A Histological Procedure to Study Fungal Infection in the Wax Moth

European Journal of Histochemistry 2014; volume 58:2428

A histological procedure to Introduction Correspondence: Dr. Elisa Borghi, Department of study fungal infection in the Health Sciences, University of Milan, via A. di wax moth Galleria mellonella In the past few decades, there has been a Rudinì 8, 20142 Milan, Italy. growing public consciousness for novel exper- Tel. +39.02.50323287. F. Perdoni,1 M. Falleni,1,2 D. Tosi,1,2 imental model hosts of human pathogens that E-mail: [email protected] D. Cirasola,1,3 S. Romagnoli,2 P. Braidotti,2 are ethically more acceptable than whole mam- Key words: Galleria mellonella, Candida albicans, malian models. However none of the inverte- E. Clementi,4 G. Bulfamante,1,2 E. Borghi1 host-pathogen interaction. brate models adopted can be representative of 1Department of Health Sciences, all the pathogenic aspects of host-pathogen Acknowledgments: the authors would like to University of Milan 1 interactions. The choice of the most appropri- thank Erika Messina and Roberto Tironi for tech- 2 Pathology Unit, San Paolo Hospital, ate model is a critical step in the experimental nical assistance, and Profs. Giulia Morace and Milan design, depending on the pathogenic feature Luciano Sacchi for critical reading of the manu- 3Specialization School in Microbiology of interest. The lepidopteran Galleria mel- script. This work was partially sponsored by a and Virology, University of Milan lonella is susceptible to infections by a large grant to EB from the European Society of Clinical 4Department of Biology and number of microorganisms also taxonomically Microbiology and Infectious Disease (ESCMID Biotechnology “Lazzaro Spallanzani”, unrelated as fungi and bacteria. This model RESEARCH GRANT 2013). has been previously successfully used as a tool University of Pavia, Italy Received for publication: 3 June 2014. to study the pathogenicity of yeast clinical iso- Accepted for publication: 29 July 2014. lates, having a similar immune response to mammals.2 The wax-moth larva model, com- This work is licensed under a Creative Commons Abstract pared to other invertebrates, offers some Attribution NonCommercial 3.0 License (CC BY- advantages such as the ability to grow at mam- NC 3.0). malian temperature (i.e., 37°C) and the possi- The invertebrate model Galleria mellonella ©Copyright F. Perdoni et al., 2014 1 is a widely used factitious host to study the bility to recover infected tissues. The latter is Licensee PAGEPress, Italy microbial pathogenesis in vivo. However, a extremely important to further evaluate host European Journal of Histochemistry 2014; 58:2428 specific procedure for the recovery and the pro- responses, and tissue invasion by the doi:10.4081/ejh.2014.2428 cessing of the infected tissues, important for a pathogen. better understanding of the host-pathogen Insects, though lacking adaptive immune model Galleria mellonella. In the present interactions, has not been reported to our response typical of vertebrates, possess well- study, we used two isolates of Candida albi- knowledge. In the present study we describe a developed innate responses including physical cans, the most common species causing inva- new procedure of fixation and processing of barriers, such as integument and gut, and, sive fungal infection11, differing in the in vitro larval tissue that allows studying the larval after this first line of defence, also host 3-5 biofilm-forming ability, and in the infected lar- topographic anatomy and assessing the mor- immune cells and antimicrobial molecules. 12 phological changes due to the fungal infection. The immune system of insects exhibits a high vae survival. To describe the infectious Lepidopteran larvae were infected with degree of structural and functional similarity process, the infected larvae were processed at Candida albicans strains displaying various with the innate immune system of mammals.6 three time points and sectioned, and several biofilm-forming abilities. The whole larvae The antifungal immunity of insects to fungal histological staining were adapted to the wax- were then examined for tissue changes by his- pathogens involves cellular and humoral com- moth larva tissues to highlight the insect tological techniques. We show that comparing ponents. The hemolymph system of wax moth, response to the yeast invasion. cutting planes, serial transversal sections of which contains different types of hemocytes, A comparison between a traditional fat-body paraffin-embedded larva result in better accu- plays a role in recognition and elimination of squeeze and a whole larva processing was also racy and