Attachment-inducing capacities of fish skin epithelial extracts on oncomiracidia of Benedenia seriolae (Monogenea: Capsalidae)

Attachment-inducing capacities of skin epithelial extracts of yellowtail, Japanese flounder and red sea bream on oncomiracidia of the monogenean Benedenia seriolae were examined. Clear differences were not detected in the capacity among the fish species, although B. seriolae infects only yellowtail and its congeners in Seriola. This suggests that either the capacity is not host specific or host-specific attachment-inducing capacity cannot be detected by the assay method. Further, the attachment-inducing capacities were suppressed by wheat-germ lectin and concanavalin A in skin epithelial extracts of Japanese flounder and yellowtail, respectively. This suggests that some sugar-related chemical substances existing in fish epithelia induce the attachment of B. seriolae oncomiracidia.     

Morphological variation in the cosmopolitan fish parasite Neobenedenia girellae (Capsalidae: Monogenea)

Intra-species morphological variation presents a considerable problem for species identification and can result in taxonomic confusion. This is particularly pertinent for species of Neobenedenia which are harmful agents in captive fish populations and have historically been identified almost entirely based on morphological characters. This study aimed to understand how the morphology of Neobenedenia girellae varies with host fish species and the environment. Standard morphological features of genetically indistinct parasites from various host fish species were measured under controlled temperatures and salinities. An initial field-based investigation found that parasite morphology significantly differed between genetically indistinct parasites infecting various host fish species. The majority of the morphological variation observed (60%) was attributed to features that assist in parasite attachment to the host (i.e. the posterior and anterior attachment organs and their accessory hooks) which are important characters in monogenean taxonomy. We then experimentally examined the effects of the interaction between host fish species and environmental factors (temperature and salinity) on the morphology of isogenic parasites derived from a single, isolated hermaphroditic N. girellae infecting barramundi, Lates calcarifer. Experimental infection of L. calcarifer and cobia, Rachycentron canadum, under controlled laboratory conditions did not confer host-mediated phenotypic plasticity in N. girellae, suggesting that measured morphological differences could be adaptive and only occur over multiple parasite generations. Subsequent experimental infection of a single host species, L. calcarifer, at various temperatures (22, 30 and 32?°C) and salinities (35 and 40‰) showed that in the cooler environments (22?°C) N. girellae body proportions were significantly smaller compared with warmer temperatures (30 and 32?°C; P?<?0.0001), whereas salinity had no effect. This is evidence that temperature can drive phenotypic plasticity in key taxonomic characters of N. girellae under certain environmental conditions.     

Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)

Neobenedenia girellae, a monogenean skin parasite, shows low host specificity. N. girellae is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. To clarify the mechanisms of host specificity, we purified and identified the attachment-inducing substances of oncomiracidia from tiger puffer fish. The attachment-inducing substances were mainly included in skin mucous extract. Skin mucous extract lost its ability to induce attachment after boiling and/or exposure to the reducing agent dithiothreitol, suggesting that attachment-inducing substances are of a proteinaceous nature. Since lectins such as Con A, WGA, PHA-L, and PSA inhibited the induction of attachment, attachment-inducing proteins were suspected to be glycoproteins. Glycoproteins specifically interacting with Con A were collected and purified by anion exchange chromatography, resulting in two active peaks (peaks 3-A and 6). The active component in peak 3-A was identified as Wap 65-2 by N-terminal amino acid sequencing, while the glycoprotein in peak 6 could not be identified. These results suggested that oncomiracidia recognised Wap 65-2 and another glycoprotein of their host.     

Insemination of the monogenean Neobenedenia girellae (Capsalidae,Benedeniinae)

In vitro spermatophore formation and insemination of Neobenedenia girellae (Monogenea: Capsalidae, Benedeniinae) were recorded on video and described for the first time. Upon contact of two individuals, the anterior adhesive discs of the donor firmly attached to the dorsal tegument of the recipient and the donor's fore body strongly contracted such that the genital pore region protruded and the penis was pushed anteriorly to protrude through the genital pore. It is hypothesised that the donor penis mechanically damaged the tegument of the recipient. The sperm and spermatophore matrix were released together through the penis, which was placed under the left anterior attachment disc immediately behind the adhesive pad. The spermatophore matrix containing the spermatozoa became solid and attached to the dorsal surface of recipient's body. When observed under scanning electron microscopy, the spermatophores were irregularly shaped, with a diameter of 52–83 μm. Under light microscopy they consisted of a proximal eosinophilic matrix portion and a distal thin-walled portion containing spermatozoa. Both parts were enclosed with a thin outer casing. Insemination occurred during and after spermatophore formation. Three types of insemination were recorded, unilateral and mutual insemination and self-insemination. The presence of self-insemination indicates that even a single N. girellae on a cultured fish may cause a significant parasite infection in the entire aquaculture system.     

