High-Resolution, 3D Imaging of the Zebrafish Gill-Associated Lymphoid Tissue (GIALT) Reveals a Novel Lymphoid Structure, the Amphibranchial Lymphoid Tissue.

The zebrafish is extensively used as an animal model for human and fish diseases. However, our understanding of the structural organization of its immune system remains incomplete, especially the mucosa-associated lymphoid tissues (MALTs). Teleost MALTs are commonly perceived as diffuse and scattered populations of immune cells throughout the mucosa. Yet, structured MALTs have been recently discovered in Atlantic salmon (Salmo salar L.), including the interbranchial lymphoid tissue (ILT) in the gills. The existence of the ILT was only recently identified in zebrafish and other fish species, highlighting the need for in-depth characterizations of the gill-associated lymphoid tissue (GIALT) in teleosts. Here, using 3-D high-resolution microscopy, we analyze the GIALT of adult zebrafish with an immuno-histology approach that reveals the organization of lymphoid tissues via the labeling of T/NK cells with an antibody directed to a highly conserved epitope on the kinase ZAP70. We show that the GIALT in zebrafish is distributed over at least five distinct sub-regions, an organization found in all pairs of gill arches. The GIALT is diffuse in the pharyngeal part of the gill arch, the interbranchial septum and the filaments/lamellae, and structured in two sub-regions: the ILT, and a newly discovered lymphoid structure located along each side of the gill arch, which we named the Amphibranchial Lymphoid Tissue (ALT). Based on RAG2 expression, neither the ILT nor the ALT constitute additional thymi. The ALT shares several features with the ILT such as presence of abundant lymphoid cells and myeloid cells embedded in a network of reticulated epithelial cells. Further, the ILT and the ALT are also a site for T/NK cell proliferation. Both ILT and ALT show structural changes after infection with Spring Viraemia of Carp Virus (SVCV). Together, these data suggest that ALT and ILT play an active role in immune responses. Comparative studies show that whereas the ILT seems absent in most neoteleosts ("Percomorphs"), the ALT is widely present in cyprinids, salmonids and neoteleosts, suggesting that it constitutes a conserved tissue involved in the protection of teleosts via the gills.

Adaptation mechanism of the adult zebrafish respiratory organ to endurance training.

In order to study the adaptation scope of the fish respiratory organ and the O2 metabolism due to endurance training, we subjected adult zebrafish (Danio rerio) to endurance exercise for 5 weeks. After the training period, the swimmer group showed a significant increase in swimming performance, body weight and length. In scanning electron microscopy of the gills, the average length of centrally located primary filaments appeared significantly longer in the swimmer than in the non-trained control group (+6.1%, 1639 µm vs. 1545 µm, p = 0.00043) and the average number of secondary filaments increased significantly (+7.7%, 49.27 vs. 45.73, p = 9e-09). Micro-computed tomography indicated a significant increase in the gill volume (p = 0.048) by 11.8% from 0.490 mm3 to 0.549 mm3. The space-filling complexity dropped significantly (p = 0.0088) by 8.2% from 38.8% to 35.9%., i.e. making the gills of the swimmers less compact. Respirometry after 5 weeks showed a significantly higher oxygen consumption (+30.4%, p = 0.0081) of trained fish during exercise compared to controls. Scanning electron microscopy revealed different stages of new secondary filament budding, which happened at the tip of the primary lamellae. Using BrdU we could confirm that the growth of the secondary filaments took place mainly in the distal half and the tip and for primary filaments mainly at the tip. We conclude that the zebrafish respiratory organ-unlike the mammalian lung-has a high plasticity, and after endurance training increases its volume and changes its structure in order to facilitate O2 uptake.

Regeneration of the gill filaments and replacement of serotonergic neuroepithelial cells in adult zebrafish (Danio rerio).

Respiratory epithelia and chemoreceptors of the gills and mammalian lung derive from the same embryonic structures. While the lung is limited to facultative regeneration, the regenerative capacity of the gill has not been adequately explored. We report regeneration of gill filaments and respiratory lamellae in adult zebrafish (Danio rerio). Gill filaments retained a constitutive population of mitotic cells identified by the proliferating cell nuclear antigen (PCNA). Within 24 h of resection, a new mass of PCNA-positive cells appeared at the filament tip. At 40 days post-resection, approximately half of resected tissue was replaced; and at 160 days post-resection, regeneration was nearly complete. Chemoreceptive neuroepithelial cells, identified by serotonin immunohistochemistry, were present in regenerates and established innervation by nerve fibres. Use of the transgenic zebrafish line Tg(fli1a:EGFP), in which the gill vasculature was labelled with enhanced green fluorescent protein, indicated that angiogenesis occurred during the regenerative process. Thus, the zebrafish is capable of substantive gill regeneration and replacement of respiratory chemoreceptors.

Differential Expression and Localization of Branchial AQP1 and AQP3 in Japanese Medaka (Oryzias latipes).

