The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) system of prokaryotes is an adaptative immune defense mechanism to protect themselves from invading genetic elements (e.g., phages and plasmids). Studies that describe the genetic organization of these prokaryotic systems have mainly reported on the Enterobacteriaceae family (now reorganized within the order of Enterobacterales). For some genera, data on CRISPR-Cas systems remain poor, as in the case of Serratia (now part of the Yersiniaceae family) where data are limited to a few genomes of the species marcescens. This study describes the detection, in silico, of CRISPR loci in 146 Serratia complete genomes and 336 high-quality assemblies available for the species ficaria, fonticola, grimesii, inhibens, liquefaciens, marcescens, nematodiphila, odorifera, oryzae, plymuthica, proteomaculans, quinivorans, rubidaea, symbiotica, and ureilytica. Apart from subtypes I-E and I-F1 which had previously been identified in marcescens, we report that of I-C and the I-E unique locus 1, I-E*, and I-F1 unique locus 1. Analysis of the genomic contexts for CRISPR loci revealed mdtN-phnP as the region mostly shared (grimesii, inhibens, marcescens, nematodiphila, plymuthica, rubidaea, and Serratia sp.). Three new contexts detected in genomes of rubidaea and fonticola (puu genes-mnmA) and rubidaea (osmE-soxG and ampC-yebZ) were also found. The plasmid and/or phage origin of spacers was also established.

The CRISPR-Cas system of Prokaryotes is an adaptive immune defense mechanism to protect themselves from invading genetic elements (e.g. phages and plasmids). Studies that describe the genetic organization of these prokaryotic systems have mainly reported on the Enterobacteriaceae family (now reorganized within the order Enterobacteriales). For some genera, data on CRISPR-Cas systems remain poor, as in the case of Serratia (now part of the Yersiniaceae family) where data are limited to a few genomes of the species marcescens. This study describes the detection, in silico, of CRISPR loci in 146 Serratia complete genomes and 336 high-quality assemblies available for the species ficaria, fonticola, grimesii, inhibens, liquefaciens, marcescens, nematodiphila, odorifera, oryzae, plymuthica, proteomaculans, quinivorans, rubidaea, symbiotic, and ureilytica. Apart from subtypes I-E and I-F1, which had previously been identified in marcescens, we report that of I-C and the variants I-ES1, I-ES2 and I-F1S1. Analysis of the genomic contexts for CRISPR loci revealed mdtN-phnP as the region mostly shared (grimesii, inhibens, marcescens, nematodiphila, plymuthica, rubidaea, and Serratia sp.). Three new contexts detected in genomes of rubidaea and fonticola (puu genes-mnmA) and rubidaea (osmE-soxG and ampC-yebZ) were also found. Plasmid and/or phage origin of spacers was also established.

The CRISPR-Cas adaptive immune system has been attracting increasing scientific interest for biological functions and biotechnological applications. Data on the Serratia marcescens system are scarce. Here, we report a comprehensive characterisation of CRISPR-Cas systems identified in S. marcescens strains isolated as secondary symbionts of Rhynchophorus ferrugineus, also known as Red Palm Weevil (RPW), one of the most invasive pests of major cultivated palms. Whole genome sequencing was performed on four strains (S1, S5, S8, and S13), which were isolated from the reproductive apparatus of RPWs. Subtypes I-F and I-E were harboured by S5 and S8, respectively. No CRISPR-Cas system was detected in S1 or S13. Two CRISPR arrays (4 and 51 spacers) were detected in S5 and three arrays (11, 31, and 30 spacers) were detected in S8. The CRISPR-Cas systems were located in the genomic region spanning from ybhR to phnP, as if this were the only region where CRISPR-Cas loci were acquired. This was confirmed by analyzing the S. marcescens complete genomes available in the NCBI database. This region defines a genomic hotspot for horizontally acquired genes and/or CRISPR-Cas systems. This study also supplies the first identification of subtype I-E in S. marcescens.

