Foregut morphology of Pseudothelphusidae and Trichodactylidae ( Decapoda : Brachyura ) from northeastern Pará , Brazil

The gross morphology of foregut was investigated and described in detail for four freshwater crab species: a pseudothelphusid, Fredius reflexifrons (Ortmann, 1897) and three trichodactylids - Sylviocarcinus pictus (H. Milne-Edwards, 1853), Valdivia serrata White, 1847, and Dilocarcinus septemdentatus (Herbst, 1783). In general, the gastric mills of freshwater crabs showed a similar degree of complexity in relation to other marine species of Anomura and Brachyura previously described in the literature. However, significant morphological differences were observed among the analyzed species, as in ossicle numbers, features and connection types. A total of 38 ossicles were described for S. pictus and V. serrata and 37 ossicles for D. septemdentatus and F. reflexifrons, respectively. The latter species can be distinguished from the others by the presence of a wide cardiac sac resembling two sacs. Most of the ossicles were connected through a rigid connection, and few ossicles were fused. The degree of calcification varied little among the studied species. The differences found among the species suggest that the morphological aspects of the foregut may be useful in the identification of similar species.

The foreguts were obtained from 10 individuals (male and females) of each species.The stomach of each individual was fixed in aqueous formalin solution (10%) for 24 hours, immersed in hydroxide solution of potassium (10%) and heated to 100ºC for one hour.They were then removed and the skeletal structure of the stomach was washed in distilled water and immersed in an ethyl alcohol 70% + glycerol (1:1) solution.Later, the foreguts were removed and transferred to another slide where they were stained with aqueous solution of Alizarinred (0.1%) according to the methodology used by BRÖSING et. al. (2002) and adapted by PILGRIM (1964PILGRIM ( , 1965PILGRIM ( , 1973)).
The stomach of each species was illustrated in lateral (right side), dorsal and ventral view under an optical microscope (Zeiss).The description of ossicles that compose the stomach (Tab.I), nomenclature and abbreviations followed MEISS & NORMAN (1977), MARTIN et al. (1998) andBRÖSING et al. (2002).
Voucher specimens are deposited in the collection of crustaceans of the Institute National of Researches of the Amazonian -INPA (Manaus, Amazonas, Brazil)

Foregut ossicles of Fredius reflexifrons
The oesophagus is connected with antero-ventral portion of the cardiac chamber; cardiac chamber wider than long with 17 ossicles; pyloric chamber with 16 ossicles, little shorter than the cardiac chamber, terminal portion slightly straight; cardiac sac quite wide as two lateral bag.
Lateral supporting cardiac ossicles (Fig. 1) Pectineal ossicle (VIII): paired, bearing three to four elongate teeth forming a small comb (accessory tooth) slightly calcified and flexible, outer margin with a pronounced cavity and a fragment well calcified connected with prepectineal (IX) and postpectineal (X) ossicles respectively.
Postpectineal ossicle (X): paired, firmly calcified, thin superior extremity curved towards the centre of the foregut, connection with the base of pectineal ossicle (VIII) base, inferior extremity entirely fused to "Quill"(posterior ending of ossicle) of the postpectineal ossicle (Xa), showing a "J"-shaped projection, firm connection of the lateral margin with inferior lateral cardiac ossicle (XIII), with posterior lateral cardiac plate (XII) and with anterior ossicle of the cardio-pyloric valve ossicles (XVI).
Anterior lateral cardiac plate (XI): paired, slightly calcified, irregular shaped, thinned towards lateral margins of the foregut, firm connection with the narrowest portion of posterior lateral cardiac plate (XII).
Subdentate ossicle (XIV): paired, firmly calcified, and showing a slight lateral curvature, superior extremity curved on the cavity of the zygocardiac ossicle (V), posterior extremity, slightly flattened and larger than anterior, showing firm connection with the inferior lateral cardiac ossicle (XIII).

