Bruno Bellisario

Genetic and Morphological Approaches Distinguish the Three Sibling Species of the Anisakis simplex Species Complex, with a Species Designation as Anisakis berlandi n. sp. for A. simplex sp. C (Nematoda: Anisakidae)

Abstract:  Numerous specimens of the 3 sibling species of the Anisakis simplex species complex (A. pegreffii, A. simplex (senso stricto)), and A. simplex sp. C) recovered from cetacean species stranded within the known geographical ranges of these nematodes were studied morphologically and genetically. The genetic characterization was performed on diagnostic allozymes and sequences analysis of nuclear (internal transcribed spacer [ITS] of ribosomal [r]DNA) and mitochondrial (mitochondrial [mt]DNA cox2 and rrnS) genes. These markers showed (1) the occurrence of sympatry of the 2 sibling species A. pegreffii and A. simplex sp. C in the same individual host, the pilot whale, Globicephala melas Traill, from New Zealand waters; (2) the identification of specimens of A. pegreffii in the striped dolphin, Stenella coeruleoalba (Meyen), from the Mediterranean Sea; and (3) the presence of A. simplex (s.s.) in the pilot whale and the minke whale, Balaenoptera acutorostrata Lacépède, from the northeastern Atlantic waters. No F1 hybrids were detected among the 3 species using the nuclear markers. The phylogenetic inference, obtained by maximum parsimony (MP) analysis of separate nuclear (ITS rDNA region), combined mitochondrial (mtDNA cox2 and rrnS) sequences datasets, and by concatenated analysis obtained at both MP and Bayesian inference (BI) of the sequences datasets at the 3 studied genes, resulted in a similar topology. They were congruent in depicting the existence of the 3 species as distinct phylogenetic lineages, and the tree topologies support the finding that A. simplex (s.s.), A. pegreffii, and A. berlandi n. sp. (=A. simplex sp. C) represent a monophyletic group. The morphological and morphometric analyses revealed the presence of morphological features that differed among the 3 biological species. Morphological analysis using principal component analysis, and Procrustes analysis, combining morphological and genetic datasets, showed the specimens clustering into 3 well-defined groups. Nomenclatural designation and formal description are given for A. simplex species C: the name Anisakis berlandi n. sp. is proposed. Key morphological diagnostic traits are as follows between A. berlandi n. sp. and A. simplex (s.s.): ventriculus length, tail shape, tail length/total body length ratio, and left spicule length/total body length ratio; between A. berlandi n. sp. and A. pegreffii: ventriculus length and plectane 1 width/plectane 3 width ratio; and between A. simplex (s.s.) and A. pegreffii: ventriculus length, left and right spicule length/total body length ratios, and tail length/total body length ratio. Ecological data pertaining to the geographical ranges and host distribution of the 3 species are updated.

Genetic identification and distribution of the parasitic larvae of Anisakis pegreffii and Anisakis simplex (s. s.) in European hake Merluccius merluccius from the Tyrrhenian Sea and Spanish Atlantic coast: implications for food safety.

The consumption of the hake Merluccius merluccius is widespread in European countries, where this fish has a high commercial value. To date, different larval species of Anisakis have been identified as parasites in M. merluccius from European waters, Anisakis pegreffii and Anisakis simplex (s. s.) being the two most common. The aim of the study is to present data on the occurrence of Anisakis spp. larvae in the viscera and flesh of M. merluccius. Consequently, the distribution and infection rates of different species of Anisakis in different sites (viscera, and dorsal and ventral fillets) were investigated in hake caught in the central Tyrrhenian Sea (FAO 37.1.3) and the NE Atlantic Ocean (FAO 27 IXa). A sample of N=65 fish individuals (length>26 cm) was examined parasitologically from each fishing ground. The fillets were examined using the pepsin digestion method. A large number (1310) of Anisakis specimens were identified by multilocus allozyme electrophoresis (MAE) and mtDNA cox2 sequence analysis; among these, 814 larvae corresponded to A. simplex (s. s.) and 476 to A. pegreffii. They were found to infect both the flesh and the viscera. The two species co-infected the same individual fish (both in the viscera and in the flesh) from the FAO 27 area, whereas only A. pegreffii was found in hake from the Tyrrhenian Sea. The average parasite burden of A. pegreffii in hake from the Tyrrhenian Sea was significantly lower to that observed from hake off the Atlantic coast of Spain, both in prevalence and in abundance. In addition, whereas no significant difference in overall prevalence values was recorded between the two Anisakis species in the viscera of the FAO 27 sample, significant differences were found in the abundance levels observed between these species in the flesh, with A. simplex (s. s.) exhibiting significantly higher levels than that observed for A. pegreffii (p<0.001). Given that the pathogenic role in relation to man is known for these two species of Anisakis, both the flesh inspection and the infection rates of the different anisakid species assume particular importance in terms of assessing the risk they pose to humans.

