Biology subjects

Alonso, C.

Publications and source records attributed to Alonso, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Genome-Scale Codon Deoptimization Enables Attenuation of Rift Valley Fever Virus

Rift Valley Fever Virus (RVFV) is a mosquito-borne zoonotic pathogen responsible for severe disease in domestic and wild ungulates as well as humans, representing a major threat to livestock production and human public health. RVFV is endemic in many African countries and has the potential to spread to new geographical regions. Current vaccines have limitations in safety and efficacy, highlighting the need for strategies to develop new vaccines candidates. In this study, we explored the use of codon deoptimization (CD) as a novel attenuation approach for the development of live-attenuated vaccine (LAV) against RVFV. CD exploits the redundancy of the genetic code by replacing frequently used codons with synonymous, less-preferred codons, thereby reducing translational efficiency without altering the amino acid sequence. We recoded parts of the M and S genome segments of RVFV using the least frequently used codons in mammalian cells, ensuring complete preservation of protein functionality and immunogenicity. Using reverse genetics, we rescued a panel of recombinant (r)RVFV encoding codon-deoptimized S-segment NSs gene (rNScd), M-segment Gn/Gc genes (rMcd), or both (rMcd/NScd). These recombinant CD viruses were characterized in vitro in mammalian and insect cell lines and in vivo using wild-type and immunocompromised mice. Results demonstrated varying degrees of attenuation among the three CD rRVFV, with the one deoptimized in both viral segments, rMcd/NScd, as a promising LAV based on the safety profiles. This study provides proof of concept for the use of CD as a rational strategy to generate attenuated RVFV, for the development of next-generation vaccines against this zoonotic threat.

microbiology↗

bioRxiv2026

Transgenerational imprint of subindividual variation: somatic epigenetic divergence within trees cascades into epigenetic and phenotypic pollen divergence

The evolutionary significance of somatic mosaicism created within plants by random epigenetic drift or localized responses to the environment will rest on its transgenerational implications. This paper tests whether the epigenetic mosaicism exhibited by individuals of a long-lived tree leaves some discernible "imprint" in the next gametophytic generation. Paired samples of leaves and pollen from five distinct branches within each of five wild-growing Pistacia terebinthus (Anacardiaceae) trees were characterized epigenetically using genome-wide DNA cytosine methylation level and multivariate epigenetic fingerprinting. Pollen grains were also characterized phenotypically by size and shape. Individual trees were somatically heterogeneous with regard to both cytosine methylation level and multilocus epigenetic fingerprints of leaf tissue, and within-tree variance in leaf epigenetic features exceeded among-tree variance. Phenotypic and epigenetic features of pollen differed significantly among branches of individual trees, and intraplant variation in phenotype and epigenetic fingerprints of pollen was predicted by intraplant variation in epigenetic features of leaves. Results provide evidence that intraindividual epigenetic heterogeneity arising in long-lived plants can leave its imprint into subsequent sporophyte generations via the production of epigenetically and phenotypically heterogeneous haploid gametophytes. Testable hypotheses for future epigenetic research on wild populations of non-model plants are suggested.

Plant Biology↗

bioRxiv2025

Understanding epigenetic regulation in non-model plants: Transcriptomic responses to seed demethylation in leaves and roots of the annual herb Erodium cicutarium (Geraniaceae)

