Neobenedenia Melleni Research Articles

Proteomic analysis of Neobenedenia sp. and Rhabdosynochus viridisi (Monogenea, Monopisthocotylea): Insights into potential vaccine targets and diagnostic markers for finfish aquaculture

Monogeneans are parasitic flatworms that represent a significant threat to the aquaculture industry. Species like Neobenedenia melleni (Capsalidae) and Rhabdosynochus viridisi (Diplectanidae) have been identified as causing diseases in farmed fish. In the past years, molecular research on monogeneans of the subclass Monopisthocotylea has focused on the generation of genomic and transcriptomic information and the identification in silico of some protein families of veterinary interest. Proteomic analysis has been suggested as a powerful tool to investigate proteins in parasites and identify potential targets for vaccine development and diagnosis. To date, the proteomic dataset for monogeneans has been restricted to a species of the subclass Polyopisthocotylea, while in monopisthocotyleans there is no proteomic data. In this study, we present the first proteomic data on two monopisthocotylean species, Neobenedenia sp. and R. viridisi, obtained from three distinct sample types: tissue, excretory-secretory products (ESPs), and eggs. A total of 1691 and 1846 expressed proteins were identified in Neobenedenia sp. and R. viridisi, respectively. The actin family was the largest protein family, followed by the tubulin family and the heat shock protein 70 (HSP70) family. We focused mainly on ESPs because they are important to modulate the host immune system. We identified proteins of the actin, tubulin, HSP70 and HSP90 families in both tissue and ESPs, which have been recognized for their antigenic activities in parasitic flatworms. Furthermore, our study uncovered the presence of proteins within ESPs, such as annexin, calcium-binding protein, fructose bisphosphate aldolase, glutamate dehydrogenase, myoferlin, and paramyosin, that are targets for immunodiagnostic and vaccine development and hold paramount relevance in veterinary medicine. This study expands our knowledge of monogeneans and identified proteins that, in other platyhelminths are potential targets for vaccines and drug discovery.

Sterol profile of Neobenedenia melleni, a marine ectoparasite fish

Neobenedenia melleni, a marine fish ectoparasite, is responsible for considerable losses in the mariculture industry. In maintaining the parasite's homeostasis, sterols are structural and functional lipids that perform vital functions. Thus, understanding the mechanisms of biosynthesis and the uptake of sterols can reveal potential pharmacological targets. The objective of this work was thereby to characterize the N. melleni sterols. The most abundant sterol found was cholesterol in either its free (47.48 ± 15.93 %) or esterified form. However, its precursors, squalene (3.53 ± 0.92 %) and desmosterol (0.25 ± 0.03 %), were also found, suggesting the uptake of these intermediates from hosts or an unusual active pathway of sterol biosynthesis, which can be further explored as pharmacological targets.

Molecular epidemiology of helminth diseases of the humpback grouper, Cromileptes altivelis, as a pattern for mapping fish diseases in the Sunda Strait, Indonesia.

Background and Aim:Neobenedenia girellae and Haliotrema epinepheli are important but neglected helminth parasites of marine fish. The humpback grouper, the most relevant definitive host, harbors several Neobenedenia and Haliotrema spp. simultaneously on body surfaces and gills. These species can be distinguished morphologically This study aimed to identify Neobenedenia and Haliotrema spp. infestations in monogenean humpback grouper by multiplex polymerase chain reaction method, which seems to be widely distributed in the study area. Data can be used as a basis for mapping disease patterns in Strait waters.Materials and Methods:Eighty humpback groupers (Cromileptes altivelis) were collected from eight different areas in the Sunda Strait and examined using scrapings from body surfaces and gill lamellae followed by multiplex PCR for identification.Results:Parasites on body surfaces were recovered from 49 fish (61.2%) and were found on gill lamellae in 72 fish (90%) by microscopic examination. Endoparasites were absent. Ectoparasites identified included, N. girellae, Neobenedenia melleni eggs, Neobenedenia pasifica, Neobenedenia longiprostata, Haliotrema eukurodai, H. kurodai, Haliotrema leporinus, Haliotrema dongshaense, Haliotrema angelopterum, Haliotrema aurigae, Haliotrema scyphovagina, and H. epinepheli.Conclusion:The distribution of trematode disease in humpback grouper in Sunda Strait waters was revealed. All parasites were from genera, Neobenedenia and Haliotrema. Risks associated with these parasites should not be overlooked. Prevention and control programs need to be extended to other marine fish. Humpback grouper should be dewormed more frequently.

