Environmental science

Since the creation of the Global Polio Eradication Initiative (GPEI) in 1988, significant progress has been made toward attaining a poliovirus-free world. This has resulted in the eradication of wild poliovirus (WPV) serotypes two (WPV2) and three (WPV3) and limited transmission of serotype one (WPV1) in Pakistan and Afghanistan. However, the increased emergence of circulating vaccine-derived poliovirus (cVDPV) and the continued circulation of WPV1, although limited to two countries, pose a continuous threat of international spread of poliovirus. These challenges highlight the need to further strengthen surveillance and outbreak responses, particularly in the African Region (AFRO). Phylogeographic visualization tools may provide insights into changes in poliovirus epidemiology, which can in turn guide the implementation of more strategic and effective supplementary immunization activities and improved outbreak response and surveillance. We created a comprehensive protocol for the phylogeographic analysis of polioviruses using Nextstrain, a powerful open-source tool for real-time interactive visualization of virus sequencing data. It is expected that this protocol will support poliovirus elimination strategies in AFRO and contribute significantly to global eradication strategies. These tools have been utilized for other pathogens of public health importance, for example, SARS-CoV-2, human influenza, Ebola, and Mpox, among others, through real-time tracking of pathogen evolution (https://nextstrain.org), harnessing the scientific and public health potential of pathogen genome data.

Plastic pollution presents a looming danger to the environment and virtually all life on planet Earth. Especially pernicious are nanoplastics (NPs), which are plastic fragments with dimensions ≤1 μm. Conventional detection methods are ineffective for NPs, while their high specific surface area renders them efficient carriers of toxic substances; additionally, they may even be inherently toxic. Although NP waste chiefly arises from environmental weathering of larger plastic fragments, most published studies employed manufactured pristine NPs of uniform size and shape. Furthermore, almost all NP effects were studied using polystyrene (PS) as a convenient model material, despite PS accounting for <6% of all plastic pollution. There is thus an urgent need to expand investigations of environmental NP pollution and effects on biota. The present work provides a comprehensive roadmap for studying the effects of “real-world” NP pollution on living systems, using, for example, lung alveolar epithelial cells on which such NPs deposit by breathing ambient air. Herein, we describe detailed in-house methods to fabricate various NPs that are weathered with UV light and O₃ gas exposure to more closely mimic real environmental NPs. We also illustrate a simple and straightforward bioelectrical method for assessing passive and active ion transport properties of primary rat lung alveolar epithelial cell monolayers as a model for the distal mammalian lung exposed to one of the generated NPs. This protocol allows researchers to rapidly and more accurately assess the biological impact of various simulated environmental NPs on a vulnerable air–blood barrier in the lung.

The mass rearing of anopheline mosquitoes under laboratory conditions is essential for advancing malaria research. It facilitates in-depth studies on mosquito biology, behavior, and genetics and their role in Plasmodium transmission. However, the colonization of Neotropical anophelines such as Anopheles darlingi—a primary malaria vector in the Amazon region—has proven particularly challenging due to its unique reproductive characteristics. Unlike other species that can initially be colonized using forced copulation methods and later adapt to natural mating, An. darlingi does not copulate under forced conditions. Recent breakthroughs in An. darlingi colonization have been achieved using flashlight induction techniques, which have enabled the establishment and maintenance of stable laboratory populations. These advancements have created new opportunities for vector control studies in Brazil, including the testing of innovative control methods and Plasmodium transmission-blocking strategies. This protocol offers a comprehensive, step-by-step guide for initiating and scaling up large laboratory colonies of An. darlingi and An. deaneorum, a secondary malaria vector. It details methods for copulation induction, colony management, and successful artificial infection of mosquitoes with Plasmodium vivax. The guide serves as a critical resource for establishing new Neotropical anopheline colonies from different populations, contributing to future malaria research and control efforts in the Amazon. Additionally, the establishment of Brazil’s first Malaria Vector Production and Infection Platform (Plataforma de Produção e Infecção de Vetores da Malária, PIVEM) has further supported the development of new control technologies and the study of P. vivax–Anopheles interaction, advancing efforts to combat malaria in the region.

