BIOMETRICAL AND MORPHOHISTOLOGICAL STUDY OF POSTPHARYNGEAL DIGESTIVE TUBE FOR SALAMANDRA SALAMANDRA SALAMANDRA L.

Analele Ştiinţifice ale Universităţii Al. I. Cuza Iaşi, s. Biologie animală, Tom LIV, 2008 BIOMETRICAL AND MORPHOHISTOLOGICAL STUDY OF POSTPHARYNGEAL DIGESTIVE TUBE FOR SALAMANDRA SALAMANDRA SALAMANDRA L. Sergiu HAIMOVICI Al. I. Cuza University Iaşi, Faculty of Biology, Bd. Carol I 20A, 700505 Iaşi, Romania Abstract. Salamander, common nowadays as a species belongs to Urodels, one of the first species who moved from the aquatic to terrestrial environment 150 million years ago. They preserved primitive characters and added some others new as well as some specific characters. This is clearly evidentiated if we take into consideration the postpharyngeal digestive tube. So, we made a biometrical, morphological and histological study of this tube of 17 mature individuals, of which 10 males and 7 females. Measurements have been executed and morphological studies have been made on the four segments of the digestive tube: esophagus, stomach, small intestine and large intestine and than, on histological sections using nine coloration types we described the characteristics as we see on microscope for fragments coming from the four segments. Some data have been outlined the most important aspects being presented as conclusions and some others being included in the paper, for supporting our affirmations. Keywords: Salamander, digestive tube, biometrical morphological histological study. Rezumat. Studiu biometric şi morfohistologic al tubului digestiv postfaringial la Salamandra salamandra salamandra L. Salamandra, astăzi comună ca specie, aparţine urodelelor, care au fost printre primele care acum 150 milioane de ani au reuşit să treacă din mediul acvatic în mediul terestru. Ele au rămas cu caractere primitive dar au căpătat şi altele noi, mai evaluate, iar unele caracteristice lor. Acest lucru apare bine evidenţiat dacă considerăm tractusul digestiv postfaringian. Noi am lucrat pe 17 exemplare mature, zece de sex mascul şi şapte de sex femel. S-au executat măsurători şi studii de morfologie pe cele patru segmente ale acestui tub: esofag, stomac, intestin mediu şi intestin posterior, iar apoi, pe secţiuni histologice folosind nouă tipuri de colorare, am descris şi caracteristicile văzute prin microscop, pe porţiuni din fiecare cele patru segmente. Au fost puse în evidenţă o serie de date, dintre care cele mai importante sunt arătate în concluziile lucrării noastre, şi altele, ce sunt prezentate în text, ceea ce arată foarte bine ceea ce am susţinut mai sus. Cuvinte cheie: Salamandra, tub digestiv, studiu biometrico morfologico histologic. Introduction We chose to study a relatively common species for the Romanian high falling leaves woods, a well known species described even from Antiquity by Aristoteles who considered it immortal, immune to fire, maybe influenced by the stories of the ancient Greek story-tellers. His words were taken as good in the Middle Ages, especially on the space of nowadays Germany, a place where it used to be known as Foer Salamander; the common people did not experimented this but the wizards were often associated with the presence of this small animal. We studied the salamander s digestive tube from scientific reasons, because firstly the urodels represent a very old group from the paleontological point of view. They are closed to the first tetrapodals who left the aquatic area for the land 150 million years ago. The urodels have not evolved too much, still preserving some primitive characters, some of them even more primitive than those of the teleostean fish, younger from the paleontological point of view but more evolved, for these fish a great diversification being specific, caused by their diverse habitat. We could name them as living fossils. Secondly, although their digestive tube have been studied but not too thoroughly by varied authors, salamander was considered as a gender, without mentioning the species or subspecies; sometimes some characters are present only with some particular species

Sergiu Haimovici (or subspecies); more than these only a small number of individuals have been studied. We should mention that in Western Europe as well as in the Mediterranean area another species is common, We studied Salamandra salamandra, salamandra subspecies common on Romanian territory and mentioned by Linné (Fuhn, 1960). We should also say that Salamander is a carnivorous insectivorous species. Material and Methods The material was collected from the falling leaves forests around the biological research area Pângaraţi, on the middle course of Bistriţa River (Neamţ county) from early spring till late in autumn. There are 17 mature individuals, 10 males and 7 females (the sex was determined by abdominal section). Both external and internal measurements have been