J Microbiol Methods. 2015 Nov;118:182-6. doi: 10.1016/j.mimet.2015.10.004.

Tenebrio molitor (Coleoptera: Tenebrionidae) as an alternative host to study fungal infections.

 

de Souza PC1, Morey AT1, Castanheira GM1, Bocate KP1, Panagio LA1, Ito FA2, Furlaneto MC1, Yamada-Ogatta SF1, Costa IN3, Mora-Montes HM4, Almeida RS5.
  • 1Department of Microbiology, State University of Londrina, Londrina, PR, Brazil.
  • 2Department of Oral Medicine and Pediatric Dentistry, State University of Londrina, Londrina, PR, Brazil.
  • 3Department of Pathological Sciences, Center of Biological Sciences, State University of Londrina, Londrina, PR, Brazil.
  • 4Department of Biology, Universidad de Guanajuato, Guanajuato, Mexico.
  • 5Department of Microbiology, State University of Londrina, Londrina, PR, Brazil. Electronic address: rikodonto@gmail.com.

 

Abstract

Models of host–pathogen interactions are crucial for the analysis of microbial pathogenesis. In this context, invertebrate hosts, including Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode) and Galleria mellonella (moth), have been used to study the pathogenesis of fungi and bacteria. Each of these organisms offers distinct benefits in elucidating host–pathogen interactions. In this study,we present a newinvertebrate infection model to study fungal infections: the Tenebrio molitor (beetle) larvae. Here we performed T. molitor larvae infection with one of two important fungal human pathogens, Candida albicans or Cryptococcus neoformans, and analyzed survival curves and larva infected tissues.We showed that increasing concentrations of inoculum of both fungi resulted in increased mortality rates, demonstrating the efficiency of the method to evaluate the virulence of pathogenic yeasts. Additionally, following 12 h post-infection, C. albicans formsmycelia, spreading its hyphae through the larva tissue,whilst GMS stain enabled the visualization of C. neoformans yeast and theirmelanin capsule. These larvae are easier to cultivate in the laboratory than G. mellonella larvae, and offer the same benefits. Therefore, this insect model could be a useful alternative tool to screen clinical pathogenic yeast strainswith distinct virulence traits or different mutant strains.

KEYWORDS: Candida albicans; Cryptococcus neoformans; Infection model; Tenebrio molitor

PMID: 26453946

 

Supplement:

Experimentation using vertebrate hosts always generated ethical conflicts, as these are capable of feeling pain and anguish. Given the social pressure, alternative hosts have been developed, being one of the most common model, the invertebrate hosts. The hosts models most used in research is the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans and the wax moth Galleria mellonella (1). The life cycle of insects is much smaller than mammals, these organisms also have reduced size and reproduce in greater quantity. Furthermore the results are provided in a shorter time (2,3).

An invertebrate host that has gained prominence is Tenebrio molitor (Coleoptera: Tenebriodae). The yellow mealworm beetle, that has worldwide distribution (4), is an important plague of grain plantations (5). Our research group was the first to study the fungal pathogenesis in T. molitor infection model system (Figure 1). Larva of this insect were infected with different inocula concentrations of Cryptococcus neoformans and Candida albicans. Afterwards, the survival curves and infected tissues were analyzed. We showed that increasing concentrations of inoculum of both fungi resulted in increased mortality rates, demonstrating the efficiency of the method to evaluate the virulence of pathogenic yeasts. C. albicans showed more virulent as C. neoformans and this result was also found in G. mellonella larvae infected with these fungi (6).

After 12 hours of infection, was possible to observe the expression of virulence factor of C. albicans that suffered a morphological transition forming mycelia and spreading its hyphae through the larva tissue. It was also possible to observe the translocation of immune cells to the site of infection. These histopathological findings are similar to those described in infected mice (7), in liver from pig (8), in the alternative chick embryonated egg model (9), and in moth larva (6). C. neoformans yeast is visualized within larvae tissue, forming its melanin capsule, as seen in infected G. mellonella and mice (6,10). Additionally, this model can also support antifungal drug evaluation, as shown by our research group, which evaluated the antifungal activity of a natural compound against Candida tropicalis (11).

FIGURE 1

 

Acknowledgement:

This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), project 486042/2013-8. HMMM issuported by CONACyt (CB2011-166860). PCS and KPB are suported by Master degree scholarships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).

 

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embryo model resembles systemic murine infections. PLoS One 2011 Jan;6(5):e19741.

10. Casadevall A. Amoeba provide insight into the origin of virulence in pathogenic fungi. Adv Exp Med Biol 2012 Jan;710:1–10.

11. Morey AT, de Souza FC, Santos JP, Pereira CA, Cardoso JD, de Almeida RSC, et al. Antifungal Activity of Condensed Tannins from Stryphnodendron adstringens: Effect on Candida tropicalis Growth and Adhesion Properties. Curr Pharm Biotechnol 2016 Jan;17(4):365–75.

 

lattesDr. Ricardo Sergio Couto de Almeida

Professor Adjunto de Microbiologia

Departamento de Microbiologia – CCB

Laboratório de Micologia e Métodos Alternativos ao Uso de Animais – NIP9

Universidade Estadual de Londrina – UEL

Fone: (43) 3371-4976

 

 

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