags can be detected and automatically recorded up to 0.5 meter away, so fish do not necessarily have to be captured to obtain information on movement, survival, and habitat use. They remain difficult to implement for very small fishes, however, as survival varies by surgical method and fish size, tags can be shed over time (Archdeacon et al. 2009) and both survival and retention can vary widely by species (Sandford et al. 2020). Passive integrated transponder tags have been used in small fish movement studies with small sample sizes (i.e. < 100 individuals, Wells et al. 2017; Pennock et al. 2018) or rely on hatchery fish tagged prior to release (e.g., Chavez et al. 2024). In situ studies employing PIT tags in small-bodied fishes with large sample sizes are lacking, likely because the methods are not feasible to implement in the field (but see Vatland and Caudron 2015). Further, the smallest PIT tags (8 mm) do not have suitable detection ranges to allow for use of passive scanning techniques; fish must be recaptured to be identified. Finally, another option for small-bodied fishes is visible implant alphanumeric (VIA) tags, which are small plastic tags with visible alphanumeric codes. Visible implant alphanumeric tag identification requires fish recapture, but tag numbers can be seen with the naked eye. However, tag retention in the few small-bodied species tested varied from 32% to 100% (Sandford et al. 2020).

Visible implant alphanumeric tags may be promising for uniquely tagging the endangered Rio Grande Silvery Minnow Hybognathus amarus. Rio Grande Silvery Minnow is a small-bodied, federally endangered freshwater fish native to the Rio Grande Basin in the southwestern United States (Bestgen and Platania 1991). These fish are well-studied; however, estimating demographic parameters has been difficult because of extreme inter- and intra-annual variation in abundance and low capture probability in a large river (Yackulic et al. 2022). The ability to uniquely mark wild Rio Grande Silvery Minnow in situ could aid in management decisions by improving estimates of abundance, survival, movement, and captive broodstock management. Because VIA tags are inexpensive and can be read without specialized equipment, they may be suitable for in situ studies on Rio Grande Silvery Minnow after evaluation under controlled laboratory conditions.

Our objectives were to determine the feasibility of VIA tag use in Rio Grande Silvery Minnow as a marking method. Feasibility for field studies is dictated by survival, retention of tags in target species, and ease of use in the field. Visible implant alphanumeric tags may be viable for field studies involving Rio Grande Silvery Minnow or other small-bodied fishes. Thus, we evaluated four responses after tagging: 1) fish survival, 2) fish growth, 3) tag retention, and 4) tag legibility. We frame our results in the context of feasibility for field studies of small-bodied fishes.

Rio Grande Silvery Minnow are regularly held in captivity as a source of broodstock for captive propagation efforts. We obtained 200 age-3 retired broodstock from the Albuquerque BioPark Aquatic Conservation Facility in Albuquerque, New Mexico, in November 2023. We held fish in a 5,100-L recirculating system consisting of seven individual fiberglass tanks, one of which contained the sump and filtration equipment. The system was filled with reverse-osmosis filtered municipal water. Water drains through a standpipe in each tank to a sump containing a sand filter (flow rate ∼215 L/h) before being circulated back to each tank. The mean volume of water in each treatment tank was ∼700 L. Fish were fed 1% of their body mass twice per day during weekdays throughout the study, and 2% once a day on weekends.

One hundred-eighty of the 200 fish were selected for the experiment, with 30 fish held in each of six tanks for the duration of the study. The treatment order was randomized, with 10 fish in each tank designated as control fish that were anesthetized but not tagged. Of the remaining fish, 10 fish in each tank were visible implant elastomeric (VIE) tagged blue and VIA tagged and the other 10 served as a sham control that were VIE tagged orange and injected with the empty VIA applicator. The VIE tags consisted of ∼5 mm orange or blue elastomer injected subcutaneously to the left of the dorsal fin. Fluorescent orange VIA tags were injected on the right side of the dorsal fin (Figure 1). Each fish was anesthetized with MS-222, weighed, measured (standard length, 1 mm), held out of the water for ∼30 seconds while tags were applied, and placed into the corresponding randomly assigned tank. The tagger was experienced with tagging fishes but had no previous experience with VIA tags. Control fish were anesthetized, weighed, and measured, but given no tags or sham tags, and held out of the water to simulate air exposure during tagging to compensate for potential effects of additional handling of tagged fish.