information recovering. Using this microorganisms. It is important to note that carried out. technique, it was possible to preserve the insects and humans share some evolutionary integrity of G. mellonella internal structures conserved families of antimicrobial peptides allowing the detailed analysis of morphological such as the defensins and specific inhibitors differences in different experimental groups against virulence associated metalloproteinas- Materials and Methods (i.e., healthy vs infected larvae). We were also es.7,8 Most of the studies using the Galleria able to study strain-related differences in the model for the understanding of microbial Final instar larvae of Galleria mellonella pathogenesis of C. albicans by observing the pathogenesis are focused on the screening of were purchased from the Allevamento Cirà, immune response elicited and the invasive- laboratory mutants by means of survival Como, Italy. Larvae were stored in wood shav- ness of two isolates within the larval tissues. curves, in order to identify genes potentially ings in the dark at 15°C prior to experiment. In general, by processing the whole larva relevant for the pathogen virulence.1,9 The All larvae were used within one week from and optimizing routinely histochemical stain- recovery of infected tissue, when performed, receipt. Two Candida albicans strains were ings, it is possible to visualize and analyse often implies the squeeze of the whole fat-body used in the study; the reference strain SC5314, infected tissues. Various degrees of patho- tissue of the larva allowing the visualization of strong biofilm-producer, and a clinical strain genicity (strain- or inoculum-related), and the the pathogens, but with an important loss of CaBA22, biofilm non-producer.12 Overnight infection time course can be described in the insect anatomy and morphology.10 YPD cultures of each isolate were washed, details. Moreover, the host immune response The purpose of our study was to set up his- counted by hemocytometer, and standardized events can be followed throughout the infec- tological procedures to study the host- to 1x107 cells/mL in PBS. G. mellonella larvae tious process leading to a comprehensive pic- pathogen interaction in vivo, by optimizing were injected with 105 CFU/larva, directly into ture of the studied phenomenon. common laboratory techniques of fixation, pro- the hemocoel via the last left proleg, using a cessing, and staining to the experimental Hamilton syringe, and incubated at 37°C in

[page 258] [European Journal of Histochemistry 2014; 58:2428] Original Paper

plastic Petri plates.9,12 Each experimental serial tissue sections was then performed: tion into the tissues (Figure 1B). The evalua- group contained 16-20 randomly chosen larvae haematoxylin and eosin (HE) was used to eval- tion of sagittal sections, obtained by cutting of appropriate weight (330±20 mg); all exper- uate tissue morphology, periodic acid Schiff the entire paraffin-block, was used mainly for iments included a control group consisting in (PAS) and Grocott Methenamine staining the assessment of the whole larva anatomy. A an equal number of larvae injected with sterile (GMS) to highlight fungi localization and host good morphology of the G. mellonella cuticle PBS to monitor the trauma, and non-injected interaction, Giemsa, Alcian blue at various pH and internal organs was obtained coupled with larvae. At 24, 48 and 72 h post-infection, larvae (1, 2.5, and 3.1) to evaluate hemocytes, and a topographical reconstruction of its anatomy were processed by two different methods: i) fat Feulgen staining to evaluate DNA. All histo- (Figure 1C). chemical stainings were performed according body collection (squeeze), and ii) whole larvae Host-fungus interaction body collection. For each time point and group, to standard laboratory protocols.14 Sagittally at least six larvae were used. sectioned larvae were routinely paraffin- We were able to follow the infectious embedded.13 A distance of 50 µm was main- process by both the used methods. In the Fat body collection (squeeze) tained between serial 4-micron-thick tissue squeezed tissues, the infectious agent, in its Infected larvae were cut from the upper part sections of the two halves, and the slides were different morphological forms of yeast and of the body, and gently pressed allowing the stained with haematoxylin and eosin. hyphae, was visualized among the tissues, hemolymph to exit. Once the hemolymph has The microscopic visualization has been per- rarely incorporated in melanized nodules been extruded and discarded, the larva was formed using a Leica Microscope DMLB, and (Figure 2 A-D). The CaBA22 biofilm non-pro- held over