Linking metacercariae and adults of Microphallus basodactylophallus (Digenea: Microphallidae), based on larval stages from ctenophores and adult parasites from aquatic birds found in Mexico

Members of the genus Microphallus Ward, 1901, are endoparasites mainly of birds and mammals distributed worldwide. Unencysted metacercariae of Microphallus sp., were collected from the mesoglea of ctenophores of the genus Pleurobrachia Fleming; adult digeneans were recovered from the intestines of Eudocimus albus Linnaeus (Threskiornithidae) and Buteogallus urubitinga Gmelin (Accipitridae), in four locations from southeastern Mexico. Adult specimens were identified as M. basodactylophallus (Bridgman, 1969) based on the following features: body pyriform entirely covered by minute spines, prepharynx short, oesophagus very long, caeca short and widely divergent, testes slightly symmetrical and excretory vesicle short and V-shaped. Sequences from D1–D3 domain of the large subunit of ribosomal DNA (LSU) were generated, aligned, and compared with those of congeneric species available in GenBank. Phylogenetic analyses indicated that the metacercariae and adults formed a clade together with an isolate identified as M. basodactylophallus from Florida, USA (GenBank: AY220628). The intraspecific genetic divergence among isolates was low ranged from 0.0% to 0.6%, allowing the link between the two stages of the life cycle. We observed phenotypic plasticity in the morphological traits of M. basodactylophallus adults in definitive hosts (mammals and birds) throughout the distribution, which ranged from the USA to southeastern Mexico. Finally, the unencysted metacercariae identified as M. basodactylophallus represent the first report of a microphallid in ctenophores.


Introduction
Digeneans are the most diverse group of parasites recorded in wildlife from Mexico.To date, 624 species have been recorded and classified into 311 genera and 78 families with a high percentage of endemicity, representing 30% of the biodiversity of digeneas.However, the inventory of digeneans in the country is far from complete (Pérez-Ponce de León et al. 2007), and Mexico could be considered a hotspot of diversity because it occupies a transitional position between Nearctic and Neotropical biogeographical regions (Morrone, 2006;Pérez-Ponce de León et al., 2007).The genus Microphallus Ward, 1901 contains approximately 45 species distributed worldwide parasitizing birds and occasionally mammals (Deblock, 2008;Galaktionov & Blasco-Costa, 2018).In Mexico, metacercariae of Microphallus sp. were recorded in the bulldog goodeid (Alloophorus robustus Bean) from lakes of central Mexico.However, the records could not be verified because the specimens are not available in any collection (see Pérez-Ponce de León et al., 2007).Two species of Microphallus were described from Mexico, i.e., M. muellhaupti Coil, 1955, from a shorebird (Tringa sp.) and M. trilobatus Cable and Kuntz, 1951, from a roadside hawk (Rupornis magnirostris Gmelin) (see Pérez-Ponce de León et al., 2007).
The species M. basodactylophallus (Bridgman, 1969) Deblock, 1971, was described from the intestine of a racoon (Procyon lotor Linnaeus) in North America (see Bridgman, 1969), and since then, this microphallid has been recorded in other definitive hosts, the marsh rice rat (Oryzomys palustris Harlan) (Heard & Overstreet, 1983).The life cycle of M. basodactylophallus is well known; adult worms live and reproduce sexually in the digestive tract of their definitive hosts.Mature eggs are expelled in the environment with the faeces of the definitive host.Later, a gastropod mollusc serves as the first intermediate host, and crabs, crayfishes, and shrimp serve as second intermediate hosts, which are eaten by the definitive hosts, completing the life cycle (see Bridgman, 1969;Heard & Overstreet, 1983;Gehman et al., 2021).
During a study of parasites infecting invertebrates and vertebrates from southeastern Mexico, unencysted metacercariae of Microphallus sp., were found in the mesoglea of ctenophores (Pleurobrachia sp.) at a single locality, and adult specimens were recovered from the intestine of two bird species at three localities.A detailed morphological study of both stages and comparative analyses based on sequences of domains D1-D3 of the large subunit of ribosomal DNA allowed the link between these two stages.The current study provides a morphological and molecular characterization of the larval and adult specimens identified as M. basodactylophallus, a generalist parasite of mammals and birds distributed from the USA to southeastern Mexico.

