Morphological and molecular characterization of Encotyllabe vallei Monticelli, 1907 (Monopisthocotylea, Monogenea) from the gilthead seabream Sparus aurata Linnaeus (Teleostei, Sparidae) from the southwestern Mediterranean and notes on host specificity of the genus Encotyllabe Diesing, 1850.

Incomplete original descriptions, the unavailability or poor conditions of specimens and the lack of detailed redescriptions have caused the validity of several species of the genus Encotyllabe Diesing, 1850 to be questioned. To date, seven of the recognized species were described upon one or two specimens, hindering study of intraspecific variations. This was made worse by considering few morphoanatomical differences sufficient to erect new species. Among Encotyllabe spp. occurring in Mediterranean waters, E. vallei was first described from the gilt-head bream Sparus aurata (Sparidae) off Italy. Although beautifully illustrated for a paper from that century, morphometric data for E. vallei from the type-host S. aurata remain unavailable. Previous records of E. vallei provided either morphometrical or molecular data, and its validity was questioned. We provide a redescription of E. vallei based on newly collected specimens from the S. aurata from the southwestern Mediterranean (off Algeria) using integrative taxonomy. Analysis of cox1 sequences of E. vallei from S. aurata, compared to sequences from other sparid hosts, mainly Pagellus bogaraveo, revealed a divergence not exceeding 2%, suggesting a stenoxenic specificity for this monogenean. Given that P. bogaraveo is the type-host for Encotyllabe pagelli, we were tempted to suggest a synonymy between E. vallei and E. pagelli. We refrained from doing so because E. pagelli was first described from the Atlantic coast off Brest, France. Morphological data for Encotyllabe from P. bogaraveo are warranted assessing the host specificity of E. vallei and whether there might be a species complex within individual sparid fish species.

Phototactic responses in four monogenean oncomiracidia.

Phototaxis is the common behavioral response exhibited by the oncomiracidia of various monogeneans. However, the changes in the oncomiracidial swimming behavior in response to light cues are not well understood. Here, we investigated the light responses of four monogeneans that are important pathogens in mariculture, namely Benedenia epinepheli, Benedenia seriolae, Neobenedenia girellae, and Heteraxine heterocerca. The swimming trajectory and speed of oncomiracidia of each species were assessed in a glass Petri dish with an LED light placed adjacent to it, based on three different light responses: LED light in the off position (normal swimming), LED light in the on position (phototactic behavior), and immediately (< 5 s) after switching the LED light off (photophobic behavior). The oncomiracidia of all four species exhibited positive phototactic and photophobic responses; however, the change in swimming speed between each response differed among the species. The oncomiracidia of three species (B. epinepheli, N. girellae, and H. heterocerca) exhibited high swimming speed, as a phototactic response; in contrast, the oncomiracidia of B. seriolae exhibited reduced swimming speed when moving toward the light source. Benedenia epinepheli and H. heterocerca exhibited the highest swimming speed during the phototaxis phase, whereas B. seriolae and N. girellae exhibited the highest swimming speed during the photophobic phase. These light responses are considered adaptive traits to increase the chance of encountering and infecting suitable hosts in nature, and such responses could potentially be applied to the control of parasite infections in aquaculture.

Effects of temperature on the life cycle of Neobenedenia sp. (Monogenea: Capsalidae) from Seriola rivoliana (Almaco jack) in Bahía de La Paz, BCS Mexico.