While studying Septobasidium interaction with Diaspididae off Laurus nobilis L., Phillyrea latifolia L. and angustifolia L. in Apulia we found small - 1 to 3 millimetre - brown/black particles on plant bark. Each particle embeds or lays one or more dead armoured scales belonging to Aonidia lauri (Bouche) or Lepidosaphes flava (Signoret), depending on the host plant. The brown-black bodies were irregularly hemispheric, lobated and dull in appearance. We report the results of observations by light microscopy, SEM and X-rays microanalysis of foamy black matters that constitute most of the particles and embed the scale. Findings of hyphae (?) inside the dead insect bodies in connection to Diaspidid scale phenology at the time of death suggest that the particles result from an entomopathogenic or saprophytic interaction. Acknowledgement: we recognise the help and competence of Pasquale Trotti, SELGE UR3. laboratory responsible, for the SEM pictures & microanalysis and Prof Claudio Ciccarone (SAFE-UNIFG) for the tentative identification of the hyphae found associated with Aonidia lauri. We also recognize the support of Applied Ecology. Aonidia lauri (Bouche) is a common Diaspididae often found on Laurel where it infests trunk and twigs but leaves also. Septobasidium grows to great epiphytic thalli on the plant bark and often infects the scale (Porcelli, 2013). On the same Septobasidium-inhabited Laurel as on Pistacia lentiscus L. or Phillyrea latifolia and angustifolia there are numerous black-dull masses embedding one or more A. lauri or Lepidosaphes flava and often sheltering several more individuals under the margin of the masses themselves. Not sheltered scales are comparatively less than those that, possibly following a tygmotactic stimulus, prefer to fix near or under the masses. Moreover, scrutinising the masses one can easily find several or many scales almost entirely embedded in the black matter or still partially exposed. Even those masses that do not show scales embedded reveal one or more individuals inside the matter once purposely dissected. SEM study reveals the presence of stromatic and possibly penetrative hyphae associate with moniliform Nostoc-like chains glued by a foamy gelatinous sheath. Hyphae, Nostoc-like chains and jelly strongly resemble a species of Collema (Fungi) often symbiotically associates with Nostoc (Cyanobacteria) to form Lichen that in the case fills the scale body. Chemical elementary semiquantitative microanalysis supports the organic origin of the black matter. Next studies will confirm the nature of the actors involved in this intriguing phenomenon.

The euonymus scale Unaspis euonymi (Comstock) (Hemiptera: Diaspididae) is a pest of spindle that exhibits a strong preference for Euonymus, although it has been detected on at least 18 genera in 13 plant families (Buxus, Camellia, Celastrus, Daphne, Eugenia, Euonymus, Hibiscus, Ilex, Jasminum, Ligustrum, Lonicera, Olea, Pachistima, Pachysandra, Perychmenum, Prunus and Syringa) (Salisbury et al., 2013). Heavy infestation by this pest may lead to the death of the host plant and consequential loss of income from the cultivation of ornamental plants (Kaygin et al., 2008). U. euonymi is an armored scale insect originally from mild Eastern Asia and probably introduced into Europe in the 20th century (Pellizzari & Germain, 2010). Its lifecycle, depending on climate conditions, comprises two-three generations a year and the control measures to limit its diffusion mainly rely on the use of insecticides or the growing of resistant cultivars. The insects can engage mutualistic interactions or symbioses with a variety of bacteria that can profoundly affect the host’s biology. Apart from obligate symbionts (maternally transmitted), a growing number of facultative or secondary symbionts (that can be horizontally transmitted) have been identified (Sandstrom et al., 2001, Moran et al., 2008). Despite not being essential for the host’s life cycle, this last type of symbiont can strongly influence their fitness (Oliver et al., 2003, Jaenike & Brekke, 2011). Additionally, the mutualistic association between insects and bacteria may play a role in the evolution of the latter as described for some groups of Entereobacteriaceae (Moran et al., 2005). A number of genomic and phylogenetic studies on mutualistic associations between Enterobacteriaceae and aphids, psyllids, scale insects, whiteflies, weevils and other insects have been reported (Lefevre et al., 2004, Thao & Baumann, 2004). Here we report the identification of Serratia symbiotica (strain UESS2016) in U. euonymi adult females collected from Sofia (Bulgaria) in 2013.