Isolation and characterization of pathology in case of massive mortality by Photobacterium damselae subsp. piscicida in Rachycentron canadum

This study aimed to investigate outbreak with high mortality in cultured juvenile cobia occurred in Southeast Brazil in 2011. Fish displayed retarded growth rates, lethargy, fin ulceration, skin depigmentation, corneal opacity, and physical deformities. Internally, livers were increased in volume and pale in different degrees. Firm whitish nodules were disseminated in the liver, kidney and spleen. A moderate number of parasites identified as Neobenedenia melleni were recovered from the body surface. Microscopically, severe hepatic steatosis and extensive granulomatous lesion were identified in all fish sampled. Microbiological analysis of moribund fish revealed the presence in pure culture of a Gram-negative bacterium identified as Photobacterium damselae subsp. piscicida using biochemical and molecular characteristics. Analysis of the partial 16S rRNA sequences confirmed the results demonstrating high identity (98%). The isolates were sensitive to chloramphenicol and enrofloxacin and resistant to ciprofloxacin, florfenicol, doxycycline hydrochloride, norfloxacin, oxytetracycline, and tetracycline. Chronic pasteurellosis was considered as the main problem in the farm, while hepatic steatosis and parasitic infestation may have contributed to the development of the process.

Genome-wide identification of ABC transporters in monogeneans

ATP-Binding Cassette (ABC) transporters are proteins that actively mediate the transport of a wide variety of molecules, including drugs. Thus, in parasitology, ABC transporters have gained attention as potential targets for therapeutic drugs. Among the parasitic Platyhelminthes, ABC transporters have been identified and classified in a few species of Trematoda and Cestoda but not in Monogenea. Monogeneans are mainly ectoparasites of marine and freshwater fish, although they can also be found on other aquatic organisms. Severe epizootics caused by monogeneans have been reported around the world, mainly in confined and/or overcrowded fish. The purpose of this study was to identify the ABC transporters in four species of monogeneans (Gyrodactylus salaris, Protopolystoma xenopodis, Eudiplozoon nipponicum and Neobenedenia melleni) for which genomic resources are publicly available. For comparative purposes, ABC transporters were also identified in endoparasitic (Schistosoma mansoni and Echinococcus granulosus) and free-living (Macrostomun lignano and Schmidtea mediterranea) platyhelminths. Thirty-two putative ABC transporters were identified in the genome of G. salaris, 40 in the genome of P. xenopodis, 46 in the transcriptome of E. nipponicum and 9 in a rather limited ESTs set available for N. melleni. Of the eight ABC subfamilies (A–H) known in metazoans, subfamily H was the only one not found in any monogenean species. In contrast, ABCC was the best represented subfamily. Phylogenetic analyses showed a few cases of one-to-one orthologous relationships, which agree with results from other metazoan species. We found some monogenean ABC members related to subfamilies B, C and G involved in drug resistance in humans. This information may be useful for future functional studies on ABC transporters in monogeneans.

Neobenedenia melleni (Monogenea: Capsalidae) in ornamental reef fish imported to Brazil.

The capsalid monogenean Neobenedenia melleni is known as a lethal pathogen for captured marine teleost ornamental fish, if left untreated. This study reports the occurrence of N. melleni parasitizing four species of ornamental reef fish imported into Brazil and maintained in quarantine: Arabian angelfish (Pomacanthus asfur ), yellowbar angelfish (Pomacanthus maculosus), regal angelfish (Pygoplites diacanthus), and bluecheek butterflyfish (Chaetodon semilarvatus). Ten days after the beginning of quarantine, some fish showed behavioral alterations, such as irritability, and corneal opacity, which were rapidly diagnosed to be caused by monogenean parasites by body surface scraping. The fish from the same batch were treated with two applications of 2 mg L-1 praziquantel each at an interval of four days. Seven days after the first treatment, the mucus surface of the fish was re-examined, which did not reveal the parasites presence being delivered for commercialization.

