Rearing and Maintenance of Galleria Mellonella and Its Application to Study Fungal Virulence

Journal of Fungi

Review Rearing and Maintenance of Galleria mellonella and Its Application to Study Fungal Virulence

1,2, 1, 1, 1,3, Carolina Firacative † , Aziza Khan † , Shuyao Duan † , Kennio Ferreira-Paim † , Diana Leemon 4 and Wieland Meyer 1,* 1 Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Faculty of Medicine and Health, Sydney Medical School, Westmead Clinical School, Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Westmead Hospital (Research and Education Network), Westmead Institute for Medical Research, Westmead 2145, NSW, Australia; cﬁ[email protected] (C.F.); [email protected] (A.K.); [email protected] (S.D.); [email protected] (K.F.-P.) 2 Studies in Translational Microbiology and Emerging Diseases Research Group (MICROS), School of Medicine and Health Sciences, Universidad del Rosario, Bogota 111221, Colombia 3 Infectious Disease Department, Triangulo Mineiro Federal University, Uberaba 38025-440, Brazil 4 Agri Science Queensland, Department of Agriculture and Fisheries and Forestry, Brisbane 4102, QLD, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-2-86273430 These authors contributed equally. † Received: 3 July 2020; Accepted: 5 August 2020; Published: 7 August 2020

Abstract: Galleria mellonella larvae have been widely used as alternative non-mammalian models for the study of fungal virulence and pathogenesis. The larvae can be acquired in small volumes from worm farms, pet stores, or other independent suppliers commonly found in the United States and parts of Europe. However, in countries with no or limited commercial availability, the process of shipping these larvae can cause them stress, resulting in decreased or altered immunity. Furthermore, the conditions used to rear these larvae including diet, humidity, temperature, and maintenance procedures vary among the suppliers. Variation in these factors can affect the response of G. mellonella larvae to infection, thereby decreasing the reproducibility of fungal virulence experiments. There is a critical need for standardized procedures and incubation conditions for rearing G. mellonella to produce quality, unstressed larvae with the least genetic variability. In order to standardize these procedures, cost-effective protocols for the propagation and maintenance of G. mellonella larvae using an artificial diet, which has been successfully used in our own laboratory, requiring minimal equipment and expertise, are herein described. Examples for the application of this model in fungal pathogenicity and gene knockout studies as feasible alternatives for traditionally used animal models are also provided.

Keywords: Galleria mellonella; Cryptococcus gattii; larvae; fungal infection; animal model

1. Introduction The greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) is a ubiquitous pest of honeybee colonies globally causing damage to wax combs in stressed beehives and stored beekeeping equipment, where the larvae feed and transform into moths [1]. G. mellonella can undergo a complete life cycle in 8 to 12 weeks under favorable conditions of temperature and humidity [1]. Eggs hatch to larvae, which undergo 7 moults (instars) before pupation and metamorphosis into adults. The cream-colored 6th instar larvae, which are about 3 cm in length and weigh approximately 300 mg, have become increasingly popular as an alternative non-mammalian model to study fungal disease and virulence [2–7]. As a model host, G. mellonella larvae have several advantages including

J. Fungi 2020, 6, 130; doi:10.3390/jof6030130 www.mdpi.com/journal/jof J. Fungi 2020, 6, 130 2 of 12 low maintenance costs, ability to obtain large quantities, ease of fungal inoculation, and use without major ethical constraints. These larvae can be kept at a range of temperatures, from 20 ◦C to 42 ◦C, allowing experimental procedures to mimic mammalian physiological conditions. Moreover, previous studies have demonstrated that results obtained by the fungal inoculation of larvae correlate with those obtained with murine model experiments [7,8]. Currently, no standardized procedures and conditions for maintenance of the larvae are available. Although they can be obtained commercially in some countries, the propagation protocols, dietary conditions, humidity, and temperature for their maintenance and anti-bacterial requirements differ among companies [9,10]. In addition, different research groups have also reported various dietary conditions for the larvae that are being tested, namely, starvation for one week before inoculating versus no starvation, and providing food after inoculation versus no food provided after inoculation [9]. Furthermore, the insect is not readily available for purchase in most countries and ordering from overseas suppliers can cause the larvae stress due to transportation conditions, resulting in altered larval immunity. These factors would decrease the reproducibility of virulence experiments using G. mellonella among laboratories [11,12]. It has also already been demonstrated that batches of larvae from different suppliers or even from the same supplier can vary in their genetics and overall health, thus affecting larval survival rates [10]. In addition, it may not be possible to obtain larvae from overseas suppliers due to import restrictions. Therefore, propagating and maintaining G. mellonella larvae within the laboratory will be useful for ongoing fungal virulence studies, while also allowing the larvae to acclimatize to optimal conditions for one or more life cycles before experimental use. The successful use of G. mellonella as a reproducible model for fungal virulence experiments relies heavily on the standardization of maintenance and propagation methods to produce the least genotypic and phenotypic variability, which is best achieved by maintaining larval cultures within a controlled laboratory environment. This will overcome any effects due to the variable methodology used by suppliers and also provide a regular, consistent diet to produce larvae of reliable quality for fungal virulence experiments. Here, simple and cost-effective methods for rearing G. mellonella through its life cycle in a laboratory using only basic equipment, even with no prior insect handling experience, is established. These methods have proven to be successful in our own laboratory where we have continually cultured over 10 years approximately 45 generations of G. mellonella. The inoculation protocol associated with the larvae and its advantage as a model for studying fungal infection and virulence, specifically in the example to study cryptococcosis, a life-threatening fungal infection of the lungs and the central nervous system (CNS), is also described.

2. Protocols

2.1. Larvae The larvae were originally obtained from the wax moth culture maintained by the Queensland Department of Agriculture and Fisheries at the Ecosciences Precinct in Dutton Park, Brisbane, Australia.

