Survival, growth and tag retention of juvenile European eel (Anguilla anguilla L.) with implanted 12 mm passive integrated transponder tags and acoustic tags.

To evaluate the efficiency of tagging juvenile European eels with implanted 12 mm passive integrated transponder (PIT) tags or Eel/Lamprey acoustic transmitters (ELATs), the authors studied tag retention, survival and growth of eels (7-25 g). Experimental eels were obtained from an eel farm, tagged and then released in a series of shallow dug-out ponds with a surface area of c. 200 m2 . Tagged and control eels were distributed evenly, with 50 tagged and 50 control eels in each of four ponds, giving a total of 200 tagged and 200 control eels mixed. After 76 days, the ponds were drained, and eels were sampled and measured. A total of 344 eels (86%) were recaptured, indicating high survival. Tag retention was 99% as only one of the recaptured PIT-tagged eels had lost the tag and none of the ELAT tagged. The results demonstrated that tagging juvenile eels >16 cm with these small tags is indeed feasible. The growth of tagged and control fish was differentiated but generally low in length and negative in mass but did not differ between the three groups.

Longer is Not Always Better: Optimizing Barcode Length for Large-Scale Species Discovery and Identification.

New techniques for the species-level sorting of millions of specimens are needed in order to accelerate species discovery, determine how many species live on earth, and develop efficient biomonitoring techniques. These sorting methods should be reliable, scalable, and cost-effective, as well as being largely insensitive to low-quality genomic DNA, given that this is usually all that can be obtained from museum specimens. Mini-barcodes seem to satisfy these criteria, but it is unclear how well they perform for species-level sorting when compared with full-length barcodes. This is here tested based on 20 empirical data sets covering ca. 30,000 specimens (5500 species) and six clade-specific data sets from GenBank covering ca. 98,000 specimens ($>$20,000 species). All specimens in these data sets had full-length barcodes and had been sorted to species-level based on morphology. Mini-barcodes of different lengths and positions were obtained in silico from full-length barcodes using a sliding window approach (three windows: 100 bp, 200 bp, and 300 bp) and by excising nine mini-barcodes with established primers (length: 94-407 bp). We then tested whether barcode length and/or position reduces species-level congruence between morphospecies and molecular operational taxonomic units (mOTUs) that were obtained using three different species delimitation techniques (Poisson Tree Process, Automatic Barcode Gap Discovery, and Objective Clustering). Surprisingly, we find no significant differences in performance for both species- or specimen-level identification between full-length and mini-barcodes as long as they are of moderate length ($>$200 bp). Only very short mini-barcodes (<200 bp) perform poorly, especially when they are located near the 5$^\prime$ end of the Folmer region. The mean congruence between morphospecies and mOTUs was ca. 75% for barcodes $>$200 bp and the congruent mOTUs contain ca. 75% of all specimens. Most conflict is caused by ca. 10% of the specimens that can be identified and should be targeted for re-examination in order to efficiently resolve conflict. Our study suggests that large-scale species discovery, identification, and metabarcoding can utilize mini-barcodes without any demonstrable loss of information compared to full-length barcodes. [DNA barcoding; metabarcoding; mini-barcodes; species discovery.].

Quantification of alizarin red S uptake in coregonid eggs after mass-marking.

An easy method to measure the uptake rate of the persistent dye alizarin red S (ARS) during marking of whitefish eggs was established and used to measure the ARS content in three different whitefish species during and at the end of the marking procedure. Those values show that only 6-10% of the ARS in the marking solution will be absorbed by the eggs (0.0061-0.0119 mg per egg). Additional analyses 6, 15 and 36 months after marking showed ARS levels below the response level (<6.9 µg kg-1 ).

Assessing DNA for fish identifications from reference collections: the good, bad and ugly shed light on formalin fixation and sequencing approaches.

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.

Differentiating three Indian shads by applying shape analysis from digital images.

Tenualosa ilisha, T. toli and Hilsa kelee are three Indian shad species of the family Clupeidae. In past, relationships among these shads have been explored in few morphological studies but still remain poorly understood. In this study, 120 specimens of three shad species were collected from the wild and a truss network system of 13 landmarks was interconnected to yield 77 size-adjusted characters. Discriminant function analysis (DFA) produced 13 discriminating variables in delineating the three species. The landmark based morphometric distance between origin of dorsal fin and posterior end of eye was the most important character in discriminating the three species. Three prominent characters extracted from DFA (distance between posterior end of operculum and posterior end of eye, distance between origin of dorsal fin and posterior end of eye and distance between insertion of pelvic fin and end of operculum) were used as traditional morphometric characters and their ratio to standard length were found significant in differentiating the three shad species. The present study strongly supports the efficiency of landmark-based morphological characters generated from digital images in discriminating the species and can be used as a rapid diagnostic tool in recognizing them.

