Visible Implant Research Articles

Zebrafish Injectable Plastic for Identification Tagging (ZIP IT) for larvae to adults using a fluorescent Visible Implant Elastomer

Distinguishing individuals or small groups is essential for many experiments. The regenerative properties of zebrafish make traditional marking methods for rodent models (e.g., tattoos, tissue clipping) ineffective. Fluorescent Visible Implant Elastomer (VIE) can permanently mark juvenile and adult zebrafish but to date no marking technique has been described for larval stage zebrafish. This protocol, Zebrafish Injectable Plastic for Identification Tagging (ZIP IT), utilizes VIE in zebrafish as early as 2 days post fertilization (dpf) using standard microinjection methods and direct injection using an insulin syringe at one month. Larval zebrafish between 2 and 7 dpf were injected in the dorsal musculature. At one month, retention and visibility of the VIE was observed in 72% of the injected fish with no effect on growth; however, a variable change in mortality was observed, generally higher than the uninjected fish. This demonstrates that VIE can be used in very early stages of fish development, providing the first procedure to track individuals or groups within a larger population. Subcutaneous injection of juvenile zebrafish starting at one month has greater than 99%-mark retention and visibility with very low mortality. The combination of larvae and juvenile VIE injections also provide a powerful tool to track and gather data from marked fish throughout their lifespan.•We present a method for tagging individuals or groups of zebrafish at most life stages (larvae or juvenile/adult) with Visible Implant Elastomer (VIE).•The larvae and juvenile injection procedures can be combined so that an individual fish can be tagged for its entire lifetime. Larvae injections become significantly less visible after one month; however, the fish can be reinjected using the juvenile procedure, thus allowing continuity of the visible mark.•This protocol was empirically built on the “Evaluation of VIE tags in Zebrafish (Danio rerio)” by Hohn and Petrie-Hanson (2013) and expanded to include larvae and a small batch elastomer mixing technique.

Visible Implant Elastomers in gilthead seabream (Sparus aurata L.) for experimental research: Preferred injection sites to optimize tag retention and minimize histological effects

Visible Implant Elastomers in gilthead seabream (Sparus aurata L.) for experimental research: Preferred injection sites to optimize tag retention and minimize histological effects

Migration and Histologic Effects of Visible Implant Elastomer (VIE) and Passive Integrated Transponder (PIT) Tags in the Marine Toad (Rhinella marina).

Simple SummaryPassive integrated transponder (PIT) and visible implant elastomer (VIE) tags are commonly used to identify reptiles, amphibians, and fish. The aim of this study was to evaluate for pathologic changes associated with these tags in the marine toad (Rhinella marina). For the 56 toads in this study, all PIT tags were functional and 95% remained at the insertion site with little to no damage to the tissue. However, only 48% of VIE tags were functional, i.e., visible through the skin. Although there was little to no damage to the skin at the site of placement, the VIE was found to have migrated to the kidneys in 98% of toads as well as along the legs and sporadically in other internal organs. VIE in the kidney caused inflammation and damage to the vasculature that progressed in severity over time. Based on these findings, the authors recommend the use of PIT tags over VIE tags for identification of adult anurans, when feasible.Passive integrated transponder (PIT) and visible implant elastomer (VIE) tags are commonly used to identify reptiles, amphibians, and fish. Despite reports of good retention rates and little to no negative effect on survival time, migration remains a concern and histologic changes have not been widely evaluated. Fifty-six wild-caught marine toads (Rhinella marina) were marked with a PIT tag in the left caudal thigh and a VIE tag over the right gastrocnemius muscle prior to transport to the North Carolina Zoo. Fourteen toads were then humanely euthanized on day 9, 15, 32, and 62 for postmortem examination and histopathology which were compared to 10 control toads with no tags. All PIT tags were functional and 95% remained at the insertion site with minimal to no histologic changes. Externally, 48% of VIE tags were visible through the skin at the original site of injection under fluorescent or UV light. Upon gross examination of the tissues, VIE tags had an overall retention rate of 62% at the injection site, with similar retention rates across time points. Migrated VIE material was visible grossly and histologically in the kidneys of 98% of toads and along the right leg, proximally and distally, in 25% of toads. VIE material was also found sporadically in mesentery, colon, and free in the coelomic cavity. Histologically, VIE material in the skin was surrounded by minimal to mild granulomatous inflammation while in the kidney it was associated with dilation of the small vessels, edema, and granulomatous nephritis that progressed in severity over time. Based on these findings, the authors recommend the use of PIT tags over VIE tags for identification of adult anurans, when feasible.

