Characterization of prevalence and genetic subtypes of Blastocystis sp. in wild and domestic Suidae of central Italy aided by amplicon NGS

Blastocystis spp. is a common single-celled intestinal symbiont, comprising several genetic subtypes (ST) and trans- missible by animal-to-animal, human-to-human, animal-to-human and, possibly, human-to-animal routes. This work was designed to explore the presence of Blastocystis in sympatric domestic and wild suids and their ability to carry zoonotic STs, in a condition of widespread opportunity to come in contact with the microorganism through their shared water and food resources, and other carriers. We sampled 42 and 37 stool samples from wild boars and domestic pigs, respectively. STs were first identified by PCR followed by Sanger sequencing. Sequences represented in double-band PCR products or in Sanger chromatograms displaying multiple peaks, were resolved by next generation sequencing (NGS). Twenty-six (61.9%) wild boar and 26 (70.2%) pig samples were PCR-positive, respectively. ST3, ST5 and ST15 were found in 3.8%, 38.4% and 80.8% of the positive wild boars and 11.5%, 88.5%, 11.5% of the positive pigs, respectively. ST1 was found only in pigs (3.8%). STs 5 and 15 were common in both groups of animals, but in reversed proportions, suggesting preferential colonization. We found significantly different ST distributions among wild boars and domestic pigs. This might indicate that lifestyle differences between the two populations influence their risk for contracting certain subtypes, or that ST5 and ST15 can colonize preferentially wild or domestic animals. Based on the STs described here, wild boars and domestic pigs can act as reservoirs with zoonotic potential. The ability of suids to carry zoonotic STs appears to be higher when using NGS than Sanger sequencing, and resolution of complex sequencing profiles is imperative before excluding the presence of STs of human concern

Three new species of Raphitoma Bellardi, 1847 (Mollusca, Gastropoda, Raphitomidae) from Croatian waters (NE Adriatic Sea)

Three new species of the neogastropod family Raphitomidae Bellardi, 1875, are described from the Croatian waters of the Adriatic Sea: Raphitoma petanii Prkić, Giannuzzi-Savelli & Pusateri n. sp., R. pusaterii Prkić & Giannuzzi-Savelli n. sp. and R. stanici Prkić, Giannuzzi-Savelli & Pusateri n. sp. The new species are diagnosed by teleoconch characters and by genetic data (COI sequences) in an integrative taxonomy framework. They belong to a group of species with microgranules on the external surface of all teleoconch whorls and all three have a multispiral protoconch indicating a planktotrophic larval phase. The first two species are so far known only from material found in Croatia. R. petanii Prkić, Giannuzzi-Savelli & Pusateri n. sp. and R. stanici Prkić, Giannuzzi-Savelli & Pusateri n. sp. are predominantly shallow-water species, whereas R. pusaterii Prkić & Giannuzzi-Savelli n. sp. has been so far collected only from deeper bottoms with presence of the red coral, Corallium rubrum (Linnaeus, 1758). Syntypes of Murex echinatusBrocchi, 1814 are figured and commented

Candidate cases of poecilogony in Neogastropoda: Implications for the systematics of the genus Raphitoma Bellardi, 1847

Poecilogony is the intraspecific variation in developmental mode, with larvae of different types produced by the same individual, population or species. It is very rare among marine invertebrates, and in gastropods has long been described only in a few opisthobranchs. The physiological and regulatory mechanisms underlying larval evolutionary transitions, such as loss of planktotrophy that occurred repeatedly in many caenogastropod lineages, are still largely unknown. We have studied the inter- v. intraspecific variation in larval development in the north-east Atlantic neogastropod genus Raphitoma Bellardi, 1847, starting with an iterative taxonomy approach: 17 morphology-based Preliminary Species Hypotheses were tested against a COI molecular-distance-based method (ABGD), and the retained species hypotheses were eventually inspected for reciprocal monophyly on a multilocus dataset. We subsequently performed an ancestral state reconstruction on an ultrametric tree of the 10 Final Species Hypotheses, time-calibrated by fossils, revealing that the interspecific changes were planktotrophy > lecithotrophy, and all have occurred in the Pleistocene, after 2.5 million years ago. This is suggestive of a major role played by Pleistocene Mediterranean oceanographic conditions-enhanced oligotrophy, unpredictable availability of water column resources-likely to favour loss of planktotrophy. Within this group of species, which has diversified after the Miocene, we identified one pair of sibling species differing in their larval development, Raphitoma cordieri (Payraudeau, 1826) and R. horrida (Monterosato, 1884). However, we also identified two Final Species Hypotheses, each comprising individuals with both larval developmental types. Our working hypothesis is that they correspond to one or two poecilogonous species. If confirmed by other nuclear markers, this would be the first documentation of poecilogony in the Neogastropoda, and the second in the whole Caenogastropoda. Although sibling species with different developmental strategies may offer good models to study some evolutionary aspects, poecilogonous taxa are optimally suited for identifying regulatory and developmental mechanisms underlying evolutionary transitions

Becoming a limpet: An ‘intermittent limpetization’ process driven by host features in the kleptoparasitic gastropod family Capulidae

