A high quality method for hemolymph collection from honeybee larvae

Author(s)

Butolo, Nicole Pavan ^[1] ; Azevedo, Patricia ^[2] ; de Alencar, Luciano Delmondes ^[2] ; Domingues, Caio E. C. ^[1] ; Miotelo, Lucas ^[1] ; Malaspina, Osmar ^[1] ; Ferreira Nocelli, Roberta Cornelio ^{[1, 3]}

Affiliation

^[1] Univ Estadual Paulista Julio de Mesquita Filho UN, Ctr Estudos Insetos Sociais CEIS, Inst Biociencias, Programa Pos Grad Biol Celular & Mol, Rio Claro, SP - Brazil

^[2] Univ Estadual Campinas UNICAMP SP, Inst Biol, Grp Genet & Genom Conservacao, Programa Pos Grad Genet & Biol Mol, Campinas, SP - Brazil

^[3] Univ Fed Sao Carlos UFSCar SP, Ctr Ciencias Agr, Dept Ciencias Nat Matemat & Educ, Araras, SP - Brazil

• Document type: Journal article
• Source: PLoS One; v. 15, n. 6 JUN 18 2020.
• Web of Science Citations: 0

Abstract

The drastic decline of bees is associated with several factors, including the immune system suppression due to the increased exposure to pesticides. A widely used method to evaluate these effects on these insects' immune systems is the counting of circulating hemocytes in the hemolymph. However, the extraction of hemolymph from larvae is quite difficult, and the collected material is frequently contaminated with other tissues and gastrointestinal fluids, which complicates counting. Therefore, the present work established a high quality and easily reproducible method of extracting hemolymph from honeybee larvae(Apis mellifera), the extraction with ophthalmic scissors. Extraction methods with the following tools also were tested: 30G needle, fine-tipped forceps, hypodermic syringe, and capillaries tubes. The hemolymph was obtained via an incision on the larvae's right side for all methods, except for the extraction with ophthalmic scissors, in which the hemolymph was extracted from the head region. To assess the purity of the collected material, turbidity analyses of the samples using a turbidimeter were proposed, tested, and evaluated. The results showed that the use of ophthalmic scissors provided the clearest samples and was free from contamination. A reference range between 22,432.35 and 24,504.87 NTU (nephelometric turbidity units) was established, in which the collected samples may be considered of high quality and free from contamination. (AU)

• FAPESP's process: 17/21097-3 - Bee-agriculture interactions: perspectives to sustainable use
• Grantee: Osmar Malaspina
• Support Opportunities: BIOTA-FAPESP Program - Thematic Grants
• FAPESP's process: 16/15743-7 - Analysis of effects of strobilurin fungicide pyraclostrobin in midgut of Africanized Apis mellifera Linnaeus, 1758 (Hymenoptera: Apidae) and Melipona scutellaris Latreille, 1811 (Hymenoptera: Apidae)
• Grantee: Caio Eduardo da Costa Domingues
• Support Opportunities: Scholarships in Brazil - Doctorate