The reference genome of Erebia palarica will provide valuable insights into evolutionary and conservation genomics. On one hand, the reference genome paves the way to unravel the speciation process, reproductive barriers, and putative hybridisation of E. palarica with its closely related sister species: Erebica meolans. On the other hand, the reference genome will play an important role in the genetic monitoring of this endemic species, facilitating the use of genomics to estimate population genomics parameters. The genome was assembled into 14 contiguous chromosomal pseudomolecules (Z chromosome included). This chromosome-level assembly encompasses 0.49 Gb, composed of 38 contigs and 17 scaffolds, with contig and scaffold N50 values of 34.2 Mb and 38.4 Mb, respectively. ### Competing Interest Statement The authors have declared no competing interest.

Introduction: Multidrug-resistant Klebsiella pneumoniae (Kpn) poses a growing global health threat due to its role in severe hospital-acquired infections and increasing resistance to antibiotics. The objective of this study was to isolate, characterize, and evaluate the therapeutic potential of two novel lytic bacteriophages targeting multidrug-resistant K. pneumoniae, aiming for the development of alternatives to conventional antibiotics. Materials and methods: Phages KpnS01BRG and KpnS02SCE were analyzed using transmission electron microscopy (TEM) and whole-genome sequencing (WGS). Replication kinetics were determined through single-step growth curves (latency period and burst size). Antibacterial efficacy was tested in vitro using a phage cocktail at a multiplicity of infection (MOI) of 10,000 over 12 h. Additionally, a genome mechanics analysis was conducted to evaluate viral DNA cyclizability and flexibility. Results: TEM revealed that both phages belong to the class Caudoviricetes with a siphovirus-like morphology, and WGS classified them within the genus Webervirus (family Drexlerviridae). Phage KpnS01BRG exhibited a latency period of 60 min and a burst size of 17.7 virions/cell, while KpnS02SCE showed a latency of 5.8 min and a burst size of 18.6 virions/cell. The phage cocktail reduced the bacterial load by approximately 97.2% after 12 h, relative to the untreated control. Genomic analysis indicated that the higher DNA flexibility in KpnS02SCE correlated with a slightly higher virion production, corroborating previous findings on genome mechanics. Conclusions: The novel phages demonstrated potent antibacterial activity (i.e., strongly reduced bacterial counts after 6 h) and favorable genomic characteristics, establishing themselves as promising candidates for phage-based therapeutic strategies against multidrug-resistant K. pneumoniae infections.

Emerging phytopathogens are a growing threat to global agriculture, undermining crop yields and jeopardizing food security. Among these, Pseudomonas coronafaciens pv. garcae (Pcg) causes coffee halo blight, a disease with serious implications for coffee production. Current control measures rely heavily on copper-based compounds and antibiotics like kasugamycin, both of which present environmental hazards and drive resistance development. In this context, bacteriophages offer a targeted and sustainable alternative. Although phage therapy has been explored for several plant pathogens, no phage-based solution has yet been tailored to control Pcg. Here, we describe an in-depth genomic characterization of four novel lytic phages (PCG-05T, PCG-06T, PCG-07T, and PCG-09T) capable of infecting Pseudomonas coronafaciens pv. garcae. Genomic sequencing and structural analysis via transmission electron microscopy revealed that phages PCG-05T, PCG-07T, and PCG-09T exhibit myovirus morphology, while phage PCG-06T displayed a siphovirus morphotype. Beyond taxonomic classification, we investigated the intrinsic cyclizability of their genomes, integrating insights from DNA biophysics to explore how sequence-dependent mechanical properties influence phage virion assembly. Our findings suggest that DNA bendability, shaped by base pair composition and helical periodicity, may correlate with phage morphogenesis efficiency. This study not only broadens the repertoire of candidate phages for biocontrol of coffee plant pathogens but also introduces an innovative hypothesis linking genome mechanics to phage virion fitness. These insights lay the groundwork for environmentally friendly phage-based strategies to combat bacterial blight in coffee plant cultivation.

