The genus Ecteinascidia (family Perophoridae) comprises colonial ascidians typically found in warm seas, known for their biomedical significance. Four species have been recorded to date in the Mediterranean, but the records remain patchy due to the similar external morphology of these species and limited taxonomic data. This study presents the first record of the ascidian Ecteinascidia turbinata from Cyprus, extending its Mediterranean range, where it is currently regarded as cryptogenic. Here, we combine morphological and genetic data to compare E. turbinata from Cyprus with congeneric Ecteinascidia thurstoni specimens from Israel, an Indo-Pacific species introduced into the Mediterranean through the Suez Canal, highlighting the key diagnostic features and genetic divergence between the two species. Although the two species resemble each other in external appearance, E. turbinata and E. thurstoni differ in key diagnostic traits, including gut loop shape (open C vs. compact C), zooid length (15-22 mm vs. 4-7 mm), and the number of stigmata rows (17-20 vs. 12-14), respectively. Genetic data from Cypriot specimens indicate close affinity with both neighboring Mediterranean and Atlantic populations, suggesting these areas as possible sources of introduction. Based on the literature, the two species contend with similar environmental conditions, suggesting a potential niche overlap. Future monitoring of Mediterranean ascidian fauna may help to clarify this, especially in the context of global change an its influence on marine biodiversity and species distribution. Our findings also resolve past misidentifications and underscore the value of integrative taxonomy for reliable tracking of ascidian bioinvasions.

Sponges harbor microbial communities that play crucial roles in host health and ecology. However, the genetic adaptations that enable these symbiotic microorganisms to thrive within the sponge environment are still being elucidated. To understand these genetic adaptations, we conducted a comparative genomics analysis on 350 genomes of Actinobacteriota, a phylum commonly associated with sponges. Our analysis uncovered several differences between symbiotic and free-living bacteria, including an increased abundance of genes encoding prokaryotic defense systems (PDSs) and eukaryotic-like proteins (ELPs) in symbionts. Furthermore, we identified GPP34 as a novel symbiosis-related gene family, found in two symbiotic Actinobacteriota clades, but not in their closely related free-living relatives. Analyses of a broader set of microbes showed that members of the GPP34 family are also found in sponge symbionts across 16 additional bacterial phyla. While GPP34 proteins were thought to be restricted to eukaryotes, our phylogenetic analysis shows that the GPP34 domain is found in all three domains of life, suggesting its ancient origin. We also show that the GPP34 family includes genes with two main structures: a short form that includes only the GPP34 domain and a long form that encompasses a GPP34 domain coupled with a cytochrome P450 domain, which is exclusive to sponge symbiotic bacteria. Given previous studies showing that GPP34 is a phosphatidylinositol-4-phosphate (PI4P)-binding protein in eukaryotes and that other PI4P-binding proteins from bacterial pathogens can interfere with phagolysosome maturation, we propose that symbionts employ GPP34 to modulate phagocytosis to colonize and persist within sponge hosts.

Myxozoans are parasitic cnidarians that can cause severe damage to fish, resulting in economic losses to aquaculture and fisheries. In Israel, only a few taxonomic studies have been conducted on Myxozoa infecting freshwater fish and none on barb parasites. Here, we describe two new myxozoan species - Myxidium grauri n. sp. and Myxidium sharmai n. sp. - from the gallbladder of the barbs Carasobarbus canis and Luciobarbus longiceps, respectively, from the Sea of Galilee (Lake Kinneret). The prevalence of infection was 42.2% (19/45) for M. grauri n. sp. and 25% (5/20) for M. sharmai n. sp. We obtained 18S rRNA sequences for both species, providing the first molecular data on Myxidium infecting barbs from the Sea of Galilee. Phylogenetic analysis confirmed that the genera Myxidium and Zschokkella are not monophyletic. The δ-statistic was used to assess the phylogenetic signal of categorical traits within the Biliary Tract Clade IV lineage, which includes the species studied. The analysis revealed a significant phylogenetic signal associated with the host clade, the parasite's geographic origin and the type of environment it inhabits. However, some of these statistical results may be influenced by sampling bias, as Percomorpha and Otomorpha fish are disproportionately represented in marine and freshwater environments, respectively. The newly obtained sequences form a distinct lineage within a clade of freshwater-infecting myxozoans. Our findings suggest that myxozoan infections are widespread in the Sea of Galilee. Given their potential impact on fisheries and the lake's ecosystem, further research is needed to assess their distribution, dynamics and ecological consequences.

