Morphology and morphometry of Paratanaisia bragai () Freitas, 1959 (Digenea, Eucotylidae) through light and scanning electron microscopy

Abstract Paratanaisia bragai is a digenetic trematode that reaches sexual maturity in the kidney collecting ducts of domestic and wild birds, while the snails Subulina octona and Leptinaria unilamellata serve as its intermediate hosts in Brazil. The present study analyzed the morphology and morphometry of P. bragai. Adult specimens of the parasite were collected from naturally infected Columba livia kidneys, fixed and prepared for observation via bright field and differential interference contrast light microscopy and scanning electron microscopy. The parasite has an elongated and flattened body, with a subterminal oral sucker located at the anterior end of the body, as observed by all techniques used. Staining the parasite with hematoxylin-eosin enabled observation of the pharynx, located posteriorly to the oral sucker, the vitelline glands, which are extra-cecal and extend anteriorly to the pre-ovarian region and later to the median region of the body, and intestinal caeca parallel to the vitelline glands. The presence and functionality of the acetabulum are controversial points in the literature, but it was observed in all specimens analyzed by scanning electron microscopy, with a major diameter of 38.36 ± 6.96 (28.77 – 45.39) and minor diameter of 31.59 ± 7.04 (21.75 - 38.16). Close to the acetabulum, scales were observed in the integument of the parasite. Scales with (1 - 5) blade divisions were identified. In the genital pore, it was possible to see the everted cirrus with rosette shape. The excretory pore (first morphometric record) is dorsal and subterminal, with major diameter of 12.27 ± 9.16 (5.79 - 18.75) and minor diameter of 3.95 ± 1.49 (2.89 - 5.00).


Materials and Methods
Adult C. livia pigeons were caught in the Irajá district of the city of Rio de Janeiro (22°49'51"S and 43°20'17"W).The birds were transported to the Helminth Biology and Ecology Laboratory of the Animal Biology Department of Rio de Janeiro Federal Rural University (UFRRJ), in the city of Seropédica, where they were examined according to Ritchie's fecal sedimentation method (De Carli, 1994).
Two infected birds were euthanized in a CO 2 chamber and necropsied to remove the kidneys.The organs were placed in Petri dishes containing a 0.85% physiological saline solution (0.85% NaCl) and then sectioned with a scalpel.Adult helminths were recovered and transferred to another Petri dish containing the same saline solution.
Light microscopy (LM) -The fixed adult helminths were mounted between slides and cover slips using the fixing agent as mounting medium and observed under an Olympus BX51 light microscope coupled to an Olympus DP12 digital camera.The images were obtained and processed using the iTEM image capture system, under bright field and differential interference contrast (DIC).
The captured images of adult helminths were used to obtain the measures, which are presented in micrometers (µm), expressed as mean ± standard deviation, with minimum and maximum values within parentheses.
Staining -The adult helminths fixed as previously described were stained with Delafield's hematoxylin, according to the regressive technique proposed by Amato and Amato (2010) and mounted on glass slides.Images of the tissues were obtained using an Olympus DP12 digital image system coupled to an Olympus BX51 light microscope.
Passive infection is established in snails by the ingestion of embryonated eggs of P. bragai eliminated with the excretion products of the definitive host.After the miracidia hatch, two generations of sporocysts, cercariae and metacercariae, develop inside the snail.The definitive host becomes infected by ingesting the parasitized snail (Maldonado, 1945;Keller and Araujo, 1992;Brandolini and Amato, 2006).
Wild birds infected with P. bragai probably act as reservoirs, because they often come into proximity by eating the same food as chickens kept outdoors (Gomes et al., 2005).Migration of wild birds infected by trematodes can also spread helminths to new areas, posing a high risk of native hosts being exposed to infection (Huffman, 2008).These factors give veterinary importance to P. bragai because infection by this parasite can cause economic losses when it results in serious lesions or death of the host.
potassium ferrocyanide and washed again in the buffer solution.The specimens were then dehydrated in an ascending ethanol series (30% to 100%) for 1 hour and dried in a critical point chamber using CO 2 (Baltec CPD, Balzers Union).The processed adult helminths were then mounted on metal bases and gold sputtered for observation under an FEI Quanta 250 scanning electron microscope operating at 20kV (Pinheiro et al., 2004).