Ossicles of cardio-pyloric valve (Figs 1, 3)
Ossicles of anterior cardio-pyloric valve (XVI): unpaired, slightly calcified, showing as a fine plate gutter-shaped with a central line firmly calcified dividing the plate in the middle and laterally connected with postpectineal (X) and inferior lateral cardiac (XIII) ossicles.
Posterior ossicle of the cardio-pyloric valve (XVII): unpaired, firmly calcified and subtriangular with a sharp carina exposed horizontally in the surface, connected to the preampullary ossicle (XXII) and cardio-pyloric valve (cpv), firm connection with the ossicles of the lateral ossicle of the cardiopyloric valve (XVIII) and with anterior inferior pyloric ossicle (XXIII).Lateral ossicle of the cardio-pyloric valve (XVIII): paired, firmly calcified, with irregular surface and a distinct lateral curvature, making protection of the cardio-pyloric valve (cpv), firmly connected through to posterior ossicle of the cardiopyloric valve (XVII), lateral cardio-pyloric (XV) and inferior lateral cardiac (XIII) ossicles, connection also with the anterior supra-ampullary ossicle (XXVIII).
Cardio-pyloric valve (cpv): moderately calcified with two prominent protuberances enclosed for long numerous simple setae (not illustrated).
Uropyloric ossicle (XXI): unpaired, moderately calcified on the anterior portion; in dorsal view of foregut, shown as an arched piece that opens, side to side, the pyloric chamber, connection in the anterior portion with posterior mesopyloric ossicle (XX).
Inferior ampullary ossicle (XXIV): par, moderately calcified, showing as a paired of fine convex plates, covered externally by discreet transverse lines in which, making outer protection of the ampullary filter (filter press), connected with preampullary (XXII) and anterior inferior pyloric (XXIII) ossicles, firm connection with the posterior inferior pyloric ossicle (XXVII).
Ampullary roof ossicle, upper portion (XXVI): paired, firmly calcified shown as a concave plate in the central region, entirely fused in the terminal portion of the pyloric chamber with the posterior pleuropyloric ossicle (XXXIII) and, with ampullary roof ossicle, lower portion (XXV), firm connection with the anterior pleuropyloric ossicle (XXXII).
Posterior supra-ampullary ossicle (XXX): paired, firmly calcified, plate-shaped, partially fused to the centre, also partially fused to the terminal portion of the posterior inferior pyloric ossicle (XXVII) and firm connection with posterior pleuropyloric ossicle (XXXIII).
Ossicles of the cardio-pyloric valve Anterior ossicle of the cardio-pyloric valve X X X X Posterior ossicle of the cardio-pyloric valve X X X X Lateral ossicle of the cardio-pyloric valve X X X X Supporting ossicles of the dorsal pyloric chamber   Posterior pleuropyloric ossicle (XXXIII): paired, firmly calcified, robust, irregular, short and slightly curved, partially fused to ampullary roof ossicle, lower portion ossicle (XXV) and ampullary roof ossicle, upper portion (XXVI), firm connection with posterior supra-ampullary ossicle (XXX).

Foregut ossicles of Sylviocarcinus pictus
Oesophagus short connected with ventral portion of the cardiac chamber; cardiac chamber wider and little longer than pyloric chamber bearing 17 ossicle; pyloric chamber with 17 ossicles, little shorter than cardiac chamber with the terminal portion slightly curved upward in the apex; cardiac sac small and heart shaped.

