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

Abstract

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.

Key words
Biodiversity; Digenea; Proterodiplostomidae; Siluriformes

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 definitive host. After hatching, a free-living larvae is release and reach the first intermediate host, a mollusk, where develops into a free-living cercariae. They must find a suitable second intermediate host, developing in a metacercariae. The life cycle is complete when the definitive host ingest the metacercariae together with the second intermediate host (Lefebvre & Poulin 2005a).

Due to the need for a high predation rate among hosts to complete the life cycle, trematodes have developed various adaptations and strategies for reaching the full cycle in the intermediate host, without even reaching the definitive host (Lagrue & Poulin 2009). Thus, metacercariae through progeny can produce viable eggs by self-fertilization in the second intermediate host, usually these eggs are released into the environment only after the host’s death (Poulin 2001, Lagrue & Poulin 2009, Herrmann & Poulin 2011). According to Poulin & Cribb (2002) the greatest challenge in progenesis is the releasing of eggs into the environment. Progenetic species expect its host to die naturally by decay or predation to release the eggs (Herrmann & Poulin 2011).

The aim of this study was to analyze morphologically and molecularly the progenetic metacercariae of Crocodilicola pseudostoma Willemoes-Suhm 1870 (Digenea: Proterodiplostomidae) collected in R. quelen.

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’-TGTAAAACGACGGCCAGTTTWCITTRGATCATAAG-3’) and MplatCOX1dR (5’-CAGGAAACAGCTATGACTGAAAYAAYAIIGGATCICCACC-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. Mesostephanus microbursa Caballero, Grocott & Zerecero 1953 (MF398316) was used as an “outgroup”.

The Geneious v. 7.1.3 (Kearse et al. 2012) with ClustalW (Larkin et al. 2007) and default settings were used to align the sequences. The analysis was performed using only positions that were unambiguously alignable across all taxa. MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003) was used to perform bayesian inference. The nucleotide substitution model used was GTR+I+G. The Markov Chain Monte Carlo (MCMC) chains were run for 10 million generations and the log-likelihood scores plotted. The ‘burn in’ was set to 25%. Phylogenetic tree was generated and edited in FigTree v. 1.4 (Rambaut 2012).

RESULTS AND DISCUSSION

Fish presented an average weight of 63.0 ± 76.3 g and standard length of 14.9 ± 4.7 cm. Sixty-eight 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. Parasitological indices of C. pseudostoma presented a prevalence of 3.3%, mean intensity 68.0 ± 12.4 and mean abundance 2.3 ± 0.4.

Figure 1
Progenetic metacercariae of Crocodilicola pseudostoma Willemoes-Suhm 1870 found encysted in the liver and free in the swimming bladder, cavity, intestine and stomach of Rhamdia quelen Quoy & Gaimard 1824. Scale bar: 200 μm.

Morphometric analysis of progenetic metacercariae of C. pseudostoma (based on 13 specimens) found in R. quelen are presented in Table I, as well as are approaches the morphometric values obtained in the description by Armas de Conroy (1986), which was studied C. pseudostoma in the host Rhamdia hilarii Valenciennes 1840 from the Instituto de Pesca, state of São Paulo, Brazil and also, the morphometric values in the description by Ferrari-Hoeinghaus et al. (2007), which was studied C. pseudostoma collected in the intermediate host Loricariichthys platymetopon Isbrücker & Nijssen 1979 from the Upper Paraná River floodplain, Brazil.

Table I
Comparative morphometric data of the progenetic metacercariae of Crocodilicola pseudostoma Willemoes-Suhm 1870 found in Rhamdia quelen Quoy & Gaimard 1824, Loricariichthys platymetopon Isbrücker & Nijssen 1979 and Rhamdia hilarii Valenciennes 1840 from Brazil.

		Present study	Ferrari-Hoeinghaus et al. (2007)	Armas de Conroy (1986)
Site of infection		Encysted in the liver and free in the swimming bladder, cavity, intestine and stomach	Digestive tract	Body cavity
Host		Rhamdia quelen	Loricariichthys platymetopon	Rhamdia hilarii
Locality		Jacaré-Pepira River, Brazil	Upper Paraná River floodplain, Brazil	Instituto de Pesca, Brazil
Body	Length	1250.0 – 3000.0 (2000.0) µm	2075.0 – 2575.0 (2364.0) µm	1336.0 – 1721.0 µm
	Width	250.0 – 580.0 (490.0) µm	500.0 – 800.0 (662.0) µm	722.0 – 845.0 µm
Oral sucker	Length	60.0 – 89.0 (71.0) µm	60.0 – 70.0 (64.0) µm	61.8 – 148.0 µm
	Width	54.0 – 86.0 (74.0) µm	50.0 – 70.0 (58.0) µm	63.0 – 80.0 µm
Pharynx	Length	61.0 – 112.0 (80.0) µm	50.0 – 70.0 (56.0) µm	54.8 – 64.8 µm
	Width	36.0 – 60.0 (48.0) µm	50.0 – 60.0 (55.0) µm	43.8 – 53.8 µm
Acetabulum	Length	100.0 – 150.0 (122.0) µm	110.0 – 150.0 (124.0) µm	102.4 – 112.8 µm
	Width	116.0 – 170.0 (136.0) µm	110.0 – 190.0 (145.0) µm	132.2 – 185.6 µm
Tribocytic organ	Length	140.0 – 252.0 (212.0) µm	170.0 – 260.0 (206.0) µm	50.4 – 78.0 µm
	Width	93.0 – 189.0 (136.0) µm	150.0 – 230.0 (182.0) µm	98.0 – 147.2 µm
Eggs	Length	99.0 – 131.0 (116.0) µm	80.0 – 120.0 (96.0) µm	111.2 – 124.0 µm
	Width	43.0 – 74.0 (60.0) µm	50.0 – 80.0 (74.0) µm	54.4 – 68.0 µm