Daphnia magna is a well-established model organism in ecotoxicology, environmental monitoring, and genetics due to its sensitivity to pollutants, its pivotal role in freshwater ecosystems, and its well-characterized genome. Despite its extensive use in these fields, there is a notable lack of established protocols for developing primary cell culture systems and conducting transgenic studies in Daphnia spp. This study addresses these gaps by optimizing a medium and standardizing protocols for primary cell culture and transgenic experiments in D. magna. Primary cell cultures were established from both D. magna embryos and whole organisms, with medium optimization verified using XTT assay. Cell viability was sustained for over two months using a modified Schneider’s insect medium enriched with FBS, glucose, MEM vitamin mix, and selenium. DNA replication and cell proliferation were confirmed through BrdU labeling. Both mechanical and enzymatic passaging methods were compared, resulting in 20% and 10% cell attachment, respectively. For transgenic applications, this study successfully standardized plasmid-mediated lipofection and baculovirus-mediated transduction, achieving success rates of 52% and 45%. These findings represent a pioneering effort in D. magna embryonic cell culture, offering a reliable in vitro platform for future biological research, including ecotoxicological and epigenetic investigations. The established protocols and optimized cell culture medium have significant implications for advancing crustacean cell line research and transgenic model development, enhancing our understanding of biological processes in controlled laboratory environments.

Wastewater-based surveillance (WBS) can provide a wealth of information regarding the health status of communities from measurements of nucleic acids found in wastewater. Processing workflows for WBS typically include sample collection, a primary concentration step, and lysis of the microbes to release nucleic acids, followed by nucleic acid purification and molecular-based quantification. This manuscript provides workflows from beginning to end with an emphasis on filtration-based concentration approaches coupled with specific lysis and nucleic acid extraction processes. Here, two WBS processing approaches are presented, one focusing on RNA-specific pathogens and the other focused on DNA-specific pathogens found within wastewater: 1) The RNA-specific approach, employed for analyzing RNA viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) couples electronegative filtration of wastewater with the placement of the filter within a lysis buffer followed by direct RNA extraction. 2) The DNA-specific approach, employed for analyzing DNA pathogens like Candida auris, uses size selection membranes during filtration, subsequently followed by a lysis buffer, bead-beating, and DNA extraction. Separate workflows for RNA versus DNA isolations have the advantage of improving the detection of the target pathogen. A novel aspect of the RNA-specific workflow is the direct extraction of nucleic acids from filter lysates, which shows enhanced recoveries, whereas the DNA-specific approach requires bead beating prior to extraction. Novelty is also provided in a new qPCR approach called Volcano 2nd Generation (V2G), which uses a polymerase capable of using RNA as a template, bypassing the reverse transcriptase step normally required for qPCR.

Campylobacter jejuni, a widespread pathogen found in birds and mammals, poses a significant risk for zoonosis worldwide despite its susceptibility to environmental and food-processing stressors. One of its main survival mechanisms is the formation of biofilms that can withstand various food-processing stressors, which is why efficient methods for assessing biofilms are crucial. Existing methods, including the classical culture-based plate counting method, biomass-staining methods (e.g., crystal violet and safranin), DNA-staining methods, those that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts and resazurin), immunofluorescence with flow cytometry or fluorescence microscopy, and PCR-based methods for quantification of bacterial DNA, are diverse but often lack specificity, sensitivity, and suitability. In response to these limitations, we propose an innovative approach using NanoLuc as a reporter protein. The established protocol involves growing biofilms in microtiter plates, washing unattached cells, adding Nano-Glo luciferase substrate, and measuring bioluminescence. The bacterial concentrations in the biofilms are calculated by linear regression based on the calibration curve generated with known cell concentrations. The NanoLuc protein offers a number of advantages, such as its small size, temperature stability, and highly efficient bioluminescence, enabling rapid, non-invasive, and comprehensive assessment of biofilms together with quantification of a wide range of cell states. Although this method is limited to laboratory use due to the involvement of genetically modified organisms, it provides valuable insights into C. jejuni biofilm dynamics that could indirectly help in the development of improved food safety measures.