Light microscopy
Differential interference contrast (DIC) light microscopy revealed that the adult P. bragai trematode has an elongated and dorsoventrally flattened body, with the presence of a subterminal oral sucker at the anterior end of the body, followed by the pharynx, along with the presence or vitellogenic glands on both sides of the parasite's body (see Figures 1A and 1C).
The specimens stained with hematoxylin-eosin (see Figure 1B), were observed by bright-field light microscopy, showing more details of the vitellogenic glands, which are outside the cecum and extend to the pre-ovarian region.
The intestinal ceca (see Figure 1B) are parallel to the vitellogenic glands, on both sides of the body, with sinuous shape, located 230.00 µm from the posterior region of the body.
The morphometric data are compiled (see Table 1), which also presents a comparison between the morphometric parameters observed in this study with those reported by other authors.
Through scanning electron microscopy, it was possible to gain an overview of the parasite, which presented an elongated and dorsoventrally flattened body, with the presence of a subterminal oral sucker at the anterior end of the body, as well as an oral opening and acetabulum (see Figure 2A).Detailed examination of the oral sucker revealed the presence of external papillae around the suction cup, arranged irregularly, along with internal papillae (see Figures 2B -2D).We observed a wall with musculature in parallel rows inside the oral cavity (see Figures 2E -2F).The external papillae had major diameter of 4.52 ± 1.20 µm (2.42 -8.33) and minor diameter of (1,600 -1,840) (1,028.17 -1,451.92) (1,620 -2,550) (1,674.The nomenclature larger diameter and smaller diameter was used in this study for structures with rounded appearance, such as oral cups, pharynx and acetabulum, the same nomenclature used by Brandolini and Amato (2007) in measuring cups.The other cited authors used the nomenclature width and length.Thus, when filling out the 3.62 ± 0.83 µm (2.35 -5.55), while the internal papillae were smaller, with major diameter of 2.24 ± 0.66 µm (1.65 -3.53) and minor diameter of 1.68 ± 0.29 µm (1.43 -2.09).
The acetabulum had major diameter equal to 38.36 ± 6.96 µm (28.77 -45.39) and minor diameter of 31.59 ± 7.04 µm (21.75 -38.16).Besides this, for the first time we measured the distance from the oral sucker to the acetabulum, finding 490.82 ± 54.71 µm (429.58 -534.88), while the distance from the acetabulum to the most apical part of the posterior end was 746.54 ± 160.19 µm (563.38 -860.46).The papillae present in the acetabulum region had major diameter of 1.79 ± 0.41 µm (1.40 -2.65) and minor diameter of 1.51 ± 0.52 µm (0.88 -2.35) and were arranged irregularly around the border.Finally, we observed the absence of scales around the acetabulum (see Figures 3A-3C).
Only one genital pore was observed, ventrally and to 109,89 µm anterior to the acetabulum with major diameter of30.59µm and minor diameter of 26.18 µm (see Figures 4A -4B).The cirrus was everted, projecting through the genital pore (see Figure 4C), with rosette ornamentations.
Scales were observed when examining the details of the region of the acetabulum and genital pore, arranged in 1 to 3 divisions (see Figures 3A, 3B and 4C).
The morphometric data are compiled (see Table 1), which also presents comparisons with the morphometric parameters reported by other authors.

Discussion
The characteristics of the adult stage of P. bragai observed in this study, such as elongated and flattened body, presence of a subterminal oral sucker at the anterior end of the body, followed by the pharynx and vitellogenic glands on both sides of the body, corroborates the observations described by various authors (Santos, 1934;Stunkard, 1945;Byrd and Denton, 1950;Freitas, 1951;Franco, 1965), besides the presence of the acetabulum, also reported by Maldonado (1945), Stunkard (1945), Freitas (1951) and Brandolini and Amato (2007).
In this study, the measurements of the adult parasite obtained by light microscopy were larger than those obtained by scanning electron microscopy for the average length, average width of the anterior region, average width of the acetabulum region and average width of the posterior region.
