Transgenerational experiment
To test the transgenerational effect of MHW and FPC on the performance of tropical copepod Pseudodiaptomus incisus , P. incisuswere studied over two generations: F1 and F2. F1 P. incisus were exposed to one of four treatments, including 2 thermal treatments (30 or 34°C) × 2 FPC (presence or absence) × 10 replicates (Fig. 1). The control temperature of 30°C was chosen as it is the mean coastal water temperature in the coastal water in southern Vietnam (see Appendix S1, Doan et al., 2018). We manipulated an experimental MHW condition with a temperature of 34°C, which is about ~2°C higher than the 90% temperature variations measured in the Cam Ranh Bay (Doan X.N., Pham, Q.H. and Dinh K.V., unpublished data).
To start the experiment, acclimatized F0 P. incisus carrying egg sacs (prosomal length = 797.43 ± 2.17 µm, clutch size = 16 ± 2 eggs) were assigned to 1.2-L plastic bottles (15 females each bottle) and fed with I. galbana for acclimation. After 30 h, 180 - 240 F1 nauplii were assigned to each experimental bottle; those bottles were pre-acclimated to experimental temperatures. FPC solution or filtered seawater (1 ml) was added to each experimental bottle. The rearing medium and FPC were renewed daily to minimize the change in the FPC concentration (Truong et al., 2020).
To generate F2, 20 F1 females carrying egg sacs in 10 bottles of each treatment (200 individuals per F1 treatment) were transferred into 20 bottles (10 F1 females per bottle) and incubated for 30 h. F2 offspring in every F1 treatment was divided into four groups, corresponding to four experimental conditions 30°C – no FPC, 30°C – FPC, 34°C – no FPC and 34°C - FPC, resulting in 16 treatments (Fig. 1).
In both generations, we analysed clutch size, hatching success, survival of males and females, cumulative nauplii and faecals of P. incisus over five days using the same procedure in our previous study (Truong et al., 2020).