Larval rearing and sampling
Ballan wrasse larvae were reared at the Marine Harvest Labrus, a commercial hatchery at Øygarden, Norway. Two batches of fertilized eggs (collected the 21.-22. and the 27.-28. August 2015) originated from natural spawning fish distributed in eight tanks (9000 L.) containing 30 females and 5 males in each tank. The eggs were incubated (900-1000L tanks) upon hatching (9-10 days at 11.3 ?C, 12/12h light/dark regime). Newly hatched larva (0 DPH) were transferred to feeding tanks (9000L; initial stock density 1.0-1.3 mill. larvae, 13.7 ?C, water flow 45 l/m). Larvae were kept under a 24h darkness until 4 DPH. From 4 DPH the larvae were kept under a 24h light regime, and feed was added four times daily into the tanks. Water flow and temperature were gradually increased to 60 l/m at 22 DPH and 16 ?C at 24 DPH respectively. Rotifers (Brachionus spp., Aquafarms, USA) and Artemia (INVE Aquaculture Inc., USA), both enriched with Multigrain (BioMar, Norway), were given as live feed to the larvae from 4-25 DPH and 25-57 DPH respectively. A co-feeding protocol (live Artemia and formulated feed Nofima, Norway) were introduced at 47 DPH and kept for 10 days. The larvae were fed purely formulated feed from 57 DPH (Otohime B1/B2 and later C1, Japan).
Larvae were sampled at six developmental stages (Stage 1-6; n=8-11 larvae per sample). Shortly after sampling; larvae were sedated (Metacaine) and studied under a microscope for quality check and photographed for SL measurement (ImageJ, NIH, USA) before fixation. Only larvae of specific standard length (SL) were further analysed.
Scanning electron microscopy
Two fixated larvae from each Stage (1-6) were dissected using razor blades and hydrated (50% ethanol) prior to secondary fixation in 1% osmium tetraoxide. One series (one larva from each stage) was prepared for the outer morphological study of the digestive system, hence the skin and skeletal muscles surrounding the abdominal cavity were carefully dissected away. The second series of larvae were cut sagittal or oblique to study the left and right inner morphology of the digestive system. After secondary fixation, larvae were rinsed in distilled water and rehydrated in acetone series and critical point dried. Larvae were sputter-coated with gold-palladium and examined using ZEISS Supra 5VP field-emission scanning electron microscope.
Light microscopy and 3D reconstructions of the digestive organs
One high-quality larva from each Stage (1-6) was prepared for light microscopy. The larvae were dehydrated in ethanol and embedded in Technovit 7100 (Heraeus Kulzer GmbH, Germany) and cut in semi-thin (2?m) serial sections. The sections were stained Toluidine blue and scanned using RS Photometric CoolSNAP-PRO mounted on either a Leica M420 macroscope with a 6:1 Leica Apozoom objective or on a Leica DMLB microscope.
Every 2nd to 13th section depending on the stage were used for 3D reconstructions. Micrographs were edited in Adobe Photoshop CC2015 (Adobe System Inc., USA) and batch converted using IrfanView (Irfan Skiljan, Austria). Alignment of image stacks was performed in AutoAligner 6.1 (Bitplane AG, Switzerland). Aligned image stacks were uploaded into Imaris 8.4 (Bitplane AG) for 3D reconstruction. One 3D model was created for each developmental stage containing the digestive tract from the pharynx until anus, and digestive glands observed at the given stage. Up to nine out of ten surfaces were observed within a stage: 1) Outer surface of the digestive tract. 2) Lumen esophagus. 3) Lumen intestine. 4) The valve between mid-and hindgut. 5) Yolk sac. 6) Liver. 7) Gallbladder with the common bile duct. 8) Exocrine pancreas. 9) Endocrine pancreas. 10) Pancreatic duct. Each surface was manually drawn polygons with nodes with specific color code for the specific organs and tissues (i.e. the liver for all stages was red). For further detailed method for 3D reconstruction, see (Kamisaka and Rønnestad, 2011, Norland, 2017). For public accessibility of the 3D models of the digestive system in ballan wrasse, PDF-files were generated, see details in (Bergum, 2016). In summary, 3D models in the Imaris software were saved as .vrl-files, size-reduced in Meshlab (Cignoni et al., 2008) and exported as .obj-files into Deep Exploration (Right Hemisphere) for scene reconstruction and surface rename. The files were exported as .u3d-files and imported into Adobe Pro (Adobe System Inc., USA) and saved as .pdf-files.
