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Received: 1 November 2017 Accepted: 7 April 2018 DOI: 10.1111/ahe.12358
ORIGINAL ARTICLE
Morphofunctional description of mucous cells in the gills of the Arapaimidae Arapaima gigas (Cuvier) during its development C. A. Ramos1
| O. T. F. da Costa1 | W. L. P. Duncan1 | M. N. Fernandes2
1 Department of Morphology, Federal University of Amazon, Coroado, Manaus, Brazil 2
Summary The gill structure of the Amazonian fish Arapaima gigas (Cuvier 1829) shows ontoge-
Physiological Sciences Department, Federal University of São Carlos, São Carlos, Brazil
netic changes during development, particularly due the transition from the aquatic to
Correspondence Cleverson Agner Ramos, Department of Morphology, Federal University of Amazon, Coroado, Manaus, Brazil. Email:
[email protected]
be found in the gills: mitochondrial rich cells, pavement cells and mucous cells (MCs).
the obligatory air breathing mode of respiration. However, three main cell types can The MCs are involved in the secretory pathway. The functions of the secreted molecules include mechanical protection of epithelia, protection against parasites and bacterial infection, and role on ion regulation. In this study, we analysed mucous cell location and mucous cell type, based on pH, during the development of A. gigas. Using samples obtained from the environment, gills were collected and fixed in buffered solution. Histological techniques for the identification of MCs were performed Alcian Blue (AB) and periodic acid-Schiff (PAS). The results showed the presence of PAS+ and AB+ cells in the whole filament in all examined fish. In animals less than 50 g, few MCs were present, and no differences were observed in AB+ and PAS+ cells. In animals weighing close to 500 g, more PAS+ cells than AB+ cells were observed, and in animals that weighed more than 1,000 g, more AB+ cells than PAS+ cells were observed. These observations may be a result of the ontogenetic changes in the gill epithelia, which can change the osmorespiratory compromise in ion regulation functions as well the glycosaminoglycans secreted by PAS cells, which in large animals can play a role in the protection against parasites and bacterial infection.
1 | I NTRO D U C TI O N
et al., 2005; Hossler, Musil, Karnaky, & Epstein, 1985; Hughes, 1984; Perry, 1997).
The gills of obligate air-breathing fish show a distinctive morphol-
Mucous cells are large cells with extensive cytoplasm and a large
ogy; two gill filaments, which are connected by one interbranchial
number of organelles that are involved in the secretory pathway and
septum, projecting from each gill arch. Along each of these fila-
contain high molecular weight molecules (Cinar, Aksoy, Emre, & Aşti,
ments, respiratory lamellae, where gas exchange takes place, are
2009). These macromolecules are known in the literature as glycos-
distributed perpendicularly. Gills are considered a multifunctional
aminoglycans (GAGs). GAGs are responsible for absorbing water and
organ (Evans, Piermarini, & Choe, 2005) because they are not only
interacting with other molecules, and they originate in areas with a
involved in gas exchange but also in ion exchange, acid-base regu-
viscous appearance that is characteristic of mucus. Thus, MCs are
lation and nitrogenous waste excretion. Such functions require a
responsible for secreting the mucosubstances on the surfaces of
typical cellular morphology, which is present in fish gills. The most
gills. According to Moron, Andrade and Fernandes (2009); Shephard
common cell type present in gills is the pavement cell (PC). Cells
(1994), in addition to gill lubrication, mucus plays an important role
with different morphologies can be observed between PCs, includ-
in gill protection, as it protects this organ against mechanical shock
ing mitochondrial rich cells (MRCs) and mucous cells (MCs) (Evans
from matter suspended in the water, microorganisms and exposure
Anat Histol Embryol. 2018;1–8.
wileyonlinelibrary.com/journal/ahe © 2018 Blackwell Verlag GmbH | 1
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RAMOS et al.
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to air and pollutants dissolved in water. Additionally, mucus may be involved in osmoregulation and is considered an adaptive mecha-
Therefore, the goal of this study is to elucidate the role of MCs in the transition from bimodal breathing to air breathing.
