A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Abstract

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1 . In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2 . Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3 . They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4,5 . Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6,7 , the mosquitoes Aedes aegypti and Anopheles gambiae 8,9 and the tobacco hornworm, Manduca sexta 10,11 . However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12 .Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13 . Video LinkThe video component of this article can be found at http://www.jove.com/video/4173/ Protocol 1. Material Preparation 1. Prepare needles (1-2 μm tip and 3-4 mm taper) using borosilicate glass capillary tubes and a micropipette puller. Instrument settings: ramp: 561; velocity: 20; heat: 560; delay: 1; pull: 100; pressure: 500. 2. Prepare the collection solution: 60% of Grace's Medium (GM) supplemented with 10% of Fetal Bovine Serum (FBS) and 20% of Anticoagulant Buffer (98 mM NaOH, 186 mM NaCI, 1.7 mM EDTA and 41 mM citric acid, pH 4.5). The above solution is prepared fresh under sterile conditions and kept on ice at all times. 3. Prepare the incubation buffer solution: 90% of GM supplemented with 10% of FBS. This solution is also prepared fresh and kept on ice at all times. 4. Prepare a total of at least 15 μl × the number of insects for both the collection a...

Hemocyte Density Increases with Developmental Stage in an Immune-Challenged Forest Caterpillar

“…The densities of hemocytes in Euclea and Lithacodes that we report here are in accordance with our previous findings in these two species as well as with values reported in other lepidopteran larvae [38]–[40]. The lack of significant differences in constitutive hemocyte density between small and large naïve caterpillars may suggest that homeostatic mechanisms keep hemocyte densities relatively constant, even as caterpillars grow, but that the ability to mount an induced response improves with size/developmental stage (at least in Lithacodes ).…”

“…Live hemocytes were extracted from Lithacodes and Euclea caterpillars by the methods described in [38]. Briefly, caterpillars were anesthetized on ice, injected with a collection solution [70% Grace’s Insect Media (GM) (Gibco), 10% heat-inactivated Fetal Bovine Serum (FBS) (Hyclone) and 20% Anticoagulant Buffer (98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA, 41 mM citric acid, pH 4.5)], returned to ice for 30 mins, and then “bled” to release hemolymph.…”

“…The hemolymph-buffer solution (approx. 10–20 µl/caterpillar) was collected, and an equal volume of incubation solution was added (90% GM, 10% FBS) [38]. Collected hemolymph was then used for functional assays and immunostaining experiments described below.…”

Use of <em>Galleria mellonella</em> as a Model Organism to Study <em>Legionella pneumophila</em> Infection

“…Previous videos have shown various methods for extracting hemocytes from insects 54,55 however, the method presented here is simple and suitable for immediate processing. Once extracted, hemocytes can be easily quantified, used for immunofluorescence, transmission electron microscopy 36 or flow cytometry 56 or cultured and infected ex vivo 3 allowing the response of the cells to infection to be investigated in detail.…”

Extraction of Hemocytes from <em>Drosophila melanogaster</em> Larvae for Microbial Infection and Analysis

“…Additionally, the infection of hemocytes in vivo , prior to extraction, allows the cells to interact with other host proteins and tissue, an advantage over extraction of hemocytes prior to ex vivo infection. A number of different methods have been utilized to obtain a sufficient number of hemocytes in a short period of time to keep the hemocytes alive 8,17,18,19 .…”