Eels

Female silver eels were obtained from a freshwater lake, Vandet, Jutland, Denmark. Male eels were obtained from a Danish commercial eel farm (Stensgård Eel Farm A/S). Experimental maturations were conducted at a DTU Aqua research facility at Lyksvad Fishfarm, Vamdrup, Denmark, where eels were housed in 300 L tanks equipped with a recirculation system [34 ]. Eels were maintained under low intensity light (~20 lux), 12 h day/12 h night photoperiod, salinity of ~36 ppt, and temperature of 20°C. Acclimatization took place over 10 days. As eels naturally undergo a fasting period from the onset of the pre-pubertal silvering stage, they were not fed during treatment. Prior to experimentation, eels were anaesthetized (ethyl p-aminobenzoate, 20 mg L^-1; Sigma-Aldrich, Missouri, USA) and tagged with a passive integrated transponder. Females used for experiments (n = 4) had a mean (± SEM) standard length and body weight of 65 ± 4 cm and 486 ± 90 g, respectively. To induce vitellogenesis females received weekly injections of salmon pituitary extract (Argent Chemical Laboratories, Washington, USA) at 18.75 mg kg^-1 body weight [11 (link), 34 ]. To stimulate follicular maturation and induce ovulation, females received 17α,20ß-dihydroxy-4-pregnen-3-one (Sigma-Aldrich, Missouri, USA) at 2 mg kg^-1 body weight [35 ] and were strip-spawned within the subsequent 12–14 h. Male eels (n = 11) had a mean (± SEM) standard length and body weight of 40 ± 3 cm and 135 ± 25 g, respectively. Males received weekly injections of human chorionic gonadotropin (Sigma-Aldrich, Missouri, USA) at 150 IU per fish [34 ]. Prior to fertilization, an additional injection was given and milt was collected by strip-spawning ~12 h after administration of hormone. Milt samples were pipetted into a P1 immobilizing medium [36 (link)] and only males with sperm motility of category IV (75–90%) were used for fertilization within 4 h of collection [37 ]. Only floating viable eggs/embryos were further used for experimentation.

In this study, we used the 3D particle-tracking scheme developed by Ohashi and Sheng [27 ], which is based on the fourth-order Runge–Kutta method [28 ]. The position of a particle is tracked from its position at time t (x→t) to a new position at time t + Δt (x→t+Δt) based on
${\overrightarrow{x}}^{t+\Delta t}={\overrightarrow{x}}^t+{\displaystyle \underset{t}{\overset{t+\Delta t}{\int }}\overrightarrow{u}(\overrightarrow{x},t)dt+\overrightarrow{\delta }}$
where $\overrightarrow{u}$ is the ocean current from the JCOPE2 reanalysis and $\overrightarrow{\delta }$ represents the additional displacement associated with a random walk during this time interval, representing unresolved sub-grid turbulent flow and other local processes [29 ]. The estimated δ(x,y) was approximately 600 m, and δ(z) was approximately 20 m. Note that the vertical velocity was not used in simulation. The integration time step was three hours. Bilinear interpolation was used to establish a continuous velocity dataset in time and space. The same tracking scheme was used by Chang et al. [26 (link)] to investigate migration of Japanese eel larvae in the western Pacific Ocean, and it was also applied to the simulation of the long-distance migration of adult eels in the Sargasso Sea [30 ].

Simple correlation analysis was used to assess association between migration depth and light intensity during daytime. Migration depth data averaged every one minute were used, since light intensity data during daytime were recorded at one minute intervals. Correlation coefficient between migration depth and light intensity was evaluated by t-test. Geolocation of freshwater eels without light data may be possible using migration behavior as noticed and applied in the European eel [17 , 18 ]. In order to determine the start points of large descent in the morning and large ascent in the evening, slope changes in the depth trajectory were first visually located. Subsequently, the depth data within 30 minutes before and after the time of this slope change were used for obtaining one minute averaged depth data. If the differences between these averaged depth data were consecutively positive for 10 minutes or longer, the behavior during this period was interpreted as an initial part of large descent, whereas it was interpreted as an initial part of large ascent if the differences were consecutively negative for 10 minutes or longer. The starting point of descent and ascent was defined as the time one minute before the start of this period. Based on the descent and ascent timings as determined above, sunrise and sunset times were estimated as described in Results and Discussion. Eels’ positions (latitude and longitude) were calculated by M-Series BASTrack software (Biotrack Ltd., Dorset, UK), which uses day/night length for latitude estimate and absolute time of local midday for longitude estimate. Sun altitude was set to -0.9. The estimated positions obtained were compared with actual eels’ positions at noon.

