New species of Temnocephala (Platyhelminthes, Temnocephalida) ectosymbiont on vulnerable species of aeglids (Crustacea, Anomura) from the Neotropical Region

A new species of the genus Temnocephala Blanchard, 1849 from southern Brazil was found on two species of anomuran crustaceans, Aegla spinipalma Bond-Buckup & Buckup, 1994 and Aegla grisella Bond-Buckup & Buckup, 1994, the latter classified as a vulnerable species by the “Lista de Referência da Fauna Ameaçada de Extinção no Rio Grande do Sul. Decreto no 41.672, de 11 junho de 2002”. The crustaceans were collected from a tributary creek of the Forqueta river, Perau de Janeiro, Arvorezinha and a tributary creek of the Fão river, Pouso Novo, Rio Grande do Sul, Brazil; both localities belong to the Sub-Basin of Forqueta River. The new species differs from seven other temnocephalans epibionts on Aegla Leach, 1820, by having the following characters: 1. a long and slightly curved cirrus, 2. two vaginal sphincters, one proximal, big and asymmetric, and one distal, smaller and symmetric, and; 3. longer than wide, elongated epidermal ‘excretory’ syncytial plates (EPs), with a almost horizontally central excretory pore, displaced to the anterior portion of the plate. The new species’ EP is the largest in total length among epibionts temnocephalans in crustaceans already registered. Regarding the similarities with the male reproductive system of Temnocephala axenos Monticelli, 1898, the new species has important differences in the female reproductive system. It has a larger proximal vaginal sphincter, located in the middle of the vagina, while the smaller distal one is at the extreme end of the organ. Besides that, the vaginal portion between the proximal and distal sphincters is conspicuous, with a strong muscular wall. This is the first record of a species of Temnocephala in the Taquari Valley, as well in the ‘Perau de Janeiro’, which is an area with a rich endemic fauna.

Temnocephala chilensis was the first species of the genus to be described and was recorded consistently after that (Dioni 1967a, Damborenea 1992).However, the authors have not updated the species description using more recent techniques.Dioni (1972) recorded T. mexicana in Aegla sp. and Parastacus sp. from Argentina.The species was described by Vayssière (1898) and redescribed by Lamothe-Argumedo (1968).Both publications were based on specimens collected from Procambarus digueti (Bouvier, 1897) and Pseudothelphusa jouyi Rathbun, 1893 (added by Lamothe-Argumedo in 1968) from Mexico.
Temnocephala axenos is the most well-studied species (Baer 1931, Dioni 1967b, 1968, Damborenea 1992, et al. 1997), but it has substantial taxonomic problems, such as a misidentification of the host type (Amato et al. 2003) and the loss of the holotype at the Berlin Natural History Museum because of war damage.The superficial description of T. axenos (Monticelli 1898) lead to the consideration of this species as a senior synonym of Temnocephala brasiliensis Merton, 1922 by Baer (1931) and Temnocephala bresslaui Pérez-González, 1949by Dioni (1967c).Volonterio (2007) stated that T. bresslaui was erroneously synonymized by Dioni (1967c) and it is, probably, still a valid species.The author re-described T. axenos, solving some of these issues.The incomplete description of T. cyanoglandula, with only data of the male reproductive system, has led Volonterio (2007) to suggest a synonymy for this species with T. axenos or T. bresslaui.However, a recent study of the female reproductive system (Seixas et al. 2015a) has confirmed T. cyanoglandula as a valid species.
While describing T. mertoni, an epibiont species on anomuran crabs, Volonterio (2007) pointed out the difficulties of distinguishing temnocephalans species on crustaceans given the similarities in the males' reproductive system.Among other features, the author highlighted the importance of describing in detail the female reproductive system of ectosymbionts hosted by crustaceans.
There are no records of temnocephalans at the Forqueta River Sub-Basin (Fig. 1), where the crustaceans fauna is less well investigated.The Forqueta and Fão Rivers, localized at the municipalities of Arvorezinha and Pouso Novo, respectively, represent the two main rivers of the sub-basin.The present study aims to describe a new species of Temnocephala ectosymbiont on Aegla spinipalma Bond-Buckup & Buckup, 1994 and Aegla grisella Bond-Buckup & Buckup, 1994 (Fig. 2a), which is classified as a vulnerable species by Rio Grande do Sul State Law (Marques et al. 2002).Both are being registered as new host species for neotropical temnocephalans.
The collections occurred monthly between August 2014 and April 2015 as part of a larger project for taxonomic and ecological studies.All crustaceans were collected with dip nets, sexed, measured, and returned to their natural habitat.Only a few specimens (≅ 10) were taken alive to the "Setor de Evolução e Ecologia, Univates" to be examined for temnocephalans.
The temnocephalans were studied through a series of techniques focusing especially on the morphology of the vagina and other female reproductive organs, as well as the morphology of the cirrus structure and the epidermal 'excretory' syncytial plates (EPs).
For general measurements, the helminths were fixed with AFA, under slight cover slip pressure, following the protocols established by Amato et al. (2007) and Seixas et al. (2010).The specimens were stained with Delafield's hematoxylin or aceto-carmine/fast green, cleared in cedar oil, and mounted as permanent slides on Canada balsam.
For the EPs studies, the specimens were dehydrated according to a protocol adapted from Kashi et al. (2014) for Scanning Electron Microscopy (SEM).The SEM preparations and photomicrographs were made at the 'Laboratório MEV (Microscopia Eletrônica de Varredura)' at Tecnovates, Univates.The images of the EPs were measured, according to Seixas et al. (2015b), using the AxioVision Zeiss LE 4.7.2 software.
Cirrus measurements were taken from extracted cirri mounted on Faure´s mounting medium (F).The terminology used to describe the male reproductive structures followed Seixas et al. (2010).
Photomicrographs of the temnocephalans were taken with the microscope Zeiss Axiolab.The line drawings and photographic images were prepared using Adobe's Fireworks ® CS6 and Adobe's Photoshop ® CC 2017.Measurements are in micrometers (μm) unless otherwise indicated, ranges are followed (between parentheses) by the mean, the standard deviation values, and the number of specimens measured for a given character (when different than 25).The ecological concepts applied to the symbiotic organisms follow Bush et al. (1997).