2.2. Housing Containers and Storage Conditions Tall wide neck glass jars (approximately 22 cm high and 10 cm in diameter) were used for rearing the larvae (Figure1a). Prior to culturing, jars were sterilized by autoclaving to prevent mold and bacterial growth. Jar lids were modiﬁed by cutting a large hole through the center and replacing it with ultra-thin stainless-steel wire mesh (sieve size of 0.1 mm2) to allow ventilation (Figure1b). Any wooden, plastic, or cotton material was avoided to store larvae as they can chew through, allowing escape. A piece of ultra-thin stainless-steel wire mesh secured by a perforated hose clamp was used to cover the whole lid as a secondary protective layer (Figure1c). Overall, the containers were well ventilated and of a clear material allowing observation while being able to contain young larvae (around 600 larvae per jar). Colonies were sporadically stressed by mold or bacterial growth. In this J. Fungi 2020, 6, x 3 of 14 wereJ. Fungi well2020 ,ventilated6, 130 and of a clear material allowing observation while being able to contain young3 of 12 larvae (around 600 larvae per jar). Colonies were sporadically stressed by mold or bacterial growth. In this case, the affected larvae (presenting increase in melanin production and/or slow movements) andcase, the the contents aﬀected of larvae the glass (presenting jars were increase disposed in melanin of appropriately, production with and/ orjars slow washed movements) and autoclaved and the beforecontents reuse. of the glass jars were disposed of appropriately, with jars washed and autoclaved before reuse.

Figure 1. StorageStorage containers containers for for rearing rearing and maintainin maintainingg the larvae. Glass Glass hous housinging jar jar approximately approximately 22 cmcm height height with with wide wide neck neck for housing for housing larval colonieslarval (coloniesa). Modiﬁed (a). jarModified lid (b). Ultra-thinjar lid (b stainless-steel). Ultra-thin stainless-steelwire mesh is ﬁxed wire bymesh a hose is fixed clamp by around a hose clamp the lid around as a secondary the lid as layer a secondary (c). layer (c).

The time taken for G. mellonella toto complete its its life cycle is affe aﬀectedcted by temperature. It can take 8–10 weeks at temperatures temperatures between 28 and 34 °C◦C (Table (Table 11),), butbut upup toto 1313 weeksweeks atat roomroom temperaturetemperature (24 °C).◦C). Larvae and pupae were reared and incubated in the insectarium of of the Westmead Hospital Animal CareCare Facility, Facility, Sydney, Sydney, Australia, Australia, under under controlled controlled conditions conditions of 26 ◦ Cof and26 60%°C and relative 60% humidity. relative humidity.The housing The equipment housing equipment was kept in was a relatively kept in darka re arealatively where dark they area would where not they be disturbed would not aside be disturbedfrom feeding aside and from larval feeding collection and larval periods. collection periods.

2.3. CultureTable 1. Medium Summary for of G. the mellonella life cycle of Galleria mellonella and maintenance requirements at each stage.

The followingLife protocol made culture medium (artificial diet) for approximately 1800 larvae (or Stage Comments Maintenance Requirements three housing jars).Span In a sterilized beaker, 58.3 g (22%) of glycerol, 58.3 g (22%) of organic honey, and 10 mL (4%) of water wereAfter combined. mating, the Then, adult thefemale mixture moths was heated in a microwave at 1000 W for usually lay their eggs and die. A single 1 min and allowed to cool to room temperature. Then, 250 g (48%)Once of cereal eggs are was hatched, mixed discard with the all liquiddead 2 female moth can lay as many as 1000 untilEgg the mixture* crusted; then, 8 g (4%) of instant dry baker’s yeastadult were moths added as well and as mixed pupating thoroughly. larvae weeks eggs in the medium and on container Farex® Original multigrain cereal—fine grains, 6+ months, H.J. Heinzthat Company, have not emerged Southbank, as moths. Victoria, walls. Eggs will then hatch at ambient Australia is recommended. If not available, an equivalent product containing a mixture of ground rice, temperature. maize flour, soy flour, vitamins [vitamin C, Niacin (B3), Thiamin (B1)], mineral (iron), and traces of It is important to feed young larvae wheat and milk could be used.After eggs The hatch, culture the medium young larvae was preparedfeed twice fresh weekly and not to ensure stored, a asmaximum this dried the mixture. on culture medium and begin to number of healthy non-pigmented produce their webbing/silk. Larvae use larvae that can be used for 2.4. Rearing of G. mellonellathis in the silk Laboratory for cocooning as a protective experiments. It is advised to divide Larva After obtaining the larvae,mechanism they wereand for placed transforming into glass into housing larval jars colonies with fresh of overcrowded culture medium jars 5 to 6 pupae. Healthy larvae are cream into separate new jars. Lids should be filled up to 5–8 cm high, and the 6th instar mature larvae that were producing silk were allowed to form weeks colored with no dark discolorations. cleaned of webbing frequently to a protective cocoon and transform into pupae. At this stage, feeding of the larvae stopped. Several jars Note that overcrowding in the jar can improve ventilation. Once they of colonies were always kept,cause to stress, prevent resulting loss inin productioninfection with due develop to mold into or bacterialmature larvae contamination, (2–3 cm in which occurred seldomly.mold When or other the adultmicroorganisms moths emerged, causing uplength) to 10 moths place healthy, were transferred cream-colored to a separate housing jar withgray fresh markings medium, and using pigmentation a male on to femalethe larvae ratio ofinto 1:1. a separate To prevent jar with the fresh moths from flying away, they were transferred tolarvae. new jars by making them flymedium into a plasticin preparation bag, which for was attached to the jar before the lid was open. Male moths have a wingspanexperimental of 10 to 15 use. mm and have Mature larvae start to spin cocoons then When the cocoons are not easily cut a lighter color with2 to faint 3 markings. Adult female moths are larger with a wingspan of up to 20 mm Pupa remain in the pupal stage until ready to open, the larvae are pupating and and are of a darkerweeks brown coloration (Table1). Note that the moth stage does not require feeding and females will die after layingemerge eggs. as adult moths. At this stage, should not be used in experiments as

J. Fungi 2020, 6, x 3 of 14 were well ventilated and of a clear material allowing observation while being able to contain young larvae (around 600 larvae per jar). Colonies were sporadically stressed by mold or bacterial growth. In this case, the affected larvae (presenting increase in melanin production and/or slow movements) and the contents of the glass jars were disposed of appropriately, with jars washed and autoclaved before reuse.

