Stephen A. Bullard

Amyloodinium ocellatum causes epizootics in marine fish hatcheries. Frozen, wild fish, often used as food in aquaculture, could introduce the disease. We investigated the parasite's ability to survive, reproduce and infect a host after freezing. Infected gills were frozen at −20°C for either 0, 24, 36, 48, or 72 h. Parasite viability was assessed first by placing thawed infected gills into tanks with naïve fish. Gills of the exposed fish were examined on day 0 and on days 4 and 7 post‐exposure. Tomonts from infected gills in all freeze durations produced infections. However, dinospore production decreased and infections took longer to establish in fish exposed to tomonts from the 72 h treatment. Second, eight tomonts from each freeze duration were placed in 5 mL of 25 ppt artificial seawater at 22°C in individual wells of a 12‐well plate and monitored for hatching. Ninety seven percent of tomonts from all treatments hatched. Tomonts frozen for up to 72 h survived, reproduced and infected fish, but dinospore production decreased as freeze duration increased. Extrapolation from the rate of reduction in dinospore production over the freeze durations tested suggests that a freeze duration of 237 h could inactivate all tomonts.

We herein describe a new species of Cathariotrematinae Bullard, 2021 (Monogenoidea: Monocotylidae Taschenberg, 1879) and propose a new genus to accommodate it. Nasigulper itsgotatao n. gen., n. sp. infects the olfactory lamellae (nose) of the deepwater gulper shark, Centrophorus granulosus (Bloch and Schneider, 1801) (Squaliformes: Centrophoridae) from the north-central Gulf of Mexico outer continental shelf. The new species was assigned to Cathariotrematinae by having a 3-part attachment organ (TAO) and by lacking open loculi that symmetrically encircle a cluster of >2 loculi in the center of the haptor. The proposal of a new genus is warranted because the new species is the only known monocotylid that has a TAO comprising 3 closed loculi with 2 relatively small hamuli flanking the posteriormost loculus. The most morphologically similar cathariotrematine genus is represented by a single species (Squalotrema llewellyni Kearn and Greene, 1983) that infects the spiny dogfish, Squalus acanthias (Squaliformes: Squalidae) in the North Sea. Nasigulper itsgotatao differs from Squalotrema llewellyni by having a TAO comprising 3 closed loculi, relatively small hamuli flanking the posteriormost loculus, and a relatively short, sinous ovovitelline duct and by lacking depressions associated with the central loculus. Squalotrema llewellyni has a TAO comprising 3 supraloculi, hamuli that are equal to the length of the posteriormost loculus, a central loculus having a central depression flanked by 2 pear-shaped depressions anterolaterally, and an extensive ovovitelline duct that is sinuous sinistral to the proximal ovary. A phylogenetic analysis of all acceptable monocotylid 28S sequences recovered the new species sister to isolates of Cathariotrema selachii (MacCallum, 1916) Johnston and Tiegs, 1922 from the Gulf of Mexico (no sequence of a species of Squalotrema exists). The new genus and species are the sixth and eighth, respectively, added to Cathariotrematinae.

We provide a supplemental description of the type species for Cryptogonimus Osborn, 1903 (Digenea: Cryptogonimidae), Cryptogonimus chili Osborn, 1903, based on newly-collected, heat-killed, formalin-fixed specimens infecting rock bass, Ambloplites rupestris (Rafinesque), and smallmouth bass, Micropterus dolomieu Lacepède (both Centrarchiformes: Centrarchidae), from the Duck River, Tennessee (USA). We emend Cryptogonimus to include features observed in the present specimens of its type species and in the descriptions of its congeners: a broad (wider than long) oral sucker, an intestine that bifurcates in the posterior half of the forebody, a bipartite seminal vesicle, a hermaphroditic duct that is dorsal to the ventral sucker, a preovarian seminal receptacle, and a Laurer's canal that opens dorsally at the level of the anterior testis. We describe Caecincola duttonae sp. n. (Cryptogonimidae) infecting largemouth bass, Micropterus salmoides (Lacepède), from Neely Henry Reservoir (Coosa River, Alabama, USA). The new species differs from its congeners by having a combination of a less elongate body, an intestine that bifurcates at the level of the ventral sucker, caeca that terminate at the level of the testes, diagonal testes in the middle of the hindbody, and a vitellarium predominantly distributed in the hindbody. We emend Caecincola Marshall et Gilbert, 1905 (type species Caecincola parvulus Marshall et Gilbert, 1905) to include features of the new species and recently-described congeners: an elongate body, an intestine that bifurcates in the posterior half of the forebody, caeca that extend posteriad beyond the testes, tandem testes, and a vitellarium that is wholly or primarily in the hindbody. Our 28S and ITS2 phylogenetic analyses recovered Caecincola and Cryptogonimus as sister taxa; Caecincola was recovered as paraphyletic with 28S but monophyletic with ITS2. This is the first phylogenetic study of Cryptogonimidae that includes a nucleotide sequence for a species of the type genus Cryptogonimus. We regard Cryptogonimus diaphanus (Stafford, 1904) Miller, 1941 as a species inquirenda.

