Table 2 Overview of experimental conditions and resulting effects on expression of bdccp genes

Continuous culture growth

2210A G2 1802A G8

Rouen G11

The initial parasitemia was set up at 0.1 % and expression of bdccp genes was analyzed daily for all five days post culture initiation (DPI); cultivation was performed without medium replacement.

increased*

Long-term cultivation

2210A G2 6903C E2 Rouen F5

B. divergens clones 2210A G2 and 6903C E2, were continuously propagated in vitro for ≈ 1 year. Samples before and after long-term cultivation were analyzed; parasitemia was equal for all analysed samples to minimize variations in the expression of bdccp genes. Expression of bdccp genes by B. divergens clone Rouen F5 was analyzed by PCR using gDNA and cDNA.

decreased

Imidocarbe treatment

2210A G2 Rouen G11

The range of efficient doses of both drugs was determined following parasite growth monitoring in vitro for 48 h [66] to select effective concentrations of drugs (imidocarbe 179.5 nM, 359 nM and 718 nM; atovaquone 10 nM, 40 nM and 75 nM). The culture without drug treatment was used as a control. The effect of drug treatment was measured 2 DPI; starting parasitemia was 2 %.

increased*

Atovaquone treatment

increased or decreased*

(concentration dependent)

Altered cultivation temperature and air environment

2210A G2

XA was added at 100 μM concentration and its effect was tested after 24 h of parasites cultivation either under standard (37 °C, 5 % CO2) or altered conditions (28 °C, air). As a control, cultures without XA were used. A starting parasitemia was set up 6 % in order to reach > 10 % parasitemia level (experiment design setting taken from [11]).

increased*

XA addition

increased*

Combination of altered cultivation and XA addition

increased*

Co-infection

2210A G2 Rouen G11 7101A D11

Different clonal lines were mixed in the same ratio and expression of bdccp genes was analysed in cultures cultivated for 24 h and 48 h. As a control, clones were cultivated independently; starting parasitemia was 2 %.

not affected

RBCs lysate addition

2210A G2 Rouen G11

Lysate of uninfected RBCs was added into the culture to simulate cultivation medium corresponding with 10 % parasitemia. Analyses were performed after 24 and 48 h of cultivation; the control was represented by a culture without lysate addition; starting parasitemia was 2 %.

not affected

Hematocrit increase

2210A G2 Rouen G11

Hematocrit increase was simulated by doubling the quantity of RBCs in the medium and analyses were performed after 24 h and 48 h of cultivation; standard in vitro culture was used as a control; starting parasitemia was 2 %.

not affected

High parasitemia maintenance

2210A G2 Rouen G11

Analyses were performed at the starting point (0 DPI), where parasitemia was starting at 10 %, and 1 and 2 DPI. Media were changed daily.

not affected

Cultivation without FCS

2210A G2 Rouen G11

Altered cultivation conditions (cultivation in medium without FCS) were maintained for 24 h in culture with 10 % parasitemia. Analyses were performed 0 and 1 DPI; starting parasitemia was 2 %.

not affected