Figure 1. Storage containers for rearing and maintaining the larvae. Glass housing jar approximately 22 cm height with wide neck for housing larval colonies (a). Modified jar lid (b). Ultra-thin stainless-steel wire mesh is fixed by a hose clamp around the lid as a secondary layer (c).

The time taken for G. mellonella to complete its life cycle is affected by temperature. It can take 8–10 weeks at temperatures between 28 and 34 °C (Table 1), but up to 13 weeks at room temperature (24 °C). Larvae and pupae were reared and incubated in the insectarium of the Westmead Hospital Animal Care Facility, Sydney, Australia, under controlled conditions of 26 °C and 60% relative humidity. The housing equipment was kept in a relatively dark area where they would not be disturbed aside from feeding and larval collection periods. J. Fungi 2020, 6, 130 4 of 12

Table 1. Summary of the life cycle of Galleria mellonella and maintenance requirements at each Table 1. Summary of the life cycle of Galleria mellonellastage. and maintenance requirements at each stage.

Life Stage Life SpanComments Comments Maintenance Maintenance Requirements Requirements Span After mating, the adult female After mating, the adult female moths moths usually lay their eggs Once eggs are hatched, discard all usually layand their die. eggs A single and femaledie. A moth Once eggs are hatched, discard all dead adult moths as well as Egg *2 2 weekssingle femalecan moth lay as can many lay as 1000many eggs dead adult moths as well as Egg * pupating larvae that have not weeks as 1000 eggs inin the the medium medium and and on on pupating larvae that have not emerged as moths. container walls.container Eggs walls. will then Eggs hatch will then emerged as moths. at ambienthatch at temperature. ambient temperature. After eggs hatch, the young It is important to feed young larvae After eggs hatch, the young larvae It is important to feed young larvae feed on culture mediumtwice weekly to ensure a maximum feed on culture medium and begin to larvae twice weekly to ensure a and begin to produce their number of healthy non-pigmented produce their webbing/silk. Larvae maximum number of healthy webbing/silk. Larvae use this non-pigmentedlarvae that can larvaebe used that for can be use this silksilk for forcocooning cocooning as a as a Larva experiments.used for experiments. It is advised It to is divide advised Larva protectiveprotective mechanism mechanism and for and for larval coloniesto divide of larval overcrowded colonies ofjars transformingtransforming into pupae. intoHealthy pupae. 5 to 6 into overcrowdedseparate new jarsjars. intoLids separate should larvae are creamHealthy colored larvae with are creamno weeks5 to 6 weeks be cleanednew jars. of webbing Lids should frequently be cleaned to colored with no dark dark discolorations. Note that of webbing frequently to improve discolorations. Note that improve ventilation. Once they overcrowding in the jar can cause ventilation. Once they develop overcrowding in the jar can develop into mature larvae (2–3 cm stress, resulting in infection with into mature larvae (2–3 cm in cause stress, resulting in in length) place healthy, mold or other microorganisms length) place healthy, infection with mold or other cream-colored larvae into a separate causing gray markings and cream-colored larvae into a microorganisms causing gray jar with fresh medium in pigmentation on the larvae. separate jar with fresh medium in markings and pigmentation preparation for experimental use. J. Fungi 2020, 6, x preparation for experimental4 of use. 14 J. Fungi 2020, 6, x 2 to 3 Mature larvae starton to the spin larvae. cocoons When the cocoons are not easily4 ofcut 14 Pupa Pupa weeks then remainMature in the pupal larvae stage start until to spin open,When the larvae the cocoons are pupating are not easilyand readyready to to emerge emerge as as adult adult moths. moths. At At shouldshould not not be be used used in in experiments experiments as as cocoons then remain in the cut open, the larvae are pupating thisthis stage, stage,pupal they they will stagewill no no until longer longer ready to thisthis causes causesand bias bias should in in survival survival not be used rates. rates. in No No 2 to 3 weeksconsumeconsumeemerge food food and and as live adultlive off off moths. the the fat fat At thisfeedingfeedingexperiments is is required required as after thisafter causes all all larvae larvae bias in in suppliessuppliesstage, in in their their they bodies. bodies. will no longer survivalthethe jar jar have have rates. cocooned. cocooned. No feeding is consume food and live oﬀ the required after all larvae in the jar

fat supplies in their bodies. have cocooned. Adult Adult TransferTransferTransfer moths moths moths at at ataa male amale male to to tofemale female female The adult moths do not feed. TheThe adult adult moths moths do do not not feed. feed. Females Females ratioratioratio of of 1:1 of1:1 1:1 into into into a a separate separate a separate jar jar jarwith with with 22 Females will usually begin 2 weekswillwill usually usually begin begin laying laying eggs eggs within within freshfreshfresh medium. medium. medium. Check Check Check once once once weekly weekly weekly weeksweeks laying eggs within a few days a few days after they emerge. forfor eggs eggs on on sides sides of of the the jar jar and and a few days afterafter they they emerge. emerge. for eggs on sides of the jar and bottombottombottom of of oflid.lid. lid.

** Eggs Eggs* Eggs are are not are not notpictured pictured pictured as as as they they they are are too tootoo small small to to to see. see. see.