Diplomonorchis micropogoni Nahhas & Cable, 1964 was considered a junior subjective synonym of Diplomonorchis leiostomi Hopkins, 1941 in 1969. Diplomonorchis leiostomi has since been widely reported from the coastal Western Atlantic between Delaware Bay and southern Brazil. Until now, taxonomically verifiable DNA sequence data for D. leiostomi has been available from an individual worm collected from the northern Gulf of Mexico. We generated a partial sequence of the 28S rRNA gene from D. leiostomi from the spot croaker, Leiostomus xanthurus Lacepède (type-host) from Beaufort, North Carolina, USA (type-locality) that differed at 31 of 1,246 bases from the available 28S sequence. This prompted a reevaluation of Diplomonorchis spp. identities from the northern Gulf of Mexico. We found D. leiostomi and D. micropogoni distinguishable by testes shape and size, and to a lesser degree by relative caecal length. Museum specimens of D. leiostomi, identified from the Gulf of Mexico represent a species complex containing D. leiostomi, D. cf. micropogoni and, a new species of Diplomonorchis. The sequences previously identified as D. leiostomi in GenBank (AY222137 & AY222252) are herein identified as D. cf. micropogoni. The new species is described from newly collected material herein. Phylogenetic analysis of 28S rRNA sequences from the species complex plus 46 species from the Monorchioidea Odhner, 1911 indicated all three Diplomonorchis spp. are closely related and form a clade with some species of Lasiotocus Looss, 1907. With the addition of the new species, and acceptance of D. micropogoni, there are currently 14 valid species in Diplomonorchis.

We propose and describe Alobophora sandrae Cajiao-Mora & Bullard n. gen., n. sp. (Digenea: Caballerotrematidae) for specimens we collected from arapaima, Arapaima gigas sensu lato (Osteoglossiformes: Arapaimidae) in the Amazon River near Leticia, Colombia. Alobophora differs from Caballerotrema Prudhoe, 1960 by lacking head collar projections and by having clustered corner spines and a narrow head collar (4–5× wider than pharynx), whereas Caballerotrema has head collar projections, lacks clustered corner spines, and has a broad head collar (7–8× wider than pharynx). We reassign Caballerotrema annulatum (Diesing, 1850) Ostrowski de Núñez & Sattmann, 2002 to the new genus, as Alobophora annulata (Diesing, 1850) Cajiao-Mora and Bullard n. comb., and provide a supplemental description of Caballerotrema brasiliense Prudhoe, 1960 based on specimens we collected from arapaima. We also examined the holotype and a paratype of Caballerotrema piscicola (Stunkard, 1960) Kostadinova & Gibson, 2001 and concluded that C. piscicola is a junior subjective synonym of C. brasiliense. Our 28S phylogeny recovered A. sandrae sister to A. annulata, with that clade sister to a clade comprising C. brasiliense and an innominate species of Caballerotrema. Caballerotrematidae was recovered sister to Echinostomatidae. We also provide a dichotomous key to caballerotrematids based on head collar projections, corner spine arrangement, proportional pharynx and head collar breadth, testes shape and arrangement, body surface spine shape and distribution, vitellarium distribution, and abundance of prostatic cells.

