PHYLOGENETIC POSITION OF NEOGOGATEA (DIPLOSTOMOIDEA; CYATHOCOTYLIDAE)
ABSTRACT
Neogogatea Chandler and Rausch, 1947, is a small genus of cyathocotylid digeneans parasitic in the intestines of birds of prey. The genus contains only 3 species: 2 from the Nearctic and 1 from the Palearctic. No sequence data from mature, properly identified representatives of Neogogatea were available until now. In the present study, we collected mature adult Neogogatea kentuckiensis (Cable, 1935) Hoffman and Dunbar, 1963, from osprey in Georgia, Florida, and Delaware, and sequenced fragments of the nuclear large ribosomal subunit (28S) gene and cytochrome c oxidase subunit I (COI) mtDNA gene. We used 28S sequence data for phylogenetic inference and COI data for comparisons at the species level. In the phylogenetic tree, N. kentuckiensis was situated in a clade with representatives of the genera Mesostephanus Lutz, 1935, Braunina Wolf, 1903, and an unidentified cyathocotylid cercaria from Australia. The observed variability in the COI gene between our isolates suggests the need for collecting fresh, well-fixed, and mature specimens from a broader geographic range to test for the presence of more than 1 Neogogatea species in New World osprey.
Neogogatea Chandler and Rausch, 1947, is a small genus of cyathocotylids parasitic as adults in the intestines of birds of prey in the Nearctic and Palearctic (Chandler and Rausch, 1947, 1948; Hoffman and Dunbar, 1963; Zazornova, 1995). Chandler and Rausch (1947) erected the genus for Neogogatea bubonis Chandler and Rausch, 1947, from great horned owl Bubo virginianus (Gmelin) collected from Wisconsin. Dwivedi (1967) considered the genus to be synonymous with Gogatea Lutz, 1935, based on morphology. Dubois (1975) maintained Neogogatea and Gogatea as separate genera. Only 3 species are currently recognized: (1) N. bubonis (type species); (2) Neogogatea kentuckiensis (Cable, 1935) Hoffman and Dunbar, 1963 (syn. Neogogatea pandionis Chandler and Rausch, 1948) from osprey Pandion haliaetus in the United States (Chandler and Rausch, 1948; Hoffman and Dunbar, 1963); and (3) Neogogatea rauschi Zazornova, 1995, from white-tailed eagle Heliaetus albicilla (L.) in Russia (Zazornova, 1995).
Achatz et al. (2019) generated the first DNA sequence attributed to a species of Neogogatea; however, it originated from an immature specimen collected from a hooded merganser Lophodytes cucullatus (L.) in Mississippi. More recently, Sokolov et al. (2024) demonstrated a close phylogenetic affinity between Neogogatea sp. of Achatz et al. (2019) and Paracoenogonimus ovatus Katsurada, 1914, which suggests that Neogogatea sp. may have been misidentified. In the present study, we collected and sequenced fragments of the nuclear large ribosomal subunit (28S) gene, internal transcribed spacer 2 (ITS2) region, and cytochrome c oxidase subunit I (COI) mtDNA gene of mature adult N. kentuckiensis from osprey in the United States. Molecular data combined with reevaluation of morphological characters, such as the presence or absence of ventral sucker, relative size of pharynx to oral sucker, and the presence or absence of the vaginal sphincter, allowed us to confirm the status of Neogogatea and Gogatea as separate genera as well as analyze relationships of Neogogatea with other cyathocotylid taxa.
MATERIALS AND METHODS
Neogogatea kentuckiensis specimens were obtained from 2 ospreys collected from Lake Seminole, Silver Lake Wildlife Management Area, Decatur County, Georgia (30°48′55.6″N, 84°45′28.9″W). Live digeneans were rinsed in saline, killed in hot water, and immediately fixed in 80% ethanol. Additional specimens were obtained from frozen carcasses of ospreys in Sussex County, Delaware (38°35′29.4″N, 75°17′28.7″W) and Sumter County, Florida (28°55′37.2″N, 82°00′13.7″W), donated by the Tri-State Bird Rescue and Rehabilitation, the Clinic of the Rehabilitation of Wildlife (CROW), and the Audubon Center of Birds of Prey. After thawing, dead digeneans were removed from the intestine and immediately fixed in 80% ethanol. Freshly fixed, high-quality specimens from Georgia were used in the morphological and molecular study, while those from Delaware and Florida were used for only the molecular study.