The literature presents variations of the size of adult specimens of P. bragai regarding length and width.Santos (1934) found that the width of adults reached up to 3,000 µm and the width up to 600 µm, but did not differentiate the body regions, i.e., anterior, acetabular and posterior.The variation observed by Santos (1934) for length was very broad but fits in the range of measures obtained in this study, both under light microscopy and scanning electron microscopy.With respect to the width, the measures presented by that author are only in accordance with those obtained by us through light microscopy.Stunkard (1945) stated that the gravid parasite's length ranged from 1,200 to 3,000 µm and width from 200 to 450 µm, similar to the ranges observed by us according to both microscopic techniques.
The measures reported by Byrd and Denton (1950), Freitas (1951) and Brandolini and Amato (2007) are similar to those found by in this study by light microscopy.Franco (1965), however, observed much larger parasites than those reported by the other authors, with length varying between 3,490 and 4,260.D'Ávila et al. ( 2017) stated that the length of the post-ovigerous adult stage varied from 1,675 to 2,250 µm, a range that fits with our measurements by light microscopy, and width varying from 191.60 to 350.00 µm, similar to our results obtained by scanning electron microscopy.Maldonado (1945) observed that P. bragai with four days adult development had length of 600 µm, as well as Stunkard (1945) who verified the same measurement for the length of the immature specimen, while specimens at eight days had length of 950 µm and the adult stage, with fully developed and functional reproductive system (between 11 and 15 days of development) had length of 1250 µm.This last value is within the variation of the specimens we observed by scanning electron microscopy, but we did not distinguish between the stages before and after oviposition.In turn, D'ávila et al. ( 2017) reported length ranging from 530 and 1,000 µm for adults before egg laying and between 1,675 and 2,250 µm for specimens afterward.These studies are the only ones to report comparisons of adults before and after oviposition, and they observed differences in the amplitude of variation.
The variations found in the literature in comparison with those found in this study with respect to the body dimensions of P. bragai adults are related to the different development stages of the helminth analyzed (before and after oviposition), and within these stages the number of days of development.They can also be related to the contraction of the parasite during the processing for the different microscopic techniques employed.
In the present study, the measures of the oral sucker by light microscopy are similar to those reported by  Byrd and Denton (1950) and Freitas (1951), while those obtained by scanning electron microscopy are similar to those observed by Stunkard (1945) and Brandolini and Amato (2007).By the two microscopic methods used by us, the values are in accordance with those reported by D'Ávila et al. ( 2017) while the measures reported by Santos (1934) and Franco (1965) are much greater than those found by the other authors.
Through detailed analysis of the oral sucker, we observed external papillae around the suction cup, arranged irregularly, as also observed by Brandolini and Amato (2007).Besides this, we observed internal papillae.According to Hockley (1973), who examined the oral sucker of specimens of both sexes of Schistosoma mansoni Sambon, 1907, the internal surface of the oral sucker of both sexes contained small spines pointing toward the mouth, but these were absent on the external surface of the suction cup.
The esophagus was not visualized in any specimen examined in this study, as also occurred in the study by Santos (1934Santos ( ). D'Ávila et al. (2017) ) did not refer to the esophagus, and according to the figures presented, this organ was not observed by them.In contrast, Stunkard (1945) found the presence of a short esophagus, visualized only when the posterior end of the parasite was extended.Byrd and Denton (1950) described an esophagus variable, as did Franco (1965) and Freitas (1951), who reported variation of the organ in the specimens examined, long esophagus, with and without dilation, as well as absence in some cases.
Through DIC and staining it was possible to observe the vitellogenic glands in more detail.They are located outside the cecum and extend to the pre-ovarian region, as also observed by Freitas (1951) and Byrd and Denton (1950).Stunkard (1945) found that the ovary is situated about a third of the body length of the anterior end.The vitellogenic glands visualized in the present study are located beyond this region, that is, they are pre-ovarian.The dimensions of the vitellogenic glands observed in this study are within the variation reported D' Ávila et al. (2017).Other authors mentioned here use the ovary as a reference for positioning the vitellogenic glands.