Bacterial blight of coffee caused by Pseudomonas coronafaciens pv. garcae (Pcg) poses a significant threat to coffee production. This study reports the isolation and characterization of four lytic bacteriophages - PCG-05T, PCG-06T, PCG-07T, and PCG-09T - as potential biocontrol agents against Pcg. Isolated using multiple Pcg strains, the phages exhibited differential host specificity, plaque morphology, and lytic efficiency. Adsorption kinetics and one-step growth analyses revealed fast binding (PCG-06T and PCG-07T) and high burst sizes (PCG-05T and PCG-09T). In addition, phages PCG-06T and PCG-09T also displayed broad host range and strong lytic activity, with low frequencies of emergence of bacterial resistant mutants (≤10−3), with all phages positioning themselves as promising candidates for phage therapy. Electrostatic and diffusion analyses suggested that adsorption efficiency was influenced by surface charge interactions, with PCG-06T showing enhanced binding due to a higher (less negative) Zeta potential. In vitro inactivation assays demonstrated MOI-dependent suppression of Pcg populations, with phage cocktails yielding superior reductions - up to 6.17 log CFU/mL - especially at MOI 10000. Ex planta assays on artificially contaminated coffee plant leaves further confirmed significant bacterial inactivation, achieving up to 4.08 log CFU/mL reduction after 36 h. Despite slight regrowth at later time points, phage persistence remained stable, highlighting the potential of high-MOI applications for effective short-term control. Genomic screening confirmed the absence of undesirable genes, reinforcing safety for agricultural use. While results are promising, further studies on formulation, environmental stability, and field efficacy are needed to optimize phage-based interventions for controlling Pcg in coffee plantations.

The reference genome of the Spanish Moon Moth, Graellsia isabellae, will be of great importance for evolutionary and conservation genomics. Firstly, this reference genome, alongside phylogenomic analyses, may finally resolve the longstanding debate regarding the scientific name of this iconic species, whether it should be Graellsia isabellae (Graells, 1849) or Actias isabellae (Graells, 1849). Secondly, the reference genome will be instrumental in the genetic monitoring of this protected species, enabling advanced methods to calculate contemporary population genomics estimates. The genome was assembled into 31 contiguous chromosomal pseudomolecules (Z chromosome included). The mitochondrial genome has also been assembled and is 15,247 bp in length. This chromosome-level assembly encompasses 0.56 Gb, composed of 38 contigs and 32 scaffolds, with contig and scaffold N50 values of 18.9 Mb and 20.4 Mb, respectively. ### Competing Interest Statement The authors have declared no competing interest.

Aims In this study, we report the use of two novel lytic polyvalent phages as a cocktail in in planta assays and their efficacy in the control of bacterial halo blight (BHB) caused by Pseudomonas coronafaciens pv. garcae (Pcg) in coffee plants.Methods and results Phages were isolated from samples of coffee plant leaves collected at two different locations in Brazil. Both phages belong to the class Caudoviricetes and present myovirus-like morphotypes, and both exhibited specificity to their host, Pcg strain IBSBF-158. The two phages were encapsulated in chitosan-coated Ca-alginate nanoparticles, which demonstrated promising performance, promoting reductions in disease severity ranging from 66.83% to 83.37%, depending on the timing of application relative to infection. Both phages were somewhat susceptible to the effects of abiotic factors when in free form, with solar radiation seriously negatively impacting their lytic activity. However, nanoencapsulation of both phages as a lytic cocktail within chitosan-coated Ca-alginate nanoparticles proved successful in fully stabilizing both phages from the deleterious action of UV radiation.Conclusions Application of such lytic nanoparticles in pre- and post-inoculated coffee seedlings in in planta greenhouse assays proved successful in controlling the phytopathogen responsible for BHB of coffee, Pcg, with a significant decrease in the progression of the disease. The results suggest that lytic nanoparticles may become an effective and sustainable strategy for coffee BHB control, as an alternative to conventional approaches relying on chemical (copper hydroxide or oxychloride or kasugamycin hydrochloride) or biological agents, but more studies are needed in the field to confirm this. The phage protection system developed represents a potential alternative treatment for bacterial plant diseases with minimum damage to the environment.

1. Conservation efforts are often limited by the lack of information on poorly understood species. Such is the case of Erebia palarica Chapman, 1905, a narrow endemic butterfly restricted to the mountain ranges of northwest Iberia and classified as of "Least Concern" by the IUCN. By combining mark-release-recapture (MRR) and genetic data, we provide an insight into the population size of this species. 2. First, we marked 707 butterflies in a relatively isolated E. palarica population-Serra do Courel (NW Spain)-and obtained census size estimates for two close localities (Alto do Couto, N-c = 303; A Cabeza Grande, N-c = 565); the vast majority of individuals dispersed less than 500 metres. 3. Second, genotyping of 120 individuals from Alto do Couto (four generations, 1999-2021) with seven nuclear microsatellites revealed a small effective population size (N-e < 100), while also depicting a steady increase in its value since 2018. We also provide a fine-scale distribution map of the species in the study area: we found E. palarica in a number of new sites with favourable habitat and confirmed its persistence in an area affected by a massive wildfire during 2022. 4. Finally, we propose that the conservation status of E. palarica should be updated to "Near Threatened", as it is remarkably close to meet several criteria of the IUCN to qualify as "Vulnerable".

