Sean A. Locke

The genus Contracaecum comprises anisakid nematodes with complex life cycles involving aquatic intermediate hosts and piscivorous birds as definitive hosts. The black-crowned night heron ( Nycticorax nycticorax ) is a widespread ardeid species known to host diverse helminth communities, including Contracaecum spp., although species-level identification is often lacking. In this study, we describe a new species, Contracaecum fioravantii n. sp., based on an integrative morphological and molecular approach. Adult nematodes were collected from 3 N. nycticorax individuals in Israel and examined using light and scanning electron microscopy. The new species is characterized by a distinct combination of morphological features, including robust, subequal spicules, a sagittate tail tip in males, and a specific arrangement of caudal papillae and phasmids, distinguishing it from closely related taxa such as C. microcephalum and members of the C. rudolphii complex. Multivariate morphometric analyses supported its separation from congeners. Molecular characterization based on ITS (Internal Transcribed Spacers) rDNA and cox2 mtDNA sequences revealed high similarity with previously unidentified Contracaecum spp. from ardeid hosts in China, and phylogenetic analyses placed the new species in a well-supported, distinct clade. These findings highlight the hidden diversity within Contracaecum and underscore the importance of combining morphological and molecular data for accurate species delimitation. The identification of C. fioravantii n. sp. contributes to a better understanding of host–parasite relationships in piscivorous birds and suggests a wider geographic distribution linked to migratory pathways of N. nycticorax .

The coffee bean weevil, Araecerus fasciculatus (Coleoptera, Curculionoidea, Anthribidae), is a cosmopolitan pest of over 100 stored agricultural commodities, with particular economic impact on coffee (Coffea arabica). Although two chromosome-level anthribid genomes have recently been released as part of the Darwin Tree of Life (DToL) project (Booth et al. 2024; Crowley et al. 2025), no functionally annotated genome has been available for the family. Here we present a draft genome assembly for A. fasciculatus, generated from PacBio HiFi long reads and processed through a three tiered metagenomic filtering pipeline to remove host plant (C. arabica) and microbial contamination. The final assembly spans 475 Mb across 3,617 scaffolds (N50 = 170 kb) with 88.5% BUSCO completeness (insecta_odb10) and only 3.1% duplication. Gene prediction with BRAKER2 identified 22,384 protein-coding genes, of which 11,783 received functional annotations through SwissProt similarity. Notably, we identified 92 cytochrome P450 (CYP) genes, including tandem gene clusters on two scaffolds (4 genes on ptg000464l, 5 genes on ptg001867l), suggestive of lineage-specific expansion through tandem duplication. Homology searches against Drosophila melanogaster caffeine-metabolizing P450s (CYP12D1, CYP6d5, CYP6a8) recovered strong matches (e-values 9.7e10-110 to 5.4e10-101, 33-38% identity). In stark contrast, comprehensive BLAST searches for bacterial caffeine N-demethylase genes (ndmA/B/C/D), which mediate caffeine degradation via horizontal gene transfer in the coffee berry borer Hypothenemus hampei (Scolytinae), returned zero hits across the A. fasciculatus genome, predicted proteome, and associated bacterial scaffolds. AlphaFold2 structure prediction of four top Araecerus P450 candidates produced high-confidence models (pLDDT 84.5-93.9, pTM 0.735-0.930) with conserved P450 catalytic motifs. Foldseek structural homology searches confirmed that all four candidates adopt cytochrome P450 folds (top hits: human CYP3A4, CYP3A7, CYP11A1; TM-scores 0.90-0.92; probability 1.000), with zero hits to bacterial Rieske-fold enzymes. Molecular docking of caffeine against these structures yielded binding affinities of -5.41 to -5.80 kcal/mol for the Araecerus candidates, comparable to or exceeding the -5.55 kcal/mol obtained for the experimentally validated Drosophila CYP6a8 and substantially stronger than the -3.70 kcal/mol for the bacterial NdmA structural outgroup (PDB: 6ICP). Phylogenetic analysis revealed that all four candidates have clear orthologs in two non-seed-feeding DToL anthribids (Pseudeuparius sepicola and Platystomos albinus), demonstrating that these P450 genes predate the dietary transition to caffeine-containing seeds. The Araecerus candidates predominantly belong to the CYP6 family (clan 3), whereas the primary Drosophila caffeine P450 CYP12D1 belongs to the mitochondrial clan, confirming convergent recruitment of different P450 subfamilies for caffeine metabolism. These results support the hypothesis that A. fasciculatus employs an insect-encoded, P450-mediated caffeine detoxification pathway fundamentally distinct from the bacterial horizontal gene transfer mechanism documented in Scolytinae. This represents convergent evolution of caffeine resistance via independent molecular strategies within Curculionoidea, and provides the first functionally annotated genomic resource for comparative studies across the Anthribidae.

