. 2021 Dec 2;12:761130. doi: 10.3389/fimmu.2021.761130

Table 3.

Relative expression of immune genes in anterior kidney and spleen tissues of olive flounder in the immersion vaccinated and NVC control groups at different time intervals post-challenge with virulent M. avidus (5 × 10⁴ cells/fish).

		Head kidney			Spleen
		24 hpc	48 hpc	96 hpc	24 hpc	48 hpc	96 hpc
IgM	NVC control	1.87 ± 0.22^x	1.53 ± 0.46^x	1.44 ± 0.60^x	1.02 ± 0.28^x	1.01 ± 0.26^x	0.90 ± 0.27^x
	Immersion	2.56 ± 1.18^a	2.05 ± 0.66^a	2.16 ± 0.81^a	1.24 ± 0.45^a	2.64 ± 0.67^a	1.80 ± 0.65^a
		p-value: time—0.798; treatment—0.292; interaction—0.988			p-value: time—0.341; treatment—0.033; interaction—0.345
IgT	NVC control	0.28 ± 0.12^x	0.42 ± 0.26^x	2.20 ± 0.90^x	3.20 ± 1.54^x	2.73 ± 1.17^x	2.29 ± 0.76^x
	Immersion	1.87 ± 1.02^a	*23.71 ± 8.30^b	*20.05 ± 5.47^ab	3.97 ± 1.19^a	4.66 ± 1.88^a	4.96 ± 1.70^a
		p-value: time—0.036; treatment—0.001; interaction—0.053			p-value: time—0.997; treatment—0.149; interaction—0.801
pIgR	NVC control	2.84 ± 0.93^x	3.80 ± 1.02^x	2.87 ± 0.59^x	1.57 ± 0.17^x	2.31 ± 0.74^x	2.64 ± 0.70^x
	Immersion	5.30 ± 1.57^a	5.19 ± 0.84^a	4.26 ± 1.92^a	2.98 ± 0.71^a	4.50 ± 1.42^a	5.47 ± 1.34^a
		p-value: time—0.755; treatment—0.108; interaction—0.882			p-value: time—0.207; treatment—0.017; interaction—0.762
IL-1β	NVC control	2.29 ± 0.11^x	2.38 ± 0.29^x	2.45 ± 0.02^x	0.87 ± 0.26^x	1.30 ± 0.28^x	1.14 ± 0.35^x
	Immersion	3.42 ± 0.67^a	*4.18 ± 0.32^a	3.54 ± 0.82^a	*2.20 ± 0.40^a	2.37 ± 0.48^a	2.70 ± 0.63^a
		p-value: time—0.659; treatment—0.004; interaction—0.702			p-value: time—0.633; treatment—0.002; interaction—0.845
IL-10	NVC control	2.76 ± 1.30^x	3.43 ± 1.53^x	3.40 ± 0.89^x	1.62 ± 0.40^x	3.18 ± 0.52^xy	3.96 ± 0.89^y
	Immersion	1.55 ± 0.65^a	1.73 ± 0.27^a	1.58 ± 0.31^a	1.30 ± 0.10^b	**0.73 ± 0.10^a	*1.24 ± 0.13^b
		p-value: time—0.895; treatment—0.065; interaction—0.945			p-value: time—0.081; treatment—0.000; interaction—0.043
TNF-α	NVC control	2.01 ± 0.15^x	2.24 ± 0.57^x	1.77 ± 0.52^x	1.29 ± 0.14^x	1.09 ± 0.14^x	1.12 ± 0.10^x
	Immersion	*3.39 ± 0.32^b	2.10 ± 0.08^a	2.81 ± 0.45^ab	1.73 ± 0.22^a	2.24 ± 0.47^a	**1.80 ± 0.08^a
		p-value: time—0.394; treatment—0.036; interaction—0.172			p-value: time—0.656; treatment—0.002; interaction—0.328
IL-8	NVC control	2.28 ± 0.27^x	3.45 ± 0.40^x	4.03 ± 1.14^x	2.60 ± 0.39^x	3.78 ± 0.19^x	3.32 ± 0.97^x
	Immersion	3.84 ± 0.55^a	4.95 ± 1.38^a	5.16 ± 1.64^a	3.17 ± 0.63^a	7.95 ± 2.34^a	6.59 ± 1.70^a
		p-value: time—0.338; treatment—0.124; interaction—0.975			p-value: time—0.098; treatment—0.026; interaction—0.377
CD-8α	NVC control	1.87 ± 0.49^x	1.70 ± 0.36^x	1.75 ± 0.14^x	0.64 ± 0.18^x	1.38 ± 0.24^xy	1.55 ± 0.24^y
	Immersion	2.33 ± 0.68^a	2.39 ± 0.65^a	2.60 ± 0.52^a	1.09 ± 0.65^a	**2.98 ± 0.24^b	2.48 ± 0.49^ab
		p-value: time—0.968; treatment—0.133; interaction—0.929			p-value: time—0.009; treatment—0.008; interaction—0.349
Caspase 3	NVC control	1.39 ± 0.25^x	1.47 ± 0.55^x	1.06 ± 0.25^x	0.87 ± 0.08^x	1.70 ± 0.30^y	2.81 ± 0.18^z
	Immersion	1.38 ± 0.26^a	2.10 ± 0.37^a	1.90 ± 0.46^a	**1.35 ± 0.03^a	3.63 ± 1.01^b	*4.05 ± 0.39^b
		p-value: time—0.553; treatment—0.139; interaction—0.514			p-value: time—0.001; treatment—0.008; interaction—0.325

Mean (n = 3) expression levels of each gene relative to the naive control are tabulated with standard error at different time intervals. Two-way ANOVA was performed with each dataset, and the p-values within the group (time-wise), between groups (treatment-wise), and for the interaction effect were evaluated to determine statistical significance (p < 0.05). Duncan’s multiple range test (homogenous subsets indicated by lowercase [x-z] for NVC control and [a-c] for Immersion group) within the groups (time-wise) and an unpaired t-test (indicated by asterisks * for significance level) between the groups at different time points were also performed to analyze the statistical differences.

* denotes significance level of t-test between the 2 groups viz., * and ** are for p < 0.05 and 0.01 respectively.