Abstract
The development of osmoregulatory and gas exchange organs was studied in larval yellowfin tuna (Thunnus albacares) from 2 to 25 days post-hatching (2.9–24.5 mm standard length, SL). Cutaneous and branchial ionocytes were identified using Na+/K+-ATPase immunostaining and scanning electron microscopy. Cutaneous ionocyte abundance significantly increased with SL, but a reduction in ionocyte size and density resulted in a significant decrease in relative ionocyte area. Cutaneous ionocytes in preflexion larvae had a wide apical opening with extended microvilli; however, microvilli retracted into an apical pit from flexion onward. Lamellae in the gill and pseudobranch were first detected ~ 3.3 mm SL. Ionocytes were always present on the gill arch, first appeared in the filaments and lamellae of the pseudobranch at 3.4 mm SL, and later in gill filaments at 4.2 mm SL, but were never observed in the gill lamellae. Unlike the cutaneous ionocytes, gill and pseudobranch ionocytes had a wide apical opening with extended microvilli throughout larval development. The interlamellar fusion, a specialized gill structure binding the lamellae of ram-ventilating fish, began forming by ~ 24.5 mm SL and contained ionocytes, a localization never before reported. Ionocytes were retained on the lamellar fusions and also found on the filament fusions of larger sub-adult yellowfin tuna; however, sub-adult gill ionocytes had apical pits. These results indicate a shift in gas exchange and NaCl secretion from the skin to branchial organs around the flexion stage, and reveal novel aspects of ionocyte localization and morphology in ram-ventilating fishes.
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Acknowledgements
This study was supported by the Inter-American Tropical Tuna Commission. We are grateful to the technical staff of the Achotines Laboratory in Panama for their assistance with measurements and larval rearing of yellowfin tuna. We thank Daniel Margulies and Vernon Scholey of the IATTC for development of yellowfin rearing methods and supervision of the spawning and larval rearing at the Achotines Laboratory. The authors would like to thank Dr. Greg Rouse for the use of microscope and camera equipment, Sabine Faulhaber for technical assistance with the scanning electron microscope, Taylor Smith for her assistance in dissection and imaging, and Johnathan Evanilla and Dan Fuller for providing sub-adult yellowfin tuna samples. We also thank William Watson (Southwest Fisheries Science Center) and Alex Da-Silva (Inter-American Tropical Tuna Commission) for helpful review comments. Lastly, we thank the editor and two anonymous reviewers for their helpful comments on an earlier draft. G.T.K. was supported by the San Diego Fellowship and the National Science Foundation Graduate Research Fellowship Program.
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Supplementary material 1: Daily mean tank pH, intake pH, temperature (°C), dissolved O2 (mg/L), and salinity (ppt) throughout the yellowfin tuna larvae sampling period (JPG 351 KB)
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Supplementary material 2: Western blot with anti-Na+/K+-ATPase (NKA) monoclonal antibodies on sub-adult yellowfin tuna gill tissue yielded a single ~ 108 kDa band, which matches the predicted size of the protein. a NKA signal in the membrane fraction (MB) was significantly stronger (exposure time: 4 s) than b the crude homogenate (CH) and cytoplasm (CYT) fraction (exposure time: 304 s). This indicates NKA is present in the basolateral membrane as expected. Western blot method: gill samples were dissected from yellowfin tuna, immediately flash frozen in liquid N2, and kept at − 80 °C until processed. Frozen gill tissue was pulverized with porcelain mortar and pestle and mixed in an ice-cold protease inhibiting buffer (250 mmol L−1 sucrose, 1 mmol L−1 EDTA, 30 mmol L−1 Tris, 10 mmol L−1 benzamidine hydrochloride hydrate, 200 mmol L−1 phenylmethanesulfonyl fluoride, 1 mol L−1 dithiothreitol, pH 7.5). Debris was removed by low-speed centrifugation (3000×g for 10 min, 4 °C), and the resulting solution was saved as the crude homogenate fraction. A subset of the crude homogenate fraction was further subjected to a medium speed centrifugation (21130×g for 30 min, 4 °C), and the supernatant and membrane pellet was saved as the cytoplasmic fraction and the plasma membrane fraction, respectively. The total protein concentration of the three fractions was determined by Bradford protein assay, and 5 µg protein was combined with 2× Laemmli buffer (90%) and 2-mercapaethanol (10%). After heating at 70°C for 5 min, proteins were separated in 7.5% polyacrylamide mini gel (60 V 15 min, 200 V 45 min). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane using a wet transfer cell (90 mA 8 h) (Bio-Rad, Hercules, CA, USA). After transfer, the PVDF membrane was incubated in blocking buffer (Tris-buffered saline, 1% tween, 10% skim milk) at room temperature (RT) for 1 h and incubated with the anti-NKA antibody (1.5 µg/mL) at 4 °C overnight. On the following day, the PVDF membrane was washed three times (10 min each) in Tris-buffered saline + 1% tween (TBS-T), incubated in goat anti-mouse HRP-linked secondary antibodies (1:10,000, Bio-Rad) at RT for 1 h and washed three times (10 min each) in TBS-T. Protein bands were made visible using ClarityTM Western ECL Substrate (Bio-Rad), and imaged and analyzed in a Bio-Rad Universal III Hood using ImageQuant software (Bio-Rad) (JPG 219 KB)
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Supplementary material 3: Method for quantification of cutaneous ionocytes in yellowfin tuna larvae and early-stage juveniles > 5 m SL. Ionocytes were identified by their intense Na+/K+-ATPase immunostaining and counted within randomly sampled boxes of the overlaid grid within the head (green), trunk (pink), and fin (blue) regions. Dashed white lines outline regions that were not sampled due to heavy pigmentation preventing accurate ionocyte counts (JPG 46748 KB)
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Supplementary material 4: Cutaneous ionocyte area relative to total skin surface area through larval yellowfin tuna development (n = 18) in relation to accumulated thermal units (linear regression: F1,16 = 42.54; p < 0.001; r2 = 0.7267). The black line shows the linear regression curve and dotted lines denote 95% confidence levels. Error bars (gray) denote standard error of the mean. Ionocyte absence in gills, presence in the gill filaments, and presence in interlamellar fusions and in the gill filaments is noted as a square, circle, and triangle, respectively (JPG 470 KB)
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Kwan, G.T., Wexler, J.B., Wegner, N.C. et al. Ontogenetic changes in cutaneous and branchial ionocytes and morphology in yellowfin tuna (Thunnus albacares) larvae. J Comp Physiol B 189, 81–95 (2019). https://doi.org/10.1007/s00360-018-1187-9
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DOI: https://doi.org/10.1007/s00360-018-1187-9