Specimens used for light microscopy were stained with aqueous alum carmine according to the protocol of Lutz et al. (2017) and studied using a DIC-equipped Olympus BX53 microscope (Olympus Corp., Tokyo, Japan). Voucher specimens are deposited in the Museum of Southwestern Biology Division of Parasites (Table I).
DNA was extracted from entire specimens according to the protocol provided by Tkach and Pawlowski (1999). The fragments of 28S and ITS2 rDNA, as well as COI mtDNA, were amplified by polymerase chain reactions (PCR). The PCR amplifications of 28S were performed using the forward primer digL2 (5′-AAG CAT ATC ACT AAG CGG-3′) and reverse primer 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) (Tkach et al., 2003); the ITS2 region was amplified using forward primer d58f (5′-GCG GTG GAT CAC TCG GCT CGT G-3′) and reverse primer 300R (5′-CAA CTT TCC CTC ACG GTA CTT G-3′) (Snyder and Tkach, 2007; Kudlai et al., 2015); the COI fragment was amplified using forward primer Dipl_Cox_5′ (5′-ACK TTR GAW CAT AAG CG-3′) and 1 of the following reverse primers Dipl650R (5′-CCA AAR AAY CAR AAY AWR TGY TG-3′) or Dipl_Cox_3′ (5′-WAR TGC ATN GGA AAA AAA CA-3′) (Achatz et al., 2021). PCRs were carried out in a total volume of 25 μl using One-Taq quick load PCR mix from New England Biolabs (Ipswich, Massachusetts) with annealing temperatures of 53 C (28S) and 45 C (COI).
The PCR amplicons were purified using an Illustra ExoProStar PCR clean-up enzymatic kit (Cytiva, Marlborough, Massachusetts) and cycle sequenced using a BrightDye Terminator Cycle Sequencing Kit (MCLAB, South San Francisco, California). Sequencing reactions were purified using a BigDye Sequencing Clean Up Kit from MCLAB, then run on an ABI 3130 automated capillary sequencer (Thermo Fisher Scientific, Waltham, Massachusetts). The PCR primers were used for sequencing reactions. Contiguous sequences were assembled using Sequencher 4.2 software (GeneCodes Corp., Ann Arbor, Michigan) and deposited in GenBank (Table I).
The 28S sequence of N. kentuckiensis was aligned with 24 previously published sequences of cyathocotylids using ClustalW as implemented in MEGA7 software (Kumar et al., 2016). The alignment was trimmed to the length of the shortest sequence included in the analysis (1,085 bp); 38 nucleotide positions with ambiguous homology were excluded from the analyses. Harmotrema laticaudae Yamaguti, 1933, was used as the outgroup in the analysis, based on the tree topology published by Pérez-Ponce de León and Hernández-Mena (2019). The general time-reversible model with estimates of invariant sites and gamma-distributed among-site variation (GTR + G + I) model was determined to be the best-fitting nucleotide substitution model using MEGA7. The phylogenetic analysis was conducted using Bayesian Inference as implemented in MrBayes v3.2.6 software (Ronquist and Huelsenbeck, 2003) with Markov chain Monte Carlo (MCMC) chains run for 3,000,000 generations with a sample frequency set at 1,000. The length of analysis was considered sufficient because the standard deviation stabilized below 0.01. Log-likelihood scores were plotted, and only the final 75% of trees were retained to produce the consensus trees.
RESULTS
Molecular phylogeny
The topology and supports of the phylogeny resulting from the analysis of 28S were essentially identical to those published in recent studies (Fig. 1; Achatz et al., 2024; Sokolov et al., 2024). Neogogatea kentuckiensis was situated in a weakly supported clade as a sister species to an unknown cyathocotylid cercaria from Australia. These 2 taxa were positioned as a clade in a 99% supported polytomy that also included the clades of Braunina spp. (100% supported) and Mesostephanus spp. (100% supported).
