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Arsenic (As) is a metalloid present in the earth's crust and widely distributed in the environment. Due to its high concentrations in the Andean valleys and its chemical similarity with phosphorus (P), its biological role in Andean Microbial Ecosystems (AMEs) has begun to be studied. The AMEs are home to extremophilic microbial communities that form microbial mats, evaporites, and microbialites inhabiting Andean lakes, puquios, or salt flats. In this work, we characterize the biological role of As and the effect of phosphate in AMEs from the Laguna Tebenquiche (Atacama Desert, Chile). Using micro X-ray fluorescence, the distribution of As in microbial mat samples was mapped. Taxonomic and inferred functional profiles were obtained from enriched cultures of microbial mats incubated under As stress and different phosphate conditions. Additionally, representative microorganisms highly resistant to As and able to grow under low phosphate concentration were isolated and studied physiologically. Finally, the genomes of the isolated Salicola sp. and Halorubrum sp. were sequenced to analyze genes related to both phosphate metabolism and As resistance. The results revealed As as a key component of the microbial mat ecosystem: (i) As was distributed across all sections of the microbial mat and represented a significant weight percentage of the mat (0.17 %) in comparison with P (0.40%); (ii) Low phosphate concentration drastically changed the microbial community in microbial mat samples incubated under high salinity and high As concentrations; (iii) Archaea and Bacteria isolated from the microbial mat were highly resistant to arsenate (up to 500 mM), even under low phosphate concentration; (iv) The genomes of the two isolates were predicted to contain key genes in As metabolism (aioAB and arsC/acr3) and the genes predicted to encode the phosphate-specific transport operon (pstSCAB-phoU) are next to the arsC gene, suggesting a functional relationship between these two elements.

The ISAV has a genome composed of eight segments of (-)ssRNA, segment 6 codes for the hemagglutinin-esterase protein, and has the most variable region of the genome, the highly polymorphic region (HPR), which is unique among orthomyxoviruses. The HPR has been associated with virulence, infectivity and pathogenicity. The full length of the HPR is called HPR0 and the strain with this HPR is avirulent, in contrast to strains with deleted HPR that are virulent to varying degrees. The molecular mechanism that gives rise to the different HPRs remains unclear. Here, we studied in vitro the evolution of reassortant recombinant ISAV (rISAV) in Atlantic salmon head kidney (ASK) cells. To this end, we rescued and cultivated a set of rISAV with different segment 6-HPR genotypes using a reverse genetics system and then sequencing HPR regions of the viruses. Our results show rapid multiple recombination events in ISAV, with sequence insertions and deletions in the HPR, indicating a dynamic process. Inserted sequences can be found in four segments of the ISAV genome (segments 1, 5, 6, and 8). The results suggest intra-segmental heterologous recombination, probably by class I and class II template switching, similar to the proposed segment 5 recombination mechanism.

Background Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages. Results The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1–4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes ( Conclusion Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen−/−). Notably, LmCen−/− is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen−/− have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen−/− immunization results in protection and an immune response comparable to leishmanization. LmCen−/− is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials., Here, the authors engineer an attenuated knock-out Leishmania (LmCen−/−) vaccine that is safe in immunocompromised mice and induces an immune response and protection similar to leishmanization with wild-type Leishmania. Since LmCen−/− is antibiotic resistant marker free, it is a candidate for clinical development.

Objectives To gain insight into the propagation of miltefosine (MIL) resistance in visceral leishmaniasis, this laboratory study explored development of resistant parasites with a defective miltefosine transporter (MT) in sand flies. Methods Infectivity, colonization of stomodeal valve and metacyclogenesis of a MIL-resistant (MIL-R) Leishmania infantum LEM3323 line with a defective MT were assessed in the natural sand fly vectors Phlebotomus perniciosus and Lutzomyia longipalpis. Given our recent description of partial drug dependency of the MT-deficient line, the impact of MIL pre-exposure on sand fly infectivity was explored as well. Results A significant reduction in sand fly infection, stomodeal valve colonization and differentiation into metacyclics (determined by a lower flagellum/cell body length ratio) was observed in both vectors for MIL-R as compared to the isogenic parent MIL-susceptible line. Re-introduction of the wildtype MT gene into MIL-R tended to partially rescue the capacity to infect sand flies. Pre-exposure to MIL did not alter infectivity of the MIL-R line. Conclusion The MIL resistant L. infantum LEM3323 line is significantly hampered in its development and transmissibility potential in two sand fly vector species. Additional studies are warranted to evaluate whether this applies to other visceral Leishmania parasites with acquired MIL-resistance., Graphical abstract Image 1

The use of plant growth regulators (PGRs) is widespread in commercial table grape vineyards. The synthetic cytokinin CPPU is a PGR that is extensively used to obtain higher quality grapes. However, the effect of CPPU on berry firmness is not clear. The current study investigated the effects of pre-anthesis applications (BBCH15 and BBCH55 stages) of CPPU on ‘Thompson Seedless’ berry firmness at harvest through a combination of cytological, morphological, and biochemical analyses. Ovaries in CPPU-treated plants presented morphological changes related to cell division and cell wall modification at the anthesis stage (BBCH65). Moreover, immunofluorescence analysis with monoclonal antibodies 2F4 and LM15 against pectin and xyloglucan demonstrated that CPPU treatment resulted in cell wall modifications at anthesis. These early changes have major repercussions regarding the hemicellulose and pectin cell wall composition of mature fruits, and are associated with increased calcium content and a higher berry firmness at harvest.

Simple Summary Edible red sea urchin (Loxechinus albus) is an endemic species of echinoderm distributed along the Chilean coasts. This resource has been overexploited in recent years, depleting their natural populations. At present, there are few reported gene sequences available in public databases, restricting the molecular studies associated with aquaculture for this species. The aim of this study was to present the first annotated reference transcriptome of L. albus using NGS technologies and the differential expression transcripts analysis of the evaluated tissues. The transcriptome data obtained in this study will serve as a reference for future molecular research in the edible red sea urchin and other sea urchin species. Abstract Edible red sea urchin (Loxechinus albus) is an endemic echinoderm species of the Chilean coasts. The worldwide demand for high-quality gonads of this species has addressed the depletion of its natural populations. Studies on this sea urchin are limited, and genomic information is almost nonexistent. Hence, generate a transcriptome is crucial information that will considerably enrich molecular data and promote future findings for the L. albus aquaculture. Here, we obtained transcriptomic data of the edible red sea urchin by Illumina platform. Total RNA was extracted from gonads, intestines, and coelomocytes of juvenile urchins, and samples were sequenced using MiSeq Illumina technology. A total of 91,119,300 paired-end reads were de novo assembled, 185,239 transcripts produced, and a reference transcriptome created with 38.8% GC content and an N50 of 1769 bp. Gene ontology analysis revealed notable differences in the expression profiles between gonads, intestines, and coelomocytes, allowing the detection of transcripts associated with specific biological processes and KEGG pathways. These data were validated using 12 candidate transcripts by real-time qPCR. This dataset will provide a valuable molecular resource for L. albus and other species of sea urchins.

