Population-level immunologic variation in wild threespine stickleback (Gasterosteusaculeatus).

Wild organisms are regularly exposed to a wide range of parasites, requiring the management of an effective immune response while avoiding immunopathology. Currently, our knowledge of immunoparasitology primarily derives from controlled laboratory studies, neglecting the genetic and environmental diversity that contribute to immune phenotypes observed in wild populations. To gain insight into the immunologic variability in natural settings, we examined differences in immune gene expression of two Alaskan stickleback (Gasterosteus aculeatus) populations with varying susceptibility to infection by the cestode Schistocephalus solidus. Between these two populations, we found distinct immune gene expression patterns at the population level in response to infection with fish from the high-infection population displaying signs of parasite-driven immune manipulation. Further, we found significant differences in baseline immune gene profiles between the populations, with uninfected low-infection population fish showing signatures of inflammation compared to uninfected high-infection population fish. These results shed light on divergent responses of wild populations to the same parasite, providing valuable insights into host-parasite interactions in natural ecosystems.

Distinct evolutionary lineages of Schistocephalus parasites infecting co-occurring sculpin and stickleback fishes in Alaska.

Sculpins (coastrange and slimy) and sticklebacks (ninespine and threespine) are widely distributed fishes cohabiting 2 south-central Alaskan lakes (Aleknagik and Iliamna), and all these species are parasitized by cryptic diphyllobothriidean cestodes in the genus Schistocephalus. The goal of this investigation was to test for host-specific parasitic relationships between sculpins and sticklebacks based upon morphological traits (segment counts) and sequence variation across the NADH1 gene. A total of 446 plerocercoids was examined. Large, significant differences in mean segment counts were found between cestodes in sculpin (mean = 112; standard deviation [s.d.] = 15) and stickleback (mean = 86; s.d. = 9) hosts within and between lakes. Nucleotide sequence divergence between parasites from sculpin and stickleback hosts was 20.5%, and Bayesian phylogenetic analysis recovered 2 well-supported clades of cestodes reflecting intermediate host family (i.e. sculpin, Cottidae vs stickleback, Gasterosteidae). Our findings point to the presence of a distinct lineage of cryptic Schistocephalus in sculpins from Aleknagik and Iliamna lakes that warrants further investigation to determine appropriate evolutionary and taxonomic recognition.

Description and pathology of a new genus and species of fish blood fluke (Digenea: Aporocotylidae Odhner, 1912) infecting white mullet, Mugil curema Valenciennes, 1836 (Mugiliformes: Mugilidae) in Mobile Bay (northern Gulf of Mexico) with a phylogenetic analysis.

Three fish blood flukes (Aporocotylidae Odhner, 1912) infect mullets (Mugiliformes: Mugilidae): Cardicola mugilis Yamaguti, 1970 and Plethorchis acanthus Martin, 1975 infect striped mullet, Mugil cephalus Linnaeus, 1758 in the Central Pacific Ocean (Hawaiian Islands) and Brisbane River (Australia), respectively; Cardicola brasiliensis Knoff & Amato, 1992 infects Lebranche mullet, Mugil liza Valenciennes, 1836 from the Southwestern Atlantic Ocean (Brazil). White mullets were cast-netted from the mouth of Deer River, a coastal saltmarsh of Mobile Bay, in the north-central Gulf of Mexico and examined for blood fluke infections. Specimens of Mugilitrema labowskiae Warren & Bullard n. gen., n. sp. were found infecting the endocardial surface and inter-trabecular spaces of the atrium, ventricle, and bulbous arteriosus. The new genus and species differ from all other aporocotylids by having the combination of two post-caecal testes, a uterus with straight ascending and descending portions, and a common genital pore. The 28S analysis recovered the new species and P.acanthus as sister taxa and Aporocotylidae as monophyletic. Carditis associated with intense infections comprised endocardial hyperplasia, resulting in a thickened cardiac endothelium. Probable dead or deteriorating eggs in the myocardium were encapsulated by granulomas composed of epithelioid histiocytes. Live eggs infected the afferent artery of gill filaments and were associated with varied hyperplasia of the overlying epithelium and haemorrhaging from the afferent artery in high-intensity infections. The new species is the first aporocotylid infecting a mullet from the northwestern Atlantic Ocean and only the second description of demonstrable endocarditis attributed to an adult fish blood fluke infection.