The location of the vitellogenic glands is used to distinguish the genera of the subfamily Tanaisiinae.The species Tanaisia bragai (Santos, 1934), Tanaisia confuse Freitas, 1951 andTanaisia robusta Freitas, 1951 have vitellogenic glands that extend anteriorly beyond the ovarian zone, a trait that differs from the other species of the genus (Freitas, 1951).Due to this particularity, Freitas (1959) described the genus Paratanaisia as including the three species, with P. bragai being the type species.The genus Paratanaisia was maintained by Kanev et al. (2002), where in the taxonomic key it uses as a characteristic that the vitellogenic fields extend pre and post testicular.According to Freitas (1959) the genus Tanaisia (Paratanaisia) has a pre-testicular ovary, which confirms the anterior position of the ovarian zone.Therefore, the study of the morphology of the adult stage of P. bragai is very important to establish its taxonomy, because besides the use of vitellogenic glands as a specific trait, the tegument can also be utilized, since according to Freitas (1951), the three species mentioned above each presents peculiarities: T. confuse has spines in its tegument, unlike T. robusta and T. bragai, which have scales and differ from each other by the appearance of these scales.According to the illustrations by Freitas (1951) P. robusta has scales divided up to the base of attachment to the tegument, while P. bragai the scales have the part attached to the tegument, not divided, and a free part that has divisions, as well as, visualized in this study.
We observed that the intestinal ceca run parallel to the vitellogenic glands, on both sides of the body, in sinuous form, and fuse at the posterior end, corroborating the observation of Santos (1934).D'Ávila et al. ( 2017) verified that in post-ovigerous adults the distance between the intestinal ceca and posterior region of the body had amplitude of variation of 200 -360 µm, which includes the values found in this study.The results of Freitas (1951) are also corroborated by our findings, since he described the presence of the intestinal ceca dorsal to the gonads more or less sinuous and fused, located100 to 450 µm from the posterior end of the body.Byrd and Denton (1950) also reported the ceca running parallel at the edges of the body, fusing near the body's posterior end, at a distance from that end of one-fifth to one-eighth of the length of the parasite, a relation similar to that found by us, as well as by Freitas (1951) andD'Ávila et al. (2017).Franco (1965) also observed that the intestinal ceca fuse at a distance of 580 to 610 µm from the posterior end of the body.These are very large measures compared to those observed by us.However, these lengths fit in the range described by Byrd and Denton (1950).The difference between the values found in this study and those presented by Franco (1965) can be attributed to the fact he analyzed larger specimens (measuring 3,490 -4,260 µm) than those analyzed in the present study.
The visualization of the acetabulum by differential interference contrast microscopy was very difficult due to its small size and by the fact that all the specimens observed in this study were in the post-ovigerous stage, in which numerous eggs were observed.The presence of the acetabulum was confirmed by scanning electron microscopy in all the specimens analyzed.Some scientific works, however, do not mention the presence of the acetabulum and others state that it was not observed in the adult parasite (Santos, 1934;Byrd and Denton, 1950;Franco, 1965).
The difficulty of observing the acetabulum in postovigerous adults of P. bragai was also reported by D 'Ávila et al. (2017), because they were unable to visualize the acetabulum of such specimens of P. bragai using light microscopy, only doing so by confocal laser scanning microscopy.In contrast, those authors were able to visualize the acetabulum of pre-ovigerous adults by light microscopy.Therefore, it is important associate the stage of the adult specimen (pre-ovigerous or post-ovigerous) with the microscopic technique so as not to err in describing the morphology of the species.Maldonado (1945) described for the first time the presence of the acetabulum in adults with eight days of development, but when the specimens reached sexual maturity, the organ was atrophied, less than 40 µm in diameter or even absent.In the present study, however, analysis of the acetabulum morphology by scanning electron microscopy indicated that although small, the acetabulum was not atrophied, and instead was still functional, as also reported by Brandolini andAmato (2007) andD'Ávila et al. (2017).
