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.     

Halogenated acetic and acrylic acids from the red alga Asparagopsis taxiformis

Nine halogenated acetic acids and nine halogenated acrylic acids have been identified in the aqueous extract of Hawaiian Asparagopsis taxiformis.     

Beyond Symbiosis: Cleaner Shrimp Clean Up in Culture

Cleaner organisms exhibit a remarkable natural behaviour where they consume ectoparasites attached to “client” organisms. While this behaviour can be utilized as a natural method of parasitic disease control (or biocontrol), it is not known whether cleaner organisms can also limit reinfection from parasite eggs and larvae within the environment. Here we show that cleaner shrimp, Lysmata amboinensis, consume eggs and larvae of a harmful monogenean parasite, Neobenedenia sp., in aquaculture. Shrimp consumed parasite eggs under diurnal (63%) and nocturnal (14%) conditions as well as infectious larvae (oncomiracidia) diurnally (26%). Furthermore, we trialled the inclusion of cleaner shrimp for preventative parasite management of ornamental fish, Pseudanthias squamipinnis, and found shrimp reduced oncomiracidia infection success of host fish by half compared to controls (held without shrimp). Fish held without cleaner shrimp exhibited pigmentation changes as a result of infection, possibly indicative of a stress response. These results provide the first empirical evidence that cleaner organisms reduce parasite loads in the environment through non-symbiotic cleaning activities. Our research findings have relevance to aquaculture and the marine ornamental trade, where cleaner shrimp could be applied for prophylaxis and control of ectoparasite infections.     

Alternative development in Polystoma gallieni (Platyhelminthes,Monogenea) and life cycle evolution

Considering the addition of intermediate transmission steps during life cycle evolution, developmental plasticity, canalization forces and inherited parental effect must be invoked to explain new host colonization. Unfortunately, there is a lack of experimental procedures and relevant models to explore the adaptive value of alternative developmental phenotypes during life cycle evolution. However, within the monogeneans that are characterized by a direct life cycle, an extension of the transmission strategy of amphibian parasites has been reported within species of Polystoma and Metapolystoma (Polyopisthocotylea; Polystomatidae). In this study, we tested whether the infection success of Polystoma gallieni within tadpoles of its specific host, the Stripeless Tree Frog Hyla meridionalis, differs depending on the parental origin of the oncomiracidium. An increase in the infection success of the parasitic larvae when exposed to the same experimental conditions as their parents was expected as an adaptive pattern of non-genetic inherited information. Twice as many parasites were actually recorded from tadpoles infected with oncomiracidia hatching from eggs of the bladder parental phenotype (1.63 ± 0.82 parasites per host) than from tadpoles infected with oncomiracidia hatching from eggs of the branchial parental phenotype (0.83 ± 0.64 parasites per host). Because in natural environments the alternation of the two phenotypes is likely to occur due to the ecology of its host, the differential infection success within young tadpoles could have an adaptive value that favors the parasite transmission over time.     

Habitat heterogeneity promotes the coexistence of exotic seaweeds

Despite the progressive accumulation of exotic species in natural communities, little effort has been devoted to elucidating the mechanisms underpinning the coexistence of invaders in environmentally and biologically heterogeneous systems. The exotic seaweeds, Asparagopsis taxiformis and Caulerpa racemosa, exhibit a segregated distribution on Mediterranean rocky reefs. A. taxiformis dominates assemblages in topographically complex habitats, but is virtually absent on homogenous platforms. In contrast, C. racemosa achieves extensive cover in both types of habitat. We assessed whether differences in their distribution were generated by biotic interactions (between invaders and/or between invaders and natives) or by environmental constraints. Three models were proposed to explain seaweed distribution patterns: (1) invaders inhibit one another; (2) native assemblages, differing between complex and simple habitats, prevent the establishment/spread of one invader, but not that of the other; and (3) environmental conditions regulate the establishment/persistence of the seaweeds in different habitats. We removed the dominant invader and resident assemblages in each type of habitat. Moreover, A. taxiformis thalli were transplanted into the habitat dominated by C. racemosa to establish whether its failure to colonize the simple habitat was due to the lack of propagules or post-recruitment mortality. C. racemosa spread in the complex habitat was not influenced by the removal of resident assemblages, but it was slightly enhanced by A. taxiformis removal. Neither C. racemosa removal nor that of resident assemblages promoted A. taxiformis colonization and survival in simple habitats. Our results suggest that heterogeneity in environmental conditions can promote invader coexistence by mitigating the effects of negative biotic interactions. Therefore, the accumulation of introduced species in native communities does not necessarily imply established invaders fostering further invasion.     