Morphology, DNA barcoding and seasonal occurrence of Ergasilus lizae Krøyer, 1863 (Copepoda: Ergasilidae) parasitizing mullets from northwestern Mexico.

Ergasilus lizae Krøyer, 1863 is a parasitic copepod known to infect mullets (Mugilidae) in different parts of the world. It was originally reported from the east coast of North America, but the original description lacks enough detail, making identification with this information difficult. In this study, we provide a redescription of E. lizae found on Mugil curema Valenciennes and M. cephalus Linnaeus, caught in two coastal lagoons of northwestern Mexico during two climatic seasons: warm/rainy and cold/dry. The prevalence of this parasite was higher in the warm season than in the cold season. To facilitate the species identification, new sequences of the barcoding gene (COI mtDNA) of E. lizae were generated and compared against unpublished sequences of E. lizae available in the Barcode of Life Database (BOLD). Our results suggest that the sequences of BOLD possibly belong to a species misidentified as E. lizae.

Frequency dependence limits divergent evolution by favouring rare immigrants over residents.

Two distinct forms of natural selection promote adaptive biological diversity. Divergent selection occurs when different environments favour different phenotypes, leading to increased differences between populations. Negative frequency-dependent selection occurs when rare variants within a population are favoured over common ones, increasing diversity within populations. These two diversifying forces promote genetic variation at different spatial scales, and may act in opposition, but their relative effects remain unclear because they are rarely measured concurrently. Here we show that negative frequency-dependent selection within populations can favor rare immigrants over locally adapted residents. We reciprocally transplanted lake and stream ecotypes of threespine stickleback into lake and stream habitats, while manipulating the relative abundance of residents versus immigrants. We found negative frequency-dependence: survival was highest for the locally rare ecotype, rather than natives. Also, individuals with locally rare major histocompatibility complex (MHC) class IIb genotypes were infected by fewer parasites. This negative frequency-dependent selection will tend to favour rare immigrants over common residents, amplifying the effect of migration and undermining the efficacy of divergent natural selection to drive population differences. The only signal of divergent selection was a tendency for foreign fish to have higher parasite loads than residents, after controlling for MHC genotype rarity. Frequency-dependent ecological interactions have long been thought to promote speciation. Our results suggest a more nuanced view in which negative frequency dependence alters the fate of migrants to promote or constrain evolutionary divergence between populations.

Host phenotype and microbiome vary with infection status, parasite genotype, and parasite microbiome composition.

A growing literature demonstrates the impact of helminths on their host gut microbiome. We investigated whether the stickleback host microbiome depends on ecoevolutionary variables by testing the impact of exposure to the cestode parasite Schistocephalus solidus with respect to infection success, host genotype, parasite genotype, and parasite microbiome composition. We observed constitutive differences in the microbiome of sticklebacks of different origin, and those differences increased when sticklebacks exposed to the parasite resisted infection. In contrast, the microbiome of successfully infected sticklebacks varied with parasite genotype. More specifically, we revealed that the association between microbiome and immune gene expression increased in infected individuals and varied with parasite genotype. In addition, we showed that S. solidus hosts a complex endomicrobiome and that bacterial abundance in the parasite correlates with expression of host immune genes. Within this comprehensive analysis we demonstrated that (i) parasites contribute to modulating the host microbiome through both successful and unsuccessful infection, (ii) when infection is successful, the host microbiome varies with parasite genotype due to genotype-dependent variation in parasite immunomodulation, and (iii) the parasite-associated microbiome is distinct from its host and impacts the host immune response to infection.

