Development of an insect model for the in vivo pathogenicity testing of yeasts

Conventional in vivo assays to determine the relative pathogenicity of yeast isolates rely upon the use of a range of mammalian species. The purpose of the work presented here was to investigate the possibility of using an insect (Galleria mellonella) as a model system for in vivo pathogenicity testing. The haemolymph of G. mellonella larvae was inoculated with PBS containing different concentrations of stationary phase yeasts of the genus Candida by injection at the last pro-leg. Larvae were incubated at 30 degrees C and monitored over 72 hours. Results indicate that G. mellonella can be killed by the pathogenic yeast Candida albicans and by a range of other Candida species but not to a significant extent by the yeast Saccharomyces cerevisiae. The kill kinetics for larvae inoculated with clinical and laboratory isolates of C. albicans indicate the former class of isolates to be more pathogenic. Differences in the relative pathogenicity of a range of Candida species may be distinguished using G. mellonella as a model. This work indicates that G. mellonella may be employed to give results consistent with data previously obtained using mammals in conventional in vivo pathogenicity testing. Larvae of G. mellonella are inexpensive to culture, easy to manipulate and their use may reduce the need to employ mammals for routine in vivo pathogenicity testing with a concomitant reduction in mammalian suffering.     

Potential of Cactoblastis cactorum as a Vector for Fungi Pathogenic to Pricklypear, Opuntia inermis

In Australia, fungi associated with larvae of the biological control agent Cactoblastis cactorum may contribute to the control of the exotic weed pricklypear (Opuntia inermis). C. cactorum larvae were assessed for their ability to vector pathogenic fungi into O. inermis by the infestation of larvae with fungal suspensions. Six fungal isolates caused disease after being carried into the host on external surfaces of larvae, and propagules of one isolate (UQ5109) initiated disease after being transferred from the cladode epidermis into the host by larvae feeding on the plant. Scanning electron microscopy revealed extensive hyphal growth on the external surfaces of larvae infested with several of the isolates. Fungi isolated from field-grown O. inermis cladodes were tested for pathogenicity to this plant in an in vivo plant assay. In total, 152 isolates were screened, 22 of which infected the host in pathogenicity tests. Only 1 (UQ5115) infected undamaged host tissue, whereas the remainder required the host to be wounded before infection could proceed. The majority of isolates were only weakly pathogenic, even when inoculated via wounds, suggesting that most were either saprophytes or weak parasites. This study demonstrates that it is possible for larvae of C. cactorum to transmit fungal pathogens into O. inermis tissue and it has provided a sound basis for future field work to determine the contribution that fungi make to the control of O. inermis.     

Galleria mellonella Infection Model Demonstrates High Lethality of ST69 and ST127 Uropathogenic E. coli

Galleria mellonella larvae are an alternative in vivo model for investigating bacterial pathogenicity. Here, we examined the pathogenicity of 71 isolates from five leading uropathogenic E. coli (UPEC) lineages using G. mellonella larvae. Larvae were challenged with a range of inoculum doses to determine the 50% lethal dose (LD₅₀) and for analysis of survival outcome using Kaplan-Meier plots. Virulence was correlated with carriage of a panel of 29 virulence factors (VF). Larvae inoculated with ST69 and ST127 isolates (10⁴ colony-forming units/larvae) showed significantly higher mortality rates than those infected with ST73, ST95 and ST131 isolates, killing 50% of the larvae within 24 hours. Interestingly, ST131 isolates were the least virulent. We observed that ST127 isolates are significantly associated with a higher VF-score than isolates of all other STs tested (P≤0.0001), including ST69 (P<0.02), but one ST127 isolate (strain EC18) was avirulent. Comparative genomic analyses with virulent ST127 strains revealed an IS1 mediated deletion in the O-antigen cluster in strain EC18, which is likely to explain the lack of virulence in the larvae infection model. Virulence in the larvae was not correlated with serotype or phylogenetic group. This study illustrates that G. mellonella are an excellent tool for investigation of the virulence of UPEC strains. The findings also support our suggestion that the incidence of ST127 strains should be monitored, as these isolates have not yet been widely reported, but they clearly have a pathogenic potential greater than that of more widely recognised clones, including ST73, ST95 or ST131.     