Halogen chemistry of the red alga Asparagopsis

Ethanol extraction of fresh Asparagopsis taxiformis and A. armata, followed by pentane partition, results in the isolation of a series of halomethanes, among them MeI, CHCl₃, and CCl₄. Under these extraction conditions, esterification readily occurs, allowing the isolation and identification of a series of polyhaloethyl acetates and acrylates. The recognition of acetone and polyhaloacetones in these extracts suggests biological halogenations occur which result in haloform reactions.     

Water-soluble natural products from seaweed have limited potential in controlling bacterial pathogens in fish aquaculture

Seaweeds producing natural products with in vitro efficacy against bacterial pathogens offer the opportunity for therapeutic services in fish aquaculture, for example inhibiting the growth of pathogens through the direct release of bioactive metabolites into the culture environment. The red alga Asparagopsis taxiformis was used to test this hypothesis because of its effective natural products and mechanisms to release these metabolites into the surrounding environment and because it can be cultivated intensively in land-based systems. Here we quantify the release, accumulation and residence time of the major halogenated metabolites from A. taxiformis in the cultivation medium and subsequently test the in vitro bioactivity of these water-soluble metabolites against multiple strains of the pathogenic bacterium Streptococcus iniae. The two major halogenated metabolites in A. taxiformis (bromoform and dibromoacetic acid) were released into the water and reaching steady-state concentrations of 27.7?±?1.5 μg L^?1 and 4.8?±?1.3 μg L^?1, respectively, under standard cultivation conditions. This water delayed the in vitro growth of the pathogenic bacteria S. iniae but did not prevent it. Increasing the levels of A. taxiformis metabolites in the water by three orders of magnitude with aqueous extracts of biomass inhibited the growth of S. iniae, but it was also toxic to fish. In an immersion challenge experiment, intermediate non-ichthyotoxic levels of A. taxiformis metabolites were ineffective as a treatment for barramundi infected by S. iniae. The evidence suggests that there is little potential for A. taxiformis bioactive metabolites to provide therapy services to fish infected with S. iniae.     

Endemic or introduced? Phylogeography of Asparagopsis (Florideophyceae) in Australia reveals multiple introductions and a new mitochondrial lineage

The red seaweed Asparagopsis taxiformis embodies five cryptic mitochondrial lineages (lineage 1–5) introduced worldwide as a consequence of human mediated transport and climate change. We compared globally collected mitochondrial cox2‐3 intergenic spacer sequences with sequences produced from multiple Australian locations and South Korea to identify Asparagopsis lineages and to reveal cryptic introductions. We report A. taxiformis lineage 4 from Cocos (Keeling) Islands, Australia, and the highly invasive Indo‐Pacific Mediterranean lineage 2 from South Korea and Lord Howe Island, Australia. Phylogeographic analysis showed a clear haplotype and geographic separation between western Australian and Great Barrier Reef (GBR) isolates belonging to the recently described lineage 5. The same lineage, however, was characterized by a substantial genetic and geographic break between the majority of Australian specimens and Asparagopsis collections from South Solitary Island, Southern GBR, Lord Howe Island, Kermadec Islands, Norfolk Island, New Caledonia and French Polynesia. The disjunct geographic distribution and sequence divergence between these two groups supports the recognition of a sixth cryptic A. taxiformis mitochondrial lineage. As climatic changes accelerate the relocation of biota and offer novel niches for colonization, periodic surveys for early detection of cryptic invasive seaweeds will be critical in determining whether eradication or effective containment of the aliens are feasible.     

Effects of Asparagopsis taxiformis metabolites on the feeding behaviour of post-larval Acanthurus triostegus

Our study highlights the effect of the macroalgae Asparagopsis taxiformis on the feeding behaviour of the tropical surgeonfish Acanthurus triostegus. The presence of A. taxiformis chemical cues reduced A. triostegus feeding, suggesting that the presence of this algae could affect not only the survival of fish in the post-larval stage, but also alter the grazing pressure on coral reefs.     