Context-dependent parasite infection affects trophic niche in populations of sympatric stickleback species.

How parasites alter host feeding ecology remains elusive in natural populations. A powerful approach to investigate the link between infection and feeding ecology is quantifying unique and shared responses to parasite infection in related host species within a common environment. Here, 9 pairs of sympatric populations of the three-spined and nine-spined stickleback fishes were sampled across a range of freshwater and brackish habitats to investigate how parasites alter host feeding ecology: (i) biotic and abiotic determinants of parasite community composition, and (ii) to what extent parasite infection correlates with trophic niche specialization of the 2 species, using stable isotope analyses (δ15N and δ13C). It was determined that parasite community composition and host parasite load varied among sites and species and were correlated with dissolved oxygen. It was also observed that the digenean Cyathocotyle sp.'s abundance, a common directly infecting parasite with a complex life cycle, correlated with host δ13C in a fish species-specific manner. In 6 sites, correlations were found between parasite abundance and their hosts' feeding ecology. These effects were location-specific and occasionally host species or host size-specific. Overall, the results suggest a relationship between parasite infection and host trophic niche which may be an important and largely overlooked ecological factor. The population specificity and variation in parasite communities also suggest this effect is multifarious and context-dependent.

The timing and development of infections in a fish-cestode host-parasite system.

The cestode Schistocephalus solidus is a common parasite in freshwater threespine stickleback populations, imposing strong fitness costs on their hosts. Given this, it is surprising how little is known about the timing and development of infections in natural stickleback populations. Previous work showed that young-of-year stickleback can get infected shortly after hatching. We extended this observation by comparing infection prevalence of young-of-year stickleback from 3 Alaskan populations (Walby, Cornelius and Wolf lakes) over 2 successive cohorts (2018/19 and 2019/20). We observed strong variation between sampling years (2018 vs 2019 vs 2020), stickleback age groups (young-of-year vs 1-year-old) and sampling populations.

Parasite identification in mullet fish (Mugil curema) from Chautengo Lagoon, Guerrero, Mexico, based on morphology and molecular analysis.

Parasite identification is crucial in areas where no sanitary inspection is conducted on fish, especially considering that parasitic zoonoses like anisakiasis and gnathostomiasis can pose a risk for human health. In this study, parasites in mullet fish (Mugil curema) from the Chautengo Lagoon, Guerrero, Mexico, were identified by morphological and molecular methods. A total of 122 specimens weighing 317 ± 51.25 g and 19.3 ± 1.14 cm in length were assessed. Their helminthofauna was classified by measuring internal structures, total length, and maximum width; a morphometric index was also calculated for larval stages. The prevalence of parasitosis in these mullets was 91.8%, with a mean infection intensity of 4.1. The acanthocephalan Floridosentis mugilis was identified by its external and internal structures. The nematodes found were of the Anisakidae family in stage 3 (L3), with a morphology consistent with Contracaecum sp. To determine the species, the ITS ribosomal gene and the mitochondrial genes COX2 and rrnS were molecularly characterized by PCR; then, they were aligned by CLUSTAL W, and a phylogenetic tree was obtained. In this analysis, the sequences were compared with those reported in GenBank. A total of 460 parasites were studied, 283 of which were nematodes (61.5%) and 177 were acanthocephalans (38.5%). The sequences of seven nematodes showed 99% homology with each other, and thus they formed an independent branch within the Contracaecum sp. group. This is the first report identifying Contracaecum multipapillatum in mullet fish in the Chautengo Lagoon, Guerrero.

Stickleback mass occurrence driven by spatially uneven parasite pressure? Insights into infection dynamics, host mortality, and epizootic variability.