a sterile tube containing 1 mL of 10% the image acquisition with the NanoZoomer- ducing strain showed a slower progression of buffered formalin, and then squeezed to collect XR C12000 series (Hamamatsu Photonics K.K., the infection (Figure 2A), with small nodules the fat body. The tube was stored at 4°C Tokio, Japan.). dispersed in the fat body. Blastocells were the overnight to block melanization, and to allow most common morphological form detected in fixation. Fixed tissue was processed by means the larval tissues. The strain showed a tropism of an automatic apparatus (Leica ASP3005, for the gut epithelium, but the lack of a strong Wetzlar, Germany), and routinely paraffin Results filamentation resulted in the sequestering of embedded as previously described.13 Briefly, the pathogen mainly outside the gut wall processing was performed overnight by means To optimize the study of host-pathogen (Figure 2B). The SC5314 isolate, biofilm-pro- of a serial steps where dehydrating reagents interactions, two tissue collection techniques ducer, resulted in more rapid progression of are injected into the station room following were compared: fat body squeeze and whole infection characterized by the presence of standard protocols. Sections were then har- larva processing (Figure 1). simultaneous foci of fungus replication (Figure 2C). The strain showed a high propen- vested with a painting brush on Super Frost Fat body collection (squeeze) Plus (Thermo Scientific, Waltham, MA, USA) sity to filament and a massive invasion of the slides, and stained. The processing by squeezing the larval tis- intestinal outer layer (Figure 2D). In the whole sues allowed the visualization of the fat body larval embedding (Figure 2 E-L), the preserva- Whole larvae processing cellular morphology, and of some intestinal and tion of the cuticle allowed observing the nod- Since the larval exoskeleton is resistant to tracheal structures (Figure 1A). An overall good ules localization into the larva. The CaBA22 known fixative reagents, we performed an ade- tissue larval morphology was achieved: frag- isolate was mainly encapsulated in small nodules localized preferentially in the subcuticular quate fixation by injecting buffered formalin ments of the fat body sometimes entrapping area (Figure 2 E,I,J). The CaBA22 was con- 10% with an insulin syringe in the last left pro- muscle fibers and short tracts of tracheae, lim- firmed to be poorly able to filament, and leg. The volume of solution injected, to have a ited segments of the intestinal tube, and some showed a lesser capability to invade larval tis- turgid consistency of the larvae, was about hemocytes were observed in each sample. sues (Figure 2F). 100μL. Larvae were then stored at 4°C for 24 Whole larva processing Differences in the capability of the two iso- h, to fix internal organs and block melaniza- lates to filament and to invade the larval tion. Whole larvae were dissected transversally The positioning of four to six different rings organs were fully evident during the progres- or sagittally into two halves by means of an in a sole paraffin block facilitated the tridimen- sion of the infection. As previously described anatomic pincers and by using a new lancet sional reconstruction of the larval anatomy, by our group,15 the SC5314 isolate showed a blade for each larva. The procedure was care- and the study of the progression of the infec- fully performed to avoid the squeeze of the larval tissues. The two halves of larvae were placed in the same BioCassette and routinely processed in the path lab. For transversally sectioned larvae, each paraffin-embedded half was further sectioned into two/three rings, after cooling the larva at room temperature for a few minutes to harden the tissues. A crucial step to avoid the paraffin block rupture during microtome sectioning was to keep the cut larval tissues in hot paraffin for one hour to sta- bilize the inclusion, and to obtain a complete merge of the cut rings in the final paraffin Figure 1. Comparison of the two methods used for larval tissues processing. Control larvae were processed in parallel, and stained with HE. A) Fat body recovery by squeezing: block. Finally, four/six rings (one in the distal segments of the gastrointestinal (GI) tract, tracheal (T) system, fat body (FB), and mus- part, two in the middle, and one in the proxi- cle fibers (MF) are visible in a section from squeezed paraffine-embedded material. B, C) mal part) were positioned in each paraffin- Whole larva processing: B) proximal, middle and distal transversal sections are visible in block. Histochemical staining on slides with a sole slide. C) Sagittal section of the larval body.