Efficacy of praziquantel and a combination anthelmintic (Adecto®) in bath treatments against Tagia ecuadori and Neobenedenia melleni (Monogenea), parasites of bullseye puffer fish

Diclidophoridae and Capsalidae are families of monogenean parasites that include some of the most pathogenic species for cultured finfish. In this study, the efficacy of praziquantel (PZQ) and a combination anthelmintic (PZQ, ivermectin, pyrantel pamoate and fenbendazole) commercialized under the name of Adecto® (Adler Pharma) were evaluated in vitro against adults and eggs of the diclidophorid Tagia ecuadori and the capsalid Neobenedenia melleni. Freshwater and formalin immersions were also evaluated against T. ecuadori for comparative purposes. In addition, the efficacy in vivo of Adecto® as a bath treatment to eliminate mixed infections as well as the median lethal concentration (LC50) of this drug in juvenile, healthy bullseye puffer fish (Sphoeroides annulatus) were determined. Triglycerides, hemoglobin, total protein and glucose levels were measured in three groups of fish: uninfected, infected and infected/treated with Adecto®. At the doses tested, PZQ did not have a concentration-dependent effect. Thus, 2.5 mg/L PZQ was 100% effective against adults of T. ecuadori after 20 h, and 3 mg/L killed 87% of N. melleni after 12 h. Adecto® had a concentration-dependent effect. The concentration required to kill all parasites in the minimum time was 20 mg/L Adecto® (12 h for T. ecuadori, and 16 h for N. melleni). Neither PZQ nor Adecto® were effective at inhibiting egg hatching. Adults of T. ecuadori were highly tolerant to freshwater; mortality was <40% after 24 h, whereas formalin was 100% effective against both adults and eggs in this species. In vivo, 20 mg/L Adecto® administered for 12 h was 100% effective against T. ecuadori; however, it was not 100% effective against N. melleni. Mixed infections provoked increases in hemoglobin and total protein levels in fish. Fish exposed to 20 mg/L Adecto® did not show signs of toxic effect after 24 h. For those fish, the 24-h LC50 of Adecto® was 30.8 mg/L. This study confirms that PZQ is effective against the parasitic phase of monogeneans but under prolonged exposure. Our results suggest that combination anthelmintics have the potential to kill all parasites in less time; however, alternative combinations should be investigated to find one that is effective under low concentrations to provide a greater safety margin.

In vivo and invitro studies using larval and adult antigens from Neobenedenia melleni on immune response in yellowtail (Seriola lalandi).

Neobenedenia melleni is a monogenean parasite that causes significant mortality and economic losses in fish aquaculture. Changes in the antigenic composition of this parasite occur during its developmental stages. In this study, we evaluated humoral parameters in serum and transcriptional immune responses of yellowtail naturally infected with N.melleni. In addition, invitro assays were performed to study the stimulatory effects of antigens from larvae and adults on spleen leucocytes from non-infected fish at 6 and 24h post-stimulation. The results showed enhanced total protein, myeloperoxidase and antiprotease activities in N.melleni-infected fish compared with non-infected ones. The induction of Toll-like receptors (TLRs) and pro-inflammatory cytokines in spleen leucocytes during natural infection with N.melleni suggests that these immune-related genes play an important role in the initiation of the immune defence mechanism for controlling parasite infection. Interestingly, the magnitude of invitro responses of spleen leucocytes was dependent on the parasitic stage. An important stimulation of gene expression by adult antigens on spleen leucocytes was observed. Differential expression patterns of TLRs and target cytokines in yellowtail leucocytes in both invivo and invitro studies suggest that the quality of yellowtail immune response is conditioned by N.melleni development.

Short report on the ectoparasitic fauna from a wild Epinephelus marginatus and osmotic shock efficiency on their removal

Short report on the ectoparasitic fauna from a wild Epinephelus marginatus and osmotic shock efficiency on their removal

Neobenedenia melleni-specific antibodies are associated with protection after continuous exposure in Mozambique tilapia.