Ethanol, a natural by-product of sugar fermentation, can be found in various fruits and nectar. Although many animals routinely consume ethanol in low concentrations as part of their natural diets, its inherent toxicity can cause severe damage. Even species particularly well adapted to ethanol consumption face detrimental effects when exposed to concentrations above 4%. Here, we investigated the metabolism of ethanol and its impact on survival and behavior in the Oriental hornet ( Vespa orientalis ), a social wasp that naturally consumes ethanol. We show that chronic ethanol consumption, even at concentrations as high as 80%, had no impact on hornet mortality, construction behavior, or agonistic behavior. Using 13 C1 labeled ethanol, we show that hornets efficiently metabolized ingested ethanol and at a much higher rate than honey bees. The presence of multiple copies of the alcohol dehydrogenase (NADP+) gene in the Vespa genera suggests a potential mechanism for ethanol tolerance. These findings support the hypothesis that the mutualistic relationship between ethanol-producing organisms and vespid hosts may be at the origin of their remarkable capacity to utilize and metabolize ethanol.

BACKGROUND:Myxozoa is a class of cnidarian parasites that encompasses over 2,400 species. Phylogenetic relationships among myxozoans remain highly debated, owing to both a lack of informative morphological characters and a shortage of molecular markers. Mitochondrial (mt) genomes are a common marker in phylogeny and biogeography. However, only five complete myxozoan mt genomes have been sequenced: four belonging to two closely related genera, Enteromyxum and Kudoa, and one from the genus Myxobolus. Interestingly, while cytochrome oxidase genes could be identified in Enteromyxum and Kudoa, no such genes were found in Myxobolus squamalis, and another member of the Myxobolidae (Henneguya salminicola) was found to have lost its entire mt genome. To evaluate the utility of mt genomes to reconstruct myxozoan relationships and to understand if the loss of cytochrome oxidase genes is a characteristic of myxobolids, we sequenced the mt genome of five myxozoans (Myxobolus wulii, M. honghuensis, M. shantungensis, Thelohanellus kitauei and, Sphaeromyxa zaharoni) using Illumina and Oxford Nanopore platforms.RESULTS:Unlike Enteromyxum, which possesses a partitioned mt genome, the five mt genomes were encoded on single circular chromosomes. An mt plasmid was found in M. wulii, as described previously in Kudoa iwatai. In all new myxozoan genomes, five protein-coding genes (cob, cox1, cox2, nad1, and nad5) and two rRNAs (rnl and rns) were recognized, but no tRNA. We found that Myxobolus and Thelohanellus species shared unidentified reading frames, supporting the view that these mt open reading frames are functional. Our phylogenetic reconstructions based on the five conserved mt genes agree with previously published trees based on the 18S rRNA gene.CONCLUSIONS:Our results suggest that the loss of cytochrome oxidase genes is not a characteristic of all myxobolids, the ancestral myxozoan mt genome was likely encoded on a single circular chromosome, and mt plasmids exist in a few lineages. Our findings indicate that myxozoan mt sequences are poor markers for reconstructing myxozoan phylogenetic relationships because of their fast-evolutionary rates and the abundance of repeated elements, which complicates assembly.

