常压室温等离子体(ARTP)诱变对凡纳对虾不同家系幼体发育及仔虾抗逆性状的影响

Effect of atmospheric and room-temperature plasma mutagenesis on the larval development of Penaeus vannamei

关键词:  凡纳对虾  诱变育种  ARTP  幼体发育

Key words:  Penaeus vannamei  Mutagenic breeding  Atmospheric and room-temperature plasma  Juvenile development

摘要:

为研究物理诱变对虾类前期幼体发育及抗逆性状的影响，探讨通过诱变技术增加选育群体遗传多样性和可利用遗传变异的可行性，本研究利用常压室温等离子体(ARTP)诱变技术对4尾来自不同家系(A、B、C和D)的凡纳对虾(Penaeus vannamei)个体所产的受精卵(原肠胚期)分别进行相同功率(360 W)、不同时间(2、4、8和12 min)的批量诱变处理，并对孵化率、肢芽期孵化畸形率、存活率、变态率以及仔虾低氧耐受性和氨氮胁迫指标进行分析。结果显示，ARTP对凡纳对虾进行诱变处理后在不同家系间有一些共性的规律，在幼体发育前期阶段，4个家系随着诱变时间的增加，肢芽期畸形率均呈上升趋势，孵化率均呈下降趋势；与未经过诱变的对照组相比，除A和D家系诱变2 min组外，其余处理组肢芽期孵化畸形率均显著升高(P<0.05)；除A家系诱变2 min组外，其余各家系处理组孵化率均显著降低(P<0.05)；肢芽期孵化畸形率和孵化率与诱变时间均呈现中度或高度相关；与对照组相比，A、B、C和D 4个家系在相同阶段的同一处理组存活率的表现具有不定向性，A家系诱变8 min组存活率显著降低(P<0.05)，而C家系诱变4和8 min组存活率显著升高，D家系诱变2和4 min组存活率显著降低，而诱变8 min组则显著升高。可能是诱变的不定向性以及诱变作用于个体造成的。通过存活率和各期变态率综合分析可得，低剂量(2 min组)诱变死亡高峰期多发生于幼体发育后期，高剂量(4、8和12 min组)诱变幼体死亡高峰期多发生于幼体发育中前期。对A家系幼体的低氧耐受性和氨氮胁迫测试结果表明，低剂量的辐射可能会在一定程度上提高低氧耐受能力；诱变2 min组和8 min组分别在氨氮胁迫实验第4天、第7天存活率很高，诱变对该家系幼体对抗氨氮胁迫的能力有一定的影响。说明ARTP诱变凡纳对虾受精卵在不同家系中幼体发育前期具有一些共性趋势，在幼体发育后期和性状的改变上具有不定向性。研究结果对凡纳对虾新种质创制及功能研究材料的制备提出了新的方法，并为凡纳对虾诱变育种提供了基础数据和科学依据。

Abstract:

This study aimed to investigate the effects of physical mutagenesis on the development and adaptive traits of early-stage shrimp larvae and to explore the feasibility of increasing genetic diversity and utilizing genetic variation in breeding populations through mutation technology. Atmospheric and room-temperature plasma (ARTP) mutagenesis technology was used to perform batch mutagenesis on fertilized eggs (gastrula stage) of four different families (A, B, C, and D) of Penaeus vannamei at the same power (360 W) and different times (2, 4, 8, and 12 min). Hatching rates, limb bud stage hatching deformity rates, survival rates, metamorphosis rates, and indicators of juvenile shrimp hypoxia tolerance and ammonia nitrogen stress were analyzed. The experimental results showed that all groups except for the 2 min mutation group showed hatching deformity rates significantly higher than those of the control group for the A and D families (P<0.05). The deformity rate of each treatment group showed no significant differences from that of the control group in Family B. The malformation rate for Family C in the 8 min mutation treatment group was significantly higher than that in the control group. However, there was no significant difference between the 2 and 4 min treatment groups. In all four families, the hatching rate of fertilized eggs decreased with an increase in mutation time, and when the mutation time was greater than or equal to 4 min, the hatching rates of all four families were less than 15%. Compared with that of the control group, all treatment groups of the families except for the 2 min mutation group from Family A showed a significant decrease in hatching rate (P<0.05). The hatching rate of the 2 min group for families A, B, C, and D was significantly higher than that of the 4, 8, and 12 min mutation groups, and the hatching rate of the 4 min mutation group from Family B was significantly higher than that of the 8 min mutation group (P<0.05). The hatching and limb bud stage hatching deformity rates were moderately and highly correlated with mutagenic time, respectively. Compared with the control group, the survival rates of families A, B, C, and D in the same treatment group at the same stage showed no significant differences. The survival rate of the 8 min mutation group from Family A was significantly reduced (P<0.05), that of the 4 and 8 min mutation groups from Family C was significantly increased, and that of the 2 and 4 min mutation groups from Family D was significantly reduced relative to the control value, whereas that of the 8 min group from Family D was significantly increased. Considering Family A as an example, metamorphosis rates in each stage were analyzed. The metamorphosis rates of the control group and the 2 min mutation group showed a significant decreasing trend during the three stages of larval development (P<0.05); however, the metamorphosis rate in the M–P stage was significantly lower than that in the N–Z and Z–M stages. The metamorphosis rate in the Z–M stage of the 4 min and 12 min groups was significantly lower than that during the N–Z and M–P stages, whereas the metamorphosis rate during the N–Z stage of the 8 min group was significantly lower than that of the Z–M stage. After low-dose (0 and 2 min) mutagenesis of fertilized eggs of P. vannamei, the peak period of death mainly occurred in the late stage of larval development. The peak period of larval death after relatively high doses of mutagenesis (4, 8, and 12 min) mainly occurred in the middle or early stages of larval development. The peak period of death varied among different treatment groups. The results caused by radiation treatment showed directionality, and this trend of peak mortality was similar between all families. Hypoxia tolerance and ammonia-nitrogen stress testing in Family A of juvenile shrimp revealed that almost all shrimp in the 4, 8, and 12 min groups died after 5.5 h of hypoxia stress. In contrast, the control group died after 9 h of stress, and the 2 min group died after 10 h of stress. The results indicate that low doses of radiation (2 min) may improve hypoxia tolerance to some extent. The survival rates of the 2 min and 8 min groups were highest on the fourth and seventh days of the ammonia nitrogen stress experiment, respectively. Mutation had a certain impact on the ability of larvae in Family A to resist ammonia-nitrogen stress. The experimental results suggest that ARTP induced the mutation of fertilized P. vannamei eggs with non-directional changes in adaptive traits. These findings suggest the feasibility of novel methods for creating new germplasms and preparing functional research materials for P. vannamei and provide basic data and a scientific basis for the mutagenic breeding of P. vannamei.