Retention of Postocular Visible Implant Elastomer in Two Sizes of Adult Brown Trout and Rainbow Trout

AbstractRetention of visible implant elastomer (VIE) was assessed during an evaluation of put‐grow‐and‐take Brown Trout Salmo trutta and Rainbow Trout Oncorhynchus mykiss stockings in Apalachia Reservoir, North Carolina. Visible implant elastomer was injected in the postocular adipose tissue of two sizes (approximately 254 [small] and 356 [large] mm TL) of Brown Trout and Rainbow Trout in November 2012–2015. Each year, 1,000 large and 1,500 small trout of each species were marked with VIE. Mark retention was assessed after 30 d in a hatchery and during poststocking collection events in Apalachia Reservoir. The recaptured trout were examined for VIE marks during annual spring electrofishing, fall gill‐net samples, and a year‐long creel survey. The 30‐d VIE retention rate for all trout and all years was 99.9%. Following stocking, both sizes of Rainbow Trout were collected up to 17 months after marking and VIE retention was 100%. Large and small Brown Trout were collected up to 33 and 34 months after marking, and the VIE retention rates were 100% and 99.3%, respectively. This study found visible implant elastomer to be an effective and simple batch mark that remained visible on Rainbow Trout for up to 17 months and Brown Trout for up to 34 months postmarking in the wild.

Ecological and growth patterns of the longsnout seahorse Hippocampus reidi inferred by mark-recapture techniques in a tropical estuary

Abstract: The population structure of the longsnout seahorse Hippocampus reidi (Ginsburg 1933) was evaluated in an estuarine system in northeastern Brazil. A total of 425 captures were performed in the Vaza-Barris estuary and 232 individuals were marked with Visible Implant Fluorescent Elastomers. The population was estimated to be composed of 428 fish. The von Bertalanffy growth curve shows a steeper growth during the first months of life, up to about 17 months, and stabilization after 16 cm and 27 months of age. The hereby study indicates that the Vaza-Barris estuary is home to resident populations of longsnout seahorses. Moreover, these populations use mangrove roots as the main substrate for attachment and present well-defined ecological characteristics, such as habitat fidelity and highly structured social organization.

Evaluation of Visible Implant Elastomer Tags in Wild Coastal Cutthroat Trout in the Marine Environment

AbstractWe evaluated the detectability of visible implant elastomer (VIE) tags in Coastal Cutthroat Trout Oncorynchus clarkii clarkii in the marine environment by comparing visually identified recaptures from VIE tags with known recaptures that were identified through genotype matching. A total of 89 individual Coastal Cutthroat Trout were marked in the lower jaw with colored VIE tags, sampled for genetics, and recaptured across 12 months in 2015. The rate of correspondence between the VIE tags and genetic matches was 92% (82/89) of the recaptured Coastal Cutthroat Trout in the nearshore marine environment. We found that red‐ and blue‐colored VIE tags were detected at a higher rate (100%) than were yellow‐ and orange‐colored tags (87.3% and 90.6%, respectively). In contrast, tag type (single or double), tag location (left or right), fish length (FL, mm), and time (days) since tagging had no effect on tag detectability during the study period. All of the tag colors were recovered for the maximum life of the study (up to 342 days). In all of the cases of nondetections (5/89), the tags were not identified upon initial recapture or subsequent capture events, suggesting that they were lost or not visible immediately upon insertion in the field—as opposed to being unidentified due to the degradation of tag detectability over time. The results of this study suggest that VIE tags have the potential to be detectable in juvenile and adult Coastal Cutthroat Trout for at least 12 months after insertion, with blue and red performing the best. Additional monitoring extending beyond 12 months after tagging would be necessary to identify the maximum life of VIE tags.

