Freeze-thawing method: a bleeding method from lepidopteran larvae utilizing a spontaneous insect body contraction after a freezing-thawing treatment

Insect haemolymph contains growth promotor(s) for cultured insect cells and is frequently used as an additive to the culture media. Insect haemolymph serves as a pool of a protein product produced by a virus vector-insect hos t system. Haemolymph collection is an essential step in the above process, which should limit the scale and cost of their performance. In the present study, a simple procedure for bleeding from lepidopteran larvae, Bombyx mor i, has been developed which utilized a spontaneous contraction of the insec t body after a freezing-thawing treatment. In the case of fifth-instar B. m ori, 60 to 80% of the total haemolymph was collected by this method. The au thors applied the method to a haemolymph collection from frozen larvae stor ed at -80 degrees C for longer than 1 month. Preservability of the frozen l arvae enabled the development of a system dealing with a huge bulk of insec ts. The bleeding method was effective under cooled condition at 0 degrees C or 4 degrees C, which was desired for protein handling. Development of a l arge system would result in a cost reduction for the insect haemolymph prod ucts such as insect cell-culture additive. Furthermore, the above bleeding method was applied to the nuclear polyhedrosis virus-infected B. mori larva e and up to 80% of the total haemolymph was collected from the virus-infect ed larvae. It suggests the bleeding method as an effective means of haemoly mph collection in the protein productive system using a virus vector and it s insect host.