Plant DNA Methylation: An Epigenetic Mark in Development, Environmental Interactions, and Evolution.

DNA methylation is an epigenetic modification of the genome involved in the regulation of gene expression and modulation of chromatin structure. Plant genomes are widely methylated, and the methylation generally occurs on the cytosine bases through the activity of specific enzymes called DNA methyltransferases. On the other hand, methylated DNA can also undergo demethylation through the action of demethylases. The methylation landscape is finely tuned and assumes a pivotal role in plant development and evolution. This review illustrates different molecular aspects of DNA methylation and some plant physiological processes influenced by this epigenetic modification in model species, crops, and ornamental plants such as orchids. In addition, this review aims to describe the relationship between the changes in plant DNA methylation levels and the response to biotic and abiotic stress. Finally, we discuss the possible evolutionary implications and biotechnological applications of DNA methylation.

Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases.

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Extending the Toolkit for Beauty: Differential Co-Expression of DROOPING LEAF-Like and Class B MADS-Box Genes during Phalaenopsis Flower Development.

The molecular basis of orchid flower development is accomplished through a specific regulatory program in which the class B MADS-box AP3/DEF genes play a central role. In particular, the differential expression of four class B AP3/DEF genes is responsible for specification of organ identities in the orchid perianth. Other MADS-box genes (AGL6 and SEP-like) enrich the molecular program underpinning the orchid perianth development, resulting in the expansion of the original "orchid code" in an even more complex gene regulatory network. To identify candidates that could interact with the AP3/DEF genes in orchids, we conducted an in silico differential expression analysis in wild-type and peloric Phalaenopsis. The results suggest that a YABBY DL-like gene could be involved in the molecular program leading to the development of the orchid perianth, particularly the labellum. Two YABBY DL/CRC homologs are present in the genome of Phalaenopsis equestris, PeDL1 and PeDL2, and both express two alternative isoforms. Quantitative real-time PCR analyses revealed that both genes are expressed in column and ovary. In addition, PeDL2 is more strongly expressed the labellum than in the other tepals of wild-type flowers. This pattern is similar to that of the AP3/DEF genes PeMADS3/4 and opposite to that of PeMADS2/5. In peloric mutant Phalaenopsis, where labellum-like structures substitute the lateral inner tepals, PeDL2 is expressed at similar levels of the PeMADS2-5 genes, suggesting the involvement of PeDL2 in the development of the labellum, together with the PeMADS2-PeMADS5 genes. Although the yeast two-hybrid analysis did not reveal the ability of PeDL2 to bind the PeMADS2-PeMADS5 proteins directly, the existence of regulatory interactions is suggested by the presence of CArG-boxes and other MADS-box transcription factor binding sites within the putative promoter of the orchid DL2 gene.

Targeting the autosomal Ceratitis capitata transformer gene using Cas9 or dCas9 to masculinize XX individuals without inducing mutations.

BACKGROUND: Females of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs. RESULTS: With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults. CONCLUSIONS: Our data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination.

Identification of sex determination genes and their evolution in Phlebotominae sand flies (Diptera, Nematocera).

BACKGROUND: Phlebotomine sand flies (Diptera, Nematocera) are important vectors of several pathogens, including Leishmania parasites, causing serious diseases of humans and dogs. Despite their importance as disease vectors, most aspects of sand fly biology remain unknown including the molecular basis of their reproduction and sex determination, aspects also relevant for the development of novel vector control strategies. RESULTS: Using comparative genomics/transcriptomics data mining and transcriptional profiling, we identified the sex determining genes in phlebotomine sand flies and proposed the first model for the sex determination cascade of these insects. For all the genes identified, we produced manually curated gene models, developmental gene expression profile and performed evolutionary molecular analysis. We identified and characterized, for the first time in a Nematocera species, the transformer (tra) homolog which exhibits both conserved and novel features. The analysis of the tra locus in sand flies and its expression pattern suggest that this gene is able to autoregulate its own splicing, as observed in the fruit fly Ceratitis capitata and several other insect species. CONCLUSIONS: Our results permit to fill the gap about sex determination in sand flies, contribute to a better understanding of this developmental pathway in Nematocera and open the way for the identification of sex determining orthologs in other species of this important Diptera sub-order. Furthermore, the sex determination genes identified in our work also provide the opportunity of future biotechnological applications to control natural population of sand flies, reducing their impact on public health.