Ossicles of the gastric mill (Figs 4, 5)
Mesocardiac ossicle (I): unpaired, firmly calcified, accentuated re-entrance in the base completely fused with urocardiac ossicle (VII), anterior portion free, with soft re-entrance, lateral margin wide with firm connection in the inner lateral margin of the pterocardiac ossicle (II).
Zygocardiac ossicle (V): paired, firmly calcified, dorsoventrally curved, left anterolateral portion connected with postpterocardiac ossicle (IIb), firm connection with exopyloric ossicle (IV), medial portion large forming a plate with depressions in the surface bearing from seven to eight small short denticles constituting the lateral teeth, ventral portion with deep cavity supporting part of the subdentate ossicle (XIV) and showing firm connection.Propyloric ossicle (VI): unpaired, firmly calcified, log glass-shaped, inferior base smaller than superior and firmly linked to urocardiac ossicle (VII) in the inner portion of foregut, superior base connected through firm connection with the pyloric (III) and exopyloric (IV) ossicles.
Urocardiac ossicle (VII): unpaired, firmly calcified, long, wide in the superior region and little narrower in the inferior region, "T"-shaped, inferior region with deep cavity forming two fine folds in the lateral side, fused to the mesocardiac ossicle (I) in the superior region and linked to propyloric ossicle (VI), inner margin with three to four protuberant teeth in screw shaped.
Lateral supporting cardiac lateral ossicles (Fig. 4) Pectineal ossicle (VIII): paired, moderately calcified, short, fine, curved to the centre, bearing three to four fine and sharp teeth forming a small comb (accessory tooth), firm connection in the left extremity with prepectineal ossicle (IX), right extremity with postpectineal ossicle (X) and posterior lateral cardiac plate (XII).
Prepectineal ossicle (IX): paired, firmly calcified, and curved stick-shaped in the inferior extremity, superior portion with connection to postpterocardiac (IIb) and in the inferior portion bearing firm connection with pectineal (VIII) ossicles, respectively.Postpectineal ossicle (X): paired, firmly calcified, superior extremity thin and curved towards the centre of the foregut with firm connection to the base of the pectineal ossicle (VIII), inferior extremity completely fused "Quill" (posterior ending) of ossicle of postpectineal (Xa) forming a projection "J"-shaped, firm connection to the inferior lateral cardiac ossicle (XIII) and posterior lateral cardiac plate (XII).
Posterior lateral cardiac plate (XII): paired, irregularly shaped, enlarged, moderately calcified in the borders, slightly calcified and concave in the centre, with firm connection to postpectineal ossicle (X) and partially fused to anterior lateral cardiac plate (XI).
Subdentate ossicle (XIV): paired, firmly calcified, and showing light lateral curvature, superior extremity curved on the cavity of the zygocardiac ossicle (V), inferior extremity slightly flat and more enlarged than superior, firm connection with the inferior lateral cardiac ossicle (XIII) in its base.
Posterior ossicle of the cardio-pyloric valve (XVII): unpaired, firmly calcified, semi-oval, with carina in the surface, connection with ossicle of lateral cardio-pyloric valve (XVIII),  Lateral ossicle of the cardio-pyloric valve (XVIII): paired, firmly calcified, irregular shaped, similar to a curved plate towards the centre of the foregut, with protection of the cardiopyloric valve (cpv), connection with posterior ossicle of the cardio-pyloric valve (XVII).
Cardio-pyloric valve: moderately calcified, showing two lobes covered by numerous and long simple setae (not illustrated).

Foregut ossicles of Valdivia serrata
Oesophagus long and slightly bent forward, connecting with the ventral region of the foregut; the cardiac chamber showing wider than long bearing 17 ossicles; pyloric chamber with 17 ossicles, little shorter than cardiac with terminal portion bent downward 90°.
Zygocardiac ossicle (V): paired, strongly calcified, ventrally curved backward, with pronounced medial depression, anterolateral margin narrow, connection with postpterocardiac ossicle (IIb), margin turned to the medial portion of the widest chamber with nine small flat teeth (lateral teeth), ventral portion with deep cavity sheltering part of the subdentate ossicle (XIV).
Lateral supporting cardiac ossicles (Fig. 8) Pectineal ossicle (VIII): paired, moderately calcified, short, robust and curved to the centre, lacking teeth but with numerous long and simple setae, outer margin with discreet cavity, connected in the left extremity through firm connection with the prepectineal ossicle (IX) and in the right extremity with the postpectineal ossicle (X), with firm connection also with posterior lateral cardiac plate (XII).
Postpectineal ossicle (X): paired, firmly calcified, long and fine, with thin and curved extremity and towards to the centre of the cardiac chamber, firm connection to the base of the pectineal ossicle (VIII), inferior extremity whole fused to the "Quill" (posterior ending of ossicle) of the postpectineal ossicle (Xa), showing a projection "J"-shaped, firm connection with inferior lateral cardiac ossicle (XIII) and with posterior lateral cardiac plate (XII).
Anterior lateral cardiac plate (XI): paired, slightly calcified, semi-rectangle-shaped, with accentuated carina in the base, firm connection with posterior lateral cardiac plate (XII), firm connection with posterior lateral cardiac plate (XII).
Posterior lateral cardiac plate (XII): paired, irregular shaped, moderately calcified in the borders and slightly calcified in the centre, enlarged and concave in the portion in which connect to postpectineal ossicle (X) in the medial area, connection with anterior lateral cardiac plate (XI) with irregular surface.
Subdentate ossicle (XIV): paired, firmly calcified, stickshaped with light lateral curvature, superior extremity curved on the cavity of the zygocardiac ossicle (V), posterior extremity more enlarged than anterior with firm connection to inferior lateral cardiac ossicle (XIII).
Lateral cardio-pyloric ossicle (XV): paired, moderately calcified and subtriangular, firm connection with inferior lateral cardiac ossicle (XIII) and lateral ossicle of the cardio-pyloric valve (XVIII), connection with the superior portion of the anterior supra-ampullary in the superior portion (XXVIII).
Ossicles of the cardio-pyloric valve (Fig. 9) Anterior ossicle of the cardio-pyloric valve (XVI): unpaired, irregular shaped, slightly calcified in the lateral margins forming a fine plate, gutter-shaped and moderately calcified to the centre forming a medium line, connected sidelong through firm connection with postpectineal (X) and inferior lateral cardiac (XIII) ossicles.
Lateral cardio-pyloric valve (XVIII): paired, firmly calcified, irregular shaped, similar to a curved plate towards the centre of the cardiac chamber, links through connection with the posterior ossicle of the cardio-pyloric valve (XVII).
Supporting ossicles of the lateral pylorus (Figs 7 and 8) Anterior pleuropyloric ossicle (XXXI): paired; firmly calcified, shown as elongate plates, irregular shaped and with a central carina along the plate, showing a triangular expansion, slightly calcified from the medial portion, firm connection with posterior mesopyloric (XX), anterior supra-ampullary ossicles (XXVIII) and partially fused to ampullary roof ossicle, upper portion (XXVI).