A coiled shell is the most evident feature of the typical Bauplan of a gastropod mollusc. However, at least 54 families independently evolved an apparently simplified shell morphology: the limpet. Species with this largely uncoiled, depressed shell morphology occur in almost every aquatic habitat and are associated to a number of different lifestyles and diets. The marine gastropod family Capulidae includes 18 recognised genera, the large majority of which are coiled, but with a number of limpet-like species. Capulid shell plasticity is also associated to a broad range of feeding ecologies, from obligate suspension feeders to kleptoparasites. To investigate the evolution of the limpet-like shell in the family Capulidae we performed an ancestral state reconstruction analysis on a time-calibrated phylogenetic tree (COI, 16S, and ITS2) including 16 species representing a good deal of its morphological diversity. Our results identified at least three capulid lineages that independently evolved limpet-like shells, suggesting that a recurrent limpetization process characterizes this family. One of the limpet-like genera was undescribed and was here named Cryocapulus n. gen. We suggest that capulids evolved from a coiled suspension feeder lineage and that the shift to kleptoparasitism, which occurred in the family ancestor, may have represented a strategy to save energy through the exploitation of the water current produced by the host. Probably the major drivers of shell evolution in capulids are related to their ecology, most of them being kleptoparasites, include the shape and the kind of host substrate, and lead to the repeated acquisition of a limpet-like shape

Molecular identity of Nemoura lacustris (Plecoptera: Nemouridae) throughout its distributional range

The recent report of Nemoura lacustris Pictet, 1865 in Great Britain has raised doubts on its identity, given the isolation with respect to the Mediterranean and continental populations of this species. Using molecular analyses, we tested if populations from the United Kingdom and the Iberian Peninsula were conspecific and tested the hypotheses of a recent colonization event versus a more ancient origin for the British populations. Phylogenetic analysis of the mitochondrial marker COI allowed us to conclude that the United Kingdom specimens morphologically ascribed to N. lacustris were conspecific with populations from France and the Iberian Peninsula. Based on the genetic divergence of the two reciprocally monophyletic clades from the Iberian Peninsula and Great Britain/France, respectively, the present distribution of N. lacustris can be postulated as a relatively recent dispersal or introduction into Great Britain from France. Finally, we note the isolated position displayed by N. lacustris in the phylogenetic tree of Nemoura species based on COI sequences, as the sister to all included species of the genus. This isolated position corresponds with the specific morphology of N. lacustris genitalia and requires additional studies to ascertain clearer generic boundaries within the Nemouridae

Revision of Mediterranean and NE Atlantic Raphitomidae (Gastropoda, Conoidea) 8: The genus Leufroyia Monterosato, 1884

The four recent species of the genus Leufroyia Monterosato, 1884 are revised based on an integrative taxonomy approach: L. leufroyi (Michaud, 1828), L. concinna (Scacchi, 1836), L. erronea Monterosato, 1884 and L. villaria (Pusateri & Giannuzzi-Savelli, 2008)

High cryptic diversity in the kleptoparasitic genus Hyalorisia Dall, 1889 (Littorinimorpha: Capulidae) with the description of nine new species from the Indo-West Pacific

Species in the family Capulidae (Littorinimorpha: Capuloidea) display a wide range of shell morphologies. Several species are known to live in association with other benthic invertebrates-mostly bivalves and sabellid worms, but also other gastropods-and are believed to be kleptoparasitic filter feeders that take advantage of the water current produced by the host. This peculiar trophic ecology, implying a sedentary lifestyle, has resulted in highly convergent shell forms. This is particularly true for the genus Hyalorisia Dall, 1889, which occurs in deep water in the Caribbean and Indo-West Pacific provinces, with two nominal species recognized so far. Combining morphological, ecological and molecular data, we assessed the diversity of the genus, its phylogenetic position inside the family and its association with its bivalve host, the genus Propeamussium de Gregorio, 1884 (Pectinoidea), resulting in the description of nine new cryptic species. When sympatric, species of Hyalorisia are associated with different host species, but the same species of Propeamussium may be the host of several allopatric species of Hyalorisia

Whelks, rock-snails, and allied: a new phylogenetic framework for the family Muricidae (Mollusca: Gastropoda)

The worldwide distributed neogastropod family Muricidae comprises more than 1800 extant species of whelks, rock-shells, murex-shells, drill-shells, and coral-shells. Despite several attempts at a taxonomic revision based on morphological characters, the systematics of this family is still largely debated. Here, we present a molecular revision of the family Muricidae based on the largest dataset analysed so far, which comprises 384 specimens representing 360 species and includes, for the first time, all the currently recognised subfamilies. A molecular dataset of cox1, 12S rRNA, 16S rRNA, and 28S rRNA genetic markers and six fossil-based calibration points were used to produce time-calibrated phylogenetic reconstructions, using a Maximum Likelihood approach. Our results confirmed the monophyly of most of the accepted subfamilies, suggested a revision of the taxonomic composition of Muricopsinae and Muricinae, and highlighted some lineages not immediately comprised in any of the recognised subfamilies. The origin and early diversification of the subfamilies of Muricidae occurred between 32 and 60 million years ago

An assessment of Raphitoma and allied genera (Neogastropoda: Raphitomidae)

The systematics of several Eastern Atlantic conoidean species, traditionally ascribed to the genus RaphitomaBellardi, 1847, are revised on the basis of DNA sequence data from three gene regions (cytochrome c oxidase subunit I, 16S rRNA and 12S rRNA). We assign genus ranking to three major lineages (Raphitoma, CyrilliaKobelt, 1905 and LeufroyiaMonterosato, 1884) and suggest that two West African species belong in the subgenus Daphnella (Paradaphne) Laseron, 1954. A new classification, based on molecular systematics and critical study of morphology, is provided for all Eastern Atlantic and Mediterranean species that are currently ascribed to Raphitoma s.l. The genus ClathromangeliaMonterosato, 1884 is confirmed as belonging to Raphitomidae. Phylogenetic relationships and genetic distances suggest that Raphitoma maculosaHøisæter, 2016 and R. obesaHøisæter, 2016 may be divergent morphotypes of R. bicolor (Risso, 1826) and Cyrillia aequalis (Jeffreys, 1867), respectively