Seriola rivoliana cultivated in Mexico are infected by Neobenedenia sp. (Monogenea: Capsalidae), resulting in dermal ulceration and subsequent bacterial invasion that can cause fish death. This study assesses the effects of temperature over hatching success, oncomiracidia longevity, and infection success. The experimental design consisted of culturing the parasite at temperatures ranging between 16 and 32 °C. The oncomiracidia infection success, time to sexual maturity, and size at sexual maturity of Neobenedenia sp. were examined only at three temperatures (20 °C, 24 °C, and 30 °C). Experiments were conducted under controlled conditions in the laboratory. The oncomiracidia development was found to be faster at warmer temperatures (4-5 days between 24 and 30 °C) than in colder treatments (7-11 days between 18 and 20 °C). Hatching success and oncomiracidia longevity were higher at 24 °C and 26 °C. At 20 °C, 24 °C, and 30 °C, infection success was greater than 90%. Additionally, the laid eggs were observed at 9, 12, and 15 days at 30 °C, 24 °C, and 30 °C, respectively. The results of this study will allow for improving the temporal schedule of applications of treatments against Neobenedenia sp. by the function of temperatures. In conclusion, it is recommended to treat fish more frequently if the temperature in cultures is higher than 24 °C, because Neobenedenia sp. development is faster. As an alternative, the fish could be moved to deeper and cooler waters.

DNA profiling reveals Neobenedenia girellae as the primary parasitic monogenean in global fisheries and aquaculture.

Accurate identification of parasite species and strains is crucial to mitigate the risk of epidemics and emerging disease. Species of Neobenedenia are harmful monogenean ectoparasites that infect economically important bony fishes in aquaculture worldwide, however, the species boundaries between two of the most notorious taxa, N. melleni and N. girellae, has been a topic of contention for decades. Historically, identifications of Neobenedenia isolates have overwhelmingly been attributed to N. melleni, and it has been proposed that N. girellae is synonymous with N. melleni. We collected 33 Neobenedenia isolates from 22 host species spanning nine countries and amplified three genes including two nuclear (Histone 3 and 28S rDNA) and one mitochondrial (cytochrome b). Four major clades were identified using Maximum Likelihood and Bayesian inference analyses; clades A-D corresponding to N. girellae, N. melleni, N. longiprostata and N. pacifica, respectively. All unidentified isolates and the majority of Neobenedenia sequences from GenBank fell into clade A. The results of this study indicate that N. girellae is a separate species to N. melleni, and that a large proportion of previous samples identified as N. melleni may be erroneous and a revision of identifications is needed. The large diversity of host species that N. girellae is able to infect as determined in this study and the geographic range in which it is present (23.8426°S and 24.1426°N) makes it a globally cosmopolitan species and a threat to aquaculture industries around the world.

Morphological and molecular characterisation of Tristoma integrum Diesing, 1850 (Monogenea, Capsalidae), including its complete mitogenome.

Capsalids are monopisthocotylean monogenean parasites found on the skin and gills of fish. Capsalines (subfamily Capsalinae) are large-sized capsalids, parasitic on highly prized gamefish, and species of Tristoma parasitise only the gills of swordfish (Xiphias gladius). We obtained specimens of Tristoma integrum Diesing, 1850 from swordfish collected off Algeria in the Mediterranean Sea. Here, we describe the specimens, including the key systematics characters of dorsolateral body sclerites. One specimen was used for a next generation sequencing analysis but a part of it, including the sclerites, was mounted on a permanent slide, drawn, and deposited in a curated collection. We characterised the complete mitogenome, the ribosomal cluster (including 18S and 28S) and additional genes such as Elongation factor 1 alpha (EF1α) and Histone 3. We also retrieved molecular information from the host tissue present in the gut of the monogenean and provide the sequence of the complete rRNA cluster of the host, X. gladius. The mitogenome of T. integrum is 13 968 bp in length and codes for 12 protein, 2 rRNA and 22 tRNA. Phylogenies of capsalids were generated from 28S sequences and concatenated mitochondrial protein-coding genes, respectively. In the 28S phylogeny, most subfamilies based on morphology were not found to be monophyletic, but the Capsalinae were monophyletic. In both phylogenies, the closest member to Tristoma spp. was a member of the Capsaloides. In an Appendix, we report the complex nomenclatural history of Tristoma Cuvier, 1817 and its species.