Foregut ossicles of Dilocarcinus septemdentatus
The oesophagus connecting with antero-ventral portion of the cardiac chamber slightly bent to anterior area; cardiac chamber showing wider than long with 17 ossicles; pyloric chamber little shorter than cardiac, terminal portion bent downward bearing 16 ossicles and cardio-pyloric valve with four ossicles.
Pterocardiac ossicle (II): paired, strongly calcified, showing an enlarged plate in the portion linking to mesocardiac ossicle (I) and narrower in the portion connected with postpterocardiac ossicle (IIb).
Zygocardiac ossicle (V): paired, firmly calcified and dorsoventrally curved downward, antero-lateral portion narrow with connection to postpterocardiac (IIb) and exopyloric (IV) ossicles, medial portion quite enlarged with nine small flat teeth forming the lateral teeth, ventral portion with deep cavity supporting part of the subdentate ossicle (XIV).
Propyloric ossicle (VI): unpaired, strongly calcified, log glass-shaped with superior region more larger than inferior, showing a inner central cavity located in the largest region, connection with pyloric (III), exopyloric ossicle (IV) and urocardiac ossicle (VII) in the inner base.
Lateral supporting cardiac ossicles (Fig. 10) Pectineal ossicle (VIII): paired, moderately calcified, short, curved in the centre, irregular shaped, bearing three to four fine and sharp teeth forming the accessory teeth, small comb slightly calcified and flexible, firm connection with prepectineal (IX) and postpectineal (X) ossicles and posterior lateral cardiac plate (XII) ossicles.
Postpectineal ossicle (X): paired, showing strong calcification, long stick-shaped enlarged in the base, superior extremity thin and curved in the cardiac chamber centre, connection with the base of pectineal ossicle (VIII), the inferior extremity entirely fused to "Quill" (posterior ending of ossicle) of postpectineal ossicle (Xa) forming a projection "J"-shaped, connection with inferior lateral cardiac ossicle (XIII) and posterior lateral cardiac plate (XIII).Anterior lateral cardiac plate (XI): paired, irregular and slightly calcified, connection with posterior lateral cardiac plate (XII).
Posterior lateral cardiac plate (XII): paired, moderately calcified in the borders and slightly calcified in the centre, showing as an irregular plate, large and concave in the centre, strong connection with pectineal (VIII) and postpectineal (X) ossicles and with the anterior lateral cardiac plate (XI).Inferior lateral cardiac ossicle (XIII): paired, strongly calcified, robust, large with rounded margins and acute in the inferior portion, firm connection with subdentate ossicle (XIV), anterior ossicle of the cardio-pyloric valve (XVI), lateral cardio-pyloric (XV) and postpectineal (X) ossicles.
Ossicles of the cardio-pyloric valve (Figs 10,12) Anterior ossicle of the cardio-pyloric valve (XVI): unpaired, moderately calcified, showing as a fine plate, guttershaped, with firmly calcified line along the plate dividing in the middle, connection with postpectineal (X) and inferior lateral cardiac (XIII) ossicles.
Lateral ossicle of the cardio-pyloric valve (XVIII): paired, firmly calcified, irregular, showing a small carina in the surface, and distinct lateral curvature with protection of the cardiopyloric valve (cpv), connection with posterior ossicle of the cardio-pyloric valve (XVII).
Cardio-pyloric valve (cpv): moderately calcified, showing as a globose cavity covered by long and numerous simple setae (not illustrated).
Uropyloric ossicle (XXI): unpaired, firmly calcified, showing a wide arch in the middle and narrow in the extremities, connection with mesopyloric ossicle (XX).
Inferior ampullary ossicle (XXIV): paired, moderately calcified, showing as a paired of fine convex plates, outer covered by fine transverse lines in which make protection of the filter press, connection with preampullary (XXII), anterior inferior pyloric (XXIII) and posterior inferior pyloric (XXVII) ossicles.
Posterior inferior pyloric ossicle (XXVII): unpaired, strongly calcified, irregular and concave, in the ventral view showing as a short stick and with extremities slightly curved upward, connection with anterior inferior pyloric (XXIII), posterior supraampullary (XXX) and inferior ampullary (XXIV) ossicles.
Posterior supra-ampullary ossicle (XXX): paired, strongly calcified, showing as plates with anterior portion fine and narrow in the central direction, connection with the terminal portion of posterior inferior pyloric (XXVII) and posterior pleuropyloric (XXXIII) ossicles.