Pituitary adenylate cyclase-activating polypeptide in the testis of the quail Coturnix coturnix: Expression, localization, and phylogenetic analysis.

To evaluate the involvement of pituitary adenylate cyclase-activating polypeptide (PACAP)/receptors system in the control of testis activity, we have investigated the expression and localization of PACAP and the distribution of its receptors in the testis of mature samples of quail Coturnix coturnix, and we have performed a phylogenetic analysis of PACAP in birds. Using histological, molecular, and bioinformatics tools, we demonstrated that (a) PACAP messenger RNA shows a high sequence identity with that reported in other birds studied so far and in other vertebrates. Furthermore, we showed that purifying selection acts on PACAP; (b) the PACAP peptide is present only in Leydig cells, whereas its receptors are localized within both Leydig and germ cells; (c) the synthesis of PACAP does not take place in seminiferous tubules. The role of PACAP in the control of spermatogenesis and steroidogenesis in birds is discussed. Finally, we talk about the phylogenetic and evolutionary relationships between PACAP in birds and in other vertebrates.

Optimisation of artemisinin and scopoletin extraction from Artemisia annua with a new modern pressurised cyclic solid-liquid (PCSL) extraction technique.

INTRODUCTION: Artemisia annua is a small herbaceous plant belonging to the Asteraceae family declared therapeutic by the World Health Organisation, in particular for its artemisinin content, an active ingredient at the base of most antimalarial treatments, used every year by over 300 million people. In the last years, owing to low artemisinin content, research of new ways to increase the yield of the plant matrix has led to the use of the total extract taking advantage from the synergic and stabilising effects of the other components. OBJECTIVE: In this work we evaluated and compared the content of artemisinin and scopoletin in extracts of A. annua collected in the Campania Region (southern Italy), by two different extraction processes. METHODOLOGY: Artemisia annua plants were extracted by traditional maceration (TM) in hydroalcoholic solution as a mother tincture prepared according to the European Pharmacopeia and by pressurised cyclic solid-liquid (PCSL) extraction, a new generation method using the Naviglio extractor. RESULTS: The results showed that the PCSL extraction technique is more effective than traditional methods in extracting both phytochemicals, up to 15 times more, reducing the extraction times, without using solvents or having risks for the operators, the environment and the users of the extracts. CONCLUSION: The Naviglio extractor provides extracts with an artemisinin and scopoletin content eight times higher than the daily therapeutic dose, which should be evaluated for its stability over time and biological properties for possible direct use for therapeutic purposes.

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.).

The aim of this study is to assess, by RT-PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis).

The OitaAG and OitaSTK genes of the orchid Orchis italica: a comparative analysis with other C- and D-class MADS-box genes.

According to the ABCDE model of flower development, the C- and D- class MADS box genes are involved in the formation of male and female reproductive organs (fused to form the column in orchids) and in ovule maturation (triggered by fertilization in orchids). In the present study, we report the isolation of the Orchis italica genes OitaAG and OitaSTK, homologs of the C-class AGAMOUS and the D-class SEEDSTICK genes of Arabidopsis, respectively. Analysis of their expression profiles reveals high levels of mRNA in columns and ovaries, particularly after pollination. However, weak expression is also detectable in the inner tepals (OitaAG) and the lip and root (OitaSTK). This expression profile is only partially overlapping with those reported in other orchid species and may be the consequence of a different evolutionary history of these functional gene classes in orchids. The genomic characterization of the OitaAG and OitaSTK genes shows that a high number of traces of mobile elements are present in introns and could have contributed to the size expansion of some of them (e.g., intron 2 and 3 of OitaAG and intron 3, 4 and 5 of OitaSTK). Nucleotide sequences of intron 1 of the OitaSTK gene and other STK-like genes do not share regulatory motifs, whereas sequence comparison of intron 2 of the OitaAG gene with that of intron 2 of other AG-like genes reveals, for the first time in an orchid species, the presence of conserved cis-regulatory boxes and binding sites for transcription factors that positively (e.g., LEAFY and WUSCHEL) or negatively (e.g., BELLRINGER) regulate the expression of the AG homologs in dicots and monocots.

