To protect biodiversity we must understand its structure and composition including the bacteria and microparasites associated with wildlife, which may pose risks to human health. However, acquiring this knowledge often presents challenges, particularly in areas of high biodiversity where there are many undescribed and poorly studied species and funding resources can be limited. A solution to fill this knowledge gap is sampling roadkill (animals that die on roads as a result of collisions with circulating vehicles). These specimens can help characterize local wildlife and their associated parasites with fewer ethical and logistical challenges compared to traditional specimen collection. Here we test this approach by analyzing 817 tissue samples obtained from 590 roadkill vertebrate specimens (Amphibia, Reptilia, Aves and Mammalia) collected in roads within the Tropical Andes of Ecuador. First, we tested if the quantity and quality of recovered DNA varied across roadkill specimens collected at different times since death, exploring if decomposition affected the potential to identify vertebrate species and associated microorganisms. Second, we compared DNA stability across taxa and tissues to identify potential limitations and offer recommendations for future work. Finally, we illustrate how these samples can aid in taxonomic identification and parasite detection. Our study shows that sampling roadkill can help study biodiversity. DNA was recovered and amplified (allowing species identification and parasite detection) from roadkill even 120 hours after death, although risk of degradation increased overtime. DNA was extracted from all vertebrate classes but in smaller quantities and with lower quality from amphibians. We recommend sampling liver if possible as it produced the highest amounts of DNA (muscle produced the lowest). Additional testing of this approach in areas with different environmental and traffic conditions is needed, but our results show that sampling roadkill specimens can help detect and potentially monitor biodiversity and could be a valuable approach to create biobanks and preserve genetic data.

Antibiotic resistance is a global threat to public health, and the search for new antibacterial therapies is a current research priority. The aim of this in silico study was to test nine new fluoroquinolones previously designed with potential leishmanicidal activity against Campylobacter jejuni, Escherichia coli, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Salmonella typhi, all of which are considered by the World Health Organization to resistant pathogens of global concern, through molecular docking and molecular dynamics (MD) simulations using wild-type (WT) and mutant-type (MT) DNA gyrases as biological targets. Our results showed that compound 9FQ had the best binding energy with the active site of E. coli in both molecular docking and molecular dynamics simulations. Compound 9FQ interacted with residues of quinolone resistance-determining region (QRDR) in GyrA and GyrB chains, which are important to enzyme activity and through which it could block DNA replication. In addition to compound 9FQ, compound 1FQ also showed a good affinity for DNA gyrase. Thus, these newly designed molecules could have antibacterial activity against Gram-negative microorganisms. These findings represent a promising starting point for further investigation through in vitro assays, which can validate the hypothesis and potentially facilitate the development of novel antibiotic drugs.

The work seeks to identify molecules with inhibitory activity against the DNA gyrase of Gram-negative microorganisms resistant to fluoroquinolones. Previously designed compounds were used to study antimicrobial potential in silico. Molecular docking was performed with nine new ciprofloxacin analog molecules, optimized through the PM6/ZDO theory level, in GyrA wild type (WT) and mutant type (MT) of C. jejuni, E. coli (6RKU PDB ID), N. gonorrhoeae, P. aeruginosa, S. enteritidis, and S. typhi. The molecule with the highest affinity for GyrA was selected based on its binding free energy and inhibition constant. In addition, a retrospective docking was carried out, to guarantee the correct affinity of the ligand to the receptor at the defined binding site. The results show a molecule with greater affinity for GyrA in five microorganisms, showing a binding free energy of less than −7.0 kcal/mol, suggesting a good antibacterial activity in silico.

Studying the microbial communities within the gastrointestinal tract of vertebrate species can provide insights into biodiversity, disease ecology, and conservation. Currently, we have very limited understanding of the composition of endogenous microbiota in wildlife, particularly in high biodiversity tropical areas. Knowledge is limited by the logistical and ethical challenges of obtaining samples for free-living animals. Roadkill carcasses offer a largely untapped source for biological material, including endogenous gut microbiota. These animals that have died on roads due to collisions with vehicles are suitable for accessible, opportunistic sampling. Here, we used metabarcoding for the V₃—V₄ region of the 16S rRNA gene in gut samples of nine roadkill samples collected from a road in Ecuador representing two vertebrate species: the speckled worm lizard (Amphisbaena bassleri) and the smooth-billed ani (Crotophaga ani). We successfully identify microbial phyla in both samples including Firmicutes, Bacteroidetes, and Proteobacteria for A. bassleri, and Firmicutes and Actinobacteria for C. ani. Our study provides the first description of the gut microbiota for these two vertebrates, and demonstrates the feasibility of studying endogenous microbial communities from roadkill material that can be opportunistically collected and preserved in biobanks.

