Silver females (n = 14; 80.1 ± 0.4 cm; 978.1 ± 19.5 g) and males (n = 8; 45.6 ± 1.4 cm; 172.5 ± 14.9 g) of New Zealand short-finned eels (A. australis) were caught in Lake Ellesmere in Christchurch, New Zealand, and transported to The Netherlands in aired plastic bags with a small amount of water, fitted into polystyrene boxes. Silver male European eels (A. anguilla) were purchased from the eel farm Royaal BV (Helmond, The Netherlands).
Anguilla australis females and males were kept together in a 1000 L tank filled with natural seawater, collected from Lake Grevelingen (30 ppt), and coupled to a 1500 L recirculation system (salinity 30 ppt, 21°C). The daily cycle was set with blue light (Philips special TLD Blue 36W/18) at 16:8 L:D. To compensate for the 11 h time difference between The Netherlands and New Zealand, the daily cycle was changed stepwise (1 h per week) to Central European Time (CET). Anguilla anguilla males (n = 15; 40.4 ± 0.6 cm; 118.8 ± 4.9 g) were kept in a 1500 L tank connected to a 2400 L recirculation system, in natural seawater (30 ppt, 21°C), under a complete dark regime. PVC pipes were introduced into both systems to provide refuges for animals. All animals were starved throughout the experiment, and treated on a weekly basis with Melafix (API aquarium pharmaceuticals, MARS Fishcare North America Inc., Chalfont, PA, USA) against infections. Prior to treatment, eels were anesthetized with 1-2 mL/L 10% clove oil (oil mixed 1:10 with absolute ethanol). At the start of this study all eels were tagged with passive transponders with unique identification numbers (Trovan, EID Aalten BV, Aalten, The Netherlands).
This experiment was approved by the animal ethical commission of the Leiden University (DEC# 08112).
Female eels were distributed into four groups, with the starting point of hormonal treatment for each group being shifted one week forward, on a weekly basis. Treatment followed a modified version of Ohta's protocol [4, 5]. On the first day of the week, females were weighed and injected intramuscularly (IM), at a point approximately 1 cm below the rostral attachment of the dorsal fin, with 20 mg salmon pituitary extract (SPE; Argent Labs, Redmond, WA, USA) per kg dissolved in 0.9% saline. When a 5% increase in body weight (BW) -- with respect to initial BW -- was reached, females were transferred to a separate 400 L tank, connected to the same system. The BW was measured the day after the transfer and/or 2 days later. When a 10% increase in BW was reached, an oocyte sample was collected by means of inserting a cannula (polyethylene tube, inner diameter 1.4 mm) through the oviduct. Oocytes were checked under a microscope to ascertain developmental stages. When migration of the germinal vesicle -- still with many oil droplets in the oocytes (stage 3/4 in European eel according to ) -- was observed, the female was primed with a single injection of SPE (20 mg/kg dissolved in 1 mL 0.9% saline). After 24 h, ovulation was induced by means of intraperitoneal injections of 17, 20β-dihydroxy-4-pregnen-3-one (DHP 2 mg/kg, Sigma-Aldrich BV, Zwijndrecht, The Netherlands) dissolved in DMSO, administered at six to eight locations.
Male New-Zealand short-finned eels were treated according to a modified version of Ohta's protocol [4, 5]. They were subjected to a weekly IM injection procedure (at a site approximately 0.5-1 cm below the rostral attachment of the dorsal fin) with 250 IU human chorionic gonadotropin (hCG, Sigma-Aldrich BV, Zwijndrecht, The Netherlands) dissolved in 0.1 mL 0.9% saline. Males were injected every week for up to 9 weeks and checked for spermiation by hand stripping. After 9 weeks, all males produced milt and injections were stopped until a female was ready to spawn. (This reduced handling stress; it was also noted that the sperm quality did not decline much during the "holding" period). On the day before the eggs were to be stripped, two to three males that demonstrated high sperm motility were selected and stimulated with a single booster dose of 500 IU dissolved in 0.1 mL saline. Sperm motility was determined visually under a microscope, after mixing a drop of sperm with a drop of seawater. Only sperm with at least 50% motility (continuous activity of > 50% of spermatozoa) was used for fertilization.
For the production of a hybrid between A. australis and A. anguilla, 15 farmed male European eels received weekly intraperitoneal (IP) hCG injections, according to the protocol for European eel , at a dose of 200 IU/male, followed by a booster dose of 1,000 IU hCG (in 0.2 ml 0.9% saline) 24 h before a fertilization trial.
Artificial fertilization and larval rearing
Two to three males per species were hand stripped 24 h after the hCG booster injection. Milt was collected in a syringe (10 mL) and kept on ice or in the refrigerator for a maximum of 48 h. Sperm motility was checked prior to fertilization by means of microscopic examination. Females were expected to ovulate between 11 and 15 h after the final injection with DHP. The artificial fertilization programme was terminated in cases when female spawning only commenced after more than 18 h after the final injection, which is indicative of low fertility and hatchability . After the final injection, females were checked hourly for egg release, by gently pressing on the abdomen near the vent. When a female showed an ovarian plug, the plug was gently removed. Eggs were collected in plastic, pre-weighed, sterilized bowls. The first flow of eggs (~50 g) was not used for fertilization. The combined egg weight was determined after all eggs had been stripped.
The collected sperm was added to dry eggs in bowls and mixed. Fresh seawater (35 ppt, 20°C) was added, and after approximately 3-4 min the eggs were transferred into buckets with fresh (sterile) seawater (~20 L). A net (of mesh size 600 μm) was used to separate floating eggs from sinking eggs. The former were transferred, after 30-45 min, to another bucket containing fresh seawater. Finally, the eggs were transferred to 1 L glass beakers and/or 200 mL Petri dishes, for observation. At this stage they were kept in complete darkness at 21°C and a salinity of 35 ppt. Approximately 24 hours after incubation, the water was refreshed by transferring the still-floating eggs into new glass beakers or Petri dishes. During the trial, all white or sunken eggs were removed. A portion of eggs from one batch were also reared at 25°C.
As described by Frankowski & Bastrop , parental species and their hybrids can be identified by means of polymorphism in nuclear 18 S rDNA. For our study we used a slightly different protocol, as described below.
Total DNA was isolated and purified from ten whole hybrid larvae and fin-clips of parental specimens, using a DNeasy Blood & Tissue Kit (Qiagen). The polymerase chain reaction (PCR) was performed using the FastStart High Fidelity PCR System protocol (Roche) and an amplification profile consisting of denaturation for 3 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C, followed by 5 min at 72°C for final extension. Amplification was carried out according to the manufacturer's instructions in 50 μL 1 × FastStart High Fidelity Reaction Buffer containing 1.8 mM MgCl2, 2.5 U FastStart High Fidelity Enzyme Blend, 1 μg chromosomal DNA, 0.4 μM of the 18 S rDNA forward and reverse primers, and 0.2 mM dNTPs.
The sense (5'-AGC ATA TGC TTG TCT CAA AGA TTA AG-3') and antisense (5'-CTG CTG CCT TCC TTG GAT GTG G-3") primers were based on NCBI accession numbers FM946133 (A. australis) and FM946070 (A. anguilla) . The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen) and 0.5 μg of the purified fragments were digested with two units of the restricting enzyme BssHII (New England Biolabs Inc) according to the manufacturer's instructions. Restriction enzyme digestion was conducted in 10 μL reaction buffer for 1 h at 37°C. DNA fragments were made visible using a 2% agarose gel.