Sequencing the mitochondrial genome of the tunicate Oikopleura dioica is a challenging task due to the presence of long poly-A/T homopolymer stretches, which impair sequencing and assembly. Here, we report on the sequencing and annotation of the majority of the mitochondrial genome of O. dioica by means of combining several DNA and amplicon reads obtained by Illumina and MinIon Oxford Nanopore Technologies with public RNA sequences. We document extensive RNA editing, since all homopolymer stretches present in the mitochondrial DNA correspond to 6U-regions in the mitochondrial RNA. Out of the 13 canonical protein-coding genes, we were able to detect eight, plus an unassigned open reading frame that lacked sequence similarity to canonical mitochondrial protein-coding genes. We show that the nad3 gene has been transferred to the nucleus and acquired a mitochondria-targeting signal. In addition to two very short rRNAs, we could only identify a single tRNA (tRNA-Met), suggesting multiple losses of tRNA genes, supported by a corresponding loss of mitochondrial aminoacyl-tRNA synthetases in the nuclear genome. Based on the eight canonical protein-coding genes identified, we reconstructed maximum likelihood and Bayesian phylogenetic trees and inferred an extreme evolutionary rate of this mitochondrial genome. The phylogenetic position of appendicularians among tunicates, however, could not be accurately determined.

MOTIVATION:Currently used methods for estimating branch support in phylogenetic analyses often rely on the classic Felsenstein's bootstrap, parametric tests, or their approximations. As these branch support scores are widely used in phylogenetic analyses, having accurate, fast, and interpretable scores is of high importance. RESULTS:Here, we employed a data-driven approach to estimate branch support values with a probabilistic interpretation. To this end, we simulated thousands of realistic phylogenetic trees and the corresponding multiple sequence alignments. Each of the obtained alignments was used to infer the phylogeny using state-of-the-art phylogenetic inference software, which was then compared to the true tree. Using these extensive data, we trained machine-learning algorithms to estimate branch support values for each bipartition within the maximum-likelihood trees obtained by each software. Our results demonstrate that our model provides fast and more accurate probability-based branch support values than commonly used procedures. We demonstrate the applicability of our approach on empirical datasets. AVAILABILITY AND IMPLEMENTATION:The data supporting this work are available in the Figshare repository at https://doi.org/10.6084/m9.figshare.25050554.v1, and the underlying code is accessible via GitHub at https://github.com/noaeker/bootstrap_repo.

Sequencing the mitochondrial genome of the tunicate Oikopleura dioica is a challenging task because of the presence of long poly-T/A homopolymer stretches, which impair sequencing and assembly. Here, we report the sequencing and annotation of the majority of the mitochondrial genome of O. dioica by combining several DNA and amplicon reads obtained by Illumina and MinIon Oxford Nanopore Technologies (ONT) with public RNA sequences. We document extensive RNA editing, since all homopolymer stretches present in the mitochondrial DNA correspond to 6U-regions in the mitochondrial RNA. Out of the 13 canonical protein-coding genes, we were able to detect eight plus an unassigned ORF, which lacked sequence similarity to canonical mitochondrial protein-coding genes. We showed that the nad3 gene has been transferred to the nucleus and acquired a mitochondria-targeting signal. In addition to two very short rRNAs, we could only identify a single tRNA (tRNA-Met), suggesting multiple losses of tRNA genes, supported by a corresponding loss of mitochondrial aminoacyl-tRNA synthetases in the nuclear genome. Based on the eight canonical protein-coding genes identified, we reconstructed maximum likelihood and Bayesian phylogenetic trees and inferred an extreme evolutionary rate of this mitochondrial genome. However, the phylogenetic position of appendicularians among tunicates could not be accurately determined. Significance Sequencing and annotating the mitochondrial genome of fast-evolving organisms is difficult because they often present unusual characteristics. The tunicate O. dioica is a model species for understanding tunicate and chordate genome evolution. However, no complete annotated mitochondrial genome for this species has been published to date. Here, we determined the major part of the mitochondrial genome of O. dioica . Our results indicate the presence of highly modified rRNA genes and the absence of all tRNAs except tRNA-Met. Moreover, we show that the mitochondrial genome undergoes editing at the RNA level. Our study demonstrates that a combination of public RNA data and DNA from long-and short-read sequencing platforms significantly improves our ability to study mitochondrial genomes with atypical characteristics. ### Competing Interest Statement The authors have declared no competing interest.

