Hybridization and extensive mitochondrial introgression among fire salamanders in peninsular Italy

Discordance between mitochondrial and nuclear patterns of population genetic structure is providing key insights into the eco-evolutionary dynamics between and within species, and their assessment is highly relevant to biodiversity monitoring practices based on DNA barcoding approaches. Here, we investigate the population genetic structure of the fire salamander Salamandra salamandra in peninsular Italy. Both mitochondrial and nuclear markers clearly identified two main population groups. However, nuclear and mitochondrial zones of geographic transition between groups were located 600 km from one another. Recent population declines in central Italy partially erased the genetic imprints of past hybridization dynamics. However, the overall pattern of genetic variation, together with morphological and fossil data, suggest that a rampant mitochondrial introgression triggered the observed mitonuclear discordance, following a post-glacial secondary contact between lineages. Our results clearly show the major role played by reticulate evolution in shaping the structure of Salamandra salamandra populations and, together with similar findings in other regions of the species’ range, contribute to identify the fire salamander as a particularly intriguing case to investigate the complexity of mechanisms triggering patterns of mitonuclear discordance in animals

Macroinvertebrates assembly in a patchy environment: centrality measures for the spatial network of detritus-based communities

Spatial patterns influence the persistence of populations and communities, giving useful insights on the mechanisms that confer robustness to ecological networks. The mechanisms that regulate the spatial distribution of species are related to the ability of populations to respond to spatio-temporal variations of ecological conditions, contributing to network structure and dynamic of persisting communities. We applied the framework of complex network to study the colonization process of Phragmites australis leaf detritus in six different pools in the patchy aquatic environment of Tarquinia saltern (central Italy). We used the colonization data of macroinvertebrates on leaf detritus assigning a link between two taxa if they shared a common pool, and measured their positional importance within the network. We found high clustering and short path lenght among nodes that is representative of small-world pattern, showing the relationship between robustness and nodes synchronicity in network attachment dynamics. Here we show how the identification of local (individual use of substrates by macroinvertebrates) and global (network properties) patterns in community structure could be the key to better understand the ecology, evolution and management of complex ecological network

Climate change promotes hybridisation between deeply divergent species

Rare hybridisations between deeply divergent animal species have been reported for decades in a wide range of taxa, but have often remained unexplained, mainly considered chance events and reported as anecdotal. Here, we combine field observations with long-term data concerning natural hybridisations, climate, land-use, and field-validated species distribution models for two deeply divergent and naturally sympatric toad species in Europe (Bufo bufo and Bufotes viridis species groups). We show that climate warming and seasonal extreme temperatures are conspiring to set the scene for these maladaptive hybridisations, by differentially affecting life-history traits of both species. Our results identify and provide evidence of an ultimate cause for such events, and reveal that the potential influence of climate change on interspecific hybridisations goes far beyond closely related species. Furthermore, climate projections suggest that the chances for these events will steadily increase in the near future

Species-specific Real Time-PCR primers/probe systems to identify fish parasites of the genera Anisakis, Pseudoterranova and Hysterothylacium (Nematoda: Ascaridoidea)

Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.), P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum, a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency (E) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers

Gene expression profiles of antigenic proteins of third stage larvae of the zoonotic nematode Anisakis pegreffii in response to temperature conditions

Anisakis pegreffii, a recognised etiological agent of human anisakiasis, is a parasite of homeothermic hosts at the adult stage and of ectothermic hosts at the third larval stage. Among distinct factors, temperature appears to be crucial in affecting parasite hatching, moulting and to modulate parasite-host interaction. In the present study, we investigated the gene transcripts of proteins having an antigenic role among excretory secretory products (ESPs) (i.e., a Kunitz-type trypsin inhibitor, A.peg-1; a glycoprotein, A.peg-7; and the myoglobin, A.peg-13) after 24 h, in A. pegreffii larvae maintained in vitro, under controlled temperature conditions. Temperatures were 37 °C and 20 °C, resembling respectively homeothermic and ectothermic hosts conditions, and 7 °C, the cold stress condition post mortem of the fish host. Primers of genes coding for these ESPs to be used in quantitative real-time PCR were newly designed, and qRT-PCR conditions developed. Expression profiles of the genes A.peg-1 and A.peg-13 were significantly up-regulated at 20 °C and 37 °C, with respect to the control (larvae kept at 2 °C for 24 h). Conversely, transcript profiles of A.peg-7 did not significantly change among the chosen temperature conditions. In accordance with the observed transcript profiles, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of the three target ESPs at 37 °C, while only A.peg-13 was observed at 7 °C. The results suggest that temperature conditions do regulate the gene expression profiles of A.peg-1 and A.peg-13 in A. pegreffii larvae. However, regulation of the glycoprotein A.peg-7 is likely to be related to other factors such as the host's immune response

Drilling down hotspots of infraspecific diversity to bring them into on-ground conservation of threatened species

Unprecedented rates of biodiversity loss raise the urgency for preserving species ability to cope with ongoing global changes. An approach in this direction is to target intra-specific hotspots of genetic diversity as conservation priorities. However, these hotspots are often identified by sampling at a spatial resolution too coarse to be useful in practical management of threatened species, hindering the long-appealed dialog between conservation stakeholders and conservation genetic researchers. Here, we investigated the spatial and temporal variation in species presence, genetic diversity, as well as potential risk factors, within a previously identified hotspot of genetic diversity for the endangered Apennine yellow-bellied toad Bombina pachypus. Our results show that this hotspot is neither a geographically homogeneous nor a temporally stable unit. Over a time-window spanning 10–40 years since previous assessments, B. pachypus populations declined in large portions of their hotspot, and their genetic diversity levels decreased. Considering the demographic trend, genetic and epidemiological data, and models of current and future climatic suitability, populations at the extreme south of the hotspot area still qualify for urgent in-situ conservation actions, whereas northern populations would be better managed through a mix of in-situ and ex-situ actions. Our results emphasize that identifying hotspots of genetic diversity, albeit an essential step, does not suffice to warrant on-ground conservation of threatened species. Hotspots should be analyzed at finer geographic and temporal scales, to provide conservation stakeholders with key knowledge to best define conservation priorities, and to optimize resource allocation to alternative management practices