A new model study species: high accuracy of discrimination between individual freckled hawkfish (Paracirrhites forsteri) using natural markings.

Variations between distinct natural markings of freckled hawkfish (Paracirrhites forsteri) could allow in situ identification of individuals from underwater photography. Receiver operating characteristic analysis was used to assess the ability of the Interactive Individual Identification System (I3 S) software to assist in discriminating between images of P. forsteri individuals. This study's results show the high discriminant ability of I3 S to differentiate between unlike individuals and identify images of the same individual. The ability to use automatic computer-aided assistance in the study of this species will enable future research to explore behaviour and movements of individuals in the wild.

Surgical implantation of radio tags in three eel species (Anguilla spp.) in South Africa.

Studies have reported poor survival of surgically tagged freshwater fishes in warm African waters. This study aimed to assess the applicability of using radio telemetry (and surgical implantation of tags) for Anguilla spp. Nineteen yellow eels (Anguilla bengalensis, A. marmorata and A. mossambica) were surgically implanted with radio tags between October 2018 and January 2019 in the Thukela River, South Africa. Most eels were alive 6 months after tagging, and recaptured eels displayed advanced or complete healing at the incision site. Therefore, this method appears suitable for African freshwater eels.

[A multiplex PCR method based on nuclear and cytoplasmic inheritance to identify the horse and donkey-derived components of Asini Colla Corii and the hide].

To identify the original components of Asini Colla Corii and its raw material hides provides a guarantee for authenticity of Asini Colla Corii. It is urgent for Asini Colla Corii production enterprises and market supervision departments to develop effective identification methods of Asini Colla Corii and hides derived from horses, donkeys, mules and hinnies. This study screened species-specific DNA sequences of nuclear and mitochondrial genomes as detection targets, designed horse and donkey specific primers and established multiple PCR identification methods for identifying the animal hides (including the horse, donkey, mule and hinny) and Asini Colla Corii containing horse-derived and donkey-derived components. Our method can identify the horse, donkey, mule and hinny hides and horse, donkey-derived components of Asini Colla Corii with high species specificity (no crossed amplification was observed ). The limit of detection was 0.2 ng DNA. The method developed in this study provides technical support for Asini Colla Corii production enterprises and market supervision departments.

A comparison of population estimation techniques for individually unidentifiable free-roaming dogs.

BACKGROUND: Measuring the size of free roaming dog populations quickly and accurately is critical in the implementation of numerous preventive health and population control interventions. However, few studies have investigated the relative performance of population size assessment tools when applied to dogs. The aim of this study was to compare the commonly used mark-resight methodology with distance sampling methods, which are less resource intensive, to estimate free-roaming dog abundance in Goa, India. Twenty-six working zones were surveyed along all roads twice by the same surveyor at the same time of day, following a vaccination campaign which included marking of vaccinated dogs with a coloured paint. The Chapman estimate was then used to evaluate the mark-resight abundance. Additionally, the number of dogs and perpendicular distance from the road for all dogs sighted was recorded. This was used to estimate dog density and abundance using distance sampling methods. The detection function was fitted based on goodness-of-fit and AIC. RESULTS: The Chapman abundance estimate for the entire study area was 5202 dogs (95%CI 4733.8-5671.0), and the distance sampling method abundance estimate was 5067 dogs (95%CI 4454.3-5764.2). For individual working zones, after taking other factors into account in a mixed effects model, the average distance sampling estimate was 35% higher (95%CI 20-53%) than the Chapman estimate. There was also evidence of a difference in estimates between surveyors of 21% (95%CI 7-37%) and between days (22% lower on day 2, 95%CI 8-38%) for individual working zones. CONCLUSION: Our study demonstrated that the distance sampling estimates were comparable overall to the Chapman method of abundance estimation of free roaming dogs across the entire study region but there was noticeable variation between the two methods when individual zones were compared. Consequently, distance sampling methods may be suitable to enumerate dogs over large areas in a more time efficient manner than the widely used mark-resight approach.

Detection of Cattle Using Drones and Convolutional Neural Networks.

Multirotor drones have been one of the most important technological advances of the last decade. Their mechanics are simple compared to other types of drones and their possibilities in flight are greater. For example, they can take-off vertically. Their capabilities have therefore brought progress to many professional activities. Moreover, advances in computing and telecommunications have also broadened the range of activities in which drones may be used. Currently, artificial intelligence and information analysis are the main areas of research in the field of computing. The case study presented in this article employed artificial intelligence techniques in the analysis of information captured by drones. More specifically, the camera installed in the drone took images which were later analyzed using Convolutional Neural Networks (CNNs) to identify the objects captured in the images. In this research, a CNN was trained to detect cattle, however the same training process could be followed to develop a CNN for the detection of any other object. This article describes the design of the platform for real-time analysis of information and its performance in the detection of cattle.