Genome size variation in eukaryotic species is largely influenced by repetitive DNA sequences such as transposable elements (TEs), simple repeats, and satellite DNAs (satDNAs), which do not necessarily correlate with organismal complexity. In insects, TEs are crucial to evolutionary processes and are correlated with variations in genome size. In this study, we describe, for the first time, the mobilome and satellitome of Drosophila amaguana , an Ecuadorian Neotropical species with a large, unexplored genome size, to assess the contribution of these repetitive DNA sequences to its genome composition. Using a draft genome assembly of approximately 455.5 Mb, generated from Illumina short-read sequences obtained from 10 wild specimens of D. amaguana collected at the Refugio de Vida Silvestre Pasochoa, we employed a de novo approach to create a manually curated TE library of 737 consensus sequences. We identified 716 novel TE families that had not been previously described, 20 TEs previously characterized in other Drosophila species, and one DNA transposon previously described in the Lepeophtheirus genus. The total TE content in the D. amaguana genome was 21.54%, distributed as follows: 6.35% Helitrons (1 superfamily), 5.13% LTR retrotransposons (5 superfamilies), 3.63% TIRs (9 superfamilies), 3.61% LINEs (7 superfamilies), 1.17% MITEs, 0.94% Maverick, 0.67% PLE, 0.02% SINEs, and 0.01% DIRS. We also identified 11.8% of simple repeats. Additionally, we estimated the satDNA content using Illumina raw reads and identified 16 satDNA families, all unique to the Drosophila genus, which comprise 4.90% of the genome. Overall, our results based on short-read data suggest that the large genome size of D. amaguana may not be the consequence of a high amount of TEs or satDNAs. Instead, its large genome size could be attributed to other factors (e.g., noncoding DNA occupying substantial portions of the genome or a high percentage of duplicated genes) that remain to be determined or explored in future studies using long-reads to overcome short-reads limitations. These findings may currently offer valuable insights into the adaptative and evolutionary processes of the mesophragmatica species group in the Andean forests.

Wildlife hosts play important roles in the ecology and transmission of vector-borne parasites, yet information on host associations remains scarce in many biodiverse tropical regions. Within a One Health framework, characterizing parasite diversity in wildlife can improve understanding of ecosystem health and disease emergence. Road-killed animals provide a non-invasive opportunity to investigate host–parasite interactions while minimizing disturbance to natural habitats. We screened 127 liver and intestinal tissue samples obtained from 76 road-killed vertebrates collected near protected areas in two Ecuadorian biodiversity hotspots, the Tropical Andes and Chocó-Darién, for trypanosomatids and other vector-borne microorganisms. Molecular analyses targeted the 18S rRNA and cytochrome b genes of trypanosomatids and included additional screening for Trypanosoma cruzi , Trypanosoma rangeli , Rickettsia spp., and piroplasmids. Twenty-nine samples were positive for kinetoplastids. We detected diverse trypanosomatids representing the genera Leishmania , Porcisia , Trypanosoma , Phytomonas , Blastocrithidia , and Obscuromonas , as well as free-living kinetoplastids of the order Neobodonida. The most frequently detected species was Leishmania amazonensis , identified in 17 samples from at least 13 species of birds, reptiles, and caecilians, predominantly in liver tissue, suggesting previously unrecognized host associations. We also identified a putatively novel species of Porcisia and three potentially undescribed avian trypanosomes belonging to the subgenus Ornithotrypanum. No evidence of T. cruzi , T. rangeli , Rickettsia spp., or piroplasmids was found. Our findings identify birds, reptiles, and caecilians as potential reservoir hosts of L. amazonensis . In addition, we substantially expanded current knowledge of kinetoplastid diversity in Ecuadorian wildlife. This study demonstrates the value of road-killed animals as a practical, non-invasive resource for wildlife pathogen surveillance and highlights the importance of integrating biodiversity research into One Health approaches to better understand parasite transmission dynamics in rapidly changing tropical ecosystems.