How Bacteria in Snails (Mollusca: Gastropoda) Differ in Relation to Digenean (Platyhelminthes) Infection: An Unfinished Study Rebecca Wolff*, Andrew McElwain*, Peter Newell**Department of Biological Sciences, State University of New York Oswego
We did not find the information we were looking for. Iâ€™m still going to go through everything we did, why it did not work, what could have been done instead, and sometimes, why we chose to do it the way we did knowing the other ways it could have been done. That being said, letâ€™s get into it.
Background: Digenea (Platyhelminthes, Trematoda) Parasitic Worms known for their two host life cycle Intermediate hosts are very commonly molluscs, particularly snails (Gastropoda)Infection is located in the gut of the snailPechenik 2015, Esch and Fernandez 1994 https://www.sciencedirect.com/topics/immunology andmicrobiology/digenea
Background: Microbiome The community of bacteria associated with an organism Help the host organism to digest food and interact with the immune systemComposition of the microbiome is shaped by the environment inside and around the organismCarroll et al 2019
Introduction There are very few studies on the interaction of between bacteria and parasites (at least in gastropods). We chose Digenea and a freshwater snail population as a model for microbiome/parasite interaction due to local availability. The snails serve as a micro ecosystem, and the parasites and bacteria should interact to some extent simply due to proximity.If parasites and bacteria interact in any capacity , there should be differences in the microbiome between snails with and without parasites.
Hypothesis Two main ways the microbiome could have differed:Composition: the same species are present between groups but in different concentrationsEx) Species A composes 95% of microbiome in parasite free snails, but 16% of infected snailsDiversity: different species are present between groupsEx) Species B is present in infected snails, but absent in parasite free snailsNull Hypothesis: there are no significant differences between the microbiota
Overarching Study Design Two main groups: infected and uninfected snail Individuals were dissected, isolated, PCR, and sequencedBacterial analysis was attempted, but ultimately failedParasites were identified genetically
Collection Two collection locations: Fallbrook and Rice Creek Green TrailA total of 74 snails were collected (with permit)Collection occurred over the summer (Late June Late August) https://www.oswego.edu/ricecreek/trails overview
Dissection Individuals were deshelled, and beheaded.Parasite infection was identified by type (vermiform, and sporocyst and/or cercaria), most infections were heavy. Sporocyst infections were coupled with cercaria. This is just how the life cycle works. Spire was used for DNA isolation, as that contained both the bacterial and parasite samples. Separate parasite sample were taken when possible for separate genetic analysis. 1 2 3 4Picture 2: Vermiform, Pictures 1 and 4: cercaria, Picture 3: sporocyst and cercaria
DNA Isolation of Snail samples Phenol chloroform protocol most efficient to remove proteins and polysaccharides Mucopolysaccharides are PCR inhibitors CTAB binds to mucopolysaccharides WORKFLOW Add T.E., 10%SDS, and homogenization beads to tissue sample. Shake vigorously. Add NaCl and CTAB and incubate for 10 minutes. Add proteinase K and incubate for 1 hour. Alternate between adding chloroform:isoamyl and phenol chloroform, putting liquid layer into a new tube each time. Add isopropanol to precipitate DNA, then add ethanol to wash. Nishiguchi et al 2002, Sokolov 2000, Theodorakis 2014 and Winnepenninckx et al 1993
DNA Isolation of Parasite samples The separated parasite samples were isolated using Qiagenâ€™s DNeasy Blood + Tissue Kit. The samples were washed twice with water to remove snail tissue remnants, then the manufacturerâ€™s protocol was followed. https://www.qiagen.com/br/products/topsellers/dneasy bloodandtissue kit/#orderinginformation
PCR of Bacteria Uses primers and enzymes to copy specific fragments of DNAUsed to amplify the 16s gene that is common to most bacterial species Was unsuccessful, mostly likely due to proportion Adapted from https://www.repertoire.co.jp/
PCR of Parasites 18s rRNA gene was targetedFirst tested primers for qPCR, this failedUsed a different primer pair to target whole gene in separated parasites, this was successful Adapted from https://www.repertoire.co.jp
Sequence Analysis and Identification The PCR result were purified using GeneJET PCR Purification Kit. Sequences were outsourcedUsed BLASTn default settings to identify the sequencesThree sequences were analyzed for each sample the forward, reverse, and joinedF: 5â€™ ATGTACAGTAGTAG 3â€™ R: 3â€™ ACTGGACATGATGA 5â€™ F: 5â€™ ATGTACAGT AGTAG 3â€™ + RC: 5â€™ AGTAG TACAGGTCA 3â€™ Joined Sequence: 5â€™ ATGTACAGT AGTAG TACAGGTCA 3â€™
Results Three digeneans were identified: Lecithodendriidae sp. PAFlukeA, Braunina condiformis, and Collyriclum fabaSome samples returned with high background, indicative of multiple PCR productsSome samples returned as Batillaria cumingi, a snail species The top blast hits of the joined sequences with the percent identity of each individual.
Lecithodendriidae sp. PAFlukeA Specimens only identified up to FamilyMainly parasitize bats and occasionally birds (Lotz, J. M. & Font, W. F., 2008) https://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&sea rch_value=56498#null
Braunina cordiformis Observed as the vermiform sporocystsMost of the literature involves dolphin hostsWe most likely found a close relative. https://www.biolib.cz/en/taxon/id82031/
Collyriclum faba Infects birdsA study by Heneberg et al. (2015) described the complete lifecycle of a Central European population.Was observed to have two intermediate hosts, a snail and a mayflyIn definitive hosts, C. faba form cysts that can cause anemia and emaciation when abundant. (Heneberg et al., 2015) https://www.researchgate.net/ publication/324601752_Collyr iclosis_in_Red backed_Shrikes_Lanius_Coll urio_from_Israel_and_Czech _Republic/figures?lo=1 Cercaria of Collyriclum (Heneberg et al., 2015)
Conclusion The bacterial communities of these snails are still unknown.The snail species and parasites that were genetically identified are most likely close relatives to the collected specimens as the known attributes of the identified species are inconsistent with the collection environment. https://taconet.pixnet.net/blog/post/47063644
Thank you to: Peter Newell and Andrew McElwain for their guidance and help. The Student Challenge Grant and RCA Small Grants for their support in this project.
If you have any questions, feel free to contact Rebecca Wolff at email@example.com or Peter Newell at firstname.lastname@example.org
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