Background Dogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown. Methodology/Principal findings We examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with mild to moderate disease showed significantly higher transmission to the vector than dogs with subclinical or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia. Conclusions/Significance Together, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies., Author summary Sand flies transmit Leishmania parasites between infected dogs and humans leading to the life-threatening tropical disease Visceral Leishmaniasis (VL). Identifying which dogs transmit parasites well to sand flies is important to curb disease spread. The offspring of both dogs and humans can also be infected vertically while in utero. Despite this, the infectiousness of dogs that receive parasites in utero to sand flies has not been thoroughly investigated. Thus, we allowed sand flies to feed on a group of vertically infected dogs at varying stages of VL disease severity and measured sand fly parasite uptake. We found vertically infected dogs were readily able to transmit parasites to the sand flies. Dogs that were most infectious had mild to moderate clinical disease and relatively high levels of parasite infection in their blood and skin. However, the level of skin infection was significantly higher than that observed in the blood, and the skin parasite load had the strongest correlation with sand fly parasite uptake. This implicates the skin may be an underappreciated driver of canine infectiousness to the sand fly vector. In addition, this work highlights that vertically infected dogs are very important parts of the transmission cycle and must be considered in all public health efforts addressing VL.

Root hairs (RH) growth is highly influenced by endogenous as well as by external environmental signals that coordinately regulate its final cell size. RHs actively expand the root surface responsible for nutrient uptake and water absorption. We have recently determined that RH growth was unexpectedly boosted when Arabidopsis thaliana seedlings are cultivated at low temperatures. It was proposed that RH growth plasticity in response to cold was linked to a reduced nutrient availability in the media. Here, we explored the molecular basis of this strong RH growth response by using the Genome Wide Association Studies (GWAS) approach on Arabidopsis thaliana natural accessions. We identified the poorly characterized PEROXIDASE 62 (PRX62) as a key protein triggering this conditional growth under a moderate low-temperature stress. In addition, we identified the related protein PRX69 as an important factor in this developmental process. The prx62 prx69 double mutant and the PRX62 and PRX69 over-expressing lines showed contrasting RH phenotypes, peroxidase activities and cyt/apoReactive Oxygen Species (ROS) levels. Strikingly, a cell wall protein extensin (EXT) reporter revealed the effect of peroxidase activity on the EXT cell wall association at 10C in the RH apical zone. EXT cell wall insolubilization was enhanced at 10C, which was completely abolished under the PRX inhibitor salicylhydroxamic acid (SHAM) treatment. Finally, we demonstrated that the Root Hair defective 6 like 4 (RSL4) transcription factor directly controls the expression of PRX69 under low-temperature. Collectively, our results indicate that both PRX62 and PRX69 are key apoplastic PRXs that modulate ROS-homeostasis and cell wall EXT-insolubilization linked to RH elongation at low-temperature. Fil: Martinez Pacheco, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Ranocha, Philippe. Université de Toulouse; Francia Fil: Kasulin, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Fusari, Corina Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Servi, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Ferrero, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Berdion Gabarain, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Peralta, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Borassi, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Marzol, Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Rodriguez Garcia, Diana Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Rondon Guerrero, Yossmayer del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Carignani Sardoy, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Botto, Javier Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina Fil: Meneses, Claudio. Universidad Andrés Bello; Chile Fil: Ariel, Federico Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Petrillo, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Dunand, Christophe. Université de Toulouse; Francia Fil: Estevez, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad Andrés Bello; Chile

Salmonid rickettsial septicemia (SRS) is the major infectious disease of the Chilean salmonid aquaculture industry caused by Piscirickettsia salmonis. Intensive farming conditions generate stress and increased susceptibility to diseases, being skeletal muscle mainly affected. However, the interplay between pathogen infection and stress in muscle is poorly understood. In this study, we perform an RNA-seq analysis on rainbow trout myotubes that are pretreated for 3 h with cortisol (100 ng/mL) and then infected with P. salmonis strain LF-89 for 8 h (MOI 50). Twelve libraries are constructed from RNA samples (n = 3 per group) and sequenced on Illumina HiSeq 4000. A total of 704,979,454 high-quality reads are obtained, with 70.25% mapped against the reference genome. In silico DETs include 175 total genes—124 are upregulated and 51 are downregulated. GO enrichment analysis reveals highly impacted biological processes related to apoptosis, negative regulation of cell proliferation, and innate immune response. These results are validated by RT-qPCR of nine candidate transcripts. Furthermore, cortisol pretreatment significantly stimulated bacterial gene expression of ahpC and 23s compared to infection. In conclusion, for the first time, we describe a transcriptomic response of trout myotubes infected with P. salmonis by inducing apoptosis, downregulating cell proliferation, and intrinsic immune-like response that is differentially regulated by cortisol.

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen−/−) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen−/− parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen−/− immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen−/− infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen−/− immunized hamsters. This study demonstrates that the LmCen−/− parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial., Karmakar et al produced a dermotropic, live attenuated centrin gene-deleted Leishmania major (LmCen−/−) vaccine against Visceral Leishmaniasis (VL). They demonstrated in hamsters that a single intradermal injection confers robust and durable protection against lethal VL that is transmitted naturally via bites of L. donovani-infected sand flies.

Background We established the first prospective cohort to understand how infection with dengue virus is influenced by vector-specific determinants such as humoral immunity to Aedes aegypti salivary proteins. Methods Children aged 2–9 years were enrolled in the PAGODAS (Pediatric Assessment Group of Dengue and Aedes Saliva) cohort with informed consent by their guardians. Children were followed semi-annually for antibodies to dengue and to proteins in Ae. aegypti salivary gland homogenate using enzyme-linked immunosorbent assays and dengue-specific neutralization titers. Children presented with fever at any time for dengue testing. Results From 13 July to 30 August 2018, we enrolled 771 children. At baseline, 22% (173/770) had evidence of neutralizing antibodies to 1 or more dengue serotypes. By April 2020, 51 children had symptomatic dengue while 148 dengue-naive children had inapparent dengue defined by neutralization assays. In a multivariate model, individuals with higher antibodies to Ae. aegypti salivary proteins were 1.5 times more likely to have dengue infection (hazard ratio [HR], 1.47 [95% confidence interval {CI}, 1.05–2.06]; P = .02), particularly individuals with inapparent dengue (HR, 1.64 [95% CI, 1.12–2.41]; P = .01). Conclusions High levels of seropositivity to Ae. aegypti salivary proteins are associated with future development of dengue infection, primarily inapparent, in dengue-naive Cambodian children. Clinical Trials Registration NCT03534245

Peach (Prunus persica) fruits have a fast ripening process and a shelf-life of days, presenting a challenge for long-distance consuming markets. To prolong shelf-life, peach fruits are stored at low temperatures (0 to 7 °C) for at least two weeks, which can lead to the development of mealiness, a physiological disorder that reduces fruit quality and decreases consumer acceptance. Several studies have been made to understand this disorder, however, the molecular mechanisms underlying mealiness are not fully understood. Epigenetic factors, such as DNA methylation, modulate gene expression according to the genetic background and environmental conditions. In this sense, the aim of this work was to identify differentially methylated regions (DMRs) that could affect gene expression in contrasting individuals for mealiness. Peach flesh was studied at harvest time (E1 stage) and after cold storage (E3 stage) for 30 days. The distribution of DNA methylations within the eight chromosomes of P. persica showed higher methylation levels in pericentromeric regions and most differences between mealy and normal fruits were at Chr1, Chr4, and Chr8. Notably, differences in Chr4 co-localized with previous QTLs associated with mealiness. Additionally, the number of DMRs was higher in CHH cytosines of normal and mealy fruits at E3; however, most DMRs were attributed to mealy fruits from E1, increasing at E3. From RNA-Seq data, we observed that differentially expressed genes (DEGs) between normal and mealy fruits were associated with ethylene signaling, cell wall modification, lipid metabolism, oxidative stress and iron homeostasis. When integrating the annotation of DMRs and DEGs, we identified a CYP450 82A and an UDP-ARABINOSE 4 EPIMERASE 1 gene that were downregulated and hypermethylated in mealy fruits, coinciding with the co-localization of a transposable element (TE). Altogether, this study indicates that genetic differences between tolerant and susceptible individuals is predominantly affecting epigenetic regulation over gene expression, which could contribute to a metabolic alteration from earlier stages of development, resulting in mealiness at later stages. Finally, this epigenetic mark should be further studied for the development of new molecular tools in support of breeding programs.