We monitored and recorded dead fish twice daily for seven weeks (49 days). At the end of the treatment period, each fish was weighed and measured. Visible implant alphanumeric tags were fluoresced with a handheld ultraviolet light and examined with a magnifying glass to determine readability. Tag loss was either recorded at the death of an individual or by visual final inspection on day 49. Tag legibility was assessed on a three-level qualitative scale: illegible, legible (reader was able to see the tag but was not confident of the tag number), and clearly legible (reader was confident of reading the correct number). Additionally, we monitored VIA-tagged fish for two weeks after reading tags to observe any mortality associated with extended handling times during tag reading.

To investigate negative effects of tagging on Rio Grande Silvery Minnow growth and how survival or tag retention was influenced by fish length or body condition, we used a randomized block design with tank as a random effect. Limited or no mortality among treatment groups and tanks prevented the use of a randomized block design analysis, as the observed variance was 0. Instead, we used a χ² test to determine the statistical significance of treatment effects on survival. To estimate the effects of body condition on tag retention, we calculated the scaled mass index following Peig and Green (2009) using the average length and slope of the standardized major axis regression from the log(weight)-log(length) relationship from all 180 fish used in the study. The average standard length was 62.4 mm, and the slope was 2.81. Control and sham control fish were not individually identifiable, so changes in length and weight were based on tank averages. Length and body condition were predicted by period (pre-experiment or post-experiment) and treatment, with tank as a blocking variable. We used a logistic regression to estimate the effect of fish length on post-tagging survival and tag retention, where tank was also a blocking variable. We used program R (ver. 4.3.2, R Foundation for Statistical Computing, Vienna, Austria, see https://www.r-project.org/) for logistic models, and R package smatr (ver. 3.4.8, see https://cran.r-project.org/package=smatr/; Warton et al. 2012) to determine the slope of the standard major axis regression.

Differences in survival among treatment groups were neither statistically significant nor biologically relevant (χ² = 4.14, P = 0.126), although we note the results of the χ² test may be unreliable as the expected cell counts for dead fish were all < 5. Overall survival was high, with 100% survival for control fish, 96.7% for VIA-tagged fish, and 93.3% for sham-tagged fish. At the beginning of the experiment, sham-tagged fish were slightly larger (2.3 mm longer, 0.3 g heavier), but the differences were marginally statistically significant (Table 1). More importantly, we observed no statistically significant interactions between treatment post-experiment lengths, weight, or scaled mass index (Table 1). All fish were longer (P < 0.001) and heavier (P = 0.007) and improved in scaled mass index (P = 0.003) at the conclusion of the experiment (Table 1). Tagging did not affect growth or body condition (Figure 2); we found no statistically significant or biologically relevant changes in length, weight, or scaled mass index among groups after tagging (Table 1). However, retention of VIA tags was low at 36.2%, with only 21 of the 58 surviving fish retaining their tag. Tag retention did not appear to be related to length or scaled mass index; however, there was a weak statistical relationship between tag retention and weight (Figure 3). Of the 21 fish that retained VIA tags, one tag was illegible, five were legible, and 15 were clearly legible. Although we did not test directly for survival of fish that were VIE tagged, fish with VIE tags had high survival and 100% retention. We observed no mortality for two weeks after the final examination for tag retention.