Parasitic infection by larval helminths in Antarctic fishes: pathological changes and impact on the host body condition index

We examined pathological changes and relationship between body condition index (BCI) and parasitic infection in 5 species of fish, including 42 icefish Chionodraco hamatus (Channichtyidae), 2 dragonfish Cygnodraco mawsoni (Bathydraconidae), 30 emerald rock cod Trematomus bernacchii, 46 striped rock cod T. hansoni and 9 dusty rock cod T. newnesi (Nototheniidae) from the Ross Sea, Antarctica. All parasites were identified by a combination of morphology and mtDNA cytochrome-oxidase-2 sequence (mtDNA cox2) analysis, except Contracaecum osculatum s.l., for which only the latter was used. Five larval taxa were associated with pathological changes including 2 sibling species (D and E) of the C. osculatum species complex and 3 cestodes including plerocercoids of a diphyllobothridean, and 2 tetraphyllidean forms including cercoids with monolocular and bilocular bothridia. The most heavily infected hosts were C. hamatus and C. mawsoni, with C. hamatus most often infected by C. osculatum sp. D and sp. E and diphyllobothrideans, while C. mawsoni was most often infected with tetraphyllidean forms. Histologically, all fish showed varying severity of chronic inflammation associated with larval forms of helminths. Diphyllobothrideans and C. osculatum spp. were located in gastric muscularis or liver and were associated with necrosis and mild to marked fibrosis. Moderate multifocal rectal mucosal chronic inflammation was associated with attached tetraphyllidean scolices. C. hamatus showed a strong negative correlation between BCI and parasite burden.

Spatial network structure and robustness of detritus-based communities in a patchy environment

The mechanisms that regulate the spatial distribution of species are an essential aid to understanding the effects of the environment on the persistence of populations and communities. The effects of spatial structure on the persistence and robustness of ecological communities can, in turn, prove useful in uncovering their functioning, e.g., in the decomposition of leaf detritus. We applied the framework of complex networks to evaluate the effects of spatial structure on the colonization process of leaf detritus in a patchy aquatic environment, with a spatial network of six pools at different salinity. We found three well-defined modules formed by groups of taxa sharing the same pools, observing an association between modularity and spatial proximity of pools. Modules maximize the number of links within modules, and minimize the number of links among modules, showing the presence of a strong site-specific association between taxa and pools. The topological characteristics of the network show robustness against random perturbations and a lower tolerance of targeted perturbations. These findings suggest that random events, such as flooding or heavy rains, slightly affect the robustness of the system, while localized perturbations on the most connected nodes could have a negative effect on the connectivity of the whole network. The consequences could lead to a structural and functional homogenization of the system, with potential effects for the entire trophic chain. Here we discuss the topological properties of the network in relation to the spatial distribution of pools, showing how network analysis can yield valuable insight for conservation and management.

Integrating Anisakis spp. parasites data and host genetic structure in the frame of a holistic approach for stock identification of selected Mediterranean Sea fish species

The unique environment of the Mediterranean Sea makes fish stock assessment a major challenge. Stock identification of Mediterranean fisheries has been based mostly from data on biology, morphometrics, artificial tags, otolith shape and fish genetics, with less effort on the use of parasites as biomarkers. Here we use some case studies comparing Mediterranean vs Atlantic fish stocks in a multidisciplinary framework. The generalized Procrustes Rotation (PR) was used to assess the association between host genetics and larval Anisakis spp. datasets on demersal (hake) and pelagic (horse mackerel, swordfish) species. When discordant results emerged, they were due to the different features of the data. While fish population genetics can detect changes over an evolutionary timescale, providing indications on the cohesive action of gene flow, parasites are more suitable biomarkers when considering fish stocks over smaller temporal and spatial scales, hence giving information of fish movements over their lifespan. Future studies on the phylogeographic analysis of parasites suitable as biomarkers, and that of their fish host, performed on the same genes, will represent a further tool to be included in multidisciplinary studies on fish stock structure.

