Morphology and histology of P . argentinus ( Crustacea , Decapoda , Caridea ) digestive tract

This work describes the morphology and histology of the P. argentinus digestive tract. The foregut comprises the mouth, oesophagus, and stomach and is lined by a simple cylindrical epithelium overlain by cuticle. There are tegumental glands in the oral region and in the first portion of the oesophagus and of the hindgut. The cardiac stomach is an oval dorsal sac in the cephalothorax and has no calcified structures. The pyloric stomach comprises an upper chamber and a lower gland filter. The filter consists of an outer row of elongated setae and an inner row of dorsally curved setae forming longitudinal channels 16-18μm wide. The midgut runs from the dorsal chamber of the pyloric stomach to the sixth abdominal somite without caeca. The hindgut runs from the sixth abdominal somite to the ventral anus. The mid-gut epithelium comprises dominant cylindrical cells and small undifferentiated cells in the first portion. The hindgut wall presents longitudinal folds, conspicuous muscular bundles, and a folded cuticle. The digestive tract of P. argentinus is basically similar to that of most of decapods. The absence of calcified structures in the stomach and the width of the longitudinal channels in the filter are related to the predominantly detritivorous diet. BIOCELL 2006, 30(2): 287-294 ISSN 0327 9545 PRINTED IN ARGENTINA


Introduction
There is a large body of information about decapods digestive tract particularly concerning economically important species such as palinurids (Cox and Bruce, 2003;Cox andJohnston, 2003, 2004), thalassinids (Pinn et al., 1998) and scyllarids (Johnston and Alexander, 1999).The gut of decapods is essentially a tube opening anteriorly at the mouthparts and posteriorly at the anus; the foregut and hindgut are ectodermally derived and are lined by cuticle, and the midgut with a glandular epithelium is derived from endoderm (Icely and Nott, 1992).Caine (1975) investigated the alimentary behaviour of Procambarus species, concluding that there are two basic methods to obtain food: the detritivorous mechanism and the manipulation of big pieces of food.In crustacean, the mouth parts and the gastric mill show different adaptations in relation to the diet (Lin, 1996).Macrophagous decapods have a well developed and calcified gastric mill, while detritivorous evidence a reduced structure (Icely and Nott, 1992).In spite of this, there is evidence that the anatomy of the foregut may be modified in closely related species with similar feeding habits (Icely and Jones, 1978).Morphology and histological structure of the digestive tract can be also altered by external factors such as environmental contamination (Meyers and Hendricks, 1985;Vogt, 1987).
Palaemonetes argentinus is one of the most widely distributed decapods in the littoral region of Argentina, Paraguay, Uruguay and southern Brazil (Morrone and Lopreto, 1995).This prawn plays an important trophic role in the littoral communities and inhabits from freshwater to brackishwater lakes and streams (Spivak, 1997).Some of the waterbodies in which this prawn can be found receive direct discharges of chemicals from terrestrial ecosystems and the individuals accumulate important amounts of organochlorine pesticides in their tissues (Gonzalez Sagrario et al., 1998;Miglioranza et al., 2002).The hepatopancreas of P. argentinus was previously studied (Sousa and Petriella, 2000;Sousa et al., 2005) and the organ undergoes histological and histochemical modifications in response to different physiological demands (moult, reproduction) (Sousa and Petriella, 2001) and environmental changes such as salinity and pollution (Sousa, 2003).
This work aims to describe the morphology and histology of the P. argentinus digestive tract, as part of a project which focuses to detect possible histological alterations in different organs of this species, such as gills, hepatopancreas and the rest of the digestive tract as a consequence of pesticides action.

Materials and Methods
Adult individuals of both sexes, at sexual rest, of initial weight between 0.100 and 0.200g were collected from Sotelo stream, tributary of Mar Chiquita lagoon (Argentina, 38°S 55°W).This stream is a sallow creek where pesticide concentrations are below toxic levels and are not related to adverse biological effects (Menone, 1999;Miglioranza et al., 2002).
For macroscopic observation of the digestive tract, some individuals fixed in formalin 5% were observed under a stereoscopic microscope.
For histological description, the cephalothorax and abdomen of 24 individuals in intermoult were fixed for 24 h in Davidson fluid (ethanol, formol, acetic acid and water) (Bell and Lightner, 1988), dehydrated in increasing concentration of ethanol, butyl alcohol (two changes of 24 h), butyl-paraffin 50:50 (for 24 h) and finally    embedded in paraffin.Sections (3μm) were stained with haematoxylin-eosin, Mallory's triple stain, PAS and Toluidine Blue.
The moult stage was determined by microscopic examination of the setae of the uropods exopodite, following the criteria established by Díaz et al. (1998).