The influence of Conidiobolus coronatus on phagocytic activity of insect hemocytes

An essential component of the insect cellular response is phagocytosis. Analyses of the in vitro phagocytosis could be useful for the studies of the relationship between insects and their pathogens. Fungal metabolites are known to inhibit phagocytosis whereas components of the fungal cell wall stimulate phagocytosis. To achieve a better understanding of fungal pathogenesis in insects, haemocyte populations of two insect species susceptible to Conidiobolus coronatus infection (Galleria mellonella, Dendrolimus pini ) were compared with haemocytes of the resistant species (Calliphora erythrocephala ). Fungal infection increased phagocytic activity of G. mellonella plasmatocytes 3.3 times and this of D. pini plasmatocytes 2.1 times. Analysis of infected C. erythrocephala larvae did not reveal any influence of C. coronatus upon phagocytic activity.     

Experimental Trichinella infection in seals

The susceptibility of seals to infection with Trichinella nativa and the cold tolerant characteristics of muscle larvae in seal meat were evaluated. Two grey seals, Halichoerus grypus, were inoculated with 5000 (100 larvae/kg) T. nativa larvae and two grey seals with 50000 (1000 larvae/kg). One seal from each dose group and two control seals were killed at 5 and 10 weeks post-inoculation (p.i.). At 5 weeks p.i., infection was established in both low and high dose seals with mean larval densities of 68 and 472 larvae per gram (lpg), respectively, using eight different muscles for analyses. At 10 weeks p.i., mean larval densities were 531 and 2649 lpg, respectively, suggesting an extended persistence of intestinal worms. In seals with high larval density infections, the distribution of larvae in various muscles was uniform, but in one seal with a low larval density infection, predilection sites of larvae included muscle groups with a relative high blood flow, i.e. diaphragm, intercostal and rear flipper muscles. Trichinella-specific antibody levels, as measured by ELISA, increased during the 10 week experimental period. Infected seal muscle was stored at 5, -5 and -18 degrees C for 1, 4 and 8 weeks. Muscle larvae released from stored seal muscle by artificial digestion were inoculated into mice to assess viability and infectivity. Larvae from seal muscle 10 weeks p.i. tolerated -18 degrees C for 8 weeks but larvae from seal muscle 5 weeks p.i. tolerated only 1 week at -18 degrees C, supporting the hypothesis that freeze tolerance increases with the age of the host-parasite tissue complex. The expressed susceptibility to infection, extended production of larvae, antibody response and freeze tolerance of T. nativa in seals are new findings from the first experimental Trichinella infection in any marine mammal and suggest that pinnipeds (phocids, otariiids or walrus) may acquire Trichinella infection by scavenging even small amounts of infected tissue left by hunters or predators.     

Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans

Larvae of the greater wax moth (Galleria mellonella) that had been subjected to physical stress by shaking in cupped hands for 2 min showed reduced susceptibility to infection by Candida albicans when infected 24 h after the stress event. Physically stressed larvae demonstrated an increase in haemocyte density and elevated mRNA levels of galiomicin and an inducible metalloproteinase inhibitor (IMPI) but not transferrin or gallerimycin. In contrast, previous work has demonstrated that microbial priming of larvae resulted in the induction of all four genes. Examination of the expression of proteins in the insect haemolymph using 2D electrophoresis and MALDI TOF analysis revealed an increase in the intensity of a number of peptides showing some similarities with proteins associated with the insect immune response to infection. This study demonstrates that non-lethal physical stress primes the immune response of G. mellonella and this is mediated by elevated haemocyte numbers, increased mRNA levels of genes coding for two antimicrobial peptides and the appearance of novel peptides in the haemolymph. This work demonstrates that physical priming increases the insect immune response but the mechanism of this priming is different to that induced by low level exposure to microbial pathogens.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Atkinsiella dubia infection in the larvae of Japanese mitten crab,Eriocheir japonicus

Heavy mortalities reaching 100% among larvae of the Japanese mitten crab,Eriocheir japonicus, occurred in Yamaguchi Prefectural Naikai Fisheries Experimental Station, Yamaguchi, Japan. Under the microscope, infected zoeal larvae were seen to be filled with numerous aseptate hyphae. An investigation was carried out to identify the pathogenic fungus and determine its pathogenicity under laboratory conditions. The pathogenic fungus was identified asAtkinsiella dubia. Its optimum growth temperature was 25°C, and it grew only at 2.5% NaCl. Under laboratory conditions, it showed pathogenicity to the larvae of the swimming crab,Portunus trituberculatus. This is the first report of mass mortality in crustaceans due toA. dubia infection in Japan.     

