Centrorhynchus spp. (Acanthocephala) in South America: new anuran record and checklist of vertebrate hosts

Abstract The aim of this study was to record Centrorhynchus sp. associated with the exotic species Aquarana catesbeiana (bullfrog) in southern Brazil and to present a checklist of vertebrate hosts in South America. Twenty-nine adults and juveniles of A. catesbeiana were collected in Capão do Leão, state of Rio Grande do Sul, Brazil, between October 2019 and December 2020. We found 275 specimens of Centrorhynchus sp. cystacanths in the stomach musculature and coelomic cavity of 55.1% of hosts (16). There was no significant differences in the prevalence and mean intensity of infection with cystacanths when compared males and females of A. catesbeiana. The prevalence was significantly higher in adults than in juveniles. The checklist presents 106 species of vertebrate hosts and 14 taxa of Centrorhynchus recorded in nine South American countries. Avian were the main definitive hosts of Centrorhynchus spp. and snakes Dipsadidae, anurans Hylidae and Leptodactylidae the main paratenic hosts in South America. This is the first record of Centrorhynchus cystacanths in A. catesbeiana in the South America. The study provides tools to help understand the parasitic relationships between species of Centrorhynchus and A. catesbeiana and other hosts in areas where bullfrog have been introduced.

Bullfrog Aquarana catesbeiana (Shaw, 1802) (Anura: Ranidae) due its large size in adulthood native to the northeastern region of the United States and Canada and has been introduced to several countries as a species of commercial interest (Both et al., 2011;Maneyro & Carreira, 2012).In Brazil, this species was introduced in the 1930s and can be found mainly in the southern and southeastern regions (Silva, 2016).Occurrences of cystacanths of Centrorhynchus in A. catesbeiana have been recorded in Texas and North Carolina in the United States (Brandt, 1936;Hollis, 1972) but there are no records for bullfrogs in South America.
The lack of knowledge about the diversity of helminths associated with the exotic A. catesbeiana in Brazil, highlights the importance of helminthological research, since the introduction of host species and parasites can cause imbalances in native ecosystems (Maneyro & Carreira, 2012).The checklist provides a basis for studies on the interactions between Centrorhynchus species and their different hosts and life cycle.The aim of the present study was to record cysthacanths parasitizing A. catesbeiana in southern Brazil, and to provide a checklist of vertebrate hosts in South America.

Host sampling, parasite collection and identification
Twenty-nine specimens of A. catesbeiana (9 males, 10 females and 10 juveniles of undetermined gender) were collected from artificial ponds located in Ichthyology Laboratory of the Department of Animal Science, Universidade Federal de Pelotas (UFPel), in the municipality of Capão do Leão (31°48'13.1"S52°25'00.8"W),state of Rio Grande do Sul (RS), Brazil, between October 2019 and December 2020.These hosts were collected using a fishing rod with a hook and bait and were euthanized in accordance with Resolution No. 1000 of the Federal Council of Veterinary Medicine (Brasil, 2012).
The bullfrog were packed in individual plastic bags and transported to the Laboratory.The hosts were refrigerated, measured for the total snout-vent length (SVL), weighted and necropsied.Five were necropsied and examined after refrigeration and the others (which had been frozen), after thawing.We examined host gender during necropsies, observing the gonads and considering external sexual dimorphism.Hosts stage of maturation (juveniles or adults) were defined according to Quiroga et al. (2015).
We examined separetely all internal organs, and the cystacanths collected were remove from cysts and transferred to cold tap water to extrovert the proboscis.Subsequently fixed in AFA solution (92% 70°GL ethanol + 5% formalin + 3% acetic acid), preserved in 70ºGL ethanol and stained with hydrochloric carmine, clarified with creosote and mounted in Canada balsam (Amato & Amato, 2010).
The measurements of six specimens are in micrometers (unless when indicated) and are presented as range of values and mean in parenthesis.Photomicrographs were prepared using an Olympus BX 41 microscope with an attached camera and figures were created using Adobe Photoshop CS5.Representative specimens were deposited in the Helminth Collection of the Laboratory of Wild Animal Parasitology (LAPASIL) of the Universidade Federal de Pelotas  and in the Helminthological Collection of the Oswaldo Cruz Institute 39948).
Prevalence (P%), mean intensity of infection (MII) and mean abundance (MA) were estimated as described by Bush et al. (1997).The influence of host gender (males and females) and stage of maturation (juvenile and adults) in prevalence of cysthacanths were tested using Fisher's exact test (p ≤ 0.05) while the effect of those host factors in MII was analyzed by t-test (p≤ 0.05) using the Quantitative Parasitology software (QPweb) (Reiczigel et al., 2019).

Checklist elaboration
The checklist used information compiled from Google Scholar, Pubmed, and Scielo.The databases used words such as: "Centrorhynchus and/or Acanthocephalans in South America"; "Centrorhynchus and/or Acanthocephalans in vertebrates of Brazil" (Argentina, Uruguay, Chile, Paraguay, Bolivia, Peru, Ecuador, Colombia, Venezuela, Guyana e Suriname); checklist of the Centrorhynchus and/or Acanthocephalans in South America".
Each record includes the name of the Centrorhynchus species, the authority and year.Host species are recorded in alphabetical order and, where possible, followed by the locality of the record in parenthesis, site of infection and life stage.The references are indicated in chronological order.The abbreviations for all localities cited in the work are:

Centrorhynchus cystacanths in Aquarana catesbeiana
Two hundred and seventy-five cystacanths of Centrorhynchus sp. were found in the stomach musculature (4 host) and celomic cavity (16 host) of the anurans.The overall prevalence was 55.1%, while the mean intensity and mean abundance of infection were 17.1 helminths/host and 9.4, respectively.There was no significant difference in the prevalence and mean intensity of cystacanth infection between males and females of A. catesbeiana (Table 1).However, the prevalence of helminths in adult bullfrogs was significantly higher than in juvenile (Table 2).