Evolutionary Plasticity in Insular Lizard, Adapting over Reproduction, Metabolism, and Color Variation.

The Italian wall lizard (Podarcis siculus) living on islets exhibits a melanic skin coloration and a suite of adaptive traits lacking in nearby mainland populations. On islets, the unpredictable environmental conditions and highly fluctuating population densities are believed to have produced reversed island syndrome (RIS). Several physiological, behavioral, and life-history changes based on the RIS could result from positive selection on increased activity of melanocortins. We hypothesize that phenotypes on islets are the product of a plastic variation depending on the regulation of specific genes. Focusing on control systems that determine the insular-adapted phenotype, we demonstrated that reproductive markers, involved in the hypothalamus-hypophysis-gonadal axis, and metabolism markers, flags for hypophysis-melanocortin receptors, are all up-regulated in island lizards under the RIS. This behavior, combined with the observed limited variation in the mitochondrial genome, agrees with the hypothesis that plasticity enables populations to persist in novel environmental conditions and that over time, natural selection will "fine-tune" the population to the environment by modifying the phenotype under selection. We believe that analysis of the transcriptome and the single gene expression, such that all the variations observed in the island populations, can be useful to shed light on evolutionary plasticity as a process affecting animals' populations in general.

Female Sex Determination Factors in Ceratitis capitata: Molecular and Structural Basis of TRA and TRA2 Recognition.

In the model system for genetics, Drosophila melanogaster, sexual differentiation and male courtship behavior are controlled by sex-specific splicing of doublesex (dsx) and fruitless (fru). In vitro and in vivo studies showed that female-specific Transformer (TRA) and the non-sex-specific Transformer 2 (TRA2) splicing factors interact, forming a complex promoting dsx and fru female-specific splicing. TRA/TRA2 complex binds to 13 nt long sequence repeats in their pre-mRNAs. In the Mediterranean fruitfly Ceratitis capitata (Medfly), a major agricultural pest, which shares with Drosophila a ~120 million years old ancestor, Cctra and Cctra2 genes seem to promote female-specific splicing of Ccdsx and Ccfru, which contain conserved TRA/TRA2 binding repeats. Unlike Drosophila tra, Cctra autoregulates its female-specific splicing through these putative regulatory repeats. Here, a yeast two-hybrid assay shows that CcTRA interacts with CcTRA2, despite its high amino acid divergence compared to Drosophila TRA. Interestingly, CcTRA2 interacts with itself, as also observed for Drosophila TRA2. We also generated a three-dimensional model of the complex formed by CcTRA and CcTRA2 using predictive approaches based on Artificial Intelligence. This structure also identified an evolutionary and highly conserved putative TRA2 recognition motif in the TRA sequence. The Y2H approach, combined with powerful predictive tools of three-dimensional protein structures, could use helpful also in this and other insect species to understand the potential links between different upstream proteins acting as primary sex-determining signals and the conserved TRA and TRA2 transducers.

A widespread picornavirus affects the hemocytes of the noble pen shell (Pinna nobilis), leading to its immunosuppression.