The number of species dispersing to higher and/or lower latitudes, in association with temperature warming, is growing exponentially with only a few described opposite cases of dispersal to warmer regions. Here we describe the dispersal of the solitary ascidian Ascidia virginea , considered native to the seas of North Europe, to disperse to warmer regions: the Eastern Mediterranean and a Red Sea marina. This case highlights an emerging trend of taxa being introduced by marine vessels and successfully establishing populations in unfavourable environmental conditions by opportunistically utilizing niche areas. We provide molecular and morphological data that facilitate the identification of A. virginea in regions in which it may have previously been overlooked. We also employ ecological-niche modeling to project habitat suitability for this species, predicting the coasts of North America, South America, the northwest Pacific, and the Red Sea as moderately and highly suitable habitats. Phylogenetic analyses based on the mitochondrial COI gene reveal that the A. virginea sequences obtained in this work belong to a well-supported clade, including previously published sequences of A. virginea from California and Florida, localities where its presence was not predicted by our model. Furthermore, publicly available COI sequences of A. malaca and A. sydneiensis are assigned to the same A. virginea clade, implying a case of synonymy or misidentification. Our results provide useful data for the accurate identification of this species when expanding into other areas in which it may become a nuisance, and highlight the need to further explore the potential of cold-water species to establish in ports and niche areas in warmer regions.

In the last decade, advances in sequencing technology have led to an exponential increase in genomic data. These new data have dramatically changed our understanding of the evolution and function of genes and genomes. Despite improvements in sequencing technologies, identifying contaminated reads remains a complex task for many research groups. Here, we introduce GenomeFLTR, a new web server to filter contaminated reads. Reads are compared against existing sequence databases from various representative organisms to detect potential contaminants. The main features implemented in GenomeFLTR are: (i) automated updating of the relevant databases; (ii) fast comparison of each read against the database; (iii) the ability to create user-specified databases; (iv) a user-friendly interactive dashboard to investigate the origin and frequency of the contaminations; (v) the generation of a contamination-free file. Availability: https://genomefltr.tau.ac.il/.

Mullets (Mugilidae) are economically important fish in Israel. Two species of mugilids (i.e., the thinlip mullet Chelon ramada and the flathead grey mullet Mugil cephalus) have been stocked in the Sea of Galilee (Lake Kinneret) in order to increase fishermen's income and lake water quality. These catadromous species do not reproduce in the lake, consequently, fingerlings have been introduced every year since 1958. Following a survey of myxozoan infections in the Sea of Galilee, we described Myxobolus pupkoi n. sp. infecting the gill arches, and reported Myxobolus exiguus from visceral peritoneum and gall bladder of C. ramada. The prevalence of infection of both Myxobolus pupkoi n. sp. and M. exiguus were 11.5% (2/23). Our study indicates that the parasites infecting C. ramada belong to a lineage of myxozoans infecting mugilids. This result suggests that the infection took place in the Mediterranean Sea, where the fingerlings were caught, before their introduction into the Sea of Galilee. Since 2018 only farm-raised fingerlings have been introduced. We thus recommend to closely monitor the presence of these parasites in the future to determine if the presence of parasites disappear with the introduction of farm-raised fingerlings.