Morphology and anatomical relations
The foregut comprises the mouth, oesophagus, and stomach.The mouth is situated on the ventral side of the cephalic region, bounding laterally by robust mandibles.The oesophagus is a short vertical structure which connects the mouth with the stomach (Fig. 1).
The cardiac stomach is dorsal in the cephalothorax and looks like an oval sac; it leads into the pyloric stomach, which is situated in a ventro-posterior position in relation to the cardiac stomach.The pyloric stomach is reduced in size, elliptically shaped and comprises two chambers: an upper chamber leading into the midgut, and a lower chamber or gland filter.Externally, the filter looks like two lateral pouches with longitudinal grooves, opening into the hepatopancreatic ducts.The hepatopancreas occupies much of the cephalothoracic cavity and is twice as big as the cardiac stomach.The hepatopancreatic lobes are dorsally located and surround completely the midgut.
The midgut runs from the dorsal chamber of the pyloric stomach to the sixth abdominal somite, curving downwards between the hepatopancreatic lobes; and in the abdomen it curves upwards adopting a dorsal position.The midgut has neither anterior nor posterior caeca, and is the longest part of P. argentinus' digestive tract.
The hindgut runs from the sixth abdominal somite to the ventral anus and has no caeca (Fig. 1).

Histology
The oral region (Fig. 2) is lined by a simple cylindrical epithelium overlain by cuticle.The epithelial cell nuclei are at different heights giving a pseudo-stratified aspect.In the labrum, a dense connective tissue can be observed with Mallory´s triple stain, with abundant connective cells, and longitudinal striated muscular fibres.In the paragnatha, the connective tissue is spongy.Tegumental glands are scattered throughout the oral region and their cells stains metacromatically with Toluidine Blue.
The oesophagus comprises a simple cylindrical epithelium overlain by cuticle, both the epithelium and the cuticle share similar characteristics to those of the oral region.The connective tissue is scarce and the striated muscle is well developed.The oesophagus wall presents deep infoldings along its length (Fig. 3) and tegumental glands in the first portion, which are similar to those observed in the oral region.
The cardiac stomach is lined by a simple cylindrical epithelium, whose height varies in the different zones; nuclei are at different heights in the cells (at medial and basal zones).The epithelium is underlain by a thin layer of dense connective tissue and surrounded by circular and longitudinal striated fibres.A serrated cuticle lays on the epithelium.The stomach wall forms many small folds projecting into the lumen, whose number and size vary according to distension degree (Figs. 3, 4).In some zones where the folds are bigger, the epithelium shows a stratified aspect and the connective tissue is well developed with big haemolymphatic lagoons.The cardiac stomach is separated from the pyloric one by the cardiopyloric valve, which is a fold of the ventroposterior wall of the cardiac stomach extending dorsally and leaving a narrow canal towards the pyloric stomach (Fig. 3).
The pyloric stomach (Fig. 3) is small comparing with the cardiac one.The wall histology is similar to that of the cardiac stomach, but the striated muscle is better developed (Fig. 5).In the upper chamber the epithelium shows numerous deep folds that reduce the lumen (Figs. 5, 6)).In the lower chamber the cuticle forms the double structure of the filter (Fig. 5).The filter consists of an outer row of elongated setae and an inner row comprising dorsally curved setae where each seta overlaps the next (Figs.5, 7).The inner row of filtration setae form longitudinal channels of 16-18μm wide (Fig. 7).In the filter zone the epithelium is underlined by scarce connective tissue.
The midgut in P. argentinus has a simple epithelium which is cubical or cylindrical in relation to the content volume.One cellular type is dominant and extends from the basal lamina to the lumen (Fig. 8).These cells have central nuclei, small subapical vacuoles and a well developed brush border; some of them have a basophilic cytoplasm and are secreting actively (Fig. 9).In the first portion, small undifferentiated cells can be observed on the basal lamina (Fig. 8).Mucopolysaccharides are evidenced by Toluidine Blue stain in the apical region of the cells, in the brush border and in the lumen.The basal lamina is well developed and is strongly PAS positive, while the brush border was   The hindgut is lined by a simple cylindrical epithelium with central nuclei; the thin basal lamina is underlain by spongy connective tissue.The hindgut wall presents deep longitudinal folds and conspicuous bundles of striated longitudinal and circular muscle; the cuticle is thicker than in the midgut and presents small folds.In the anterior portion, there are numerous tegumental glands that share similar characteristics with those of the oral region and of the oesophagus.The posterior portion shows a great development of striated circular muscle towards the anus.

