Glass eels collection (Anguilla anguilla, L.)
Three hundred European glass eels (< 7 cm) were collected in two French rivers, at Saujon in July 2016, at Pas de Bouc in June 2018 and in the Gironde Estuary in February 2018 and 2019 (Supplementary Fig. S1). A Hundred of glass eels from the Gironde Estuary were marked by Fish-Pass in a bath of 150 ppm of alizarin red S for 3 h (see details of the marking protocol in12). Before being sampled, especially the marked and control glass eels from the Gironde estuary were transferred at INRAE Saint-Seurin experimental station (animal experimentation approval number A33-478-001) for one month of rearing. After two hours of acclimation, these glass eels were divided equally into 4 baskets of control or marked glass eels set into an outdoor 14 m3-protected pool, continuously aerated and filled with well water. In each basket, the mean density was 40 g m−2. These young eels were fed ad libitum. After one month, alive young eels were collected for FRI analyses. In total, the analyses were conducted on 85 individuals with control (n = 35) and marked (n = 50) glass eels.
2D Fluorescence reflectance imaging (FRI)
In vivo FRI is an innovative and non-intrusive technique for fluorescence detection on live animals. This technique was tested for the first time on fish. In vivo FRI quantifies the fluorescence signals of live animals that are imaged under excitation and emission filter sets. Depth of penetration for FRI light in tissue is several centimeters, allowing for whole body imaging of small animals. The FRI analyses were conducted at Vivoptic platform (ANR-11-INBS-006, Univ. Bordeaux, CNRS, INSERM, TBM-Core, UMS 3427, US 5, F-33000 Bordeaux).
Marked (n = 8) and control live glass eels (n = 8) were anaesthetised with an eugenol solution (0.03 mL.L-1) before being placed one by one into a slot of an holder (a dish drainer, Sticks, Lékué, Espagne). The holder with live glass eels (4 marked, 4 control glass eels × 2) were placed into Lumina LT optical system (Perkin Elmer Inc., Boston, USA) equipped with a CDD camera (maximal field of view in the machine: 12.5 × 12.5 cm). All alive glass eels were imaged by FRI (1 s time of exposition) with two sets of excitation and emission wavelength filter: 1) 450 nm–480 nm at excitation, 515–575 nm at emission; 2) 485 nm–515 nm at excitation, 515–575 nm at emission. The imaging of glass eels was photographed (100 ms time of acquisition) and the 2D images were analysed with Living Image software (Perkin Elmer Inc., Boston, USA). The glass eels awaken were then transferred to the INRAE laboratory for the samplings.
Prior to the analyses, an alizarin drop (alizarin red S, 150 ppm, SigmaAldrich, China) was imaged (Supplementary Fig. S2b) to select the wavelengths of the excitation and emission filters. The holder was imaged with the filters selected and no autofluorescence was detected (Supplementary Fig. S2a).
Sampling
Biometrics and fin clipping were conducted in the dark for marked ones at INRAE. Glass eels were anaesthetized with eugenol solution (0.03 mL.L-1) before being euthanized with eugenol solution overdose (0.3 mL. L-1). Total body length (± 0.01 mm), wet mass (± 0.1 mg) were measured. Pigmentation stage was determined according to31. A total of 85 individuals were sampled with control (n = 35, 64.82 ± 1.20 mm, 221.55 ± 18.80 mg, glass eels stage from VIA0 to VIB) and marked (n = 50, 64.81 ± 1.27 mm, 235.7 ± 17.09 mg, glass eels stage from VIA0 to VIA4) glass eels.
The tips of fins were clipped on a total of 69 glass eels (n control = 27, n marked = 42). For fluorescence spectroscopy analyses, EEM, clips of caudal fin were placed into 1.5 mL Eppendorf tubes and kept frozen (–20 °C). For epifluorescence microscopy, EPI, clips of caudal and pectoral fins were mounted between a glass slide and a cover slip. The glass slides were dried overnight at room temperature.
Excitation-emission matrix (EEM) fluorescence spectroscopy
The analysis by EEM fluorescence spectroscopy of fish fin clips was tested for the first time. This technique provides rapidly the 3D total fluorescence spectrum of a small piece of tissue over a range of UV–visible to near IR wavelengths by creating an excitation-emission-fluorescence intensity matrix. The analyses were conducted at EPOC laboratory.