Title: Caractérisation morphologique et moléculaire de Tristoma integrum Diesing, 1850 (Monogenea, Capsalidae) y compris son mitogénome complet. Abstract: Les Capsalidae sont des monogènes Monopisthocotylea parasites de la peau et des branchies des poissons. Les Capsalinae sont des Capsalidae de grande taille qui parasitent les poissons de pêche sportive, très prisés, et les espèces de Tristoma ne parasitent que les branchies de l'espadon (Xiphias gladius). Nous avons obtenu des spécimens de Tristoma integrum Diesing, 1850 à partir d'espadons collectés au large de l'Algérie en Méditerranée. Nous décrivons les spécimens, y compris les caractères systématiques clés des sclérites dorsolatéraux du corps. Un spécimen a été utilisé pour une analyse de séquençage de nouvelle génération, mais une partie de celui-ci, qui comprend des sclérites, a été montée sur une lame permanente, dessinée et déposée dans une collection. Nous avons caractérisé le mitogénome complet, le cluster ribosomal (y compris 18S et 28S) et des gènes supplémentaires tels que le facteur d'élongation 1 alpha (EF1α) et histone 3. Nous avons également récupéré des informations moléculaires à partir du tissu de l'hôte présent dans l'intestin du monogène et fournissons la séquence du cluster d'ARNr complet de l'hôte, X. gladius. Le mitogénome de T. integrum a une longueur de 13 968 pb et code pour 12 gènes de protéines, 2 ARNr et 22 ARNt. Des phylogénies des Capsalidae ont été générées, respectivement à partir des séquences 28S et des gènes codant pour les protéines mitochondriales concaténés. Dans la phylogénie 28S, la plupart des sous-familles basées sur la morphologie n'ont pas été trouvées monophylétiques, à l'exception notable des Capsalinae. Dans les deux phylogénies, les membres les plus proches de Tristoma spp. étaient des espèces de Capsaloides. Dans une annexe, nous rapportons l'histoire nomenclaturale complexe de Tristoma Cuvier, 1817 et de ses espèces.

Capsalid monogeneans of fishes from the Seto Inland Sea, Japan: Description of Benedenia kobudai n. sp. parasitic on Semicossyphus reticulatus (Perciformes: Labridae).

The Seto Inland Sea, the largest inland sea in Japan, is a rich fishery with high biodiversity and productivity. Monogeneans have been studied for more than 120 years, and 58 nominal species have been recorded in the Seto inland Sea. This study provided DNA information on five species of Benedenia sensu lato (Capsalidae) from marine fishes from the sea, and one of them, Benedenia kobudai n. sp., is described from Semicossyphus reticulatus (Perciformes: Labridae). This new species differs from the other congeners by the hooded appearance between the anterior attachment organs, the morphology of the penis, the absence of the lobe near the vaginal pore and the common genital pore, the position of the vaginal pore, the germarium lying near the slightly hexagonal testes, the morphology of the haptor, and the shape and position of the hamuli. Phylogenetic analysis showed B. kobudai n. sp. in a separate clade from the other Benedenia species, B. epinepheli, B. hoshinai, B. sekii, and B. seriolae collected from the sea. Each of the newly provided DNA sequences (28S rDNA, ITS1-5.8S-ITS2, cox1) of the above four species are based on specimens obtained from the type hosts and/or type localities and are considered important for future taxonomic re-examination and confirmation of the reliability of the registered sequences. Furthermore, these four species are important causes of fish diseases in aquaculture, and it is expected that information on the distribution, host range, and occurrence of fish diseases for each capsalid secured by molecular identification will be accumulated. The Life Science Identifier (LSID): urn:lsid:zoobank.org:pub:26C15D17-CFD1-450D-9FCE-EFFF692D2133.

Neobenedenia melleni from reef ornamental fish species in a retailer of Southeastern Brazil and its possible role as a mechanical vector of bacterial infection.