Introduction: The widespread mass mortality of the noble pen shell (Pinna nobilis) has occurred in several Mediterranean countries in the past 7 years. Single-stranded RNA viruses affecting immune cells and leading to immune dysfunction have been widely reported in human and animal species. Here, we present data linking P. nobilis mass mortality events (MMEs) to hemocyte picornavirus (PV) infection. This study was performed on specimens from wild and captive populations. Methods: We sampled P. nobilis from two regions of Spain [Catalonia (24 animals) and Murcia (four animals)] and one region in Italy [Venice (6 animals)]. Each of them were analyzed using transmission electron microscopy (TEM) to describe the morphology and self-assembly of virions. Illumina sequencing coupled to qPCR was performed to describe the identified virus and part of its genome. Results and discussion: In 100% of our samples, ultrastructure revealed the presence of a virus (20 nm diameter) capable of replicating within granulocytes and hyalinocytes, leading to the accumulation of complex vesicles of different dimensions within the cytoplasm. As the PV infection progressed, dead hemocytes, infectious exosomes, and budding of extracellular vesicles were visible, along with endocytic vesicles entering other cells. The THC (total hemocyte count) values observed in both captive (eight animals) (3.5 × 104-1.60 × 105 ml-1 cells) and wild animals (14 samples) (1.90-2.42 × 105 ml-1 cells) were lower than those reported before MMEs. Sequencing of P. nobilis (six animals) hemocyte cDNA libraries revealed the presence of two main sequences of Picornavirales, family Marnaviridae. The highest number of reads belonged to animals that exhibited active replication phases and abundant viral particles from transmission electron microscopy (TEM) observations. These sequences correspond to the genus Sogarnavirus-a picornavirus identified in the marine diatom Chaetoceros tenuissimus (named C. tenuissimus RNA virus type II). Real-time PCR performed on the two most abundant RNA viruses previously identified by in silico analysis revealed positive results only for sequences similar to the C. tenuissimus RNA virus. These results may not conclusively identify picornavirus in noble pen shell hemocytes; therefore, further study is required. Our findings suggest that picornavirus infection likely causes immunosuppression, making individuals prone to opportunistic infections, which is a potential cause for the MMEs observed in the Mediterranean.

Orchid NAC Transcription Factors: A Focused Analysis of CUPULIFORMIS Genes.

Plant transcription factors are involved in different developmental pathways. NAC transcription factors (No Apical Meristem, Arabidopsis thaliana Activating Factor, Cup-shaped Cotyledon) act in various processes, e.g., plant organ formation, response to stress, and defense mechanisms. In Antirrhinum majus, the NAC transcription factor CUPULIFORMIS (CUP) plays a role in determining organ boundaries and lip formation, and the CUP homologs of Arabidopsis and Petunia are involved in flower organ formation. Orchidaceae is one of the most species-rich families of angiosperms, known for its extraordinary diversification of flower morphology. We conducted a transcriptome and genome-wide analysis of orchid NACs, focusing on the No Apical Meristem (NAM) subfamily and CUP genes. To check whether the CUP homologs could be involved in the perianth formation of orchids, we performed an expression analysis on the flower organs of the orchid Phalaenopsis aphrodite at different developmental stages. The expression patterns of the CUP genes of P. aphrodite suggest their possible role in flower development and symmetry establishment. In addition, as observed in other species, the orchid CUP1 and CUP2 genes seem to be regulated by the microRNA, miR164. Our results represent a preliminary study of NAC transcription factors in orchids to understand the role of these genes during orchid flower formation.

Expression pattern of two paralogs of the PI/GLO-like locus during Orchis italica (Orchidaceae, Orchidinae) flower development.