made on the individuals. The whole digestive tube as well as its fragments has been measured (in mm) using a thread to follow all the sinuosities. By cutting, the internal morphology of the digestive tube fragments was studied (using a 3x magnifying glass): the esophagus, stomach, the larger area we called fundic and the tight one we called pilorical, the small intestine and large intestine till the cloacum. Small fragments from the walls of every segment have been prepared according to histological technique for 87 slides. General colorants have been used to give color the histological fragments and selective colorants for histochemistry or evidence the presence or absence of some formations; nine color blends have been used. For studying the histological material we used a microscope with 10x15 or 15x15 magnifying indices and for some details with an increased magnifying index. Results and Discussion External morphology The digestive tube, immediately after the pharyngeal fragment, is made of a relatively short and thick esophagus which continues with the stomach, longer with a proximal part as thick as the esophagus and growing larger in the medium part (the fundus area) and then it becomes narrow again, so its final part (the pyloric area) is even tighter than the esophagus. This fragment makes a curve, as some authors say, a well circumscribed swelling but just on palpation it seems harder and its aspect is a little whiter (Pernkopf & Lehner, 1937). Next follows the middle intestine, much tighter with three sometimes four intestinal ansa well packed in order to get into the belly. We may observe that there is a slight narrowing from the proximal to distal area so we can distinguish three fragments: the proximal (let us call it duodenum), the middle and the distal ones. Then, we have identified the large intestine, much larger (similar to the fundus area of the stomach) with a tight area to the opening in the cloacum. Biometrical study The table contains measurements (in mm). There are 17 individuals, seven females and ten males; one of the males (number 5) is much smaller than the others. We may say that the digestive tube is three times longer than the body length (from the anterior part to the opening in the cloacum) an average of about 297.50 mm. This indicates superiority as we said before salamander is a carnivorous species and as we know this animal group usually has a smaller digestive tube. By studying the postpharyngean digestive tract by segments we may present the following results: esophagus length is about 5.05%; stomach about 11.24%; middle intestine 75.45%; large intestine 7.87% of the digestive tube. We may say that the greatest percentage belongs to the middle intestine as a proof of its absorption function importance. This fragment as well as the whole digestive

Analele Ştiinţifice ale Universităţii Al. I. Cuza Iaşi, s. Biologie animală, Tom LIV, 2008 tube is a little bit longer for the females and this is not only because their body length is bigger. Internal morphology The internal morphology was studied for the whole digestive tube longitudinally sectioned. We shall take it fragment after fragment. The esophagus is relatively narrow and short longitudinally oriented and often placed towards the dorsal (back) part of the individual s body. We may observe 7-11 longitudinal prominent folds that may be seen without a microscope. Towards the inferior part of the esophagus they become smaller and less prominent. The passing through the stomach is gentle, with no sphincter. Stomach with its large lumen in the middle part has also 3 to 9 folds, some of them as a kind of elongation of the ones in the esophagus; the others originate in the stomach and they do not cover the whole stomach length. The stomach intestine passage is gentle and on a segment of about 3 mm, the color is whiter and the surface is harder, there are no more folds and a sphincter like structure appears but with no covering valva. The middle intestine has two to five folds not too prominent in its anterior part, and only 2 in its posterior part (the intestine gets narrower). The passing middle to large intestine is abrupt with no specific formation, from 1.5 mm in diameter to 6-7 mm. The large intestine with its representative diameter has many folds, sometimes 12 but very small distinguishable only with a magnifying glass. There is no gender characteristic evidenced by the internal morphology. Structure (microscopical) study We should say from the beginning that histological material belongs to three individuals, a male and two females. On the studied slides there was no characteristic specific to a certain gender. We shall follow the structure of each segment of the postpharyngeal tube. Esophagus. We took fragments from two different parts of the esophagus: the one noted with A, from the proximal area, immediately