Annually, more than 2500 ornamental fish species are traded worldwide. Forty percent of these are from marine water. Some 98% of marine species are wild-caught from their natural habitat, and the majority subsequently exported. Wild fish frequently carry pathogens, which could induce diseases after the stress of capture. Neobenedenia melleni is a platyhelminth that mainly attaches to the skin and eyes of the host. It provokes dermal inflammation, epidermal loss, skin depigmentation, reduction in the number of mucous cells, and, consequently, decreased mucus protection, and declining immunological barriers. This makes fish susceptible to secondary infections. A total of 47 wild reef fish from a retailer were examined, suspected to be infected with ectoparasites. The morphological identification revealed N. melleni as a monogenean agent. One monogenean specimen was collected from the eye of each of the 40 fish analyzed to evaluate possible bacterial secondary infections using the matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) technique. The MALDI-TOF MS identified that 59% of monogenean collected from the eyes had bacteria, including some pathogenic to fish. This led us to believe that the ectoparasite can be a possible mechanical vector of pathogenic bacteria for fish culture and maintenance. The use of praziquantel as an antiparasitic agent is also discussed.

Complete Mitochondrial Genome of Two Ectoparasitic Capsalids (Platyhelminthes: Monogenea: Monopisthocotylea): Gene Content, Composition, and Rearrangement.

The capsalid monogeneans are important pathogens that generally infect marine fishes and have a substantial impact on fish welfare in aquaculture systems worldwide. However, the current mitogenome information on capsalids has received little attention, limiting the understanding of their evolution and phylogenetic relationships with other monogeneans. This paper reports the complete mitochondrial genomes of Capsala katsuwoni and Capsala martinieri for the first time, which we obtained using a next-generation sequencing method. The mitogenomes of C. katsuwoni and C. martinieri are 13,265 and 13,984 bp in length, respectively. Both species contain the typical 12 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region. The genome compositions show a moderate A+T bias (66.5% and 63.9% for C. katsuwoni and C. martinieri, respectively) and exhibit a negative AT skew but a positive GC skew in both species. One gene block rearrangement was found in C. katsuwoni in comparison with other capsalid species. Instead of being basal to the Gyrodactylidea and Dactylogyridea or being clustered with Dactylogyridea, all species of Capsalidea are grouped into a monophyletic clade. Our results clarify the gene rearrangement process and evolutionary status of Capsalidae and lay a foundation for further phylogenetic studies of monogeneans.

Novel molecular data for monogenean parasites of sparid fishes in the Mediterranean and a molecular phylogeny of the Microcotylidae Taschenberg, 1879.

During a study of the monogeneans of four sparid fishes (Diplodus vulgaris, Pagellus bogaraveo, Pagrus pagrus and Sparus aurata) from the Western Mediterranean off Algeria, a large collection of parasites was characterised molecularly (28S rRNA and cox1 genes). A total of 46 partial sequences (23 for each gene) were generated from 38 isolates of monogeneans which included four species (Atrispinum acarne, Microcotyle erythrini (sensu stricto), Sparicotyle chrysophrii and Prostatomicrocotylinae gen. sp.) of the family Microcotylidae, two putative species of the family Capsalidae (Encotyllabe spp.), and one species (Choricotyle chrysophryi) of the family Diclidophoridae. Our study provides (i) the first molecular data for the Capsalidae in the Mediterranean; (ii) the first record of a member of the Prostatomicrocotylinae in the Mediterranean and in a sparid fish (D. vulgaris); (iii) the first cox1 sequences for A. acarne; (iv) the second record of M. erythrini (s.s.) from P. pagrus; and (v) the second confirmed by molecular data record of S. chrysophrii in wild populations of S. aurata. The first phylogenetic hypotheses for the family Microcotylidae developed here, revealed the monophyly of the subfamily Prostatomicrocotylinae and the genus Microcotyle but the relationships among the subfamilies were still largely unresolved with the best represented subfamily Microcotylinae being polyphyletic. Our results highlight the importance of molecular methods in the assessment of monogenean diversity and the need for a thorough taxon-sampling approach to increase the accuracy of phylogenetic reconstruction of the relationships of the large and taxonomically complex polyopisthocotylean family Microcotylidae.