Since 2012, a massive invasion of the three-spined stickleback (Gasterosteus aculeatus) has taken place into the pelagic area of Lake Constance. This species, which had previously been restricted to the littoral zone, is now the dominant pelagic fish and the previously dominant whitefish (Coregonus wartmanni) has suffered severe reductions in growth and recruitment. In this study, in total, 2871 sticklebacks were collected via monthly sessions over a 4-year period in pelagic and benthic areas of Lake Constance and examined for signs of infection with Schistocephalus solidus, a parasite known to be potentially fatal. The infection risk to sticklebacks increases throughout the course of the year and is size- and sex-dependent. Habitat has only a marginal impact. All parasite-induced harm is imparted after stickleback spawning and parental care is over. The results did not support the hypothesis that the invasion of the pelagic area might be driven by parasite-avoiding behaviour. Furthermore, the impact of the parasite is likely to be limited to post-reproductive adults, thereby ensuring stable reproduction of the hosts despite high rates of transmission and mortality. In consequence, stickleback stock development is independent of S. solidus infection, leading to secure coexistence of host and parasite even at extraordinary high host levels.

How cunning is the puppet-master? Cestode-infected fish appear generally fearless.

Trophically transmitted parasites have life cycles that require the infected host to be eaten by the correct type of predator. Such parasites should benefit from an ability to suppress the host's fear of predators, but if the manipulation is imprecise the consequence may be increased predation by non-hosts, to the detriment of the parasite. Three-spined sticklebacks (Gasterosteus aculeatus) infected by the cestode Schistocephalus solidus express reduced antipredator behaviours, but it is unknown whether this is an example of a highly precise manipulation, a more general manipulation, or if it can even be attributed to mere side effects of disease. In a series of experiments, we investigated several behaviours of infected and uninfected sticklebacks. As expected, they had weak responses to simulated predatory attacks compared to uninfected fish. However, our results suggest that the parasite induced a general fearlessness, rather than a precise manipulation aimed at the correct predators (birds). Infected fish had reduced responses also when attacked from the side and when exposed to odour from a fish predator, which is a "dead-end" for this parasite. We also tested whether the reduced anti-predator behaviours were mere symptoms of a decreased overall vigour, or due to parasite-induced hunger, but we found no support for these ideas. We propose that even imprecise manipulations of anti-predator behaviours may benefit parasites, for example, if other behaviours are altered in a way that increases the exposure to the correct predator.

First morphological and phylogenetic data on Ligophorus kaohsianghsieni (Platyhelminthes: Monogenea) from the Black Sea and the Sea of Japan and molecular evidence of deep divergence of sympatric Ligophorus species parasitizing Planiliza haematocheilus.

Ligophorus kaohsianghsieni (Gusev, ) Gusev, was collected from the so-iuy mullet Planiliza haematocheilus (Temminck & Schlegel, 1845) from the Black Sea and the Sea of Japan. DNA sequences data for L. kaohsianghsieni, as well as its morphological characters from the Sea of Japan were obtained for the first time. Significant morphometric and genetic diversity between specimens of L. kaohsianghsieni from the Black-Azov Sea region and the Sea of Japan were not found. For the first time, the molecular phylogeny of L. kaohsianghsieni based on three fragments of the nuclear DNA ribosomal cluster (18S, internal transcribed spacer 1 and 28S) was reconstructed. Molecular analysis of Ligophorus species from the Atlantic and Pacific Oceans revealed a significant phylogenetic distance between L. kaohsianghsieni and two others species (Ligophorus pilengas and Ligophorus llewellyni) from the same host (P. haematocheilus) and region. This result indicates the lack of correspondence between the phylogenetic and geographical closeness of the hosts and the relation of their parasites from the genus Ligophorus.

Marine parasite in a freshwater wetland: new host and geographical records of Progrillotia dasyatidis (Cestoda: Trypanorhyncha) from Gasterosteus aculeatus (Actinopterygii: Gasterosteidae) in Bulgaria, with comments on its life-cycle.