[European Journal of Histochemistry 2014; 58:2428] [page 259] Original Paper strong tropism for the gut epithelium (Figure 2 G,H), with some fungal elements reaching the intestinal wall even in the first phase of the infection (24 h). The growth of the fungus and its filamentation led to midgut walls invasion at 48 h post infection (Figure 2 G,H). The SC5314 isolate induced nodules with stronger melanization (Figure 2K), and with the tendency to converge in large aggregates (Figure 2 K,L). The nodules were localized more deeply within the fat body, around the intestinal wall and, although more rarely, close to the tracheal system (Figure 2 K,L). The Feulgen staining, initially performed to assess the possible presence of extracellular DNA in the fungal biofilm, was very useful to visualize the melanin distribution. Figure 2. Host-pathogen interaction study by processing larval tissue by squeezing (A-D) and by whole larva analysis (E-L). A,B,E,F,I,J) Larvae infected with the biofilm non-pro- Cellular immune response ducing C. albicans CaBA22 (48 h post-infection). The fungus showed a tropism for the The whole larva processing allowed also the intestinal tract; the presence of pseudohyphae is sporadic whereas the most common morphological form is the blastocells. F) The number of fungal elements able to reach the understanding of the time-dependent hemo- gut lumen is low and appreciable only at post-infection 72 h. I,J) The nodules are small- cytes recruitment in response to the C. albi- er in dimension and usually isolated into the fat body. C, D, G, H, K, L) larva infected cans infection. Uninfected larvae, processed in with the biofilm-producing C. albicans SC5314 (48 h post-infection): a high degree of the same way, were used for comparative pur- fungal filamentation is demonstrated as well as the tendency to invade the gut wall (C, poses. Histochemical stainings were of great D, G, and H). K,L) The nodules are big in dimension and confluent. A-H) PAS staining. I) Alcian Blue pH 2.5. J,K) Feulgen. L) Giemsa. E,G) Middle sections of the larva. Hy, use in the hemocytes characterization: differ- hyphae and pseudohyphae; Y, yeast cells; Mx, biofilm matrix; *, site of infection. ences in shape, cytoplasmic staining affinity and inclusions were detected. Morphological aspects of host-pathogen interactions appeared particularly detailed: fungal pattern of growth at infections sites and the host organ damage were well depicted by PAS, GMS (not showed because it was overlapping with PAS results) and Feulgen stainings, whereas different hemocytes involved in response were well observed by Giemsa, and Alcian blue staining, giving information about the entity of infection and the virulence of pathogen. Morphologically distinct immune cells were visualized in different tissues, and in nodules (Figure 3). Hemocytes localization in control larvae were mainly subcuticolar (Figure 3A), dispersed in the fat body (Figure 3B), and in discrete aggregates around intestinal and tracheal walls (Figure 3C). After the infection with both strains, in the first 24 hours, an increase of hemocytes was observed primarily at the inoculum site Figure 3D). During the infection progression (48 h), we observed, compared to control larvae, an increase number of immune cells in the fat body (Figure 3E), and inside sparse small melanized nodules containing fungal elements. In the late Figure 3. Cellular immune response in control larvae (A-C) and in infected larvae (D-I) phases of infection (72 h), and in relation to in various phases of the infection (24, 48, and 72h). A) Hemocytes monolayer localized sub-cuticularly (Giemsa staining). B) Scattered hemocytes in the normal fat body (PAS). the pathogenicity degree of C. albicans iso- C) small aggregates of hemocytes surrounding the tracheal walls in normal conditions lates, the yeast reached and progressively sur- (Feulgen). D) Hemocytes increase in the subcuticular area close to the injection area rounded the larval intestinal walls. A large (Giemsa). E) Hemocytes increment in the fat body during the infection process (Giemsa). number of hemocytes were then visualized F) A small melanized nodule, and hemocytes recruitment close to tracheae in the later next to intestinal and tracheal walls (Figure phases (72 h) of infection (Giemsa). G) Hemocytes aggregation and mucins characterization of a GI tract (Alcian blue pH 2.5). H) Melanine deposition in nodule with spindle 3F). Cellular characterization of hemocytes, cells clotting, and newer round hemocytes (§) recruited from the hemolymph (Feulgen). with different cytoplasmic granules, inclu- I) Mature nodule formed by hemocytes with different mucins-affinity acquisition (Alcian sions, and mucins production, were further blue pH 3.1). Hemocytes are highlighted by arrows. CT, cuticle; FB, fat body; T, trachea; visualized with the Alcian blue staining at pH GI, gastro intestinal tract; Nd, nodule; Mel, melanin.