Morphological analyses
The specimens were stained with Mayer's paracarmine (Merck, Darmstadt, Germany), dehydrated in an ethanol series, cleared in methyl salicylate and mounted in Canada balsam for morphological analysis.Specimens were then examined using a compound microscope equipped with a bright field (Leica DM 1000 LED microscope, Leica, Wetzlar, Germany).Measurements were taken using Leica Application Suite microscope software (Leica Microsystems GmbH, Wetzlar, Germany) and are given in micrometres as range followed by mean in parentheses.Some specimens from each locality were dehydrated in ethanol series, critical-point dried, sputter coated with gold, and examined with a Hitachi Stereoscan Model S-2469N scanning electron microscope operating at 15 kV.Adults and metacercariae were deposited in the Colección Nacional de Helmintos (CNHE) of the Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), México City (CNHE. No. 12063-12066).

Morphological identification
Adult digeneas found in two bird species in southeastern Mexico were identified as M. basodactylophallus, based on similar morphology present in the original description of the species, by Bridgman (1969), which includes, (i) body pyriform; (ii) tegument covered entirely with minute spines; (iii) prepharynx short or absent; (iv) oesophagus very long; (v) caeca short, widely divergent; (vi) testes slightly symmetrical; and (vii) excretory vesicle short, V-shaped.The present specimens were somewhat divergent form the type material of M. basodactylophallus regarding the morphometry of body, oral sucker, ventral sucker, pharynx, oesophagus, testes and eggs (see Table 1).

Phylogenetic analyses
The newly generated sequences (five adults and three metacercariae) from the LSU were analysed together with 35 published sequences from 29 species, forming an alignment of 1,279 sites.The best evolutionary model was GTR+I+G.This dataset included species of the genera Microphallus, Maritrema Nicoll, 1907, Levinseniella Ward, 1901and Longiductotrema Deblock & Heard, 1969 belonging to Microphallidae, plus sequences of three representatives belonging to Haematoloechus Looss, 1899, Plagiorchis Lühe, 1899 and Telorchis Lühe 1899 used as outgroups (Fig. 5), since they have a common ancestry with microphallids (see Kudlai et al., 2015;Galaktionov & Blasco-Costa, 2018).The newly obtained LSU sequences formed a fully supported, monophyletic assemblage, with that of M. basodactylophallus (GenBank: AY220628), recovered from the marsh rice rat (Oryzomys palustris) in the USA.This clade was sister to a lineage formed by several other congeners with high nodal support (Fig. 5).The interspecific genetic divergence estimated among the species of Microphallus spp., ranged from 1.1 to 7%, whereas the genetic divergence estimated among adults and metacercariae identified as M. basodactylophallus ranged from 0.0 to 0.06%.

