Investigation of Nematocyst Discharge in Parasitic Cnidarians (Myxozoa)

Myxozoans are an enigmatic group of obligately parasitic, microscopic cnidarians. They diverged from their free-living relatives over 600 million years ago and have highly reduced genomes. However, they have retained nematocyst stinging cells which characterize the phylum Cnidaria. Free-living cnidarians utilize this cellular weaponry for defense and predation whereas the myxozoans use it to anchor to their hosts as the first step to infection. In this thesis, I explore similarities and differences in the structure and function of nematocysts from myxozoans and free-living cnidarians via a “double-bladed” approach consisting of wet-lab techniques and bioinformatic analyses. In chapter 2, I report my results from two wet-lab assays that I developed to test the effect of chemical treatments on nematocyst discharge and infection, using the model myxozoan Myxobolus cerebralis. I found that Na+ significantly increased discharge and reduced infection to rainbow trout. This finding aligned with prior work using free-living Cnidaria, suggesting divalent cations bound to poly-gamma glutamate inside the nematocyst can be exchanged with monovalent ions from the environment through the capsule wall to promote discharge. In Appendix A, I report my initial results from bioinformatic analyses of proteomic and transcriptomic datasets from the myxozoan Ceratonova shasta, in searches for genes for voltage-gated ion channels. In free-living Cnidarians, these channels play a role in signaling nematocyst discharge and exocytosis of Ca2+. I found that in C. shasta, genes for these channels were co-expressed with nematocyst-specific genes during sporogenesis in the fish host. Taken together, the results of my wet-lab and computational “double-bladed” approach demonstrate structural and functional homologies between the nematocysts of myxozoans and their free-living cnidarian relatives

The cnidarian parasite Ceratonova shasta utilizes inherited and recruited venom-like compounds during infection

Background Cnidarians are the most ancient venomous organisms. They store a cocktail of venom proteins inside unique stinging organelles called nematocysts. When a cnidarian encounters chemical and physical cues from a potential threat or prey animal, the nematocyst is triggered and fires a harpoon-like tubule to penetrate and inject venom into the prey. Nematocysts are present in all Cnidaria, including the morphologically simple Myxozoa, which are a speciose group of microscopic, spore-forming, obligate parasites of fish and invertebrates. Rather than predation or defense, myxozoans use nematocysts for adhesion to hosts, but the involvement of venom in this process is poorly understood. Recent work shows some myxozoans have a reduced repertoire of venom-like compounds (VLCs) relative to free-living cnidarians, however the function of these proteins is not known. Methods We searched for VLCs in the nematocyst proteome and a time-series infection transcriptome of Ceratonova shasta, a myxozoan parasite of salmonid fish. We used four parallel approaches to detect VLCs: BLAST and HMMER searches to preexisting cnidarian venom datasets, the machine learning tool ToxClassifier, and structural modeling of nematocyst proteomes. Sequences that scored positive by at least three methods were considered VLCs. We then mapped their time-series expressions in the fish host and analyzed their phylogenetic relatedness to sequences from other venomous animals. Results We identified eight VLCs, all of which have closely related sequences in other myxozoan datasets, suggesting a conserved venom profile across Myxozoa, and an overall reduction in venom diversity relative to free-living cnidarians. Expression of the VLCs over the 3-week fish infection varied considerably: three sequences were most expressed at one day post-exposure in the fish’s gills; whereas expression of the other five VLCs peaked at 21 days post-exposure in the intestines, coinciding with the formation of mature parasite spores with nematocysts. Expression of VLC genes early in infection, prior to the development of nematocysts, suggests venoms in C. shasta have been repurposed to facilitate parasite invasion and proliferation within the host. Molecular phylogenetics suggested some VLCs were inherited from a cnidarian ancestor, whereas others were more closely related to sequences from venomous non-Cnidarian organisms and thus may have gained qualities of venom components via convergent evolution. The presence of VLCs and their differential expression during parasite infection enrich the concept of what functions a “venom” can have and represent targets for designing therapeutics against myxozoan infections

How Do Parasites Infect Salmon? Comparative Transcriptomics Reveal Myxozoan Adaptations for Parasitism