Contamination of water with mercury constitutes a serious public health problem, especially in locations where the use of Hg occurs improperly/illegally and negligently, as is the case in the Amazon region (Brazil). The riverside populations in the Amazon are frequently invaded by illegal mining, exposing these populations to significant risks, of which contamination by heavy metals such as mercury (Hg2+) has the potential to cause serious illnesses. Furthermore, exposure to this metal causes neurological, cardiovascular, immune and digestive system disorders, in addition to damaging the lungs, kidneys, skin and eyes. The aquatic biome is extremely important for the local economy and population, being drastically affected by Hg2+ contamination and its effects. Therefore, it is necessary to develop bioremediation/biomitigation methods that are effective and less harmful to the environment, aiming to remove Hg2+ from water. Hence, when we think about new methodologies that can lead to the reduction of mercury in water, the use of protein entities is a potential option and, for this reason, we can highlight the possibility of using bacteriophage virions to remove Hg2+ ions from water by biosorption using their negative Zeta Potential for this purpose. In this sense, the main goal of the research work undertaken was to test the possibility of mitigating the presence of mercury (II) ions in water through the immobilization of a bacteriophage isolated and already characterized by our research group (EcoM021, T4 myovirus of the Straboviridae family and genus Tequatrovirus), on a chitosan-coated Ca-alginate microparticle support, through which water contaminated with Hg2+ ions was percolated. The system developed in microparticle form integrating trapped phage virions showed to be very promising for retaining mercury ions through biosorption (electrostatic attraction), thus enabling the removal of ionic mercury from water.

Traditionally, control of coffee plant bacterial halo blight (BHB) caused by the phytopathogen Pseudomonas coronafaciens pv. garcae (Pcg) involves frequent spraying of coffee plantations with non-environmentally friendly and potentially bacterial resistance-promoting copper products or with kasugamycin hydrochloride. In this study we report a leap forward in the quest for a new ecofriendly approach, characterizing (both physicochemically and biologically) and testing both in vitro and ex vivo a new lytic phage for Pcg. An in-depth molecular (genomic and DNA structural features) characterization of the phage was also undertaken. Phage PcgS01F belongs to the class Caudoviricetes, Drexlerviridae family and genus Guelphvirus, and presents a siphovirus-like morphotype. Phage PcgS01F showed a latency period of 40min and a burst size of 46 PFU/host cell, allowing to conclude that it replicates well in Pcg IBSBF-158. At Multiplicity Of Infection (MOI, or the ratio of phage to bacteria) 1000, the performance of phage PcgS01F was much better than at MOI 10, promoting increasing bacterial reductions until the end of the in vitro inactivation assays, stabilizing at a significant 82% bacterial load reduction. Phage PcgS01F infected and killed Pcg cells ex vivo in coffee plant leaves artificially contaminated, with a maximum of Pcg inactivation of 7.66log CFU/mL at MOI 1000 after 36h of incubation. This study provides evidence that the isolated phage is a promising candidate against the causative agent of BHB in coffee plants.

This review aims at presenting the main strategies that are currently available for the delivery of bacteriophages to combat bacterial infections in humans, animals, and plants. It can be seen that the main routes for phage delivery are topical, oral, systemic, and airways for humans. In animals, the topical and oral routes are the most used. To combat infections in plant species, spraying the plant’s phyllosphere or drenching the soil are the most commonly used methods. In both phage therapy and biocontrol using phages, very promising results have been obtained so far. However, more experiments are needed to establish forms of treatment and phage doses, among other parameters. Furthermore, in general, there is a lack of specific standards for the use of phages to combat bacterial infections.

The goal of this research was to create an antibacterial biopolymeric coating integrating lytic bacteriophages against Salmonella enterica for use in ripened cheese. Salmonella enterica is the main pathogen that contaminates food products and the food industry. The food sector still uses costly and non-selective decontamination and disease control methods. Therefore, it is necessary to look for novel pathogen biocontrol technologies. Bacteriophage-based biocontrol seems like a viable option in this situation. The results obtained show promise for food applications since the edible packaging developed (EdiPhage) was successful in maintaining lytic phage viability while preventing the contamination of foodstuff with the aforementioned bacterial pathogen.

Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5′ end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A–H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies.