Use of Galleria mellonella larvae to evaluate the in vivo anti-fungal activity of [Ag2(mal)(phen)3]

Larvae of the insect Galleria mellonella were employed to assess the in vivo antifungal efficacy of ([Ag₂(mal)(phen)₃]), AgNO₃ and 1,10-phenanthroline. Larvae pre-inoculated with these compounds were protected from a subsequent lethal infection by the yeast Candida albicans while larvae inoculated 1 and 4 h post-infection showed significantly increased survival (P < 0.01) compared to control larvae. Administration of these compounds resulted in an increase over 48 h in the density of insect haemocytes (immune cells) but there was no widespread activation of genes for antimicrobial peptides. This work demonstrates that G. mellonella larvae may be employed to ascertain the antifungal efficacy of silver(I) compounds and offers a rapid and effective means of assessing the in vivo activity of inorganic antimicrobial compounds.     

A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera; Buprestidae)

We used a newly developed bioassay method to demonstrate for the first time the potential of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to be used for the control of neonate larvae of Capnodis tenebrionis, a major threat to stone-fruit orchards in several countries. Four B. bassiana and four M. anisopliae isolates were all pathogenic for neonate larvae of C. tenebrionis; mortality rates 10 days after inoculation by dipping in a suspension with 10(8)conidia/ml varied from 23.5% to 100%. Three of the four M. anisopliae isolates caused 100% mortality. In most cases, postmortem hyphal growth and sporulation of M. anisopliae or B. bassiana was observed covering the larvae in their galleries. The eight isolates were also evaluated for pathogenicity to C. tenebrionis eggs at the same dosage. Only two B. bassiana isolates caused significant egg hatching reduction of 84.5% and 94.5%. Our results indicate that M. anisopliae and B. bassiana may be considered as promising for a new approach to prevent larval infestations by C. tenebrionis.     

A study for minisatellitic markers of Conidiobolus coronatus' pathogenicity to Galleria mellonella larvae

A selected panel of 13 colonies of entomopathogenic fungus Conidiobolus coronatus representing 6 variants of pathogenicity to Galleria mellonella larvae (ranged from 100 to 10% of efficiency), derived from single spores, were tested for the presence of hypervariable loci in their genomes by hybridization with Jeffreys' human minisatellite probe 33.6. The probe produced informative fingerprints and revealed slight differences among colonies analyzed. Up to 20 variable bands per colony were recognized in the size range of 2-20 kb. The band sharing within groups with the same pathogenicity ranged from 0.966 to 0.800. The genetic distance between different variants ranged from 0.026 to 0.282. A few characteristic bands for high and low pathogenicity to the larvae were found.     

Fusarium pathogenesis investigated using Galleria mellonella as a heterologous host

Members of the fungal genus Fusarium are capable of manifesting in a multitude of clinical infections, most commonly in immunocompromised patients. In order to better understand the interaction between the fungus and host, we have developed the larvae of the greater wax moth, Galleria mellonella, as a heterologous host for fusaria. When conidia are injected into the haemocoel of this Lepidopteran system, both clinical and environmental isolates of the fungus are able to kill the larvae at 37 °C, although killing occurs more rapidly when incubated at 30 °C. This killing was dependent on several other factors besides temperature, including the Fusarium strain, the number of conidia injected, and the conidia morphology, where macroconidia are more virulent than their microconidia counterpart. There was a correlation in the killing rate of Fusarium spp. when evaluated in G. mellonella and a murine model. In vivo studies indicated G. mellonella haemocytes were capable of initially phagocytosing both conidial morphologies. The G. mellonella system was also used to evaluate antifungal agents, and amphotericin B was able to confer a significant increase in survival to Fusarium-infected larvae. The G. mellonella-Fusarium pathogenicity system revealed that virulence of Fusarium spp. is similar, regardless of the origin of the isolate, and that mammalian endothermy is a major deterrent for Fusarium infection and therefore provides a suitable alternative to mammalian models to investigate the interaction between the host and this increasingly important fungal pathogen.     