Neobenedenia melleni is a significant monogenean pathogen of fish in aquaculture facilities and public aquaria. Immunity after exposure to live N. melleni is well established, but the mechanisms of immunity remain unclear. In this study, tilapia (Oreochromis mossambicus) were continuously exposed to N. melleni over a four-month period and assessed for immunity as determined by a reduction in the number of parasites dislodged from the experimental animals during freshwater immersion. Specific mucosal and systemic antibody levels were by determined via enzyme-linked immunosorbent assay. At 45 days postexposure (DPE), fish displayed high parasite loads and baseline levels of mucosal antibodies. At 102 and 120 DPE parasite loads were significantly decreased, and antibody levels were significantly increased for mucus and plasma samples. The correlation between immunity (reduction in parasite load) and an increased humoral antibody response suggests a key role of antibody in the immune response. This is the first report of immunity against N. melleni that is associated with specific mucosal or systemic antibodies.

First record of Neobenedenia “melleni” – like species (Monogenea: Capsalidae) in Goliath grouper Epinephelus itajara in Brazil

Neobenedenia melleni (MacCallum, 1927) (Monogenea) is a pathogen widely spread in marine teleost cultures around the world. The present paper recorded this parasite species in the Goliath grouper Epinephelus itajara for the first time Brazil. The use of freshwater bath for parasite control was successful for the Goliath grouper.

Outbreak of mortality among cage-reared cobia (Rachycentron canadum) associated with parasitism

This study reports a disease outbreak among juvenile cobia (Rachycentron canadum) farmed in cages in the state of Rio de Janeiro, Brazil, caused by the dinoflagellate Amyloodinium ocellatum and the monogenean Neobenedenia melleni. Two thousand five hundred fish were stocked at 0.4 kg/m3 in a set of 12 m3 tanks, in autumn (mean weight 15.0 ± 7.3 g) and in winter (mean weight 43.0 ± 5.6 g). Freshwater baths were administered as a routine treatment, as the symptoms were detected followed by two collection samples. Firstly in May 2011 (n = 5) and secondly in September 2011 (n = 10). In the first sample, the prevalence of N. melleni on the body surface was 100% and the mean intensity was 42.0 ± 1.7, while in the second sample the prevalence was 60% with a mean intensity 3.0 ± 0.2 and mean abundance 1.8 ± 0.4. Amyloodinium ocellatum was only found in the second sample, at a prevalence 100% and mean intensity 46.8 ± 3.4. The cause of fish mortality was possibly associated with a decrease in fish resistance after the first contact with monogenean parasites, allied with respiratory difficulty caused by the presence of A. ocellatum in the gills.

Parasite fauna of wild and cultured dusky-grouper Epinephelus marginatus (Lowe, 1834) from Ubatuba, southeastern Brazil.

This study aimed at identifying and quantifying the parasites of wild and cultured dusky grouper Epinephelus marginatus. During a year and thereby all four seasons, 20 wild and 20 cultured groupers were examined for the presence of parasites, except in the last season, in which 19 wild and 20 cultured fish were examined, totalling 159 groupers analysed from Ubatuba, southeastern Brazil. Prevalence, mean intensity of infection, mean abundance and mean relative dominance were calculated. Five species of parasites were identified in fish from both origins: Pseudorhabdosynochus beverleyburtonae (Monogenea), Neobenedenia melleni (Monogenea), Pseudempleurosoma sp. (Monogenea), Helicometrina nimia (Digenea) and larvae of Contracaecum sp. (Nematoda). The prevalence of ectoparasites, in most cases, was higher than endoparasites. The most abundant parasite was the monogenea Pseudorhabdosynochus beverleyburtonae in both wild and cultured fish, along all seasons. Neobenedenia melleni was observed in wild and cultured fish in all seasons, with a gradual increase in the number of parasites from the coldest to the hottest seasons, with the highest prevalence and mean intensity in the summer. Helicometrina nimia was found in all seasons in both wild and cultured fish, except for summer, where its presence was detected only in wild fish. Pseudempleurosoma sp. and larvae of Contracaecum sp. showed low prevalence occurring in wild and cultured fish in the autumn and spring, respectively. This study revealed high intensities of potentially pathogenic parasites that could favour disease outbreaks in culture conditions.