The class B MADS-box genes belong to two distinct functional groups: the AP3/DEF-like and the PI/GLO-like sub-families. In orchids, AP3/DEF-like genes are present in four copies, each with a different role in floral organ formation, which is described in the "orchid code" model. Interestingly, the orchid PI/GLO-like genes are present in two copies in Orchidinae, whereas they are described as single copy in the other orchid lineages. The two PI/GLO-like paralogs have site-specific different selective constraints; in addition, they show relaxation of purifying selection when compared to the single-copy lineages. In this study, we present a comparative analysis of the expression patterns of the two PI/GLO-like paralogs, OrcPI and OrcPI2, in floral tissues of Orchis italica in different developmental stages using real-time PCR. The two genes show similar expression profiles in the tissue examined, with differences detectable between immature and mature inflorescence. In all cases, OrcPI2 is expressed at a higher level than OrcPI. Real-time PCR results reveal that the co-expression of the two duplicated loci could have a fully or partially redundant function. The possible evolutionary fate of OrcPI and OrcPI2 is discussed as well as their involvement in ovary development.

Correction: Fixation of genetic variation and optimization of gene expression: The speed of evolution in isolated lizard populations undergoing Reverse Island Syndrome.

[This corrects the article DOI: 10.1371/journal.pone.0224607.].

Isolation and phylogenetic footprinting analysis of the 5'-regulatory region of the floral homeotic gene OrcPI from Orchis italica (Orchidaceae).

The nucleotide sequences of regulatory elements from homologous genes can be strongly divergent. Phylogenetic footprinting, a comparative analysis of noncoding regions, can detect putative transcription factor binding sites (TFBSs) shared among the regulatory regions of 2 or more homologous genes. These conserved motifs have the potential to serve the same regulatory function in distantly related taxa. We isolated the 5'-noncoding region of the OrcPI gene, a MADS-box transcription factor involved in flower development in Orchis italica, using the thermal asymmetric interlaced polymerase chain reaction technique. This region (comprising 1352 bp) induced transient beta-glucuronidase expression in the petal tissue of white Rosa hybrida flowers and represents the 5'-regulatory sequence of the OrcPI gene. Phylogenetic footprinting analysis detected conserved regions within the 5'-regulatory sequence of OrcPI and the homologous regions of Oryza sativa, Lilium regale, and Arabidopsis thaliana. Some of these sequences are known TFBSs described in databases of plant regulatory elements. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the following accession numbers: AF198055 promoter region of the PISTILLATA (PI) gene of A. thaliana; AB094985 cDNA of OrcPI (PI/GLOBOSA [PI/GLO] homologue) of O. italica; AB378089 5'-regulatory region of the OrcPI gene of O. italica; AP008211 putative promoter region of OSMADS2 (PI/GLO homologue) of O. sativa; AP008207 putative promoter region of OSMADS4 (PI/GLO homologue) of O. sativa; and AB158292 putative promoter region of the PI/GLO homologue of L. regale.

Radial or Bilateral? The Molecular Basis of Floral Symmetry.

In the plant kingdom, the flower is one of the most relevant evolutionary novelties. Floral symmetry has evolved multiple times from the ancestral condition of radial to bilateral symmetry. During evolution, several transcription factors have been recruited by the different developmental pathways in relation to the increase of plant complexity. The MYB proteins are among the most ancient plant transcription factor families and are implicated in different metabolic and developmental processes. In the model plant Antirrhinum majus, three MYB transcription factors (DIVARICATA, DRIF, and RADIALIS) have a pivotal function in the establishment of floral dorsoventral asymmetry. Here, we present an updated report of the role of the DIV, DRIF, and RAD transcription factors in both eudicots and monocots, pointing out their functional changes during plant evolution. In addition, we discuss the molecular models of the establishment of flower symmetry in different flowering plants.

Epidemiology of Noble Pen Shell (Pinna nobilis L. 1758) Mass Mortality Events in Adriatic Sea Is Characterised with Rapid Spreading and Acute Disease Progression.