Each caudal fin clip from marked (n = 12) and control (n = 12) glass eels was prepared into a homogenate by a 20 min-sonication in 2 mL ultra-pure water (Milli-Q, Millipore, USA). Then, the homogenates were filtered (0.70 µm Whatman GF/F precombusted glass-fiber filters) and diluted to a maximum UV–vis absorbance of 0.1 (V-560 UV–VIS spectrophotometer, Jasco Corporation, Japan) to avoid inner filter effects during fluorescence analysis. The homogenates were placed into a 850 µL quartz micro-cuvette (111.057-QS, Hellma Analytics, Germany) thermo-stated at 20 °C and analysed with an Aqualog spectrofluorometer (Jobin Yvon technology, Horiba Scientific, France). The EEM fluorescence spectra (n = 24) were obtained between the wavelengths 240–800 nm at excitation (2 s integration time, 5 nm intervals) and 250–810 nm at emission (high CCD detector gain, 0.58 nm intervals). Each EEM was subtracted from the EEM spectrum of an ultrapure water blank to eliminate Rayleigh and Raman scatter peaks and corrected for instrumental biases. Prior to the analyses, the EEM spectrum of a eugenol solution (0.3 mL. L-1) was acquired and the solution did not fluoresce. The EEM spectrum of an alizarin solution (alizarin red S, 150 ppm, A5533, Sigma-Aldrich, China) was acquired to determine the wavelengths of the fluorescence peak of alizarin.
Based on the EEM spectra of glass eels, 15 main fluorescence emission spectra were studied in all glass eels at five fixed excitation wavelengths (Table 2). These 15 fluorescence emission spectra were acquired for each homogenate between the emission wavelengths 360–600 nm and with high resolution (0.5 s integration time, 1 nm interval) using a Fluorolog fluorometer (FL3-22 SPEX, Jobin Yvon technology, Horiba Scientific, France; 950 V) as described in32,33. Each fluorescence emission spectrum was subtracted from an ultrapure water blank and corrected for instrumental biases.
Epifluorescence microscopy (EPI)
EPI analyses were conducted at INRAE. The glass slides of pectoral (n = 45) and caudal (n = 45) fin tips of marked (n = 30) and control (n = 15) glass eels (n total = 45) were photographed with an epifluorescence binocular magnifier (SMZ25, Nikon, Japan; camera: DS-Ri2, Nikon, Japan) equipped with a B-2A fluorescence filter (Nikon, excitation bandpass: 450–490 nm; dichromatic mirror cut-on: 500 nm longpass; barrier filter: 515 nm longpass). The fluorescence of each photograph was analysed using NIS-Elements BR software (version 5.02).
The fluorescence intensity observed on the photographs was assessed using a scale of 0–3 (0, no fluorescence; 1, weak autofluorescence around fin rays and at the edge of the fin as described in15; 2, bright and heterogeneous fluorescence of alizarin stain on fin rays and tissue; 3, very bright and homogeneous fluorescence of alizarin stain on fin tip) (Fig. 6b). Fluorescence scores were analysed independently and by blinding by three research operators. The final score was then determined by selecting the value that more than one operator had allocated to the photo. A score ≥ 2 was judged to be an acceptable and good detection of the alizarin stain.
Statistical analyses
Statistical analyses were performed using R software (v3.6.1).
FRI provided images of the levels of radiance (photons/secondes/cm2/streradian) of the live glass eels. The levels of radiance between the marked and control glass eels were presented. Wilcoxon-Mann–Whitney tests (α = 0.05) were used to assess the differences in radiance between the two images obtained with the two excitation filter sets and between the marked and control groups for each image. The fluorescence quenching percentage Q (%) was evaluated with the radiance of each group for each image.
The fluorescence quenching percentage Q (%) was evaluated as follows:
$$Q left(%right)=left(1-frac{{F}_{M}}{{F}_{C}}right)times 100$$
with FM, the fluorescence intensity of marked fish and FC, the fluorescence intensity of control fish. High positive values of Q (%) indicate a high fluorescence intensity quenching of the marked group compared to the control.