The study of minute cuticular details of small delicate insects is possible by slide mounting of the entire exoskeleton or part of it. This technique requires whole insect body clearing or tissue bleaching and washing. Lesser sclerotized insect body greatly suffers for such treatments and looses its natural body shape by shrinkage or by flattening, consequently. The aim of this study is to suggest an effective, fast and cheap technique to image less sclerotized insects that are prone to shrink or to wrinkle their bodies because of desiccation. Actual availability of desktop Cryo- SEM (Hitachi TM 3000 series) suggested us to experiment the opportunity to preserve natural body shape of the minute, delicate and lesser sclerotized insects in their living attitude. The technique bases on freezing the specimen, either living or previously EtOH-preserved but moved in water for the preparation, in the water down to -40°C on the SEM Cryo-stage and setting it for observation in SEM vacuum chamber. Once in the vacuum a proper T°C increase at about -28/-22°C allows external ice sublimation and exposes the frozen insect to direct SEM imaging. The technique appears promising because of the overall quality of results, the resolving power, the opportunity to measure the specimens. In fact, delicate specimens as Phylloxera ilicis Grassi (Hemiptera: Phylloxeridae, a representative of Phylloxera quercus Boyer de Fonscolombe group), the Italian grape mealybugs Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) and Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) maggots that are all usually ruined by desiccation during direct SEM observation, beautifully retain their natural body shape by this technique allowing the study and imaging of external morphology. As a further advantage, there is no need to critical point drying or metal coating, and the same sample can be submitted to conventional slide mounting later, after being studied by Cryo-SEM. Finally, we present a table of the running time/cost per observation of the proposed technique.

Recurrent Nidularia pulvinata outbreaks off Quercus ilex L. (Fagaceae) led us to scrutinize large amount of infested and damaged trunks, branches and twigs. A long series of detailed observations suggested studying the attitude of the Kermesidae to induce phloem/xylem disorganization by saliva injection, possibly. Evidence show that woody plants tissues near or under the scale insects swell considerably giving the organs a prominent and rising appearance. Infested bark and other plant surfaces are prone to produce crevices and other possible shelters for future crawlers that will find a nice living site nearby. In vivo transverse section, accurate observations corroborated with polarized light microscopy and Scanning Electron Microscopy suggests that the scale stylets strongly disturb the explored plant tissues. Plant tissues reply to the injury with considerable overgrowing and necrosis. By observations and evidenced we discuss the opportunity to consider Nidularia pulvinata a gall-making species, also comparing its attitude with that of other Kermesidae and Asterolecaniidae. Nidularia pulvinata infestation starts from crawlers that set into natural bark crevices. Scales feeding elicits the plant reaction that results in progressive widening of infested crevices. That, in consequence, offers more room for the subsequent broods of the Kermesidae. To evaluate the action of Nidularia feeding on host plant wood, we fell down two small Quercus ilex infested to death. We chose and mark several points before to cut the infested trunks and branches transversally. The action of Nidularia pulvinata feeding on host plant wood is clear in the left and central figure in comparison to right picture of a not infested trunk. Each log was pictured from the transversal section and the corresponding side. Red dashed lines connect corresponding points of the wood. The red arrow points to wood overgrowths for Nidularia infestation, while a blue arrow target a lateral branch bud. Red lines encircle wood overgrowths due to hyperplasia/hypertrophy stimulated by the Kermesidae. On twigs the Nidularia pulvinata feeding results in wood swelling, depression/overgrowth and necrosis.

Pest outbreaks often give to insect pathogens the opportunity to infect their host species eventually leading them to death. Recent Nidularia outbreaks off urban Quercus ilex L. showed some cases of entomopathogenic fungi virulence, apparently sustained by species of Fusarium and other fungi. Infection is apparent on the Nidularia population because fungi provoke the scale color shifting to orange or dull-green in medium in large patches. After the isolation in an axenic culture of the Kermesid-associate Mycota, ITS genomic regions amplified by PCR using the universal ITS5/ITS4 primers were sequenced by external service (Macrogen, Seoul, South Korea) for molecular identification. Blast analysis (http://blast. ncbi. nlm. nih. gov/Blast. cgi) of the ITS sequence showed a high homology with Quambalaria cyanescens (de Hoog & G. A. de Vries) Z. W. de Beer, Begerow & R. Bauer 2006 (Fungi: Basidiomycota), (identity: 97-96%; e-value 0. 0; coverage 82-75%), Fusarium acuminatum Ellis & Everh. 1895 (Fungi: Ascomycota) and Fusarium avenaceum (Fr. ) Sacc. 1886 (Fungi: Ascomycota) (identity: 99%; e-value 0. 0; coverage 82-80%), and Penicillium sumatraense Svilv. 1936 (Fungi: Ascomycota) (identity: 100%; e-value . 0; coverage 95-91%). We investigate the biological and ecological role of the above-recorded fungi by correlating the age of infected scale and their age. Mass culturing of the most promising pathogen will lead to semi-field trials to demonstrate the isolate entomopathogenic ability. Finally, we discuss the case of homonymy between the fungal Taxon named Nidularia pulvinata (Schwein.) (Gasteromycetes) and the scale Nidularia pulvinata (Planchon) that can lead to some confusion.