Parasite Communities of Icefish (Chionodraco hamatus) in the Ross Sea (Antarctica): Influence of the Host Sex on the Helminth Infracommunity Structure

Parasite communities of Chionodraco hamatus were investigated from Terra Nova Bay (Ross Sea, Antarctica) during host spawning time. Special attention was given to helminth infracommunities and effect of host sex on its structure. A total of 21 taxa including 5 ecto-parasites and 16 endo-parasites were identified. The number of ecto and endo-parasite species per individual host ranged from 1 to 3 and 3 to 10, respectively, while the mean numbers of parasite specimens per individual host were 4.7 and 1309.7, respectively. The rich abundance of infection suggests a rich concentration of helminth intermediate/paratenic hosts in the coastal waters of Terra Nova Bay. Chionodraco hamatus serves as a definitive host for 10 helminth taxa, while it acts as an intermediate/paratenic host for 6 helminth taxa. Larvae of 6 helminth taxa for which C. hamatus serves as intermediate/paratenic host represented 98.7% of all specimens found. Of these, the tetraphyllidean and diphyllobothridean cestodes and the nematode Contracaecum osculatum s.l. were the most prevalent and abundant. ‘Larval’ infracommunities had significantly higher species richness, total abundance and diversity than ‘adult’ infracommunities, suggesting the important role of C. hamatus in supporting the life cycles of those parasites in the study area as a paratenic/intermediate host. Significant differences in the pattern of helminth infracommunities of larval forms between male and female fish were found. These differences could be caused by physiological, and most probably by behavioral differences between sexes suggesting that sex is an important factor influencing parasite burden in C. hamatus during reproductive season.

The interplay between network structure and functioning of detritus-based communities in patchy aquatic environment

Understanding models of networks formation is fundamental to explore the role of the structure in the functioning of the systems they describe, and their ability to respond to change. In this work, we aimed to understand whether and how the modular (or compartmented) structure of a network composed by macroinvertebrates and leaf detritus in six pools of aquatic system was related with the decomposition process. First, we evaluated the relationship between the temporal patterns of leaf detritus colonization and the modular subdivision of the network. Modularity was then related with the clustering of the pools based on convergent traits of leaf detritus consumption and environmental conditions. A significant relationship between the colonization patterns and the distribution of taxa and pools in well-defined modules was found. The modular and clustering subdivision of the pools showed a significant overlap, which revealed the intimate linkage between the structure and functioning of the system. Modularity shapes the functional architecture of the network, by increasing the spatial differences of leaf litter decomposition over time and the diversity of functional traits among detritus feeder. As a consequence, modularity influences the variability of communities’ responses to disturbance, increasing the diversity and robustness of functional processes. Our results have also implications from a conservation point of view, showing the importance of habitat heterogeneity for the robustness of ecosystem functioning, potentially enhancing biodiversity with positive, long-term effect on the whole food web.

Habitat overlap between bottlenose dolphins and seabirds: a pilot study to identify high-presence coastal areas in the Tyrrhenian Sea

The identification of foraging hotspots able to support the co-existence of multiple top predators provides a potential approach to addressing protection measures for marine ecosystems. In this study, we conducted visual surveys in the central Tyrrhenian Sea to determine areas with simultaneous presence of bottlenose dolphins, four species of seabirds (Audouins gull, Yellow-legged gull, Yelkouan shearwater and Corys shearwater), and baitballs occurring at the surface, indicating the presence of potential prey items. We also analysed their occurrence in relation to topography (depth, slope and distance from the shore) and seabed types. Kriging analysis identified areas with simultaneous presence of several marine top predators. Dolphin distribution appeared to be linked to gentle slope (6–10 m) and muddy seabed, possibly associated with prey distribution, whereas the four seabird species were more frequent in areas with a water depth of 100–150 m, gentle slope and muddy seabed, apart from Audouins gull, which preferred a depth of 10–20 m. Baitball distribution was linked to depth (20–40 m), gentle slope (6–10 m) and muddy seabed. The overlapping presence of bottlenose dolphins, seabirds and baitballs allowed the identification of foraging areas, presumably representing biodiversity and productivity hotspots, located in waters of 50–100 m depth at the mouths of two rivers. This approach provides a promising tool for identifying highly productive coastal areas, and should also be recommended for wider-scale surveys.