Posthodiplostomum minimum (MacCallum, 1921) Dubois, 1936 (Digenea, Diplostomidae) was described from adult worms from a great blue heron ( Ardea herodias ; Ardeidae) in New York. Metacercariae have been widely reported from diverse freshwater fishes, despite early experimental work and recent molecular data indicating exclusive infection of minnows (Leuciscidae) in North America. We compared the type specimens of P. minimum to adults taken from domestic chicks ( Gallus gallus domesticus ) fed metacercariae from fathead minnow ( Pimephales promelas ; Leuciscidae), as well as adults in naturally infected ardeid hosts. Sequences of cytochrome c oxidase I (CO1) showed these specimens were conspecific with worms from leuciscids and ardeids sampled across North America. However, adult morphology of the species with metacercariae in North American leuciscids differed markedly from the type specimens of P. minimum. Consequently, we resurrect and transfer to Posthodiplostomum Dubois, 1936, the earliest name for the lineage infecting leuciscids in North America: Posthodiplostomum vancleavei (Agersborg, 1926) n. comb. In North America, the four most likely junior synonyms of P. minimum are Posthodiplostomum aztlanensis González-García, López-Jiménez, Ortega-Olivares, Sereno-Uribe, Pérez-Ponce de León et García-Varela, 2024, Posthodiplostomum dawnsherryae Achatz, Von Holten, Pritchard et Keel, 2025, Posthodiplostomum nanum Dubois, 1937, and Posthodiplostomum seminolense Achatz, Von Holten, Pritchard et Keel, 2025. Sequences of CO1 indicate P. vancleavei n. comb. has been introduced in Japan and we discuss the high likelihood of its establishment in Europe. The complete rDNA operon and near-complete mitochondrial genome sequence of P. vancleavei show expected phylogenetic affiliation with Posthodiplostomum centrarchi Hoffman, 1958.

The family Didymozoidae is a speciose group of fish parasites with unusual morphologic and life-history characters. In the Caribbean, juvenile didymozoids are common in demersal fishes such as snappers (Lutjanidae), but few deoxyribonucleic acid (DNA) sequences are available from these developmental stages in this region. In the present study, 1 complete and 1 partial juvenile didymozoid from a snapper (Lutjanus sp.) from Puerto Rico were characterized morphologically, and the complete ribosomal DNA operon and mitochondrial genome were sequenced. The specimens resembled the allomonilicecum morphotype, and phylogenetic analysis indicated membership in the Didymodiclininae, which comprises the genera Didymodiclinus and Paragonapodasmius. The presence of a ventral sucker excludes Paragonapodasmius, which has also not been recorded in the Atlantic region. Identification of the specimen as a member of Didymodiclinus, which is known from the broader region of our study, was generally supported in phylogenetic analysis. Mitogenome phylogenies recovered Didymodiclinus with Azygiidae, consistent with the classification of these taxa within Hemiurata. However, the position of the Hemiurata within the Digenea fluctuated radically in mitogenome trees obtained with different analytic approaches, particularly depending on whether alignments were based on amino acid translations or nucleotides. The present data have value for the study of the distribution, life history, and diversity of these parasites in the Caribbean, particularly given the negative effects of didymozoids on highly valued fishes such as tunas and groupers.