Occurrence of Neobenedenia girellae (Monogenea: Capsalidae) in Gilthead Seabream Sparus aurata (Actinopterygii: Sparidae) Cultured in Portugal.

Monogenean capsalids of the genus Neobenedenia are widespread parasites of wild and farmed marine fish, and represent a potential threat to mariculture due to their pathogenicity and ability to cause mortality in fish maintained in controlled conditions. The identification of Neobenedenia species and, consequently, the definition of their host specificity is often problematic due to their highly conserved morphology; therefore, in order to establish their specific identity, microscopic observation should be complemented with molecular analysis. The present work aims at characterizing Neobenedenia specimens infecting the skin of cage reared gilthead seabream Sparus aurata from Portugal. Parasite samples obtained from caged fish were processed for morphological analysis, through observation in light and scanning electron microscopy, and for molecular analysis, through amplification and sequencing of 28S rDNA and cytB, aimed at identifying them to the species level. Our results showed that the collected parasites belonged to the species Neobenedenia girellae; the susceptibility of S. aurata towards this pathogenic capsalid monogenean highlighted in the present work represents an important risk in the farming of this valuable fish species.

Temperature alters reproduction and maternal provisioning in a fish ectoparasite.

This study quantified the effects of temperature on reproduction and maternal provisioning of the ectoparasite, Neobenedenia girellae (Platyhelminthes: Monogenea), a species known to cause detrimental impacts to aquaculture fishes in tropical and subtropical environments worldwide. At 20 and 25 °C, parasites exhibited relatively slower production of larger eggs that were energy-dense. In contrast, parasites at 30 °C attained sexual maturity faster, were reproductively active over a shorter period, grew to a smaller size and laid smaller, less energy-rich eggs at a faster rate. As such, parasites exhibited two distinct reproductive patterns in response to temperature: parasites at lower temperatures produced larger eggs with higher energy content, while those at the higher temperature had a higher rate of egg production. Larger eggs produced under cooler conditions were better provisioned with energetic reserves and important, membrane-bound lipids that would likely facilitate larval longevity and development success. This is commensurate with previous observations of epizootics of this parasite species in aquaculture systems during winter. Meanwhile, eggs produced at 30 °C contained higher proportions of saturated fatty acids compared with polyunsaturated fatty acids, likely reflecting metabolic regulation of cell membrane fluidity, which is necessary for larvae to survive warm conditions. This study demonstrates that fish ectoparasites have evolved substantial reproductive and metabolic flexibility to maximise infection success under variable environmental conditions.

Biochemical composition of marine monogenean parasite eggs.

This study on the eggs of the tropical monogenean Neobenedenia girellae presents the first detailed quantitative biochemical information of a marine parasite species' eggs. Moisture and protein composed the majority of the contents of freshly laid eggs (79.12±0.82 and 11.51±0.49% respectively) followed by lipid (2.50±0.15%). Lipids were composed of approximately equal amounts of saturated, monounsaturated, and polyunsaturated fatty acids and the predominant lipid class was triacylglycerol (33.82±1.20%). This study represents a fundamental step towards a better understanding of the early life biology of this important species of parasite.

New record of monogeneans (Platyhelminthes: Monogenea) infecting some marine fishes from the Peruvian coastal zone