Epigenetic regulation has emerged as a significant element in adaptation to heterogeneous and stressful environments, with modifications in DNA methylation being particularly relevant in plants. DNA methylation inhibitors have been used to investigate the relationship between DNA methylation and plastic plant phenotypes. However, their effect in gene expression regulation along lifetime remains understudied in non-model plants. Here, we analyze the effects of seed exposure to 5-azacytidine (5-azaC) in plant gene regulation. Scarified seeds from a single inbred line of Erodium cicutarium were soaked for 48 h in either water or a low concentration solution of 5-azaC before sowing. Subsequently, RNA was extracted from juvenile roots, juvenile leaves and adult leaves, and their transcriptomes were sequenced. Differential gene expression analysis was performed between treatments (control vs. treated) for all tissues together and separately. Beforehand, a draft genome of E. cicutarium was assembled to use it as reference for the transcriptome analysis, and its DNA methyltransferase genes were characterized. We found that 5-azaC up-regulated chromomethylase CMT1 across all treated samples, and the domain rearranged DNA methyltransferase DRM2 in juvenile roots. Furthermore, adult leaves showed more differentially expressed genes between control and 5-azaC treated samples compared to juvenile leaves, supporting long term transcriptomic effects of a short exposure to 5-azaC at seed germination. At adult stage, leaves of individuals treated with 5-azaC exhibited up-regulation of genes involved in seed dormancy release, photoinhibition, and osmotic stress. Finally, gene co-expression network analysis revealed a module of co-expressed genes with differential gene expression linked to 5-azaC treatment in juvenile roots, that was enriched with genes involved in retrotransposon activity and in anthocyanin metabolism. Altogether, this study illustrates how the experimental treatment with 5-azaC at seed stage generates tissue- and age-specific transcriptional shifts, directly affecting gene regulation and potentially broadening phenotype variation in this fast-growing annual plant.

genomics↗

bioRxiv2025

Somatic epigenetic mosaicism within Pistacia terebinthus (Anacardiaceae) trees leaves signature in reproductive cells

Propagation of epimutations over the branching topology of individuals often produces epigenetic mosaicism in long-lived plants, but whether such epigenetic mosaics can bear some evolutionary relevance remains largely unknown. We test here whether epigenetic reprogramming during gametogenesis completely erases intraplant epigenetic variation or, alternatively, some "signature" of the somatic mosaicism persists into reproductive cells. Intraplant variation in global cytosine methylation of leaf and pollen DNA samples was studied in five adult males of the dioecious tree Pistacia terebinthus (Anacardiaceae). Paired samples of fresh expanding leaves and pollen grains were taken from five different twigs in each tree, and percent cytosine methylation estimated using HPLC. Trees were internally heterogeneous in global cytosine methylation of both leaf and pollen. Within-tree variance accounted for 49.7% and 30.5% of population-wide variance in methylation for leaves and pollen, respectively. Tree means and within-tree variances in pollen methylation were directly correlated, respectively, with tree means and within-tree variances in leaf methylation. As a consequence of the epigenetic variability accumulated in somatic tissues over the individual plants lifetimes being only weakly eroded by gametogenesis, intraplant epigenetic heterogeneity became a natural source of epigenetic gametic diversity in the wild-growing population of P. terebinthus trees studied.

evolutionary biology↗

bioRxiv2025

Identification of key bacteria for ecological dynamics in a coastal marine observatory

Bacteria are essential for ecosystem maintenance, drive biogeochemical cycles, and influence responses to climate change. Additionally, many are involved in blooms and toxin production, affecting environmental quality. Despite their crucial role, some of their characteristics are only now being understood with the development of molecular techniques independent of cultivation. Thus, the recent effort to unify classical and microbial ecology studies has led to a better understanding of their dynamics. Here, we investigate the temporal processes that influence bacterial abundance and persistence, as well as their role in promoting or inhibiting the presence of certain organisms in the core of a metacommunity. Using molecular approaches such as amplicon and metagenomic analyses, we retrieved information on key bacteria (those with high abundance and/or persistence) from the South Atlantic Microbial Observatory (SAMO) to assess their interactions with environmental and biological factors. Bimodality was hardly observable, while the community structuring reflected the influence of large scale environmental processes in a recurrent pattern that clusters winter-spring and summer-autumn communities, reinforcing previous findings of intense influence of marine currents in this region. Among the key organisms, SAR11 and Flavobacteriaceae stand out. Flavobacteriaceae is known for its adaptive capacity and large genomes, while SAR11 has a reduced genome, making it dependent on compounds produced by other organisms. We identified an annual fluctuation in its abundance linked to Synechococcus blooms. The significant co-occurrence of ASVs from both groups reinforces the evidence of a biological interaction that sustains SAR11 through the exchange of these compounds. Through this study, we contribute to clarifying the factors that locally influence the geographic patterns of marine bacteria and identifying the pathways that promote their dynamics and functions in the ecosystem.

ecology↗

bioRxiv2025

Flower yeasts obfuscate intrinsic individual variation in nectar sugar concentration in a bumble bee-pollinated plant

