CC BY
31ISSN 2223-4047
Вестник магистратуры. 2020. № 3-2 (102)
Б И О Л О Г И Ч Е С К И Е
НАУКИ
UDC 574.21:574.24:592:577.19
K. T. Maenzanise
A COMPARATIVE STUDY FOR THE DETERMINATION OF A MORE EFFICIENT METHOD TO USE FOR COLLECTING HEMOLYMPH FROM GALLERIA MELLONELLA LARVAE
The aim of this article is to outline an effective and efficient method to collect hemolymph from the larvae of Galleria mellonella and the inhibition of mellanization under three different conditions from the collected hemolymph samples. Under sterile conditions we designed a protocol for an easy and quick collection of clear hemolymph from the larvae of G.mellonella. The results showed that use of Insect phosphosaline buffer helped to prevent mellamization from the collected hemolymph while the second sample which had both insect phosphosaline buffer and urea showed signs of mellanization as well as the third collected sample which only had hemolymph with no buffer. From each larvae we were able to extract approximately of hemolymph. This method can be used to study hemocyte types of Lepidopteran species harvestedfrom the field, or it can be readily engaged in hemolymph proteomic profiling paving way for biotesting at molecular level.
Key words: hemolymph, Galleria mellonella, Lepidoptera, bioassays, protocol, mellanization, insectphosphosaline buffer.
Introduction. Insect hemolymph has many fundamental roles in the physiology of an insect and these include, transportation of nutrients and hormones to the cells and tissues, regulation of body temperature, maintenance of body structure and immune response [11]. In order to fully understand the characterization of the insect species there is need to thoroughly investigate the biochemical components of hemolymph. For insects such as the honeybee, the antennae method for hemolymph sampling (AMHS) has been used and considered as the quickest and most reliable method for hemolymph collection [12]. However, hemolymph collection and analysis for many
© Maenzanise, 2020.
Научный руководитель: ШУРАЛЕВ ЭДУАРД АРКАДЬЕВИЧ - кандидат ветеринарных наук, доцент, Казанский (Приволжский) федеральный университет, Россия.
BecmnuK Maeucmpamypu. 2020. № 3-3 (102)
ISSN 2223-4047
insects has been a challenge owing to their tiny body sizes relative to the honeybee thus the need to design more efficient protocols inorder to resolve this issue [1,5,8].
In insects, the innate immune system is comprised mainly of cellular and humoral immunity [14]. Cellular immunity includes phagocytosis of small invading microbes and the encapsulation of large parasites by circulating hemocytes [14]. Humoral immunity is induced by humoral antibacterial peptides (AMP) produced via the Toll and/or immune deficiency (Imd) pathways, as well as many other immunity proteins [7]. PPO is a humoral protein that can induce melanization around invading pathogens after activation, and induces cellular and humoral immunity simultaneously [7]. Intermediates produced in the melanization process can kill bacteria directly [15].
One lepidopteran model in particular, the greater wax moth, Galleria mellonella, attracts attention because this species can be reared at temperatures to which human pathogens are adapted and which are essential for the production of many microbial toxins [4]. For example, G. mellonella was recently established as a suitable host model to study the pathogenesis of bacterial and yeast species causing diseases in humans, such as Pseudomonas aeruginosa [9], Staphylococcus aureus [4], Bacillus cereus [3], Cryptococcus neoformans [10], and Can-dida albicans [2]. Galleria mellonella is widely used for scientific and industrial purposes, for example, it is used in laboratory experiments on the study of developmental biology, physiology and toxicology of insects; it is used as a host of parasites-entomophages (chalcid, ichneumonid, braconid, tachin).
Hemolymph like blood in vertebrates has antagonistic functions in the physiology of insects playing roles such as transporting nutrients and hormones throughout the body, waste removal, thermal regulations as well as fighting infections [10]. Studies with genetic or physiological insect models have made use of hemolymph extracted from fruit fly, Drosophila melanogaster [11], the red flour beetle, Tribolium custaneum [7], Diatrea sac-choralis [5] and the mulberry silkworm, Bombyx mori [6]. However, little research on the classification, morphology, function and diversity of hemocytes in non-model insect species such as those belonging to the order Lepi-doptera found in the wild have been carried out [12]. Thus the aim of this article is to outline an effective and efficient method to collect hemolymph from the larvae of Galleria mellonella and the inhibition of mellanization under three different conditions from the collected hemolymph samples.
Materials and methods. Larvae of G. mellonella (Lepidoptera: Pyralidae) were reared on a natural diet -fragments of a honeybee's nest at 30°C in the dark. Larvae of the last age stage (250-300 mg by weight) were used throughout the study. Prior to the experiment the larvae were stored in a perforated plastic container which was placed in the fridge (+4°C) overnight. Two insect phosphosaline buffers were also prepared prior to the day of hemolymph extraction with one buffer containing urea while the other had no urea.
Preparation of Insect phosphate buffer solutions. Two solutions of insect phosphosaline buffer (50ml each) were prepared, one with urea and the other one without urea. Each buffer solution consisted of 150mM NaCl, 5mM KCl, 10mM EDTA, 30Mm Sodium citrate ,10mM Urea (for one solution requiring urea), 25ml distilled water and Tris HCl (pH 6.9). The buffer solutions were stored in separate airtight bottles clearly labelled and placed in the refrigerator (+4°C) until use.