From May to October 2019, multiple mass mortality events (MMEs) of Pinna nobilis were observed along Croatian coastline starting from the south-east and rapidly progressing in north-western direction. Time dynamics of the MMEs closely followed general speed and direction patterns of surface sea-currents, advancing approximately 350 km in less than 3 months. Surveillance, clinical evaluation, and sample collection were performed on multiple sites with various degrees of mortality rates. Moribund P. nobilis individuals were collected and subjected to pathological, molecular, and microscopical investigation. Affected animals were positive for Mycobacterium in 70% of the individuals, and Haplosporidium pinnae was present in 58% of the cases. Observed pathological lesions were most severe where concurrent presence of both pathogens was confirmed (in 45.8% of moribund individuals). Moderate to strong lesions were observed in animals positive for Mycobacterium only (25% of cases), and lesions were absent or minor to moderate when only H. pinnae was confirmed (16% of cases). Considering the rapid and severe spread of the MMEs, the areas less exposed to major sea currents appeared to be at lower risk of pathogen transmission. Surveillance activities along the Croatian coastline identified several P. nobilis populations in such "lower risk" areas without apparent mortality or clinical symptoms. Such areas are of particular interest as source of potentially healthy individuals to support active recovery actions.

Diagnosis of Centrocestus formosanus Infection in Zebrafish (Danio rerio) in Italy: A Window to a New Globalization-Derived Invasive Microorganism.

: Centrocestus formosanus is a digenetic trematode with a complex life cycle, involving invertebrate and vertebrate hosts, humans included. In particular, it causes gill lesions and mortality in freshwater fish species, and gastrointestinal symptoms in infected humans. Here, we describe the occurrence of C. formosanus infection in zebrafish imported in Italy and propose a newly designed species-specific primer pair to ameliorate the diagnostic investigations for C. formosanus. Gill arches of 30 zebrafish were examined for the presence of encysted metacercariae under a stereomicroscope and processed through molecular analyses targeting the ribosomal internal transcribed sequence 2 (ITS2). Although C. formosanus distribution was originally restricted to Asia, it has been subsequently reported in new countries, revealing itself as an invasive species and raising important concerns for biodiversity, economy, scientific research, as well as animal and public health. Given the crucial role played by the ornamental fish industry in spreading this parasite, there is an urgent need for control measures to prevent the introduction and establishment of C. formosanus in non-endemic areas, including Europe. We also suggest developing new strategies in microbiology and epidemiology to better explore this new globalization-derived invasive species.

The MADS-box genes expressed in the inflorescence of Orchis italica (Orchidaceae).

The Orchidaceae family, which is one of the most species-rich flowering plant families, includes species with highly diversified and specialized flower shapes. The aim of this study was to analyze the MADS-box genes expressed in the inflorescence of Orchis italica, a wild Mediterranean orchid species. MADS-box proteins are transcription factors involved in various plant biological processes, including flower development. In the floral tissues of O. italica, 29 MADS-box genes are expressed that are classified as both class I and II. Class I MADS-box genes include one Mß-type gene, thereby confirming the presence of this type of MADS-box genes in orchids. The class II MIKC* gene is highly expressed in the column, which is consistent with the conserved function of the MIKC* genes in gametophyte development. In addition, homologs of the SOC, SVP, ANR1, AGL12 and OsMADS32 genes are expressed. Compared with previous knowledge on class II MIKCC genes of O. italica involved in the ABCDE model of flower development, the number of class B and D genes has been confirmed. In addition, 4 class A (AP1/FUL) transcripts, 2 class E (SEP) transcripts, 2 new class C (AG) transcripts and 1 new AGL6 transcript have been identified. Within the AP1/FUL genes, the sequence divergence, relaxation of purifying selection and expression profiles suggest a possible functional diversification within these orchid genes. The detection of only two SEP transcripts in O. italica, in contrast with the 4 genes found in other orchids, suggests that only two SEP genes could be present in the subfamily Orchidoideae. The expression pattern of the MIKCC genes of O. italica indicates that low levels at the boundary of the domain of a given MADS-box gene can overlap with the expression of genes belonging to a different functional A-E class in the adjacent domain, thereby following a "fading borders" model.