Aquaporins (AQPs) facilitate transmembrane water and solute transport, and in addition to contributing to transepithelial water transport, they safeguard cell volume homeostasis. This study examined the expression and localization of AQP1 and AQP3 in the gills of Japanese medaka (Oryzias latipes) in response to osmotic challenges and osmoregulatory hormones, cortisol, and prolactin (PRL). AQP3 mRNA was inversely regulated in response to salinity with high levels in ion-poor water (IPW), intermediate levels in freshwater (FW), and low levels in seawater (SW). AQP3 protein levels decreased upon SW acclimation. By comparison, AQP1 expression was unaffected by salinity. In ex vivo gill incubation experiments, AQP3 mRNA was stimulated by PRL in a time- and dose-dependent manner but was unaffected by cortisol. In contrast, AQP1 was unaffected by both PRL and cortisol. Confocal microscopy revealed that AQP3 was abundant in the periphery of gill filament epithelial cells and co-localized at low intensity with Na+,K+-ATPase in ionocytes. AQP1 was present at a very low intensity in most filament epithelial cells and red blood cells. No epithelial cells in the gill lamellae showed immunoreactivity to AQP3 or AQP1. We suggest that both AQPs contribute to cellular volume regulation in the gill epithelium and that AQP3 is particularly important under hypo-osmotic conditions, while expression of AQP1 is constitutive.

Salmon gill poxvirus disease in Atlantic salmon fry as recognized by improved immunohistochemistry also demonstrates infected cells in non-respiratory epithelial cells.

Gill diseases cause serious losses in farming of Atlantic salmon and the number of agents involved increases. Salmon gill poxvirus (SGPV) and the gill disease in causes where SGPV apparently was the only disease-causing agent were initially characterized. Recently, it was further shown that SGPV can be a common denominator in widely different multifactorial gill diseases. Here, we present the challenge of diagnosing gill disease with SGPV in salmon fry of 0,3-5 grams. Apoptosis of gill lamellar epithelial cells and hemophagocytosis was also observed in fry similar to findings in smolts and grow-out fish. Using our newly developed immunohistochemistry method, we further demonstrate that some of the apoptotic epithelial cells covering the oral cavity were positive for SGPV. Thus, SGPV is not restricted to respiratory epithelium alone and may infect the fish at very early life stages. Furthermore, as the cases examined here are from Norway, Faroe Island and Scotland, we show that SGPV is more widespread than previously reported.

Morphology of a Hidden Tube: Resin Injection and CT Scanning Reveal the Three-dimensional Structure of the Spiracle in the Japanese Bullhead Shark Heterodontus japonicus (Chondrichthyes; Heterodontiformes; Heterodontidae).

The spiracle of elasmobranchs (sharks, skates, and rays) is a gill-slit-derived tube located behind the eye. Its inner structure was well studied in the late nineteenth to early twentieth century, but its entire morphology has rarely been characterized and is poorly understood. The present study shows the three-dimensional morphology of the spiracular tube for the first time, using resin injection and CT scanning, in the Japanese bullhead shark. The spiracular tube is characterized by the presence of two caeca (dorsal and ventral spiracular caeca) on the medial wall of the spiracular tube and the presence of a pseudobranch on the anterior wall. This study provides a basis for further studies on the morphological diversity, function, and evolution of spiracles in elasmobranch fishes. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Ontogeny of juvenile freshwater pearl mussels, Margaritifera margaritifera (Bivalvia: Margaritiferidae).

The gills of juvenile freshwater bivalves undergo a complex morphogenesis that may correlate with changes in feeding ecology, but ontogenic studies on juvenile mussels are rare. Scanning electron microscopy was used to examine the ultrastructure and ontogeny of 117 juvenile freshwater pearl mussels (Margaritifera margaritifera) ranging in age from 1-44 months and length from 0.49-8.90 mm. Three stages of gill development are described. In Stage 1 (5-9 inner demibranch filaments), only unreflected inner demibranch filaments were present. In Stage 2 (9-17 inner demibranch filaments), inner demibranch filaments began to reflect when shell length exceeded 1.13 mm, at 13-16 months old. Reflection began in medial filaments and then proceeded anterior and posterior. In Stage 3 (28-94 inner demibranch filaments), outer demibranch filaments began developing at shell length > 3.1 mm and about 34 months of age. The oral groove on the inner demibranch was first observed in 34 month old specimens > 2.66 mm but was never observed on the outer demibranch. Shell length (R2 = 0.99) was a better predictor of developmental stage compared to age (R2 = 0.84). The full suite of gill ciliation was present on filaments in all stages. Interfilamentary distance averaged 31.3 µm and did not change with age (4-44 months) or with size (0.75-8.9 mm). Distance between laterofrontal cirri couplets averaged 1.54 µm and did not change significantly with size or age. Labial palp primordia were present in even the youngest individuals but ciliature became more diverse in more developed individuals. Information presented here is valuable to captive rearing programmes as it provides insight in to when juveniles may be particularly vulnerable to stressors due to specific ontogenic changes. The data are compared with two other recent studies of Margaritifera development.