2.3.2.3. Culture CultureAdult Medium Medium female moths for for G. G. beganmellonella mellonella laying eggs for 1 to 2 weeks at 26 ◦C. Eggs could be found at the edge of the lid or rim of the jar and were scraped gently with a scalpel blade and dropped into the culture TheThe following following protocol protocol made made culture culture medium medium (artificial (artificial diet) diet) for for approximately approximately 1800 1800 larvae larvae (or (or medium. When newly laid eggs did not lift oﬀ the surface easily, one more day was left before moving threethree housing housing jars). jars). In In a a sterilized sterilized beaker, beaker, 58.3 58.3 g g (22%) (22%) of of glycerol, glycerol, 58.3 58.3 g g (22%) (22%) of of organic organic honey, honey, and and them. Eggs hatched in 1 to 2 weeks, and tiny larvae were gravitating to the bottom of the glass jar 1010 mL mL (4%) (4%) of of water water were were combined. combined. Then, Then, the the mi mixturexture was was heated heated in in a a microwave microwave at at 1000 1000 W W for for 1 1 where they could be seen on their thin trail marks. All moths were discarded once hatched larvae minmin and and allowed allowed to to cool cool to to room room temperature. temperature. Then, Then, 250 250 g g (48%) (48%) of of cereal cereal was was mixed mixed with with the the liquid liquid were evident in the same housing jar. Live moths were disposed by placing them into plastic bags, untiluntil thethe mixturemixture crusted;crusted; then,then, 88 gg (4%)(4%) ofof instantinstant drydry baker’sbaker’s yeastyeast werewere addedadded andand mixedmixed which were stored for one day in a fridge and then took for incineration. Female moths can lay 400 to thoroughly.thoroughly. FarexFarex®® OriginalOriginal multigrainmultigrain cerealcereal——finefine grains,grains, 6+6+ months,months, H.J.H.J. HeinzHeinz Company,Company, 1000 eggs at a time. Southbank,Southbank, Victoria, Victoria, Australia Australia is is recommended. recommended. If If not not available, available, an an equivale equivalentnt product product containing containing a a Larvae were reared for 5 to 6 weeks, during which fresh culture medium was added twice mixturemixture ofof groundground rice,rice, maizemaize flour,flour, soysoy flour,flour, vitaminsvitamins [vitamin[vitamin C,C, NiacinNiacin (B3),(B3), ThiaminThiamin (B1)],(B1)], weekly. Separating larvae into new jars ensured an appropriate ratio of food per larvae and avoided mineralmineral (iron), (iron), and and traces traces of of wheat wheat and and milk milk coul couldd be be used. used. The The culture culture medium medium was was prepared prepared fresh fresh overcrowding, which can cause stress and the discoloration of larval cuticles. Old and hardened andand not not stored, stored, as as this this dried dried the the mixture. mixture. medium was discarded regularly. Lids were frequently cleaned of webbing and pupating larvae to 2.4.2.4.improve Rearing Rearing cross-ventilation. of of G. G. mellonella mellonella G.in in mellonellathe the Laboratory Laboratorypupated for 2 to 3 weeks, and a new cycle began when adult moths emerged. AfterAfter obtaining obtaining the the larvae, larvae, they they were were placed placed into into glass glass housing housing jars jars with with fresh fresh culture culture medium medium filledfilled up up to to 5 5––88 cm cm high, high, and and the the 6th 6th instar instar mature mature larvae larvae that that were were producing producing silk silk were were allowed allowed to to formform aa protectiveprotective cocooncocoon andand transformtransform intointo pupae.pupae. AtAt thisthis stage,stage, feedingfeeding ofof thethe larvaelarvae stopped.stopped. SeveralSeveral jarsjars ofof coloniescolonies werewere alwaysalways kept,kept, toto preventprevent lossloss inin productionproduction duedue toto moldmold oror bacterialbacterial contamination,contamination, whichwhich occurredoccurred seldomly.seldomly. WhenWhen thethe adultadult mothsmoths emerged,emerged, upup toto 1010 mothsmoths werewere transferredtransferred toto aa separateseparate housinghousing jarjar withwith freshfresh medium,medium, usingusing aa malemale toto femalefemale ratioratio ofof 1:1.1:1. ToTo preventprevent thethe mothsmoths fromfrom flyingflying away,away, theythey werewere trantransferredsferred toto newnew jarsjars byby makingmaking themthem flyfly intointo aa plasticplastic bag, bag, which which was was attached attached to to the the jar jar before before th thee lid lid was was open. open. Male Male moths moths have have a a wingspan wingspan of of 10 10 toto 1515 mmmm andand havehave aa lighterlighter colorcolor withwith faintfaint markings.markings. AdultAdult femalefemale mothsmoths areare largerlarger withwith aa wingspanwingspan of of up up to to 20 20 mm mm and and are are of of a a darker darker brown brown coloration coloration (Table (Table 1). 1). Note Note that that the the moth moth stage stage doesdoes not not require require feeding feeding and and females females will will die die after after laying laying eggs. eggs. AdultAdult female female moths moths began began laying laying eggs eggs for for 1 1 to to 2 2 we weekseks at at 26 26 °C. °C. Eggs Eggs could could be be found found at at the the edge edge ofof the the lid lid or or rim rim of of the the jar jar and and were were scraped scraped gently gently with with a a scalpel scalpel blade blade and and dropped dropped into into the the culture culture medium.medium. WhenWhen newlynewly laidlaid eggseggs diddid notnot liftlift offoff ththee surfacesurface easily,easily, oneone moremore dayday waswas leftleft beforebefore movingmoving them.them. EggsEggs hatchedhatched inin 11 toto 22 weeks,weeks, andand tinytiny larvaelarvae werewere gravitatinggravitating toto thethe bottombottom ofof thethe glassglass jar jar where where they they could could be be seen seen on on their their thin thin trail trail marks. marks. All All moths moths were were discarded discarded once once hatched hatched larvaelarvae were were evident evident in in the the same same housing housing jar. jar. Live Live moths moths were were disposed disposed by by placing placing them them into into plastic plastic bags,bags, which which were were stored stored for for one one day day in in a a fridge fridge an andd then then took took for for incinerati incineration.on. Female Female moths moths can can lay lay 400400 to to 1000 1000 eggs eggs at at a a time. time. LarvaeLarvae werewere rearedreared forfor 55 toto 66 weeks,weeks, duringduring whichwhich freshfresh cultureculture mediummedium waswas addedadded twicetwice weekly.weekly. Separating Separating larvae larvae into into new new jars jars ensured ensured an an appropriate appropriate ratio ratio of of food food per per larvae larvae and and avoided avoided overcrowding,overcrowding, whichwhich cancan causecause stressstress andand thethe discdiscolorationoloration ofof larvallarval cuticles.cuticles. OldOld andand hardenedhardened mediummedium was was discarded discarded regularly. regularly. Lids Lids were were freque frequentlyntly cleaned cleaned of of webbing webbing and and pupating pupating larvae larvae to to improveimprove cross-ventilation.cross-ventilation. G.G. mellonellamellonella pupatedpupated forfor 22 toto 33 weeks,weeks, andand aa newnew cyclecycle beganbegan whenwhen adultadult moths moths emerged. emerged.

