# An Easy-to-Use Histological Technique for Small Biological Samples of Senegalese Sole Larvae
## 3 Results  
\(S.\)_senegalensis Morphological Features_  
Figure 3 shows the photomicrographs of the \(S.\)_senegalensis_ larvae stained sections of the whole larva (Figure 3A), and the multiple tissues and cellular structures (Figure 3B-D). Considering the methodology used in this work, the morphological features of \(8\) dph \(S.\)_senegalensis_ larva could be perfectly observed (Figure 3). At this age, the larva is at stage three of development, a few days before metamorphosis and eye migration start (approximately on the \(11\) dph). At this phase, the yolk sac was resorbed entirely (i.e., it is no longer a lecithotrophic larva), and the dorsal finfold covered the head retracted back, leaving the eyes uncovered. Some authors reported a visible swim bladder above the gut, which was observed in this study (Figure 3B) [31]. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Solution** & **Time** & **Temperature (\({}^{\circ}\)C)** & **Local** \\ \hline Ethanol 80\% & 45 min & - & \\ Ethanol 95\% & 45 min & - & \\ Ethanol 95\% & 1 h & - & \\ Ethanol 100\% & 1 h & - & Fame hood \\ Ethanol 100\% & 1 h & - & Fame hood \\ Xylene & 1 h & - & Fame hood \\ Xylene & 1 h & - & Fame hood \\ Paraffin & 2 h & 60 & Laboratory drying oven \\ Paraffin & 2 h & 60 & Laboratory drying oven \\ Paraffin & 2 h & 60 & Laboratory drying oven \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Dehydration and Paraffin embedding steps, including the solution and exposure time, temperature and handling place, for correct procedures. \begin{table}
\begin{tabular}{c c} \hline \hline
**Solution** & **Time** \\ \hline Xylene & 5 min \\ Xylene & 5 min \\ Ethanol 100\% & 3 min \\ Ethanol 95\% & 10 s \\ Ethanol 80\% & 10 s \\ Ethanol 50\% & 10 s \\ Distilled water & 5 s \\ Harris Hematoxylin & 4–5 min \\ Tap water & 6 s \\ Eosin yellowish 3\% & 15 s–3 min \\ Ethanol 95\% & 10 s \\ Ethanol 100\% & 10 s \\ Ethanol 100\% & 10 s \\ Ethanol 100\% & 10 s \\ Xylene & 10 s \\ Xylene & 10 s \\ Xylene & 10 s \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Hematoxylin and Eosin (H&E) staining steps, including the solution and exposure time, and details for correct procedures. Figure 3: Photomicrographs of 8-day-old _Solea senegalensis_ larvae applying the easy-to-use histological technique developed in this study. (**A**) Whole larva. (**B**) Abdominal area. (**C**) Tail region. (**D**) Cephalic area. ai (anterior intestine); e (eyes); h (heart); l (liver); m (mouth); nt (notochord); ol (olfactory lobe); pb (pseudobranch); pi (posterior intestine); pt (pronephric tubule); sb (swim bladder); sc (spinal cord); tm (tail muscle); (\(\blacktriangleright\)) chloride cell; (\(\blacktriangleright\)) sacciform cell; (\(\lx@paragraph\)) thyroid follicles. H&E staining. Scale bar 20 \(\upmu\)m.  
This early larval pelagic stage is characterized by a rapid development of sensory, mainly vision, and digestive organs, including the liver and intestine (Figure 3B), in agreement with [PERSON] et al. [31]. The parenchymal architecture of the liver exhibits a normal shape, i.e., polygonal hepatocyte with a spherical and centralized nucleus. This development is related to an acceleration of larval growth and survival, leading to the initiation of the metamorphosis [39]. The eye development is remarkably fast, with the retinal structure distinguished at 1 dph. The larva displays active hunting behavior at 3 dph, with the mouth and anus opened and the eyes fully pigmented (Figure 3A,D); the metamorphosis and the eye migration are completed at 30 dph [31]. The hunting behavior of sole larvae, highly associated with vision maturing, occurs only during this early development phase. During the transition from pelagic to benthic life, associated with morphological, physiological, behavioral, and ecological changes, the sole larvae show typical nocturnal feeding habits after 8 dph [40]. They can feed on dead and inert prey from 12 dph, probably relying on chemical stimuli [41; 42].