Collection of hemolymph. Galleria mellonella larvae were placed in a petri dish and washed under running cold tap water to remove debris before being placed in the freezer (-20°C) to anaesthetize them. Sterilization of the larvae was carried out by dipping a Q-tip in 70% ethanol and gently rubbing it on the body surface of each larvae. In order to collect hemolymph from each larvae, a sterile syringe was used to pierce the hind legs of the larvae resulting in the flow of hemolymph which was then extracted using a micropipette. Using a 10 microliter pipette extract hemolymph and placed in Eppendorf tubes as shown in the table below:
Table 1
Allocation ratios for buffer and hemolymph in each sample
Eppendorf tube Sample content
1 10^l IPS buffer + 3 ^l hemolymph
2 10 ^l IPS buffer (with urea) + 3 ^l hemolymph
3 15 ^l hemolymph (No buffer)
The larvae from which hemolymph had been extracted was placed in the refrigerator (-20°C) for one hour before disposal.
Sample treatment. The collected samples in Eppendorf tubes 1, 2 and 3 were placed in a refrigerator (+4°C) for two hours. After two hours a visual analysis of the samples was carried out to note any colour change. Using a centrifuge (Microspin FV-2400), the 3 samples were centrifuged for 10 minutes at room temperature. After cen-trifugation, the supernatant was pipetted into clean Eppendorf tubes and stored in a refrigerator (-20°C) until the day for use in further bioassay experiments.
Results. We successfully developed an efficient modified method for collecting hemolymph from the larvae of Galleria mellonella. Our particular interest lay in the prevention of melanization from extracted hemolymph
ISSN 2223-4047
BecmnuK Maeucmpamypu. 2020. № 3-2 (102)
and as indicated by the results in Table 2 the absence of pigmentation in the first sample (Containing IPS buffer and hemolymph only) while the black pigmentation observed in samples 2 and 3 indicate that melanization took place. In normal Galleria mellonella larvae, the hemolymph is faint yellowish in color, but can be rapidly melanized in the air, which is known as spontaneous melanization coming from plasma melanization [12]. The results as shown in Table 2 above indicate that there were color changes in samples 2 and 3 while 1 showed no change in hemolymph color.
Table 2
Results from visual observation of samples
Eppendorf tube Observations
1 No colour change
2 Black pigmentation
3 Black pigmentation
Fig. 1. Galleria mellonella larvae showing signs of melanization
Conclusion. Hemolymph melanization is a conserved immune response in insects and other arthropods. Our method allows for the conduction of further experiments such as the proteomic profiling of hemolymph as an indicator for biotesting at the molecular level.
References
1. Casanova-Torres A.M., Goodrich-Blair H. Immune signaling and antimicrobial peptide expression in Lepidoptera // Insects. 2013. 4: 320-338.
2. Brennan M., Thomas D., Whiteway M., Kavanagh K. Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae // FEMS Immunol. Med. Microbiol. 2002. 13: 65-72.
3. Fedhila S., Daou N., Lereclus D., Nielsen-Leroux C. Identifica-tion of Bacillus cereus internalin and other candidate virulence genes specifically induced during oral infection in insects // Mol. Microbiol. 2006. 62: 339-355.
4. Garci a-Lara J., Needham A., Foster S. Invertebrates as animal models for Staphylococcus aureus pathogenesis: a window into host-pathogen interaction // FEMS Immunol. Med. Microbiol. 2005. 43: 311-323.
5. Yi H.Y., Chowdhury M., Huang Y.D., Yu X.Q. Insect antimicrobial peptides and their applications // Appl. Microbiol. Biotechnol. 2014. 98: 5807-5822.
Вестник магистратуры. 2020. № 3-3 (102)
ISSN 2223-4047
6. Kajiwara H, Imamaki A, Nakamura M, Mita K, Xia Q, Ishizaka M. Proteome analysis of silkworm 2. Hemolymph // J Electrophoresis. 2009. 53: 27-31.
7. Lemaitre B., Hoffmann J. The host defense of Drosophila melanogaster // Annu. Rev. Immunol. (2007). 25: 697743.
8. Los A, Strachecka A. Fast and cost-effective biochemical spectrophotometric analysis of solution of insect "blood" and body surface elution // Sensors (Basel). 2018. 18(5): 1494.
9. Miyata S., Casey M., Frank D.W., Ausubel F.M., Drenkard E. Use of the Galleria mellonella caterpillar as a model host to study the role of the type III secretion system in Pseudomonas aeruginosa pathogenesis // Infect. Immun. 2003. 71: 2404-2413.
10. Mylonakis E., Moreno R., El Kohoury J., Idnurm A., Heitmann J., Calderwood S., Ausubel F., Diener A. Galleria mellonella as a model system to study Cryptococcus neoformans pathogensis // Infect. Immun. 2005. 73: 3842-3850.
11. Nation J.L Insect physiology and biochemistry. 3rd ed. Florida: CRC Press; 2016.
12. Sampson C.J., Williams M.J. Protocol for ex vivo incubation of Drosophila. Primary post-embryonic haemocytes for real-time analyses // Methods Mol Bio. 2012. 827: 359-367.
13. Stoepler T.M., Castillo J.C., Lill J.T., Eleftherianos I. A simple protocol for extracting hemocytes from wild caterpillars // J Vis Exp. 2012. 69: e4173.
14. Strand M.R. The insect cellular immune response // Insect Sci. 2008. 15: 1-14.
15. Zhao P., Li J., Wang Y., Jiang H. Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase // Insect Biochem. Mol. Biol. 2007. 37: 952-959.
МАЕНЗАНИСЕ КЕРЕН ТСИТСИ - магистрант, Казанский (Приволжский) федеральный университет, Россия.