Cortisol is a critical neuroendocrine regulator of the stress response in fish. Cortisol practically affects all tissues by interacting with an intracellular receptor and modulating target gene expression. However, cortisol also interacts with components of the plasma membrane in a nongenomic process that activates rapid signaling. Until now, the implication of this novel cortisol signaling for the global transcriptional response has not been explored. In the present work, we evaluated the effects of the membrane-initiated actions of cortisol on the in vivo transcriptome of rainbow trout ( Oncorhynchus mykiss) skeletal muscle. RNA-Seq analyses were performed to examine the transcriptomic changes in rainbow trout stimulated by physiological concentrations of cortisol and cortisol coupled with bovine serum albumin (cortisol-BSA), a membrane-impermeable analog of cortisol. A total of 660 million paired-ends reads were generated. Reads mapped onto the reference genome revealed that 1,737; 897; and 1,012 transcripts were differentially expressed after 1, 3, and 9 h of cortisol-BSA treatment, respectively. Gene Ontology analysis showed that this novel action of cortisol modulates several biological processes, such as mRNA processing, ubiquitin-dependent protein catabolic processes, and transcription regulation. In addition, a KEGG analysis revealed that focal adhesion was the main signaling pathway that was upregulated at all the times tested. Taking these results together, we propose that the membrane-initiated cortisol action contributes significantly in the regulation of stress-mediated gene expression.

Upon imbibition, epidermal cells of Arabidopsis thaliana seeds release a mucilage formed mostly by pectic polysaccharides. The Arabidopsis mucilage is composed mainly of unbranched rhamnogalacturonan-I (RG-I), with low amounts of cellulose, homogalacturonan, and traces of xylan, xyloglucan, galactoglucomannan, and galactan. The pectin-rich composition of the mucilage and their simple extractability makes this structure a good candidate to study the biosynthesis of pectic polysaccharides and their modification. Here, we characterize the mucilage phenotype of a mutant in the UDP-rhamnose/galactose transporter 2 (URGT2), which exhibits a reduction in RG-I and also shows pleiotropic changes, suggesting the existence of compensation mechanisms triggered by the lack of URGT2. To gain an insight into the possible compensation mechanisms activated in the mutant, we performed a transcriptome analysis of developing seeds using RNA sequencing (RNA-seq). The results showed a significant misregulation of 3149 genes, 37 of them (out of the 75 genes described to date) encoding genes proposed to be involved in mucilage biosynthesis and/or its modification. The changes observed in urgt2 included the up-regulation of UAFT2, a UDP-arabinofuranose transporter, and UUAT3, a paralog of the UDP-uronic acid transporter UUAT1, suggesting that they play a role in mucilage biosynthesis. Mutants in both genes showed changes in mucilage composition and structure, confirming their participation in mucilage biosynthesis. Our results suggest that plants lacking a UDP-rhamnose/galactose transporter undergo important changes in gene expression, probably to compensate modifications in the plant cell wall due to the lack of a gene involved in its biosynthesis., The transcriptome of urgt2 plants is used to identify compensation mechanisms in response to mutation in the UDP-rhamnose/galactose transporter to discover new genes involved in mucilage biosynthesis and/or modifications.

Antarctic vascular plants such as Deschampsia antarctica (Da) could generate more suitable micro-environmental conditions for the establishment of other plants like Colobanthus quitensis (Cq). Although positive plant–plant interactions have been shown to contribute to plant performance and establishment, little is known about how microorganisms might modulate those interactions, particularly in stressful environmental conditions. Several reports have focused on the possible ecological roles of microorganisms on vascular plants, but if rhizospheric microorganisms can impact positive interactions among Antarctic plants has been seldom studied. Here, we assessed the physical–chemical characteristics of rhizospheric soils from Cq growing alone or associated with Da (Cq + Da). In addition, we compared the rhizosphere microbiomes associated with Cq, either growing alone or associated with Da (Cq + Da), using a shotgun metagenomic DNA sequencing approach and using eggNOG for comparative and functional metagenomics. Overall, there were no differences among rhizospheric soils in terms of physical–chemical characteristics. On the other hand, our results show significant differences in terms of taxonomic diversity between rhizospheric soils. Functional annotation and pathway analysis showed that microorganisms from rhizospheric soil samples also have significant differences in gene abundance associated with several functional categories related to environmental tolerance and in metabolic pathways linked to osmotic stress, among others. Overall, this study provides foundational information which will allow to explore the biological impact of the rhizobiome and its functional mechanisms and molecular pathways on plant performance and help explain the concerted strategy deployed by Cq to inhabit and cope with the harsh conditions prevailing in Antarctica.

Ripening heterogeneity of Hass avocados results in inconsistent quality fruit delivered to the triggered and ready to eat markets. This research aimed to understand the effect of a heat shock (HS) prior to controlled atmosphere (CA) storage on the reduction of ripening heterogeneity. HS prior to CA storage reduces more drastically the ripening heterogeneity in middle season fruit. Via correlation network analysis we show the different metabolomics networks between HS and CA. High throughput proteomics revealed 135 differentially expressed proteins unique to middle season fruit triggered by HS. Further integration of metabolomics and proteomics data revealed that HS reduced the glycolytic throughput and induced protein degradation to deliver energy for the alternative ripening pathways. l-isoleucine, l-valine, l-aspartic and ubiquitin carboxyl-terminal hydrolase involved in protein degradation were positively correlated to HS samples. Our study provides new insights into the effectiveness of HS in synchronizing ripening of Hass avocados.

Epigenetic modifications can provide information about the connection between genotype and phenotype and which can be directly affected by environmental conditions. In temperate perennial fruit species like sweet cherry, prolonged exposition to cold temperatures is required for dormancy release and flowering in spring. Besides genetic diversity, epigenetic regulation is believed to contribute to different chilling requirements and phenotypic plasticity among cultivars. We set out to profile methylation patterns of floral buds from sweet cherry cultivars contrasting in chilling requirement. We performed a sequence-based genome-wide differential methylation analysis using whole genome sequencing of bisulfite-treated DNA of a high chill cultivar (‘Kordia’: 1637 CH for budbreak) at four time points of cold accumulation during dormancy. As a result, we obtained global methylation levels and methylation-demethylation patterns at 0, 443, 1295 and 1637 CH. A total of 50% of the reads mapped uniquely to the reference genome of ‘Kordia’ (untreated genome). Approximately a 25-30% of total cytosines were methylated in all the conditions, corresponding to a 10-12, 9-10 and 7-8% of specific methylations at the CpG, CHG and CHH sites, respectively. Annotation of differentially methylated regions (DMRs) is under development, focusing on the distinctive patterns of transposon and gene body methylation, in an attempt to describe epigenetic marks that may be contributing to contrasting chilling requirement.

Here we present the construction of a partial draft genome of the sweet cherry Prunus avium 'Karina' (2n=2x=16) taking advantage of the NGS technologies available and by implementing a de novo assembly strategy. The estimated assembly size was 352.5 Mb, comprising 904,813 contigs with a N50 of 9,188 bp. The contig merging process resulted in 898,488 scaffolds with a N50 of 13,235 bp and a maximum scaffold length of 419,819 bp. We performed a search for known genes with agronomical importance such as the dormancy-associated MADS-box transcription factors (DAM) and two members of the eIF4G gene subfamily. The search results showed that six putative DAM genes are present in an apparent tandem array with a mean identity of 89.14% with the genomic sequences of Prunus persica. Similar results were achieved for the eIFG genes, obtaining 89.76% of identity with the genomic sequences of Prunus persica and 60.36% of identity with the genomic sequences of Arabidopsis thaliana. With this we are hoping to obtain valuable data to perform genomic studies and to contribute to the better understanding of the genetic background of this cultivar and to obtain a tool to perform genomic studies in sweet cherry.

Firm berries are highly appreciated by table grape consumers. Cell wall composition is one of the main factors influencing the firmness of table grape berries. Nevertheless, the biological factors driving changes in berry firmness remain unclear. In the present work, we evaluated the firmness of berries of Vitis vinifera cv. Thompson Seedless. We selected two orchards displaying contrasting berry firmness and evaluated polar metabolites and cell wall composition. Our results suggest that berries from the soft orchard exhibited a higher accumulation of sugars at veraison whereas berries from the hard orchard accumulated the same sugars at harvest plus a higher amount of glucose monosaccharide at the cell wall. Thus, this study opens new insights about a connection between metabolic and cell wall changes with fruit firmness in a table grape variety, suggesting that it is possible to use metabolomic tools to identify metabolic biomarkers associated with table grape berry firmness.

Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P, Immune mimicry has been shown in chemokine like moieties from bacteria and viruses. Here, the authors characterise a sand fly salivary protein that induces neutrophil chemotaxis and explore its impact in a model of parasitic infection.

Kiwifruit (Actinidia chinensis var. deliciosa (A Chev) Liang et Ferguson) is a sub-tropical vine species from the Actinidiaceae family native to China. This species has an allohexaploid genome (from diploid and autotetraploid parents), contained in 174 chromosomes producing a climacteric and fleshy fruit called kiwifruit. Currently, only a small body of transcriptomic and proteomic data are available for A. chinensis var. deliciosa. In this low molecular knowledge context, the main goal of this study is to construct a tissue-specific de novo transcriptome assembly, generating differential expression analysis among these specific tissues, to obtain new useful transcriptomic information for a better knowledge of vegetative, floral and fruit growth in this species. In this study, we have analyzed different whole transcriptomes from shoot, leaf, flower bud, flower and fruit at four development stages (7, 50, 120 and 160 days after flowering, DAF) in kiwifruit obtained through RNA-seq sequencing. The first version of the developed A. chinensis var. deliciosa&nbsp, de novo transcriptome contained 142,025 contigs (x¯ = 1044 bp, N50 = 1133 bp). Annotation was performed with BLASTX against the TAIR10 protein database, and we found an annotation proportion of 35.6% (50,508), leaving 64.4% (91,517) of the contigs without annotation. These results represent a reference transcriptome for allohexaploid kiwifruit generating a database of A. chinensis var. deliciosa genes related to leaf, flower and fruit development. These results provided highly valuable information identifying over 20,000 exclusive genes including all tissue comparisons, which were associated with the proteins involved in different biological processes and molecular functions.

The metallophyte Imperata cylindrica inhabits copper (Cu) polluted soils in large areas from Central Chile. Here, we subjected clonal vegetative plantlets to 300 mg Cu kg−1 of substrate for 21 days to identify the main molecular pathways involved in the response to Cu stress. Transcriptomic analyses were performed for shoots and roots, with and without Cu supply. RNA-Seq and de novo transcriptome assembly were performed to identify the gene response associated with molecular mechanisms of Cu tolerance in I. cylindrica. De novo transcriptome revealed a total of 200,521 transcripts (1777 bp) comprising ~91% complete ultra-conserved genes in the eukaryote and Plantae database. The differentially expressed genes (DEGs) in roots were 7386, with 3558 of them being up-regulated and the other 3828 down-regulated. The transcriptome response in shoots was significantly less, showing only 13 up-regulated and 23 down-regulated genes. Interestingly, DEGs mainly related with actin and cytoskeleton formation, and to a minor degree, some DEGs associated with metal transporters and superoxide dismutase activity in root tissues were found. These transcriptomic results suggest that cytoskeleton could be acting as a mechanism of Cu-binding in the root, resulting in a high Cu tolerance response in this metallophyte, which deserve to be analyzed ultra-structurally. Our study contributes to reinforcing the potential of I. cylindrica as a candidate plant species to be used as a phytoremediation agent in Cu-contaminated environments.

Kiwifruit are climacteric fruit, so they must be harvested before they are fully ripe, allowing for the extension of their shelf-life via cold storage. Therefore, an adequate knowledge about how ethylene-induced fruit senescence is required to avoid significant economic losses. The main goal of the present study was to investigate the kiwifruit ripening process at the physiological and molecular levels by RNA-seq after 1-methylcyclopropene (1-MCP, ethylene inhibitor) and Ethrel® (ethylene stimulator) treatments. The results showed that Ethrel® (ethephon) treatment induced more accelerated fruit ripening, leading to rapid fruit senescence, meanwhile 1-MCP caused a slowing flesh softening, and thus a longer shelf-life period. The RNA-seq was carried out on the fruit after 4 and 13 days, considering day 4 as the most determinant in terms of differentially expressed genes (DEGs). The sequencing achieved 70.7% alignment with the `Hongyang¿ genome, obtaining 18,036 DEGs. The protein-protein interaction (PPI) network shows the interaction between different pathways in two main clusters: (1) pentose and glucoronate interconversions, citrate cycle, glycolysis and gluconeogenesis or starch, and sucrose metabolism and (2) porphyrin and chlorophyll metabolism. The first cluster is mainly interconnected by G6PD1 (pentose pathway); E1 ALPHA and ACLB-2 (citrate cycle); Achn209711 (pentose and glucoronate); LOS2 (glycolysis); HKL1 and HXK1 (glycolysis¿starch and sucrose); and PHS2 (starch and sucrose). In the second cluster, GUN5 through PORA is interacting with CRD1 and NYC1 which were overexpressed by 1-MCP in the porphyrin and chlorophyll metabolism. In addition, genes linked to PSBY and PSBP photosynthesis-linked proteins in photosystem 2 were overexpressed by 1-MCP which is undoubtedly related to chlorophyll degradation and fruit senescence. These results suggest that in kiwifruit, the main pathways that are regulated by ethylene-induced senescence comprise sugar catabolism and chlorophyll degradation., The authors would like to thank the National Commission for Scientific and Technological Research (CONICYT) of Chile government through the project FONDEF N° D09i-1136. Additionally, we thank the Seneca Foundation in the region of Murcia (Spain) through the Regional subprogram “Saavedra Fajardo” (20397/ SF/17) and Ministry of Science and Innovation through the project “Juan de la Cierva Incorporación” N° IJC2018-0366-I

Visceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen−/−). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen−/− against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen−/− did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen−/−-immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen−/− immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-γ and TNF-α, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen−/− which showed equal protection as laboratory-grade LmCen−/− parasites in hamsters. Importantly, GLP-grade LmCen−/− parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen−/− parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to Leishmania parasites. However, the permissiveness of other sand flies with respect to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector, Lutzomyia longipalpis. We found that LPG-mediated midgut binding persists in late-stage parasites. This observation is of relevance for the understanding of vector-parasite molecular interactions and suggests that only a subset of infective metacyclic-stage parasites (metacyclics) lose their ability to attach to the midgut, with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in Leishmania infantum retention in the midgut of Lutzomyia longipalpis, a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be a complex issue and should be investigated on a strain-specific basis., The major surface lipophosphoglycan (LPG) of Leishmania parasites is critical to vector competence in restrictive sand fly vectors in mediating Leishmania attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of Leishmania remains elusive. We tested binding of Leishmania infantum wild-type (WT), LPG-defective (Δlpg1 mutants), and add-back (Δlpg1 + LPG1) lines to sand fly midguts in vitro and their survival in Lutzomyia longipalpis sand flies in vivo. Le. infantum WT parasites attached to the Lu. longipalpis midgut in vitro, with late-stage parasites binding to midguts in significantly higher numbers than were seen with early-stage promastigotes. Δlpg1 mutants did not bind to Lu. longipalpis midguts, and this was rescued in the Δlpg1 + LPG1 lines, indicating that midgut binding is mediated by LPG. When Lu. longipalpis sand flies were infected with the Le. infantum WT or Le. infantum Δlpg1 or Le. infantum Δlpg1 + LPG1 line of the BH46 or BA262 strains, the BH46 Δlpg1 mutant, but not the BA262 Δlpg1 mutant, survived and grew to numbers similar to those seen with the WT and Δlpg1 + LPG1 lines. Exposure of BH46 and BA262 Δlpg1 mutants to blood-engorged midgut extracts led to mortality of the BA262 Δlpg1 but not the BH46 Δlpg1 parasites. These findings suggest that Le. infantum LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, but that it is not necessary to prevent Le. infantum evacuation along with the feces in the permissive vector Lu. longipalpis. IMPORTANCE It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to Leishmania parasites. However, the permissiveness of other sand flies with respect to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector, Lutzomyia longipalpis. We found that LPG-mediated midgut binding persists in late-stage parasites. This observation is of relevance for the understanding of vector-parasite molecular interactions and suggests that only a subset of infective metacyclic-stage parasites (metacyclics) lose their ability to attach to the midgut, with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in Leishmania infantum retention in the midgut of Lutzomyia longipalpis, a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be a complex issue and should be investigated on a strain-specific basis.

Background Berry size is considered as one of the main selection criteria in table grapes breeding programs, due to the consumer preferences. However, berry size is a complex quantitive trait under polygenic control, and its genetic determination of berry weight is not yet fully understood. The aim of this work was to perform marker discovery using a transcriptomic approach, in order to identify and characterize SNP and InDel markers associated with berry size in table grapes. We used an integrative analysis based on RNA-Seq, SNP/InDel search and validation on table grape segregants and varieties with different genetic backgrounds. Results Thirty SNPs and eight InDels were identified using a transcriptomic approach (RNA-Seq). These markers were selected from SNP/InDel found among segregants from a Ruby x Sultanina population with contrasting phenotypes for berry size. The set of 38 SNP and InDel markers was distributed in eight chromosomes. Genotype-phenotype association analyses were performed using a set of 13 RxS segregants and 41 table grapes varieties with different genetic backgrounds during three seasons. The results showed several degrees of association of these markers with berry size (10.2 to 30.7%) as other berry-related traits such as length and width. The co-localization of SNP and /or InDel markers and previously reported QTLs and candidate genes associated with berry size were analysed. Conclusions We identified a set of informative and transferable SNP and InDel markers associated with berry size. Our results suggest the suitability of SNPs and InDels as candidate markers for berry weight in seedless table grape breeding. The identification of genomic regions associated with berry weight in chromosomes 8, 15 and 17 was achieved with supporting evidence derived from a transcriptome experiment focused on SNP/InDel search, as well as from a QTL-linkage mapping approach. New regions possibly associated with berry weight in chromosomes 3, 6, 9 and 14 were identified.

Here, we report the results from PCR and sequencing of bacterial 16S rRNA and fungal internal transcribed spacer 1 (ITS1) genes from needle, branch, trunk, and root samples of Araucaria araucana, plus soil and associated insects, collected along the entirety of its geographic distribution in Chile (January 2017 and 2018).

Indexación: Scopus. Temperate deciduous fruit tree species like sweet cherry (Prunus avium) require long periods of low temperatures to trigger dormancy release and flowering. In addition to sequence-based genetic diversity, epigenetic variation may contribute to different chilling requirements among varieties. For the low chill variety ‘Royal Dawn’ and high chill variety ‘Kordia’, we studied the methylome of floral buds during chilling accumulation using MethylC-seq to identify differentially methylated regions (DMRs) during chilling hours (CH) accumulation, followed by transcriptome analysis to correlate changes in gene expression with DNA methylation. We found that during chilling accumulation, DNA methylation increased from 173 CH in ‘Royal Dawn’ and 443 CH in ‘Kordia’ and was mostly associated with the CHH context. In addition, transcriptional changes were observed from 443 CH in ‘Kordia’ with 1,210 differentially expressed genes, increasing to 4,292 genes at 1,295 CH. While ‘Royal Dawn’ showed approximately 5,000 genes differentially expressed at 348 CH and 516 CH, showing a reprogramming that was specific for each genotype. From conserved upregulated genes that overlapped with hypomethylated regions and downregulated genes that overlapped with hypermethylated regions in both varieties, we identified genes related to cold-sensing, cold-signaling, oxidation-reduction process, metabolism of phenylpropanoids and lipids, and a MADS-box SVP-like gene. As a complementary analysis, we used conserved and non-conserved DEGs that presented a negative correlation between DNA methylations and mRNA levels across all chilling conditions, obtaining Gene Ontology (GO) categories related to abiotic stress, metabolism, and oxidative stress. Altogether, this data indicates that changes in DNA methylation precedes transcript changes and may occur as an early response to low temperatures to increase the cold tolerance in the endodormancy period, contributing with the first methylome information about the effect of environmental cues over two different genotypes of sweet cherry. © Copyright © 2020 Rothkegel, Sandoval, Soto, Ulloa, Riveros, Lillo-Carmona, Cáceres-Molina, Almeida and Meneses. https://www.frontiersin.org/articles/10.3389/fpls.2020.01115/full

The major surface lipophosphoglycan (LPG) of Leishmania parasites is critical to vector competence in restrictive sand fly vectors by mediating Leishmania attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of Leishmania remains elusive. We tested binding of Leishmania infantum wild type (WT), LPG-defective (Δlpg1 mutants) and add-back lines (Δlpg1 + LPG1) to sand fly midguts in vitro and their survival in Lutzomyia longipalpis sand flies in vivo. Le. infantum WT parasites attached to the Lu. longipalpis midgut in vitro with late-stage parasites binding to midguts in significantly higher numbers compared to early-stage stage promastigotes. Δlpg1 mutants did not bind to Lu. longipalpis midguts, and this was rescued in the Δlpg1 + LPG1 lines, indicating that midgut binding is mediated by LPG. When Lu. longipalpis sand flies were infected with either Le. infantum WT, Δlpg1, or Δlpg1 + LPG1 of the BH46 or BA262 strains, the BH46 Δlpg1 mutant, but not the BA262 Δlpg1 mutant, survived and grew to similar numbers as the WT and Δlpg1 + LPG1 lines. Exposure of BH46 and BA262 Δlpg1 mutants to blood engorged midgut extracts led to the mortality of the BA262 Δlpg1 but not the BH46 Δlpg1 parasites. These findings suggest that Le. infantum LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, however, it is not necessary to prevent Le. infantum evacuation along with the feces in the permissive vector Lu. longipalpis.IMPORTANCEIt is well established that LPG is sufficient to define the vector competence of restrictive sand fly vectors to Leishmania parasites. However, the permissiveness of other sand flies to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector Lutzomyia longipalpis. We found out that LPG-mediated midgut binding persists in late-stage parasites. This observation is paradigm-changing and suggests that only a subset of infective metacyclics lose their ability to attach to the midgut with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in Leishmania infantum retention in the midgut of Lutzomyia longipalpis, a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be complex and should be investigated on a strain-specific basis.