Nectar-dwelling yeasts modulate the ecology of interactions between flowers and pollinators. We evaluate here the hypothesis that floral nectar traits, which are a central element in most plant-pollinator relationships, can largely represent the extended fungal phenotypes of nectar-dwelling yeasts. The following specific question is addressed, Do local genotypes of the specialist nectar yeast Metschnikowia reukaufii have the ability to obfuscate intrinsic individual variation among Helleborus foetidus plants in nectar sugar concentration ? An array of paired plant-yeast genotypes mimicking a natural field situation was created in the laboratory by inoculating field-collected nectar from different plant individuals with distinct yeast genotypes following a factorial design. Chemical analyses of nectar sugars before and after exposure to yeasts were performed using ion-exchange high performance liquid chromatography. Plant individual, yeast genotype, and their interaction all had strong, significant effects on sucrose concentration of H. foetidus nectar. Yeasts abolished 79% of intrinsic variance among plants in nectar sucrose concentration and profoundly reshaped patterns of intrinsic among-plant variation. Our results support the hypothesis that among-plant variation in nectar sugar concentration found by pollinators in the field can sometimes reflect the extended phenotypes of nectar yeasts more closely than intrinsic differences among the plants themselves.

ecology↗

bioRxiv2025

The hyper-attenuated RVFV 40Fp8 strain can be safely administered to pregnant ewes to protect them from a virulent challenge

In the present study, we evaluated the immunogenicity, safety and protective efficacy of the attenuated RVFV-40Fp8 strain in natural hosts (non-pregnant ewes) and in a highly susceptible host infection model such as pregnant ewes in the first third of pregnancy. Our results confirm the immunogenicity of 40Fp8 administration in non-pregnant and pregnant ewes, as well as the absence of foetal damage even after a high-dose vaccination regime in pregnant ewes. In addition, the ewes and their foetuses were protected against a virulent RVFV-56/74 strain challenge, as shown by comparative histopathological evaluation of tissue samples from vaccinated and non-vaccinated pregnant ewes. These results confirm the potential use of 40Fp8 as a RVF live-attenuated vaccine candidate and pave the way for further clinical developments.

microbiology↗

bioRxiv2024

Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic disease that causes severe disease in both domestic and wild ungulates and humans, making it a significant threat to livestock and public health. The RVFV genome consists of three single-stranded, negative-sense RNA segments differing in size: Small (S), Medium (M) and Large (L). Segment S encodes the virus nucleoprotein N and the virulence-associated factor non-structural (NSs) protein in opposite orientations, separated by an intergenic region (IGR). To overcome the current need of using secondary techniques to detect the presence of RVFV in infected cells, we used T7-driven polymerase plasmid-based reverse genetics to generate replication-competent recombinant (r)RVFV expressing Nanoluciferase (Nluc) or Venus fluorescent proteins. These reporter genes were used as valid surrogates to track the presence of RVFV in mammalian and insect cells. Notably, we explored the genome plasticity of RVFV and compared four different strategies by modifying the viral segment S in order to introduce the reporter gene foreign sequences. The reporter-expressing rRVFV were stable and able to replicate in cultured mammalian and insect cells, although to a lesser extent than the recombinant wild-type (WT) counterpart. Moreover, rRVFV expressing reporter genes were validated to identify neutralizing antibodies or compounds with antiviral activity. In vivo, all mice infected with the reporter-expressing rRVFV displayed an attenuated phenotype, although at different levels. These rRVFV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of RVFV, and represent an excellent biotechnological tool for developing new therapeutics against RVFV infections. IMPORTANCERift Valley fever virus (RVFV) is a mosquito-borne virus and zoonotic agent threat that can be deadly to domestic or wild ungulates, and humans. In this work, we used reverse genetics approaches to explore the genome plasticity of RVFV by generating a set of recombinant rRVFV that express fluorescent or luminescent proteins to track viral infection. All the generated reporter-expressing rRVFV were able to propagate in mammalian or insect cells, and in a mouse model of infection. Our studies may contribute to advances in research on RVFV and other bunyaviruses and pave the way for the development of novel vaccines and the identification of new antivirals for the prophylactic and therapeutic treatment, respectively, of RVFV infections.