Complete drawn surfaces were 3D reconstructed by the Imaris software, while Imaris MeasurementPro (extension of Imaris 3D rendering software) provided statistical data of the 3D models (surface area and volume). The data were analysed for morphometric scaling and relate the scaling to functional capacity of the digestive system. Specific growth rate (SGR) for each organ was calculated from the volume data using Equation 1 (where V2 is the volume of the organ at time 2 (t2), V1 as the initial volume at t1).
Equation 1 SGR = (ln V2 – ln V1) (t2 – t1)-1
Morphological development of the digestive system
The development of the digestive system in ballan wrasse was studied from Stage 1 (onset of exogenous feeding) until Stage 6 (juvenile ballan wrasse). The digestive tract in Stage 1 (4 DPH) ballan wrasse larvae were a straight tube, flat (no villies), and located dorsally for the yolk sac (who was fully consumed between Stage 2 (10 DPH) and Stage 3 (18 DPH)). The pharynx and intestine were distinguished from each other from Stage 1 and on by an esophageal-intestinal constriction, the common bile duct ended in the proximal midgut, and the presence of microvilli on the apical end of the enterocytes (brush border). The digestive tract in Stage 2 could be compartmentalized into the esophagus (mucoid esophageal mucosa), and non-mucoid intestine with a proximal enlargement of the midgut diameter (bulbus), and distal midgut and hindgut with leaf-like folded mucosa separated from each other by the ileorectal valve. Stage 3 demonstrated the initial rotation of the gut into one loop and pronounced presence of villi in the esophagus and intestine, and with an intestinal mucoid epithelium from Stage 4 (29 DPH). During the late ontogeny of the digestive tract (Stage 5-6; 71-102 DPH), the presence bulbus of the proximal midgut and Z-shaped rotation of the gut persisted while the mucosal folding of the intestine became progressively shorter from the bulbus towards the anus.
The layers of the digestive tract were closer studied. The epithelium in Stage 1 consisted more or less of simple columnar epithelium throughout the tract but became differentiated into simple cuboidal in the esophagus and simple columnar in the rest of the tract. The surface area of the enterocytes increased from Stage 1 to Stage 6 with 44.43±8.36 ?m to 69.6±11.07 ?m (mean±s.d.). Closer examination of the outer muscle layer (muscularis externa) showed the formation of prominent striated outer muscle layer of the esophagus during the development. However, it did not show muscular changes in the region of the bulbus nor post-bulbus region compared to the rest of the midgut, indicating no functional pseudogaster. The outer muscular layer increase in the region of the ileorectal valve and in the hindgut during the development.
Stage 1 revealed the presence of liver located anteriorly in the abdomen, exocrine pancreas posterior for the liver and the gallbladder in between liver and pancreatic tissue and ventrally for the intestine. The liver maintained the shape of a compact organ located anteriorly in the abdomen until after metamorphosis (Stage 5) when it became elongated ventrally for the rotated midgut all the way to the hindgut. Low fat accumulation in the hepatocytes with very small changes during the ontogeny indicates a low-fatty liver in ballan wrasse. The gallbladder ended in the lumen of the proximal midgut from the ventral side and was located mainly on the right side of the digestive tract during the development.