There is little previous research on the efficacy of VIA tags used to uniquely mark small cyprinids, and this study is the first using Rio Grande Silvery Minnow. Although we found acceptable effects on growth and survival and tags were generally legible, tag retention was extremely low, limiting the usefulness of this method for Rio Grande Silvery Minnow. Despite differing tag placement, our retention rates were like those observed for the congeneric Western Silvery Minnow H. argyritis tagged in the flesh over the operculum (45%, Neufeld et al. 2015). Tag placement in the operculum of Western Silvery Minnow required use of larger fish (>80 mm fork length), which would preclude the tagging of smaller individuals used in this study. Overall, poor tag retention is a common theme for VIA-based marking of small-bodied fishes (Griffiths 2002; Olsen et al. 2004; Davis et al. 2014), though there have been some limited successes with Delta Smelt (Lindberg et al. 2013). Consequently, we agree with Neufeld et al. (2015) that VIA tags could only be marginally useful in small-bodied cyprinids. Despite finding poor retention of VIA tags in Rio Grande Silvery Minnow, our study offers some insights for tagging small fishes.

Although formal evaluation of VIE tagging was not the objective of this study, we observed very low mortality in sham-tagged Rio Grande Silvery Minnow. Tagging-induced mortality and tag retention of VIE tags in Rio Grande Silvery Minnow has been studied (Archdeacon et al. 2023) but lacked a true control group. Here, the combination of VIE and sham-tagging had very little effect on survival and all sham-tagged fish retained their VIE tags. Typically, survival after VIE tagging is high among small-bodied fishes (Bangs et al. 2013; Jungwirth et al. 2019; Long et al. 2024). We provide further evidence of the utility of VIE as a batch-mark for small-bodied fishes.

We chose to use hatchery fish instead of collecting wild fish. Aside from not harming source populations through over-collection, use of hatchery fish allowed us to design controlled experiments on presumably healthy fish. In a tagging experiment on Western Silvery Minnow, mortality was high across all experimental groups (20%), with >20% mortality in control groups, which the authors attributed to confinement and handling stress (Neufeld et al. 2015). In contrast, we observed 100% survival in control fish and low mortality in VIA and sham-tagged fish, which required similar handling times to record length and weight. Our observations on survival agree with other tagging studies that used large numbers of hatchery fish to evaluate survival and retention after tagging of small fishes (Olsen et al. 2004; Bolland et al. 2009; Turek et al. 2014). We suggest using a large sample size of healthy fish for experimental designs where growth or mortality are key components for evaluating feasibility. However, we caution extrapolating survival estimates from laboratory studies to field studies. Laboratory studies can be used to compare different tagging methods under controlled conditions, but robust in situ estimates of tag-induced mortality for field studies will inform analyses.

We noted several other issues with the feasibility of VIA tags for small-bodied fishes. Insertion of VIA tags required precise injection into a readable location under the skin, which proved difficult and time-consuming with small-bodied fish, even in a controlled setting. The tags were sometimes difficult to load into the injector, which could be exacerbated by conditions in the field. There is also evidence to suggest VIA tag readability degrades over time and tags may become illegible within four months of tag injection (Knapp et al. 2023). Although VIA tags are less expensive and tagged fish exhibited high survival, the low retention and difficulty of insertion disqualify them as a viable alternative for PIT tags when used in large-scale tagging events. Investigation into other tagging methods for uniquely marking small-bodied fishes is needed. Designing field studies of Rio Grande Silvery Minnow around batch marks, such as VIE, remains the most feasible marking method.

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Data S1. Microsoft Excel spreadsheet of survival and tag retention data collected during an examination of the use of visible implant alphanumeric tags in captive Rio Grande Silvery Minnow Hybognathus amarus in 2024.

Available: https://doi.org/10.3996/JFWM-24-047.S1 (42.7 KB xlsx)

We thank Chasity Barnes for assistance with data collection and fish husbandry during the experiment, Lyle Thomas for assistance with fish husbandry and equipment maintenance, and the U.S. Bureau of Reclamation for funding this project. We thank two anonymous reviewers for the comments to help improve the manuscript. Fish were held under U.S. Fish & Wildlife Service permit TE676811-0 and New Mexico Department of Game and Fish permit 3764.

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