Cavanilles Institute for Biodiversity and Evolutionary Biology, Science Park, University of Valencia, P.O. Box 22 085, 46071, Valencia, Spain.
Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic.
Parasit Vectors. 2019 Mar 12;12(1):92. doi: 10.1186/s13071-019-3351-9.
Despite their complex life-cycles involving various types of hosts and free-living stages, digenean trematodes are becoming recurrent model systems. The infection and penetration strategy of the larval stages, i.e. cercariae, into the fish host is poorly understood and information regarding their entry portals is not well-known for most species. Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982 (Digenea, Strigeidae) uses the gilthead seabream (Sparus aurata L.), an important marine fish in Mediterranean aquaculture, as a second intermediate host, where they encyst in the brain as metacercariae. Labelling the cercariae with in vivo fluorescent dyes helped us to track their entry into the fish, revealing the penetration pattern that C. longicollis uses to infect S. aurata.
Two different fluorescent dyes were used: carboxyfluorescein diacetate succinimidyl ester (CFSE) and Hoechst 33342 (NB). Three ascending concentrations of each dye were tested to detect any effect on labelled cercarial performance, by recording their survival for the first 5 h post-labelling (hpl) and 24 hpl, as well as their activity for 5 hpl. Labelled cercariae were used to track the penetration points into fish, and cercarial infectivity and later encystment were analysed by recording brain-encysted metacercariae in fish infected with labelled and control cercariae after 20 days of infection.
Although the different dye concentrations showed diverse effects on both survival and activity, intermediate doses of CFSE did not show any short-term effect on survival, permitting a brighter and longer recognition of cercariae on the host body surface. Therefore, CFSE helped to determine the penetration points of C. longicollis into the fish, denoting their aggregation on the head, eye and gills region, as well as on the dorsal fin and the lower side. Only CFSE-labelled cercariae showed a decreased number of encysted metacercariae when compared to control.
Our study suggests that CFSE is an adequate labelling method for short-term in vivo studies, whereas NB would better suit in vivo studies on long-term performance. Cardiocephaloides longicollis cercariae seem to be attracted to areas near to the brain or those that are likely to be connected to migration routes to neuronal canals.
尽管复殖类吸虫的生命周期复杂，涉及多种宿主和自由生活阶段，但它们正成为反复出现的模式系统。幼虫阶段（即尾蚴）感染和穿透鱼类宿主的策略知之甚少，大多数物种的进入门户信息也不为人知。Cardiocephaloides longicollis（Rudolphi，1819）Dubois，1982（复殖类，Strigeidae）将金头鲷（Sparus aurata L.）用作第二中间宿主，在那里它们在脑中形成囊蚴。用体内荧光染料标记尾蚴有助于我们追踪它们进入鱼类的过程，揭示了 C. longicollis 感染 S. aurata 的穿透模式。
使用了两种不同的荧光染料：羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）和 Hoechst 33342（NB）。测试了每种染料的三个递增浓度，以检测它们对标记尾蚴性能的任何影响，方法是记录标记后前 5 小时（hpl）和 24 hpl 的存活率以及 5 hpl 的活性。用标记的尾蚴追踪进入鱼类的穿透点，并通过记录感染标记和对照尾蚴 20 天后感染鱼脑中的囊蚴来分析尾蚴的感染力和随后的囊化。
尽管不同的染料浓度对存活和活性都有不同的影响，但 CFSE 的中间剂量对存活没有任何短期影响，允许在宿主体表上更亮、更长时间地识别尾蚴。因此，CFSE 有助于确定 C. longicollis 进入鱼类的穿透点，表明它们在头部、眼睛和鳃区聚集，以及在背鳍和下方。与对照相比，只有 CFSE 标记的尾蚴显示囊蚴数量减少。
我们的研究表明，CFSE 是短期体内研究的合适标记方法，而 NB 更适合体内长期性能研究。Cardiocephaloides longicollis 尾蚴似乎被吸引到靠近大脑的区域或可能与向神经元管迁移的路线相关的区域。