Indexación: Scopus. Clostridium difficile B1/NAP1/RT027/ST01 has been responsible for outbreaks of antibiotic-associated diarrhoea in clinical settings worldwide and is associated with severe disease presentations and increased mortality rates. Two fluoroquinolone-resistant (FQR) lineages of the epidemic B1/NAP1/RT027/ST01 strain emerged in the USA in the early 1990s and disseminated trans continentally (FQR1 and FQR2). However, it is unclear when and from where they entered Latin America (LA) and whether isolates from LA exhibit unique genomic features when compared to B1/NAP1/RT027/ST01 isolates from other regions of the world. To answer the first issue we compared whole-genome sequences (WGS) of 25 clinical isolates typed as NAP1, RT027 or ST01 in Costa Rica (n=16), Chile (n=5), Honduras (n=3) and Mexico (n=1) to WGS of 129 global isolates from the same genotype using Bayesian phylogenomics. The second question was addressed through a detailed analysis of the number and type of mutations of the LA isolates and their mobile resistome. All but two B1/NAP1/RT027/ST01 isolates from LA belong to the FQR2 lineage (n=23, 92 %), confirming its widespread distribution. As indicated by analysis of a dataset composed of 154 WGS, the B1/NAP1/RT027/ST01 strain was introduced into the four LA countries analysed between 1998 and 2005 from North America (twice) and Europe (at least four times). These events occurred soon after the emergence of the FQR lineages and more than one decade before the first report of the detection of the B1/NAP1/RT027/ST01 in LA. A total of 552 SNPs were identified across all genomes examined (3.8–4.3 Mb) in pairwise comparisons to the R20291 reference genome. Moreover, pairwise SNP distances were among the smallest distances determined in this species so far (0 to 55). Despite this high level of genomic conservation, 39 unique SNPs (7 %) in genes that play roles in the infection process (i.e. slpA) or antibiotic resistance (i.e. rpoB, fusA) distinguished the LA isolates. In addition, isolates from Chile, Honduras and Mexico had twice as many antibiotic resistance genes (ARGs, n=4) than related isolates from other regions. Their unique set of ARGs includes a cfr-like gene and tetM, which were found as part of putative mobile genetic elements whose sequences resemble undescribed integrative and conjugative elements. These results show multiple, independent introductions of B1/NAP1/RT027/ ST01 isolates from the FQR1 and FQR2 lineages from different geographical sources into LA and a rather rapid accumulation of distinct mutations and acquired ARG by the LA isolates. https://www.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.000355

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal, resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163+CD91+ professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppresses skin inflammation. This indicates that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens.

The peach is the third most important temperate fruit crop considering fruit production and harvested area in the world. Exporting peaches represents a challenge due to the long-distance nature of export markets. This requires fruit to be placed in cold storage for a long time, which can induce a physiological disorder known as chilling injury (CI). The main symptom of CI is mealiness, which is perceived as non-juicy fruit by consumers. The purpose of this work was to identify and compare the metabolite and lipid profiles between two siblings from contrasting populations for juice content, at harvest and after 30 days at 0 °C. A total of 119 metabolites and 189 lipids were identified, which showed significant differences in abundance, mainly in amino acids, sugars and lipids. Metabolites displaying significant changes from the E1 to E3 stages corresponded to lipids such as phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG) and lysophosphatidylcholines (LPC), and sugars such as fructose 1 and 1-fructose-6 phosphate. These metabolites might be used as early stage biomarkers associated with mealiness at harvest and after cold storage.

Peach is the third most important temperate fruit crop considering fruit production and harvested area in the world. Exporting peaches represents a challenge due to the long-distance export markets. This requires fruit to be placed in cold storage for a long time, which can induce a physiological disorder known as chilling injury (CI). The main symptom of CI is mealiness which is perceived as non-juicy fruit by consumers. The purpose of this work was to identify and compare the metabolic and lipid profile between two siblings from a contrasting population for juice content, at harvest and after 30 days at 0°C. A total of 119 metabolites and 189 lipids were identified, which showed significant differences of abundance including mainly in amino acids, sugars and lipids. Our results indicate that some of the top metabolites and lipids could be used as biomarkers associated with mealiness at harvest and after cold storage.

Background Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking. Methodology/Principal findings We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum. Conclusions Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies., Author summary One of the major gaps in Leishmania research is the lack of stage-specific markers to differentiate the Leishmania promastigote stages in the sand fly vector as well as in culture. In this study, we report the transcriptional boundaries amongst Leishmania infantum promastigote stages developing in the sand fly vector Lutzomyia longipalpis. Despite the policystronic nature of Leishmania mRNAs, we were able to detect 10- to 100 fold differences in transcriptional abundance between genes throught RNA sequencing. Significant number of genes were differentially expressed between subsequent stages, showing enrichment of stage specific up-regulated genes even across chromosomes. In light of such differences, we identified potential stage-specific markers for each promastigote stage of Leishmania with potential use in epidemiological studies as well as in further characterization the developmental differences amongst the promastigote stages in the vector.

Background Gibberellins (GA3) are the most sprayed growth regulator for table grape production worldwide, increasing berry size of seedless varieties through pericarp cell expansion. However, these treatments also exacerbate berry drop, which has a detrimental effect on the postharvest quality of commercialized clusters. Several studies have suggested that pedicel stiffening caused by GA3 would have a role in this disorder. Nevertheless, transcriptional and phenotypic information regarding pedicel responses to GA3 is minimal. Results Characterization of responses to GA3 treatments using the lines L23 and Thompson Seedless showed that the former was up to six times more susceptible to berry drop than the latter. GA3 also increased the diameter and dry matter percentage of the pedicel on both genotypes. Induction of lignin biosynthesis-related genes by GA3 has been reported, so the quantity of this polymer was measured. The acetyl bromide method detected a decreased concentration of lignin 7 days after GA3 treatment, due to a higher cell wall yield of the isolated fractions of GA3-treated pedicel samples which caused a dilution effect. Thus, an initial enrichment of primary cell wall components in response to GA3 was suggested, particularly in the L23 background. A transcriptomic profiling was performed to identify which genes were associated with these phenotypic changes. This analysis identified 1281 and 1787 genes differentially upregulated by GA3 in L23 and cv. Thompson Seedless, respectively. Concomitantly, 1202 and 1317 downregulated genes were detected in L23 and cv. Thompson Seedless (FDR 3 application. Twelve genes were characterized by qPCR and striking differences were observed between genotypes, mainly in genes related to cell wall synthesis. Conclusions High levels of berry drop are related to an early strong response of primary cell wall synthesis in the pedicel promoted by GA3 treatment. Genetic backgrounds can produce similar phenotypic responses to GA3, although there is considerable variation in the regulation of genes in terms of which are expressed, and the extent of transcript levels achieved within the same time frame.