In this paper, we present a detailed protocol for microinjecting DNA, RNA, or protein solutions into fertilized eggs of the multicolored Asian ladybird beetle, Harmonia axyridis, under a stereomicroscope equipped with an injection apparatus. H. axyridis is an emerging model organism for studying various biological fields, showing intraspecific polymorphisms exhibiting highly diverse color patterns on the elytra. Here, we describe how to rear ladybird beetles in a laboratory and obtain fertilized eggs for microinjection experiments. We also provide a constant fluid flow injection method, which enhances the efficiency of microinjection and improves throughput. Our step-by-step protocol is applicable to generating transgenic or genome-edited ladybird beetles, facilitating functional genetics in H. axyridis; the microinjection method should be applicable to other insect eggs.

The ectoparasites of rodents and other small mammals usually involve five categories of arthropods—fleas, sucking lice, gamasid mites, chigger mites, and occasionally, ticks. These ectoparasites are medically important, serving as vectors for diseases such as plague, murine typhus, scrub typhus, forest encephalitis, Lyme disease, and other zoonoses. Field surveys, collection, and specimen preparation of ectoparasites are crucial for studying taxonomy, faunistics, ecology, and epidemiology. They are also essential for vector surveillance. The present protocol summarizes the on-site monitoring and specimen-making of ectoparasites of rodents and other sympatric small mammals. Besides the collection and specimen preparation of small mammal hosts, the protocol describes in detail the collection, fixation, specimen-making, and taxonomic identification of ectoparasites and provides some monitoring indices. The on-site monitoring indices include the host density index and the infestation indices of ectoparasites (prevalence, mean abundance, mean intensity). The methodologies outlined in this protocol provide technical guidance and references for vector monitoring (surveillance) and control.

Morphology underpins key biological and evolutionary processes that remain elusive. This is in part due to the limitations in robustly and quantitatively analyzing shapes within and between groups in an unbiased and high-throughput manner. Geometric morphometrics (GM) has emerged as a widely employed technique for studying shape variation in biology and evolution. This study presents a comprehensive workflow for conducting geometric morphometric analysis of fish morphology. The step-by-step manual provides detailed instructions for using popular free software, such as the TPS series, MorphoJ, ImageJ, and R, to carry out generalized Procrustes analysis (GPA), principal component analysis (PCA), discriminant function analysis (DFA), canonical variate analysis (CVA), mean shape analysis, and thin plate spline analysis (TPS). The Momocs package in R is specifically utilized for in-depth analysis of fish outlines. In addition, selected functions from the dplyr package are used to assist in the analysis. The full process of fish outline analysis is covered, including extracting outline coordinates, converting and scaling data, defining landmarks, creating data objects, analyzing outline differences, and visualizing results. In conclusion, the current protocol compiles a detailed method for evaluating fish shape variation based on landmarks and outlines. As the field of GM continues to evolve and related software develops rapidly, the limitations associated with morphological analysis of fish are expected to decrease. Interoperable data formats and analytical methods may facilitate the sharing of morphological data and help resolve related scientific problems. The convenience of this protocol allows for fast and effective morphological analysis. Furthermore, this detailed protocol could be adapted to assess image-based differences across a broader range of species or to analyze morphological data of the same species from different origins.

Many studies on mosquito biology rely on laboratory-reared colonies, emphasizing the need for standardized protocols to investigate critical aspects such as disease biology, mosquito behavior, and vector control methods. While much knowledge is derived from anthropophilic species from genera like Anopheles, Aedes, and Culex, there is a growing interest in studying mosquitoes that feed on non-human hosts. This interest stems from the desire to gain a deeper understanding of the evolution of diverse host range use and host specificity. However, there is currently a limited number of comprehensive protocols for studying such species. Considering this gap, we present a protocol for rearing Uranotaenia lowii, a mosquito species specialized in feeding on anuran amphibians by eavesdropping on host-emitted sound cues. Additionally, we provide instructions for successfully shipping live specimens to promote research on this species and similar ones. This protocol helps fill the current gap in comprehensive guidelines for rearing and maintaining colonies of anuran host–biting mosquitoes. It serves as a valuable resource for researchers seeking to establish colonies of mosquito species from the Uranotaeniini tribe. Ultimately, this protocol may facilitate research on the evolutionary ecology of Culicidae, as this family has recently been proposed to have originated from a frog-feeding ancestor.