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).

Table II
Species of Proterodiplostomidae, Diplostomidae and Strigeidae presented in the molecular phylogenetic analysis with details of host, locality and reference.

Parasite	Host	Locality	Reference
(MF398317) Crocodilicola pseudostoma	Rhamdia guatemalensis	Mexico	Hernández-Mena et al. 2017
(MF398318) Crocodilicola pseudostoma	Rhamdia guatemalensis	Mexico	Hernández-Mena et al. 2017
(GQ292490) Diplostomum huronense	Multiple species	Canada	Locke et al. 2010a
(FJ477196) Diplostomum indistinctum	-	Canada	Moszczynska et al. 2009
(HM064679) Diplostomum sp.	Multiple species	Canada	Locke et al. 2010b
(JQ639170) Diplostomum pseudospathaceum	Perca fluviatilis	Germany	Behrmann-Godel 2013
(JX986909) Tylodelphys clavata	Perca fluviatilis	Germany	Georgieva et al. 2013
(KC685329) Tylodelphys mashonensis	Clarias gariepinus	Tanzania	Chibwana et al. 2013
(KM115882) Austrodiplostomum ostrowskiae	Cichlasoma trimaculatum	Mexico	García-Varela et al. 2015
(KR271494) Tylodelphys jenynsiae	Cnesterodon decemmaculatus	South America	Locke et al. 2015
(FJ477223) Tylodelphys scheuringi	-	Canada	Moszczynska et al. 2009
(KR271481) Tylodelphys immer	Multiple species	South America	Locke et al. 2015
(JX977780) Apharyngostrigea cornu	Nyctycorax nyctycorax	Mexico	Hernández-Mena et al. 2014
(HM064887) Apaharyngostrigea pipientis	-	Canada	Locke et al. 2010b
(JX977731) Parastrigea diovadena	Eudocimus albus	Mexico	Hernández-Mena et al. 2014
(JX977776) Parastrigea plataleae	Platalea ajaja	Mexico	Hernández-Mena et al. 2014
(JX977760) Parastrigea cincta	Eudocimus albus	Mexico	Hernández-Mena et al. 2014
(JX977781) Cotylurus gallinulae	Aythya affinis	Mexico	Hernández-Mena et al. 2014
(KY513232) Cotylurus cornutus	Gyraulus acronicus	Norway	Soldánová et al. 2017
(KY513232) Cotylurus cornutus	Radix balthica	Norway	Soldánová et al. 2017
(KT831347) Cotylurus gallinulae	Physella gyrina	Canada	Gordy et al. 2016
(JX977784) Cardiocephaloides sp.	Larus occidentalis	Mexico	Hernández-Mena et al. 2014
(JX977782n) Cardiocephaloides medioconiger	Larus sp.	Mexico	Hernández-Mena et al. 2014
(JX977782) Cardiocephaloides medioconiger	Larus sp.	Mexico	Hernández-Mena et al. 2014

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).

Figure 2
Bayesian inference analysis on partial COI sequences showing the position of Crocodilicola pseudostoma Willemoes-Suhm 1870 among other Digenea. Numbers at the nodes represent Bayesian posterior probability gaining more than 0.6 posterior probability. Values lower than 0.6 are represented by dashes. Scale bar is given under the tree.

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 abundance, which ends up exhibiting a low predation rate. The fourth factor is related to the prolonged duration of the metacercaria stage in the intermediate hosts (Lefebvre & Poulin 2005b).

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.

ACKNOWLEGMENTS

We thank to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - Process num. 2016/01587-3 and 2015/24901-2) for the scientific and financial support; and the Laboratório de Parasitologia de Animais Silvestres (LAPAS), Department of Parasitology, UNESP, for the support in the carrying out of the molecular biology.

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Publication Dates

• Publication in this collection
  12 Aug 2020
• Date of issue
  2020

History

• Received
  26 Dec 2018
• Accepted
  17 Oct 2019