Evolutionary modularity and morphological integration in the haptoral anchor structures of Ligophorus spp. (Monogenea: Dactylogyridae).

An important question in the study of phenotypic evolution is whether characters are independent of each other or behave and evolve as integrated modules. Morphological integration and modularity provide a powerful framework for the analysis of the evolution of morphological traits. We used geometric morphometrics and phylogenetically independent contrasts (PIC) to test four different modularity hypotheses in the haptoral anchors of 14 monogenean species of Ligophorus. Integration between the modular units identified was further evaluated with two-block partial least squares analysis. Roots and points represented two modules in the dorsal and ventral anchors, but modularity was not statistically supported when parasite phylogeny was accounted for, which may indicate convergent evolution related to host characteristics and gill morphology. In contrast, PIC revealed medial and lateral modules in ventral anchors only. Moreover, we found evidence for ventral and dorsal anchor pairs forming two modules, supporting the notion that they play different functional roles. Integration between all identified modules was strong. We conclude that there is modular structure in the anchors of Ligophorus spp., accounted by adaptive and phylogenetic factors acting at different levels, and ventral and dorsal anchors evolve as integrated modules with specific roles in attachment.

Nanomaterials to Combat NO(x) Pollution.

The presence of NO9x) gases (NO+NO2) in the atmosphere is a major concern of society because of their associated adverse and harmful effects. In order to remove the NO(x) gases from the air, photocatalysis arises as an innovative and promising technique. Through the use of photochemical oxidation processes the NO and NO2 gases are oxidised to NO3- form and thus removed from the air. In recent years new nanomaterials are being developed by researchers with the aim to enhance their photocatalytic activity to combat the NO(x) pollution. The main focus is devoted to preparing new TiO2 based compounds with the highest specific surface area (SSA), different morphology and chemical modifications. In order to increase the SSA, different substrates were used to disperse the TiO2 nanoparticles: organic and carbon fibres, mesoporous materials, clays composites and nanoporous microparticles. In the other hand, high photocatalytic performances were obtained with nanotubes, self-orderer nano-tubular films and nanoparticles with the lowest size. Conversely, when TiO2 is doped with ions the oxide exhibited a better photocatalytic performance under visible light, which is related to the creation of intermediate energy states between the conduction band and the valence band. Alternatively, visible light photocatalysts different from titanium oxide have been studied, which exhibit a good De-NO(x) efficiency working under λ > 400 nm visible light irradiation.

Disentangling the determinants of symbiotic species richness in native and invasive gammarids (Crustacea, Amphipoda) of the Baltic region.

Dispersal of alien species is a global problem threatening native biodiversity. Co-introduction of non-native parasites and pathogens adds to the severity of this threat, but this indirect impact has received less attention. To shed light on the key factors determining the richness of microorganisms in native and invasive host species, we compared symbiotic (parasitic and epibiotic) communities of gammarids across different habitats and localities along the Baltic coast of Poland. Seven gammarid species, two native and five invasive, were sampled from 16 freshwater and brackish localities. Sixty symbiotic species of microorganisms of nine phyla were identified. This taxonomically diverse species assemblage of symbionts allowed us to assess the effect of host translocation and regional ecological determinants driving assembly richness in the gammarid hosts. Our results revealed that (i) the current assemblages of symbionts of gammarid hosts in the Baltic region are formed by native and co-introduced species; (ii) species richness of the symbiotic community was higher in the native Gammarus pulex than in the invasive hosts, probably reflecting a process of species loss by invasive gammarids in the new area and the distinct habitat conditions occupied by G. pulex and invasive hosts; (iii) both host species and locality were key drivers shaping assembly composition of symbionts, whereas habitat condition (freshwater versus brackish) was a stronger determinant of communities than geographic distance; (iv) the dispersion patterns of the individual species richness of symbiotic communities were best described by Poisson distributions; in the case of an invasive host, the dispersion of the rich species diversity may switch to a right-skewed negative binomial distribution, suggesting a host-mediated regulation process. We believe this is the first analysis of the symbiotic species richness in native and invasive gammarid hosts in European waters based on original field data and a broad range of taxonomic groups including Microsporidia, Choanozoa, Ciliophora, Apicomplexa, Platyhelminthes, Nematoda, Nematomorha, Acanthocephala and Rotifera, to document the patterns of species composition and distribution.

Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain.

Infections with betanodavirus affect a wide range of wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to develop and validate real-time RT-PCR assays for sensitive and specific detection of nodavirus in diseased or carrier fish. The new detection assay was used to study the transmission and development of nodavirus infection in juvenile sea bass, Dicentrarchus labrax (L.), challenged by different routes, and also to screen for nodavirus in various farmed fish species. On average, the sensitivity was 10-100 times higher than a standard RT-PCR, and the assay was able to detect asymptomatic carrier fish that otherwise could have been classified as free of infection. Clinical signs of nodavirus infection were reproduced in fish infected following bath exposure or intramuscular injection, demonstrating horizontal transmission of the disease. Nodavirus was always detected in the brain of diseased fish but also in many recovered fish. The new assay enables us to confirm the presence of the virus at an early phase in the production cycle and may represent a useful tool to prevent or slow down the spread of nodavirus to new locations.

Molecules and morphology reveal cryptic variation among digeneans infecting sympatric mullets in the Mediterranean.

We applied a combined molecular and morphological approach to resolve the taxonomic status of Saccocoelium spp. parasitizing sympatric mullets (Mugilidae) in the Mediterranean. Eight morphotypes of Saccocoelium were distinguished by means of multivariate statistical analyses: 2 of Saccocoelium obesum ex Liza spp.; 4 of S. tensum ex Liza spp.; and 2 (S. cephali and Saccocoelium sp.) ex Mugil cephalus. Sequences of the 28S and ITS2 rRNA gene regions were obtained for a total of 21 isolates of these morphotypes. Combining sequence data analysis with a detailed morphological and multivariate morphometric study of the specimens allowed the demonstration of cryptic diversity thus rejecting the hypothesis of a single species of Saccocoelium infecting sympatric mullets in the Mediterranean. Comparative sequence analysis revealed 4 unique genotypes, thus corroborating the distinct species status of Saccocoelium obesum, S. tensum and S. cephali and a new cryptic species ex Liza aurata and L. saliens recognized by its consistent morphological differentiation and genetic divergence. However, in spite of their sharp morphological difference the 2 morphotypes from M. cephalus showed no molecular differentiation and 4 morphotypes of S. tensum were genetically identical. This wide intraspecific morphological variation within S. tensum and S. cephali suggests that delimiting species of Saccocoelium using solely morphological criteria will be misleading.

Anisakid larvae in the musculature of the Argentinean hake, Merluccius hubbsi.

We report the infection levels of third-stage anisakid larva in the muscle of the Argentinean hake, Merluccius hubbsi, in relation to fish size and location in the musculature. The musculature of 42 hake was separated into hypaxial (ventral) and epiaxial (dorsal) parts and surveyed for nematode larvae. Two anisakid species were detected: Anisakis sp. (prevalence, 52.4%; mean +/- SD abundance, 1.2 +/- 1.7) and Pseudoterranova sp. (prevalence, 9.5%; mean +/- SD abundance, 0.2 +/- 0.7). Since the fish were gutted after capture, the occurrence of anisakids in the flesh indicates that the worms had migrated into the muscle before capture. The number of Anisakis sp. in muscle was not correlated with fish length or weight. Therefore, fish size cannot be used as a predictor of parasite loads in the muscle. Only one Anisakis sp. and one Pseudoterranova sp. appeared in the epiaxial musculature. The density of Anisakis sp. in the hypaxial muscles was significantly higher than that in the epiaxial ones. This suggests that removal of the hypaxial musculature can reduce the risk of anisakid-induced allergies and gastrointestinal anisakidoses among consumers.

Growth and emigration of third-stage larvae of Hysterothylacium aduncum (Nematoda: Anisakidae) in larval herring Clupea harengus.