after the pharynx and the one noted with B from the distal area, closer to the stomach. For the A fragment (Fig. 1) we may see some folds mucous formation, some longer and sharp and sometimes blunt with shorter or longer, thinner or larger side ramifications, like a little pine tree along the folds axe with its leaves up. There are also smaller and simpler folds. So the section lumen appears multi-star shaped. The mucous formation is represented by a stratified and cylindrical epithelium with 3-5 cell rows on the basal membrane. There are cylindrical, narrow cells, with an elongated, oval nucleus. To the structure s apical end, they become cylindrical cubical, the nucleus shortens but it does not become round. The last cell row to the lumen, presents short ciliated cells, and starting with the epithelium middle bigger and oval goblet cells become more and more numerous, opening in the lumen among the ciliated cells; for some areas they are placed next to each other; there are no blood vessels in the epithelium. The corium of the mucous formation (lamina propria) consists of conjunctive tissue with a bigger quantity of conjunctive fibers, also fibrocytes (fiber cells) to the basal membrane as well as elastic fibers. The passing to the submucous formation is gentle, the number of fibers and cells diminishing slowly. There is no muscularis mucose in the mucous formation. The sub mucous formation consists of conjunctive tissue, almost lax with some bigger vessels surrounded by elastic fibers. Here and there lymphoid infiltrations come from lamina propria. The corium contains ductal - alveolar glands, slightly swell at the inferior part and not with a typical alveolus; they have cubic or cylindrical - cubical cells with round nucleus usually at the basis, with slightly acidophilus

Sergiu Haimovici grains cytoplasm (named by the German authors oxiphil granulierente zellen ); the cells become polymeric or oval shaped with flattened nuclei and the cytoplasm turns white, their apical end is larger and without membrane, their acid secretion leaking out. The muscle formation is made of a circle layer of long muscle fibers; the longitudinal one looks like discontinuous formations made of long fibers too. We have also found two striated muscle fibers. The so-called B fragment (Fig. 2) has a structure similar to the A fragment. The folds are lower, distally widened and less prominent. The glands in the corium of the mucous formation are more numerous white colored, with an acid secretion. The muscle formation is circular, integer with no striated fibers. The longitudinal one is discontinuous. Stomach. For the amphibians the stomach has a wide part, let us call it fundus (noted with C) (Fig. 3) and another part, tighter, placed just before the middle intestine, let us call it pyloric (noted with D) (Fig. 4). Like all the vertebrates, the mucous epithelium is made of a single cell type; here and there the epithelium goes back inside creating holes. The C part (fundus) The mucous formation folds are small and large and the epithelium is simple. There are prismatic, high cells packed together. The oval, elongated nucleus is placed to the cell basis, strongly colorable and with many nucleoli. The cytoplasm colors well too; about a third of it near the apex contains a mucous type with positive Schiff reaction that makes it quite distinct from the rest of the cell. The cell height and the layer thickness diminish to the end of those holes. The mucus drains by a kind of dialysis and not by the apical end breaking; that is why some authors consider them as closed mucous cells. The mucous formation corium is completely covered with dense tubular glands (very closed to each other) so lamina propria is represented by conjunctive inter - granular walls. All these glands are placed on line. They have two cell types: the main cells with a relatively round nucleus and a relatively granulated and acid cytoplasm ( oxyphil gekornte zellen, in German) at the basis; on top to the apical part to the gland opening there is a layer made of some cells (the so called throat cells cellules a mucus du collet, in French), slightly colored with allogenous blue; some authors consider that they replace the continuous destroying special cells from the epithelium in the holes; the main cells produce a white acid mucus. Under these glands there is a fiber layer that splits the corium from the submucous formation as there is no muscularis mucose; there are some muscle cells here and there. The submucous formation is made of lax conjunctive tissue with some bigger vessels surrounded by elastic fibers. This is very narrow. The muscle membrane is a circular, thicker strip (about 1/3 of mucous and sub mucous membrane); the longitudinal strip is so thin that it seems almost discontinuous. We identified a MAST cell (metachromatic coloration