microbiology↗

bioRxiv2024

Decoupled genetic and epigenetic variation at local scale in an endemic and a widespread Erodium (Geraniaceae) species

Epigenetic states offer an additional layer of variation besides genetic polymorphism that contribute to phenotypic variation and may arise either randomly or in response to environmental factors. We hypothesize that closely related species with different life-histories and habitat requirements could show distinct patterns of intraspecific epigenetic variation. We used Restriction-site Associated DNA sequencing (RADseq) and its bisulfite-converted variant (bsRADseq) to investigate in Sierra de Cazorla (SE Spain) the genetic and epigenetic population structure of two congeneric species, the endemic woody perennial Erodium cazorlanum and the widely distributed annual herb E. cicutarium. Population genomics analyses revealed no structure in either E. cazorlanum and E. cicutarium, suggesting substantial gene flow between the study populations. In contrast, we found that the mean proportion of global DNA methylation was different between populations and species, with E. cazorlanum DNA showing higher methylation average and across-individuals variation. For each species, we searched for the loci with the strongest epigenetic differentiation between populations (differentially methylated cytosines, DMCs), and summarized them across regions (differentially methylated regions, DMRs). Multivariate analysis and hierarchical clustering of single cytosines methylation percentages did not group individuals by population, pointing to high epigenetic variation within populations. DMRs were enriched in Copia transposable elements, putatively associated with stress response in plants. Our results suggest that variation at both genetic and epigenetic levels in our study area occur mainly within natural populations of E. cazorlanum and E. cicutarium, with stronger population structure in E. cicutarium, and highlight the relevance of analyzing short distance spatial patterns.

genomics↗

bioRxiv2024

A dynamic epigenetic perspective on above and below-ground phenotypic responses to drought: insights from global DNA methylation in Erodium cicutarium

O_LIPremise of the study. Mounting evidence supports the view that the responses of plants to environmental stress are mediated by epigenetic factors, including DNA methylation. Understanding the relationships between DNA methylation, plant development and individual fitness under contrasting environments is key to uncover the potential impact of epigenetic regulation on plant adaptation. Experimental approaches that combine a controlled alteration of epigenetic features with exposure to some relevant stress factor can contribute to this end. C_LIO_LIMethods. We combined the experimental application of a demethylating agent (5-azacytidine) with recurrent drought, and recorded their effects on above- and below-ground phenotypic traits related to early development, phenology and fitness in Erodium cicutarium from two provenances. C_LIO_LIKey results. We found that 5-azacytidine significantly reduced DNA methylation in leaf and root tissues. Moreover, it slowed plant development, delayed flowering, and reduced the number of inflorescences produced, and such detrimental effects occurred independently of water regime. Recurrent drought reduced final above- and below-ground biomass and total inflorescence production, and such negative effects were unaffected by artificial changes in DNA methylation. Increased fruit and seed-set were the only adaptive responses to drought observed in E. cicutarium, together with an increased number of flowers per inflorescence recorded in water stressed plants previously treated with 5-azacytidine. C_LIO_LIConclusion. Epigenetic effects can desynchronize plant growth, flowering and senescence among individual plants in both favourable and adverse environments. Future studies should focus on understanding intraspecific variation in the ability to change plant methylome in response to stress. C_LI

ecology↗

bioRxiv2024

The Rift Valley fever (RVF) vaccine candidate 40Fp8 shows an extreme attenuation in IFNARKO mice following intranasal inoculation