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163+CD91+ professional iron recycling subpopulation, produces HO-1 after insect bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number; whereas CO, an end-product of the HO-1 enzymatic reaction suppresses skin inflammation. This indicates that HO-1 induction by blood feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods thus promoting early-stage tolerance to vector-borne pathogens.

Leishmaniasis is a spectrum of diseases transmitted by sand fly vectors that deposit Leishmania spp. parasites in the host skin during blood feeding. Currently, available treatment options are limited, associated with high toxicity and emerging resistance. Even though a vaccine for human leishmaniasis is considered an achievable goal, to date we still do not have one available, a consequence (amongst other factors) of a lack of pre-clinical to clinical translatability. Pre-exposure to uninfected sand fly bites or immunization with defined sand fly salivary proteins was shown to negatively impact infection. Still, cross-protection reports are rare and dependent on the phylogenetic proximity of the sand fly species, meaning that the applicability of a sand fly saliva-based vaccine will be limited to a defined geography, one parasite species and one form of leishmaniasis. As a proof of principle of a future vector saliva-based pan-Leishmania vaccine, we engineered through a reverse vaccinology approach that maximizes translation to humans, a fusion protein consisting of immunogenic portions of PdSP15 and LJL143, sand fly salivary proteins demonstrated as potential vaccine candidates against cutaneous and visceral leishmaniasis, respectively. The in silico analysis was validated ex vivo, through T cell proliferation experiments, proving that the fusion protein (administered as a DNA vaccine) maintained the immunogenicity of both PdSP15 and LJL143. Additionally, while no significant effect was detected in the context of L. major transmission by P. duboscqi, this DNA vaccine was defined as partially protective, in the context of L. major transmission by L. longipalpis sand flies. Importantly, a high IFN¿ response alone was not enough to confer protection, that mainly correlated with low T cell mediated Leishmania-specific IL-4 and IL-10 responses, and consequently with high pro/anti-inflammatory cytokine ratios. Overall our immunogenicity data suggests that to design a potentially safe vector-based pan-Leishmania vaccine, without geographic restrictions and against all forms of leishmaniasis is an achievable goal. This is why we propose our approach as a proof-of principle, perhaps not only applicable to the anti-Leishmania vector-based vaccines’ field, but also to other branches of knowledge that require the design of multi-epitope T cell vaccines with a higher potential for translation. The research leading to these results has received funding from the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This research was also supported in part by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. PC was supported by Foundation for Science and Technology (FCT), Portugal, through the individual Grant SFRH/BD/121252/2016. PC was also supported by Fulbright Portugal. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Chilling injury represents a major constrain for crops productivity. Prunus persica, one of the most relevant rosacea crops, have early season varieties that are resistant to chilling injury, in contrast to late season varieties, which display chilling symptoms such as mealiness (dry, sandy fruit mesocarp) after prolonged storage at chilling temperatures. To uncover the molecular processes related to the ability of early varieties to withstand mealiness, postharvest and genome-wide RNA-seq assessments were performed in two early and two late varieties. Differences in juice content and ethylene biosynthesis were detected among early and late season fruits that became mealy after exposed to prolonged chilling. Principal component and data distribution analysis revealed that cold-stored late variety fruit displayed an exacerbated and unique transcriptome profile when compared to any other postharvest condition. A differential expression analysis performed using an empirical Bayes mixture modeling approach followed by co-expression and functional enrichment analysis uncover processes related to ethylene, lipids, cell wall, carotenoids and DNA metabolism, light response, and plastid homeostasis associated to the susceptibility or resistance of P. persica varieties to chilling stress. Several of the genes related to these processes are in quantitative trait loci (QTL) associated to mealiness in P. persica. Together, these analyses exemplify how P. persica can be used as a model for studying chilling stress in plants.

The potato is susceptible to water stress at all stages of development. We examined four clones of tetraploid potato, Cardinal, Desir&eacute, e, Clone 37 FB, and Mije, from the germplasm bank of the National Institute of Agricultural Research (INIA) in Chile. Water stress was applied by suspending irrigation at the beginning of tuberization. Stomatal conductance, and tuber and plant fresh and dry weight were used to categorize water stress tolerance. Cardinal had a high susceptibility to water stress. Desir&eacute, e was less susceptible than Cardinal and had some characteristics of tolerance. Mije had moderate tolerance and Clone 37 FB had high tolerance. Differential gene expression in leaves from plants with and without water stress were examined using transcriptome sequencing. Water stress-susceptible Cardinal had the fewest differentially expressed genes at 101, compared to Desir&eacute, e at 1867, Clone 37 FB at 1179, and Mije at 1010. Water stress tolerance was associated with upregulation of the expression of transcription factor genes and genes involved in osmolyte and polyamine biosynthesis. Increased expression of genes encoding late embryogenesis abundant (LEA) and dehydrin proteins along with decreased expression of genes involved in nitrate assimilation and amino acid metabolism were found for clones showing water stress tolerance. The results also show that a water deficit was associated with reduced biotic stress responses. Additionally, heat shock protein genes were differentially expressed in all clones except for highly susceptible Cardinal. Together, the gene expression study demonstrates variation in the molecular pathways and biological processes in response to water stress contributing to tolerance and susceptibility.

Indexación: Scopus Salmonella Typhimurium, a bacterial pathogen with high metabolic plasticity, can adapt to different environmental conditions; these traits enhance its virulence by enabling bacterial survival. Neutrophils play important roles in the innate immune response, including the production of microbicidal reactive oxygen species (ROS). In addition, the myeloperoxidase in neutrophils catalyzes the formation of hypochlorous acid (HOCl), a highly toxic molecule that reacts with essential biomolecules, causing oxidative damage including lipid peroxidation and protein carbonylation. The bacterial response regulator ArcA regulates adaptive responses to oxygen levels and influences the survival of Salmonella inside phagocytic cells. Here, we demonstrate by whole transcriptomic analyses that ArcA regulates genes related to various metabolic pathways, enabling bacterial survival during HOCl-stress in vitro. Also, inside neutrophils, ArcA controls the transcription of several metabolic pathways by downregulating the expression of genes related to fatty acid degradation, lysine degradation, and arginine, proline, pyruvate, and propanoate metabolism. ArcA also upregulates genes encoding components of the oxidative pathway. These results underscore the importance of ArcA in ATP generation inside the neutrophil phagosome and its participation in bacterial metabolic adaptations during HOCl stress. © Copyright © 2019 Pardo-Esté, Castro-Severyn, Krüger, Cabezas, Briones, Aguirre, Morales, Baquedano, Sulbaran, Hidalgo, Meneses, Poblete-Castro, Castro-Nallar, Valvano and Saavedra. https://www.frontiersin.org/articles/10.3389/fmicb.2019.02754/full

Salmonella Typhimurium is an intracellular pathogen that is capable of generating systemic fever in a murine model. Over the course of the infection, Salmonella faces different kinds of stressors, including harmful reactive oxygen species (ROS). Various defence mechanisms enable Salmonella to successfully complete the infective process in the presence of such stressors. The transcriptional factor SlyA is involved in the oxidative stress response and invasion of murine macrophages. We evaluated the role of SlyA in response to H2O2 and NaOCl and found an increase of slyA expression upon exposure to these toxics. However, the SlyA target genes and the molecular mechanisms by which they influence the infective process are unknown. We hypothesised that SlyA regulates the expression of genes required for ROS resistance, metabolism, or virulence under oxidative stress conditions. Transcriptional profiling in wild type and ΔslyA strains confirmed that SlyA regulates the expression of several genes involved in virulence [sopD (STM14_3550), sopE2 (STM14_2244), hilA (STM14_3475)] and central metabolism [kgtP (STM14_3252), fruK (STM14_2722), glpA (STM14_2819)] in response to H2O2 and NaOCl. These findings were corroborated by functional assay and transcriptional fusion assays using GFP. DNA-protein interaction assays showed that SlyA regulates these genes through direct interaction with their promoter regions.