Reproductive Strategies of the Insidious Fish Ectoparasite,Neobenedenia sp. (Capsalidae: Monogenea)

Fish monogeneans are lethal parasites in aquaculture. We provide the first experimental evidence that a notorious fish monogenean, Neobenedenia sp., can produce viable eggs in isolation for three consecutive generations. We infected individual, isolated, farmed barramundi, Lates calcarifer (Bloch) with a single oncomiracidium (larva) of the hermaphroditic monogenean Neobenedenia sp. Isolated parasites reached sexual maturity at day 10 post-hatch (24°C, 35‰) and laid ∼3,300 embryonated eggs over 17 days. Egg production rapidly increased following sexually maturity on day 10 (58±15 eggs) and peaked on day 15 (496±68 eggs) before gradually decreasing. Neobenedenia sp. exhibited egg laying and egg hatching rhythms. Parasites laid eggs continuously, but egg production increased in periods of darkness (64.3%), while the majority of oncomiracidia (81%) emerged from eggs in the first three hours of light. Eggs laid by isolated ‘parent’ parasites hatched and individual emerging oncomiracidia were used to infect more individual, isolated fish, with three consecutive, isolated, parasite generations (F1, F2 and F3) raised in the laboratory. Infection success and egg hatching success did not differ between generations. Our data show that one parasite, in the absence of a mate, presents a severe threat to captive fish populations.     

Neobenedenia pargueraensis n. sp. (Monogenea: Capsalidae) from the red hind, Epinephelus guttatus, and comments about Neobenedenia melleni.

Neobenedenia pargueraensis n. sp. from Epinephelus guttatus of Puerto Rico differs from all known species in possessing scoop-shaped accessory sclerites with pointed tips. It is most similar to Neobenedenia melleni and Neobenedenia longiprostata but differs from the former in having a fenestrate ovary and from the latter in that the ducts of the accessory glands are short, terminating just anterior to the vitelline reservoir rather than reaching the caudal end of the body. It differs from both species in having smooth rather than lobate testes. Neobenedenia melleni occurs throughout the tropical and subtropical western North Atlantic including Bermuda. It kills aquarium and aquaculture fishes with massive infestations and is a severe restraint on the culture of tilapia in sea water in the Caribbean.     

Habitat and species differences in prevalence and intensity of Neobenedenia melleni (Monogenea: Capsalidae) on sympatric Caribbean surgeonfishes (Acanthuridae)

We quantified Neobenedenia melleni from the skin of Caribbean surgeonfishes (Acanthuridae) from June through October 2005 and 2007. Prevalence, or mean intensity of infection, or both, varied significantly among the 3 species, and among sites and between years for the most heavily infected species, blue tang (Acanthurus coeruleus). Among 6 sites sampled, no more than 12% of ocean surgeonfish (Acanthurus bahianus) were infected, compared with 10 to 100% of A. coeruleus. The prevalence of infection among doctorfish (Acanthurus chirurgus), collected at only 1 of the sites, was intermediate between the other 2 species (46%). Mean intensity (range) of infection for the few infected A. bahianus was 1 (1) to 3 (1-8), compared with 1.3 (1-2) to 14.3 (1-59) for A. coeruleus, and 2.5 (1-8) for A. chirurgus. Expected abundance of N. melleni on A. coeruleus from shallow bay sites was greater than for those from non-bay sites. Higher infections on A. coeruleus may be attributable to differences in habitat use, or susceptibility to infection, or both, compared to other species. Among site and between-year differences may be associated with differences in benthic habitat, or water conditions, or both. This system seems ideal for future comparative studies on the relationship between environmental variables and parasites on Caribbean coral reefs.     