Results
Description.Based on 63 temnocephalans specimens collected from A. grisella and 34 specimens from A. spinipalma: 13 whole mounted adults from A. grisella, 12 whole mounted adults from A. spinipalma, 3 dissected cirri from A. grisella, and 2 dissected cirri from A. spinipalma measured.
Reproductive system.Female.Vitellarium arborescent and thin (Fig. 3a   muscular wall of the prostatic bulb (11 µm thick on average).The cirrus showed intraspecific variation of the curvature from straight to slightly curve.The introvert portion of the cirrus has a small variation in length (25-30 µm) but the same measure in width at base (15 µm) in all specimens measured.The total length of the cirrus is three times bigger than the maximum width of the shaft at its base.The posterior pair of the testes is two times bigger than the anterior pair.

Discussion
Temnocephala chilensis, T. axenos, T. talicei and T. mertoni present cirrus measuring, on average, between 123-149 µm long (Damborenea & Cannon 2001, Volonterio 2007), while T. cyanoglandula has the largest cirrus among anomuran crabs temnocephalans, having an average length of 256 µm (Amato et al. 2003).Among these species, T. grisella sp.nov.has a cirrus of intermediate size, measuring 179 µm on average (Table 1).Dioni (1967c) studied specimens of T. axenos ectosymbiont on species of Aegla and Parastacus Huxley, 1879 from Uruguay and Brazil, finding a great cirrus' size variability (125-150 µm), although, due to the lack of data on female reproductive system, is impossible to compare with the new species described in the present work.Nonetheless, both cirri measurements presented by Dioni (1967c) and Volonterio (2007), on their description of Uruguayan specimens of T. axenos, differ from that of T. grisella sp.nov.(Table 1).
Lamothe-Argumedo (1968) also found a great cirrus' size variability (144-206 µm) while re-describing T. mexicana.Although the range was similar to T. grisella sp.nov., the shape of the cirrus differed.In fact, the cirrus' shape of T. mexicana presented by the author differs greatly from the original description of the species.Vayssière (1898) describe the cirrus as a little curved, with an "exsertile" portion at its outer end, meaning that it have a portion projected beyond the organ, which is visible in the diagram provided by the author.The cirrus described by Lamothe-Argumedo (1968) doesn't have this characteristic introvert and was similar to T. mertoni by having a slightly sinuous portion in the shaft.The single vaginal sphincter of T. mexicana, evidenced by the diagram presented, is also similar to T. mertoni.These cirrus and vaginal sphincter characteristics differs T. mexicana and T. mertoni from T. grisella sp nov.Damborenea & Cannon (2001), on a neotropical temnocephalans revision, pointed out the absence of any muscular structure (sphincter) in T. chilensis vagina.They also recorded a conic cirrus with a swollen introvert measuring an average of 149 µm in length.All these characteristics differs from T. grisella sp nov.The authors also assert the lack of sphincter on T. talicei.Volonterio (2009), while re-describing T. talicei, has shown the existence of a single conspicuous and asymmetric distal sphincter, pointing out similarities of this species with T. mertoni.Likewise T. talicei and T. mertoni, T. cyanoglandula also have only one vaginal sphincter, which is also distal and asymmetric (Seixas et al. 2015a).
Temnocephala dionii have a unique cirrus with a "groove between introvert and the shaft" and a single vaginal sphincter (Ponce de León et al. 2015), which differentiates T. dionii from the new species.This cirrus' shape characteristics have some similarities with the original description of T. mexicana' cirrus, pointing out the importance of a future revision of T. mexicana.