Technical note: Use of an automated grooming brush by heifers and potential for radiofrequency identification-based measurements of this behavior.

Healthy cattle readily use grooming brushes but this behavior subsides when animals become ill. Tracking use of a brush may provide an opportunity for health monitoring, especially if the process could be automated. We assessed how healthy heifers groom themselves on a brush and hypothesized that radiofrequency identification (RFID) could be used to accurately and automatically record this behavior. Angus and Hereford heifers (n = 16) were fitted with 2 ultra-high-frequency RFID ear tags and monitored in groups of 8 while housed in a pen with an electronic brush, video cameras, and 4 RFID antennas. Each heifer was observed for a 6-h period using continuous video recordings, and brush contact was characterized in terms of anatomic region involved (head/neck, trunk, or posterior) and when not touching the brush but within 1 body length of it. The RFID data were collected for the same period and then processed such that intervals of up to 16 s with no detections but contained between 2 recordings were also considered positive (animal in brush proximity). Brush proximity (RFID) was regressed against brush contact duration (video) and the sensitivity and specificity for each individual heifer calculated. Across heifers, the majority of brush use involved the head/neck, although a few heifers demonstrated relatively large amounts of posterior contact, which contributed to false-negative readings when antennas failed to read the ear tags. Furthermore, for the majority of time that animals were near the brush, they were not in contact with it but rather standing or lying nearby, resulting in false-positive readings. It follows that the ability of the RFID system to accurately detect brush contact varied widely across individual heifers (sensitivity 0.54-1.0; specificity 0.59-0.98), with RFID generally overestimating the duration of brush proximity relative to actual time spent in brush contact. The implication is that RFID-based ear tag recording of brush proximity relative to continuous video observations of contact does not yield accurate results in certain heifers and therefore, as currently configured, is not a reliable representation of this type of grooming behavior.

Effect of topical vapocoolant spray on perioperative pain response of unweaned calves to ear tagging and ear notching.

OBJECTIVE: To evaluate the efficacy of a vapocoolant spray to provide local anaesthesia for calves during ear tagging and ear notching. STUDY DESIGN: Randomized study. ANIMALS: Twenty Black Angus calves aged 4-16 weeks old. METHODS: Temperature validation studies using thermocouples and a temperature data logger were conducted on dead and live tissue to determine optimal spray distance and duration to achieve tissue anaesthesia (<10 °C). A behavioural trial was conducted to assess efficacy for ear tagging and ear notching. Calves (n = 20) were randomly assigned to the vapocoolant spray (VS) or the water spray (WS) group. A 3 second spray was administered from 10 cm to both sides of the ear immediately prior to ear tagging and ear notching. A numerical rating scale (NRS) was used to score behavioural response to each procedure, with responses categorized from 0 (no response) to 3 (severe). RESULTS: Temperature and tissue validation studies indicated that a vapocoolant spray reduced dead and live tissue temperature to below nociceptive threshold levels (10 °C) for 10-16 seconds. Univariate analysis indicated that ear notching was more painful than ear tagging [odds ratio (OR) = 19.2, 95% confidence interval (CI): 5.34-68.99, p < 0.001]. When adjusted for the multivariate model, there was a significant effect of treatment, with WS calves showing higher pain response scores than VS calves (OR = 4.08, 95% CI: 1.34-12.42, p = 0.011). Ear notching resulted in greater pain response scores than ear tagging (OR = 23.19, 95% CI: 6.18-87.05, p < 0.001). CONCLUSIONS: and clinical relevance Vapocoolant spray induced local anaesthesia and significantly reduced the pain response to ear tagging and ear notching in calves. Ear notching is more painful than ear tagging. Cryoanaesthesia is an effective option for reducing the perioperative pain associated with these simple husbandry procedures.

Myxozoans as biological tags for stock identification of the Argentine hake, Merluccius hubbsi (Gadiformes: Merlucciidae).

Myxozoans have been successfully used as tags for fish stock identification around the world. However, few studies using myxozoan tags have been carried out in the Southern Atlantic, a region with complex oceanography that constitutes a potentially suitable scenario for testing the utility of myxozoans as indicators. Its usefulness was tested using six samples of Merluccius hubbsi in two different regions of the Argentine Sea. Generalized linear models were performed to assess the effects of fish size and sex, and year and region of capture and selected using the Information Theoretic approach. Three myxozoan species were recorded: Kudoa rosenbuschi, Myxoproteus meridionalis and Fabespora sp. Results of modelling species individually showed differential capabilities for detecting geographical population structure at different spatial scales, with K. rosenbuschi and Fabespora sp. allowing the discrimination of northern and southern stocks, but Fabespora sp. also as a promissory indicator of intrapopulation sub-structure due to different migratory routes during non-reproductive periods. This work confirms that myxozoans offer a set of suitable markers at different spatial scales, which can be selected individually or in any combination, depending on the geographical extent of the study, constituting tools adaptable to the objectives of further research on fish population structure.