High angle annular dark-field (HAADF) and EELS spectrum imaging were performed on a Nion UltraSTEM 100 operated at 100 kV with a 30 mrad convergence semi-angle and a fifth-order aberration corrector. Spectra were acquired with a Gatan PEELS 666 spectrometer retrofitted with an Andor iXon 897 electron-multiplying charge-coupled device (EMCCD) camera. A dispersion of 1.2 eV per channel and an exposure time of 50 ms per spectra were used to record the oxygen K-edge, Mn and Ni L_2,3 edges and La M_4,5 edge in a 32 by 128 pixel spatial grid.

For the experiments
reported, we used a copper 100 Mesh PELCO grid. The grid was tilted
by 45° with respect to the z direction (parallel
to the TEM column axis) in order to expose the corner of a rectangular
copper rod with a cross section of ∼50 × 25 μm² (see Figure S1 in SI). The edge
of the rod corner exhibited a radius of curvature of 4 μm, as
estimated from SEM micrographs. The sample was positioned such that
only one of the edges of the rectangular rod was illuminated by the
laser pulse.
To generate the charged plasma, we irradiated the
copper rod with near-infrared laser pulses of 1.55 eV central photon
energy (800 nm) and 50 fs temporal duration at a repetition rate of
100 kHz, which corresponds to ≃2.5 TW/cm². In the
reported experiments, light polarization was vertical (i.e., along
the propagation direction of the probe electron). Light entered the
microscope through the zero-angle port and was focused under normal
incidence on the copper rod via an external plano-convex lens. In
such a geometry, the light beam was also perpendicular with respect
to the electron propagation direction.
The dynamics of the photoemitted
electrons was then probed by means
of electron pulses with a temporal duration of about 600 fs and with
a controlled delay between electron and laser pulses. All the experiments
were performed in a modified JEOL 2100 TEM microscope at an acceleration
voltage of 200 kV.^{42 (link),43 (link)} The probe electrons were generated
by illuminating a LaB₆ cathode with third-harmonics UV
light at 4.65 eV photon energy.
Our transmission electron microscope
was equipped with EELS capabilities
coupled to real-space imaging. Energy-resolved spectra were recorded
using a Gatan-Imaging-Filter (GIF) camera operated with a 0.05 eV-per-channel
dispersion setting and typical exposure times of the CCD sensor from
30 to 60 s. For the acquisition of space-energy maps (see Figure 3), special care was
devoted to sample alignment. The copper rod was adjusted to be parallel
to the energy dispersion direction and placed at the edge of the spectrometer
entrance aperture.
The acquired position-dependent spectra were
analyzed as a function
of delay between the laser and electron pulses, with the time zero
being determined as the peak of PINEM signal observed within 100 nm
close to the sample surface at relatively low fluence (≃50
mJ/cm²). Camera noise and signal from cosmic events were
reduced by applying median filtering. Distortions of the spectrometer
were corrected by aligning the spectrum according to the negative
delay energy-space spectrographs (−2 ps). The first and second
moments of the spectrum were calculated in a reduced energy window,
which was taken 10 eV larger than the region in which the electron
signal was above 10% of the peak value (i.e., the maximum value among
all delays and positions measured for a given fluence). This procedure
helped to reduce contributions from the CCD background noise.
Regarding sample stability, special care was taken to ensure experimentally
reproducible results and a controlled environment. Standard TEM grids
from the same batch were used for all the experiments. The oxide layer
was removed from the surface by washing the grids in acetic acid for
approximately 5 min. Among other reasons, relatively fine-pitch grids
were selected to avoid resonant vibrations due to a large periodic
thermal load. In experiments, the smearing of the sample edge did
not exceed the resolution of ≃50 nm defined by the magnification
settings and aberrations in the photoelectron mode of TEM operation.
At the highest measured fluence, we observed a degradation of the
signal of the order of ≃10% of the peak acceleration over 2
h of experimental time. We made sure to expose a fresh part of the
sample to laser illumination at least every 60 min. Sample edge images
were realigned for each measurement during data analysis. At fluences
above 500 mJ/cm², we observed ablation of the sample on
a time scale of several minutes.