Title

Bacterial cell wall components affect hemocyte composition and gene expression in the blue crab, Callinectes sapidus: An insight into effectors of immune function

Date of Award

2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Coastal Sciences, Gulf Coast Research Laboratory

First Advisor

Marius Brouwer

Advisor Department

Coastal Sciences, Gulf Coast Research Laboratory

Abstract

The blue crab, Callinectes sapidus (Rathbun), is a commercial and recreational valuable crustacean species which lives in a microbially rich environment. In contrast to vertebrates, invertebrates lack the large repertoire of antigenic-recognition receptors and immunological memory that define the essential characteristics of acquired immunity in mammals. In crustaceans, circulating hemocytes are essential in immunity and perform functions such as phagocytosis of non-self particles, encapsulation and melanization of foreign materials, production of reactive oxygen species and lysis of foreign cells. Despite supporting valuable recreational and commercial fisheries and their potential as an aquaculture species, studies on the immune function in blue crabs are limited and no large-scale gene expression work has been conducted to elucidate immune effectors of the blue crab. To better understand the immune response of C. sapidus to bacterial infection. We conducted a series of in-vivo experiments to identify the effects of injected LPS to identify a sublethal concentration of LPS capable of inducing significant hemocytopenia (hemocyte depletion); and to identify the time required for THC to recover to pre-LPS Injection levels (Chapter II). Based on our findings we then identified cytological changes occurring within hemocytes: (1) immediately after LPS injection (<5min), and (2) use DHC to assess longer term changes in population structure of circulating hemocytes. This lead to the construction of LPS-responsive SSH libraries to identify temporal changes in gene expression in response to LPS. In crustaceans, exposure to LPS elicits rapid and massive hemocyte degranulation followed by hemocytopenia. Hemocytopenia induced during infection constitutes a serious threat to the health of an animal by compromising its ability to fight off invading microorganisms. We investigated the effects of injected lipopolysaccharide (LPS) on the circulating hemocytes of the blue crab to determine: (1) the concentration which induced the most severe hemocytopenia, (2) the time to reach maximum hemocytopenia, and (3) the time course of recovery from hemocytopenia at sub-lethal concentrations. Our results showed a sublethal concentration of 0.1 μg/μl LPS (injected at 1μl/g body weight), induced maximum hemocytopenia (43%) at 30 min pi. Within 2 hours, THC had begun to recover (32%) with THC no longer being substantially different from preinjection levels within 6 hours (20%). However, THC only provides information on the total number of cells in circulation and does not provide information regarding possible changes in cell population structure. We used transmission electron microscopy (TEM) combined with differential hemocyte counts (DHC) to examine changes in hemocyte types in Saline and LPS injected individuals to: (1) identify cytological changes occurring within hemocytes immediately after (1, 3 & 5min) LPS injection, and (2) to assess longer term changes in population structure of circulating hemocytes (30 min, 2 h & 6 h pi). We show for the first time that exposure of blue crab hemocytes exposed to LPS in vivo results in immediate morphological and cytological changes. Longer term systemic effects, resulted in substantial changes in the structure of hemocyte populations at: (1) maximum hemocytopenia (30 min pi), (2) shortly after maximum hemocytopenia as THC begins to recover (2 h pi) and, (3) when THC between LPS and Saline injected controls were no longer significantly different (6 h pi). Based on the information obtained in Chapters II & III we injected blue crabs with 0.1μg/μl of either Saline or LPS (injected at 0.1μl/g) to constructed LPS-responsive SSH libraries from hemocytes and hepatopancreas at 0.5, 2 & 6 h post injection to identify and monitor possible temporal changes in gene expression after LPS injection. After stringent quality control procedures, 1282 (53%) sequences were retained for further analysis and were queried in NCBIs non-redundant protein databse (Blastx). Contigs and unassembled singlet sequences yielded seven genes categorized as being involved in immune function: Ferritin peptide, C-Type lectin, proPO activating factor, Anti-LPS factor, Crustin, serpin proteinase inhibitor 14, Annexin. Gene specific sense and anti-sense primers for each of the genes of interest (GOI) were designed to validate the results of SSH libraries using real-time RT-PCR. Although we observed changes in mean expression over time, two-way ANOVAs for each gene of interest (GOI) across time showed no significant differences. This is most likely due to high amounts of variation seen between individual blue crabs. Our results have shown, that on exposure to LPS, THC dramatically decreases within the first 30 mins after exposure to LPS. At 2 h pi, THC of LPS injected crabs begin to recover and at 6 h pi THC are no longer substantially different from their Saline injected counter parts. Secondly, blue crab hemocytes consist of a single cell lineage and transition from one stage to the next. We also know from the literature that, unlike insects, immune factors in crustacean hemocytes exist constitutively in the large and small electron dense granules and do not require synthesis in response to the detection of a pathogen. As such, it is likely that the altered transcription observed in our GOI is related to dramatic changes in the hemocyte population structure immediately after exposure to LPS due to the maturation of hemocytes as they transition from one stage to another.