J. Fungi 2020, 6, 130 5 of 12

J. FungiSixth 2020,instar 6, x larvae, which were lightly colored with no graying or dark marks visible and weighed5 of 14 around 300 mg and were 3 cm long, were selected for inoculation (see below). 2.5. G. mellonella Inoculation 2.5. G. mellonella Inoculation To assess the performance of G. mellonella larvae as a model system to study fungal virulence, threeTo Cryptococcus assess the performancegattii strains previously of G. mellonella studiedlarvae in a as murine a model model system of infection to study were fungal selected virulence, for threeinoculationCryptococcus into G. gattii mellonellastrains larvae previously reared studied and maintained in a murine in modelour insectarium. of infection The were tested selected strains for inoculationwere CDCR265 into (VGIIa)G. mellonella and CDCR272larvae reared (VGIIb) and maintainedfrom the Vancouver in our insectarium. Island outbreak, The tested which strains have werebeen CDCR265characterized (VGIIa) as highly and CDCR272 and low (VGIIb)virulent, from respectively the Vancouver [13], and Island the outbreak,strain DMST20767, which have which been characterizedwas previously as found highly to andbe of low comparable virulent, respectivelyvirulence to CDCR265 [13], and thein a strain murine DMST20767, model [14]. which was previouslyTo prove found the tovalue be of of comparable G. mellonella virulence larvae as toa screening CDCR265 tool in a for murine the impact model of [14 gene]. knockouts on virulenceTo prove profiles, the value two ofsamplesG. mellonella of knockoutlarvae asmu atants screening were toolalso for tested: the impact the is-cDNA of gene knockouts26, which oncorresponds virulence to profiles, the putative two collagen samples binding of knockout domain mutants of a collagenase were also gene tested: present the in is-cDNA the Pacific 26, whichNorth West corresponds VGIIa genotype, to the putative and the collagen is-cDNA binding 2, which domain corresponds of a collagenase to the endoribonuclease gene present in gene the Pacificpresent North in the West VGIIa/c VGIIa genotypes genotype, [15]. and These the is-cDNA genes were 2, which disrupted corresponds in the high-virulence to the endoribonuclease reference genestrain presentCDCR265 in theusing VGIIa overlap-PCR/c genotypes with [15 ].the Theseselective genes marker were disruptednourseothricin in the acetyltransferase high-virulence reference(NAT) [16] strain and CDCR265transformed using using overlap-PCR a biolistic with shotgu then selective to generate marker knockout nourseothricin mutants acetyltransferase by homologous (NAT)recombination. [16] and transformedBoth knockouts using Δcoll a biolistic and Δendo shotgun were tocompared generate with knockout the wild-type mutants CDCR265 by homologous strain recombination.in the G. mellonella Both larvae. knockouts ∆coll and ∆endo were compared with the wild-type CDCR265 strain in the G.Groups mellonella of 10larvae. similar-sized 6th instar larvae were selected and placed in a Petri dish. Fungal strainsGroups were ofgrown 10 similar-sized on Sabouraud’s 6th instar Dextrose larvae agar were at selected 27 °C andfor 48 placed h prior in a to Petri inoculation. dish. Fungal The strains yeast wereinoculums grown were on prepared Sabouraud’s in phosphate-buffered Dextrose agar at sali 27 ne◦C (PBS), for 48 and h priorthe concentration to inoculation. was adjusted The yeast to inoculums1.0 × 108 cells/mL were prepared using a Neubauer in phosphate-bu chamber.ffered Injections saline (PBS),were performed and the concentration with 10 µL of was the adjusted inoculum to 1.0 108 cells/mL using a Neubauer chamber. Injections were performed with 10 µL of the inoculum using× a 50 U insulin syringe with a 29-gauge needle. For inoculation, each larva is held over the usingmiddle a 50finger U insulin and protected syringe with with a 29-gaugea rubber needle.thimblette For, inoculation,exposing the each larval larva pro-legs is held (Figure over the 2a). middle The fingerforefinger and and protected thumb with are used a rubber to stabilize thimblette, the insect exposing and the create larval a bend pro-legs for the (Figure syringe2a). needle The forefinger to enter andthe last thumb left arelarval used pro-leg to stabilize (Figure the 2b). insect Tenand uninoculated create a bend and for 10 thePBS-inoculated syringe needle larvae to enter were the included last left larvalas controls pro-leg to (Figure ensure2b). that Ten uninoculatedenvironmental and cond 10 PBS-inoculateditions and physical larvae wereinjuries included by inoculation, as controls torespectively, ensure that do environmental not affect the conditionssurvival rates. and Post physical inoculation, injuries larvae by inoculation, were placed respectively, into a clean do Petri not adishffect without the survival food and rates. incubated Post inoculation, at 37 °C. Mortality larvae were was placedmonitored into at a clean24-h intervals Petri dish over without a period food of and10 days incubated by gently at 37probing◦C. Mortality the larva was for monitoredmovement. at Dark 24-h coloration intervals over of the a periodcuticle ofwas 10 also days monitored by gently probingfor severity the of larva infection. for movement. Survival curves Dark colorationwere graphed of the and cuticle analyzed was by also Log-Rank monitored (Mantel–Cox) for severity test of infection.using the SurvivalGraph Pad curves Prism were 6.04 graphed software and analyzed(La Jolla, byCA, Log-Rank USA), and (Mantel–Cox) median survival test using times the Graph were Padobtained Prism for 6.04 each software strain. (La Jolla, CA, USA), and median survival times were obtained for each strain.

Figure 2. InoculationInoculation of the larvae. Exposure Exposure of the the pro-legs over the middle ﬁngerfinger (a). Inoculation of the larvae into the last left pro-leg ((b).).