Symbiotic associations, widespread in terrestrial and marine ecosystems, are of considerable ecological importance. Many tropical coral species are holobionts, formed by the obligate association between a cnidarian host and endosymbiotic dinoflagellates of the family Symbiodiniaceae. The latter are abundant on coral reefs from very shallow water down to the upper mesophotic zone (30-70 m). The research on scleractinians has revealed that the photosymbiont lineages present in the cnidarian host play an important role in the coral's ability to thrive under different environmental conditions, such as light regime and temperature. However, little is known regarding octocoral photosymbionts, and in particular regarding those found deeper than 30 m. Here, we used ribosomal (ITS2) and chloroplast (23S) markers to uncover, for the first time, the dominant Symbiodiniaceae taxa present in 19 mesophotic octocoral species (30-70 m depth) from the Gulf of Aqaba/Eilat (northern Red Sea). In addition, using high-throughput sequencing of the ITS2 region we characterized both the dominant and the rare Symbiodiniaceae lineages found in several species across depth. The phylogenetic analyses of both markers were in agreement and revealed that most of the studied mesophotic octocorals host the genus Cladocopium. Litophyton spp. and Klyxum utinomii were exceptions, as they harbored Symbiodinium and Durusdinium photosymbionts, respectively. While the dominant algal lineage of each coral species did not vary across depth, the endosymbiont community structure significantly differed between host species, as well as between different depths for some host species. The findings from this study contribute to the growing global-catalogue of Cnidaria-Symbiodiniaceae associations. Unravelling the Symbiodiniaceae composition in octocoral holobionts across environmental gradients, depth in particular, may enable a better understanding of how specialized those associations are, and to what extent coral holobionts are able to modify their photosymbionts.

Bupropion is an efficacious non-nicotine medication for smoking cessation; however, its cessation-mediating mechanism is unclear. This randomized, placebo-controlled trial examined the effect of bupropion SR (300 mg/day for 6 weeks) on plasma cotinine and on the subjective effects of smoking in 24 current daily smokers who were not trying to quit or reduce smoking. Subjective effects of smoking, as well as cue-elicited responses were assessed at bi-weekly experimental sessions using validated scales. Several indices of cigarette consumption were measured. Plasma cotinine decreased from 280 (± 133) μg/l at baseline to 205 (± 108) μg/l at end of treatment in the bupropion group (p = 0.036), but no significant change was found in the placebo group. Daily cigarette count and puff topography did not significantly change in either group. In contrast to placebo, bupropion increased post-smoking satiety (p = 0.045). Both groups reported higher craving (p = 0.025) and withdrawal (p = 0.014) after exposure to smoking-related pictures, compared to neutral pictures. This biased reactivity was not significantly affected by treatment condition (p > 0.1). Therefore, bupropion does not appear to impact the smokers' response to conditioned smoking-related cues but influences the unconditioned subjective effects of smoking in unmotivated smokers. This study is among the first to systematically investigate the effect of chronic bupropion administration, free from the confounding effect of the smoker's motivation to quit smoking.

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.

We described two novel myxozoan parasite species Ceratomyxa argentina n. sp. and Ceratomyxa raneyae n. sp. from the gall bladder of Raneya brasiliensis (Kaup) from the Patagonian coast of Argentina. Both species can be distinguished from other ceratomyxids by myxospore and polar capsule (nematocyst) morphology and morphometry, fish host and geographic locality. Phylogenetic reconstruction using ssrDNA gene sequences showed that the two new species are placed in a long-branching ceratomyxid clade which also include Ceratomyxa appendiculata Thélohan, 1892, Ceratomyxa anko Freeman, Yokoyama and Ogawa, 2008, Ceratomyxa pantherini Gunter, Burger and Adlard, 2010 and Pseudoalataspora kovalevae Kalavati, MacKenzie, Collins, Hemmingsen and Brickle, 2013. This study documents additional biodiversity of marine myxozoans in the South Atlantic, a region still largely unexplored for this group of parasitic cnidarians.

Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications. To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer's barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer's fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer's fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.

Extensive regenerative ability is a common trait of animals capable of asexual development. The current study reveals the extraordinary regeneration abilities of the solitary ascidian Polycarpa mytiligera. Dissection of a single individual into separate fragments along two body axes resulted in the complete regeneration of each fragment into an independent, functional individual. The ability of a solitary ascidian, incapable of asexual development, to achieve bidirectional regeneration and fully regenerate all body structures and organs is described here for the first time. Amputation initiated cell proliferation in proximity to the amputation line. Phylogenetic analysis demonstrated the close affinity of P. mytiligera to colonial species. This evolutionary proximity suggests the ability for regeneration as an exaptation feature for colonial lifestyle. P. mytiligera’s exceptional regenerative abilities and phylogenetic position highlight its potential to serve as a new comparative system for studies seeking to uncover the evolution of regeneration and coloniality among the chordates.