Migration of Tundra Swans (Cygnus columbianus) Wintering in Japan Using Satellite Tracking: Identification of the Eastern Palearctic Flyway.

Migration through the Eastern Palearctic (EP) flyway by tundra swans (Cygnus columbianus) has not been thoroughly documented. We satellite-tracked the migration of 16 tundra swans that winter in Japan. The objectives of this study were 1) to show the migration pattern of the EP flyway of tundra swans; 2) to compare this pattern with the migration pattern of whooper swans; and 3) to identify stopover sites that are important for these swans' conservation. Tundra swans were captured at Kutcharo Lake, Hokkaido, in 2009-2012 and satellite-tracked. A new method called the "MATCHED (Migratory Analytical Time Change Easy Detection) method" was developed. Based on median, the spring migration began on 18 April and ended on 27 May. Autumn migration began on 9 September and ended on 2 November. The median duration of the spring and autumn migrations were 48 and 50 days, respectively. The mean duration at one stopover site was 5.5 days and 6.8 days for the spring and autumn migrations, respectively. The number of stopover sites was 3.0 and 2.5 for the spring and autumn migrations, respectively. The mean travel distances for the spring and autumn migrations were 6471 and 6331 km, respectively. Seven migration routes passing Sakhalin, the Amur River, and/or Kamchatka were identified. There were 15, 32, and eight wintering, stopover, and breeding sites, respectively. The migration routes and staging areas of tundra swans partially overlap with those of whooper swans, whose migration patterns have been previously documented. The migration patterns of these two swan species that winter in Japan confirm the importance of the Amur River, Udyl' Lake, Shchastya Bay, Aniva Bay, zaliv Chayvo Lake, zal Piltun Lake, zaliv Baykal Lake, Kolyma River, Buyunda River, Sen-kyuyel' Lake, and northern coastal areas of the Sea of Okhotsk.

A minimally invasive technique to assess several life-history characteristics of the endangered great hammerhead shark Sphyrna mokarran.

A dorsal-fin photo-identification technique paired with a non-invasive parallel laser photogrammetry technique was used to non-invasively identify individual Sphyrna mokarran over time. Based on the data collected over a duration of 59 days, 16 different S. mokarran (mean ± S.D. pre-caudal length: 220·82 ± 13·66 cm; mean ± S.D. cephalofoil width: 71·38 ± 7·94 cm) were identified using dorsal-fin photo-identification, with a mean ± S.D. shark re-sighting frequency of 4·05 ± 3·06 at-sea days. The results illustrate a high S. mokarran sighting rate and therefore, the utilization of parallel laser photogrammetry and dorsal-fin photo-identification may be a plausible multi-year approach to aid in non-invasively determining the growth rate and inter-annual site fidelity of these animals.

An appraisal of the Maltese national livestock database with regard to bovines.

The creation of a centralised national livestock database for the islands of Malta and Gozo is of crucial importance for the identification and traceability of bovines. It is also important for compliance with the legal obligations that followed Malta's accession to the European Union in May 2004. This paper describes how the processes of identification, registration and traceability of bovines have changed since Malta's accession. The validation and integration of data originating from different departmental sections (such as the identification and registration section), the slaughterhouse and the National Veterinary Laboratory, ensures that any discrepancies are highlighted and can be investigated. Events recorded in the database enable the compliance and eligibility of bovine producers to be cross-checked when applications for European Union benefits are made. The main drawbacks and weak points of the system include financial costs for the government department, potentially late notification of the births and deaths of newborn calves, and insufficient uptake among bovine producers of the latest technology for notification of events such as births, deaths and movement of bovines.

Modeling structured population dynamics using data from unmarked individuals.

The study of population dynamics requires unbiased, precise estimates of abundance and vital rates that account for the demographic structure inherent in all wildlife and plant populations. Traditionally, these estimates have only been available through approaches that rely on intensive mark-recapture data. We extended recently developed N-mixture models to demonstrate how demographic parameters and abundance can be estimated for structured populations using only stage-structured count data. Our modeling framework can be used to make reliable inferences on abundance as well as recruitment, immigration, stage-specific survival, and detection rates during sampling. We present a range of simulations to illustrate the data requirements, including the number of years and locations necessary for accurate and precise parameter estimates. We apply our modeling framework to a population of northern dusky salamanders (Desmognathus fuscus) in the mid-Atlantic region (USA) and find that the population is unexpectedly declining. Our approach represents a valuable advance in the estimation of population dynamics using multistate data from unmarked individuals and should additionally be useful in the development of integrated models that combine data from intensive (e.g., mark-recapture) and extensive (e.g., counts) data sources.