Plerocerci of the trypanorhynch cestode Progrillotia dasyatidis Beveridge, Neifar & Euzet, (Progrillotiidae) were isolated from the gallbladder of the three-spined stickleback Gasterosteus aculeatus from a small freshwater marsh in the Lake Atanasovsko Wetlands, a coastal area adjacent to the Black Sea coast, Bulgaria. The parasite was recorded in five out of 134 fish individuals studied (prevalence 3.73%, intensity 1-7, mean intensity 2.40 ± 1.17 and mean abundance 0.09 ± 0.06). A description of the plerocerci is presented, expanding data on intraspecific variation. The present report is the first record of P. dasyatidis from G. aculeatus (new host record) and from Bulgaria (new geographical record). Recording only plerocerci with evaginated scoleces in the sticklebacks is consistent with the hypothesis that teleosts are paratenic hosts and not intermediate hosts of P. dasyatidis.

Experimental Parasite Infection Causes Genome-Wide Changes in DNA Methylation.

Parasites are arguably among the strongest drivers of natural selection, constraining hosts to evolve resistance and tolerance mechanisms. Although, the genetic basis of adaptation to parasite infection has been widely studied, little is known about how epigenetic changes contribute to parasite resistance and eventually, adaptation. Here, we investigated the role of host DNA methylation modifications to respond to parasite infections. In a controlled infection experiment, we used the three-spined stickleback fish, a model species for host-parasite studies, and their nematode parasite Camallanus lacustris. We showed that the levels of DNA methylation are higher in infected fish. Results furthermore suggest correlations between DNA methylation and shifts in key fitness and immune traits between infected and control fish, including respiratory burst and functional trans-generational traits such as the concentration of motile sperm. We revealed that genes associated with metabolic, developmental, and regulatory processes (cell death and apoptosis) were differentially methylated between infected and control fish. Interestingly, genes such as the neuropeptide FF receptor 2 and the integrin alpha 1 as well as molecular pathways including the Th1 and Th2 cell differentiation were hypermethylated in infected fish, suggesting parasite-mediated repression mechanisms of immune responses. Altogether, we demonstrate that parasite infection contributes to genome-wide DNA methylation modifications. Our study brings novel insights into the evolution of vertebrate immunity and suggests that epigenetic mechanisms are complementary to genetic responses against parasite-mediated selection.

Shining a light on parasite behaviour: daily patterns of Argulus fish lice.

Parasites display a wide range of behaviours that are frequently overlooked in favour of host responses. Understanding these behaviours can improve parasite control through a more precise application or development of new behaviour-based strategies. In aquaculture fish lice are an ongoing problem, infections reduce fishery production and control options are limited. Fish lice are distinct in their ability to survive and swim off hosts, allowing the transmission to multiple fish hosts across their lifespan. Here we assessed the off-host behaviour of Argulus foliaceus (a freshwater fish louse) and observed a diurnal rhythmical pattern in their behaviour. This pattern was lost when lice were exposed to constant darkness, indicating that the behaviour is not endogenously driven. Males were consistently active in light with reduced activity in darkness. In contrast, females were active during light and dark phases with peak activity at the start of dark periods. A. foliaceus was also strongly attracted to a light stimulus, preferring white- and blue-coloured lights over green- or red-coloured lights. Light is a strong driver of fish louse activity and could be used to trap parasites. Aquaculture light regimes could also be altered to reduce parasite attraction and activity.

A new freshwater leech species from Asian Swamp Eel stocks in China.

