Molecular characterization of the progenetic metacercariae Crocodilicola pseudostoma parasitizing Rhamdia quelen (Siluriformes, Heptapteridae) in Brazil.

The trematodes have developed several adaptations and strategies to complete their life cycle in the intermediate host, without even reaching the definitive host. Thus, metacercariae through progeny can produce viable eggs by self-fertilization in the second intermediate host. We analyzed 30 specimens of Rhamdia quelen Quoy & Gaimard 1824 (Siluriformes, Heptapteridae) collected in the Jacaré-Pepira River, Ibitinga. Among the specimens analyzed, only one host was parasitized by the progenetic metacercariae of Crocodilicola pseudostoma Willemoes-Suhm 1870 (Digenea: Proterodiplostomidae) presenting prevalence of 3.3%, mean intensity of 68.0 ± 12.4 and mean abundance of 2.3 ± 0.4. This is the first record of progenesis in the metacercariae of C. pseudostoma in the Jacaré-Pepira River, as well as the first partial sequence of COI gene obtained from this species in Brazil.


INTRODUCTION
Rhamdia quelen Quoy & Gaimard 1824 belongs to the Siluriform order and Heptapteridae family. Popularly known as "Jundiá", has nocturnal habit and occupy calm and deep places of the rivers, having an omnivorous habit with piscivorous tendency. This species can be found in Central and South America, as well as in southern Brazil and is of great importance for consumption (Gomes et al. 2000, Eschmeyer et al. 2017, Froese & Pauly 2017. The Jacaré-Pepira River is one of the main rivers that composes the Tietê-Jacaré Hydrographic Basin, located in the center of the state of São Paulo and it is considered one of the cleanest rivers in the state. This area is known as "Pantaninho", presenting an ecosystem with characteristics similar to those of Pantanal Matogrossense (Estado de São Paulo & Secretaria do Meio Ambiente 2013).
According to Mouritsen & Poulin (2002) parasites may interfere in the natural animal communities and the level of impact of the parasites on the hosts will depend of the prevalence and intensity of infection or infestation. Trematodes usually have a life cycle involving three hosts. Eggs produced by adults parasites are firstly released into the environment through the feces of the defi nitive host. After hatching, a free-living larvae is release and reach the fi rst intermediate host, a mollusk, where develops into a free-living cercariae. They must fi nd a suitable second intermediate host,

MATERIALS AND METHODS
A total number of 30 specimens of R. quelen were collected in the Jacaré-Pepira River, in the city of Ibitinga (21°53'30.8"S 48°48'46.0"W) between January and May 2017, under authorization to capture (SISBio, number 55914-1) and under the Ethics Commission in the Use of Animals (CEUA No. 9530230816). Fish were captured with a simple mesh fishing net and were packed in individual plastic bags to avoid any alteration of the parasitic fauna. At the time of necropsy, data on collection date, standard length (cm), weight (g) and host sex were recorded. After necropsy, the organs were separated and observed in stereomicroscope (Bel Photonics) for the collection of the parasites. A portion of the collected metacercariae were stored in 70°GL ethanol until the staining procedure and another part of the metacercariae were fixed in absolute ethanol until the molecular biology procedure.
For identification, specimens were stained with Mayer's Carmalumen and diaphanized using Eugenol, later were mounted in permanent blade using Canada Balsam (Eiras et al. 2006) and analyzed with the aid of a microscope (Nikon E200). The image software (Moticam 5.0MP) was used to perform morphometric analysis and the bibliographies of Armas de Conroy (1986) and Ferrari-Hoeinghaus et al. (2007) were used to verify if the species of this study is the same studied by these authors. Parasitological indices of prevalence, mean intensity and mean abundance were calculated according to Bush et al. (1997). A representative specimen of C. pseudostoma collected from the intestine of R. quelen was deposited in the Helminthological Collection of the Institute of Biosciences of Botucatu (CHIBB), at the Universidade Estadual Paulista "Julio de Mesquita Filho", Botucatu campus, state of São Paulo, Brazil, under deposit number: 357 L.
For molecular analysis, The DNeasy Blood & Tissue Kit (Qiagen®, Germany) was used for the extraction of deoxyribonucleic acid (DNA) from one specimen of C. pseudostoma found from the liver of the fish, following the animal tissue protocol. The COI (Cytochrome C Oxidase Subunit 1) gene was amplified using the primers MplatCOX1dF (5'-TGTAAAACGACGGC CAGTTTWCITTRGATCATAAG-3') and MplatCOX1dR (5'-CAGGAAACAGCTATGACTGAAAYAAYAIIGGATCI CCACC-3') ( Moszczynska et al. 2009). Polymerase chain reaction (PCR) was performed using Ready-to-Go PCR beads (Pure Taq™Ready-to-Go™ beads, GE Healthcare, Chicago, USA) which consists of buffers BSA, dATP, dCTP, dGTP, Dttp and ±2.5 units of puReTaq DNA polymerase. Were added 3 μl DNA extraction, 1 μl of each primer and deionized water to complete the final volume of 25 μl. Amplification reactions were performed using Bio-Rad Mycycler (Bio-Rad Laboratories Pty Ltd., Gladesville, Australia) thermo cycler, with initial denaturation at 94 °C for 3 min, followed by 5 cycles of 94 °C for 40 s, 45 °C for 40 s for annealing temperature, 72 °C for 1 min. After, 35 cycles were performed with 94 °C for 40 s, 51 °C for annealing temperature, 72 °C for 1 min and a final extension at 72 °C for 5 min. Results of the amplifications of the DNA were analyzed in agarose gel at 1% in TAE buffer by electrophoresis. PCR product was purified using QIAquick PCR Purification Kit (Qiagen®, CA, USA).
Sequence was run using Applied Biosystems ABI 3500 DNA genetic analyzer. The sequence obtained from parasite was edited in Sequencher™ v. 5.2.4 (Gene Codes, Ann Arbor, MI) and to confirm the identity, this sequence was subjected to BLAST analyse (http://blast. ncbi.nlm.nih.gov). The partial sequence obtained from the COI gene (GenBank Acc. Num. MN516738) was aligned with related sequences previously obtained from species of trematodes recorded in Genbank.