Molecular cloning, sequence analysis and stress-related changes of the heat shock protein 60 gene in &lt;I&gt;Neobenedenia melleni&lt;/I&gt;

Heat shock protein 60 is an essential chaperone that can maintain the natural structure and function of mitochondrial proteins. Here, we successfully cloned the full length cDNA of HSP60 from Neobenedenia melleni, designated as NmHSP60. Real-time quantitative PCR and Western blot were used to analyze the expression change of NmHSP60 under different temperature and salinity. Compared with the typical 25 Degrees Celsius, expressions decreased dramatically in eggs and adults at 18 Degrees Celsius. Conversely, at 32 Degrees Celsius, expression increase dramatically in adults. Compared with salinity 24, expressions were significantly down-regulated in adults at salinity 18, and up-regulated in eggs at salinity 30. Experimental results suggest that NmHSP60 may play an significant role in N. melleni's adaptation to adverse environmental conditions.

Can barber gobyElacatinus figarocontrolNeobedenia melleniinfections on dusky grouperEpinephelus marginatus?

The interaction between cultured barber goby Elacatinus figaro (cleaner fish) and the dusky grouper Epinephelus marginatus, and the efficiency of the cleaner fish in removing ectoparasites were evaluated. When the interaction between these two species was observed, cleaner fish showed a preference for the largest groupers. In a second trial, treatments: TWC (Control) – two groupers without a cleaner fish, T1C – two groupers with one cleaner fish, T3C – two groupers with three cleaner fish and T6C – two groupers with six cleaner fish were tested in four replicates. After 8 days, monogeneans were removed and identified as Neobenedenia melleni. The highest mean abundance of parasites was found on the groupers in the TWC group (37 parasites per host) and the lowest on those in the T6C group (4.1 parasites per host). By increasing the number of cleaner fish, a higher cleaning efficiency was obtained, as observed in T6C, where almost 90% of the parasites were removed. Possibly, this removal would have been complete if the number of cleaners had not been reduced in the treatments due to the mortalities observed. This study demonstrates the possibility of using gobies to remove monogeneans and in improving grouper health.

Molecular cloning and characteristic analysis of a thioredoxin from Neobenedenia melleni

Thioredoxin (Trx) can regulate disulfide bond reduction of target proteins to maintain the reduced intracellular state in various organisms. Here, we cloned a cDNA sequence of thioredoxin from Neobenedenia melleni , which is a kind of platyhelminth parasite infecting many fishes of great economic value. The deduced N. melleni Trx (NmTrx) contained 170 amino acid residues with an active site consisting of four amino acid motif CPGC. Sequence comparison and phylogenetic tree analysis confirmed NmTrx as a distinct member of thioredoxin. Real-time quantitative polymerase chain reaction (PCR) revealed a significantly higher expression of NmTrx transcript in the adult stage compared with the egg and oncomiracidium stages. In the egg and adult stages, the NmTrx transcript level in the 32°C group was higher than those in the 18 and 25°C groups. NmTrx was expressed and purified from Escherichia coli , and antiserum against NmTrx was prepared. Western blot confirmed the higher NmTrx expression of the egg and adult stages in the 32°C group with respect to the other temperature groups. Recombinant NmTrx was able to reduce the disulfide bond in insulin, and its antioxidant capacity was determined to be 5.12 U/mg protein, similar to the classic thioredoxins. Trx activity was lower in the oncomiracidium stage and higher in the adult stage compared with the egg stage. These results indicate that NmTrx could function as an important antioxidant molecule under physiological conditions. Keywords: Thioredoxin, Neobenedenia melleni , redox regulation, mRNA expression, prokaryotic expression

First record of Neobenedenia melleni (Monogenea: Capsalidae) in sea-farmed cobia (Rachycentron canadum) in Brazil

Neobenedenia melleni (MacCallum, 1927) (Monogenea) is a widespread pathogen in marine teleost cultures all over the world. The present paper reports this parasite species in farmed cobia (Rachycentron canadum) in Brazil, for the first time. Some comments on preventive actions for avoiding the disease are made.