nism in aquatic environments (Shephard, 1982, 1994). Therefore, it is common to find greater or fewer MCs in fish gills depending on the health of the fish and the environment in which it lives (R. D. Handy, Eddy, & Romain, 1989; Paulino, Sakuragui, & Fernandes, 2011). The macromolecules present in the granules of MCs have
2 | M ATE R I A L S A N D M E TH O DS 2.1 | Animal collection
different chemical properties, which may vary according to the an-
Arapaima gigas specimens weighing from 1.67 to 5,010 g (Table 1)
imal’s habitat and/or health to meet its physiological needs. GAGs
were collected using trawls and sieves in the Jacaré Lake system
may be acidic, sulphated or neutral. In addition, according to Shepard
(S03°31′537″ W60°40′181″), a set of small lakes that drain directly
(Shephard, 1994), macromolecules other than GAGs are found in the
into Solimões River, near the city of Manacapuru, approximately
mucus, such as lysozymes, immunoglobulins, carbonic anhydrase,
100 km from Manaus. Animal collection was conducted with an en-
complement system components, cytokines, C- reactive protein
vironmental permit from the Brazilian Institute of Environment and
and proteolytic enzymes. GAGs are linear polysaccharides com-
Renewable Natural Resources (Instituto Brasileiro do Meio Ambiente
posed of repetitive disaccharide units (one of the units is invariably
e dos Recursos Naturais Renováveis–IBAMA/MMA) No 15/2004.
a hexosamine, either D-glucosamine or D-galactosamine, and the other is uronic acid, glucuronic or iduronic acid or galactose) assembled in an unbranched sequence with sulphate groups in different
2.2 | Processing of gill samples After capture, the animals were irreversibly anesthetized with benzo-
positions of the polysaccharide chain. Environmental changes can induce changes in the production
caine (0.9 ml/L). The gill arches were removed, washed in 0.9% NaCl
of mucopolysaccharides. Such changes can also be found in animals
and fixed in 2.5% glutaraldehyde (GTA) in 0.1 M phosphate buffer, pH
exhibiting alterations in the basic morphology of the gill epithe-
7.3, at 4°C. After 24 hr, the samples were transferred to a 0.5% GTA
lium. In the latter case, Arapaima gigas (Cuvier 1829, Arapaimidae)
solution of the same buffer. The samples were sent to the Laboratory
is considered an excellent animal model for studies assessing such
of Zoophysiology and Comparative Biochemistry (Laboratório de
modifications.
Zoofisiologia e Bioquímica Comparativa–DCF/UFSCar) for process-
Arapaima gigas, also known as pirarucu, is a fish that belongs to
ing and analysis. All the procedure was approved by the Brazilian
the Arapaimidae family, which is endemic to the Amazon region and
Institute for the Environment and Renewable Resources (IBAMA),
is easily found throughout the watershed region. This species pres-
licence number 15/2004, protocol number 02005.000449/04-14.
ents bimodal breathing, which means that these animals are aquatic breathers or obligate air-breathers in different stages of their lives (Graham, 1997). In the first week of life, after hatching, A. gigas breathes exclusively through the gills, which are similar to the gills
2.3 | Sample processing for light microscopy in Historesin
of exclusively aquatic breathing teleost fish. In contrast, in adult
The samples were dehydrated in a graded ethanol series (from 50%
A. gigas, the gills are not able to obtain enough O2 to meet the ani-
to 95%) for 1 hr in each dehydration step and embedded in moulds
mal’s physiological needs, and O2 absorption occurs mainly through
using commercially available Historesin (Leica Historesin Embedding
the modified swim bladder. Thus, there is a change in gill function
Kit) for sagittal sectioning using a microtome (HM 360 MICROM).
so that from the fry stage to the adult stage, following remodelling
The sections (3 μm thickness) were placed on slides, and histochemi-
of the gill epithelium, gills become mainly involved in ion regulation
cal staining was performed (periodic acid-Schiff [PAS] and Alcian
(Gonzalez et al., 2010; Ramos, 2008; Ramos, Fernandes, Costa, &
blue [AB]). Imaging was performed using an Olympus-Micronal BX51
Duncan, 2013).
microscope connected to a video camera and a computer with CAST
Given that MCs play a role in the protection of gill epithelium and
System software (Olympus, Denmark).
ion regulation, the action of these cells in the osmorespiratory compromise during the ontogenetic development of A. gigas may provide a better understanding of the management of this species and tools
2.4 | Quantitative analysis of MCs
for optimizing aquaculture techniques as well as elucidating the bio-
Cells containing mucosubstances composed of neutral hex-
geographic history of the species.