Discoveries of new Hemiclepsis species (Hirudinea: Glossiphoniidae) in East and Southeast Asia were expected. A peculiar freshwater leech was found on the body surface of Asian Swamp Eel Monopterus albus (Synbranchiformes: Synbranchidae) in Jingzhou, Hubei Province, China. Here, we describe this leech species as Hemiclepsis yangtzenensis sp. nov. It could be distinguished from other congeners based on a combination of morphological features such as very small size, translucent body with dense green reticulate markings throughout dorsum, very large posterior sucker with a characteristic reticulate pattern, and two pairs of well-developed drop-like eyespots. It also represents a phylogenetic lineage that is distant from other members of the genus based on the COI and 18S rRNA sequences. This leech species seems to be a specialized fish parasite that could negatively affect farming and wild stocks of Asian Swamp Eel in China.

Morphological and molecular characterization of Neoechinorhynchus (N.) cephali n. sp. (Acanthocephala: Neoechinorhynchidae) Stiles and Hassall 1905 infecting the flathead grey mullet Mugil cephalus (Linnaeus, 1758) from the southwest coast of India.

The present paper describes Neoechinorhynchus (Neoechinorhynchus) cephali n. sp., an acanthocephalan parasite infecting the intestine of the flathead grey mullet Mugil cephalus from the southwest coast of India. The parasite exhibited a prevalence of 7.40%, mean intensity of 18.5 and abundance of 18-19 worms/infected host. Morphologically, N. (N.) cephali n. sp. is sexually dimorphic, small, cylindrical, slightly curved and creamy white in colour. Females are larger than males, measured 8.87 × 0.88 mm and 5.65 × 0.66 mm, respectively. Proboscis is armed with three circles of six hooks each, which progressively decreases in size posteriorly. Hooks are backwardly curved and robust and tapering with a sharp, pointed tip, striations on the surface and a manubrium at its base. The body is aspinose, trunk surface with micropores and pits and proboscis surface with papilliform structures. The body wall is with five dorsal and two ventral hypodermal nuclei, along with lacunar canals connected by circular anastomoses. Lemnisci are subequal, small lemnisci are uninucleated, and large ones are binucleated. The cement gland is oval, with four giant nuclei; bursa is with many sensory cells. Eggs are elliptical, with concentric shells, and polar prolongation is absent. In the molecular and phylogenetic analyses based on the 18S ribosomal DNA region, the present species stands out with a high bootstrap value and is positioned as a sister branch of N. (N.) dimorphospinus. Based on the differences in morphology, morphometry and molecular and phylogenetic analyses, the present species of acanthocephalan infecting M. cephalus is considered as new, and the name Neoechinorhynchus (Neoechinorhynchus) cephali n. sp. is proposed.

Co-occurrence and niche overlap among gill parasites of the white mullet (Mugil curema Valenciennes, 1836) (Osteichthyes: Mugilidae) from the western Atlantic, Brazil.

Different groups parasitize the gills of fishes, and the organisms can interact in positive, negative, or neutral ways, depending on parasitic abundance, environmental conditions, and the availability of resources. Here, we studied the distribution of ectoparasites on the gills of the mullet Mugil curema in the Cabedelo port region (Brazil), asking if (1) the gill parasites of M. curema fix on specific host gill arches and/or (2) co-occur in the same gill arches within a host individual; (3) if the abundance of one parasite taxon affects the abundance of another on the same gill arches; and (4) considering distinct major groups, if parasites present patterns of co-occurrence on hosts and gill arches different from the expected at random. We used generalized linear models, generalized linear mixed models, and models with zero inflation to define the microhabitats (arches) with the highest abundances of parasites and null models to analyze the co-occurrences of gill parasite groups. Three taxa of Monogenea (Ligophorus brasiliensis Abdallah, Azevedo & Luque, 2009, Ligophorus sp. 1 and Ligophorus sp. 2) and six species of Copepoda were recorded (Bomolochus nitidus Wilson, 1911; Caligus praetextus Bere, 1936; Ergasilus atafonensis Amado & Rocha, 1997; Ergasilus bahiensis Amado & Rocha, 1997; Ergasilus caraguatatubensis Amado & Rocha, 1997 and Ergasilus lizae Krøyer, 1863). We found that, in general, the gill parasites were more abundant/prevalent on specific arches (I and II) of the hosts, shared such preferences and co-occurred on the gill arches. The presence of one taxon influenced the occurrence of another only in the arches I and II; as a result, the infracommunities were characterized as interactive in the gills of this host species.

