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

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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 host 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 mori, has been developed which utilized a spontaneous contraction of the insect body after a freezing–thawing treatment. In the case of fifth-instar B. mori, 60 to 80% of the total haemolymph was collected by this method. The authors applied the method to a haemolymph collection from frozen larvae stored at −80°C for longer than 1 month. Preservability of the frozen larvae enabled the development of a system dealing with a huge bulk of insects. The bleeding method was effective under cooled condition at 0°C or 4°C, which was desired for protein handling. Development of a large system would result in a cost reduction for the insect haemolymph products such as insect cell-culture additive. Furthermore, the above bleeding method was applied to the nuclear polyhedrosis virus-infected B. mori larvae and up to 80% of the total haemolymph was collected from the virus-infected larvae. It suggests the bleeding method as an effective means of haemolymph collection in the protein productive system using a virus vector and its insect host.

Document Type: Research Article


Affiliations: National Institute of Sericultural and Entomological Science (NISES), Ohwashi, Tsukuba, Ibaraki 305–8634, Japan

Publication date: August 1, 1999

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