Temporal stability of parasite distribution and genetic variability values of Contracaecum osculatum sp. D and C. osculatum sp. E (Nematoda: Anisakidae) from fish of the Ross Sea (Antarctica).

The Ross Sea, Eastern Antarctica, is considered a “pristine ecosystem” and a biodiversity “hotspot” scarcely impacted by humans. The sibling species Contracaecum osculatum sp. D and C. osculatum sp. E are anisakid parasites embedded in the natural Antarctic marine ecosystem. Aims of this study were to: identify the larvae of C. osculatum (s.l.) recovered in fish hosts during the XXVII Italian Expedition to Antarctica (2011–2012); perform a comparative analysis of the contemporary parasitic load and genetic variability estimates of C. osculatum sp. D and C. osculatum sp. E with respect to samples collected during the expedition of 1993–1994; to provide ecological data on these parasites. 200 fish specimens (Chionodraco hamatus, Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi) were analysed for Contracaecum sp. larvae, identified at species level by allozyme diagnostic markers and sequences analysis of the mtDNA cox2 gene. Statistically significant differences were found between the occurrence of C. osculatum sp. D and C. osculatum sp. E in different fish species. C. osculatum sp. E was more prevalent in T. bernacchii; while, a higher percentage of C. osculatum sp. D occurred in Ch. hamatus and T. hansoni. The two species also showed differences in the host infection site: C. osculatum sp. D showed higher percentage of infection in the fish liver. High genetic variability values at both nuclear and mitochondrial level were found in the two species in both sampling periods. The parasitic infection levels by C. osculatum sp. D and sp. E and their estimates of genetic variability showed no statistically significant variation over a temporal scale (2012 versus 1994). This suggests that the low habitat disturbance of the Antarctic region permits the maintenance of stable ecosystem trophic webs, which contributes to the maintenance of a large populations of anisakid nematodes with high genetic variability.

Infaunal macrobenthic community dynamics in a manipulated hyperhaline ecosystem: a long-term study

BackgroundUnderstanding the responses of ecological communities to human-induced perturbations is crucial for establishing conservation goals. Ecological communities are dynamic entities undergoing fluctuations due to their intrinsic characteristics as well as anthropogenic pressures varying over time. In this respect, long-term studies, based on large spatial and temporal datasets, may provide useful information in understanding patterns and processes influencing the communities’ structure. Theoretical evidence suggests that a role of biodiversity is acting as a compensatory buffer against environmental variability by decreasing the temporal variance in ecosystem functioning and by raising the level of community response to perturbations through the selection of better performing species. Therefore, the spatial and temporal changes in the specialization of the community components may be used as an effective tool to monitor the effects of natural and anthropogenic alterations of the environment in dynamic systems. We examined the temporal dynamics of macroinvertebrate community structure in the hyperhaline habitat of Tarquinia Saltworks (central Italy). We aimed at: (i) investigating the relationships between the level of community specialization and the alterations of the environment across fourteen years; (ii) comparing the ability of aggregate community parameters such as the average abundance vs. species specialization in describing patterns of community composition.ResultsWe arranged the data in three sub-sets according to three periods, each characterized by different environmental conditions. The mean abundance of sampled macroinvertebrates showed a significant change (p < 0.01) only in the community inhabiting the saltwork basin closely connected to the sea, characterized by the highest environmental variation (i.e. the coefficient of variation, CV, of the aggregate environmental variability over the study period, CVrange = 0.010 - 0.2). Here we found marine species like Modiolus adriaticus (Lamarck, 1819), Neanthes irrorata (Malmgren, 1867), and Amphiglena mediterranea (Leydig, 1851), which inhabited the saltworks during the halt period but disappeared during the subsequent eutrophication phase. Conversely, species specialization showed a significant decrease for each sampled community in the presence of habitat degradation and a recovery after ecological restoration. The widest fluctuations of specialization were recorded for the community inhabiting the saltwork basin with the highest long-term environmental variability.ConclusionsRecent advances have shown how the increased temporal and spatial variability of species’ abundance within the communities may be a signal of habitat disturbance, even in the absence of an apparent decline. Such approach could also be used as a sensitive monitoring tool, able to detect whether or not communities are subjected to increasing biotic homogenization. Also, the increased functional similarity triggered by habitat degradation may impact on species at higher trophic levels, such as the waterbirds wintering in the area or using it as a stopover during migration.