The use of visible implant elastomer to permanently identify caecilians (Amphibia: Gymnophiona)

Identifying individual animals is important for studying populations and for the optimal management of individual animals in captivity. In the absence of natural markings that discriminate individuals, such identification may require animals to be marked by researchers. Amphibians are challenging subjects to mark due to their small size and sensitive, permeable and frequently shed skin. Visible Implant Elastomer (VIE) has been widely used to mark amphibians, but no long-term study has validated this technique in caecilian amphibians. We anaesthetised and attempted to VIE mark seven Herpele squalostoma and one Microcaecilia unicolor held at ZSL London Zoo. No specimens suffered ill effects of anaesthesia or VIE injection, but mean persistence of marks was 191 days in H. squalostoma suggesting that this marking technique is not suitable for identifying individuals of this species in the long-term. We were unable to inject VIE into the M. unicolor and/or the elastomer was not visible through the darkly pigmented skin. Further research is required to develop methods for long-term marking of a diversity of caecilians.

Visible Implant Alphanumeric (VIA) as a marking method in the lesser snouted treefrog Scinax nasicus

In this study we assessed the efficacy of Visible Implant Alphanumeric (VIA) for marking adults and juveniles of the Neotropical treefrog Scinax nasicus. We evaluated the success of this technique in the identification of individuals and the prevalence of tags in the field. As a control, we marked the same individuals through toe-clipping. Of 196 marked individuals, 57 were recaptured in a 7-month study period. Only one mark was unreadable because it was located too deep in the skin. We found one case of tag expulsion and two inverted tags. Almost complete regeneration of the adhesive disk was observed by the fifth month of the study in all recaptured frogs. We suggest VIA tagging method as suitable for S. nasicus over long term studies. Even though, a hybrid method for marking (VIA + toe-clipping) is recommended for species with dark and/or loose skin, or large frogs.

Effect of different stock types and dietary protein levels on key enzyme activities of glycolysis‐gluconeogenesis, the pentose phosphate pathway and amino acid metabolism inMacrobrachiumrosenbergii

A feeding trial was conducted to study the effect of dietary protein (DP) levels on the enzyme activities of different stocks of Macrobrachium rosenbergii juveniles, fed two diets prawn type diets either at optimum (ODP, 32%) or sub-optimum (SDP, 27%) dietary protein. The activity of key enzymes: glycolysis, gluconeogenesis, amino acid catabolism, pentose phosphate pathway, and energy metabolism was examined on different stocks of M. rosenbergii juveniles in various tissues (muscle, hepatopancreas, and gill). Wild juveniles of M. rosenbergii were collected from the west coast of India, Gujarat (G), Maharashtra (M) and from the east coast of India, Andhra Pradesh (A), and raised in culture ponds of 200 m2 at 1 juvenile m-2. The experiment was conducted for a period of 60 days. A Visible Implant Elastomer (VIE) Tag was injected into different prawn juveniles’ stocks A, (3.06–3.10 g), M, (0.80–1.01 g) and G (0.90–1.06 g) for the individual identification of each class. All the animals were acclimatized for 7 days before being released into the pond. Each of the two diets, the first with 27% SDP, termed the sub-optimum level (S), and the second 32% DP, termed the optimum level (O), was fed in triplicate ponds. The weight gain of stocked prawn was significantly higher for G stock (3.03 ± 0.18 g) compared to M stock (2.53 ± 0.25 g) and A stock (1.33 ± 0.10 g). In addition weight gain in ODP was significantly higher (3.05 ± 0.22 g) compared to SDP (2.11 ± 0.17 g). Furthermore, both protein level and stock type had a significant (p ≤ 0.05) effect on the survival rate, specific growth rate, and feed conversion ratio of the prawn. A significant interaction was recorded between metabolic enzyme activities and the variation of dietary protein levels and differences in M. rosenbergii stocks, in which stock “G” and “M” exhibited healthier enzyme physiological status than stock “A”. The findings presented here may conclude that the enzymes quickly respond to dietary protein manipulations of cultured shrimp. The results can be useful in identifying the optimum stock (genotypes) in a given culture condition and subsequently higher production may be achieved.

The Tagging Procedure of Visible Implant Elastomers Influences Zebrafish Individual and Social Behavior.