The exocrine pancreas with zymogen granules was located posterior to the liver from Stage 1, but from Stage 3 it became scattered within the abdominal cavity, as well within the liver tissue close to larger blood vessels (hepatopancreas) after metamorphosis (Stage 5). An elevated level of immune cells infiltrating the connective tissue of the pancreas were observed in Stage 6. One primary islet (endocrine pancreas) was identified within the exocrine pancreas close to the gallbladder from Stage 2 until Stage 5 when several islets were observed. The endocrine pancreas after metamorphosis was organized as one larger islet (on the site of primary islet) and several smaller islet close to the largest islet. The pancreatic duct in ballan wrasse was observed from Stage 3. The duct ended in the lumen of the proximal midgut (bulbus) next to the common bile duct, but structured as two separate openings.
Growth and morphometric scaling of the digestive system
The surface area and volumes of all organs were retrieved from Imaris MeasurementPro. The surface area of the outer surface of the digestive tract increased 59 times from Stage 1 until Stage 6 (0.99-58.40 mm2), while the esophageal and intestinal lumen increased 184 and 464 times respectively (microvilli not included). Esophageal mucosa surface area grew gradually from 0.02 to 3.64 mm2 from Stage 1 to 6, while the intestinal mucosa increased from 0.23 to 106.75 mm2. The surface of the liver for Stage 1-5 increased from 0.20 to 38.81 mm2 and falling to 30.67 mm2 at Stage 6. Exocrine pancreatic tissue grew from 0.15-34.47 mm2 from Stage 1-6, while endocrine pancreas for Stage 2-6 at 0.02-0.65 mm2.
The volume (tissue mass) from Stage 1-6 grew from 0.01-8.69 mm3 for the outer surface of the digestive tract, 0.00005-0.06 mm3 for esophageal lumen and 0.0006-3.76 mm3 for the intestinal lumen (microvilli not included). The volume of the gut tissue was measured as the volume of esophageal and intestinal lumen subtracted from the surface of the digestive tract, and it rose from 0.01 at Stage 1 to 4.87 mm3 at Stage 6. The volume of the liver had similar development as the surface area; increased from 0.001-3.10 mm3 from Stage 1-5, and decreased to 2.09 mm3 at Stage 6. Exocrine pancreas expanded from 0.001-0.92 mm3 for Stage 1-6, and endocrine pancreas from 0.0001-0.01 mm3 from Stage 2-6.
Relative volume (percentage %, of the different tissues and organs for each stage) were calculated from gut tissue, liver, exocrine and endocrine pancreas. The gut tissue always displayed highest relative volume, although decreasing from Stage 1-6 from 78.9-61.6%. The liver and exocrine pancreas increased from 13.5-26.5% and (5-7.5)-11.7% from Stage 1-6, while endocrine pancreas remained stable at 0.1-0.2% throughout the development.
Specific growth rate (SGR) was calculated for each organ between each stage (1-2, 2-3, 3-4, 4-5, 5-6) and from the start until the end of the study (Stage 1-6) and were given as percentage increase per day. All organs displayed strong growth between Stage 1-2 and gradually decline towards Stage 6. From Stage 1-6, gut tissue, liver and exocrine pancreas displayed a growth of 6.72 ± 0.39 % day-1.
BERGUM, H. O. T. 2016. A morphological study of the parasitic barnacle, Anelasma squalicola (Lovén, 1844). Master Thesis, Department of Biology, University of Bergen.
CIGNONI, P., CALLIERI, M., CORSINI, M., DELLEPIANE, M., GANOVELLI, F. & RANZUGLIA, G. 2008. MeshLab: an Open-Source Mesh Processing Tool. Sixth Eurographics Italian Chapter Conference.
KAMISAKA, Y. & RØNNESTAD, I. 2011. Reconstructed 3D models of digestive organs of developing Atlantic cod (Gadus morhua) larvae. Marine Biology, 158, 233-243.
NORLAND, S. 2017. Reconstructed 3D-models of the digestive organs of ballan wrasse (Labrus bergylta) during ontogeny. Master thesis, University of Bergen.
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