A quantitative study of blood circulation in the developing adult ascidian tunicate Ciona savignyi (Cionidae).

Development of the adult ascidian tunicate starts when the tadpole larvae attaches to a surface. In approximately two days the solid tadpole will metamorphose into two joined concentric hollow cylinders. The outer cylinder is the body and the inner cylinder is the branchial basket containing openings (stigmata) lined with cilia that pump water through a mucus net that traps food. In six days a heart and circulatory system has formed and blood is pumped through the branchial basket and a smaller visceral cavity containing the heart, stomach, intestine, and gonads. At this stage the animal is quite transparent and moving blood cells are easily distinguished from the fixed cell network of the animal body. The human eye-brain is good at identifying moving cells, but the area of high resolution is limited as is the ability to remember multiple events. However, sequential video frames obtained using a consumer grade camera mounted on a low power microscope, contains the information needed to identify and document moving cells using free open source software described in this report. Subtraction of sequential frames results in a blank difference image if the frames are the same, but produces positive-negative image pairs of cells that have moved during the frame interval. The collection of many sequential difference images thus produces a map of the circulatory system. At six days the circulatory system consists of two perpendicular loops. The larger longitudinal (sagittal) loop runs from the heart along the ventral edge of the branchial basket to a loop around the oral siphon, then back along the dorsal edge of the basket, through three branches in the small visceral cavity, and returns to the heart. One or more transverse loop(s) transports blood from the ventral to the dorsal vessel across the sides of the branchial basket and around the stigmata. Blood cells traverse the longitudinal loop in about 11 sec. As the tunicate matures the number of stigmata increases and the transverse loops develop branches. The branch points then migrate to the dorsal and ventral vessels to form a series of parallel transverse vessels. In the brachial basket blood cells move in both transverse and longitudinal direction around the stigmata.