Evidence of complement-mediated killing of Discocotyle sagittata (Platyhelminthes, Monogenea) oncomiracidia

Discocotyle sagittata oncomiracidia were rapidly killed when incubated in na?ve plasma and immune sera from both rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta), the killing proceeding at a faster rate with blood material from the latter fish species. The lethal activity of na?ve plasma and immune sera was comparable. This was abolished after incubation at 45 degrees C for 30 min and by the addition of EDTA but not EGTA supplemented with Mg(2+), indicating that complement acting via the alternative pathway is responsible for the parasiticidal effect observed. Scanning electron micrographs showed varying degrees of surface disruption in larvae exposed to fish plasma, suggesting that complement acts by breaching the oncomiracidial tegument. Control (untreated) oncomiracidia showed no damage. Ultrastructural damage was more extensive in oncomiracidia exposed to brown trout plasma than to rainbow trout plasma for equal periods, suggesting that the complement cascade may be involved in mediating host susceptibility.     

First published record of the pathogenic monogenean parasite Neobenedenia melleni (Capsalidae) from Australia

The monogenean Neobenedenia melleni (MacCallum, 1927) Yamaguti 1963 is a well-known and virulent pathogen in culture conditions recorded from the skin of many teleost fish species worldwide. Until now, N. melleni has not been reported from wild or cultured fish in Australian waters. This study documents a recent outbreak of N. melleni that occurred on Lates calcarifer (barramundi) cultivated in sea cages in Hinchinbrook Channel between Hinchinbrook Island and mainland Queensland, Australia, which resulted in the loss of 200,000 fish (50 tonnes). The origin of this outbreak is unclear because N. melleni has not been recorded from any wild host species in Australia and strict quarantine regulations exclude the possibility of its introduction on imported fish. We propose that N. melleni occurs naturally on wild populations of some teleost species in Australian waters and that the few surveys of wild fish conducted along the east coast have failed to report this species. The possibility that uncharacteristically low water temperatures led to the outbreak is discussed.     

Serotonin and dopamine in the tissue extracts of Polystoma integerrimum (Monogenea)

Spectrofluorometric analysis has revealed substances with spectral characteristics identical to those of serotonin and dopamine in tissue extracts of Polystoma integerrimum. A possible function of the substances is discussed.     

Lipid content and fatty acid composition of the monogenean Neobenedenia girellae and comparison between the parasite and host fish species

Neobenedenia girellae, a capsalid monogenean, is a destructive fish parasite. We studied the lipid content and fatty acid composition of N. girellae and the skin and cutaneous mucus of a host fish, the amberjack Seriola dumerili (Carangidae). The lipid content of adult N. girellae was less than one-fourth that of both the skin and cutaneous mucus of its host. Adult N. girellae, S. dumerili skin and mucus had a relatively high weight-percentage of C16:0, C18:1(n-9), C18:0 and C22:6(n-3) fatty acids. When S. dumerili were fed a diet supplemented with [13C] fatty acids, [13C] fatty acids were detected in S. dumerili skin and adult N. girellae on S. dumerili, but no [13C] fatty acids were detected in the S. dumerili cutaneous mucus. In addition, the epidermis of S. dumerili, attached with N. girellae, was markedly thin. These results suggest that N. girellae feeds primarily on host epithelial cells. We then infected 2 host fishes, S. dumerili and the spotted halibut Verasper variegatus (Pleuronectidae; a host less susceptible to N. girellae infection), and compared the fatty acid composition of N. girellae with that of the skin and cutaneous mucus of the hosts. The fatty acid profiles from all samples were qualitatively and quantitatively similar. Thus, the fatty acid composition of the host may not contribute to the difference in susceptibility between S. dumerili and V. variegatus. These results may serve to develop new strategies for the control of N. girellae infections.     

Comparative susceptibility of two different genetic types of tilapia to Neobenedenia sp. (Monogenea)

Two different genetic types of tilapia, Mozambique tilapia Oreochromis mossambicus (MT), and Pargo-UNAM (PU; a synthetic hybrid whose genetic composition is 50% Florida red tilapia, 25% Rocky Mountain tilapia, and 25% red variant Oreochromis niloticus), were acclimatized to salinity and exposed to seawater from the Gulf of Mexico off the port of Veracruz, Mexico. Both fish types were infected by the monogenean ectoparasite Neobenedenia sp. and were killed within 2 to 3 wk. A crude worm extract was prepared from whole specimens collected during the original outbreak and used to immunize naive hosts of the same 2 types of tilapia. Immunized fish were then exposed to seawater, which resulted in Neobenedenia sp. infection. Immunization did not confer any protection against Neobenedenia sp. infection. However, the experiment enabled detailed analysis of the dynamics of infection and comparison of the effects of the parasite on the 2 host types. Although both tilapia types exhibited similar resistance to infection (as they harbored similar parasite burdens in the early phase of infection), PU is less tolerant to Neobenedenia sp., as a mean parasite abundance of ca. 50 worms fish-' killed all hosts within a fortnight, while 22% of MT survived up to 3 wk, harboring a mean parasite abundance of ca. 900 worms fish-'. Our results suggest that, as reported elsewhere, Neobenedenia sp. could negatively affect mariculture off the Mexican coast of the Gulf of Mexico.     