La Laguna Cartagena es uno de los pocos cuerpos de agua dulce lacustre en la isla de Puerto Rico, y ha experimentado impactos ecológicos por especies invasoras y degradación del paisaje circundante. En este estudio, se recolectaron de la laguna los moluscos, peces y sus macroparásitos. Se encontraron caracoles de cinco familias, siendo Marisa cornuarietis (Linnaeus, 1758) la más abundante y Tarebia granifera (Lamarck, 1816) la predominante en un canal de entrada. Parachromis managuensis (Günther, 1867) fue el pez más abundante, seguido de Amatitlania nigrofasciata (Günther, 1867) y Poecilia sphenops (Valenciennes, 1846). Se identificaron ocho macroparásitos, incluidos Clinostomum cichlidorum Sereno-Uribe, García-Varela, Pinacho-Pinacho & Pérez-Ponce de León, 2018 y Centrocestus formosanus (Nishigori, 1924), que representan nuevos registros para Puerto Rico. Todos los organismos identificados en la laguna no eran nativos, lo que resalta la condición perturbada de este cuerpo de agua. Aunque los datos anteriores son escasos, la biota de la laguna parece haber experimentado cambios radicales en las últimas décadas. Este estudio proporciona datos de referencia útiles para el monitoreo y la restauración de este hábitat, así como para el manejo de especies invasoras.

Hysteromorpha Lutz, 1931 is a small but broadly distributed genus of diplostomoidean digeneans parasitic as adults primarily in cormorants, but also reported from some other fish-eating birds. Their metacercariae were found in a variety of freshwater fishes as second intermediate hosts. Prior to this study, the genus included only 3 nominal species, 2 of them distributed in the Old World and 1 in the New World. We obtained sequences of partial mitochondrial cox1 gene and nuclear rDNA operon from new specimens collected in Europe, North and South America and used them for species comparisons and phylogenetic analysis. We also examined morphology of our newly collected specimens as well as museum specimens. Our analysis has demonstrated that at least 2 (likely 3) species of Hysteromorpha are distributed in the Americas. Hysteromorpha sp. previously sequenced from larval stages, clearly represents a new species named herein Hysteromorpha ostrowskiae n. sp. Achatz, Locke et Tkach. Morphology of adults of the new species was earlier described in sufficient detail under the name Hysteromorpha triloba (Rudolphi, 1819). Our analyses also suggest the presence of another unknown Hysteromorpha sp. in North America represented by cox1 sequence from southeastern Canada (GenBank JF769473), but no morphological vouchers are available for this species.

In this study, we describe a new species of Pseudoparacreptotrema (Allocreadiidae) from the mugilid Dajaus monticola collected in western Puerto Rico, where no allocreadiid has previously been reported, bringing the number of species in this genus to seven (five in D. monticola, two in Profundulus spp.). The new parasite species is distinguished from congeners by its overall size, oral-to-ventral sucker size ratio, pharynx size, cirrus sac, and oral lobe morphology, and by 0.64%–3.45% divergence in a 1019-bp alignment of 28S. We build on prior suggestions that the current concept of P. agonostomi likely includes multiple species and provide the first mitochondrial data (whole mitochondrial genome) as well as the complete nuclear rDNA array from Pseudoparacreptotrema to facilitate future phylogenetic work. Within the Allocreadiidae, phylogenetic analysis of mitochondrial genomes and 28S provides conflicting topologies for the placement of Pseudoparacreptotrema and Allocreadium. The 28S phylogeny of six species of Pseudoparacreptotrema resembles that of four lineages of D. monticola in that in both host and parasite, Pacific coastal lineages branch earliest, and a Caribbean lineage is more recently evolved.

Members of the genus Scaphanocephalus mature in accipitrids, particularly osprey, Pandion haliaetus, with metacercaria causing Black Spot Syndrome in reef fishes. In most of the world, only the type species, Scaphanocephalus expansus (Creplin, 1842) has been reported. Recent molecular studies in the Western Atlantic, Mediterranean and Persian Gulf reveal multiple species of Scaphanocephalus, but have relied on 28S rDNA, mainly from metacercariae, which limits both morphological identification and resolution of closely related species. Here we combine nuclear rDNA with mitochondrial sequences from adult worms collected in osprey across North America and the Caribbean to describe species and elucidate life cycles in Scaphanocephalus. A new species described herein can be distinguished from S. expansus based on overall body shape and size. Phylogenetic analysis of the whole mitochondrial genome of Scaphanocephalus indicates a close relationship with Cryptocotyle. We conclude that at least 3 species of Scaphanocephalus are present in the Americas and 2 others are in the Old World. Specimens in the Americas have similar or identical 28S to those in the Mediterranean and Persian Gulf, but amphi-Atlantic species are unlikely in light of divergence in cytochrome c oxidase I and the lack of amphi-Atlantic avian and fish hosts. Our results provide insight into the geographic distribution and taxonomy of a little-studied trematode recently linked to an emerging pathology in ecologically important reef fishes.