A parasitological survey searching monogeneans infesting marine fish was carried out during June 2018 and January 2020 from the coastal zone of Puerto Pizarro, Tumbes (northern Peru) and from the coastal zone of Chorrillos, Lima (central Peru). The gills, skin, nasal cavities, or branchial gill-cover of seven species were sampled. Ten monogenean species assigned to six families and nine genera were identified. The monogeneans Callorhynchocotyle callorhynchi (Manter, 1955); Capsala biparasiticum (Goto, 1894) Price, 1938; Euryhaliotrema sagmatum Kritsky & Boeger, 2002; Listrocephalos kearni Bullard, Payne &Braswell, 2004; Magniexcipula lamothei Bravo-Hollis, 1981; Nasicola klawei (Stunkard, 1962) Yamaguti, 1968; and Pseudorhabdosynochus anulus Violante-Gonzalez & Rojas-Herrera, 2011 are registered for the first time in Peru. While Capsala gregalis (Wagner & Carter, 1967) Chisholm & Whittington, 2007; Heterocotyle margaritae Chero, Cruces, Sáez, Santos & Luque, 2020; and Monocotyle luquei Chero, Cruces, Iannacone, Sanchez, Minaya, Sáez & Alvariño, 2016 have been previously registered in Peruvian waters, however, the region of Tumbes (northern Peru) represent a new locality record for these species.

Durante junio de 2018 y enero de 2020 se realizó un levamiento parasitológico para buscar monogeneos que infectan peces marinos en la zona costera de Puerto Pizarro, departamento de Tumbes (norte de Perú) y en la zona costera de Chorrillos, departamento de Lima (centro de Perú). Se analizaron las branquias, la piel, las cavidades nasales o el opérculo de siete especies. Se identificaron diez especies de monogeneos, asignadas a seis familias y nueve géneros. Los monogeneos Callorhynchocotyle callorhynchi (Manter, 1955); Capsala biparasitica (Goto, 1894) Price, 1938; Euryhaliotrema sagmatum Kritsky y Boeger, 2002; Listrocephalos kearni Bullard, Payne y Braswell, 2004; Magniexcipula lamothei Bravo-Hollis, 1981; Nasicola klawei (Stunkard, 1962) Yamaguti, 1968; y Pseudorhabdosynochus anulus Violante-Gonzalez & Rojas-Herrera, 2011 son registrados por primera vez en Perú. Mientras que, Capsala gregalis (Wagner y Carter, 1967) Chisholm y Whittington, 2007; Heterocotyle margaritae Chero, Cruces, Sáez, Santos & Luque, 2020; y Monocotyle luquei Chero, Cruces, Iannacone, Sanchez, Minaya, Sáez & Alvariño, 2016 han sido previamente registrados en aguas peruanas, sin embargo, la región de Tumbes (norte de Perú) representa un nuevo registro de localidad para estas especies.

Tracking transparent monogenean parasites on fish from infection to maturity.

The infection dynamics and distribution of the ectoparasitic fish monogenean Neobenedenia sp. (Monogenea: Capsalidae) throughout its development was examined on barramundi, Lates calcarifer (Bloch) (Latidae), by labelling transparent, ciliated larvae (oncomiracidia) with a fluorescent dye. Replicate fish were each exposed to approximately 50 fluorescent oncomiracidia and then examined for parasites using an epifluorescence stereomicroscope at 10 time intervals post-exposure (15, 30, 60, 120 min, 24, 48 h, four, eight, 12, and 16 days). Fluorescent labelling revealed that parasites attached underneath and on the surface of the scales of host fish. Parasite infection success was 20% within 15 min, and peaked at 93% two days post-exposure, before gradually declining between four and sixteen days. Differences in parasite distribution on L. calcarifer over time provided strong evidence that Neobenedenia sp. larvae settled opportunistically and then migrated to specific microhabitats. Parasites initially attached (<24 h) in greater mean numbers on the body surface (13 ± 1.5) compared to the fins (4 ± 0.42) and head region (2 ± 0.41). Once larvae recruitment had ceased (48 h), there were significantly higher mean post-larvae counts on the head (5 ± 3.4) and fins (12 ± 3) compared to previous time intervals. Neobenedenia sp. aggregated on the eyes, fins, and dorsal and ventral extremities on the main body. As parasites neared sexual maturity, there was a marked aggregation on the fins (22 ± 2.35) compared to the head (4 ± 0.97) and body (9 ± 1.33), indicating that Neobenedenia sp. may form mating aggregations.