An annotated cDNA library of juvenile Euprymna scolopes with and without colonization by the symbiont Vibrio fischeri

Abstract

Background: Biologists are becoming increasingly aware that the interaction of animals, including humans, with their coevolved bacterial partners is essential for health. This growing awareness has been a driving force for the development of models for the study of beneficial animal-bacterial interactions. In the squid-vibrio model, symbiotic Vibrio fischeri induce dramatic developmental changes in the light organ of host Euprymna scolopes over the first hours to days of their partnership. We report here the creation of a juvenile light-organ specific EST database.

Results: We generated eleven cDNA libraries from the light organ of E. scolopes at developmentally significant time points with and without colonization by V. fischeri. Single pass 3' sequencing efforts generated 42,564 expressed sequence tags (ESTs) of which 35,421 passed our quality criteria and were then clustered via the UIcluster program into 13,962 nonredundant sequences. The cDNA clones representing these nonredundant sequences were sequenced from the 5' end of the vector and 58% of these resulting sequences overlapped significantly with the associated 3' sequence to generate 8,067 contigs with an average sequence length of 1,065 bp. All sequences were annotated with BLASTX (E-value < -03) and Gene Ontology (GO).

Conclusion: Both the number of ESTs generated from each library and GO categorizations are reflective of the activity state of the light organ during these early stages of symbiosis. Future analyses of the sequences identified in these libraries promise to provide valuable information not only about pathways involved in colonization and early development of the squid light organ, but also about pathways conserved in response to bacterial colonization across the animal kingdom.

Figure 1

The light organ system of E. scolopes. A. A swimming adult animal. The light organ (internal to the boxed area) is located in the center of the mantle cavity. Bar, 1 cm. B. A confocal image of a light organ (labeled with CellTracker, Molecular Probes) within a diagram of a newly hatched animal. The juvenile organ has a complex, superficial ciliated epithelium (sce) that facilitates colonization by the symbioint. Bar, 200 microns. C. A confocal image of the sites of V. fischeri entry into host tissues (labeled with acridine organ, AO). A set of three pores, into which aggregated V. fischeri cells will migrate, are located at the base of each lateral field of ciliated epithelial cells. In response to interactions with colonizing V. fischeri, host hemocytes (h) migrate into the sce, which will be lost during symbiont-induced light organ morphogenesis. Also visible in this image is the condensed chromatin characteristic of symbiont-induced apoptosis (arrows), which stains vividly with AO. Bar, 20 microns. D. A histological section revealing the path traversed by colonizing symbionts. Once aggregated in mucus outside the light organ, the symbionts enter the pores (p), travel up long ciliated ducts (d) and enter the crypt spaces where they interact with two cell types, the polarized epithelium that lines the crypts (ce) and a transient population of host hemocytes (h). Bar, 30 microns.

Figure 2

The time points chosen for cDNA library construction. The diagram illustrates when, in the context of the symbiont-induced developmental program of the host light organ and major milestones in V. fischeri colonization, tissues for library construction were dissected from juvenile animals. As the symbionts aggregate, migrate into host tissues and take up residence in the light organ crypts, they induce the diagrammed series of developmental changes in the various components of the organ. Most of these changes, including the irreversible loss of the SCE, which doesn't culminate until 96 hours, are triggered by or around 12 hours following first exposure to environmental V. fischeri. In addition, around 12 hours, the symbionts usually fully colonize the crypt spaces and their growth is attenuated and luminescence induced. By 48 hours, significant changes in the proteome occur (Lemus and McFall-Ngai, 2000). In addition, mutants of V. fischeri incapable of persisting in the light organ lose the ability to colonize the organ beginning at around 48 hours.

Figure 3

Novel gene discovery. Large increases in sequence contributions are evident from each set of libraries, which are illustrated as follows: sequences from 5 non-normalized libraries (approximately the first 10,000 ESTs); sequences from 5 normalized libraries (approximately from 10,000 to 30,000 ESTs); and sequences from pooled, subtracted libraries (after 30,000 ESTs) (Table 1). Novelty rates, calculated as the ratio of the total number of clusters identified over the total number of ESTs generated, were 39%, 54%, and 75%, for the non-normalized, normalized and subtracted libraries, after production of a total of 10,000 (3,920 clusters), 20,190 (10,822 clusters), and 5,171 (3,917 clusters) ESTs, respectively.

Figure 4

Annotation of 13,962 unique E. scolopes light organ sequences. A. A pie chart indicating the number of genes and the percent of ESTs annotated as a known protein, unknown protein or a hypothetical protein. All sequences that are annotated as a known protein or as significant (sig) have a BLASTX hit below the E-value threshold of e-03. B. A histogram illustrating the total number of unique E. scolopes ESTs, the total number of ESTs annotated as a known protein with an E-value threshold of e-03, the total number of ESTs annotated with at least one category of Gene Ontology (GO) and the number of genes annotated in each of the 3 major GO categories, biological process, molecular function and cellular component.