2.6. Histopathology At the end point of the experiment, dead infected larvae were collected and immediately placed in 10% bubufferedffered formalinformalin fixativefixative for aa minimumminimum ofof fourfour weeks.weeks. The long fixationfixation period produced better results, since the larval exoskeleton has low permeability to most fixative reagents [17]. Then, the larvae were transferred into increasing concentrations of ethanol (70%, 80%, 90%, and 100%) for one hour each, embedded in paraffin, cross-sectioned, and stained by histochemical techniques with hematoxylin and eosin (HE) and special stain Mayer’s Mucicarmine (MM) to stain the cryptococcal

J. Fungi 2020, 6, 130 6 of 12 better results, since the larval exoskeleton has low permeability to most ﬁxative reagents [17]. Then, the larvae were transferred into increasing concentrations of ethanol (70%, 80%, 90%, and 100%) for one hour each, embedded in paraﬃn, cross-sectioned, and stained by histochemical techniques with hematoxylin and eosin (HE) and special stain Mayer’s Mucicarmine (MM) to stain the cryptococcal cell capsules red. Photographs were taken of the prepared slides using an Olympus BX43 microscope.

3. Results Results from the murine experiments were obtained from our previous study [14]. To show the G. mellonella capacityJ. Fungi of 2020 the, 6, x larvae model to study fungal virulence, the larvae were inoculated6 of 14 with the high-virulence C. gattii strain CDCR265, which began dying 96 h after inoculation, and with the straincell DMST20767, capsules red. with Photographs which the were larvae taken began of the dying prepared 72 h slides post inoculationusing an Olympus (Figure BX433a). However, the medianmicroscope. survival times calculated indicated that statistically, there was no significant difference between3. Results the survival curves for the two strains (p = 0.2841), which was similar to the results obtained in mice inoculated with these two strains (p = 0.1375) (Figure3b) [ 14]. Strain CDCR272 did not cause Results from the murine experiments were obtained from our previous study [14]. To show the mortality in either G. mellonella larvae or mouse models (Figure3a,b). capacity of the G. mellonella larvae model to study fungal virulence, the larvae were inoculated with theTo showhigh-virulence the applicability C. gattii strain of the CDCR265,G. mellonella whichlarvae began modeldying 96 to h characterize after inoculation, mutant and strains,with the the C. gattii knockoutstrain DMST20767, strains ∆coll withand which∆endo thewere larvae screened began dying for 72 enhanced, h post inoculation reduced, (Figure or similar 3a). However, pathogenicity in G. mellonellathe medianlarvae survival compared times calculated to the wild-type indicated that strain statistically, CDCR265. there The was survival no significant curve difference for ∆coll showed a significantlybetween the lower survival virulence curves ( pfor= the0.0030) two strains (Figure (p 3=c), 0.2841), whilst which∆endo waswas similar found to the to beresults of equal obtained pathogenicity in comparisonin mice inoculated to the with wild-type these two strain strains CDCR265 (p = 0.1375) (p (Figure= 0.0645) 3b) [14]. (Figure Strain3d). CDCR272 did not cause mortality in either G. mellonella larvae or mouse models (Figure 3a,b). G. mellonella MelanizationTo show the isapplicability a primary of immunethe G. mellonella response larvae to model infection to characterize in mutant strains,larvae the and C. gives an indicationgattii knockout of the severity strains ∆coll of infection and ∆endo [were1]. Strains screened CDCR265 for enhanced, and reduced, DMST20767 or similar showed pathogenicity similar degrees of melanizationin G. mellonella five dayslarvaepost compared infection to the (Figure wild-type4a,b). strain Larvae CDCR265. infected The withsurvival the curve low-virulence for ∆coll C. gattii strainshowed CDCR272 a significantly presented lower higher virulence median (p = 0.0030) survival (Figure times 3c), with whilst little ∆endo to was no melanizationfound to be of equal in comparison to thepathogenicity high-virulence in comparison strains (CDCR265 to the wild-type and strain DMST20767), CDCR265 ( asp = expected0.0645) (Figure (Figure 3d).4 c).

Figure 3. Log-Rank survival curves and median survival times (MST) of 10 Galleria mellonella larvae (a) Figure 3. Log-Rank survival curves and median survival times (MST) of 10 Galleria mellonella larvae and six female BALB/c mice based on data previously obtained by our group [13,14](b) inoculated (a) and six female BALB/c mice based on data previously obtained by our group [13,14] (b) with diﬀerent Cryptococcus gattii strains. G. mellonella inoculated with the knockout mutant strains ∆coll inoculated with different Cryptococcus gattii strains. G. mellonella inoculated with the knockout (c) andmutant∆endo strains(d) wereΔcoll ( comparedc) and Δendo with (d) were the high-virulencecompared with the strain high-virulence CDCR265 strain and theCDCR265 low-virulence and strain CDCR272.the low-virulence The MST strain foreach CDCR272. strain The is givenMST for in ea brackets.ch strain is Larvae given in (a brackets.,c,d) and Larvae mice ( (ab,c),d inoculated) and with phosphate-bumice (b) inoculatedﬀered saline with (PBS)phosphate-buffered and uninoculated saline controls(PBS) and are uninoculated shown (dotted controls line). are shown (dotted line).

Melanization is a primary immune response to infection in G. mellonella larvae and gives an indication of the severity of infection [1]. Strains CDCR265 and DMST20767 showed similar degrees of melanization five days post infection (Figure 4a,b). Larvae infected with the low-virulence C. gattii

J. Fungi 2020, 6, 130 J. Fungi 2020, 6, x 7 of 14 7 of 12 strain CDCR272 presented higher median survival times with little to no melanization in comparison to the high-virulence strains (CDCR265 and DMST20767), as expected (Figure 4c).

