Ethics statement
Experiments in zebrafish were conducted in strict accordance with the recommendations of the European Guide for the Care and Use of Laboratory Animals. Fish were housed and experiments were conducted at an experimental facility (Catalonia Institute for Energy Research [IREC], Ciudad Real, Spain) with the approval and supervision of the Ethics Committee on Animal Experimentation of the University of Castilla La Mancha (PR-2021-09-14) and the Department of Agriculture, Environment and Rural Development of Castilla La Mancha (REGA code ES130340000218).
Experimental design
The experiment was designed to characterize tick saliva components associated with allergic reactions to mammalian meat consumption in the zebrafish model of AGS.
Tick saliva protein and non-protein fractions
Tick saliva (135 µl) was diluted 1:1 in PBS, and 255 µl was filtered twice through an Amicon 3 kDa unit (Merck & Co., Inc., Kenilworth, NJ, USA). Of this, 200 µl passed through the Amicon membrane and was considered the non-protein fraction. The 50 µl that did not pass through the Amicon membrane was considered the protein fraction.
Glycosidase treatment of tick saliva
For protein deglycosylation, 20 µl of tick saliva was incubated under denaturing conditions with a cocktail of α-Gal-free glycosidases using the EDEGLY enzymatic protein deglycosylation kit (Merck & Co., Inc.) and following the manufacturer’s recommendations. After deglycosylation, the tick saliva sample was diluted 1:20 in PBS for filtration.
Tick saliva protein fractionation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and characterization by mass spectrometry analysis
To obtain the different tick saliva protein fractions, 70 µg from the saliva protein fraction was mixed in 1:1 proportion with Laemmli sample buffer and applied onto two 10% SDS-PAGE gels. In one gel, protein bands were visualized by staining with GelCode Blue Stain Reagent (Thermo Fisher Scientific, Waltham, MA, USA), excised, cut into small cubes, and digested overnight at 37 °C with trypsin. The resulting tryptic peptides from each band were extracted and analyzed by reversed-phase liquid chromatography coupled to mass spectrometry (RP-LC–MS/MS) using an EASY-nLC II system coupled online to an LTQ Linear Ion Trap mass spectrometer (Thermo Fisher Scientific).
Tick protein annotations
Tick proteins identified after proteomics analysis were annotated for Gene Ontology in UniProt and VectorBase. Biological processes include annotations in Drosophila or human proteins that may be related to AGS when information is not available in tick species.
Quantitation of tick saliva proteins and α-Gal content
Protein and α-Gal content in tick saliva were determined in whole saliva and protein, non-protein, and deglycosylated fractions. The α-Gal levels were determined by an in-house enzyme-linked immunosorbent assay (ELISA) using tick saliva fractions in comparison with pig kidney (α-Gal-positive control) and human promyelocytic leukemia HL60 cells (α-Gal-negative control).
Zebrafish
Wild-type adult AB strain zebrafish were used for the experiment. Zebrafish were maintained in a flow-through water system and fed fish feed during pretreatment and dog food containing mammalian meat from day 2 until the end of the experiment at day 8.
Characterization of anti-tick protein IgM antibody titers in zebrafish
Tick salivary gland protein extracts were prepared from salivary glands of I. ricinus ticks and used to quantify anti-tick protein IgM antibody titers in zebrafish serum samples.
Characterization of anti-α-Gal IgM antibody titers in zebrafish
The ELISA was conducted to quantify anti-α-Gal IgM antibody titers in zebrafish serum samples using α-Gal-coated plates.
Characterization of anti-glycan IgM antibody response in zebrafish
The glycochip array containing glycans and bacterial polysaccharides was used to characterize the anti-glycan IgM antibody response in zebrafish serum samples.
Statistical analyses
Statistical analyses were performed to compare the incidence of allergic reactions, abnormal behavior and feeding patterns, and mortality in zebrafish between treatments. One-way ANOVA tests with post hoc Tukey HSD tests were used for comparisons. One-way ANOVA tests with Bonferroni–Holm multiple comparisons were used to compare anti-tick proteins and anti-α-Gal IgM antibody titers between treatments.
SDGs, Targets, and Indicators
SDGs Addressed:
- SDG 3: Good Health and Well-being
- SDG 12: Responsible Consumption and Production
- SDG 15: Life on Land
Targets Identified:
- Target 3.9: By 2030, substantially reduce the number of deaths and illnesses from hazardous chemicals and air, water, and soil pollution and contamination.
- Target 12.4: By 2020, achieve the environmentally sound management of chemicals and all wastes throughout their life cycle, in accordance with agreed international frameworks, and significantly reduce their release to air, water, and soil in order to minimize their adverse impacts on human health and the environment.
- Target 15.5: Take urgent and significant action to reduce the degradation of natural habitats, halt the loss of biodiversity, and protect and prevent the extinction of threatened species.
Indicators:
- Indicator 3.9.1: Mortality rate attributed to household and ambient air pollution
- Indicator 12.4.1: Number of parties to international multilateral environmental agreements on hazardous waste, hazardous chemicals, and other pollutants
- Indicator 15.5.1: Red List Index
Explanation:
1. SDG 3: Good Health and Well-being is addressed as the article discusses experiments conducted on zebrafish to study allergic reactions to tick saliva components associated with mammalian meat consumption.
2. Based on the content of the article, the specific targets that can be identified are:
– Target 3.9: By 2030, substantially reduce the number of deaths and illnesses from hazardous chemicals and air, water, and soil pollution and contamination.
– Target 12.4: By 2020, achieve the environmentally sound management of chemicals and all wastes throughout their life cycle, in accordance with agreed international frameworks, and significantly reduce their release to air, water, and soil in order to minimize their adverse impacts on human health and the environment.
– Target 15.5: Take urgent and significant action to reduce the degradation of natural habitats, halt the loss of biodiversity, and protect and prevent the extinction of threatened species.
3. The indicators mentioned or implied in the article are:
– Indicator 3.9.1: Mortality rate attributed to household and ambient air pollution.
– Indicator 12.4.1: Number of parties to international multilateral environmental agreements on hazardous waste, hazardous chemicals, and other pollutants.
– Indicator 15.5.1: Red List Index.
These indicators can be used to measure progress towards the identified targets by monitoring mortality rates related to air pollution, the number of parties involved in environmental agreements on hazardous waste and chemicals, and the status of threatened species.
Table: SDGs, Targets, and Indicators
| SDGs | Targets | Indicators |
|---|---|---|
| SDG 3: Good Health and Well-being | Target 3.9: By 2030, substantially reduce the number of deaths and illnesses from hazardous chemicals and air, water, and soil pollution and contamination. | Indicator 3.9.1: Mortality rate attributed to household and ambient air pollution |
| SDG 12: Responsible Consumption and Production | Target 12.4: By 2020, achieve the environmentally sound management of chemicals and all wastes throughout their life cycle, in accordance with agreed international frameworks, and significantly reduce their release to air, water, and soil in order to minimize their adverse impacts on human health and the environment. | Indicator 12.4.1: Number of parties to international multilateral environmental agreements on hazardous waste, hazardous chemicals, and other pollutants |
| Target 15.5: Take urgent and significant action to reduce the degradation of natural habitats, halt the loss of biodiversity, and protect and prevent the extinction of threatened species. | Indicator 15.5.1: Red List Index |
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Source: parasitesandvectors.biomedcentral.com
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