Chatton-Lwoff silver impregnation: an improved technique for the study of oncomiracidia (Platyhelminthes: Monogenea) chaetotaxy

Summary Difficulty was experienced in using direct silver nitrate impregnation techniques in the study of the chaetotaxy of the oncomiracidia of several freshwater ancyrocephalines. Eventually the Chatton-Lwoff method of silver impregnation, which was devised to study the cilia and basal bodies of ciliated protozoa, was utilized successfully following certain modifications. The improved technique is described in detail and photomicrographs showing the pattern of the sensillae are presented. ac]19800820     

Expression of vasa (vas)-related genes in germ cells and specific interference with gene functions by double-stranded RNA in the monogenean, Neobenedenia girellae

Neobenedenia girellae, a monogenean, is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. Aiming to establish a new control method by interfering with the gametogenesis of N. girellae, we focused on vasa (vas)-related genes that are expressed exclusively in the germline granules in Drosophila, Caenorhabditis elegans and other animals. Three vas-related genes (N. girellae vasa-like gene, Ngvlg1, Ngvlg2 and Ngvlg3) were isolated by PCR and Ngvlg1 and Ngvlg2 were shown to be expressed only in germ cells. We demonstrated that introduction of double-stranded Ngvlg1 or Ngvlg2 RNA by soaking resulted in partial or complete loss of germ cells. Moreover, the hatching rate of eggs from animals showing partial loss of germ cells decreased significantly. These results suggest that Ngvlg1 and Ngvlg2 are essential genes for germ cell quantity and quality. The possibility that a new control method can be developed by controlling gametogenesis of N. girellae was proven, because sterilised N. girellae could be produced.     

Effect of water temperature on reproductive development of Benedenia seriolae (Monogenea: Capsalidae) from Seriola lalandi in Australia

The monogenean Benedenia seriolae (Yamaguti, 1934) Meserve, 1938 is a major pathogen of farmed yellowtail kingfish Seriola lalandi in South Australia. To control parasite populations in commercial farms, an understanding of the effect of water temperature on parasite development is vital. This study investigated the effect of water temperature (14, 18, 22 and 26 +/- 0.5 degrees C) on development, growth rates and age at sexual maturity of B. seriolae. Five distinct developmental stages, defined by the progressive development of reproductive organs, were used to describe development of B. seriolae from recently invaded larvae to sexually mature adults. Parasite age at the first sign of sexual maturity (ability to lay eggs) was strongly influenced by water temperature and was attained at 41, 24, 16 and 14 d post infection (p.i.) at 14, 18, 22 and 26 +/- 0.5 degrees C respectively. Four parameters (parasite total length, maximum parasite width, accessory sclerite length and anterior hamulus length) were examined for suitability as an index of parasite age. Growth rates for each parameter increased with water temperature; however, mean anterior hamulus length was identified as the most reliable index of parasite age. Equations derived from these data can be used to estimate parasite age and time to sexual maturity at water temperatures ranging from 14 to 26 degrees C. The equations provide a simple tool to assist implementation of strategic treatment plans for B. seriolae infections in commercial kingfish farms, not only in South Australia but in other localities worldwide where S. lalandi is farmed.     

New species of the genera Pseudallobenedenia Yamaguti, 1966 and Lagenivaginopseudobenedenia Yamaguti, 1966 (Monogenea: Capsalidae) in the Indo-Pacific

Two new capsalid species, Pseudallobenedenia arabica n. sp. from the gills of Pristipomoides filamentosus (Lutjanidae) from the Arabian Sea and Lagenivaginopseudobenedenia tinrowi n. sp. from the gills of Etelis carbunculus (Lutjanidae) from the eastern Pacific Ocean are described and illustrated. P. arabica differs from the most similar species, P. opakapaka Yamaguti, 1966, in the greater width of the body, its larger haptor and anterior adhesive discs, the form and sizes of the testes and ovary, and differences in host and locality. L. tinrowi differs from its congener, L. etelis Yamaguti, 1966, in the larger size of the body and its organs, the absence of eye-spots and Goto's glands, and differences in the arrangement of the genital organs. All of the species in these two genera differ from other capsalids by the presence of a voluminous uterus and an extremely long penis forming a loop. All species in these two genera are parasites of fishes of the family Lutjanidae and are reported from Indo-Pacific subtropical areas.     