Effects of dehydration rate on physiological responses and survival after rehydration in larvae of the anhydrobiotic chironomid

Strategies to combat desiccation are critical for organisms living in arid and semi-arid areas. Larvae of the Australian chironomid Paraborniella tonnoiri resist desiccation by reducing water loss. In contrast, larvae of the African species Polypedilum vanderplanki can withstand almost complete dehydration, referred to as anhydrobiosis. For successful anhydrobiosis, the dehydration rate of P. vanderplanki larvae has to be controlled. Here, we desiccated larvae by exposing them to different drying regimes, each progressing from high to low relative humidity, and examined survival after rehydration. In larvae of P. vanderplanki, reactions following desiccation can be categorized as follows: (I) no recovery at all (direct death), (II) dying by unrepairable damages after rehydration (delayed death), and (III) full recovery (successful anhydrobiosis). Initial conditions of desiccation severely affected survival following rehydration, i.e. P. vanderplanki preferred 100% relative humidity where body water content decreased slightly. In subsequent conditions, unfavorable dehydration rate, such as more than 0.7 mg water lost per day, resulted in markedly decreased survival rate of rehydrated larvae. Slow dehydration may be required for the synthesis and distribution of essential molecules for anhydrobiosis. Larvae desiccated at or above maximum tolerable rates sometimes showed temporary recovery but died soon after.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

The Selection of an Isolate of the Hyphomycete Fungus,Metarhizium anisopliae,for Control of Termites in Australia

Ninety-three isolates ofMetarhizium anisopliae,mostly derived from a survey of termite material, were screened for activity againstNasutitermes exitiosusandCoptotermes frenchiorC. acinaciformisusing a grooming assay technique. Twenty-six of the most promising isolates were further evaluated by bioassay againstN. exitiosusandC. acinaciformis.All isolates were pathogenic withCoptotermesspp. being more susceptible thanN. exitiosus.A group of nine isolates, chosen for their level of pathogenicity for one or other genus of termites and to represent a genetically diverse group, was finally compared in a minicolony test using termite colonies in 1 liter jars. The isolate, code-named FI-610 (derived from nest-mound material ofC. lacteusin SE New South Wales), was one of the most effective isolates against termites from both of the two colonies tested. This isolate also grew relatively well on agar plates at 36°C. FI-610 was thus selected for field trials and was found to be effective in killing colonies ofC. acinaciformiswhen 10 g (=3 × 10¹¹conidia) or more of conidial powder was blown into the center of the large mound colonies.     

Effects of Heliothis virescens ascovirus (HvAV-3e) on a novel host, Crocidolomia pavonana (Lepidoptera: Crambidae)

Ascoviruses are double-stranded DNA viruses which cause fatal disease in lepidopteran host larvae. They induce a unique pathology, causing cleavage of host cells into virion-containing vesicles. With the single exception of Diadromus pulchellus ascovirus, all ascoviruses have been exclusively reported from the Noctuidae. To investigate whether Heliothis virescens AV (HvAV-3e) has a broader host range at the family level, larvae of Crocidolomia pavonana F. (Lepidoptera: Crambidae), a major pest of brassica crops in tropical and sub-tropical regions of the Old World and Australasia, were inoculated with HvAV-3e. Larvae were readily infected by the ascovirus and feeding, growth and survival were significantly affected. However, the milky white discolouration of the haemolymph which is characteristic of ascovirus infection in noctuid hosts was not apparent. In further contrast to infected noctuid host larvae that do not develop to the pupal stage, a significant proportion of infected C. pavonana larvae pupated but all were killed at this stage. Thus, C. pavonana appears to be a semi-permissive host of the ascovirus, the presence of such hosts in the field might be an explanation for the conundrum for the ascovirus-noctuid-wasp relationship, helping explain the persistence of the ascovirus.     

Humoral immune response upon mild heat-shock conditions in Galleria mellonella larvae

Larvae of Galleria mellonella exposed to mild heat-shock (38 degrees C) showed an enhanced humoral immune response after microbial infection in comparison to infected animals grown at 28 degrees C. This enhanced response was manifested by increased expression of antimicrobial peptide (AMP) genes leading to enhanced antimicrobial activity in the hemolymph. We found an increased level of Hsp90 and changes in the level of a 55kDa protein recognized by anti-Hsp90 antibodies in fat bodies of infected animals reared at 28 degrees C as well as in uninfected animals exposed to elevated temperature. Pre-treatment of animals with an inhibitor of Hsp90, 17-DMAG, prior to immunization resulted in increased expression of AMP genes encoding gallerimycin and cecropin at 38 degrees C. This observation was correlated with the changes in Hsp90 protein and increased level of 55kDa protein. Also G. mellonella larvae pre-treated with 17-DMAG and exposed to mild heat-shock for 30min showed an increased survival rate after infection with entomopathogenic bacteria Pseudomonas aeruginosa. We also show the effect of 17-DMAG on the phosphorylation state of ERK MAP kinase. We postulate that Hsp90 may play a significant role in converging pathways involved in the insect immune response and heat-shock.     