The growth and emigration of Hystherothylacium aduncum in laboratory-reared herring larvae Clupea harengus was studied. Experimental infections of 36-day-old herring larvae resulted in 126 hosts infected with 306 H. aduncum larvae. Regression analyses showed a significant worm emigration from the rectum to the head of the fish, accompanied by an increase in worm body length. The emigration was independent of worm intensity, which suggests an ontogenetic process. Some worms departed from this pattern by moving posteriorly or by penetrating into the muscle, and in 5 cases, the larvae were observed to leave living fish. This individual variation has not been observed in previous studies and might be explained by host signals related to condition or development stage. Indirect evidence suggested parasite-induced mortality in the tanks due to the emigrations because only 4 of the 126 infected fish survived 8 days postinfection; the emigration of H. aduncum affected vital organs, such as the heart and brain, and the larvae penetrating or leaving the host's tissues can cause extensive damage to the delicate herring larvae.

Ontogenetic habitat selection by Hadwenius pontoporiae (Digenea: Campulidae) in the intestine of franciscanas (Cetacea).

The linear habitat selection of 4 sequential maturity stages (1, 2, 3, and 4) of the trematode Hadwenius pontoporiae in the intestines of 26 South American dolphins Pontoporia blanvillei was investigated. The franciscana is a suitable host for H. pontoporiae because all 26 hosts were infected, the infrapopulations being composed mostly of gravid (stage 4) worms. Most trematodes were found in the first third of the intestine. The niches of the maturity stages decreased from stage 1 to 4. Gravid worms favored the most anterior part of the duodenum, whereas stages 1, 2, and 3 occurred more posteriorly in every host. The distributions of the maturity stages showed a narrow site fidelity and were ordered following the developmental sequence. The positions of stages 2, 3, and 4 covaried positively when the effect of intensity was controlled. All this evidence suggests that H. pontoporiae undergoes an anteriad ontogenetic migration from the posterior to the most anterior duodenum. At high intensities, the distribution of stages 2, 3, and 4 expanded and shifted posteriorly. According to the maturity structure of H. pontoporiae at the infrapopulation level, it is proposed that there might be more developing worms at high intensities, and, therefore, the expansion and shift might result, at least partly, from the presence of more migrating worms along the fundamental niche of H. pontoporiae. Other Hadwenius species show a similar general pattern of distribution to H. pontoporiae. This raises the possibility that the habitat selection behavior of H. pontoporiae may be due to the inheritance of prior phylogenetic constraints.

Corynosoma cetaceum in the stomach of franciscanas, Pontoporia blainvillei (Cetacea): an exceptional case of habitat selection by an acanthocephalan.

Adult acanthocephalans are typically found in the intestine of vertebrates, where they can readily absorb nutrients. However, Corynosoma cetaceum has been frequently reported in the stomach of cetaceans from the Southern Hemisphere. The ecological significance of this habitat was investigated by examining data on number, sex ratio, maturity status, biomass, and fecundity of C. cetaceum in different parts of the digestive tract of 44 franciscanas Pontoporia blainvillei. Individual C. cetaceum occurred in the pyloric stomach (PS) and, to lesser degrees, in the duodenal ampulla (DA) and the main stomach (MS). Females outnumbered males in all chambers, although the sex ratio was closer to 1:1 in the MS; there also was a higher proportion of nongravid females, with a smaller biomass in the MS than in the PS and the DA. This evidence suggests that cystacanths are released from prey tissues in the MS, where entire prey are reduced to semi-fluid chyme. The 3 chambers harbored gravid females that did not differ significantly in mean biomass or fecundity. The maturity status of females was nearly identical between the PS and the DA. In the MS, the higher proportion of non-gravid females is probably due to the occurrence of newly recruited females to this site. Mean biomass and fecundity of gravid females covaried strongly and positively among chambers within hosts. These results suggest that there are no major differences between the 3 chambers with respect to the suitability for reproduction by C. cetaceum. However, although the MS is the largest chamber, it harbored the smallest number of gravid females. Interestingly, worms were largely restricted to the aboral portion of the MS, a sheltered region where a concentration of chyme, and thus nutrient availability, likely occurs. Linear distribution differences of gravid female C. cetaceum at increasing intensities suggest that reproductive females occupy chambers according to available space. In summary, the stomach should be considered the main habitat for C. cetaceum. The choice of this habitat is puzzling because other Corynosoma species occur in the intestine, and because the stomach of cetaceans is not an absorptive site.