Phylogenetic relationships between the families Capsalidae and Dionchidae (Platyhelminthes, Monogenea, Monopisthocotylea) indicated by the comparative ultrastructural study of spermiogenesis

Comparative ultrastructural observations were carried out on the spermiogenesis of the capsalid Caballerocotyla manteri Price and the dionchid Dionchus remorae MacCallum. At the beginning of spermiogenesis the zones of differentiation (ZD) jut out in all directions. A large mitochondrion shaped like a perforated bead, and through which the elongated nucleus passes, is found facing each ZD. Later the ZD become parallel and are embedded within the common cytoplasmic mass. Cortical longitudinal microtubules are present in the ZD at the outset of spermiogenesis, but they later disappear. The spermatozoon is long and filiform. It shows two parallel axonemes of the 9 +"1" flatworm pattern, the nucleus and mitochondrion, and no cortical microtubule. The ultrastructure of spermiogenesis and spermatozoon is remarkably similar in the two species studied, as in other capsalids previously described. Two characteristics, the perforated bead shape of the spermatid mitochondrion and the progressive disappearance of the microtubules of the ZD, may be considered as synapomorphies which indicate close phylogenetic relationships between the families Dionchidae and Capsalidae. This interpretation coincides with Llewellyn's (1971) scheme of the evolution of the monogeneans.     

Monogenea (platyhelminthes) as hazards for fish in confinement

Occurrences of large-scale, parasite-related fish diseases have increased as the number and size of hatcheries, mass culture facilities, and public and private desplay aquaria have grown. This increase is due to concentration of hosts and parasites in confined quarters resulting in enhanced opportunity for infestations and epizootics. Monogeneans, usually well accommodated to their fish hosts in nature and causing few easily detectable effects, frequently cause severe epizootics in cultured or aquarium-held populations. In such cases, the short, direct, one-host life cycles of monogeneans enable them to reach epizootic levels very quickly when hosts and parasites are confined closely together. Also, despite their high host-specificity in the wild some monogeneans may infest numbers of other hosts when several or many fish species are confined together. Further, debilitating attacks by these helminths may be accompanied by invasions of viruses, bacteria, or fungi. Such invasion is usually via portals of entry caused by trauma of feeding or attachment by the worms. Blood-feeding monogeneans may transmit disease directly.When single or mixed-species host populations are under stress, monogeneans, like other normally benign parasites, may become pathogenic. Stress in hosts results from any single factor, or combinations of factors including: poor sanitation, poor nutrition, poor water quality, overcrowding, behaviorally incompatible species, or improperly designed culture systems or aquaria.Whatever the cause or route of infestation by monogeneans or associated flora and fauna, health and growth of hosts is usually impaired by heavy parasite attack. Deaths are not uncommon. Culturists and aquarists alike must take precautions against monogenean-related disease. Foremost among the precautions is design and thoughtful utilization of adequate holding and display facilities, and adherence to adequate acquisition and introduction procedures, such as the ICES (International Council for the Exploration of the Seas) methods. Careful quarantine and introduction procedures, with thorough monitoring of water quality are paramount. Chemical prophylaxis is possible and should be practiced as needed. When infestations occur, as they often do despite precautions, chemical treatments must be introduced. When epizootics persist, stocks must be harvested quickly or discarded. In aquaria, isolation and treatment of infested individuals or systems may be useful. If this fails, destruction is recommended. The best measure against parasitic monogeneans is avoidance or prevention. Effective sanitation is critical and must be rigorously applied. These and other aspects of monogenean infestation, associated pathology, and prevention or treatment are described and discussed.     

Diplectanidae (Monogenea) parasites of fish of the Kerkennah Islands (Tunisia)

Eleven Diplectanidae (Monogenea) gill parasites from various Sparidae and Serranidae of the Kerkennah Islands are noted. Three new species: Lamellodiscus hilii, L. bidens, L. impervius, parasites of Puntazzo puntazzo (Sparidae) are described.