Rift Valley fever (RVF) is an important zoonotic viral disease affecting several species of domestic and wild ruminants, causing major economic losses and dozens of human deaths in various geographical areas of Africa, where it is endemic. Although it is not present in Europe, there is a risk of its introduction and spread linked to globalisation and climate change. At present, the only measure that could help to prevent the disease is vaccination of flocks in areas at risk of RVF. Available live attenuated vaccines are an effective means of controlling the disease, but their use is often questioned due to residual virulence, particularly in susceptible hosts such as pregnant sheep. On the other hand, no vaccine is currently licensed for use in humans. The development of safe and effective vaccines is therefore a major area of research. In previous studies, we selected under selective mutagenic pressure a highly attenuated RVFV 56/74 virus variant called 40Fp8. This virus showed an extremely attenuated phenotype in both wild-type and immunodeficient A129 (IFNARKO) mice, yet was still able to induce protective immunity after a single inoculation, thus supporting its use as a safe, live attenuated vaccine. To further investigate its safety, in this work we have analysed the attenuation level of 40Fp8 in immunosuppressed mice (A129) when administered by the intranasal route, and compared it with other attenuated RVF viruses that are the basis of vaccines in use or in development. Our results show that 40Fp8 has a much higher attenuated level than these other viruses and confirm its potential as a candidate for safe RVF vaccine development. Author SummaryVaccines that use attenuated viruses are highly effective in terms of providing protection, duration, breadth, and quality of the immune response. However, they may pose a risk to immunosuppressed individuals or in certain situations, such as during pregnancy. It is therefore important to analyze the residual virulence of attenuated vaccines to ensure their safety. In this work, we analysed the safety of the RVF virus vaccine variant 40Fp8 developed in our laboratory. We studied its effect on immunodeficient mice and used a more aggressive route of administration such as the intranasal delivery route. To assess the degree of attenuation of 40Fp8, we compared it with other attenuated prototype RVF vaccines. The results clearly demonstrate that 40Fp8 is highly attenuated, and its residual virulence level is extremely low compared to other vaccine viruses tested.

pathology↗

bioRxiv2023

Assessing the links between pollinators and the genetic and epigenetic features of plant species with contrasting distribution ranges

In flowering plants, pollinators contribute to gene flow while they also respond to variation in plant traits together determined by genetic, epigenetic and environmental sources of variation. Consequently, a correlation between abundance and diversity of pollinators and the genetic and epigenetic characteristics of plant populations such as diversity or distinctiveness is to be expected, yet no study has explored such long-term dimensions of plant-pollinator interactions. Mediterranean narrow endemics often exhibit unexpectedly high levels of population (epi)genetic diversity. We hypothesize that pollinators may contribute to explain this pattern. Specifically, we expect a stronger association of pollinators with population (epi)genetic variability in narrow endemics than in widely distributed congeners. We studied five pairs of congeneric plant species, consisting of one narrow endemic with a restricted distribution and one widespread congener, found in the Sierra de Cazorla mountains (SE Spain). In up to three populations per species, a comprehensive characterisation of pollinators was carried out to estimate pollinator diversity and visitation rate. Genetic and epigenetic diversity and distinctiveness of each population was calculated using AFLP markers and methylation-sensitive AFLP markers (MSAP) respectively. The relationships with pollinator diversity and visitation rate were assessed. The diversity of pollinators did not vary according to the plant distribution range, whereas visitation rate was higher in widespread species. As predicted, only narrow endemics showed a significant association between pollinators and their population genetic and epigenetic characteristics. Specifically, higher pollinator diversity and visitation rates entailed higher population genetic diversity and lower epigenetic distinctiveness. This work shows the value of exploring the relationships between pollinator abundance and diversity and population (epi)genetics for understanding the evolution of plant rarity.

ecology↗

bioRxiv2023

Comparative epigenetic and genetic spatial structure in Mediterranean mountain plants: a multispecific comparison

AO_SCPLOWBSTRACTC_SCPLOWEpigenetic information can be heritable, but also respond to key environmental variables in situ, endowing individuals with an additional capacity to adapt to environmental changes. Thus, it is likely that in sesile organisms such as plants, part of the spatial epigenetic variation found across individuals will reflect the environmental heterogeneity of populations. Analysing the departure of the spatial epigenetic structure from the baseline genetic variation can help in understanding the value of epigenetic regulation in species with different breath of optimal environmental conditions. We performed a multispecies study that considered seven pairs of congeneric plant species, each encompassing a narrow endemic with habitat specialization and a widespread species. In three populations per species we used AFLP and methylation-sensitive AFLP markers to characterise the spatial genetic and epigenetic structures. In contrast to widespread species, narrow endemics showed a significant and generalised lower epigenetic than genetic differentiation across species. Within most populations of narrow species, epigenetic variation was less spatially structured than the genetic variation. This pattern resulted from a lack of correlation between epigenetic and genetic information in populations of narrow endemics. We argue that the differences found between narrow endemics and widespread congeners reflect contrasting breaths of environmental requirements. We pose the hypothesis that in species with a narrow niche breath, epigenetic variation may be more similar across populations and among individuals within a population given the expected higher similarity in environmental requirements.