Unexpected morphological and molecular diversity of trematode (Haploporidae: Forticulcitinae) parasites of mullets from the ocean Pacific coasts in Middle America.

Two new genera and four new species from subfamily Forticulcitinae are described from the intestines of white mullet (Mugil curema) and flathead grey mullet (Mugil cephalus) collected in 27 localities across a wide geographical range on Pacific Ocean slopes comprising three countries in Middle America: Mexico, Guatemala and Costa Rica. The new genera Ekuarhuni n. gen. and Overstreetoides n. gen. had to be erected to accommodate two new species, Ekuarhuni papillatum n. sp. and Overstreetoides pacificus n. sp., with unique morphological traits that differentiate them from the two genera described previously. In addition, two new species, Forticulcita minuta n. sp. and Forticulcita isabelae n. sp., were described, which were characterized as exhibiting a small body size (< 1100 µm long). These new species were classified as the diminutive morphotype, together with three other congeneric species of Forticulcita. Forticulcita minuta n. sp. is distinguished by being the smallest species within the genus (< 305 µm). Meanwhile, Forticulcita isabelae n. sp. is distinguished by its body size and testis length. In specimens of the four new species, sequencing was performed with two molecular markers, the large subunit (LSU) and the internal transcribed spacer 2 (ITS2) of nuclear rDNA, and the results were compared with other sequences available in GenBank. Phylogenetic analyses performed with the combined dataset of the two nuclear molecular markers (LSU + ITS2) placed all the analysed species within the clade of Forticulcitinae with strong bootstrap support (100%) and a high Bayesian posterior probability (1.0). The four new species showed differences in abundance in their definitive hosts and were widely distributed along the Pacific Ocean coasts of Mexico, Guatemala and Costa Rica, in Middle America.

First next-generation sequencing data for Haploporidae (Digenea: Haploporata): characterization of complete mitochondrial genome and ribosomal operon for Parasaccocoelium mugili Zhukov, 1971.

The first data on a whole mitochondrial genome of Haploporidae, Parasaccocoelium mugili (Digenea: Haploporata: Haploporidae) was generated using the next-generation sequencing (NGS) approach. We sequenced the complete mitochondrial DNA (mtDNA) and ribosomal operon of Parasaccocoelium mugili, intestine parasite of mullet fish. The mtDNA of P. mugili contained 14,021 bp, including 12 protein-coding genes, two ribosomal genes, 22 tRNA genes, and non-coding region. The ribosomal operon of P. mugili was 8308 bp in length, including 18S rRNA gene (1981 bp), ITS1 rDNA (955 bp), 5.8S rRNA gene (157 bp), ITS2 rDNA (268 bp), 28S rRNA gene (4180 bp), and ETS (767 bp). We used the mtDNA protein-coding regions to make phylogenetic reconstructions of Haploporidae. Additionally, we performed the sequence cluster analysis based on codon usage bias of most of currently available mitochondrial genome data for trematodes. The observed gene arrangement in mtDNA sequence of P. mugili is identical to those of Plagiorchis maculosus (Rudolphi, 1802). Results of maximum likelihood (ML) phylogenetic analysis showed that P. mugili was closely related to Paragonimus species from the suborder Xiphidiata. The results of sequence cluster analysis based on codon usage bias showed that P. mugili has the highest similarity with Plagiorchis maculosus (Xiphidiata). Our results do not contradict to proposing a new suborder for Haploporoidea-Haploporata. On the basis of obtained results, the relationship between mitochondrial protein-coding gene rearrangements and synonymous nucleotide substitutions in mitochondrial genomes has been suggested.