Abstract
Bio-analysis of insects is increasingly dependent on highly sensitive methods that require high quality biological material, such as hemolymph. However, it is difficult to collect fresh and uncontaminated hemolymph from adult bees since they are very active and have the potential to sting, and because hemolymph is rapidly melanized. Here we aimed to develop and test a quick and easy method for sterile and contamination-free hemolymph sampling from adult Apidae. Our novel antennae method for hemolymph sampling (AMHS), entailed the detachment of an antenna, followed by application of delicate pressure to the bee's abdomen. This resulted in the appearance of a drop of hemolymph at the base of the detached antenna, which was then aspirated using an automatic pipetter. Larger insect size corresponded to easier and faster hemolymph sampling, and to a greater sample volume. We obtained 80–100 μL of sterile non-melanized hemolymph in 1 minute from one Bombus terrestris worker, in 6 minutes from 10 Apis mellifera workers, and in 15 minutes from 18 Apis cerana workers (+/−0.5 minutes). Compared to the most popular method of hemolymph collection, in which hemolymph is sampled by puncturing the dorsal sinus of the thorax with a capillary (TCHS), significantly fewer bees were required to collect 80–100 μL hemolymph using our novel AMHS method. Moreover, the time required for hemolymph collection was significantly shorter using the AMHS compared to the TCHS, which protects the acquired hemolymph against melanization, thus providing the highest quality material for biological analysis.
Highlights
Apidological research is increasingly reliant on modern highly sensitive analytical methods
Compared to the thorax with capillary of hemolymph sampling (TCHS), antennae method of hemolymph sampling (AMHS) enabled the collection of 80–100 μL of sterile hemolymph within a shorter time and from a smaller number of individuals, which minimizing risk of melanization
In addition to the shortened collection time, the reduced use of insects is another substantial advantage of the AMHS over TCHS
Summary
Apidological research is increasingly reliant on modern highly sensitive analytical methods. Methods for collecting apian hemolymph include decapitation, cutting the tibia or femur, puncturing the heart, puncturing the dorsal or ventral sinus of the thorax, or puncturing the dorsal aorta between the head and thorax [3,6,7] These sampling methods require specialized equipment, such as glass micro-capillaries, specially prepared plastic Pasteur pipettes [1,3,6]. Mayack and Naug [8] describe a method, hemolymph is acquired by clipping bee antennae and and spinning bees in centrifuge tubes at 16,000 RCF for 30 s to obtain hemolymph The use of such tools carries a risk of contaminating the samples with crop or intestinal contents during collection or increase risk of melanization by extending its processing time. The “ideal method” for hemolymph sampling is controversial [9], and this still remains a problem “of the general interest” within entomological studies [9]
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