Taxonomy is the science of defining and naming groups of biological organisms based on shared characteristics and, more recently, on evolutionary relationships. With the birth of novel genomics/bioinformatics techniques and the increasing interest in microbiome studies, a further advance of taxonomic discipline appears not only possible but highly desirable. The present work proposes a new approach to modern taxonomy, consisting in the inclusion of novel descriptors in the organism characterization: (1) the presence of associated microorganisms (e.g.: symbionts, microbiome), (2) the mitochondrial genome of the host, (3) the symbiont genome. This approach aims to provide a deeper comprehension of the evolutionary/ecological dimensions of organisms since their very first description. Particularly interesting, are those complexes formed by the host plus associated microorganisms, that in the present study we refer to as “holobionts”. We illustrate this approach through the description of the ciliateEuplotes vanleeuwenhoekisp. nov. and its bacterial endosymbiont “CandidatusPinguicoccus supinus” gen. nov., sp. nov. The endosymbiont possesses an extremely reduced genome (~ 163 kbp); intriguingly, this suggests a high integration between host and symbiont.

Most of the microorganisms responsible for vector-borne diseases (VBD) have hematophagous arthropods as vector/reservoir. Recently, many new species of microorganisms phylogenetically related to agents of VBD were found in a variety of aquatic eukaryotic hosts; in particular, numerous new bacterial species related to the genus Rickettsia (Alphaproteobacteria, Rickettsiales) were discovered in protist ciliates and other unicellular eukaryotes. Although their pathogenicity for humans and terrestrial animals is not known, several indirect indications exist that these bacteria might act as etiological agents of possible VBD of aquatic organisms, with protists as vectors. In the present study, a novel strain of the Rickettsia-Like Organism (RLO) endosymbiont “Candidatus (Ca.) Trichorickettsia mobilis” was identified in the macronucleus of the ciliate Paramecium multimicronucleatum. We performed transfection experiments of this RLO to planarians (Dugesia japonica) per os. Indeed, the latter is a widely used model system for studying bacteria pathogenic to humans and other Metazoa. In transfection experiments, homogenized paramecia were added to food of antibiotic-treated planarians. Treated and non-treated (i.e. control) planarians were investigated at day 1, 3, and 7 after feeding for endosymbiont presence by means of PCR and ultrastructural analyses. Obtained results were fully concordant and suggest that this RLO endosymbiont can be transiently transferred from ciliates to metazoans, being detected up to day 7 in treated planarians’ enterocytes. Our findings might offer insights into the potential role of ciliates or other protists as putative vectors for diseases caused by Rickettsiales or other RLOs and occurring in fish farms or in the wild.

Rheinheimera sp. strain EpRS3, isolated from the rhizosphere of Echinacea purpurea, is already known for its ability to produce antibacterial compounds. By use of culture experiments, we verified and demonstrated its harmful effect against the ciliated protist Euplotes aediculatus (strain EASCc1), which by FISH experiments resulted to harbor in its cytoplasm the obligate bacterial endosymbiont Polynucleobacter necessarius (Betaproteobacteria) and the secondary endosymbiont “Candidatus Nebulobacter yamunensis” (Gammaproteobacteria). In culture experiments, the number of ciliates treated both with liquid broth bacteria-free (Supernatant treatment) and bacteria plus medium (Tq treatment), decreases with respect to control cells, with complete disappearance of ciliates within 6 h after Tq treatment. Results suggest that Rheinheimera sp. EpRS3 produces and releases in liquid culture one or more bioactive molecules affecting E. aediculatus survival. TEM analysis of control (not treated) ciliates allowed to morphologically characterize both kind of E. aediculatus endosymbionts. In treated ciliates, collected soon after the arising of cell suffering leading to death, TEM observations revealed some ultrastructural damages, indicating that P. necessarius endosymbionts went into degradation and vacuolization after both Supernatant and Tq treatments. Additionally, TEM investigation showed that when the ciliate culture was inoculated with Tq treatment, both a notable decrease of P. necessarius number and an increase of damaged and degraded mitochondria occur. FISH experiments performed on treated ciliates confirmed TEM results and, by means of the specific probe herein designed, disclosed the presence of Rheinheimera sp. EpRS3 both inside phagosomes and free in cytoplasm in ciliates after Tq treatment. This finding suggests a putative ability of Rheinheimera sp. EpRS3 to reintroduce itself in the environment avoiding ciliate digestion.

Hamatopeduncularia was erected with Hamatopeduncularis arii as the type species. This genus comprises monogenoidean species mostly found as ectoparasites of marine catfishes belonging to the Ariidae. There is a significant taxonomic ambiguity among Hamatopeduncularia species due to their morphological similarity, but so far only a few morphological studies have succeeded in addressing interspecific variation and relationships. Moreover, little molecular data is available for this genus. A multidisciplinary, integrated study consisting of morphological, morphometric and molecular analyses was conducted on different species of Hamatopeduncularia recovered from the gills of two marine catfishes, Arius jella Day and Plicofollis dussumieri (Valenciennes). Five species of Hamatopeduncularia, two of which represent new species, were investigated: H. arii, H. elongatum, H. thalassini, H. madhaviae sp. nov. and H. bifida sp. nov. Phylogenetic analysis was performed using the 18S rDNA sequence as a molecular marker. The most important results of the present work are: (1) the multidisciplinary description of two novel species; (2) the multidisciplinary redescription of two species and of the type species of the genus; (3) the first molecular characterisation of 18S rDNA sequences of five species of genus Hamatopeduncularia; and (4) molecular support for the monophyly of the genus. http://zoobank.org/urn:lsid:zoobank.org:act:1333F4CC-E497-4D0A-AD7D-276D44AE6413 http://zoobank.org/urn:lsid:zoobank.org:act:43D18F75-6F4A-4F9B-8C00-6234E5BA6526

In 1991 Margulis defined holobionts as the assemblage of “two or more organisms, members of different species” which remain associate “throughout a significant portion of the life history”. In recent times, holobionts have been described among many and far-related groups of living beings, such as plants, algae, insects, corals, and even humans. These studies have arisen an increasing interest in different contexts but, to our knowledge, the holobiont concept has not been applied in taxonomy. Here we propose a new approach to modern taxonomy, aimed to integrate the holobiont concept and genomic and bioinformatic analyses with the classical/morphological tools traditionally used in taxonomy. The inclusion of symbiont morphology, and of mitochondrial and symbiont genomes will allow the discipline to move toward what could become the “next generation taxonomy”. As an example of this new paradigm in the characterization of holobionts, we herein provide the taxonomic description of the ciliate protist Euplotes vanleeuwenhoeki sp. nov. (Euplotia, Ciliophora) and its bacterial endosymbiont “ Candidatus Pinguicoccus supinus” gen. nov., sp. nov. ( Opitutae, Verrucomicrobia ). Interestingly, we found that this endosymbiont has an extremely reduced genome (~163 Kbp), which is suggestive of a high integration with the host and represents the first case of such an extreme reduction in Verrucomicrobia , and the first case in a protist host.