genetics↗

bioRxiv2023

Long-term methylome changes after experimental seed demethylation and their interaction with recurrent water stress in Erodium cicutarium (Geraniaceae)

O_LIThe frequency and length of drought periods are increasing in subtropical and temperate regions worldwide. Epigenetic responses to water stress could be key for plant resilience to this largely unpredictable challenge. Experimental DNA demethylation together with application of a stress factor stands as a suitable strategy to uncover the contribution of epigenetics to plant responses to stress. C_LIO_LIWe analysed leaf cytosine methylation changes in adult plants of the Mediterranean weed, Erodium cicutarium, after seed demethylation with 5-Azacytidine and/or recurrent water stress in a greenhouse. We used bisulfite RADseq (BsRADseq) and a newly reported reference genome for E. cicutarium to characterize methylation changes in a 2x2 factorial design, controlling for plant relatedness. C_LIO_LIIn the long-term, 5-Azacytidine treatment alone caused both hypo and hyper-methylation at individual cytosines, with substantial hypomethylation in CG contexts. In control conditions, drought resulted in a decrease in methylation level in all but CHH contexts. In contrast, the genome of plants that experienced recurrent water stress and had been treated with 5-Azacytidine increased DNA methylation level by ca. 5%. C_LIO_LISeed demethylation and recurrent drought exhibited a highly significant interaction in terms of global and context-specific cytosine methylation supporting an epigenetic contribution in response to stress at molecular level. C_LI

ecology↗

bioRxiv2022

Isolation and genome characterization of Lloviu virus from Italian Schreibers bent-winged bats

Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers bent-winged bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its discovery. Studies with recombinant and wild-type LLOV isolates confirmed the susceptibility of human-derived cell lines and primary human macrophages to LLOV infection in vitro. Based on these data, LLOV is now considered as a potential zoonotic virus with unknown pathogenicity to humans and bats. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding sequence of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this article is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection. These results further confirms the role of these bats as the host of this virus, possibly throughout their entire geographic range. This is an important result to further understand the role of bats as the natural hosts for zoonotic filoviruses.

microbiology↗

bioRxiv2022

Body mass decline in a Mediterranean community of solitary bees supports the size shrinking effect of climatic warming

The long-known, widely documented inverse relationship between body size and environmental temperature ("temperature-size rule") has recently led to predictions of body size decline following current climatic warming ("size shrinking effect"). For keystone pollinators such as wild bees, body shrinking in response to warming can have pervasive effects on pollination processes, but there is still little evidence of the phenomenon because adequate tests require controlling for climate-linked confounding factors (e.g., urbanization, land use change). This paper tests the shrinking effect in a diverse community of solitary bees from well-preserved habitats in the core of a large nature reserve undergoing fast climatic warming but not disturbances or habitat changes. Long-term variation in mean body mass was evaluated using data from 1,186 individual bees (108 species, 25 genera, 6 families) sampled over 1990-2022. Climate of the region warmed at a fast rate during this period and changes in bee body mass verified expectations from the size shrinking effect. Mean individual body mass of the regional community of solitary bees declined significantly, shrinking being particularly intense for female individuals and large-bodied species. As a consequence, the pollination and mating systems of many bee-pollinated plants in the region are likely undergoing important alterations.

ecology↗

bioRxiv2022

Seasonality of pollinators in Mediterranean montane habitats: cool-blooded bees for early-blooming plants