Quantifying horizontal transmission of Nosema lymantriae, a microsporidian pathogen of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in field cage studies

Nosema lymantriae is a microsporidian pathogen of the gypsy moth, Lymantria dispar that has been documented to be at least partially responsible for the collapse of L. dispar outbreak populations in Europe. To quantify horizontal transmission of this pathogen under field conditions we performed caged-tree experiments that varied (1) the density of the pathogen through the introduction of laboratory-infected larvae, and (2) the total time that susceptible (test) larvae were exposed to these infected larvae. The time frame of the experiments extended from the early phase of colonization of the target tissues by the microsporidium to the onset of pathogen-induced mortality or pupation of test larvae. Upon termination of each experiment, the prevalence of infection in test larvae was evaluated. In the experiments performed over a range of pathogen densities, infection of test larvae increased with increasing density of inoculated larvae, from 14.2 ± 3.5% at density of 10 inoculated per 100 larvae to 36.7 ± 5.7% at 30 inoculated per 100 larvae. At higher densities, percent infection in test larvae appeared to level off (35.7 ± 5.5% at 50 inoculated per 100 larvae). When larval exposure to the pathogen was varied, transmission of N. lymantriae did not occur within the first 15 d post-inoculation (dpi) (11 d post-exposure of test larvae to inoculated larvae). We found the first infected test larvae in samples taken 20 dpi (16 d post-exposure). Transmission increased over time; in the cages sampled 25 dpi (21 d post-exposure), Nosema prevalence in test larvae ranged from 20.6% to 39.2%.     

Stage-specific mortality of Rhagoletis indifferens (Diptera: Tephritidae) exposed to three species of Steinernema nematodes

Mortality of larval, pupal, and adult western cherry fruit fly, Rhagoletis indifferens (Tephritidae) exposed to the steinernematid nematodes Steinernema carpocapsae, Steinernema feltiae, and Steinernema intermedium, was determined in the laboratory and field. Larvae were the most susceptible stage, with mortality in the three nematode treatments ranging from 62 to 100%. S. carpocapsae and S. feltiae were equally effective against larvae at both 50 and 100 infective juveniles (IJs)/cm². S. intermedium was slightly less effective against larvae than the other two species. Mortalities of R. indifferens larvae at 0, 2, 4, and 6 days following their introduction into soil previously treated with S. carpocapsae and S. feltiae at 50 IJs/cm² were 78.6, 92.5, 95.0, and 77.5% and 87.5, 52.5, 92.5, and 70.0%, respectively, and at 100 IJs/cm² were 90.0, 92.0, 100.0, and 84.0% and 90.0, 50.0, 42.0, and 40.0%, respectively. There was no decline in mortality caused by S. carpocapsae as time progressed, whereas there was in one test with S. feltiae. Larval mortalities caused by the two species were the same in a 1:1:1 vermiculite:peat moss:sand soil mix and a more compact silt loam soil. In the field, S. carpocapsae and S. feltiae were equally effective against larvae. Pupae were not infected, but adult flies were infected by all three nematode species in the laboratory. S. carpocapsae was the most effective species at a concentration of 100 IJs/cm² and infected 11–53% of adults that emerged. The high pathogenicity of S. carpocapsae and S. feltiae against R. indifferens larvae and their persistence in soil as well as efficacy in different soil types indicate both nematodes hold promise as effective biological control agents of flies in isolated and abandoned lots or in yards of homeowners.     

Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae

Candida albicans is a dimorphic human pathogen in which the yeast to hyphal switch may be an important factor in virulence in mammals. This pathogen has recently been shown to also kill insects such as the Greater Wax Moth Galleria mellonella when injected into the haemocoel of the insect larvae. We have investigated the effect of previously characterised C. albicans mutations that influence the yeast to hyphal transition on virulence in G. mellonella larvae. There is a good correlation between the virulence of these mutants in the insect host and the virulence measured through systemic infection of mice. Although the predominant cellular species detected in G. mellonella infections is the yeast form of C. albicans, mutations that influence the hyphal transition also reduce pathogenicity in the insect. The correlation with virulence measured in the mouse infection system suggests that Galleria may provide a convenient and inexpensive model for the in vivo screening of mutants of C. albicans.