The planktonic Oikopleura dioica belongs to Tunicata, the probable sister taxon to Craniota, and might show plesiomorphic characters, conserved from the common lineage of Tunicata and Craniota. In O. dioica a pericardium in a position similar to other chordates but also to the heart and pericardium of craniates is found. Surprisingly, little is known about the ultrastructure of the pericardium in O. dioica . Here, we show based on electron microscopy that the pericardium is completely lined by a single layer of 16 epithelial cells: 6 epithelial myocardial cells on the left side of the pericardium and 10 peritoneal cells constituting the right side. One of the peritoneal cells, situated at the ventral border between peritoneal cells and myocardial cells has an extension that anchors the pericardium to the basal lamina beneath the latero-ventral epidermis. The primary body cavity of O. dioica appears quite uniformly clear in electron microscopic aspect but several sheets, resembling the basal lamina of the pericardium cross the larger spaces of the body cavity and connect to the pericardial basal lamina. This is the first detailed description of two distinct cell types in the epithelial lining of the pericardium of O. dioica. In comparison with other chordates, we conclude that two cell types can be reconstructed for the last common ancestor of Chordata at least. The position of the pericardium at the intersection of trunk and tail in combination with the basal-lamina like sheets spanning the hemocoel is probably of importance for the function of the circulation of the hemocoelic fluid. Similar to the tail, the axis of the pericardium is shifted through 90 degrees to the left as compared to the main body axis of the trunk and we infer that this shift is an apomorphic character of Appendicularia.

The number of shrew species in Israel has been and still is the subject of debate. In this work we used for the first time a molecular marker, the cytochrome b gene, to investigate the number and identity of shrew species in Israel. Our molecular results confirmed the presence of four species: Crocidura leucodon, Crocidura suaveolens gueldenstaedtii, Crocidura ramona, and Suncus etruscus. The C. ramona sequences were found to differ from all other Crocidura species sequenced to date, supporting its status as a distinct species. Whether it is conspecific with Crocidura portali (described in 1920 from Israel and usually synonymized with C. suaveolens), will require additional study. The sequences of Israeli C. suaveolens were found to be very similar to those of Iran, Turkey, and Georgia (i.e., C. suaveolens gueldenstaedtii), in agreement with previous studies. The Israeli C. leucodon sequences, however, formed a distinct clade among C. leucodon. Finally, the S. etruscus sequences clustered with sequences from France, Italy, and Iran.

Although aerobic respiration is a hallmark of eukaryotes, a few unicellular lineages, growing in hypoxic environments, have secondarily lost this ability. In the absence of oxygen, the mitochondria of these organisms have lost all or parts of their genomes and evolved into mitochondria-related organelles (MROs). There has been debate regarding the presence of MROs in animals. Using deep sequencing approaches, we discovered that a member of the Cnidaria, the myxozoan Henneguya salminicola, has no mitochondrial genome, and thus has lost the ability to perform aerobic cellular respiration. This indicates that these core eukaryotic features are not ubiquitous among animals. Our analyses suggest that H. salminicola lost not only its mitochondrial genome but also nearly all nuclear genes involved in transcription and replication of the mitochondrial genome. In contrast, we identified many genes that encode proteins involved in other mitochondrial pathways and determined that genes involved in aerobic respiration or mitochondrial DNA replication were either absent or present only as pseudogenes. As a control, we used the same sequencing and annotation methods to show that a closely related myxozoan, Myxobolus squamalis , has a mitochondrial genome. The molecular results are supported by fluorescence micrographs, which show the presence of mitochondrial DNA in M. squamalis , but not in H. salminicola. Our discovery confirms that adaptation to an anaerobic environment is not unique to single-celled eukaryotes, but has also evolved in a multicellular, parasitic animal. Hence, H. salminicola provides an opportunity for understanding the evolutionary transition from an aerobic to an exclusive anaerobic metabolism.