Understanding the factors that drive community-wide assembly of plant-pollinator systems along environmental gradients has considerable evolutionary, ecological and applied significance. Variation in thermal environments combined with intrinsic differences among pollinators in thermal biology (tolerance limits, thermal optima, thermoregulatory ability) have been proposed as drivers of community-wide pollinator gradients, but this suggestion remains largely speculative. We test the hypothesis that seasonality in bee pollinator composition in montane habitats of southeastern Spain, which largely reflects the prevalence during the early flowering season of mining bees (Andrena), is a consequence of the latters thermal biology. Quantitative information on seasonality of Andrena bees in the whole plant community (275 plant species) was combined with field and laboratory data on key aspects of the thermal biology of 30 species of Andrena (endothermic ability, warming constant, relationships of body temperature with ambient and operative temperatures). Andrena bees were a conspicuous, albeit strongly seasonal component of the pollinator assemblage of the regional plant community, visiting flowers of 153 different plant species (57% of total). Proportion of Andrena relative to all bees reached a maximum among plant species which flowered in late winter and early spring, and declined precipitously from May onwards. Andrena were recorded only during the cooler segment of the annual range of air temperatures experienced at flowers by the whole bee assemblage. These patterns can be explained by features of Andrenas thermal biology: null or negligible endothermy; ability to forage at much lower body temperature than endothermic bees (difference ~10{degrees}C); low upper tolerable limit of body temperature, beyond which thermal stress presumably precluded foraging at the warmest period of year; weak thermoregulatory capacity; and high warming constant enhancing ectothermic warming. Our results demonstrate the importance of lineage-specific pollinator traits as drivers of seasonality in community-wide pollinator composition; show that exploitation of cooler microclimates by bees does not require endothermy; falsify the frequent assumption that endothermy and thermoregulation apply to all bees; and suggest that medium- and large-sized ectothermic bees with low upper thermal limits and weak thermoregulatory ability can actually be more adversely affected by climate warming than large endothermic species.

ecology↗

bioRxiv2021

Endosomal proteins NPC1 and NPC2 at African swine fever virus entry/fusion

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. The virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. As several other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. Endosomal maturation entails luminal acidification and the lowering of pH acting on the multi-layered virion structure dissolves the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Egress from endosome is related to cholesterol efflux, but it remains an intriguing process albeit essential for infection, specifically for the viral nucleic acid exit to the cytoplasm for replication. ASFV proteins E248R and E199L, with structural homology to the VACV proteins of the fusion complex, seem to have similar functions in ASFV. A direct interaction between these ASFV proteins with the cholesterol transporter protein NPC1 (Niemann-Pick C type 1) was observed, which was also shared by the E248R homologous protein L1R of VACV. Binding occurs between the transmembrane domain of E248R with the loop C of NPC1 at the same domain than EBOV binding site. These interactions suggest that these ASFV proteins are crucial for membrane fusion. CRISPR NPC1 KO Vero cells lacking NPC1 protein that were resistant to EBOV, reduced ASFV infection levels significantly. Reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by lesser viral factories of smaller size and lacking the typical cohesive morphology between endosomes and viral proteins. We observed a compensatory effect in NPC1 KO cells, elevating NPC2 levels while silencing NPC2 in Vero cells with shRNA, also reduced ASFV infection. Our findings pave the way to understand the role of these proteins at the membrane viral fusion step for several viruses. Author SummaryAfrican swine fever virus (ASFV) causes a deadly disease of pigs and wild boars that was endemic in Africa but have extended over the last years to Europe, Asia and Oceania with high socioeconomic impact. ASFV enters the cell by endocytosis and has adapted to the endosomal conditions to acquire infectivity. Viral infectivity is dependent on cholesterol traffic at the endosomes, especially at the fusion step. Fusion of the internal viral membrane with the endosomal membrane is required for the exit of the viral DNA to the cytoplasm to start replication. ASF virion internal membrane proteins E248R and E199L were found to bind the Niemann Pick C1 (NPC1) receptor at the endosome. These proteins are highly conserved among ASFV isolates and resemble proteins of the VACV entry/fusion complex. The function of NPC1 is to regulate the efflux of dietary cholesterol efflux from the endosome to the endoplasmic reticulum, which appears to be necessary for viral fusion. NPC1 knockout cells by CRISPR reduced infection affecting infectivity and early replication. Also, removing the associated endosomal protein NPC2, further declined infectivity. These results show the relevance of NPC1 receptor in the viral infection actually shared by unrelated important viral families.

microbiology↗