The dimensions of the acetabulum measured in the present study are in accordance with the range of variation reported by Freitas (1951), with length of 30 -100 µm and width of 30 -80 µm; Stunkard (1945), with diameter of 40.00 -50.00 µm; Brandolini and Amato (2007), with average of 40.83 µm (37.70 -43.96) for the major diameter and 20.41 µm (18.30-22.52) for the minor diameter; and D 'Ávila et al. (2017), with average length of 39.91 µm (25-55 µm) and width of 36.70 µm (25-50 µm).The last authors carried out measurements of pre-ovigerous adults but did not report measurements of the acetabulum in post-ovigerous adults, although affirming that the acetabulum of post-ovigerous adults was smaller than that in pre-ovigerous adults.The reduction of size of the acetabulum described by D'Ávila et al. ( 2017) can be seen in the figures of that work, which show that the opening of the acetabulum cavity is smaller in post-ovigerous adults.However, the acetabulum cavity's size might have changed due to contraction of the parasite, as mentioned by Maldonado (1945) for development of the acetabulum of P. bragai cercariae.Maldonado (1943) previously found that during the larval development, P. bragai cercariae had a well-developed acetabulum, but it then atrophied with development of the parasite.In later work Maldonado (1945) verified that in adult specimens in the eighth day of development, the acetabulum had grown, although the enlargement was not in the same proportion as the entire body, and that the acetabulum had atrophied when the specimens reached the adult stage.So, it is important to conduct further studies regarding the size of the acetabulum of P. bragai during its development to elucidate these questions.However, the presence of this suction cup in the adult stage of P. bragai is unquestionable.
The measures of the distances between the oral sucker and acetabulum and between it and the apical part of the posterior end carried out in this study corroborate the observations of Stunkard (1945) and Brandolini and Amato (2007), that the acetabulum is located in the middle third of the body.Our finding of the presence of papillae arranged irregularly around the acetabulum corroborates the observation of Brandolini and Amato (2007), who also reported the presence of protuberances in the tegument that surrounds the acetabulum.That structure was observed in the present study in other regions of the parasite's tegument, denominated papillae.Hockley (1973) also reported the presence of sensory papillae along with spines, but only on the internal surface of the acetabulum, in S. mansoni of both sexes, with spines being absent on the external surface of the suction cups.The absence of scales near the region of the acetabulum found in the present study is in accordance with the images presented by Brandolini and Amato (2007).However, those authors did not discuss this fact in the text.
The types of scales observed in this study, with variation of 1 to 3 divisions, are in accordance with the findings of Brandolini and Amato (2007), who reported single and bifid scales, i.e., scales divided into two elements with a free end.Stunkard (1945) reported the presence of three to eight fused elements.In turn, D'ávila et al. ( 2010) reported that the scales had grooves that delineated two to four teeth.How the teeth develop is controversial, since the literature reports different terms to classify them, such as fused elements as described by Stunkard (1945), and division of scales as expressed by Brandolini and Amato (2007).Therefore, new studies are necessary to clarify the ontogenesis of these structures, due to the importance of the scales as a distinctive trait of species of the family Eucotylidae, as noted by Freitas (1951).
With application of scanning electron microscopy, we noted the presence of the genital pore, located above the acetabulum.Its location in the middle and ventral region of the body is corroborated by Stunkard (1945), who observed it below the anterior region of the ovary, ventral in the middle region of the body.Byrd and Denton (1950) observed that besides the ventral and median position, the genital pore is located below the anterior end of the ovary or at the same level as the cephalic margin of the ovary, and that the genital atrium is common to the male and female genital apparatus.Freitas (1951) stated that the genital pore is median, as also reported by Franco (1965) and Brandolini (2000).Furthermore, we observed the presence of an everted cirrus with rosette ornamentations projecting through the genital pore.Byrd and Denton (1950) stated that the cirrus of T. bragai is short and robust (corroborated by our observation), and is contained in the cirrus pocket, located posterior to the genital pore.D'ávila et al. ( 2010) reported that the cirrus of Tanaisia inopina Freitas, 1951 is highly muscular and cylindrical, and that the reproductive system of T. bragai is normally similar to that described in T. inopina.Besides this, they observed that both the cirrus of T. inopina and the distal part of the uterus open into a genital atrium that leads to a common gonopore.Besides this, the cirrus is the copulatory organ of digenetic trematodes, which may or may not be contained in the cirrus pocket and is a muscular tube that everts during copulation and projects outward (Rey, 2008).However, D'Ávila et al. ( 2017) did not report any ornamentation in the cirrus, as observed in the present study, which can be related to the species analyzed, T. inopina, demonstrating that with respect to these reproductive elements, T. bragai and T. inopina are not similar.