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2.5 (Figure 3 G) and Giemsa. Furthermore, dif- interaction, immune cell maturation and be involved in clotting and formation of nod- ferent histochemical properties of hemocytes migration at the infection sites, and the pro- ules with encapsulation at different infectious cellular membranes were appreciated particu- gressive organ damage, were also fully docu- sites.7 Indeed, the hypothesis of a multistep larly with Alcian blue pH 3.1. Hemocytes mented. On slides obtained by processing the maturation process during host immune recruitment at the infection site, and spindle larvae at different time points of infection (24 response was supported; further studies with cells forming the clots were observed in nod- to 72 h), the fungus (yeast cells or hyphae) histochemical stainings are needed to better ules entrapping fungal elements (Figure 3 was easily monitored according to the different characterize morpho-functional aspects of H,I). Specifically, round hemocytes acquired a strain-related behavior. The filamentation of hemocytes. Far away from areas where the spindle shape to stratify around the infectious the biofilm-forming strain allowed the fungus infectious agent could be seen, hemocytes agent in the external part of melanized nod- to invade progressively deep tissues of the appeared as round or oval cells of various ules, thus underlining their role in encapsula- insect.18 Differences in fungal filamentation dimension, sometimes resembling small tion (Figure 3H). and biofilm matrix, detected as amorphous immature cells, with a variable number and weak eosinophilic interhyphal material, were type of cytoplasmic granules and vacuoles. In evident, with filamentous fungi showing a infection sites, hemocytes formed multicellu- more aggressive invasion of host digestive lar aggregates, i.e., nodules, to entrap the Discussion walls within a shorter time. invading yeast. We observed the morphological We were able to draw a comprehensive eval- switch from round hemocytes into spindle cells In vivo research addressing virulence of uation of hemocytes recruitment, and their (Figure 3H) that participate in the encapsula- human pathogens is influenced by experimen- dynamic organization into the hemolymph and tion process. During the infection progression, tal costs, ethical concerns, and by the new other tissues during the immune cell response nodules increased in number and in dimen- European guiding principles16 focused on to fungal infection. Specifically, we could sion, depending on biological characteristics reducing, replacing, and refining the use of observe larva immune cells tropism at differ- of the C. albicans strain. Encapsulation takes mammals as model hosts. Galleria mellonella ent sites of infection, their aggregation in nod- place in cases where the invading organism is is a widely adopted insect model to investigate ules and tendency to confluence, which too big to be phagocytized, and involves cover- the virulence of a broad range of human appeared strictly related to the different behav- ing the parasite with multiple layers of hemo- pathogens.8-9,17 Its response to infections, ior of the C. albicans isolate used. This gave cytes and/or a melanin coat. This feature was based only on innate immune response, is con- precious information about possible sites of highly noticeable in larvae infected with sidered a useful model for understanding the hemocytes production, mobilization, and SC5314, where the pronounced filamentation first steps of human host-pathogens interac- recruitment at infectious sites. In line with resulted in large hyphae aggregates that make tions.7 In