The phylum Cnidaria contains three main branches: Anthozoa (corals, sea anemones) Medusozoa (jellyfish, hydra), and Endocnidozoa. This latter branch is characterized by parasitism and contains the microscopic fish-parasites Myxozoa. Myxozoa are highly simplified, consisting of only a few cell types, however they have retained the nematocyst stinging cells and complex life cycles of their free-living relatives. Myxozoans utilize vertebrate (usually fish) and invertebrate (usually annelid) hosts and alternate between two discrete spore stages. Globally, myxozoan infections of fish contribute to declines in wild populations and cause economic losses in aquaculture. Whereas free-living cnidarians use nematocysts for predation or defense, myxozoan parasites use their nematocysts to attach to fish in the early stages of infection. Understanding the molecular composition of myxozoan nematocysts and associated sensory features is essential to understanding myxozoan diseases, and for designing prevention strategies against them. In free-living cnidarians, molecular components related to nematocyst discharge have been explored for decades with almost no analogous studies in Myxozoa. The application of high-throughput sequencing and bioinformatic methods has furthered understanding of the unique biology of Myxozoa, however most studies have been restricted to the fish-infection period of the life cycle that produces worm-infective stages. In this dissertation, I explore myxozoan features for infection including nematocysts, sensory devices, and venom compounds. We develop new myxozoan transcriptomic datasets from infections in both hosts and utilize a variety of bioinformatic techniques to compare these features between life stages, species, and free-living and parasitic Cnidaria

Water system is a controlling variable modulating bacterial diversity of gastrointestinal tract and performance in rainbow trout

<div><p>A two-phase feeding study evaluating performance of rainbow trout and comparing luminal and mucosal gastrointestinal tract (GIT) bacterial community compositions when fed two alternative protein diets in two rearing systems was conducted. Alternative protein diets (animal protein and plant protein diets) balanced with crystalline amino acids: lysine, methionine and threonine or unbalanced, were fed to rainbow trout in two separate water systems (recirculating (RR) and flow-through (FF)) for a period of 16 weeks. The four diets, each contained 38% digestible protein and 20% fats, were fed to rainbow trout with an average weight of 12.02 ± 0.61 g, and sorted at 30 fish/tank and 12 tanks per dietary treatment. Phase 1 lasted for 8 weeks after which fish from each tank were randomly divided, with one-half moved to new tanks of the opposing system (i.e. from RR to FF and vice versa). The remaining halves were retained in their initial tank and system, and fed their original diets for another 8 weeks (phase 2). After the 16^th week, 3 fish/tank were sampled for each of proximate analysis, body indexes and 16S rRNA analysis of GIT microbiota. Fish weight (P = 0.0008, P = 0.0030, P<0.0010) and body fat (P = 0.0008, P = 0.0041, P = 0.0177) were significantly affected by diet, diet quality (balanced or unbalanced) and system, respectively. Feed intake (P = 0.0008) and body energy (P<0.0010) were altered by system. Body indexes were not affected by dietary treatment and water systems. Compositional dissimilarities existed between samples from the rearing water and GIT locations (ANOSIM: (R = 0.29, P = 0.0010), PERMANOVA: R = 0.39, P = 0.0010), but not in dietary samples (ANOSIM: R = 0.004, P = 0.3140, PERMANOVA: R = 0.008, P = 0.4540). Bacteria were predominantly from the phyla <i>Proteobacteria</i>, <i>Firmicutes</i> and <i>Bacteroidetes</i>. Their abundance differed with more dissimilarity in the luminal samples (ANOSIM: R = 0.40, P = 0.0010, PERMANOVA: R = 0.56, P = 0.0010) than those from the mucosal intestine (ANOSIM: R = 0.37, P = 0.0010, PERMANOVA: R = 0.41, P = 0.0010). Bacteria generally associated with carbohydrate and certain amino acids metabolism were observed in the mucosal intestine while rearing water appeared to serve as the main route of colonization of <i>Aeromonas</i> and <i>Acinetobacter</i> in the rainbow trout.</p></div

Comparison of bacterial diversity indexes of water samples from trout fed animal and plant protein diets.

<p>Comparison of bacterial diversity indexes of water samples from trout fed animal and plant protein diets.</p

Bar chart of the relative abundance of bacterial community compositions at genus level at GIT locations.

<p>Bar chart of the relative abundance of bacterial community compositions at genus level at GIT locations.</p

Bacterial composition at phylum level of rainbow trout fed animal and plant protein diets.

<p>Digesta and water samples are grouped by dietary treatments. Dietary treatments: Balanced Animal protein diet (APD), Unbalanced animal protein diet (UnAPD), Plant protein diet (PPD),Unbalanced plant protein diet) (UnPPD), Luminal samples by Balanced APD diet (Lum_APD), Luminal samples by Balanced PPD diet (Lum_PPD), Mucosal samples by Balanced APD diet (Muc_APD), Mucosal samples by Balanced PPD diet Muc_PPD); and Water samples: Samples from APD diet (W_APD), Samples from PPD diet (W_PPD), Samples from Unbalanced APD diet (W_UnAPD), Samples from Unbalanced PPD diet (W_UnPPD).</p

Chemical analysis (% dry weight) and apparent digestibility coefficients (ADCs) of nutrients in unbalanced or balanced APD and PPD.

<p>Chemical analysis (% dry weight) and apparent digestibility coefficients (ADCs) of nutrients in unbalanced or balanced APD and PPD.</p

Ingredients and chemical compositions of experimental diets.

<p>Ingredients and chemical compositions of experimental diets.</p