A comparative study of the structure of the oncomiracidia of the monogenean gill parasites Diclidophora merlangi,D. luscae and D. denticulata

The structure of the oncomiracidia of gill parasites Diclidophora merlangi, D. luscae and D. denticulata is described. The three larvae differ in body length and in the size and arrangement of the hooks but the glandular components and excretory systems are similar. There is some evidence that glandular secretions aid hatching. In newly hatched larvae of D. merlangi marginal hook III is displaced and a pouch of tissue occurs between hooks II and IV. The pouches are presumptive clamp tissue. Many larvae fail to hatch in vitro but form one pair of clamps while within the egg capsule.     

Red algal exotics on North Sea coasts

A total of ten red seaweed species are recognized as introduced into the North Sea from other parts of the world. These areAsparagopsis armata andBonnemaisonia hamifera (Bonnemaisoniales),Grateloupia doryphora (Halymeniales),Antithamnionella spirographidis, Antithamnionella ternifolia, Anotrichium furcellatum, Dasya baillouviana, ?Dasysiphonia sp.,Polysiphonia harveyi andPolysiphonia senticulosa (Ceramiales). The oldest of these isB. hamifera, introduced prior to 1890, while the most recent,?Dasysiphonia sp., was first found in 1994 and still requires taxonomic investigation. A variety of distribution patterns is seen, with geographical ranges varying from general within the North Sea to very restricted. The diversity of introduced red algae on eastern coasts of the North Sea is much greater than in the west. The most likely explanation for this pattern is that French coasts were the initial site of introduction for many of the seaweeds, which were then distributed northwards by the residual surface currents. Their increasing success in the Netherlands has probably been promoted by the drastically changed local hydrodynamic conditions which have also permitted the recent introduction of many native European species. Of the biological features of species that may favour their success as introductions, clonal vegetative propagation, often with specialized propagules or fragmentation mechanisms, is almost ubiquitous. Low-temperature tolerances can be inferred, but data are sparse. Many of the alien red algae in the North Sea contain anti-grazing compounds such as bromophenols, which may contribute to their invasive potential by deterring grazing sufficiently to permit establishment of an inoculum.     

The Phenolic Contents and Antimicrobial Activities of Some Marine Algae from the Mediterranean Sea (Algeria)

In this study, three marine algae collected from western coast of algerian mediteranean sea (Ulva lactuca, Dictyota dichotoma, and Corallina elongata) were tested using the agar-well diffusion method for their production of antibacterial and antifungal agents on various organisms that cause diseases of humans and plants (Eschirichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Salmonella sp, Candida albicans, and Penicillium sp.). The total phenol content and antimicrobial activity were determined using different crude seaweeds extracts (methanol, diethylether, and chloroform). The results show that the chloroform extracts of (Ulva lactuca and Corallina elongata) had the highest activity against E. coli and Salmonella sp. The methanol extract obtained from (Ulva lactuca, Dictyota dichotoma, and Corallina elongata) showed antifungal activity for Candida albicans. The results of the study revealed that the seaweeds from Algeria appear to have immense potential as a source of antibacterial and antifungal compounds; they can be used in treating diseases caused by these organisms.     

Assessing global range expansion in a cryptic species complex: insights from the red seaweed genus Asparagopsis (Florideophyceae)

The mitochondrial genetic diversity, distribution and invasive potential of multiple cryptic operational taxonomic units (OTUs) of the red invasive seaweed Asparagopsis were assessed by studying introduced Mediterranean and Hawaiian populations. Invasive behavior of each Asparagopsis OTU was inferred from phylogeographic reconstructions, past historical demographic dynamics, recent range expansion assessments and future distributional predictions obtained from demographic models. Genealogical networks resolved Asparagopsis gametophytes and tetrasporophytes into four A. taxiformis and one A. armata cryptic OTUs. Falkenbergia isolates of A. taxiformis L3 were recovered for the first time in the western Mediterranean Sea and represent a new introduction for this area. Neutrality statistics supported past range expansion for A. taxiformis L1 and L2 in Hawaii. On the other hand, extreme geographic expansion and an increase in effective population size were found only for A. taxiformis L2 in the western Mediterranean Sea. Distribution models predicted shifts of the climatically suitable areas and population expansion for A. armata L1 and A. taxiformis L1 and L2. Our integrated study confirms a high invasive risk for A. taxiformis L1 and L2 in temperate and tropical areas. Despite the differences in predictions among modelling approaches, a number of regions were identified as zones with high invasion risk for A. taxiformis L2. Since range shifts are likely climate‐driven phenomena, future invasive behavior cannot be excluded for the rest of the lineages.     