Kermes vermilio and Nidularia pulvinata severe outbreaks are lethal to young holly oaks and strongly damage adult trees. Such infestations are common in several South Italian urban and peri-urban areas and have been reported for a while. Recently we have been observing in Bari (Apulia region) a new considerable invasion of both Nidularia and Kermes on Q. ilex not scale-infested hitherto. The phenomenon is possibly in connection with the new northward expansion of Nidularia that is presently recorded in Italian areas with a continental climate as Veneto and Romagna regions. City surveys and damage estimations revealed the entity of kermesidae impact regarding direct and indirect costs for the maintenance of urban parks, public gardens and leisure areas, avenues and boulevards. Moreover, we discuss the synecology of the two species evaluating the prevalence of mixed infestations on the same plant or the reciprocal competitive displacement. Possible causes of the actual outbreak may lie in ecological disturbance along the rural-urban transition zone, changes in the available range of insecticides, wrong or absent tree maintenance, changes or fluctuation in climatic parameters. Mixed infestations by Kermes vermilio and Nidularia pulvinata off Quercus ilex are not truly mixed; this is evident as we realise that K. vermilio prefers to infest host twigs while N. pulvinata mostly sets on the trunk and the main branches. The two species seems to share a single host plant occupying two near but distinct niches. Damages are similar, being both the Kermesidae capable of inducing die back, but Nidularia actively damages the trunk, also. In a case of infested public greeneries, the officers are usually unable to check the pests, i. e. by an IPM strategy or by chemical control. Possibly because of the extended swarming of crawlers or the missing of effective insecticide authorised for urban area use. Moderate control is given by tree formulate injection but results often less useful than expected. Another phytosanitary “pest cleaning” measure applied by the officers consists of cutting down symptomatic dried twigs as massive pruning every four-five years. Intense pruning in spring and the consequent plant re-sprouting appears favourable to plant health but for a very short lapse. New sprouts are quickly colonised by wandering crawlers and became intensely infested. Moreover, twigs and small branches cut down leave on the plant much more of the Nidularia population while prune down most of the Kermes out the plant. Intense and not timed pruning favours Nidularia by displacing Kermes from the host plant in such a way that urban Q. ilex are heavily infested by the most damaging and lethal of the two pest species. We classified from 1 to 5 the staging of the die back induced by Kermesidae, as reported in table below.

Thrombocytopenia-absent-radius (TAR) syndrome is a rare condition characterized by thrombocytopenia and bilateral absence of the radii with presence of both thumbs. The phenotype has a variable expression. A 200 kb minimal deletion at 1q21.1 is present in all patients. However, the microdeletion, ranging up to 1100 kb in length, is not sufficient to cause the disease. Indeed it is present in 75-80% of unaffected parents. It is assumed that the phenotype develops only in the presence of one or more additional, as-yet-unknown, deletion modifiers (mTARs).We report here on a child affected by TAR syndrome associated with Langerhans cell histiocytosis. Unexpectedly, he showed a 2.029 kb deletion at 1q21.1, almost twice that of the unaffected mother (957 kb). Interestingly, the mother-to-son increased size of the deleted region was already observed in two cases of constitutional diseases, although both resulting as chromosomal terminal deletions.Noteworthy, qPCR experiments, never before performed for patients with TAR syndrome, disclosed that the proband had a statistically significant downregulation of the majority of the genes mapping inside the part of the deletion shared with the mother. The mother, on the contrary, did not show the same downregulation.In summary, the present report adds new insights on the pathogenesis of TAR syndrome, that may represent fruitful directions for future research. (C) 2011 Elsevier Masson SAS. All rights reserved.

Sequence analyses of endangered orang-utan species The genome of the Southeast Asian great ape or orang-utan has been sequenced — specifically a draft assembly of a Sumatran female individual and short-read sequence data from five further Sumatran and five Bornean orang-utan, Pongo abelii and Pongo pygmaeus , respectively. Orang-utan species appear to have split around 400,000 years ago, more recent than most previous estimates suggested, resulting in an average Bornean–Sumatran nucleotide identity of 99.68%. Structural evolution of the orang-utan genome seems to have proceeded much more slowly than that of other great apes, including chimpanzees and humans. With both orang-utan species on the endangered list, the authors hope that knowledge of the genome sequence and its variation between populations will provide a valuable resource for conservationists.

