Resistance of Biomphalaria glabrata 13-16-R1 snails to Schistosoma mansoni PR1 is a function of haemocyte abundance and constitutive levels of specific transcripts in haemocytes

Abstract

Continuing transmission of human intestinal schistosomiasis depends on the parasite's access to susceptible snail intermediate hosts (often Biomphalaria glabrata). Transmission fails when parasite larvae enter resistant individuals in wild snail populations. The genetic basis for differences in snail susceptibility/resistance is being intensively investigated as a means to devise novel control strategies based on resistance genes. Reactive oxygen species produced by the snail's defence cells (haemocytes) are effectors of resistance. We hypothesised that genes relevant to production and consumption of reactive oxygen species would be expressed differentially in the haemocytes of snail hosts with different susceptibility/resistance phenotypes. By restricting the genetic diversity of snails, we sought to facilitate identification of resistance genes. By inbreeding, we procured from a 13-16-R1 snail population with both susceptible and resistant individuals 52 lines of B. glabrata (expected homozygosity ~87.5%), and determined the phenotype of each in regard to susceptibility/resistance to Schistosoma mansoni. The inbred lines were found to have line-specific differences in numbers of spreading haemocytes; these were enumerated in both juvenile and adult snails. Lines with high cell numbers were invariably resistant to S. mansoni, whereas lines with lower cell numbers could be resistant or susceptible. Transcript levels in haemocytes were quantified for 18 potentially defence-related genes. Among snails with low cell numbers, the different susceptibility/resistance phenotypes correlated with differences in transcript levels for two redox-relevant genes: an inferred phagocyte oxidase component and a peroxiredoxin. Allograft inflammatory factor (potentially a regulator of leucocyte activation) was expressed at higher levels in resistant snails regardless of spread cell number. Having abundant spreading haemocytes is inferred to enable a snail to kill parasite sporocysts. In contrast, snails with fewer spreading haemocytes seem to achieve resistance only if specific genes are expressed constitutively at levels that are high for the species.

Keywords: Biomphalaria; Expression; Haematocrit; Haemocyte; Host–parasite; Oxidative; Resistance; Schistosoma.

Fig. 1

Susceptibility (S) phenotypes of 19 inbred Biomphalaria glabrata snail lines and counts of their spread haemocytes/μL (mean + S.E.M.). Data points between the dotted lines all represent %S = 0 and are staggered to show S.E.M. There is a significant and moderately strong correlation (P = 0.0235, r² = 0.2670) between susceptibility and cell count (Linear Regression Analysis). Resistant snails are more likely to have spread haemocyte counts over 100 cells/μL (Fisher Exact Test, P = 0.0172, two-tailed).

Fig. 2

Expression stability measures (M values) for seven candidate reference genes (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (ywhae); actin (act); elongation factor-1 alpha (ef1); glyceralde-hyde-3-phosphate dehydrogenase 3 (gapdh); guanine nucleotide binding protein beta polypeptide (gnb); 60S ribosomal protein L32 (L32); and myoglobin (myo)) in Biomphalaria glabrata. Using geNorm (Vandesompele et al., 2002), M values were calculated from the average pair-wise variation of each candidate gene in comparison with all other genes. A higher M value represents higher variability in expression. Iterative removal of the least stable gene and recalculation of M values for the remaining genes allows a ranking of genes from least to most stable. Since the calculations are based on ratios, the protocol does not differentiate between the two most stable genes. In our system the most steadily expressed genes are L32 and myo.

Fig. 3

Constitutive expression of three genes implicated in resistance to Schistosoma mansoni, in haemocytes of Biomphalaria glabrata. (A) Allograft inflammatory factor (aif) is expressed at a significantly higher level in resistant inbred snail lines (P = 0.0456 two-tailed T-test). (B) The gene for the inferred phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (infPhox) is expressed at 2.3-fold higher levels in haemocytes from resistant lines compared with susceptible lines of equally low spread cell numbers (P = 0.0261, two-tailed T-test). (C) Peroxiredoxin 1 (prx1) expression is 2.4-fold higher in resistant snails from low spread cell lines (P = 0.0004, two-tailed T-test).

Fig. 4

The log transformed gene expression levels among all inbred Biomphalaria glabrata lines, calculated for each gene independently, were divided into three ranges. For each gene, low, medium and high groups are illustrated. Inbred lines are listed from most resistant (i170) to most susceptible (i163), and the spread cell counts (Cell #) and % susceptibility (%S) are shown for each. (A) Heat map for all genes and all inbred lines. (B) Heat map of NADPH oxidase components and peroxiredoxin 1 (prx1) from resistant (R) and susceptible (S) lines with similar spread cell counts. Expression of the gene for the inferred phox subunit (infPhox) is significantly higher in R than S lines. Two other components of the NADPH oxidase complex which were measured, nox2 and phox²², were not significantly higher in R lines but show trends towards higher expression in the R lines. Genes are abbreviated as follows, where bold numbers refer to Contig numbers in a preliminary draft of the snail genome (https://www.vectorbase.org/organisms/biomphalaria-glabrata): aif, allograft inflammatory factor; arg, arginase; c42, catalase 42; d584, dual oxidase 584; d303, dual oxidase 303; frep3, fibrinogen-related protein 3; g2402, glutathione peroxidase 2402; g97, glutathione peroxidase 97; g65, glutathione peroxidase 65; mif, macrophage migration inhibitory factor; nox2, NADPH oxidase 2; phox22, phagocyte oxidase 22; infPhox, inferred phox subunit; prx6, peroxiredoxin 6; prx4, peroxiredoxin 4; prx621, peroxiredoxin 621; prx1, peroxiredoxin 1; tal-1, T-cell acute lymphocytic leukaemia-1.

Fig. 5

Phenotypic traits of inbred Biomphalaria glabrata lines that correlate with susceptibility to Schistosoma mansoni. Traits were measured in 12 mm snails. Trait values were calculated as a percentage of the highest mean of all the lines. (A) The four most susceptible inbred lines (i36, i93, i159, i163) have less than 40% of the highest average spread cell numbers (Sprd Hem) and low allograft inflammatory factor (aif) expression which is consistent among the lines. (B) Four resistant lines (i7, i31, i113, i156) with the lowest numbers of spread cells consistently expressed high peroxiredoxin 1 (prx 1) and similar mid-levels of aif and the inferred phox subunit (infPhox). (C) Three resistant lines (i84, i147, i170) with high numbers of spread cells showed mid to lower levels of expression in nearly all depicted genes.