RESULTS AND DISCUSSION
Fish presented an average weight of 63.0 ± 76.3 g and standard length of 14.9 ± 4.7 cm. Sixtyeight metacercariae of C. pseudostoma ( Figure  1) were collected, some specimens were found encysted in the liver and others free in the swimming bladder, cavity, intestine and stomach of a single female host. According to Armas de Conroy (1986), the infection caused by progenetic metacercariae of C. pseudostoma can cause sterility in female catfish species, because the eggs that are eliminated by these parasites can change the normal functioning of the gonads.
Morphometric analysis of progenetic metacercariae of C. pseudostoma (based on 13 specimens) found in R. quelen are   Table I The primers used successfully amplified a COI gene partial sequence of 440 base pairs. The partial sequence obtained, after edited and aligned, coincided and showed 95.5% similarity in the COI gene with the partial sequence of the diplostomidae C. pseudostoma available in Genbank and is the first partial sequence of the COI gene obtained from this species in Brazil (Table II). The small difference in relation to the sequence similarity obtained with the already deposited sequence of the COI gene of the C. pseudostoma species in Genbank by Hernández-Mena et al. (2017) possibly is related to the fact that the parasite was collected in another host species (Rhamdia guatemalensis Günther 1864) and also in another country (Mexico).
Phylogenetic analysis with COI gene showed the formation of three major clades, divided among families within the Diplostomoidea superfamily. In the clade of the family Proterodiplostomidae only the sequences referring to species C. pseudostoma was observed (Figure 2). Our study corroborated with Hernández-Mena et al. (2017) which showed that Proterodiplostomidae is the sister group of the Diplostomidae, within the superfamily Diplostomoidea. Mitochondrial genes are recognized for their usefulness in solving phylogenies at a deeper level, especially for flatworms (Littlewood et al. 2015).
The authors Armas de Conroy (1986), Pérez-Ponce de León et al. (1992) and Guidelli et al. (2003) have already registered progenetic metacercariae of C. pseudostoma in fish species representatives of the Siluriformes. Guidelli et al. (2003) also analyzed a prevalence of 84.6%, mean intensity of 6.5 and mean abundance of 5.5 of C. pseudostoma in the host Hemisorubim platyrhynchos Valenciennes 1840, but the values obtained were different regarding the present study due to the number of analyzed (136) and parasitized (115) fish and also the number of parasites collected (742).
According to Herrmann & Poulin (2011) the metacercariae are considered progenetic when they mature and reproduce by self-fertilization within the second intermediate host before normal time, that is, these metacercariae become an adult parasite in an early stage and in this case no need of a definitive host, therefore the life cycle of this parasite is determined to be incomplete. According to Poulin (2001), progenetic metacercariae present a lower level of infection compared to normal metacercariae, in order to keep the intermediate host alive longer. There are four factors that can cause this change in the life cycle of the parasite: 1) internal resources of the host, 2) environmental instability, 3) unavailability of the definitive host and 4) time of development of the parasite. The first factor is related to the production of eggs that depends on the feeding of the hosts or the organs in which the metacercariae are present. The second factor determines that progenesis development is related to habitat characteristics, such as water levels with unpredictable increases and decreases, salinity and water temperature. According to the third factor, progenetic metacercariae are common when definitive hosts are absent or temporarily unavailable in the study areas, or even when definitive hosts are present, but have low The alligators and crocodiles are definitive hosts of C. pseudostoma and according to Lefebvre & Poulin (2005b) the possible cause of the presence of the progenetic metacercariae may be the extinction or reduction the definitive hosts. In this study the progenesis may be related due reduction of alligator specimens in the Jacaré-Pepira River. Similarly, the development time of the parasite in the intermediate host is a crucial factor for the development of the progenesis.
According to information on conservation of the Estado de São Paulo & Secretaria do Meio Ambiente (2013), the Jacaré-Pepira River houses species of alligators that are threatened with extinction. One of the reasons for this reduction may be related to socioeconomic activities, due to the extensive livestock and local agriculture activity, which makes the intensive use of agrochemicals, as well as the deforestation of ciliary forest for ranch construction. Another factor may be related with the population present in this region, which may be hunting the alligator for consumption.

CONCLUSION
Firstly, it is difficult to determine the cause of progenesis in this study, but with environmental changes in ecosystems, it is likely that organisms will adapt by creating some life strategies. The progenetic metacercariae shows how the parasites can adapt to a shorter life cycle due to environmental conditions, not needing the definitive host to develop. The metacercariae can also mature early and produce eggs due to some alteration of their own development as a form of adaptation, thus obtaining the flexibility needed to adjust for changes, related or not to the absence of the host.
Although there is no certainty about the development of this progenetic metacercaria of C. pseudostoma in Rhamdia quelen, this is a study that will contribute to other morphological and phylogenetic studies, as well, it is collaborating with the study of biodiversity in the Jacaré-Pepira River, being that C. pseudostoma a new record for this river.