During a parasitological survey of freshwater fishes in the Mekong River Delta, Vietnam, disporic plasmodia containing myxospores morphologically consistent with Ellipsomyxa Køie, 2003 (Bivalvulida) were observed infecting the gall bladder of Pangasius macronema Bleeker (Siluriformes: Pangasiidae). Herein, we use morphology and small subunit ribosomal DNA (SSU rDNA, 18S) sequence data to describe Ellipsomyxa intravesica Ksepka & Bullard n. sp. and relate it to other myxozoans. The new species resembles Ellipsomyxa adlardi Whipps & Font, 2013, which infects the naked goby, Gobiosoma bosc (Lacepede) (Gobiiformes: Gobiidae) in Lake Pontchartrain, Louisiana, but differs from it by having a longer myxospore (mean = 13.3; range = 12.0–15.0 vs. 12.4; 11.3–14.4) and shorter polar capsules (3.7; 3.0–4.0 vs. 4.3; 3.9–4.9). The 18S phylogenetic analysis recovered the sequence of the new species sister to those ascribed to Ellipsomyxa ariusi Chandran, Zacharia, Sathianandan & Sanil, 2020 and Ellipsomyxa sp. (MK561979); both of which infect the gall bladder of the threadfin sea catfish, Arius arius (Hamilton) (Siluriformes: Ariidiae) from the southwest coast of India. Consistent with previous phylogenetic analyses of Ellipsomyxa spp., Ellipsomyxa was recovered as monophyletic. The new species is the first species of Ellipsomyxa reported from a freshwater fish in Asia and the first myxozoan reported from P. macronema.

Many hexabothriid genera require renewed taxonomic attention to provide additional detailed comparative data from new material to resolve existing species quandaries. Our current study describes and provides additional museum material for three hexabothriid species from three genera from South Africa: Erpocotyle catenulata (Guberlet), which is confirmed as a valid species, Heteronchocotyle gymnurae Neifar, Euzet and Ben Hassine, and Hypanocotyle bullardi Chero, Cruces, Sáez, Carolina, Camargo, Portes Santos and Luque. Evidence of these three hexabothriid species from South Africa suggests that hexabothriids have more globally widespread distributions, and that they are less host-specific than previously considered. A detailed summary of hexabothriids reported from the triakid hosts, Galeorhinus galeus and Mustelus mustelus is provided, and the host-specificity of hexabothriids is discussed.

Herein, we use scanning and transmission electron microscopy to describe the foregut (mouth, pharyngeal canal, and associated epithelia and musculature) of an adult freshwater fish blood fluke, Sanguinicola volgensis (Rašín, 1929) McIntosh, 1934, infecting the blood of sabre, Pelecus cultratus Linnaeus, 1758 (Cypriniformes: Leuciscidae) from the Volga River, Russia. Our results indicate that S. volgensis has a pharynx and lacks an oral sucker and that its pharyngeal canal acts as a peristaltic pump that sucks blood into the esophagus, whereupon digestion commences with granules secreted from the esophageal epithelium. We saw no evidence of longitudinal muscle fibers beneath the pharyngeal canal epithelium, pharyngeal glands, or pharyngeal epithelial cells or muscle cells within the pharyngeal muscular complex; collectively indicating the presence of a pharynx rather than an oral sucker. The specialized epithelial lining associated with the mouth and pharyngeal canal evidently is unique among neodermatans; it is smooth, ∼40 nm thick anteriorly, and thickens (∼250–700 nm) posteriorly as the mouth cavity transitions into the pharyngeal canal. The pharyngeal canal epithelium has lumps of dense material resembling those of the basal lamina and fibrous coat of the tegument. The actin-like material within the pharyngeal cavity epithelium could provide structural support to the pharynx.