New data on the early development of Hysterothylacium aduncum (Nematoda, Anisakidae).

This note reports on incidental observations of the early development of the third-stage larvae of Hysterothylacium aduncum from gadid fishes. Gravid H. aduncum females were collected from Pollachius virens, Pollachius pollachius. Gadus morhua, and Molva molva in Norwegian waters. The eggs were incubated at 20 per thousand salinity and 5 C. Spontaneous hatching of third-stage larvae was observed 10-25 days after egg deposition. These larvae were long lived and could infect Acartia tonsa copepods, the infections being maintained for up to 34 days. The morphology of the third-stage larvae in the copepods and some traits of the life cycle were similar to those reported in previous studies. However, our results disagree with evidence suggesting that H. aduncum eggs rarely hatch, and hatched larvae have lower survival and a poorer ability to infect the first intermediate host than unhatched ones. It is difficult to account for these discrepancies because information on the early development of Hysterothylacium species is incomplete. However, we tentatively suggest that differences in the early development of H. aduncum may indicate the existence of at least 2 different taxonomic entities in the North Atlantic, which is consistent with previous evidence based on morphological traits.

Population structure of Anisakis simplex (Nematoda) in harbor porpoises Phocoena phocoena off Denmark.

The population structure and habitat selection of Anisakis simplex in 35 harbor porpoises off Denmark are described. The nematodes were collected from the stomach and duodenal ampulla and were categorized as third-stage larvae, fourth-stage larvae, subadults, and adults. The porpoises harbored 8,043 specimens of A. simplex. The proportion of adults and subadults increased with infrapopulation size. The number of development stages across infrapopulations covaried significantly (Kendall's test of concordance). Concordance was higher in hosts with the highest intensities than in those with low and medium intensities. All stages occurred mainly in the forestomach, but this trend was stronger for the adults. Adult and subadult sex ratios did not depart significantly from 1:1. Our data suggested that recruitment and duration of each stage were the main factors accounting for infrapopulation structure. The preference of A. simplex for the forestomach conformed with previous studies, but the narrower distribution of adults relative to other stages might indicate a strategy to enhance mating opportunities. Information on sex ratios of A. simplex is scarce and contradictory. We suggest that the discrepancies might partly reflect differences in categorization criteria and statistical methods.

The impact of parasites on marine mammals: a review.

The design and implementation of conservation plans for marine mammals is a matter of public concern. However, very little is known about the role of parasites in the dynamics of marine mammal populations. This is probably due to methodological constraints concerning sampling biases, poor knowledge of the biology of the hosts and parasites and difficulty and costy of experimental studies. However, current evidence supports the theory that parasites may regulate marine mammal populations. Crassicauda species in cetaceans and Uncinaria lucasi in pinnipeds seem good candidates as regulating agents. In addition, parasite-induced mass mortalities may be important in marine mammal populations. Well documented cases are the PDV virus which decimated the European common seal (Phoca vitulina) populations in 1988 and the Mediterranean striped dolphin (Stenella coeruleoalba) morbillivirus infection of 1990-1992. Due to the social organisation patterns of marine mammals it is possible that such die-offs occur at very low densities, representing a potential threat to endangered species like the Mediterranean monk seal (Monachus monachus), the Hawaiian monk seal (M. schauinslandi) or the Finish Saimaa seal (Phoca hispida saimensis). It is concluded that parasites can play an important role in marine mammal populations not only at the ecological scale but at the evolutionary one too.

Kudoa unicapsula n. sp. (Myxosporea: Kudoidae) a parasite of the Mediterranean mullets Liza ramada and L. aurata (Teleostei: Mugilidae).