Antioxidant activities and phenolics content of eight species of seaweeds from north Borneo

The antioxidant activity of eight edible species of Malaysian North Borneo seaweeds obtained from Sabah waters (Kudat, Tanjung Aru and Semporna) consisting of three red seaweeds (Eucheuma cottonii, E. spinosum and Halymenia durvillaei), two green seaweeds (Caulerpa lentillifera and C. racemosa) and three brown seaweeds (Dictyota dichotoma, Sargassum polycystum and Padina sp.) were determined. Methanol and diethyl ether were used as extraction solvent. The antioxidant activities were determined by two methods, TEAC (trolox equivalent antioxidant capacity) and FRAP (ferric reducing antioxidant power) assays. The total phenolic content of the extract was determined according to the Folin–Ciocalteu method and results were expressed as phloroglucinol equivalents. The methanolic extracts of green seaweeds, C. lentillifera and C. racemosa, and the brown seaweed, S. polycystum showed better radical-scavenging and reducing power ability, and higher phenolic content than the other seaweeds. The TEAC and FRAP assays showed positive and significantly high correlation (R ² = 0.89). There was a strong correlation (R ² = 0.96) between the reducing power and the total phenolic content of the seaweeds methanolic dry extracts. These seaweeds could be potential rich sources of natural antioxidants.     

Monogenoidean parasites of the gill lamellae of the sheepshead Archosargus probatocephalus (Walbaum) (Perciformes: Sparidae) from the Indian River Lagoon, Florida, with descriptions of four new species of Euryhaliotrema Kritsky & Boeger, 2002 (Dactylogyridae)

Examination of the gill lamellae of three sheepshead Archosargus probatocephalus (Walbaum) from the Indian River Lagoon in Florida revealed six species of Monogenoidea: Microcotyle archosargi MacCallum, 1913 (Microcotylidae); Neobenedenia sp. (Capsalidae); and four new species of Euryhaliotrema Kritsky & Boeger, 2002 (Dactylogyridae). The prevalence of all helminths was 100%, except for Neobenedenia sp., which was represented by a single immature specimen. The four new species, Euryhaliotrema carbuncularium n. sp., E. dunlapae n. sp., E. amydrum n. sp. and E. spirulum n. sp., are described and with E. carbunculus (Hargis, 1955) Kritsky & Boeger, 2002 apparently constitute a monophyletic clade of Euryhaliotrema spp. that parasitise sparid hosts in the western hemisphere. The Indian River Lagoon in Florida represents a new locality record for M. archosargi, and the sheepshead is apparently a new host record for a member (Neobenedenia sp.) of the Capsalidae.     

Attachment-inducing capacities of fish tissue extracts on oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)

When oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae) were incubated in wells with lyophilized extracts of fish skin epithelia on the bottom, some attached to the well bottom with the haptor unfolded and shed the ciliated epidermal cells. Based on these morphological changes in oncomiracidia, we developed a new assay method to examine the attachment-inducing capacities of various fish extracts for oncomiracidia. Attachment-inducing capacities were found only in extracts of fish skin epithelium and not in other fish extracts. No significant difference in capacities was observed among extracts of skin epithelia of 4 fish species. Wheat germ lectin and concanavalin A suppressed capacities in extracts of skin epithelia of Japanese flounder and yellowtail, respectively. Suppressed capacities were recovered by adding sugars that bound specifically to these lectins. These results indicate that some sugar-related chemical substances that exist specifically in fish epithelium induce the attachment of N. girellae oncomiracidia.     