Montane biodiversity is particularly vulnerable to rapid oscillations in environmental conditions. Recent modelling showed that only three of the 19 butterfly species of Erebia (Nymphalidae, Satyrinae) currently present in Iberia would persist in a worst-case scenario for 2070. One of them is Erebia palarica, endemic to Northwest Spain. We combined genetics, morphometrics and ecological niche modelling to reconstruct its evolutionary history. First, sequences of the cytochrome oxidase I (COI) mitochondrial gene obtained from 305 specimens revealed a double star-like network that suggests a demographic expansion from two ancestral populations. This finding is congruent with the larger and disjunct putative distribution revealed by climatic modelling during the Last Glacial Maximum. Second, genotyping of 262 samples with seven microsatellite markers produced a large cluster at the centre-East of the Cantabrian range, mostly affected by isolation by distance, and revealed different levels of structure in the western localities. Lastly, we reviewed the intraspecific taxonomy of the species. The westernmost site (Queixa) stands as a clearly separated unit according to genetic and morphometric analyses, which further supports its vague former description as subspecies E. p. castroviejoi. Altogether, the case of E. palarica exemplifies the complexity that narrow endemics can show and highlights that understanding the evolutionary history of species is crucial for designing adequate conservation strategies.

Coffee plants have been targeted by a devastating bacterial disease, a condition known as bacterial blight, caused by the phytopathogen Pseudomonas syringae pv. garcae (Psg). Conventional treatments of coffee plantations affected by the disease involve frequent spraying with copper- and kasugamycin-derived compounds, but they are both highly toxic to the environment and stimulate the appearance of bacterial resistance. Herein, we report the molecular characterization and mechanical features of the genome of two newly isolated (putative polyvalent) lytic phages for Psg. The isolated phages belong to class Caudoviricetes and present a myovirus-like morphotype belonging to the genuses Tequatrovirus (PsgM02F) and Phapecoctavirus (PsgM04F) of the subfamilies Straboviridae (PsgM02F) and Stephanstirmvirinae (PsgM04F), according to recent bacterial viruses' taxonomy, based on their complete genome sequences. The 165,282 bp (PsgM02F) and 151,205 bp (PsgM04F) genomes do not feature any lysogenic-related (integrase) genes and, hence, can safely be assumed to follow a lytic lifestyle. While phage PsgM02F produced a morphogenesis yield of 124 virions per host cell, phage PsgM04F produced only 12 virions per host cell, indicating that they replicate well in Psg with a 50 min latency period. Genome mechanical analyses established a relationship between genome bendability and virion morphogenesis yield within infected host cells.

Abstract Montane biodiversity is particularly vulnerable to rapid oscillations in environmental conditions. Recent modelling showed that only three of the 19 butterfly species of Erebia (Nymphalidae, Satyrinae) currently present in Iberia would persist in a worst‐case scenario for 2070. One of them is Erebia palarica, endemic to Northwest Spain. We combined genetics, morphometrics and ecological niche modelling to reconstruct its evolutionary history. First, sequences of the cytochrome oxidase I (COI) mitochondrial gene obtained from 305 specimens revealed a double star‐like network that suggests a demographic expansion from two ancestral populations. This finding is congruent with the larger and disjunct putative distribution revealed by climatic modelling during the Last Glacial Maximum. Second, genotyping of 262 samples with seven microsatellite markers produced a large cluster at the centre‐East of the Cantabrian range, mostly affected by isolation by distance, and revealed different levels of structure in the western localities. Lastly, we reviewed the intraspecific taxonomy of the species. The westernmost site (Queixa) stands as a clearly separated unit according to genetic and morphometric analyses, which further supports its vague former description as subspecies E. p. castroviejoi. Altogether, the case of E. palarica exemplifies the complexity that narrow endemics can show and highlights that understanding the evolutionary history of species is crucial for designing adequate conservation strategies.

The bioengineering of phages to display ligands leads to specific targeting, reduced toxicity, enhanced cellular uptake of an incorporated drug/gene and, ultimately, increased therapeutic efficacy.