Discussion
To the best of our knowledge, two species of the genus Microphallus (M.muellhaupti Coil, 1956, andM. trilobatus Cable &Kuntz, 1951) have been recorded in Mexico (see Pérez-Ponce de León et al., 2007).The present finding of metacercaria and adults of M. basodactylophallus infecting ctenophores (Pleurobrachia sp.), white ibises (E.albus) and great black hawks (B.urubitinga) represent new hosts and locality records for this species of parasite expanding its geographic occurrence distribution from USA to southeastern Mexico.The species M. basodactylophallus was originally described in the racoon (Procyon lotor) from southern Louisiana (Bridgman, 1969).However, type specimens of M. basodactylophallus were obtained through experimental infection of a rat (Rattus norvegicus albinus Berkenhout) (Bridgman, 1969) and, since its description, the parasite has been recorded in Florida, Mississippi, Alabama and Georgia, (all USA) (Heard & Overstreet, 1983), which may explain the intraspecific morphometric variation observed.
The life cycle of M. basodactylophallus is well known; i.e., the snails Lydores parvula (Guilding), Littoridinops monroensis Frauenfeld and Hydrobia sp., serve as the first intermediate hosts.The cercaria emerge, swim, and penetrate into crabs (blue crab Callinectes sapidus Rathbun, red-jointed fiddler crab Uca minax (Le Conte), Atlantic marsh fiddler crab Minuca pugnax (Smith) and gulf marsh fiddler crab (Minuca longisignalis) Salmon & Atsaides), where metacercaria develop and encyst.Finally, the second intermediate host with metacercariae is eaten by the definitive host, in which the adult stage occurs Vol.: (0123456789) (Bridgman, 1969;Heard & Overstreet, 1983).In the present study, unencysted metacercariae identified as M. basodactylophallus were found in the mesoglea of ctenophores of Pleurobrachia sp., with a prevalence value of 7% (376 examined hosts/26 hosts parasitized) and intensity of 1-6 per infected host.Metacercariae occur in a wide variety of planktonic and benthonic animals, such as ctenophores and hydromedusas, which serve as second intermediate hosts for digeneas of the superfamilies Gymnophalloidea, Hemiuroidea and Lepocreadioidea (Marcogliese, 1995;Martorelli, 2001;Cribb et al., 2003;Moradini et al., 2005).The evidence found in the present study suggests that the cercaria actively penetrate the ctenophore, losing their tails and persisting inside host's mesoglea as unencysted metacercaria.The unencysted metacercaria of M. basodactylophallus in ctenophores could be consider as an accidental infection.However, is well known that ctenophores may be an important food in fish diet (Mianzan et al., 1996(Mianzan et al., , 2001) ) and most likely, act as a trophic link for the parasites in the way to reach its definitive host.
The inclusion of molecular data in this study was crucial to link metacercariae and adults of M. basodactylophallus parasitizing invertebrates, mammals and aquatic birds from USA to southeastern Mexico.It was demonstrated that the body size, oral sucker, ventral sucker, pharynx, oesophagus, testes and eggs exhibit some morphometric variations (see Table 1).Therefore, the use of molecular markers is necessary to link development stages of digeneas, as well as for the description and delimitation of species of Microphallus.The present phylogenetic analyses performed with the LSU datasets confirmed that Microphallus is monophyletic and shares a common ancestor with Maritrema with low values of support.However, the near relationship between both genera is consistent with the results of previous phylogenetic analyses of microphallids (Kudlai et al., 2015;Galaktionov & Blasco-Costa, 2018).
The ecological evidence suggests that microphallids show low host specificity regarding the definitive host (see Caveny & Etges 1971;Deblock, 2008).In the present study, was found that M. basodactylophallus can parasitize both mammals and aquatic birds, favouring its distribution and dispersion along coasts in the Neotropical region.The unencysted metacercariae of M. basodactylophallus represent the first report of a microphallid in ctenophores from southeastern Mexico.special thanks to Dra.Alejandra López Jimenez, Dr. Leopoldo Andrade Gómez and M. C. Tonatiuh González García for their help during the fieldwork.We thank to Dra.Mirza Patricia Ortega Olivares for her help with the drawing of the specimens.JLNS thank the support CONACHyT (CVU.No. 96544) for granting a scholarship to complete his PhD program.

Fig. 4
Fig. 4 Scanning electron micrographs of Microphallus basodactylophallus.Adult forms (A, E, I); specimens collected from the intestine of Buteogallus urubitinga from Playa Paraiso, Tabasco (A) and from Tupilco, Tabasco (E); specimen collected from the intestine of Eudocimus albus from Tlacotalpan, Veracruz (I); unencysted metacercariae collected from the

Fig. 5
Fig. 5 Consensus Bayesian inference and Maximum likelihood trees inferred with the LSU dataset.Numbers near internal nodes show ML bootstrap percentages/Bayesian posterior probabilities.◂

Table 1
Comparative metrical data of Microphallus basodactylophallus; number of host examined/infected (prevalence of infection)