Turtle blood flukes (“spirorchiids”) comprise a paraphyletic assemblage including the monophyletic Schistosomatidae Stiles and Hassall, 1898 as a crown group. We herein morphologically diagnose the natural groups of turtle blood flukes and propose family names for them. Spirorchiidae Stunkard, 1921 (Spirorchis MacCallum, 1919 [type]; Spirhapalum Ejsmont, 1927; Plasmiorchis Mehra, 1934; Monticellius Mehra, 1939; Vasotrema Stunkard, 1928; provisionally Uterotrema Platt and Pichelin, 1994) has a ventral sucker (lost in Spirorchis), an esophageal gland surrounding the entire esophagus, lateral esophageal diverticula (plicate organ and medial esophageal diverticulum present or absent), a glandular mass at the esophagus base, paired ceca surrounded by vitelline follicles for their entire length, a non-filamented, ovoid egg, and typically a Manter's organ. Baracktrematidae n. fam. (Baracktrema Roberts, Platt, and Bullard, 2016 [type]; Unicaecum Stunkard, 1925; Neospirorchis Price, 1934) has no ventral sucker, a single cecum or cyclocoel, a coiled or looped testis extending into the anterior body half, a convoluted post-cecal and post-gonadal uterus, a uterine pouch, no metraterm, no Manter's organ, and non-filamented eggs. Plattidae n. fam. (Platt Roberts and Bullard, 2018 [type]; Hapalorhynchus Stunkard, 1922; Coeuritrema Mehra, 1933; Enterohaematotrema Mehra, 1940; Cardiotrema Dwivedi, 1967; Ruavermis Dutton and Bullard, 2020) has the anatomical sequence of a ventral sucker, external seminal vesicle, cirrus sac, anterior testis, ovary, transverse vitelline duct, and posterior testis (except Enterohaematotrema) plus a pars prostatica, dorsal genital pore, globular excretory vesicle, and no Manter's organ. Carettacolidae Yamaguti, 1958 (Carettacola Manter and Larson, 1950 [type]) has a spinose oral sucker, a spinose and contractile ventral sucker (transverse cavity present), numerous testes distributing in a post-ovarian, inter-cecal column, and an ovary and transverse vitelline duct that are pre-testicular. Hapalotrematidae (Stunkard, 1921) Poche, 1926 (Hapalotrema Loos, 1899 [type]; Amphiorchis Price, 1934; Learedius Price, 1934; Cheloneotrema Simha and Chattopadhyaya, 1980; Neocaballerotrema Simha, 1977; Satyanarayanotrema Simha and Chattopadhyaya, 1980; Shobanotrema Simha and Chattopadhyaya, 1980) has the anatomical sequence of a ventral sucker, anterior testis(es), external seminal vesicle and cirrus sac, ovary, and posterior testis(es). Atamatamidae n. fam. (Atamatam Bullard and Roberts, 2019 [type]; Paratamatam Bullard and Roberts, 2019; Pitiutrema Dutton and Bullard, 2019) has an oral sucker with anteroventral spines, vasa efferentia connecting to a pre-ovarian external seminal vesicle, a cirrus sac directed postero-laterad and having an internal seminal vesicle and glandular luminal wall, and a dendritic ovary flanked by the external seminal vesicle and oviducal seminal receptacle. A 28S phylogenetic analysis failed to reject the monophyly of these families.

We revise monotypic Leuceruthrus Marshall and Gilbert, 1905 (Azygiidae Lühe, 1909) by emending its generic diagnosis, redescribing its type species (Leuceruthrus micropteri Marshall and Gilbert, 1905), reassigning 2 species (Leuceruthrus stephanocauda [Faust, 1921] n. comb., Leuceruthrus ocalana [Smith, 1935] n. comb.), describing 2 new congeners (Leuceruthrus ksepkai n. sp. and Leuceruthrus blaisei n. sp.), and providing a phylogenetic analysis based on the internal transcribed spacer 2 (ITS2). Leuceruthrus is unique by having oblique preovarian testes, a vitellarium that does not extend anteriad into the forebody, and a uterus that is intercecal and between the ovary and ventral sucker. We describe the cercaria of Leuceruthrus cf. stephanocauda from cercariae shed from Elimia cf. carinifera and Elimia cf. modesta from Big Canoe Creek, Alabama. Leuceruthrus ksepkai n. sp. is described from cercariae shed from rasp elimia, Elimia floridensis (Reeve, 1860) from Holmes Creek, Florida, and Elimia sp. 1 from the Chocktawhatchee River, Florida. It differs from its congeners by the combination of having broadly rounded furcae with slight marginal pigmentation in live cercariae, no spines on the tail stem, distinct anterior and posterior ridges that flank the tail stem portion accommodating the withdrawn distome, minute protuberances occupying the lateral margin of the tail stem for its entire length, and protuberances that encircle the anterior third of the posterior tail stem (immediately posterior to the tail stem portion containing the withdrawn distome). Leuceruthrus blaisei n. sp. infects Elimia sp. and is the only known congener having proportionally small furcae (>tail stem maximum width) with a nipple-like distal projection and numerous minute projections on the tail stem surface. Our phylogenetic analysis included all of our new sequences plus all publicly available ITS2 sequences for Leuceruthrus spp. and Proteromera spp., and supported the monophyly of Leuceruthrus. It recovered L. ksepkai and L. cf. ksepkai in a polytomy, and Leuceruthrus blaisei as monophyletic (identical sequences), with L. micropteri and L. cf. stephanocauda recovered as sister taxa. The present study comprises the first systematic treatment of Leuceruthrus in over a century, the first description of a new species of Leuceruthrus in 117 yr, and the first taxonomic characterization of a species of Leuceruthrus from the Mobile River Basin. It also increases the number of accepted congeners from 1 to 5 and provides new host records (E. cf. carinifera and E. floridensis) for Leuceruthrus spp.