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Figure legends

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Figure 1. Expression profile of T cell markers in MACS-sorted leukocytes from the

695

kidney and gill cells of ginbuna crucian carp. DN indicates CD8 and CD4 double-negative

696

lymphocytes; NC indicates non-template control. EF1α was used as an internal control.

697

Numbers to the right indicate PCR cycles. Data from one fish are shown as representative

29

698

of the three fish analyzed.

699

700

Figure 2. Time course of cytotoxic activity of kidney (A) and gill (B) leukocytes against

701

I. multifiliis. Kidney and gill leukocytes from naïve ginbuna crucian carp were incubated

702

with I. multifiliis at various E:T ratios (2000:1, 1000:1, 300:1, and 100:1). Data from one

703

fish are shown as representative of the three fish analyzed.

704

705

Figure 3. Cytotoxic activity of unsorted and sorted effector cells from the kidneys (A)

706

and gills (B) against I. multifiliis. The mean activities of CD4+ cells, CD8+ cells, DN cells,

707

and unsorted cells are shown (gill, n = 3; kidney, n = 4). The effector cells were incubated

708

for 2 h with I. multifiliis at E:T ratio of 1000:1. Error bars indicate SD. Different letters

709

on each bar indicate significant differences among the groups (P < 0.05).

710

711

Figure 4. Contact of CD8+ cells from the kidneys and gills to the surface of I. multifiliis.

712

Arrows indicate CD8+ cells (red) and I. multifiliis (green). Kidney leukocytes and I.

713

multifiliis are shown in the upper panels (A, B, C). Scale bars indicate 10 µm.

714

715

Figure 5. Cytotoxic activity of the effector cells from the kidney (A and B) and the gill

716

(C and D) against I. multifiliis with or without the culture insert. CD8+ cells (A and C)

717

and CD8− cells (B and D) were used as the effector cells. CD8+ cells were co-cultured

718

with I. multifiliis at E:T ratio of 500:1. The results from the three individual fish are shown

719

as the mean of cell killing activities. White and black bars indicate the activities of the

720

effector cells in the absence (contact) and presence (non-contact) of the insert,

721

respectively. The asterisks indicate significant differences of the activities between non30

722

contact and contact (*P < 0.05).

723

724

Figure 6. Effect of DCI on the cytotoxic activity of CD8+ cells from the kidney (A) and

725

the gill (B). CD8+ cells were pre-incubated with various concentrations (0, 20, 40 µM) of

726

DCI. CD8+ cells were co-cultured with I. multifiliis at E:T ratio of 300:1. Data are shown

727

as the means of the activities from three (kidney) or four (gill) individual fish, respectively.

728

Error bars indicate SD. Asterisks indicate significant differences from control group (0

729

µM) (P < 0.05).

730

731

Figure 7. Effect of CMA on the cytotoxic activity of CD8+ cells from the kidney (A) and

732

the gill (B). CD8+ cells were pre-incubated with various concentrations (0, 0.5, 1.0 µM)

733

of CMA. CD8+ cells were co-cultured with I. multifiliis at E:T ratio of 300:1. Data are

734

shown as means of the activities from three (kidney) or four (gill) individual fish. Error

735

bars indicate SD. Asterisks indicate significant differences from control group (0 µM) (P

736

< 0.05).

31

...