Long noncoding RNAs (lncRNAs) are a heterogeneous class of transcripts, longer than 200 nucleotides, 5'-capped, polyadenylated, and poorly conserved among mammalian species. Several studies have shown the contribution of lncRNAs to different cellular processes, including regulation of the chromatin structure, control of messenger RNA translation, regulation of gene transcription, regulation of embryonic pluripotency, and differentiation. Although limited numbers of functional lncRNAs have been identified so far, the immense regulatory potential of these RNAs is already evident, indicating that a functional characterization of lncRNAs is needed. In this study, mouse preosteoblastic cells were induced to differentiate into osteoblasts. At 3 sequential differentiation stages, total RNA was isolated and libraries were constructed for Illumina sequencing. The resulting sequences were aligned and transcript abundances were determined. New lncRNA candidates that displayed differential expression patterns during osteoblast differentiation were identified by combining bioinformatics and reverse transcription polymerase chain reaction analyses. Among these, lncRNA-1 that exhibited increased expression during osteogenesis and was downregulated during myogenesis. Importantly, knockdown of lncRNA-1 expression in primary mouse preosteoblasts was found to inhibit osteogenic differentiation, reflected by a reduced transcription of the Runx2/p57 and Sp7 bone master genes. Together, our results indicate that lncRNA-1 represents a new regulatory RNA that plays a relevant role during the early stages of osteogenesis.

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.

Sand flies, similar to most vectors, take multiple blood meals during their lifetime1-4. The effect of subsequent blood meals on pathogens developing in the vector and their impact on disease transmission have never been examined. Here, we show that ingestion of a second uninfected blood meal by Leishmania-infected sand flies triggers dedifferentiation of metacyclic promastigotes, considered a terminally differentiated stage inside the vector 5 , to a leptomonad-like stage, the retroleptomonad promastigote. Reverse metacyclogenesis occurs after every subsequent blood meal where retroleptomonad promastigotes rapidly multiply and differentiate to metacyclic promastigotes enhancing sand fly infectiousness. Importantly, a subsequent blood meal amplifies the few Leishmania parasites acquired by feeding on infected hosts by 125-fold, and increases lesion frequency by fourfold, in twice-fed compared with single-fed flies. These findings place readily available blood sources as a critical element in transmission and propagation of vector-borne pathogens.

The fine flounder (Paralichthys adspersus) is a highly quoted flatfish species in the international and Chilean aquaculture markets. Despite presenting extraordinary nutritional value and quality white flesh, P. adspersus farming is limited by slow growth rates. This limitation can be offset with fast-refeed protocols, which result in a phenomenon termed compensatory growth. The aim of the present study was to assess the responses of the P. adspersus skeletal muscle transcriptome to a compensatory growth trial. Skeletal muscle sequence reads were obtained using Illumina Mi-Seq RNA technology for fine flounder under Control, Fasting, and Refeeding conditions. A total of 48,020,133 paired-ends reads were generated. Quality-controlled reads were de novo assembled, resulting in a reference transcriptome of 75,037 transcripts with an N50 of 2033 base pairs and average read coverage of 48X. Reads mapped onto the assembled reference transcriptome revealed 109 and 177 differentially expressed transcripts under the Fasting and Refeeding conditions, respectively. The up-regulated transcripts under Fasting were related to organic substance catabolic process, whereas down-regulated transcripts were associated with organic substance biosynthetic process. For Refeeding, significant up- and down-regulations were respectively recorded for mitotic nuclear division and muscle system processes. These results were validated by real-time qPCR analysis of 16 candidate transcripts. The obtained dataset will serve as an important transcriptomic resource for the fine flounder, with this study providing the first global gene expression analysis of a flatfish during compensatory growth.

Stress is a primary contributing factor of fish disease and mortality in aquaculture. We have previously reported that the red cusk-eel (Genypterus chilensis), an important farmed marine fish, demonstrates a handling-stress response that results in increased juvenile mortality, which is mainly associated with skeletal muscle atrophy and liver steatosis. To better understand the systemic effects of stress on red cusk-eel immune-related gene expression, the present study assessed the transcriptomic head-kidney response to handling-stress. The RNA sequencing generated a total of 61,655,525 paired-end reads from control and stressed conditions. De novo assembly using the CLC Genomic Workbench produced 86,840 transcripts and created a reference transcriptome with a N50 of 1426bp. Reads mapped onto the assembled reference transcriptome resulted in the identification of 569 up-regulated and 513 down-regulated transcripts. Gene ontology enrichment analysis revealed a significant up-regulation of the biological processes, like response to stress, response to biotic stimulus, and immune response. Conversely, a significant down-regulation of biological processes is associated with metabolic processes. These results were validated by RT-qPCR analysis for nine candidate genes involved in the immune response. The present data demonstrated that short term stress promotes the immune innate response in the marine teleost G. chilensis. This study is an important step towards understanding the immune adaptive response to stress in non-model teleost species.

Sweet cherry (Prunus avium L.) has stylar gametophytic self-incompatibility, which is controlled by the multi-allelic S-locus and encompasses the highly polymorphic genes for the S-ribonuclease (S-RNase) and S-haplotype-specific F-box (SFB), which are female and male determinants, respectively. The self-compatible mutant SFB4′ corresponds to an allele variant of SFB4 and presents a frameshift mutation. Even though male-determinant molecular markers can discriminate between SFB4 and SFB4′ alleles, the methods required are laborious, time-consuming and expensive, and not suitable for massive analysis and integration into breeding programmes. Our aim was to develop molecular markers for the evaluation of self-compatibility alleles in sweet cherry, that could be used as a high-throughput screening strategy to identify SFB4 and SFB4′ alleles, based on a marker for male determinacy. Our results were consistent using primers flanking the mutation responsible for the SFB4′ allele. We designed a specific molecular marker and confirmed it in sweet cherry commercial varieties. This new molecular marker is feasible for self-compatibility alleles in the male determinant in sweet cherry-assisted breeding programs.

Hass avocado postharvest ripening heterogeneity creates several logistics problems at destination markets. Although several approaches mainly based on targeted analysis have been undertaken to broaden our understanding of the potential causes of this postharvest ripening heterogeneity, still the secret is not yet unveiled. In this study biopsies from individual fruit displaying contrasting ripening behavior (slow vs fast) and subjected to different storage conditions (immediately after harvest, 30 d regular air at 5 °C and 30 d at 5 °C and 4 kPa O 2 and 6 kPa CO 2 ) were analyzed for targeted fatty acid analysis and cell wall enzyme activity combined with a more integrative approach based on gel free proteomics LC–MS/MS. The high throughput proteomics was capable of discriminating between slow and fast ripening avocado fruit. Proteins that were mainly less abundant in the slow ripening phenotype were annotated to correct protein folding, translation and de novo synthesis and higher abundant proteins were annotated to amino sugar and nucleotide metabolism, detoxification and stress response and proteolysis. Our study opens new insights into the causes of heterogeneous ripening and may provide new markers for avocado ripening at harvest and after prolonged storage.