Exosomal miR-224 contributes to hemolymph microbiota 4 homeostasis during bacterial infection in crustacean 5 6

8 9 1Guangdong Provincial Key Laboratory of Marine Biology, Shantou University, 10 Shantou 515063, China 11 2Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou, 12 China 13 3Institute of Marine Sciences, Shantou University, Shantou 515063, China 14 4Division of Genetics and Molecular Biology, Institute of Biological Science, Faculty 15 of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

Unfortunately, uncontrol proliferation of hemolymph microbiota could result in host . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

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The involvement of exosomes in anti-bacterial response in mud crab . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in To explore the involvement of exosomes from mud crab in bacterial infection, 137 exosomes isolated from the hemolymph of V. parahaemolyticus-challenged mud crabs 138 (i.e., exosome-Vp) and PBS (Phosphate buffer saline)-injected control crabs (i.e., 139 exosome-PBS). The typical cup-shaped structures of isolated exosomes were observed 140 under an electron microscope (Fig. 1A) and their sizes measured by Nanosight particle 141 tracking analysis (Fig. 1B). The isolated particles were further ascertained as exosomes 142 by determining the exosomal protein markers Flotillin-1, TSG101 and the cytoplasmic 143 marker Calnexin using Western blot analysis (Fig. 1C). These results indicate 144 successful isolation of exosomes from mud crabs challenged with V. parahaemolyticus 145 and PBS. 146 Next, the ability of the isolated exosomes to be internalized by mud crab 147 hemocytes was analyzed by labeling the isolated exosomes with DiO (green) before 148 being injected into mud crabs. When hemocytes from the injected crabs were collected 149 and labeled with DiI (red) before being examined with a confocal laser scanning 150 microscope, the results showed that the isolated exosomes could be internalized in 151 hemocytes (Fig. 1D). To explore the involvement of exosomes in mud crab during 152 pathogenic bacteria infection, the isolated exosomes (exosome-Vp and exosome-PBS) 153 were mixed with V. parahaemolyticus or PBS (control) before being injected into mud 154 crabs to determine the mortality rate. As shown in Fig. 1E, there was significant 155 reduction in the mortality rate of mud crabs injected with exosome-Vp mixed with V. 156 parahaemolyticus compared with mud crabs injected with exosome-PBS mixed with 157 V. parahaemolyticus, which suggest that exosomes isolated from V. parahaemolyticus-158 challenged mud crabs have an effect on pathogenic bacteria infection and therefore 159 affecting the mud crab mortality rate. Moreover, when the relative abundance of 160 hemolymph bacteria in mud crabs was determined, the results revealed that exosome-. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423222 doi: bioRxiv preprint Vp was able to inhibit the rapid increase in hemolymph bacteria during infection (Fig.   162 1F). Taken together, these results suggest that exosomes secreted by V. 163 parahaemolyticus-challenged mud crabs play a role in anti-bacterial response in mud 164 crabs, probably by helping to maintain homeostasis of hemolymph microbiota.

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Exosomes modulate hemolymph microbiota homeostasis 166 To ascertain the regulatory function of exosomes in modulating the mud crab  Similarly, transcript levels of ALF1, ALF4 and ALF5 were significantly increased in 173 mud crabs treated with exosome-Vp (Fig. 2C). Next, the bacteria species and 174 composition of hemolymph microbiota were analyzed using 16S rDNA sequencing. As  when mud crabs were treated with exosome-Vp, microbiota homeostasis was 183 maintained in mud crabs during the infection (Fig. 2E). A similar trend was observed 184 when the top 35 genera of hemolymph microbiota was analyzed (Fig. 2F), which is in 185 agreement with our data found in V. parahaemolyticus-resistant crabs collected from . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423222 doi: bioRxiv preprint the field (unpublished results). All these results suggest that during pathogenic bacteria 187 infections in mud crabs, exosomes modulate pathways that maintain the homeostasis of 188 hemolymph microbiota by regulating the levels of mROS and ALFs.

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Functional miRNA screening in exosomes 190 To determine the functional exosomal miRNAs that are crucial in modulating 191 hemolymph microbiota homeostasis, miRNA microarray analysis was carried out using   and 3F). A similarly trend was observed in the expression levels of ALF1, ALF4 and . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423222 doi: bioRxiv preprint 211 ALF5 for these mud crab samples (Fig. 3G). In addition, 16S rDNA sequencing 212 analysis revealed a disruption in the exosome-Vp-mediated hemolymph microbiota 213 homeostasis upon miR-224 silencing ( Fig. 3H and 3I). These results suggest that miR- fluorescence microscope, both miR-224 and HSP70 mRNA were found to co-localize . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423222 doi: bioRxiv preprint in hemocytes of the mud crabs (Fig. 4I). All these results suggest that HSP70 is the 237 direct target gene of miR-224 in the mud crabs.  CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in   These results indicate that released TRAF6 translocate to the mitochondria to form a . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423222 doi: bioRxiv preprint 286 complex with Ecsit, which then mediates mROS production. Ecsit is not only found in . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in pathogens infection (Fig. 8).             The authors declare no conflicts of interest.
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020.  . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in     . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423222 doi: bioRxiv preprint  CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423222 doi: bioRxiv preprint proteins that bind to HSP70. Mud crab hemocytes lysates were subjected to Co-. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423222 doi: bioRxiv preprint