Differences in Gene Expression Profiles of Seven Target Proteins in Third-Stage Larvae of Anisakis simplex (Sensu Stricto) by Sites of Infection in Blue Whiting (Micromesistius poutassou)

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The Mediterranean European hake, Merluccius merluccius: Detecting drivers influencing the Anisakis spp. larvae distribution

The European hake Merluccius merluccius is one of the most commercially important and widely distributed fish species, occurring both in European and Mediterranean Sea fisheries. We analyzed the distribution and infection rates of different species of Anisakis in M. merluccius (N = 1130 hakes), by site of infection in the fish host (viscera, dorsal and ventral fillets) from 13 different fishing grounds of the Mediterranean Sea (FAO area 37). The fillets were examined using the UV-Press method. A large number of Anisakis specimens (N = 877) were identified by diagnostic allozymes, sequence analysis of the partial EF1 α-1 region of nDNA and mtDNA cox2 gene. Among these, 813 larvae corresponded to A. pegreffii, 62 to A. physeteris, 1 to A. simplex (s. s.), whereas one resulted as a F1 hybrid between A. pegreffii and A. simplex (s. s.). Remarkably high levels of infection with A. pegreffii were recorded in hakes from the Adriatic/Ionian Sea compared to the fish of similar length obtained from the western Mediterranean fishing grounds. A positive correlation between fish length and abundance of A. pegreffii was observed. Concerning the localization of A. pegreffii larvae in the fish, 28.3% were detected in the liver, 62.9% in the rest of the viscera, 6.6% in the ventral part of the flesh, whereas 2.1% in the dorsal flesh

Population genetic structure of the parasite Anisakis simplex (s. s.) collected in Clupea harengus L. from North East Atlantic fishing grounds

The Atlantic herring is a schooling, pelagic species that inhabits both sides of the North Atlantic Ocean. Herring stock identification is usually based on several approaches, including fish meristic characters, population genetic analysis and the use of parasite species composition. A total of 654 Anisakis spp. larvae collected from herring of four fishing grounds in the Norwegian Sea, Baltic Sea, North Sea, and the English Channel off the French coast, was identified to species level using diagnostic allozymes and sequence analysis of EF1 α−1 nDNA and the mtDNA cox2 genes. Population genetic differentiation of Anisakis simplex (s. s.) among the different fishing areas was estimated, at the intraspecific level, on the basis of mtDNA cox2 sequences analysis. Spatial comparison based on molecular variance analysis and Fst values was performed for the collected specimens (among regions). Haplotype network construction showed relevant differences in haplotype frequencies between samples of A. simplex (s. s.) from the different geographical areas. Results indicate a genetic sub-structuring of A. simplex (s. s.) obtained from herring in different areas, with the population from the Norwegian Sea being the most differentiated one, and with North Sea and Baltic Sea populations being most similar. The population genetic structure of A. simplex (s. s.) was in accordance with the herring population genetic structure throughout the host’s geographical range in the NE Atlantic. Results suggest that mtDNA cox2 is a suitable genetic marker for A. simplex (s. s.) population genetic structure analysis and a valuable tool to elucidate the herring stock structure in the NE Atlantic Ocean

CORYNOSOMA AUSTRALE JOHNSTON, 1937 AND C. CETACEUM JOHNSTON & BEST, 1942 (ACANTHOCEPHALA: POLYMORPHIDAE) FROM MARINE MAMMALS AND FISH IN ARGENTINIAN WATERS: ALLOZYME MARKERS AND TAXONOMIC STATUS

Genetic and morphological studies were carried out on acanthocephalans belonging to Corynosoma Luhe, 1904 and referable to the species C. cetaceum Johnston & Best, 1942 and C. australe Johnston, 1937, which were recovered from both definitive and intermediate hosts in Argentinian waters. The aims were to estimate the level of genetic differentiation between the two taxa at any stage of their life-cycle, to provide genetic ( allozyme) markers for their recognition and to analyse the systematic status of both taxa. Acanthocephalans were collected from the stomach and intestine of Arctocephalus australis (Zimmerman), the intestine of Mirounga leonina (Linnaeus) and the stomach of Pontoporia blainvillei Gervais & D'Orbigny (definitive hosts) in Argentinian waters. Alternative alleles at all the 13 enzymatic loci studied were observed for C. australe and C. cetaceum. The specimens from the stomach of both P. blainvillei and A. australis were identified, on the basis of the great number of diagnostic loci found, as C. cetaceum; those from intestine of both A. australis and M. leonina as C. australe. A high level of genetic differentiation (D-Nei= infinity: I-Nei= 0.00) between the two taxa was found, suggesting a generic distinction between the two species. Cystacanths of the two species from the body-cavity of the fish Cynoscion guatucupa (Cuvier) collected from the same geographical area were identified genetically. Morphological patterns, such as the number of hooks and hook rows on the proboscis, the distribution of somatic and genital armature, and other morphometric and meristic differences, in addition to ecological data, enabled the identification of these two species at cystacanth, juvenile and adult stages. However, a number of morphological and morphometric features of the Argentinian material were different to those of C. australe and C. cetaceum described from other regions of the world