The presence of ornamentations in the cirrus of P. bragai is in accordance with Dawes (1968), who stated that the cirrus of digenetic trematodes generally has a wall with circular and longitudinal musculature along with spines.The presence of spines was verified in Fasciola gigantic Cobbold, 1855 by Srimuzipo et al. (2000).They visualized the cirrus in the shape of a sausage with spines of varying lengths and sharpness.Naem et al. (2012) observed that the cirrus of Fascioloides magna Bassi, 1875 has a smooth surface, with small pores in the organ's dorsal region, and that certain zones between the cirrus folds contain small pores and groups of small spines.
The excretory pore is located in the posterior end, in a subterminal position in the dorsal region of the body, as described by Brandolini (2000), but unlike observed by Stunkard (1945), who classified the excretory pore as terminal.In contrast, D'Ávila et al. ( 2017) described the presence of an excretory canal and measured its length in pre-ovigerous specimens of P. bragai, so our study contains the first morphometric record of the excretory pore of adult specimens of P. bragai.
The use of electron microscopy has enabled considerable advances in knowledge about the morphology of trematode species, not only regarding taxonomy, but also biological and immunological aspects, as well as physiological and biochemical processes of the host-parasite relationship.New morphological traits canal so be elucidated, including their functional roles, thus allowing the inclusion of these traits in the taxonomy of trematode groups.
The results presented in our study shed light on some important aspects of the morphology and taxonomy of P. bragai.Contrary to what some parasitologists claim, P. bragai is not a parasite that only poses a regional problem.Brazil is one of the main countries from which smugglers obtain wild birds for sale worldwide.This illegal activity, by circumventing health inspection, contributes to the dissemination of not only P. bragai, but several other species of parasites that are hosted in these birds.In the literature, there are records of the occurrence of P. bragai in birds from various countries, which is only discovered by postmortem diagnosis, indicating the lethality of infection by this trematode and the difficulty of identifying infection before death of the vertebrate host.In addition, P. bragai has been found parasitizing pet birds, causing their death.
In spite of this great veterinary, economic, and public health importance, there are few studies about this parasite.Additionally, its controversial taxonomy further hampers the identification and correct diagnosis of parasitosis.For this reason, the present study updates previous findings about the P. bragai taxonomy and adds new information about the morphology of this parasite, enriching the morphological tools for better identification of the parasites of this species.
in parentheses represent the observed minimum and maximum values.X ± SD= mean ± standard deviation.SEM= Scanning electron microscopy.-= Data not provided by the authors.Observation:

Figure 2 .
Figure 2. Adult stage of Paratanaisia bragai observed by scanning electron microscopy.A. Overview to the helminth with highlight on the oral sucker (os), oral opening (oo) and acetabulum (a).Scale bar = 250 µm.B. Detail of the oral sucker (os), with highlight on the presence of external papillae (exp) surrounding the suction cup and internal papillae (ip), inside the oral opening (oo).Scale bar = 50 µm.C. Detail of the internal papillae (ip) in the region of the oral sucker.Scale bar = 10 µm.D. Oral sucker (os) with external papillae (exp) surrounding it and oral opening (oo).Scale bar = 100 µm.E and F. Detail of the internal region of the oral opening (oo) of the oral sucker (os), with visualization of the lateral wall with musculature in parallel rows.Scale bar = 50 µm and 20µm, respectively.

Figure 3 .
Figure 3. Middle third region of the adult stage of Paratanaisia bragai observed by scanning electron microscopy.A, B and C. Different views of the acetabulum (a), indicating the presence of papillae (pa) surrounding it and an adjacent peripheral area (*) without the presence of scales.Scale bar = 50 µm, 30 µm and 40µm, respectively.

Figure 4 .
Figure 4. Adult stage of Paratanaisia bragai observed by scanning electron microscopy.A. Overview of the helminth, in the ventral region, indicating presence of the oral sucker (os), location of the genital pore (gp) and acetabulum (a).Scale bar = 500 µm.B. Detail of the genital pore (gp) and acetabulum (a).Scale bar = 200 µm.C. Detail of the genital pore (pg) with presence of the everted cirrus (c).Scale bar = 30 µm.D. Excretory pore (ep) in the posterior dorsal region.Scale bar= 100 µm.E. Detail of the posterior dorsal region indicating the excretory pore.Scale bar= 50 µm.
table, the largest values were inserted in the longest length and the smallest values in the shortest length, regardless of whether width or length was considered by the other authors.