DISCUSSION
The gross morphology of the gastric mill of the freshwater crabs analyzed in this study is quite complex.Its structural organization is well-developed and features the specialized brachyuran food trituration mechanism.According to DALL & MORIARTY (1983), this complex foregut may be linked to macrophagy and predation, being associated with the ingestion of larger food particles.The functional morphology of the foreguts was extensively investigated for other brachyurans by FACTOR (1982), ABRUNHOSA et al. (2003) and CECCALDI (1989).In addition to the contribution of these studies to the knowledge of the biology of those respective taxa, the results are especially important for the successful culture of the economically interesting species.
To understand the function of the decapod foreguts, a correct preparation of ossicle structures is of primary importance; this includes the application of specific methods such as staining and an adequate agent for tissue removal that does not damage the ossicles.BRÖSING et al. (2002), describing the ossicle system of the foregut of three species of Dromiidae, demonstrated a new coloration method that allowed the identification of five ossicles that had not been recognized in previous descriptions of the same species by NAUCK (1880) and PATWARDHAN (1934): pterocardiac, postpterocardiac, lateral mesopyloric, posterior uropyloric and the anterior process of the ampullary roof ossicle, upper portion.The method of BRÖSING et al. (2002) was used in the preparation of the four foreguts described here, making it possible to detect the presence of the postpterocardiac, lateral mesopyloric and posterior uropyloric ossicles.
In general, the foreguts of F. reflexifrons, S. pictus, V. serrata and D. septemdentatus are apparently similar to those described for brachyuran Libinia emarginata, Uca sp. and Calappa sulcata Rathbun, 1898 by MEISS & NORMAN (1977).They show similarities mainly in the degree of calcification, size and number of ossicles.On the other hand, all of these foreguts are quite different morphologically when compared to those of shrimps described previously, e.g.Farfantepenaeus dourarum (Burkenroad, 1938) (NISHIDA et al. 1990).
Similarities related to the pattern and presence of gastric mill ossicles were found between the brachyurans of the present The structure of the anterior pleuropyloric ossicle (XXIX) also varied among species.This ossicle had the form of an irregular plate with re-entrance in the anterior margin in F. reflexifrons, a narrow and irregular plate in S. pictus; it was short and little enlarged in V. serrata and consisted of a small, rounded and concave plate in D. septemdentatus.
Detailed descriptions of all ossicles may be useful for discriminating morphologically similar species, as exemplified by those of the genus Valdivia, where difficulties in the identification of some species arise due to changes in the external morphology during growth, misidentification led in consequence to much confusion in taxonomic treatments of this group, including multiple descriptions of the same species under different synonyms (MAGALHÃES 2003).We hope that the morphological analyses presented here will be useful also to the taxonomist and will possibly help to avoid a similar situation with the genera in question.

Table I .
Ossicles found in the foregut of the freshwater crabs F. reflexifrons, S. pictus, V. serrata and D. septemdentatus.