Citation: The Journal of Parasitology 111, 6; 10.1645/24-152
Similar to the results of Sokolov et al. (2024), in our phylogenetic tree, the Neogogatea sp. of Achatz et al. (2019) was positioned away from N. kentuckiensis in a 100% supported clade together with P. ovatus. These 2 species were situated in a 99% supported larger clade that also included Holostephanoides ictaluri (Vernberg, 1952) and Gogatea spp. (Fig. 1).
DISCUSSION
Neogogatea kentuckiensis was originally described as Cercaria kentuckiensis Cable, 1935, based on cercariae from Pleurocera semicarinata (Say) (referred to as Goniobasis semicarinata) in Kentucky (Cable, 1935). Cercaria kentuckiensis was later considered N. kentuckiensis. Several subsequent authors (Anderson, 1944; Vernberg, 1952; Hoffman, 1959; Myer, 1960; Hoffman and Dunbar, 1963) have collected and described all life stages of N. kentuckiensis from various hosts (first intermediate host: pleurocerid snails Pleurocera and Leptoxis spp.; second intermediate host: centrarchid, cyprinid, and salmonid fishes; definitive host: osprey). Hoffman and Dunbar (1963) reported moderate to severe pathogenicity as well as mortality caused by N. kentuckiensis in centrarchid and salmonid fishes, particularly young individuals.
We would like to address some of the controversies related to certain morphological features of Neogogatea spp. and their taxonomic value. Chandler and Rausch (1947, 1948) stated that Neogogatea spp. lack a ventral sucker. In these digeneans, the holdfast organ often partially or entirely overlaps the ventral sucker, which could lead researchers to mistakenly assume the ventral sucker is missing. Hoffman and Dunbar (1963) provided descriptions of cercarial, metacercarial, and adult N. kentuckiensis and noted that the ventral sucker was apparent in metacercariae, but only feebly developed (but present) in adults. Similarly, our specimens have a feebly developed, or essentially absent, ventral sucker (Fig. 2a, b, d). This situation is reminiscent of that in the closely related genus Gogatea. Although Gogatea spp. typically have a ventral sucker, at least 1 species, Gogatea anacetabulata Achatz, Von Holten, Binh and Tkach, 2024, does not have it (Achatz et al., 2024). Hence, the presence or absence of the ventral sucker cannot be considered among the features that can be used to reliably distinguish between these genera.
Citation: The Journal of Parasitology 111, 6; 10.1645/24-152
Dubois (1975) noted that Gogatea spp. tend to have a pharynx similar in size or smaller than the oral sucker, while the pharynx in Neogogatea spp. is larger than the oral sucker. However, in the drawings by Hoffman and Dunbar (1963), 1 of the specimens (shown laterally) has pharynx larger than the oral sucker, while in the other specimen (positioned dorso-ventrally) these structures are similar in size. Likewise, the oral sucker and pharynx of our specimens of N. kentuckiensis are similar in size (Fig. 2a–c). Thus, similar to the presence or absence of the ventral sucker, this character also cannot the used to clearly differentiate between Gogatea and Neogogatea.
Furthermore, Dubois (1975) wrote that Gogatea spp. lack a vaginal sphincter (see Achatz et al., 2024), while Neogogatea spp. have a weakly developed but visible vaginal sphincter (our Fig. 2f). Although this structure is difficult to observe unless specimens are of high quality, it seems to be the main morphological feature that allows for reliable differentiation between the 2 genera. Otherwise, the adults of Gogatea and Neogogatea are morphologically similar. Despite their morphological similarity, Neogogatea spp. are known to parasitize only birds as definitive hosts, while all adult Gogatea spp. are parasitic in snakes (Chandler and Rausch, 1947, 1948; Dubois, 1975; Achatz et al., 2024).