IntroductionSolid organ transplant (SOT) recipients display weak seroconversion and neutralizing antibody (NAb) responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and remain at risk of severe coronavirus disease 2019 (COVID-19). While B-cell memory is the hallmark of serological immunity, its role in driving successful vaccine responses and providing immune protection in SOT patients remains unclear.MethodsWe investigated the function and interplay of SARS-CoV-2-specific memory B cells (mBc), different cytokineproducing T cells, and cross-reactive NAb in driving seroconversion and protection against COVID-19 in two cohorts. First, we studied a large cohort of 148 SOT recipients and 32 immunocompetent individuals who underwent several vaccinations. Subsequently, we assessed 25 SOT patients participating in a randomized controlled trial to compare two different immunosuppressive strategies for allowing successful seroconversion and memory-cell responses after booster vaccination.ResultsWe corroborate previous findings that B- and T-cell memory responses are weaker and more delayed in SOT patients than in immunocompetent (IC) individuals; however, within the SOT cohort, we found that these responses are relatively stronger and more robust in patients not receiving mycophenolate mofetil (MMF)-based therapies. Anti- spike IgG titers strongly correlated with RBD-specific IgG-producing mBc, with both displaying broad viral cross reactivity. Prebooster SARS-CoV-2-specific mBc and IL-2- producing T cells accurately predicted Nab seroconversion (AUC, 0.828) and protection against severe COVID-19. While switching unresponsive SOT patients from calcineurin inhibitors (CNI)/MMF to a low-exposure CNI/mTOR-i regimen favored wider SARS-CoV-2-specific immune responses after a fourth booster vaccination, preformed RBD-specific mBc predicted NAb seroconversion.DiscussionOur study adds new insights into the pathobiology of immune memory and highlights the pivotal role of SARS-CoV-2-specific mBc in promoting immune protection inSOT patients.

Coffee canker, or bacterial halo blight (BHB) of coffee, is a disease caused by the phytopathogenic bacterium Pseudomonas syringae pv. garcae (Psg), having been found for the first time in 1955, in the Garça region (State of São Paulo), and which has stood out in the Brazilian coffee plantations in recent years, leading to severe economic losses that seriously affect coffee trade. The treatments available are still scarce, involving frequent spraying of coffee plantations with either copper derivatives or the antibiotic kasugamycin. However, these compounds should be avoided due to environmental toxicity and the development of bacterial resistances. Herein we report the isolation and physical/biological characterisation of two novel lytic phages and their efficacy in the control of Psg. Phages ph002F and ph004F were isolated from coffee plant leaves in Brazil (Sorocaba/SP and Itu/SP cities), using Psg IBSBF-158 as the host. According to the transmission electron microscopy analyses, both phages belong to the class Caudoviricetes and present myovirus-like morphotypes. Phages ph002F and ph004F showed eclipse times of 5 min and 20 min, respectively, and a burst size of 123 PFU/host cell and 12 PFU/host cell, respectively, allowing to conclude they replicate well in Psg IBSBF-158 with latency periods of 50 min. Phage ph002F (reduction of 4.59 log CFU/mL, compared to uninfected culture) was more effective in inactivating Psg than phage ph004F (reduction of 3.85 log CFU/mL) after 10 h of incubation at a MOI of 10. As a cocktail, the two phages were highly effective in reducing the bacterial load (reduction of 5.26 log CFU/mL at a MOI of 0.1 or reduction of 5.03 log CFU/mL at a MOI of 10, relative to untreated culture), after 12 h of treatment. This study provides evidence that the isolated phages are promising candidates against the causative agent of BHB in coffee plants.

Some chemical elements released in nature due to anthropogenic actions are harmful to living beings, and finding efficient and low-cost ways to measure their presence is a challenge. The major goal of this work was to use the barks of urban trees as bioindicators of the presence of these elements. For this purpose, tree barks of sixteen individual trees were collected, including Ipê (Bignoniaceae Family); Sibipiruna (Fabaceae Family); Pine (Pinaceae Family), in the city of Sorocaba, SP, Brazil, in three different districts. Two samples, one of Ipê and another of Sibipiruna, collected in the Mata Atlântica forest in Juquitiba, SP, Brazil, were used as control samples. They were also analyzed; six soil samples were collected in the same places as the tree barks in Sorocaba. The samples were analyzed using the Energy Dispersion X-Ray Fluorescence Spectroscopy technique. The elements studied ranged from Al to Bi. The results were submitted to univariate and multivariate statistical analysis showing that Sibipiruna presented a high concentration of the element Ca. At the same time, Ipê and Pine showed high concentrations of K. In the identified elements, the probable sources of contamination were pointed out, such as elements from the dust of braking automobiles (Al, Si, S, Ti, Fe, Cu, and Ba), elements from the paint used to paint the asphalt (Si, Ca, Cr and Pb) and elements from the tire tread wear (Al, S, Ca and Zn). From the analysis of soil samples and trees, it was found that there was high pollution by the element Pb in the specimens collected in front of the old Saturnia battery factory, located in the district of Éden in the city of Sorocaba, SP, Brazil (Coordinates: Lat 23K253141 m E; Long 23K7405583 m S).