Due to the low incidence of precipitation attributed to climate change, many high-altitude Andean lakes (HAALs) and lagoons distributed along the central Andes in South America may soon disappear. This includes La Brava–La Punta, a brackish lake system located south of the Salar de Atacama within a hyper-arid and halophytic biome in the Atacama Desert. Variations in the physicochemical parameters of the water column can induce changes in microbial community composition, which we aimed to determine. Sixteen sampling points across La Brava–La Punta were studied to assess the influence of water physicochemical properties on the aquatic microbial community, determined via 16S rRNA gene analysis. Parameters such as pH and the concentrations of silica, magnesium, calcium, salinity, and dissolved oxygen showed a more homogenous pattern in La Punta samples, whereas those from La Brava had greater variability; pH and total silica were significantly different between La Brava and La Punta. The predominant phyla were Proteobacteria, Bacteroidetes, Actinobacteria, and Verrucomicrobia. The genera Psychroflexus (36.85%), Thiomicrospira (12.48%), and Pseudomonas (7.81%) were more abundant in La Brava, while Pseudospirillum (20.73%) and Roseovarius (17.20%) were more abundant in La Punta. Among the parameters, pH was the only statistically significant factor influencing the diversity within La Brava lake. These results complement the known microbial diversity and composition in the HAALs of the Atacama Desert.

A new species of mealybug from Chile, Pseudococcus meridionalis Prado sp. n., is described and illustrated based on the morphological and molecular characterization of adult females. This species belongs to the “Pseudococcus maritimus” complex and displays a wide host plant range, including Japanese pear, persimmon, pomegranate, pear and grape.

In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on ‘Yagana-INIA’, a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus. This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/μl to 1 × 10^-6 pg/μl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.

Many genetic studies in insects require sex identification of individuals in all developmental stages. The most common sex chromosome system in lepidopterans is WZ/ZZ; the W chromosome is present only in females. Based on two W chromosome-specific short sequences (CpW2 and CpW5) described in Cydia pomonella (L.) (Lepidoptera: Tortricidae), we identified homologous female-specific sequences in Lobesia botrana Den. & Schiff, a polyphagous and very harmful species present in Chile since 2008. From this starting point, we extended the sequence information using the inverse PCR method, identifying the first W-specific sequences described up to now for the moth. Finally, we developed a duplex PCR method for rapid and sensitive determination of sex in L. botrana from larva to adult. The method showed a detection limit of 1 pg of genomic DNA; a blind panel of samples exhibited exact correspondence with the morphological identification. These results will be very useful for studies requiring sex-specific analyses at any developmental stage, contributing also to the understanding of gene expression in the insect, as well as to the eventual development of control protocols against the moth, such as the development of genetic sexing strains for the implementation of the sterile insect technique.

Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of ‘Valencia’ and hybrid genotypes (CsH1–CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L⁻¹ GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 µM) significantly enhanced rooting, increasing efficiencies to 37.1% in ‘Valencia’ and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure.