The photomicrographs of stained sections of multiple tissues and cellular structures of the Solea senegalensis larvae are shown in Fig. 3. In this work, we described an accessible protocol (detailed in Section 2) to apply histopathological techniques in small sized organisms in order to encourage laboratories working in ecotoxicology to perform such analyses without the need of expensive and specialized equipment, by deploying common equipment, such as fume hoods and laboratory drying ovens. The handmade box-shaped aluminum sheets for modeling the paraffin blocks are another example of successful adaptation of easy to handle and cheap material (Fig. 2C). However, the use of fresh solutions of ethanol and xylene are essential to ensure processing quality (Copper et al. 2018). Additionally, the fixation process preserves the specimens over time, however, we recommend that all the histological processing of the samples should be performed within a maximum of 6 months after fixation to avoid the samples degradation, such as excessive dryness and distortion of the tissues. In this study, the final paraffin blocks were kept in a freezer at -20°C for a few minutes before being cut to avoid the paraffin slices from curling. Soaking the blocks in ice water prior to their sectioning produced optimal sections, as advised by Copper et al. (2018). Another suggestion is that although the sections were stretched over the flame of a Bunsen burner glass alcohol lamp, it is safer to use a water bath (at approximately 40°C) lightly sprinkled with colorless gelatin (to ensure that the paraffin slices are firmly adhered to the slides), always taking care to not melt or overstretch the floating sections. The slides produced according to the proposed method were efficient for the histological analyses. Finally, the photomicrographs can be registered with a photographic equipment (e.g., a cell phone fitted to the microscope’s eye lens), and the frequency and occurrence of the histopathological alterations can be identified and classified into index, as proposed by Schwaiger et al. (1997) and Bernet et al. (1999).
To minimize the difficulty of handling the extremely small sized samples, the method of pre-embedment in agarose gel before sectioning was used herein to study the histologic features of 8 days post hatch (dph) S. senegalensis larvae. Although literature already had documented the pre-embedding technique in agar since 1960s for small tissue biopsies, this is not a routine technique in worldwide histopathology laboratories (Ridolf et al. 2019), especially in environmental studies. Some authors have applied this technique for studying zebrafish larvae (Tsao-Wu et al. 1998; Sabaliauskas et al. 2006; Copper et al. 2018), but, to our knowledge, this is the first time that the pre-embedding agar is used in Solea larvae. The advantages of the use of molecular biology grade agarose for embedding small biological samples include the softness for an easier block section, the substance hardness is similar to the fixed sample, and the positive impact on orientation before tissue sectioning. This technique is also inexpensive, safe, and easy to use (Ridolf et al. 2019), and this agarose embedding process is applicable for the examination of any small sized organisms or tissues (Tsao-Wu et al. 1998) such as fish, mollusks, annelids, crustaceans, and others, commonly used for ecotoxicological purposes. In this study, the histological methods for fixation, agarose pre-embedding, paraffin embedding, sectioning, staining and digital image capture (Fig. 1) were appropriate for flatfish larvae. It is useful to note that some stiffer samples must be decalcified (after fixed) for adequate sectioning. For example, the fixed samples can be immersed in 7% EDTA (15 min; 60°C) or in 10% nitric acid (6 h) (Plaul et al. 2017), and then proceeded to pre-soak in agarose.
Despite the fact that nowadays the histology practice is mainly restricted to automated specialized labs that obviously enable a quicker and more standardized work flow, some steps within the process are still completely dependent on the manual performance, accuracy and capability of laboratory personnel (Ridolf et al. 2019). Previous works have designed a method to embed fish larvae producing simultaneous large-scale sections on a single glass microscope slide (Tsao-Wu et al. 1998; Sabaliauskas et al. 2006). However, it is preferable to individually embed samples because it requires more specialized staff and accurate equipment to produce adequate large-scale sections.
