Studies of invertebrates have shown that the internal environment of crustaceans is not always sterile in normal conditions, and in many species, it can be populated by microorganisms even in the absence of any visible pathological processes in the body. This observation raises the question of whether genetically modified indigenous hemolymph microorganisms can be used for biotechnological purposes inside the crustacean either as local producers of some compounds or as sensors to physiological parameters. In this study, we tested the ability of the bacteria isolated from the hemolymph of the amphipod Eulimnogammarus verrucosus to hide from the cellular immune response of the host as the most important feature for their potential long-term application in vivo. 16S rDNA amplicon sequencing revealed five common bacterial genera in all analyzed samples of the amphipod hemolymph, among which Pseudomonas is most easily subjected to genome modification and, thus, the most prospective for biotechnological application. Cultivation of Pseudomonas gave us a number of strains undoubtedly derived from the amphipod hemolymph, and one of them (belonging to the Pseudomonas fluorescens group) was chosen for further tests. The primary culture of amphipod hemocytes was used to analyze the immunogenicity of the strain and showed a pronounced reaction of the immune cells to a high amount of the bacteria within six hours. This result indicates that modulation of cellular immune response to metabolically active bacterial cells is not mandatory for the survival and wide distribution of these microorganisms in the hemolymph of numerous amphipod individuals.
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We acknowledge Dr. Denis Axenov-Gribanov and Evgenii Protasov for help and advice on microbiological issues, as well as Dr. Polina Drozdova for help in the preparation of the manuscript.
The study was supported primarily by the Russian Foundation for Basic Research (#19–34-90137) and additionally by the Ministry of Science and Higher Education of Russia (#FZZE‐2020‐0026). AN was financially supported by the program «Mikhail Lomonosov» of the German Academic Exchange Service (DAAD) and the Ministry of Science and Higher Education of Russia (application #2300–21).
All experimental procedures with amphipods were conducted in accordance with the EU Directive 2010/63/EU for animal experiments and were approved by the Animal Subjects Research Committee of the Institute of Biology at Irkutsk State University.
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Below is the link to the electronic supplementary material.
Supplementary file1: Table S1. The full list of the strains isolated from hemolymph and chitin surface of E. verrucosus with data on MALDI-TOF MS identification (XLSX 48 KB)
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Shchapova, E., Nazarova, A., Vasilyeva, U. et al. Cellular Immune Response of an Endemic Lake Baikal Amphipod to Indigenous Pseudomonas sp. Mar Biotechnol (2021). https://doi.org/10.1007/s10126-021-10039-2