Multi-technique approach to characterise the effects of cryopreservation on larval development of the Pacific oyster (Crassostrea gigas)

Figures & data

Figure 1 Diagram of experimental design, including treatments with two cryoprotectant solutions (10% ethylene glycol + 1% polyvinylpyrrolidone plus either 0.2 M or 0.4 M trehalose [final concentration]) and three freezing rates (0.5, 1 and 2 °C min⁻¹) and control (no cryopreservation).

Figure 2 Mean survivability (±SE) of oyster larvae during the 22-day rearing period within control and treatments, including two cryoprotectant solutions (10% ethylene glycol + 1% polyvinylpyrrolidone plus either 0.2 M or 0.4 M trehalose [final concentration]) and three freezing rates (0.5, 1 and 2 °C min⁻¹), n = 3.

Table 1 Repeated measures ANOVA of survivability, feeding consumption and shell length data, including control (C) and treatments (0.2 M trehalose, 0.5 °C min⁻¹, 0.2 M trehalose, 1 °C min⁻¹, 0.2 M trehalose, 2 °C min⁻¹, 0.4 M trehalose, 0.5 °C min⁻¹, 0.4 M trehalose, 1 °C min⁻¹, and 0.4 M trehalose, 2 °C min⁻¹) over the 22-day rearing period.

	df	MS	F	P	Significant Tukey tests
Survivability
Between-subject
Treatment (T)	6	2114.6	15.4	0.001	Control with all treatments
Error	13	136.8
Within-subject
Day (D)	10	4506.0	222.9	0.001	All day pairs significant except:
T × D	60	69.6	3.4	0.001	D9 with D11, D13, D15, and D17
Error (Day)	130	20.2			D11 with D13, D15, D17, and D19
					D13 with D15, D17, D19, and D21
					D15 with D17, D19, D21, and D22
					D17 with D19, D21, and D22
					D19 with D21, and D22
					D21 with D22
Feeding consumption
Between-subject
Treatment (T)	6	0.2	6.2	0.002	Control with all treatments, except with 04 M trehalose, 2°C min^−1
Error	13	0.1
Within-subject
Day (D)	10	0.7	97.9	0.001	All day pairs significant except:
T × D	60	0.1	2.5	0.001	D9 with D11, D13, D15, and D17
Error (Day)	130	0.1			D11 with D17
					D13 with D15, D17, D19, and D21
					D15 with D17, D19, and D21
					D19 with D21
Shell length (control, 0.4 M trehalose, 1 °C min^−1 and 0.4 M trehalose, 2 °C min^−1 only)
Between-subject
Treatment (T)	2	3,63,744	397.0	0.001	Control with all treatments
Error	161	916
Within-subject
Day (D)	9	3,01,510	370.3	0.001	All day pairs significant except:
T × D	18	7734	9.5	0.001	D19 with D21 and D22
Error (Day)	1449	814			D21 with D22

Figure 3 Mean percent feeding consumption (±SE) of larvae over 21 days within control and treatments, including two cryoprotectant solutions (10% ethylene glycol + 1% polyvinylpyrrolidone plus either 0.2 M or 0.4 M trehalose [final concentration]) and three freezing rates (0.5, 1 and 2 °C min⁻¹).

Figure 4 Mean shell length (±SE) of larvae over 21 days within control and treatments, including two cryoprotectant solutions (10% ethylene glycol + 1% polyvinylpyrrolidone plus either 0.2 M or 0.4 M trehalose [final concentration]) and three freezing rates (0.5, 1 and 2 °C min⁻¹).

Figure 5 SEM images of C. gigas larvae over the rearing period within controls (1A–1F) and larvae exposed to 0.2 M trehalose (2A–2C) and 0.4 M trehalose (3A–3F). A, B, C, D, E and F represent larvae that were 1, 5, 9, 15, 17 and 21 days old, respectively. Abbreviations: PI, prodissoconch I; PII, prodissoconch II. Scale bars = 20 µm.

Figure 6 Light microscopy images of D-stage to late umbo stage C. gigas larvae showing overall organogenesis and microalgal content in stomachs over the rearing period within controls (1A–1F) and larvae exposed to 0.2 M trehalose (2A–2C) and 0.4 M trehalose (3A–3F). A, B, C, D, E and F represent larvae that were 1, 3, 5, 9, 13, 17 and 21 days old, respectively. Abbreviations: A, anus; AA, anterior adductor muscle; Ci, cilia; DD, digestive diverticulum; E, oesophagus; F, foot; I, intestine; M, mouth; S, stomach; U, umbo; Ve, velum; VR, velum retractor muscles. Scale bars = 20 µm.