with toluidine blue) in a fold axes. The D (pyloric) part is shorter and tighter making connection with the intestine. On cross section, a very thick circle muscle tissue is observed, its thickness being similar to the mucous and submucous formation altogether. The mucous formation folds are of two types: some simple, elongated and some elongated but ramificated ones; all these folds continue to the central part so there is no more lumen. The mucous formation has a simple epithelium but those typical cells have their apical part narrower, almost undistinguishable, and the cells become very elongated and with high and thin nuclei; the glands are shorter and rare, some of them with an almost acinous formation; the so-called main cells do not turn into acid mucous anymore. The corium is better represented inside folds, but at their basis is rather thin. There is no

Analele Ştiinţifice ale Universităţii Al. I. Cuza Iaşi, s. Biologie animală, Tom LIV, 2008 muscularis mucose in the mucous formation. The submucous made of conjunctive tissue is thin too. The thicker part is represented by circle muscles that by constriction push the fundus stomach content into intestine. In the muscle formation there is a thinner but well represented longitudinal muscle structure. Middle intestine.this represents the most elongated part of the digestive tube with the same aspect all along from the histological point of view. So, we describe thoroughly the first part, a little larger ansa noted with E (Fig. 5), some authors naming it the duodenum. The folds are relatively long and narrow, very rare branched, leaving in the middle a quite large lumen. The mucous formation has a simple epithelium but different from the one in the stomach; this is also typical for the vertebrates. They are called cells with striated plate or enterocytes. The cells from the basal membrane are high and cylindrical, of about 20-30 μ. The nucleus is oval and placed in the middle; in their apical area there is an obvious plate with a PNS positive. Among these enterocytes there are mucous producer goblet cells (rarely at the folds basis). The corium is well represented by a dense conjunctive tissue made of a connected anastomosed fibrocytes, with different leucocytes and collagen cells. In the corium, to the folds basis there are some big nuclei cells cell nests (Fig. 6) - each of them having three to five cells with big nuclei and full of chromatin splitting continuously; the nest is surrounded by thick conjunctive tissue with few elastic fibers. This is characteristic for amphibians, both urodels and anures (Andrew, 1959) even for tadpoles having the part of the epithelium cells substitution (Fig. 5); this is almost certitude as we discovered a cell turning into a goblet cell in a nest. Very rarely, to the folds basis there are some big cells of triangle shape with round nucleus and acidophili granulated cytoplasm maybe homologous to Paneth cells. It is no muscularis in the mucous formation yet. The submucous formation is very thin being made of lax conjunctive tissue, similar to the others that have been described so far. Although the muscle formation is very thin we still can distinguish the two formations, the longitudinal one being easily detectable. The second ansa (noted with F) (Fig. 7) has shorter and larger folds (maybe as a result of its fineness) but resembles to the E one. There is a parasite animal inside the lumen, a flat worm with a very thick cuticle for its protection. We could consider it a comensal as it does no harm to the salamander, as there are no lymphoid infiltrations in the corium. The immune system must have developed for the urodels. The last ansa is similar to the others. It seems the striated plate is a little bit thinner and narrower. Large intestine. The large intestine (noted with G) (Fig. 8) which is called terminal and due to its aspect we could also call it the thick intestine, has very thin walls. There are no prominent folds. The entorococytes are very low with an almost round nucleus and the striated plate is very thin or seems to disappear. The goblet cells are less than we could expect as for the mammals they are quite well represented. There is no clear distinction between lamina propria and the submucous membrane and there is no muscularis mucose. The muscle formation is very thin but the two stripes are distinguishable. The passing from the middle to the large intestine is very simple with no adjacent formation; the same thing is available for the cloacum too.

Sergiu Haimovici Figure 1. Esophagus, proximal area. Figure 2. Esophagus, distal area. Figure 3. Stomach, fundus area. Figure 4. Stomach, pyloric area. Figure 5. Middle intestine, general structure. Figure 6. Middle intestine, cell nests. Figure 7. Middle intestine, parasite inside lumen. - 264 - Figure 8. Large intestine, general structure.

