Development of a new method for collecting hemolymph and measuring phenoloxidase activity in Tribolium castaneum

• Published in: BMC research notes Vol. 12; no. 1; p. 7
• Main Authors: Tabunoki, Hiroko, Dittmer, Neal T, Gorman, Maureen J, Kanost, Michael R
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 07-01-2019; BioMed Central; BMC
• Subjects: Bacteria; Bees; Beetles; Cardiotonic agents; Dopamine; Flour; Free radicals; Genomes; Gram-positive bacteria; Hemolymph; Immune response; Innate immune response; Insects; Measurement; Melanin; Melanization; Methods; Phenoloxidase; Phenols (Class of compounds); Physiological aspects; Physiology; Research Note; Small insects; Tribolium castaneum; United States > US; Phenoloxidase; Dopamine; Hemolymph; Tribolium castaneum; Melanization; Innate immune response; Melanin; Small insects
• ISSN: 1756-0500; 1756-0500
• DOI: 10.1186/s13104-018-4041-y

Hemolymph plays many important roles in the physiology of an insect throughout its lifetime; however, for small-bodied insects, studies are lacking because of the difficulties encountered while collecting hemolymph. The objective of our study was to develop a method to collect hemolymph plasma from various stages of Tribolium castaneum and to evaluate phenoloxidase activity in the plasma samples. We first designed a procedure for easily and quickly collecting clear hemolymph plasma from T. castaneum.
By using this method, we collected approximately 5 µl plasma from 30 individuals at the larval, pupal or adult stages. And then, we studied the expression of phenoloxidase by performing western blot analysis of the plasma samples and found that phenoloxidase is present in hemolymph in each developmental stage. We also measured phenoloxidase activity in control plasma and plasma treated with Gram-positive bacteria, Micrococcus luteus. Phenoloxidase activity was greater in some of the M. luteus-treated plasma samples compared with control samples. Thus, we developed a method to collect hemolymph plasma that is suitable for studies of phenoloxidase activity.