Time series analysis of the transcriptional responses of Biomphalaria glabrata throughout the course of intramolluscan development of Schistosoma mansoni and Echinostoma paraensei

Abstract

Successful colonization of a compatible snail host by a digenetic trematode miracidium initiates a complex, proliferative development program requiring weeks to reach culmination in the form of production of cercariae which, once started, may persist for the remainder of the life span of the infected snail. How are such proliferative and invasive parasites able to circumvent host defenses and establish chronic infections? Using a microarray designed to monitor the internal defense and stress-related responses of the freshwater snail Biomphalaria glabrata, we have undertaken a time course study to monitor snail responses following exposure to two different trematode species to which the snail is susceptible: the medically important Schistosoma mansoni, exemplifying sporocyst production in its larval development, or Echinostoma paraensei, representing an emphasis on rediae production in its larval development. We sampled eight time points (0.5, 1, 2, 4, 8, 16 and 32 days p.i.) that cover the period required for cercariae to be produced. Following exposure to S. mansoni, there was a preponderance of up-regulated over down-regulated array features through 2 days p.i. but by 4 days p.i. and thereafter, this pattern was strongly reversed. For E. paraensei, there was a preponderance of down-regulated array features over up-regulated features at even 0.5 days p.i., a pattern that persists throughout the course of infection except for 1 day p.i., when up-regulated array features slightly outnumbered down-regulated features. Examination of particular array features revealed several that were up-regulated by both parasites early in the course of infection and one, fibrinogen related protein 4 (FREP 4), that remained significantly elevated throughout the course of infection with either parasite, effectively serving as a marker of infection. Many defense-related transcripts were persistently down-regulated, including several fibrinogen-containing lectins and homologs of molecules best known from vertebrate phagocytic cells. Our results are consistent with earlier studies suggesting that both parasites are able to interfere with host defense responses, including a tendency for E. paraensei to do so more rapidly and strongly than S. mansoni. They further suggest mechanisms for how trematodes are able to establish the chronic infections necessary for their continued success.

Fig. 1

Graph showing the total number of up- (black) and down-regulated (white) transcripts in Biomphalaria glabrata after infection with Schistosoma mansoni for 32 days. Snails infected with S. mansoni were collected at 0.5, 1, 2, 4, 8, 16 and 32 days p.i. and analyzed using a B. glabrata microarray targeting immune and stress associated transcripts. Each time point represents data collected from 15 individual snails, pooled into three experimental replicates and analyzed using significance analysis of microarrays to determine statistical significance compared with uninfected controls.

Fig. 2

Graph showing the total number of up- (black) and down-regulated (white) transcripts in Biomphalaria glabrata after infection with Echinostoma paraensei for 32 days. Snails infected with E. paraensei were collected at 0.5, 1, 2, 4, 8, 16 and 32 days p.i. and analyzed using a B. glabrata microarray targeting immune and stress associated transcripts. Each time point represents data collected from 15 individual snails, pooled into three experimental replicates and analyzed using significance analysis of microarrays to determine statistical significance compared with uninfected controls.

Fig. 3

Comparison of the shared up- and down-regulated transcripts over the course of a Biomphalaria glabrara 32 day infection with Schistosoma mansoni or Echinostoma paraensei. Numbers encompassed by only one circle represent the number of up- or down-regulated transcripts in B. glabrata unique to each trematode infection (S. mansoni in grey, E. paraensei in white). Numbers encompassed by both circles represents the number of common transcripts up- or down-regulated by each trematode infection. To the right of each time point is a list of the shared transcripts separated by a line for each time point. FREP; fibrinogen related protein, FreM; fibrinogen related molecule, MAP; mitogen activated protein, CREB; cyclic AMP response element binding, FReD; fibrinogen related domain,

Fig. 4

Confirmation of microarray results using quantitative reverse transcription PCR (qRT-PCR). Transcript expression measured as fold change compared with control is shown for microarray and qRT-PCR analysis methods. Data depicted is from a selected time point for each experimental group. BgLBP/BPI (2 days p.i.), Spondin (16 days p.i.), BGC03601 (8 days p.i.), Lysozyme (0.5 days p.i.), C1q-like lectin (0.5 days p.i.), Macrophage migration inhibition factor (2 days p.i.), FREP4 (1 days p.i.), Serpin B4 (16 days p.i. (Schistosoma mansoni), 1 days p.i. (Echinostoma paraensei).