One commonly used method to preserve individual identity in the study of social behavior of zebrafish is through silicone-based visible implant elastomers (VIEs), which represent a safe and durable tagging procedure. While the effects of VIE tagging on welfare and general health have been addressed in detail, whether this procedure influences social behavior remains unclear. In this study, we compared individual and group behaviors exhibited by shoals composed of three individuals: two nontagged and one (focal subject) that was either nontagged (control condition) or sham-, purple-, blue-, or yellow tagged. Traditional behavioral parameters of activity, shoaling, and schooling (speed, polarization, and interindividual distances), along with an information-theoretic measure of social interaction (transfer entropy), were used to study the effect of tagging. Our findings indicate that tagging procedure per se significantly increased individual speed of the tagged subjects and of the group. The tagging procedure also altered the level of interaction between individuals, measured by transfer entropy. Conversely, tagging procedure did not influence shoaling and schooling tendencies. These findings suggest that VIE tagging may elicit some level of stress, which may affect some behavioral responses more than others. We recommend use of alternative methods such as multitracking systems when possible.

Influence of geographic origin on post-stocking survival and condition of European grayling (Thymallus thymallus) in a small river

The post-release survival and condition of 1+ year old European grayling Thymallus thymallus reared in a local (Husinec) and two 250–300 km distant, hatcheries (Pardubice and Hynčice) were evaluated by the recapture of tagged fish five months after release into the Blanice River, Czech Republic, the fish population of which was depleted by cormorant predation during several previous winters. The fish were marked using Visible Implant Elastomer tags and released into six sites along the river in May 2014: 100 locally sourced fish and 100 of a strain from a distant source per site. Significantly higher recapture of Husinec (33%) than Pardubice (20%) was found at one site but was offset by results in the remaining two sites into which they were stocked. Significantly higher site fidelity was shown by Husinec (12%) than Hynčice (7%) fish in the three sites. Lower initial weight and condition factor of the Husinec fish was equal to or higher than fish from the distant hatcheries at recapture. Some differences in final weight, length, and condition factor were found among groups regardless of release site. Lower recapture rate, growth, and condition were displayed in fish released into the three downstream sites (Husinec 1 and Hynčice). The results indicated the potential for successful use of non-autochthonous stock for grayling population recovery.

Do small barriers affect the movement of freshwater fish by increasing residency?

River fragmentation due to artificial barriers directly impacts fish communities by limiting migratory movements. This work aims to understand how small barriers affect the movements of a potamodromous cyprinid species - the Iberian barbel (Luciobarbus bocagei (Steindachner, 1864)) - in a 5.6km upstream segment of a stream impacted only by the presence of physical barriers. Fish were marked with Visible Implant Elastomer tags (VIE), and barriers were seasonally characterized. A total of 683 fish were tagged, with 104 recaptures, during five sampling seasons (spring 2012 - early summer 2013). Eleven of the recaptured fish moved past a barrier, without any preference in terms of direction of movement. There were no differences in length, either between migrants and residents, or between upstream and downstream migrants. The results show that although barbel are able to negotiate small barriers, part of the population did not move between fragmented reaches - an indication that under such conditions, fish species populations may adjust their life-history strategy to augment residency as it was hypothesized from the results.

Artificial Visible Implant Elastomer (VIE) tags of different colour and symmetry do not influence mate choice in a cichlid

Experimental individuals are frequently marked with coloured tags for individual identification. Except for birds, the consequences of such artificial tagging on mate choice have been rarely investigated even though individuals often prefer naturally brightly coloured or symmetrically ornamented mates. We tested whether differently coloured Visible Implant Elastomer (VIE) tags influence female mate choice in rainbow kribs,Pelvicachromis pulcher. Females were allowed to simultaneously choose between a control and a VIE-marked male. The VIE-marked male carried two tags of the same colour (red, blue, green or white) set symmetrically or asymmetrically. Females did not show a preference for or avoidance of males carrying any of the colours compared to control males, no matter if the tags had been set symmetrically or asymmetrically. Although we found no discrimination for or against colour-tags, we highlight the importance of considering potential influences of colour-marks on mate choice in behavioural and evolutionary studies.