Effects of three Caribbean cleaner shrimps on ectoparasitic monogeneans in a semi-natural environment

Most research on cleaning symbioses on coral reefs has focused on fish clients being cleaned by smaller fishes. While many shrimps and other crustaceans are reported as cleaners, whether they remove parasites from fish hosts and can effectively regulate populations of ectoparasites is unclear. The effects of Pederson shrimp (Periclimenes pedersoni), spotted shrimp (P. yucatanicus), and banded coral shrimp (Stenopus hispidus), on the parasitic monogenean Neobenedenia melleni on a host reef fish, blue tang (Acanthurus coeruleus), were investigated. The abundance and size of N. melleni from fish with and without access to shrimps in a semi-natural macrocosm was quantified. P. pedersoni had a strong effect on both the abundance and size of parasites. In contrast, P. yucatanicus and S. hispidus had no effect on the abundance of parasites but had a small yet statistically significant effect on average size. These data suggest that P. pedersoni can play a significant role in the biological regulation of at least some ectoparasites on Caribbean reef fishes, but further suggest that some other shrimps regarded as “cleaners” may have little or no effectiveness at removing parasites and underscore the need for further verification before this term is applied.

Ocorrência de Neobenedenia melleni (Monogenea; Capsalidae) em Trichiurus lepturus (Perciformes; Trichiuridae), naturalmente infestados, no litoral do Rio de Janeiro, Brasil

Neobenedenia melleni (MacCallum, 1927) is a widely spread pathogen in teleost maricultures with a very low host specificity. Between July of 2006 to June of 2007, 120 specimens of Trichiurus lepturus Linnaeus, 1758 were acquired of local fishermen in the Guanabara Bay (22 degrees 57' S and 42 degrees 41' W), State of Rio de Janeiro, Brazil, to the study of the parasite fauna. The surface of the host body was scraped and the monogeneans obtained were identified as N. melleni, which showed prevalence of 8.3% and mean intensity of infection 1.2+/-0.42. Data about morphology of the specimens collected were included herein. Neobenedenia melleni is recorded by the first time parasitizing on a wild host from the Brazilian coastal zone.

Parasite fauna of seabass (Lates calcarifer) under mariculture conditions in Lampung Bay, Indonesia

Summary Fish parasites have been repeatedly reported to be a major threat to the developing industry of finfish mariculture in Indonesia, due to severe parasite and disease outbreaks. The aim of this study was to identify the metazoan parasite fauna and trichodinid ciliates that infect Lates calcarifer in a representative mariculture farm in Indonesia. Examined were 105 L. calcarifer (seabass) for the metazoan parasite fauna and trichodinid ciliates. Thirty-five specimens each from the net cages of the National Sea Farming Development Centre (Balai Budidaya Laut, BBL) in Lampung Bay, South Sumatra, Indonesia were investigated in three consecutive seasons (two dry and one rainy season from 2002 to 2003). Nineteen parasite species were identified; all fish specimens were infected with two to 10 parasite species, demonstrating a species-rich parasite fauna. Protozoans (1 species), myxozoans (1), digeneans (3), monogeneans (5), cestodes (3), nematodes (5) and acanthocephalans (1) were found, including 11 new host records in cultured L. calcarifer from Indonesia. Larval and adult parasite stages were isolated, demonstrating that this fish species, although kept inside the net cages, still functions as an intermediate and final host for marine fish parasites. During all seasons, the six detected monoxenous (single host life cycle) parasite species showed a higher prevalence than the 13 heteroxenous (multiple hosts) species. Most abundant were the fish pathogenic monogeneans Pseudorhabdosynochus epinepheli, Pseudorhabdosynochus lantauensis, Benedenia epinepheli and Neobenedenia melleni with a high prevalence. Most heteroxenous parasites (Digenea, Cestoda, Nematoda and Acanthocephala) occurred with a low prevalence below 26%, caused by the specific culture conditions. Diversity of the heteroxenous parasites was higher in the dry seasons than in the rainy season. Though some seasonality could be observed for the fish pathogenic monogeneans, severe disease outbreaks of these ectoparasites cannot be excluded in either the dry or rainy season.