Figure 4. Larvae showing high melanization at day 6 post infection with Cryptococcus gattii strains Figure 4. Larvae showingCDCR265 (a high) and DMST20767 melanization (b), and littleat melanization day 6 of post infected infection larva with CDCR272 with (cCryptococcus). gattii strains

CDCR265 (a) and DMST20767Histopathological (b ),findings and littleof uninfected melanization and infected oflarvae infected indicated larva that skeletal with muscle CDCR272 (c). damage and high cryptococcal cell numbers and spread within the organism are correlated with Histopathologicalvirulence ﬁndings (Figure of5a–j). uninfected The larvae infected andinfected with the high-virulence larvae indicated strains CDCR265 that skeletaland muscle damage and high cryptococcalDMST20767 cell numbersshowed skeletal and muscle spread damage and within large nodular the organismlesions (Figure 5h–j), are while correlated the with virulence larvae infected with the low virulent strains CDCR272 shows little muscle damage and small (Figure5a–j). The larvaenodular infected lesions (Figure with 5c–g). the high-virulence strains CDCR265 and DMST20767 showed skeletal muscle damage and large nodular lesions (Figure5h–j), while the larvae infected with the low virulent strainsJ. CDCR272Fungi 2020, 6, x shows little muscle damage and small nodular lesions (Figure8 of 14 5c–g).

Figure 5. Hematoxylin and eosin (HE) and Mayer’s Mucicarmine (MM) stained sections of Galleria Figure 5. Hematoxylinmellonella. Uninfected and control eosin larvae (HE) stained and withMayer’s HE (a) and MM Mucicarmine (b) are shown. MM (MM) stained stainedlarvae sections of Galleria mellonellainfected. Uninfected with the low-virulence control larvae Cryptococcus stained gattii with strain, HE CDCR272, (a) and show MM cryptococcal (b) are shown. cells MM stained larvae infectedcontained with the within low-virulence small nodular lesionsCryptococcus surrounded by gattii hemocytesstrain, (c,d). CDCR272, Little muscle damage show apart cryptococcal cells from nodular regions is seen. HE (e) and MM (f,g) stained larvae infected with the high-virulence C. contained withingattii small strain, nodular CDCR265, lesions show skeletal surrounded muscle damage by hemocytesand large nodular (c,d lesions.). Little Giant muscle cryptococcal damage apart from nodular regionscells, is seen.which are HE too (largee) and to be MM engulfed (f, gby) hemocytes, stained larvaeare indicated infected by arrows. with HE ( theh) and high-virulence MM (i,j) C. gattii stained larvae infected with the strain DMST20767 show extensive skeletal muscle damage. strain, CDCR265,Cryptococcal show skeletalcells are found muscle in nodular damage regions an andd diffused large throughout nodular the lesions. surrounding Giant tissue cryptococcaland cells, which are too largehemocoel. to be engulfed by hemocytes, are indicated by arrows. HE (h) and MM (i,j) stained larvae infected with the strain DMST20767 show extensive skeletal muscle damage. Cryptococcal cells are found in nodular regions and diﬀused throughout the surrounding tissue and hemocoel.