Centrorhynchus species in South America
We found 14 taxa of Centrorhynchus and 106 species of vertebrate hosts recorded in nine South American countries.The first records date back to the beginning of the 20 th century, with a greater number of reports published from the 2000s (see checklist).
We list 28 species of birds in association with at least one Centrorhynchus species.Accipitridae and Strigidae have been reported as hosts for five taxa (Table 3), while Accipitridae (nine taxa) is the group with the greatest diversity of birds recorded in association with Centrorhynchus spp.(Figure 2).Mammals (eight species) have were recorded as hosts for five Centrorhynchus taxa (Table 3).Centrorhynchus tumidulus (Rudolphi, 1819) was found in different groups of definitive hosts (6 birds families and 1 mammal family) predominating in number of host species in South American countries (Figure 3 and Table 3).

Discussion
Centrorhynchus spp.have been recorded as parasites of A. catesbeiana in the Americas only within the native range of distribution the bullfrog (Brandt, 1936;Hollis, 1972).The rates of infection with Centrorhynchus cystacanths in A. catesbeiana in southern Brazil were higher than those observed by Hollis (1972) (P % = 2.89; MII = 1) in the bullfrog's native range in Texas (USA).However, the prevalence was lower than that recorded by Brandt (1936) (P % = 81.8;MII = 13.90) in the eastern USA.The variations observed in infection rates may be associated with the characteristics of each location, where different factors (biotic and abiotic) may or may not favor transmission and infection rates in A. catesbeiana.
The higher prevalence of cystacanths in adult anurans may be associated with the predatory potential of larger frogs, and due to the fact that older anurans had longer exposure to infective forms and have larger body area for parasite colonization (Muzzall, 1991;Quiroga et al., 2015).Muzzall (1991) and Andrews et al. (1992) observed in native areas that larger individuals of A. catesbeiana showed greater parasite diversity.
Predators of adult specimens of the bullfrog are scarce in the regions where it has been introduced because of its large size (Maneyro & Carreira, 2012).However, in south America, carnivorous mammals such as Lycalopex gymnocercus and Cerdocyon thous (Canidae), and birds such as Pitangus sulphuratus (Tyrannidae), Rupornis magnirostris (Accipitridae), Guira guira (Cuculidae) and Athene cunicularia (Strigidae) are species that which include medium to large-sized anurans as part of their diet (Gatti et al., 2006;Vieira & Teixeira, 2008;Repenning et al., 2009;Rocha et al., 2011;Rodrigues et al., 2011;Soave et al., 2008;Maneyro & Carreira, 2012;Schalk & Morales, 2012;Corrêa et al., 2013) and that have been recorded as definitive hosts of Centrorhynchus spp.(Table 3).This suggests that these mammals and birds may prey on adults specimens A. catesbeiana and thus contribute to the continuity of Centrorhynchus species' life cycle.Aquarana catesbeiana can also participate in the Centrorhynchus sp.cycle as a primary transport host, which can be preyed upon by a secondary transport host, and the latter is then consumed by the definitive host.Silva & Ribeiro (2009) reported occurrences of A. catesbeiana being preyed upon by L. latrans, which has been recorded as a host for Centrorhynchus sp.cystacanths (Table 4).Leptodactylus latrans is a food resource for birds such as Rupornis magnirostris (Accipitridae) and Tyto furcata (Strigidae) (Maneyro & Carreira, 2012;Brentano et al., 2020), which are definitive hosts for Centrorhynchus species (Table 3).In this context, anurans play an important role in the transmission chain of species within this group of Acanthocephala.
The checklist presented here has expanded the information of previously published lists (Travassos, 1926;Smales, 2007a, b;Ávila & Silva, 2010;Lunaschi & Drago, 2010;Smales, 2013;Campião et al., 2014;Fugassa, 2015;Cuellar et al., 2022), as it has brought together new records for South America, where birds are the definitive hosts most frequently parasitized by Centrorhynchus spp.The reports of cysthacanths of Centrorhynchus sp.predominated in paratenic hosts in this region, which do not reach the adult stage in amphibians and reptiles, and do not have all their body structures fully formed, which makes identification at the species level difficult (Cabrera-Guzmán & Garrido-Olvera, 2014).The records show that many species of Centrorhynchus have yet to be described in South America and, in this regard, use of molecular tools and integrative taxonomic studies are important for the understanding of the diversity of species in this group.

Figure 2 .
Figure 2. A -Number of species reported as definitive hosts of Centrorhynchus spp.per host family.General Falconidae shape represent the avian and general Dasypodidae shape represent Mammal families; B -Number of species reported as paratenic hosts of Centrorhynchus spp.per host family in South America.General Tropiduridae shape represent the lizard families, snake shape represent snake families and Hylidae shape represent anuran families.Ranidae -record of the present study.

Figure 3 .
Figure 3. Species of Centorhynchus reported in definitive hosts by number of host families and number of host species.

Table 3 .
Records of bird and mammal hosts of Centrorhynchus(Lühe, 1911)species in South America.