Size structure and dynamics of an invasive population of lineage 2 of Asparagopsis taxiformis (Florideophyceae) in the Alboran Sea

In this study, we present basic population data of the red macroalga Asparagopsis taxiformis, widely recognized as invasive in the Mediterranean Sea. A 13‐month field study was carried out on a population located in southern Spain, addressing its phenology, population dynamics and demography. We further tested whether biomass variations were related to environmental variables at the study site. Gametophytes were present year‐round while tetrasporophytes were only found in spring and summer. Recruitment capacity and vegetative growth of the gametophytes are discussed as important modulators for the population structure and enhancers of its persistence. Thallus size‐time histograms revealed a high prevalence of small shoots that showed high mortality that was not related to self‐thinning. Biomass of A. taxiformis was higher from March to July. Vegetative growth was the main way the gametophyte population was maintained, although the presence of tetrasporophytes and fertile gametophytes in the field confirms that sexual reproduction also occurs. Its continuous and high recruitment, in terms of the number of smallest shoots, makes this southern population of A. taxiformis a source of future invasive populations due to the intensive maritime traffic in the region.     

Identification of reproductive sex-biased gene expression in Asparagopsis taxiformis (lineage 6) gametophytes

The sub-tropical red seaweed Asparagopsis taxiformis is of significant interest due to its ability to store halogenated compounds, including bromoform, which can mitigate methane production in ruminants. Significant scale-up of aquaculture production of this seaweed is required; however, relatively little is known about the molecular mechanisms that control fundamental physiological processes, including the regulatory factors that determine sexual dimorphism in gametophytes. In this study, we used comparative RNA-sequencing analysis between different morphological parts of mature male and female A. taxiformis (lineage 6) gametophytes that resulted in greater number of sex-biased gene expression in tips (containing the reproductive structures for both sexes), compared with the somatic main axis and rhizomes. Further comparative RNA-seq against immature tips was used to identify 62 reproductive sex-biased genes (59 male-biased, 3 female-biased). Of the reproductive male-biased genes, 46% had an unknown function, while others were predicted to be regulatory factors and enzymes involved in signaling. We found that bromoform content obtained from female samples (8.5 ± 1.0 mg·g⁻¹ dry weight) was ~10% higher on average than that of male samples (6.5 ± 1.0 mg·g⁻¹ dry weight), although no significant difference was observed (p > 0.05). There was also no significant difference in the marine bromoform biosynthesis locus gene expression. In summary, our comparative RNA-sequencing analysis provides a first insight into the potential molecular factors relevant to gametogenesis and sexual differentiation in A. taxiformis, with potential benefits for identification of sex-specific markers.     

Antioxidant properties and total phenolic contents of some tropical seaweeds of the Brazilian coast

Many types of macroalgae contain a wide range of bioactive compounds that have antioxidant potential. However, in contrast to terrestrial plants, only a few studies have reported the antioxidant activity of seaweeds. Therefore, extracts from 26 marine macroalgae species from the south and southeast coasts of Brazil were evaluated for their antioxidant activity, using the 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) method and β-carotene/linoleic acid assay, and their total phenolic contents, through Folin–Ciocalteu method. Padina gymnospora, Sargassum vulgare, and Osmundaria obtusiloba presented the highest values of total phenolic content. Using β-carotene bleaching assay, Colpomenia sinuosa, Dictyota sp., Dichotomaria marginata, Ganonema farinosum, and Spyridia clavata presented up to 65 % of antioxidant activity. Some of the extracts showed more than 60 % of inhibition of DPPH in the lowest concentration (0.01 mg/mL), including Amansia sp., Bostrychia tenella, Cryptonemia seminervis, Hypnea musciformis, Plocamium brasiliense (1), and S. clavata. Both Amansia sp., and C. seminervis presented the most relevant antioxidant potential, with percentage of inhibition greater than 70 % in the three tested concentrations. These two species were then analyzed by nuclear magnetic resonance spectroscopy (NMR) and were selected for guided fractionation bioassay. They both presented lipid compounds, fatty acids, esters of fatty acids, triglycerides, and sterols as major components. The fractionation of extracts revealed that the organic fractions were responsible for the antioxidant activity. The results obtained through this work indicate that the analyzed seaweeds are a promising source of compounds with high antioxidant potential.