EEM fluorescence spectroscopy provided matrices of fluorescence intensities (in arbritary unit). The intensity at each excitation and emission wavelengths obtained by EEM were extracted to calculate within the alizarin peak (2), every 10 nm, the fluorescence intensity means, standard deviations, maximum, minimum for each group as well as the Q(%) means and standard deviation. The EEM spectra of alizarin, marked and control glass eels were described. Then, Wilcoxon-Mann–Whitney tests (α = 0.05) were used to assess the differences in the fluorescence intensities between the marked and the control group. The Q (%) means and standard deviations were evaluated with the fluorescence intensities of each group. Wilcoxon-Mann–Whitney tests (α = 0.05) were used also to assess the difference in the fluorescence intensities minima and maxima bewteen the groups in order to determine the fluorescence thresholds of each group. Patrimat analyses were conducted with the three quartiles, means, standard deviations, minima and maxima of the fluorescence intensities of each group, in order to investigate their discriminant effect. Then, quadratic nonlinear discriminant models (QDA) were assessed with the fluorescence intensities first, second and third quartiles of each glass eels group (tenfold cross-validated correctness rate, 468 observations, 3 variables, 2 classes), the most discriminant variables from Patrimat analyses. The QDA with the best correctness rate was selected and the correct classification mean percentages of each group presented.
The fluorometer provided in high resolution fluorescence peaks (in arbitrary units) whithin fluorescence emission spectra at a fixed excitation wavelength. Thus, the other approach tested to detect the alizarin signal in marked eels was to select ratios of fluorescence peaks for which their fluorescence rates was higher than for the control group. The analysis of fluorescence ratios enabled the comparison of fluorescence peaks intensities in the alizarin signal to those in the fish signal. 15 main fluorescence peaks were selected (Table 2): nine were whitin the alizarin signal (FARS) and six other within the natural fish signal (FFISH) (Table 2). The alizarin fluorescence ratios, Rn, were computed for each combination of FARS and FFISH as follows:
$${R}_{{n };_{n:1to 54} }=frac{{F}_{{ARS}_{{i};_{ i : 1to 9} } }}{{F}_{{FISH}_{{j };_{j: 1to 6}}}}$$
with FARS, the fluorescence intensity of a peak (at a fixed excitation_emission wavelength) within the alizarin signal; FFISH, the fluorescence intensity of a peak (at a fixed excitation_emission wavelength) within the natural signal of the fish.
When fluorescence rates of the marked group was 1% higher than for the control group, the Rn was selected. Wilcoxon-Mann-Withney test (α = 0.05) was used to evaluate the difference in the values of the selected Rn between glass eels groups. Then, QDA were run with the rates values of these Rn (tenfold cross-validated correctness rate, 38 observations, 19 variables, 2 classes) to assess whether they were adequate to assign individuals to their group. The correct classification mean percentages of each group were presented.
Epifluorescence analyses provided qualitative measures of fluorescence intensity for which a score was assigned. The variability of the assigned score amomg the three operator-researchers was evaluated. Next, Chi-square tests (α = 0.05) were used to assess differences in fluorescence scores between caudal and pectoral fins in the same group and between the marked and control groups for each fin.
Ethics declaration
The French Ministry of the Territories and the Sea of Gironde issued authorizations to INRAE (animal experimentation approval number A33-478–001) for the capture of glass eels for biological and scientific examinations, including the killing of eels by overdosing with eugenol for finning or imaging (decree n°2018-03-13, decree n°2018-05-11). Anaesthesia and euthanasia respected the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). The captures were carried out in coordination with the Departmental Federation of Fishing of Gironde and Migado, both in charge of monitoring and managing migratory fish species, such as eels from Gironde. All methods were in strict accordance with the National Guidelines for Animal Care of the French Ministry of Agriculture (decree n°2013–118) and the EU regulations concerning the protection of animals used for scientific research (Directive 2010/63/EU). The study complied with (Animal Research) guidelines34. The main operator, M. G., has the diploma of fish welfare and ethics experimentation (decree 1988-04-19) to ensure direct scientific responsibility for animal experiments, delivered by ONIRIS veterinary school (France). Imaging was done at Vivoptic platform, ANR-11-INBS-006, Univ. Bordeaux, CNRS, INSERM, TBM-Core, UMS 3427, US 5, F-33000 Bordeaux, France. Vivoptic is a France Life Imaging (FLI) labelled platform (ANR-11-INBS-006).
Source: Ecology - nature.com