Analele Ştiinţifice ale Universităţii Al. I. Cuza Iaşi, s. Biologie animală, Tom LIV, 2008 Table 1. Measurements (in mm) for Salamandra salamandra salamandra. Dimensions and indices 1 2 3 4 Full length (1) 170 185 160 158 115 155 170 160 175 145 165 Body length (2) 115 95 105 90 60 20 90 90 95 80 100 Digestive tube length (3) 370 305 360 220 155 250 355 315 325 240 280 Esophagus length (4) 10 10 20 15 6 10 15 16 18 12 20 Stomach length (5) 35 30 45 25 25 22 35 38 40 24 30 Middle intestine length (6) 290 241 270 160 118 200 277 241 237 186 212 Large intestine length (7) 35 24 25 20 12 18 28 20 30 18 18 (4x100)/3 Index 2.73 3.27 5.55 6.81 3.87 4.00 4.22 5.07 5.53 5.00 7.14 (5x100)/3 Index 9.45 9.85 12.50 11.36 16.12 8.80 9.85 12.06 12.30 10.00 10.71 (6x100)/3 Index 78.87 72.62 75.00 72.72 75.80 80.00 78.02 76.50 74.48 77.50 75.71 (7x100)/3 Index 9.45 7.86 6.99 9.09 7.74 7.20 7.88 6.39 9.23 7.50 6.42 5 6 7 8 9 10 11 12 Table 1 (sequel). Measurements (in mm) for Salamandra salamandra salamandra. 13 14 15 16 17 Variation Variation General Variation Average Average General Average 165 175 185 165 178 168 115-175 160-185 115-185 158.1 172.28 165.19 100 100 105 95 100 93 60-100 90-115 60-115 89.3 101.42 95.35 310 290 306 216 272 205 155-325 272-370 155-370 252.6 321.14 288.87 15 12 20 12 18 10 6-18 10-20 6-20 12.6 16.14 14.37 39 30 44 26 28 23 22-40 28-45 22-45 29.2 35.28 32.24 231 233 212 160 206 257 118-241 206-290 118-290 192.3 244.00 218.15 25 15 30 18 20 15 12-30 18-30 12-30 20.1 25.71 22.90 4.83 4.13 6.53 5.55 6.61 4.87 3.87-6.81 2.73-7.14 2.73-7.14 4.96 5.15 5.05 12.58 10.34 13.07 12.03 10.29 11.21 8.80-16.12 9.45-13.07 8.80-16.12 11.68 10.81 11.24 74.51 80.34 69.28 74.07 75.59 76.58 72.62-80.34 69.28-78.37 69.28-80.34 76.25 74.65 75.45 8.06 5.17 3.92 8.33 7.35 7.31 5.17-9.23 3.92-9.45 3.92-9.45 8.60 7.12 7.86 Conclusions We may say that the females are a little bit longer than the males and their average digestive tube length is bigger too. The segments of the Salamander postpharyngeal digestive tube are well differentiated: esophagus, stomach, middle and large intestine. For all the levels the mucous, submucous and the muscle formation are relatively well represented but on the contrary to the superior vertebrates the muscularis mucouse is missing. Although for the esophagus the proximal and distal part are well outlined and for the stomach the fundic and piloric areas are different both by structure and aspect, for the middle intestine the ansa structure is unchanged. The internal folds are prominent except for the large intestine and the sphincters and valves between the digestive tube fragments are still missing. There are still ciliated cells in the esophagus, a primitive character. The fragments of the digestive tube have generally the same characteristics as for the other vertebrates, but for the salamander, as well as for all the amphibians, there are some cells placed in nest cells formations in the mucous corium of the middle intestine, cells meant to substitute the glands. Similar to superior vertebrates Mast cells have been

Sergiu Haimovici identified here and there and in the middle intestine the so called Paneth cells have been observed. By comparison with the superior vertebrates the defense activity of the white cells is rather low. Almost all cell secretions have an acid character. Translated by, Monica Popa References Andrew, W., 1959. Textbook of Comparative Histology, The Alimentary Tract of Vertebrates, Amphibiens, New York, Oxford University. Fuhn, I., 1960. Fauna Republicii Populare Române. Amfibieni, XIV (1), Editura Academiei Republicii Populare Române. Pernkopf, E., Lehner, J., 1937. E. Pernkopf und J. Lehner in Hanbuch der Vergleihenden Anatomie der Wibeltiere, III Banch, Darmsystem, 4 Amphibien, Urban et Scgwarzenberg Berlin und Wien.