Temnocephala axenos, alike T. grisella sp.nov., has two vaginal sphincters, one proximal asymmetric and a symmetric distal, but they greatly differ on size.In the re-description made by Volonterio (2007), T. axenos presented both vaginal sphincters with similar average sizes (43 µm proximal e 45.5 µm distal), whereas T. grisella sp.nov.presents one proximal large vaginal sphincter (83 µm on average) and a smaller distal one (56.5 µm on average).The author also pointed out that both sphincters of T. axenos are located at the final portion (distal) of the vagina, very close to one another.In contrast, in T. grisella sp.nov., the vaginal portion between the proximal and distal sphincters is quite long with a strong muscular wall (Fig. 4a -aps), measuring 38 μm in length on average.Therefore, in T. grisella sp.nov., the proximal sphincter is located in the middle of the vagina, while the distal one is at the tip end of the organ.The total vaginal length of T. grisella sp.nov. is larger than T. axenos, and both species have a vesicula intermedia, rather than seminal receptacles, that it is also slightly larger in specimens of T. grisella sp.nov.(Table 1).
Volonterio (2007) described T. axenos' EPs like "elliptical excretory syncytia, small, extends from base of external tentacles to level of anterior portion of intestine", however she did not provide character measurements.Temnocephala grisella sp.nov.has elongate EPs, wider in the area surrounding the excretor pore.The excretory pore is central and in the anterior portion of the plates.Temnocephala grisella sp.nov.' EP is the larger in total length (397.5 μm on average) among epibionts temnocephalans on crustaceans already registered, T. cyanoglandula's being the second largest, with a total length of 284.4 μm on average (Seixas et al. 2015b).The larger than wide EPs of T. grisella sp.nov.are evidenced with the ratio of total length of the EPs/total body length.Six EPs, approximately, could occupy the total length of the body while in the wider than long EPs' species, such as Temnocephala trapeziformis Amato, Amato & Seixas, 2006(Seixas et al. 2015b), 17 EPs are necessary to occupy the total length of the body.Temnocephala mertoni' EPs have a similar shape to the new species described in the present work, however their measurements, as well as T. axenos, have not been provided.Observing the illustrations of the species description, T. mertoni' EPs extend from the limit of the tentacles up to the intestine (Volonterio 2007), while in T. grisella sp.nov., the EPs start before the limit of the tentacles and extend until almost the half of the intestine, suggesting a larger overall size.Amato et al. (2003) distinguished T. cyanoglandula by the unique appearance of its cyanophilous glands, which form "two irregular-shaped, grape-like bunches of cells, located in the anterior portion of the body, at the level of mouth and pharynx".The same structures are visible in T. grisella sp.nov.(Figs 3a and 8d), but, even utilizing the same staining methods, they do not appear to be as conspicuous nor its ducts visible, as they are in T. cyanoglandula.According Volonterio (2007), T. axenos has four paranephrocytes and T. mertoni, one pair.Similar to T. mertoni, T. grisella sp.nov.also have one pair of large, round, more central paranephrocytes (Fig. 3a -head arrows).
The host A. grisella was included in the list of endangered species of the State of Rio Grande do Sul in the category of vulnerable, according to IUCN (The World Conservation Union) criteria (Marques et al., 2002).In addition, the sampling point at the municipality of Arvorezinha, although well preserved, has recently been threatened by the construction of a hydroelectric power plant.The 'Perau de Janeiro' is home to some records of highly restricted and endemic species, such as the amphibian Melanophryniscus admirabilis Di Bernardo, Maneyro & Grillo, 2006, a critically endangered species (Fonte et al. 2014).Due to these threats, the records of associated fauna of these crustaceans at this locality becomes even more necessary and can aid in the conservation policy of aeglids and its epibionts, as well as the environment that has undergone a series of actions that threaten the original biodiversity of the region.The accelerated process of degradation becomes even more worrying in a region where a great part of the fauna is still unknown, thus, it is essential to carry out taxonomic, ecological and environmental studies whose results make the elaboration of conservation actions possible.

Figure 1 .
Figure 1.Map of Rio Grande do Sul showing the Forqueta River Sub-Basin and collection points in the municipalities of Arvorezinha and Pouso Novo.

Figure 6 .
Figure 6.Diagram of the epidermal 'excretory' syncytial plate of Temnocephala grisella sp.nov., showing the limit of the plate and the excretory pore (ep).Scale bar = 100μm.