Field symptoms of chlorotic mottling and leaf deformations were observed on the cv Pinot gris (PG) in the Trentino region (Italy). Extensive assays excluded the presence of widely distributed nepo-, ampelo- and vitiviruses. An analysis of small RNA populations from two PG grapevines showing or not symptoms was carried out by Illumina high throughput sequencing. The study disclosed the virus and viroids contents of the two vines that was composed by Grapevine rupestris stem pitting-associated virus (GRSPaV), two viroids Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1), the marafiviruses Grapevine rupestris vein feathering virus (GRVFV) and Grapevine Syrah virus 1 (GSyV-1), and a hitherto unrecorded virus. This virus had a genome organization identical to that of Grapevine berry inner necrosis virus (GINV), a trichovirus reported only from Japan, with which it grouped in phylogenetic trees constructed with sequences of the RdRp domain and the coat protein gene. However, molecular differences with GINV are wide enough to warrant classification of the virus in question as a new species, for which the provisional name of Grapevine Pinot gris virus (GPGV) is proposed. A limited field survey for the presence of GPGV in diseased and symptomless plants from three different cultivars did not allow to clearly associating the virus to the observed symptoms.

We report of a spinocerebellar ataxia (SCA)27 in a daughter and her mother whose karyotype is 46, XX t(5;13)(q31.2;q33.1). The translocation breakpoint is identical in both patients, disrupting the gene-encoding fibroblast growth factor 14 isoform b (FGF14-1b). Clinically, both show signs of SCA, although the daughter is the most affected with early onset cerebellar ataxia, microcephaly, and severe mental retardation. FGF14-1b is the predominant isoform in brain, where it interacts with the voltage gated Na channel. Fgf14−/− mice develop ataxia and paroxysmal dyskinesia and have cognitive deficits. One missense and one non-sense mutation in FGF14 have previously been linked to SCA27. Truncation of one allele in our patients suggests that haploinsuffiency of FGF14 can cause SCA27.

BACKGROUND:The subtelomeric regions of human chromosomes exhibit an extraordinary plasticity. To date, due to the high GC content and to the presence of telomeric repeats, the subtelomeric sequences are underrepresented in the genomic libraries and consequently their sequences are incomplete in the finished human genome sequence, and still much remains to be learned about subtelomere organization, evolution and function. Indeed, only in recent years, several studies have disclosed, within human subtelomeres, novel gene family members.RESULTS:During a project aimed to analyze genes located in the telomeric region of the long arm of the human X chromosome, we have identified a novel transcript family, DDX11L, members of which map to 1pter, 2q13/14.1, 2qter, 3qter, 6pter, 9pter/9qter, 11pter, 12pter, 15qter, 16pter, 17pter, 19pter, 20pter/20qter, Xpter/Xqter and Yqter. Furthermore, we partially sequenced the underrepresented subtelomeres of human chromosomes showing a common evolutionary origin.CONCLUSION:Our data indicate that an ancestral gene, originated as a rearranged portion of the primate DDX11 gene, and propagated along many subtelomeric locations, is emerging within subtelomeres of human chromosomes, defining a novel gene family. These findings support the possibility that the high plasticity of these regions, sites of DNA exchange among different chromosomes, could trigger the emergence of new genes.

In 1992 the Japanese macaque was the first species for which the homology of the entire karyotype was established by cross-species chromosome painting. Today, there are chromosome painting data on more than 50 species of primates. Although chromosome painting is a rapid and economical method for tracking translocations, it has limited utility for revealing intrachromosomal rearrangements. Fortunately, the use of BAC-FISH in the last few years has allowed remarkable progress in determining marker order along primate chromosomes and there are now marker order data on an array of primate species for a good number of chromosomes. These data reveal inversions, but also show that centromeres of many orthologous chromosomes are embedded in different genomic contexts. Even if the mechanisms of neocentromere formation and progression are just beginning to be understood, it is clear that these phenomena had a significant impact on shaping the primate genome and are fundamental to our understanding of genome evolution. In this report we complete and integrate the dataset of BAC-FISH marker order for human syntenies 1, 2, 4, 5, 8, 12, 17, 18, 19, 21, 22 and the X. These results allowed us to develop hypotheses about the content, marker order and centromere position in ancestral karyotypes at five major branching points on the primate evolutionary tree: ancestral primate, ancestral anthropoid, ancestral platyrrhine, ancestral catarrhine and ancestral hominoid. Current models suggest that between-species structural rearrangements are often intimately related to speciation. Comparative primate cytogenetics has become an important tool for elucidating the phylogeny and the taxonomy of primates. It has become increasingly apparent that molecular cytogenetic data in the future can be fruitfully combined with whole-genome assemblies to advance our understanding of primate genome evolution as well as the mechanisms and processes that have led to the origin of the human genome.