this work we described an optimiza- previous observations,7,19 we observed few the phagocytosis worthless. tion of the invertebrate model tissues analysis morphologically distinct immune cells in All the above-described aspects were notice- to better investigate microbial pathogenic healthy G. mellonella larvae. Up to date, differ- able in both the sagittal and transversal sec- mechanisms using standard laboratory tech- ent types of circulating hemocytes comprising tions of the whole larva processing methods, niques. Different conditions and experimental plasmatocytes, granulocytes, spherulocytes, which are easily achievable in a routine groups were analyzed and compared: G. mel- oenocytes, and adipohemocytes have been pathology laboratory. However, transversal pro- lonella larvae infected with Candida albicans characterized in the insect hemoplymph. cessing is less time- and money-consuming, strains and healthy controls were differently These cells are believed to origin from an allowing the visualization in a single slide of processed: fat body collection (squeeze) and immature prohemocyte and are known to be multiple anatomical levels. The G. mellonella whole larva embedding (sagittal and multiple involved in phagocytosis, nodule formation model could support several fungal infections transversal sections). Furthermore, to evaluate and encapsulation during the immune such as Cryptococcus spp.20 and Aspergillus different morphological aspects of host- response.7,17 The characterization of these spp.,9,21 and a variety of different environmen- pathogen interactions, various histochemical blood cells is up to now mainly based on their tal and experimental conditions. Moreover, it stainings were used, in order to select the morphology, but it could represent an oversim- allows choosing the route of infection (sys- most informative ones. plification. A better classification based on temic injection, oral inoculation, or topical Our studies documented that whole larva molecular markers on their cell surface will application) more representative for the infec- processing is the best method to investigate improve our knowledge on hemocytes matura- tious disease studied. host-pathogen reactions and the virulence of tion and immune recruitment. This aspect is Given the relevance of the G. mellonella an infecting agent. Even though both the of particular relevance for tissue hemocytes. model to investigate human infections, the methods can be easily and quickly performed In healthy larvae, we observed the presence standardization of procedures for handling in a pathology lab, with only minimal precau- of a small number of hemocytes in the subcu- this model need to be implemented. The tions to achieve an optimal fixation and inclu- ticular area, in the hemolymph, and in close described technique adds crucial hints to its sion, the whole larva processing gave more association with aero-digestive tracts. This use for tissue recovering and analysis of host- information concerning host immune localization displayed an anatomical distribu- pathogen interaction. response in relation to the pathogen virulence. tion that peculiarly reminds the human BALT Transversal and sagittal whole sectioning of (bronchial associated lymphoid tissue) and larvae facilitated the topographical reconstruc- MALT (mucous associated lymphoid tissue) tion of both external and internal organs. On systems. After the initial phases of infection, References the contrary, microscopic visualization of the we found the number of hemocytes to increase squeezed material showed only segments of in the previous-mentioned sites, being the 1. Desalermos A, Fuchs BB, Mylonakis E. digestive and respiratory tracts, and some immune cells attracted where fungi proliferat- Selecting an invertebrate model host for hemocytes interspersed in the fat body, failing ed and began to invade, whereas the circulat- the study of fungal pathogenesis. PLoS to visualize the cuticle and subcuticular areas. ing hemocytes were reduced. In line with pre- Pathog 2012;8:e1002451. The chronological aspects of host-pathogen vious reports, hemocytes were documented to 2. Brennan M, Thomas DY, Whiteway M,

[European Journal of Histochemistry 2014; 58:2428] [page 261] Original Paper

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