Considering the methodology used in this work, morphological and physiological features of 8 dph S. senegalensis larva could be perfectly observed. At this age, the S. senegalensis larva is in the stage 3 of development, few days before metamorphosis and eye migration start (approximately on the 11th dph). At this phase, the yolk sac was completely resorbed (i.e., it is no longer a lecithotrophic larvae), and the dorsal finfold covered the head retracted back leaving the eyes uncovered (Padrós et al. 2011). Some authors reported a visible swim bladder above the gut, which was observed in this study (Fig. 3A) (Padrós et al. 2011).
This early larval pelagic stage is characterized by a rapid development of sensory, mainly vision, and digestive organs (Fig. 3A and 3C), in agreement with Padrós et al. (2011). This development is related with an acceleration of larval growth and survival, leading to the initiation of the metamorphosis (Youson 1988). The eye development is remarkably fast, with the retinal structure distinguished at 1 dph. The larva displays active hunting behavior 3 dph, with mouth (Fig. 3C) and anus opened, and the eyes fully pigmented (Fig. 3C); the metamorphosis and the eye migration completed at 30 dph (Padrós et al. 2011). The hunting behavior of sole larvae, highly associated with the vision maturing, occur only during this early phase of development. During the transition from the pelagic to the benthic life, associated with morphological, physiological, behavioral and ecological changes, the sole larvae show typical nocturnal feeding habit after 8 dph (Bayarri et al. 2004) and are able to feed on dead and inert prey from 12 dph, probably relying on chemical stimuli (Villalta et al. 2008).
As in others teleost larvae, the sole’s skin includes protective, osmoregulatory, respiratory and sensory structures. During early developmental stages, the epidermis of S. senegalensis is the major component of the organisms’ sheathing, because dermis is practically absent and a large subdermal space associated with buoyancy is observed; this structure disappears when the larval fins are developed and their swimming and floating ability are complete. This space is also associated with the chloride cells present in the skin (Fig. 3A), which possess osmoregulatory role in the early developmental stages. As metamorphosis takes place, the skin of post-metamorphic juveniles S. senegalensis is still formed by a simple bi-epithelial cell layer with scattered mucous cells, a basal lamina and some fibroblasts at the base of the epidermis. Changes in the number, size and the histochemical properties of the secretions of the mucous cells can be indicators of pathological or inflammatory processes induced by adverse environmental conditions. Only after the larva settles in the bottom, they became tolerant to a wide range of salinities and able to face stress-inducing conditions in the surrounding environment (Padrós et al. 2011).
After hatching, gas exchange in S. senegalensis is performed over the whole-body surface, by the chloride cells (Fig. 3A) dispersed over the epidermis, which are responsible for ion balance. The ability of cutaneous gas exchange became limiting to satisfy the metabolic demand for oxygen as larvae grow (Padrós et al. 2011). At 8 dph, an almost complete pseudobranch can be seen (Fig. 3C), comprised of five filaments and agglutination of lamellae and vascular structure with the chondrogenesis process occurring in the filaments. Gills form relatively late along the developmental stage and gradually displace the skin as the site of most exchange activities (Rombough 2004). Until 11 dph, gill structures do not have a major role in respiration, but as chloride cells are already present in the rudimentary gill arches, they might play a role in osmoregulatory activities together with the skin.
The notochord is formed by highly vacuolated cells within membranes arranged in “honeycomb” shape, surrounded by a collagen layer (Fig. 3B). This structure is already well-developed at hatching, and its general structure does not change substantially throughout larval development (Padrós et al. 2011). The muscle tissue was observed in the tail region (Fig. 3B). The olfactory lobes and cavities were present (Fig. 3C), with the olfactory bulbs differentiated from the telencephalon of the brain (Padrós et al. 2011).
At 8 dph, the poorly heart developed was observed below the cephalic area (Fig. 3A). In this stage, the mesonephric tubules and rudimentary renal corpuscles are still not evident in the dorso-posterior area of the kidney, comprising of pronephric tubules (Fig. 3A). Until this stage, thyroid is found as follicles (Fig. 3A and 3C), as observed by Padrós et al. (2011).