Creation of a voxel phantom of the ICRP reference crab.

The International Commission on Radiological Protection (ICRP) has modeled twelve reference animal and plant (RAP) species using simple geometric shapes in Monte-Carlo (MCNP) based simulations. The focus has now shifted to creating voxel phantoms of each RAP in order to estimate doses to biota with a higher degree of confidence. This paper describes the creation of a voxel model of a Dungeness crab from CT images with shell, gills, gonads, hepatopancreas, and heart identified and segmented. Absorbed fractions were tabulated for each organ as a source and target at twelve photon and nine electron energies: 0.01, 0.015, 0.02, 0.03, 0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, and 4.0 MeV for photons and 0.1, 0.2, 0.4, 0.5, 0.7, 1.0, 1.5, 2.0 and 4.0 MeV for electrons. AFs whose error exceeded 5% are marked with an underline in the data tables; AFs whose error was higher than 10% were excluded, and are shown in the tabulated data as a dashed line. A representative sample of the data is shown in Figs. 3-8; the entire data set is available as an electronic appendix. The results are consistent with previous small organism studies (Kinase, 2008; Stabin et al., 2006), and suggest that AF values are highly dependent on source organ location and mass.

Biomarkers as diagnostic tools for evaluating effects of unknown past water quality conditions on stream organisms.

The following biomarkers were investigated in stream populations of juvenile brown trout (Salmo trutta f. fario) and gammarids (Gammarus pulex) to determine if crayfish mortality could have been confounded by pollutants: (1) alterations of fish liver ultrastructure, (2) fish gill and kidney histopathology, (3) stress protein (hsp70) expression in fish liver and gills and in gammarids, and (4) changes in various blood parameters of brown trout. In addition, the following measurements were conducted in parallel with the biological sampling: (a) chemical analyses including several pesticides, organochlorines, PCBs, and PAHs in sediment and tissue samples of brown trout and crayfish (Astacus astacus), and (b) limnochemical analyses of nutrients, electrolytes, dissolved oxygen content, temperature and pH. Biomarkers together with chemical and limnochemical analyses concomitantly indicated moderate pollution of the stream at all sampling sites. Biological data indicated a transient, episodic event at one sampling site resulting (a) in altered stress protein levels in gills and livers of trout and in whole gammarids as well as (b) in elevated numbers of macrophages in liver tissue. Biomarker responses provided spatial and temporal evidence that a contaminant release was associated with the crayfish mortalities observed in this stream system.

Branchial blood flow distribution in the blue shark (Prionace glauca) and the leopard shark (Triakis semifasciata).

Electromagnetic flow (EMF) quantification of total cardiac stroke flow is not feasible for most elasmobranchs because the vascular anatomy precludes probe placement adjacent to the heart and proximal to all afferent branchial arteries (aba). Most previous studies report a fractional cardiac flow, made with the EMF probe placed on the ventral aorta between the innominate arteries and aba 3. Estimation of total cardiac stroke flow from such data requires a flow correction factor obtained by sacrificing the fish, and carrying out a two step in situ/in vitro flow calibration procedure which is based on tenuous assumptions. Ventral aortic blood flow measurements using the EMF techniques were carried out on large blue sharks, and radiographic imaging studies of ventral aortic and branchial blood flow were done on leopard sharks to verify previously estimated fractional cardiac stroke flow correction factors. The innominate flow fraction determined for both species in these studies are similar and agree with previous estimates for elasmobranchs. EMF data for Prionace show 38% of cardiac stroke flow goes to the innominate arteries, 23% into aba 3, 12% into aba 4, and 27% into aba 5. Radiographic analyses with Triakis reveal that 32% of its cardiac stroke volume flows into the innominate arteries which is in agreement with the in situ/in vitro fractional flow estimate (33%).

Accumulation of cadmium and lead in the gills of Mytilus edulis: X-ray microanalysis and chemical analysis.

The accumulation of Cd and Pb in the gills of the lamellibranch mollusc Mytilus edulis has been studied by electron microscopy, X-ray microanalysis, atomic absorption spectroscopy and radionuclide monitoring. The patterns of accumulation of the two elements differ markedly as do the sites of deposition whithin the gills. Lead is found extracellularly as crystalline deposits in the basal lamina which forms the capillary walls of the gill lamellae. The Pb is found associated with Ca in equiatomic ratios and occurs either as a mixed or complex carbonate. Cadmium is always associated with S and frequently with P in membrane bound vesicles within the cells of the gill epithelium and in the amoebocytes. The S is probably attributable to the presence of cysteine residues in a metal binding protein which can be extracted from the gills. Analysis of the metal binding protein shows that it binds Ag, Cd, Cu, Fe, Hg, Sn and Zn. Its amino acid composition is similar to that reported for eels and limpets but has a lower cysteine content than mammalian metal binding protein.