J. Fungi 2020, 6, 130 8 of 12

4. Discussion The use of non-mammalian models in virulence studies overcomes important limitations that come with murine models, including ethical considerations associated with mammalian model systems, housing expenses, and time spent on maintenance. To use the model in an ethical way, the number of larvae used per experiment should still be carefully considered. The G. mellonella insect model has proven useful in numerous fungal pathogenesis studies that include yeasts, such as Candida spp. [18,19] and Trichsoporon spp. [20]; molds, such as Aspergillus spp. [6,21], Fusarium oxysporum [22], Scedosporium aurantiacum [23], Madurella mycetomatis [24], and Mucorales species [25], as well as the dimorphic fungi Histoplasma capsulatum and Paracoccidiodes lutzii [26]. Moreover, the G. mellonella model has the additional advantage of being inexpensive to propagate within the laboratory in comparison to mammalian models and easy to manipulate for experimental procedures. In addition, apart from studying the pathophysiology of different fungal species, more recently, this invertebrate model has been successfully used for testing the in vivo efficacy of conventional and novel antifungal drugs [27]. In the case of Cryptococcus neoformans and C. gattii, several approaches using mainly vertebrate but also invertebrate models have been used to study fungal pathogenicity [10,28–33], recognize genes involved in pathogenicity, identify strain virulence, virulence factors (including capsule, melanin production and biofilm formation), and undertake antifungal susceptibility testing of existing and new compounds [34–39]. Although wax moth larvae are a very common pest found in apiaries, especially on old wax combs in storage, and it may be possible to source the initial larvae to set up a moth breading colony directly from a beekeeping operation, for laboratories routinely ordering G. mellonella larvae for ongoing experiments, there are likely to be a range of issues. These may include the cost, availability, and permissions requiring importation and transportation in unfavorable conditions, which can affect insect survival rates. To overcome these issues, cost-effective and simple methods for the propagation and maintenance of G. mellonella using inexpensive equipment (Table S1), with minimal expertise, were developed. The larval cultures are self-contained, with development from egg to adult moth in the same environment allowing a continuous supply of larvae from multiple jars of G. mellonella at various life stages (Table1). In addition, the diet described here took into account the results reported in a previous study, which compared three different dietary conditions [40], but simplified even more the diet composition, still containing all nutrients required for the healthy and fast growth of the larvae, including carbohydrates, protein, and lipids, as previously reported. Using the herein described rearing method, the G. mellonella colony has been continuously maintained for approximately 45 life cycles since the colony was started in 2011. This report also provides an artificial diet protocol for researchers who have ordered larvae for experimental use within 1 to 2 weeks. Most studies suggest utilizing such larvae within 7 days of receiving them. However, if this is not possible, our protocol can be used to provide sustenance for the larvae. It has been shown that larvae deprived of food for 7 days have a reduced expression of a range of antimicrobial peptides and immune proteins causing an increased susceptibility to infection [11]. Other factors, such as temperature, agitation, and transportation conditions have also been shown to increase stress and infectivity [10,12,18,40]. This may result in a variation in results between laboratories working with the same fungal strains, thus highlighting the importance of having standardized procedures for both the maintenance of larvae to allow acclimatization and subsequent experimental protocols, as reported previously [9]. G. mellonella larvae are relatively large in size, which facilitates easy inoculation and the collection of hemolymph and tissue samples for further analysis. Training to perform systemic inoculation of the larvae takes a few hours. Therefore, it is suggested to first practice the inoculation procedure with PBS. Other methods for the delivery of fungi have been previously described [5]. However, injecting offers the benefit of direct delivery of known fungal cell concentration into the larval hemocoel [4,41]. There are a number of advantages for using the G. mellonella model in fungal virulence studies. They are easily maintained under temperatures from 25 to 37 ◦C, and they are also suitable to mimic J. Fungi 2020, 6, 130 9 of 12 the physiological conditions in humans and other mammals, enabling temperature-related virulence studies [7,42]. By contrast, other non-mammalian models currently used for fungal studies do not possess this advantage and can only survive at temperatures lower than 27 ◦C[41]. However, not all fungal virulence factors necessary for the pathogenesis of human fungal infections are activated below human physiological temperatures. For example, C. neoformans demonstrates a higher degree of virulence at 37 ◦C than at 30 ◦C in larvae. Many of the same virulence traits involved in mammalian pathogenesis (such as polysaccharide capsule and various C. neoformans genes; GPA1, PKA1, and RAS1) were also associated with larval mortality [7]. Furthermore, in many aspects, the immune response against pathogens in insects is similar to the innate immunity in mammals [38,43]. Hemocytes in G. mellonella larvae use a similar mechanism to human neutrophils to recognize fungal cells using factors, such as pathogen recognition receptors, phagocytosis, and the production of superoxide, in the oxidative burst pathway [44]. The structural and functional similarities of the larval innate immune system to mammalian innate immunity makes G. mellonella an exceptional screening model for fungal disease, phagocytic cell studies, and screening antifungal agents with results correlating to those obtained in mammalian models [20,22]. To show two examples for potential applications of the G. mellonella model, we have applied it to fungal virulence and gene knockout impact studies. Our own virulence experiments showed comparable results, using survival curves (Figure3), degree of melanization (Figure4), and histopathology (Figure5) of larvae infected with C. gattii strains that have been previously characterized using the BALB/c mice model [13,14]. The strains CDCR265 and DMST20767 were highly virulent, and strain CDCR272 was low virulent in both the G. mellonella larvae (data obtained herein) and the mouse model (data previously obtained by our group [13/14]). However, the main differences between both models is the duration of the experiments, with the Galleria model cutting down the time to less than two weeks, while the mouse model takes almost two months (Figure3). Similarly, larval tissue damage was more evident in larvae inoculated with high-virulence strains, as seen previously in murine models, in which rodents inoculated with strains of higher virulence presented a higher fungal burden [31–33]. Skeletal damage at the tissue level and nodular lesions consist typically of an accumulation of yeast masses that can be observed causing tissue disorganization, which resembles the formation of granulomas or lesions in the brain parenchyma in pulmonary or meningeal cryptococcosis, respectively [45]. The pathogenicity of fungal knockout mutant strains has also been successfully assessed in the Galleria larvae model, and these strains have been found to have similar virulence when tested in murine models [8,21]. Here, the low virulence of the knockout mutant strain ∆coll was probably related to a weak attachment in the larva soft tissues, as collagen is an essential protein widely distributed among living organisms, including G. mellonella. Pre-treatment of G. mellonella with peptides that inhibit the cell wall adhesion of Paracoccidiodes brasiliensis and P. lutzii increased the survival of larvae from 64% and 60% respectively, in addition to the hemocytes count [46]. The lack of difference in the knockout mutant strain ∆endo could be related with redundancy in the pathways controlled by this large family of proteins. Virulence experiments with fungal mutants in larvae can also assist in the selection of mutant strains for further analysis in mammalian models, thereby reducing the number and unnecessary killing of experimental animals, as well as obtaining preliminary data for funding applications. However, the G. mellonella larvae model does not give insights into the adaptive immune response with regard to antibody generation and cannot be used to model organ specific pathologies [4]. Although the first draft genome sequence of G. mellonella was reported in 2018 [47], studies on transcriptional genetics and complex innate immune responses and homology studies between G. mellonella and human, mouse, and other model hosts are still needed. Thus, this model is still used best for screening virulent fungal strains, knockout mutant strains, or antifungal agents for subsequent mammalian model experiments [3,8,21,28]. Finally, a recent study described some variation between the results obtained using the G. mellonella model when compared with the traditionally used mouse model and concluded that it does not predict murine survival for all studied strains, indicating that some caution should be used when interpreting the results [48]. J. Fungi 2020, 6, 130 10 of 12

As the popularity of G. mellonella as a virulence model for both fungi and bacteria has increased, standardized propagation methods and experimental procedures are needed to make G. mellonella larvae a successful virulence model. In this study, this issue was addressed by describing simple methods for successfully rearing these G. mellonella larvae through all life cycle stages using an artiﬁcial diet and standardized conditions. Our protocols also provide quality G. mellonella larvae with the minimal genetic and phenotypic variability needed for reproducible virulence studies.

Supplementary Materials: The following are available online at http://www.mdpi.com/2309-608X/6/3/130/s1, Table S1: Equipment and average cost involved in propagating Galleria mellonella in the laboratory. Author Contributions: Conceptualization, W.M.; methodology, C.F., A.K., S.D., D.L. and K.F.-P.; validation, A.K., and K.F.-P.; formal analysis, C.F. A.K. and K.F.-P.; investigation, C.F., A.K., S.D. and K.F.-P.; resources, W.M. and D.L.; data curation, C.F.; writing—original draft preparation, C.F., A.K. and S.D.; writing—review and editing, C.F., A.K., S.D., K.F.-P., D.L. and W.M.; visualization, A.K. and K.F.-P.; supervision, W.M.; project administration, W.M.; funding acquisition, W.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Health and Medical Research Council (NHMRC) Australia project grant APP1031943 to W.M. Acknowledgments: The authors gratefully acknowledge the support of Mark Krockenberger and Elaine Chew (Veterinary Pathology Diagnostic Services, The University of Sydney) for their help and technical work on the histopathology and Alex Khan, Medical Mycology Research Laboratory, CIDM, USYD, for taking the pictures of the life cycle. The authors thank Geoff Brown, Department of Agriculture and Fisheries, for supplying the original Galleria mellonella larvae for the establishment of the insect colony. Conflicts of Interest: The authors declare no conflict of interest.

References

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