Retention Rates of Passive Integrated Transponder Tags, Visible Implant Elastomer Tags, and Maxillary Marks in Wild Trout

AbstractTagging or marking is a common method to identify individuals or groups of fish, but these tools are compromised if tags or marks are shed or deteriorate over time. We evaluated retention rates of passive integrated transponder (PIT) tags, visible implant elastomer (VIE) tags, and maxillary clips in stream‐dwelling Yellowstone Cutthroat Trout Oncorhynchus clarkii bouvieri and Rainbow Trout O. mykiss of spawning size. We tagged 2,891 fish ≥150 mm TL with PIT tags in the body cavity, muscle tissue posterior to the cleithrum, or muscle tissue ventral to the dorsal fin. Retention of PIT tags in the body cavity of fish at large for 1 year (72.1%) was lower than those implanted in the dorsal musculature (94.1%) or cleithrum (83.5%) locations. For PIT tags implanted in the body cavity, retention was lower for females (59.4%) than males (89.7%), suggesting that PIT tags implanted in the body cavity were expelled with eggs during spawning events. Retention of PIT tags implanted in the cleithrum and dorsal musculature were unaffected by sex, but retention rates decreased as body size increased for the body cavity tagging location. Annual growth rates were not affected by any PIT tag location. We observed a PIT tag failure rate of 1.7%. The likelihood of a PIT tag being retained in muscle tissue after filleting the fish was highest for the dorsal musculature location (65.1%), followed by the cleithrum (64.9%) and body cavity (4.0%) locations. One‐year retention rates were 95.4% for VIE tags and 93.0% for maxillary clips. While our results demonstrate higher PIT tag retention in the musculature than the body cavity, human consumption concerns may prohibit the use of musculature implantation where angler harvest is possible.Received January 28, 2016; accepted May 11, 2016 Published online August 31, 2016

Evaluation of Three External Marking Methods of Farmed Atlantic Salmon for the Future Use of Differentiating it From Wild Atlantic Salmon

We evaluated different external marking methods for farmed salmon to differentiate it from wild salmon without any special tools. Three marking methods were tested: 1) Adipose fin (AF) removal, 2) Freeze branding (FB) and, 3) Visible Implant Elastomer (VIE). Location of the marking method on the fish, combination of marking methods and degree of AF removal were tested in three experiments. Atlantic salmon parr weighing 20 g were marked either with individual marks or in combination of two. Further all the fish were also PIT tagged. They were kept in freshwater tanks for 4 months and later after smoltification, smolts were transferred to sea cages and kept for another 4 months. At the end of four (freshwater phase) and ten (sea cages) months, growth, survival and mark retention were recorded. All these methods had no significant effects on growth and survival compared to the control (no mark but only PIT tagged). Our results showed that of these methods, only complete removal of the adipose fin met the requirements for mark retention and was the cheapest and easiest method to automate. However, a larger commercial scale long-term testing of the AF clipping is required prior to implementing it. Further development of an automated fin clipping in combination with vaccination and an open discussion with consumers, buyers, and environmental groups are also warranted.

Assessment of avoidance behaviour by earthworms (Lumbricus rubellus and Octolasion cyaneum) in linear pollution gradients

Avoidance behaviour by earthworms is recognised as a valuable endpoint in soil quality assessment and has resulted in the development of a standardised test (ISO 17512-1, 2008) providing epigeic earthworms with a choice between test and control soils. This study sought to develop and evaluate an avoidance test utilising soil-dwelling earthworms in linear pollution gradients with Visible Implant Elastomer (VIE) tags used to identify individual organisms. Sequential experiments were established in laboratory-based mesocosms (0.6m×0.13m×0.1m) that determined the relative sensitivities (in terms of associated avoidance behaviour) of Octolasion cyaneum and Lumbricus rubellus at varying levels of polluted soil and also assessed the influence of introduction point on recorded movement within gradients. In an initial gradient (0%, 25%, 50%, 75%, 100% polluted soil), both species exhibited a clear avoidance response with all surviving earthworms retrieved (after 7 days) from the unpolluted soil. In a less polluted gradient (0%, 6.25%, 12.5%, 18.75%, 25%) L. rubellus were retrieved throughout the gradient while O. cyaneum were located within the 0% and 6.25% divisions, suggesting a species-specific response to polluted soil. Results also showed that the use of a linear pollution gradient system has the potential to assess earthworm avoidance behaviour and could provide a more ecologically relevant alternative to the ISO 17512: 2008 avoidance test. However, further work is required to establish the effectiveness of this procedure, specifically in initial chemical screening and assessment of single contaminant bioavailability, where uptake of pollutants by earthworms could be measured and directly related to the point of introduction and retrieval.