A new multivalvulid myxozoan parasite, Kudoa unicapsula n. sp., is described from the intestinal mesentery, intestine and pyloric caeca of the thin-lipped grey mullet Liza ramada (Risso 1826) and the golden grey mullet L. aurata (Risso, 1810) from the Mediterranean coastal waters of Spain. It is characterized by the presence of elongated, rice corn-like white cysts of 0.47-0.56 x 0.18-0.38 mm, filled with tetracapsulate, slightly asymmetric spores, rectangular in apical view and tear-shaped in lateral view with four polar capsules of considerably different size and slightly unequal spore valves with rounded edges, overlapping each other on the apex of the spore. One large polar capsule includes a polar filament coiled in two to three turns, and the other three polar capsules, which are very small, posses only a rudimental filament. Both light and electron microscopy data showed that this species differs from all previously described Kudoa spp. with unequal polar capsules. The molecular analysis based on 18S and 28S ribosomal deoxyribonucleic acid DNA sequence data of K. unicapsula n. sp. indicates a close relationship and thus phylogenetic clustering together with K. trifolia, a myxozoan from the same host and the same geographical location.

Kudoa trifolia sp. n. - molecular phylogeny suggests a new spore morphology and unusual tissue location for a well-known genus.

A new species of myxozoan, Kudoa trifolia sp. n., was found in various organs of the golden grey mullet, Liza aurata (Risso), and the thinlip mullet, L. ramada (Risso), from the western Mediterranean. Spores developed in subspherical plasmodia of 0.28-1 mm diameter within connective tissue, predominantly in the spleen, the outer wall of the gall bladder and the gut, the mesenteries and occasionally also in the gills. The spores of K. trifolia differ from the commonly known shape of Kudoa by considerable enlargement of one of the four valve cells, thus forming a 'spore body', which contains the major part of the binucleate sporoplasm. Scanning electron microscopy of the spores revealed the presence of grape-like appendages, which occur in bundles terminally on the valve cells. Phylogenetic analysis based on the 18S rDNA sequence of K. trifolia showed that this species is deeply embedded in the genus Kudoa despite its aberrant morphology and host tissue location. This suggests important amendments to the morphological diagnosis of the genus Kudoa.

Distribution of Pholeter gastrophilus (Digenea) within the stomach of four odontocete species: the role of the diet and digestive physiology of hosts.

We compared the distribution of the digenean Pholeter gastrophilus in the stomach of 27 harbour porpoises, Phocoena phocoena, 27 striped dolphins, Stenella coeruleoalba, 18 bottlenose dolphins, Tursiops truncatus, and 100 long-finned pilot whales, Globicephala melas. The stomach of these species is composed of 4 chambers of different size, structure and function. In all species, P. gastrophilus was largely restricted to the glandular region of the stomach, but the parasite tended to favour the fundic chamber in bottlenose dolphins and harbour porpoises, the pyloric chamber in pilot whales, and none in striped dolphins. However, predictability at infrapopulation level was generally low, suggesting a weak preference of P. gastrophilus for any of the chambers. Three hypotheses were tested to investigate a common cause for the distribution of P. gastrophilus in all host species, namely, colonization of chambers was (1) sequential, (2) dependent on chamber size, or (3) dependent on the passage time of food through the whole stomach. The latter hypothesis was indirectly tested by assuming, based on previous evidence from other vertebrates, that the greater the size of the stomach and/or the energy content of prey, the greater the delay of food passage. We found no compelling evidence that chamber colonization was sequential, or related to chamber size in any species. However, the distribution of P. gastrophilus was significantly more anteriad when the host species had larger stomachs and, particularly, when hosts fed on prey with higher caloric content. Accordingly, the stomach distribution of P. gastrophilus at this scale seems to be passively driven by features of the diet and digestive physiology of each host species. This study provides a general framework to formulate null hypotheses in future studies on microhabitat choice by parasites.

Robinia aurata n. g., n. sp. (Digenea: Hemiuridae) from the mugilid Liza aurata with a molecular confirmation of its position within the Hemiuroidea.