Myer (1960) considered N. kentuckiensis to belong to Mesostephanus Lutz, 1935, based on adult morphology, notably the presence of a vaginal sphincter. However, other authors (e.g., Hoffman and Dunbar, 1963; Dubois, 1989) rejected this transfer. In our phylogenetic analysis, Neogogatea is well separated from both Gogatea and Mesostephanus (Fig. 1). Neogogatea appeared in a polytomy that otherwise consisted of marine cyathocotylids (i.e., Braunina and Mesostephanus spp.; Fig. 1); the unknown cercariae positioned in the polytomy were collected from the channeled cerith, Clypeomorus batillariaeformis Habe and Kosuge, 1966, a marine mollusk (Huston et al., 2018). The topology in this clade suggests either (1) a single transition from marine to freshwater habitats in the case of Neogogatea, or, less likely, (2) 3 independent transitions to marine habitats in the case of Braunina and Mesotephanus spp., as well as the unknown cercaria.
In our molecular phylogeny (Fig. 1), N. kentuckiensis appeared to be well separated from Neogogatea sp. of Achatz et al. (2019), which strongly suggests that these species represent separate genera. Recently, Sokolov et al. (2024) redescribed and sequenced P. ovatus (the type species of the genus) from Russia. Based on their data, Neogogatea sp. of Achatz et al. (2019) and P. ovatus differ by only 0.8% in partial 28S sequences, similar to the intrageneric variation exhibited by other cyathocotylid genera, e.g., Braunina Wolf, 1903 (up to 0.2%), Mesostephanus (up to 2.7%), or Gogatea (up to 3.7%) (Achatz et al., 2019, 2024). Both P. ovatus and Neogogatea sp. of Achatz et al. (2019) have overall similar anatomy; however, P. ovatus possesses a ventral sucker, while Neogogatea sp. of Achatz et al. (2019) lacks a ventral sucker. Our phylogeny and pairwise comparison suggest that Neogogatea sp. of Achatz et al. (2019) is likely a member of Paracoenogonimus. It is almost certainly a new species because no Paracoenogonimus has been previously reported in the New World, nor do any known Paracoenogonimus lack a ventral sucker. Mature adults need to be collected to formally describe the species.
Based on comparison of partial COI sequences (Table II), our adult specimens are certainly conspecific with metacercariae from pumpkinseed Lepomis gibbosus (L.) and black crappie Pomoxis nigromaculatus (Lesueur) in Canada identified as Mesostephanus sp. by Locke et al. (2010). We conclude that the metacercariae collected and sequenced by Locke et al. (2010) represent N. kentuckiensis.
No variation was detected among our 28S or ITS2 sequences. However, we were unable to amplify 28S fragment from specimens of N. kentuckiensis collected in Florida and obtained only a shorter 28S sequence from specimens collected in Delaware; at the same time, we were able to obtain COI sequences from these isolates. Among 13 sequences of the barcoding COI region available for our comparison (Table II), our sequence of adult digeneans from Florida (PV755401) and a sequence of a metacercaria from Canada (HM064651) appear to represent a genetic lineage distinct from the remaining sequences. The 2 lineages identified in the present study exhibited 0.2 − 1.7% intra-lineage variation (lineage 1) and 1.1% intra-lineage variation (lineage 2) in the standard ‘barcoding’ region and 2.4–3.7% difference in the same DNA region between the lineages (Table II). At the same time, the longer COI fragment (1,089 bp) from the Delaware and Georgia (lineage 1) and Florida (lineage 2) isolates differed by only 2.0 − 2.1% of nucleotide positions. A collection of fresh, high-quality specimens of N. kentuckiensis lineage 2 is necessary for morphological and additional molecular study. DNA sequences of N. bubonis and N. rauschi are needed to explore the interrelationships among members of the genus.
Phylogenetic interrelationships among cyathocotylids based on Bayesian Inference analysis of partial 28S rDNA gene sequences. Posterior probabilities are provided above internodes; values below 80% are not shown. The new 28S sequence generated in this study is in bold. The scale bar indicates the number of substitutions per site. GenBank accession numbers are provided after the names of taxa. *Formerly identified as Neogogatea sp. of Achatz et al. (2019).
Neogogatea kentuckiensis collected in the present study. (a, b) Photographs of entire adult specimens, ventral view. (c) Anterior part of prosoma. (d) Weakly developed ventral sucker. (e) Posterior end of opisthosoma with the terminal part of the female reproductive tract shown. Note the weakly developed vaginal sphincter.
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