Retention and Stress Effects of Visible Implant Tags When Marking Western Silvery Minnow and Its Application to Other Cyprinids (Family Cyprinidae)

AbstractVisible implant tags are commonly used in fisheries research to mark individuals or batches of fish as part of movement and mark–recapture studies. To be effective, these tags generally need to have high retention rates and little impact on the behavior or physiology of the marked individuals. We tested the retention rates of both visible implant elastomer (VIE) and visible implant alphanumeric (VIA) tags in 80–139‐mm Western Silvery Minnow Hybognathus argyritis over a 104‐d period. We also measured plasma cortisol and lactate to determine the impact of tagging on stress levels. We found that VIE tags had 100% retention and did not cause a significant increase in plasma cortisol levels, though plasma lactate levels were elevated in VIE treatment groups. Overall, VIE tags were found to be suitable for marking Western Silvery Minnow and similar species. Visible implant alphanumeric tags had 36% retention over 104 d and did not cause significant increases in plasma cortisol, though plasma lactate was elevated in the treatment group with both VIE and VIA tags. Given the high retention rates and low stress effects, VIE tags appear to be more beneficial for use with Western Silvery Minnow and similar‐sized cyprinids.Received April 16, 2015; accepted July 29, 2015

Visible Implant Elastomer as a Viable Marking Technique for Common Mistfrogs (Litoria rheocola)

Mark–recapture studies are often used to quantify rates of recruitment and survival, growth rates and patterns, and individual movement, among other things. Marking techniques must satisfy several criteria—marks should: (1) not affect the behavior or survival of individuals; (2) not cause undue pain or stress; (3) last as long as the study; and (4) be easily identifiable. Visible implant elastomer (VIE) tags can be used to mark vertebrates, including amphibians, but can move under the skin, potentially affecting readability and resulting estimates of dispersal and survival. We examined the effectiveness of VIE as a marking technique for Common Mistfrogs (Litoria rheocola) in the field over 1 yr. We determined the readability of marks over time, and evaluated effects of marking on measurements of frog movement. If marking affected mobility of the marked rear limb, we expected that subjects marked in both legs would be more strongly affected (thus, individuals should move less often), or should move shorter distances, than subjects marked in only one leg. We marked 1392 animals under the skin of the thighs in either the right, left, or both legs and recaptured 255 subjects at least once. We found that 84% of marks remained readable after 1 yr. Movement parameters were very similar between frogs marked in one or two legs. We conclude that VIE is a safe and effective marking technique for long-term amphibian studies, especially for stream-associated hylid frogs.

In situUnderwater Tagging of Aquatic Organisms: A Test Using the Cave-Dwelling Olm,Proteus anguinus

In situ ecological studies on obligate cave-dwelling aquatic animals are scarce at best. This is particularly true for capture-mark-recapture (CMR) studies that form the basis of understanding population structure and dynamics. Here, we report on the in situ underwater application of the Visible Implant Elastomer (VIE) tagging system on the olm, Proteus anguinus, an obligate cave-dwelling aquatic amphibian. We tagged seven adult individuals and monitored the population during 31 dive transects during four years. We found that VIE tagging is applicable underwater. Based on our recaptures, the tags were recognisable after four years and the recaptured three individuals exhibited extreme site-fidelity. Our results indicate that CMR studies are feasible in underwater cave ecosystems without even temporarily removing individuals. In situ underwater tagging also holds great potential for studies of other aquatic ecosystems, where removing animals from water, their habitat or territory is problematic for ethical, logistic or scientific reasons.

Visible Implant Elastomer tagging influences social preferences of zebrafish (Danio rerio)

Identification of individuals is a prerequisite in many behavioural studies. Visible Implant Elastomer (VIE) colour tags are a well-established way to mark animals. VIE tagging does not seem to affect individual growth or survival. However, studies verifying their neutrality during social interactions are less common. Here, individual male and female zebrafish Danio rerio were simultaneously given the choice between two shoals, each consisting of six fish. Members of one shoal were uniformly marked with one VIE tag of a given colour, whereas the other shoal was sham-tagged. In total, 10 different colours were used. Test fish spent significantly more time near the tagged shoal (56%) than near the sham-tagged shoal (44%). Tag colour did not significantly influence the preferences. The results highlight the importance of confirming the neutrality of colour tags before using them in behavioural studies. Especially, our study advises caution when using marked and unmarked fish simultaneously.