Robinia aurata n. g., n. sp. is described from Liza aurata (Mugilidae), the golden grey mullet, from the Ebro Delta, Spanish Mediterranean. The new genus differs from all other hemiurid genera in the combined possession of muscular flanges and a vestigial ecsoma. Within the Bunocotylinae, which currently accommodates 2 genera, Bunocotyle and Saturnius, the new genus exhibits a unique combination of blind caeca, Juel's organ, post-ovarian bulk of the uterus in the hind-body, and tegumental papillae surrounding the oral and ventral sucker apertures. Furthermore, Robinia n. g. differs from both Bunocotyle and Saturnius in the nature of the muscular extensions around the oral sucker, with the shape of a muscular belt in the latter and numerous muscular papillae in the former. The phylogenetic hypothesis for the Bunocotylinae developed from sequence data analyses based on partial lsrDNA and complete ssrDNA combined (22 species) and V4 domain of the ssrRNA gene (37 species) supports the erection of the new genus and confirms its position within the Hemiuroidea. Both molecular analyses confirmed the monophyly of the Hemiuroidea, its division into 2 major clades and the polyphyly of the Derogenidae, as in previous studies, and suggest that the Gonocercinae (with 2 genera, Gonocerca and Hemipera), may require a distinct familial status. Finally, there was poor support for the distinct status of the Lecithasteridae and Hemiuridae, following previous suggestions based on different sequence data sets. A key to genera of the Bunocotylinae is presented.

Saturnius minutus n. sp. and S. dimitrovi n. sp. (Digenea: Hemiuridae) from Mugil cephalus L. (Teleostei: Mugilidae), with a multivariate morphological analysis of the Mediterranean species of Saturnius Manter, 1969.

Three species of the bunocotyline genus Saturnius Manter, 1969 are described from the stomach lining of mugilid fishes of the Mediterranean and Black Seas. Two of the species are new: S. minutus n. sp. occurs in Mugil cephalus off the Mediterranean coast of Spain; and S. dimitrovi n. sp., a parasite of M. cephalus off the Bulgarian Black Sea coast and the Spanish Mediterranean coast, was originally described as S. papernai by Dimitrov et al. (1998). In addition, S. papernai Overstreet, 1977 is redescribed from M. cephalus off the Spanish Mediterranean coast and from Liza aurata and L. saliens off the Bulgarian Black Sea coast. The three species are distinguished morphometrically using univariate and multivariate analyses. These results were verified using Linear Discriminant Analysis which correctly allocated all specimens to their species designations based on morphology (i.e. 100% successful classification rate) and assigned almost all specimens to the correct population (locality). The following variables were selected for optimal separation between samples: the length of the forebody, ventral sucker and posterior testis, the length and width of the posteriormost pseudosegment, and the width of the muscular flange at ventral sucker level.

Intestinal helminth communities of the long-finned pilot whale (Globicephala melas) off the Faroe Islands.

The intestines of 170 long-finned pilot whales, Globicephala melas, caught off the Faroe Islands (N.E. Atlantic) were examined for helminth parasites. Eight species were detected but only 4 occurred in at least 10% of the sample. No core or recurrent group of species were identified and no correlations between abundances of species were significant. Diversity values were far below those reported for other endotherms. Colonization by helminths was random, whales not being readily colonized. These features point to largely unpredictable, isolationist infracommunities, there being little potential for inter-specific interactions. Older hosts tended to harbour more diverse infracommunities, offering more opportunities for such interactions. Two hypotheses, which might also apply to other cetaceans, are proposed to account for the depauperate helminth communities of the pilot whale: (i) some ancestral helminth species failed to adapt their cycles to the marine habitat and (ii) the hosts' isolation from land prohibited new infections with helminths of mammals.

Lung-worms (Nematoda: Pseudaliidae) of harbour porpoises (Phocoena phocoena) in Norwegian waters: patterns of colonization.

We studied the component community of lung-worms of the harbour porpoise, attempting to establish the relative importance of ecological and evolutionary factors on its development. The lungs of 64 porpoises by-caught in Norwegian waters were examined for helminths. Three pseudaliid species were detected. The porpoises appear to be readily colonized by lung-worms, the structure of the component community of calves being fairly similar to that of the remainder. Prenatal and/or transmammary infections might partly account for these early infections. However, host age was correlated with the number of lung-worm species, suggesting that the lung-worms may have heteroxenous cycles similar to other metastrongyloids. The lung-worm species tended to co-occur more often than expected by chance. This pattern is commonly observed in communities formed by phylogenetic relatives. Mean species richness of lung nematodes differed significantly among mammalian orders. However, species richness of marine species seemed very similar to those of most terrestrial species. This evidence suggests that phylogenetic factors seem more important than ecological ones in determining the number of lung-worm species in mammals.