oses, sugars and/or sialic acids were identified using the PAS
TA B L E 1 Collected specimens in the Jacaré lakes system Specimen collected Total Length (cm)
6.9
17.3
19.8
19.0
25.1
31.0
Body mass (g)
1.675
34
46.5
54.5
105.6
211.9
47.2 575
53.5 1,343
79.5 4,995
81 5,020
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F I G U R E 1 Cells with secretory granules positive for PAS staining. PAS+ cells (black arrows) are observed in interlamellar regions in animals weighing 46 g (a) and 105 g (b). PAS+ cells were located in the outer layers of the gill epithelium in all analyzed animals; 212 g (c), 575 g (d), 1,343 g (e) and 4,995 g (f). An increase in the number of PAS+ cells was observed throughout the developmental stages of Arapaima gigas
(a)
(b)
(c)
(d)
(e)
(f)
staining method. MCs presenting mucopolysaccharides con-
additional trimming, ultrathin sections were obtained using an
taining carboxyl and sulphate ester groups were identified
ultramicrotome (Leica UltraCut R) and placed on a copper grid
using AB staining, pH 2.5. Following histochemical staining,
(Specimen Grid 3.05 μm diameter, 0.8 μm thickness, Copper 200
the slides were analysed using a light microscope. For each
mesh, T200-C u). Then, these samples were stained with satu-
animal, twenty non-c ontiguous fields (magnification 200x)
rated uranyl acetate and Reynolds solution (lead citrate). The
were randomly selected. Cells in each field were identified
samples were examined with a JEOL JEM1200-E XII transmission
and counted.
electron microscope.
2.5 | Transmission electron microscopy (TEM) analysis
2.6 | Statistical analysis
To examine the ultrastructural alterations in the cells of A. gigas
Statistical data analysis was performed using GraphPad Instat 3.0
during fish growth, samples were selected and processed for
software. Bartlett’s test was used to determine data homogene-
TEM. Samples fixed in buffered GTA were washed in the same
ity and the application of statistical tests. When data did not pass
buffer (same osmolarity and pH). The samples were post-f ixed in
Bartlett’s test, conversion to a log10 scale was performed to im-
1% OsO 4 , stained en bloc with 10.56% uranyl acetate and saccha-
prove data homogeneity. Statistical analysis was performed using
rose, dehydrated in a graded acetone series (from 30% to 100%),
analysis of variance (ANOVA) to test for significant differences.
and embedded in SPURR Low Viscosity- Embedding Kit resin
When statistically significant differences were found, Tukey’s post
(EMS) in a sagittal position. After embedding, block trimming was
hoc test for multiple comparisons (confidence interval 95%) was
performed. Semi-thin sections were obtained and stained with
used to determine where these statistically significant differences
toluidine blue to select a region to be examined by TEM. After
occurred.
Data are presented as the mean + standard error of the mean (SEM).
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(a)
(b)
(c)
(d)
(e)
(f)
3 | R E S U LT S 3.1 | Overview of MCs in A. gigas (histochemical analysis using PAS and AB staining of MCs) Histochemical staining showed that both PAS- p ositive (PAS+) cells and AB-p ositive (AB+) cells were found in the gills of A. gigas. Animals weighing less than 500 g still had lamellae in their gill filaments; in these animals, PAS+ and AB+ cells were found in the interlamellar region, as shown in Figures 1 and 2. In animals weighing more than 500 g, we observed that gill filaments were uniform structures with lamellae embedded in the filament tissue of gills. In these animals, microridges can be observed in the gill epithelium throughout the entire filament. MCs are present from the outer layers of the gill epithelium to the deeper regions in the epithelial microridges. Such folds are located close to the system of pillar cells, which corresponds to the interlamellar region observed in animals weighing less than 500 g that still have lamellae, as observed in Figures 1 and 2. Both PAS+ and AB+ cells were observed along the gill epithelium and were prominent in the apex region of gill filaments. Despite using one animal for each size comparison, after randomly
F I G U R E 2 Presence of cells with secretory granules positive for Alcian Blue staining AB+ (black arrows). Similar to PAS+ cells, AB+ cells are frequently localized in interlamellar regions in animals weighing 46 g (a) and 105 g (b). In all analyzed animals, AB+ cells were present in the outer layers of the gill epithelium; 212 g (c), 575 g (d), 1,343 g (e) and 4,995 g (f). The number of AB+ cells increases throughout the developmental stages of Arapaima gigas
selecting 20 non-contiguous fields under a light microscope, a quantitative analysis of the number of cells per animal was performed (cells∙(mm2)−1 x 102), and statistical tests were performed to check the significative differences in positive staining cells for each animal. These procedures allow to make inferences on branchial mucous pattern secretion along the ontogenetic development of A. gigas, as observed in Figure 3. We observed that the cell density was low in the animal that weighed approximately 50 g, but no statistically significant differences existed between PAS+ and AB+ cells (p