Discussion
The main features of P. argentinus digestive tract are coincident with the general plan of decapods; however, it presents certain particular characteristics.The oesophagus is a simple muscular tube which drives the food into the cardiac stomach.The wall folds permit to change the distension degree in relation to the size and amount of ingested food.Contrasting with that observed in big Decapoda (Barker and Gibson, 1978;Factor, 1995) and in Mysidacea (De Jong and Casanova, 1997), no oesofagic valve was observed.This fact suggests that non ingested food could be regurgitated.The absence of this valve was also mentioned for some braquiurans such as Menippe rumphii (Erri Babu et al., 1982).
The cardiac stomach of P. argentinus is a simple sac without calcified structures in the interior, which is quite common in caridean prawns (Boschi and Angelescu, 1962;Icely and Nott, 1992).Yet, the cardiac chamber of the caridean prawn Macrobrachium borelli has calcified elements in the anterior wall (Boschi, 1981).The reduction of the gastric mill in P. argentinus is compensated by the presence of complex and quitinized mandibles, with enough rigidity to reduce in size big pieces of food (Boschi, 1981).As other palaemonids, this species triturates the food with its powerful mandibles before entering the mouth parts and being mixed with the mucus (Collins, 1999).Coincidentally, Pathwardan (1935) suggested that the gastric mill efficiency is inversely correlated to the mandibles efficiency.The gastric mill reduction and the wall folds make P. argentinus cardiac stomach to be an extensible sac, which would permit food storage.Muscular development in the stomach wall suggests that, as in other species, contractions of the wall contribute to mix food with digestive enzymes from the hepatopancreas and make possible the mechanical action of the serrated cuticle.
P. argentinus pyloric stomach shares similar general characteristics with other decapods (Icely and Nott, 1992).The double structure of the filter ensures that only the smallest particles pass into the hepatopancreas, increasing the filter efficiency.The outer filter setae press the finest particles towards the inner filter and also eliminate the big particles towards the midgut (Kunze and Anderson, 1979;Lin, 2000).The width of the longitudinal channels is approximately 16-18μm in P.argentinus and this width is within the range found in species feeding on fine particles.In coincidence, Lin (2000) indicated that the width of longitudinal channels in Penaeus monodon and Metapenaeus ensis is 16-18μm at different body lengths.However, in macrophagus species the longitudinal channels width is about 30μm (Schaefer, 1970).In coincidence, Caine (1976) observed that the space between setae has a direct correlation with diet, with spacing increasing with increased macrophagy.
In the midgut, the final phases of the digestive cycle take place: digestion of products coming from the cardiac stomach, absorption and processing of digestive products, and removal of residual wastes that will form the faeces (Icely and Nott, 1992).The midgut epithelium is considered an active epithelium that regulates water flux and ion transport (Icely and Nott, 1992).In the present study, the midgut epithelium comprises mainly one cell type, and little basal cells appear scattered between the others.These characteristics are coincident with that described for other decapods (Pillai, 1960;Icely and Nott, 1992).The predominant cells were observed at different phases of secretory activity.Some authors suggests that the basal cells function as replacement elements; however, Mycles (1979) found that cell division was confined to the distal end of the gut caeca.On the other hand, the origin of replacement cells is unknown in species lacking midgut caeca (Icely and Nott, 1992), such as P. argentinus; in this study the basal cells were not observed in mitosis, for this fact they can not be affirmed as replacement elements.
The passage of solid materials into the midgut produces a volume increment that is accommodated by the epithelium and basal lamina folds.The mucopolysaccharides detected on the epithelium and lumen may lubricate the indigestible food bolus as it passes towards the hindgut.In agreement with this, the presence of these macromolecules has been mentioned for several decapod species, and they seem not to be associated to digestive processes (Lovett and Felder, 1990;Johnston and Alexander, 1999).P. argentinus hindgut shares similar histological features with the rest of decapods, and is involved in defecation which is facilitated by contractions of the wall muscles (Barker and Gibson, 1978;Icely and Nott, 1992;Factor, 1995;Johnston and Alexander, 1999).The wall folds permit to resist great volume changes; the thick cuticle and the secretion of the tegumental glands that lubricate the surface protect the epithelium from abrasion.In some decapods, the hindgut epithelium is lined by spines projected posteriorly to prevent back flow of material (Hopkin and Nott, 1980;Johnston and Alexander, 1999).In contrast, these spines were not observed in P. argentinus.As in other species, the well developed circular muscle around the anus of P. argentinus is probably involved in constricting the lumen to force the faeces through the anus to the exterior (Johnston and Alexander, 1999).
The general features of P. argentinus digestive tract are similar to those of most of decapods.As a special feature, the lack of a well structured gastric mill can be mentioned, which as in some other carideans would be reduced to numerous wall folds and a serrated cuticle.On the other hand, many Caridea and all the Penaeoidea have complex gastric mills and mandibles (Felgenhauer and Abele, 1985).Some studies showed that the digestive tract morphology depends primarily on the species phylogeny; however, other factors such as dietary preferences can modify its anatomy (Suh, 1990;Icely and Nott, 1992;De Jong, 1996).Collins (1999) studied the natural feeding of P. argentinus determining that it is an omnivorous / detritivorous species with the predominant presence of phytoplankton and benthic microinvertebrates in the diet.Consequently, the reduction of the gastric mill and the width of the longitudinal channels in the inner valve of the pyloric filter are related to the predominantly detritivorous diet.
Several authors described important pathological changes in the hepatopancreas, gills, midgut, oesophagus, stomach and hindgut of different decapod crustaceans exposed to pesticides and heavy metals (Nimmo and Blackman, 1972;Doughtie and Rao, 1984;Meyers and Hendricks, 1985).Further work, particularly, chemical and histological studies on the digestive tract of P. argentinus will be carried out to detect the effect of pesticides on the functional cytology of the different organs.