The present work is the first to describe the occurrence of microsporidiosis in an insect colony from the second generation and in field populations of eucalyptus snout beetles collected in commercial Eucalyptus plantations, with the disease being confirmed in two species present in Brazil, G. platensis and G. pulverulentus (Coleoptera: Curculionidae). Microsporidian infections have been reported in other Curculionidae17,18 as well as other coleopterans19,20,21,22, which demonstrate that this fungal pathogen is more common than previously thought infecting this diverse insect order. Adult beetles collected across the three Brazilian tested positive for the presence of this pathogen. Prevalence of natural infection in these field populations varied from 0 to 65% and 70% infection was detected in the lab colony, indicating that this pathogen is spreading with expansion of the pest in Brazil and is established in mass reared lab colonies of ESB.
More than 1400 microsporidian species have been described so far and new species are being discovered each year23,24. There are several reports of these microorganisms infecting lepidopterans25, hymenopterans26 and orthopterans27, demonstrating the high genetic plasticity of this group of pathogens. Even more important is the frequent occurrence of microsporidian epizootics in laboratory colonies, in which there is high aggregation and population density of insects facilitating pathogen spread and new infections28.
Microsporidiosis is considered an important problem in the life cycle of insects29 because of the reduction in pupal size, number and viability, along with a longer duration of the pupal stage30. We also described conspicuous morphological abnormalities in infected G. platensis and G. pulverulentus beetles, and these symptoms are good indicators of microsporidiosis diagnostic alongside the presence of spores in the haemocoel. We also observed that adult beetles showcasing wing deformities were unable to fly and had shorter life expectancy relative to noninfected individuals (data not shown).
Spore numbers in G. platensis adult beetles reached an average concentration of 2.15 (± 0.40) × 107 spores in symptomatic insects. This is similar to other microsporidia infections, Nosema cerane in honey bees yielded 1.15 × 107 spores/bee at 18 days post-inoculation31. However, diet had a considerable effect on the spore load observed in honey bees31. This difference illustrates the specific interaction between microsporidium and its host insect in regard to spore density for the development of microsporidiosis.
The morphological similarity between microsporidian species, particularly based on spore measurements in isolation, makes identification to species difficult. Therefore, other methods are needed to confirm identification. Classification based on spore morphology can be difficult and inconsistent because some microsporidia have complex life cycles and form various types of spores. In some cases, different sporulation cycles occur at different stages of the host. Some species can also form different types of spores in the same host and sometimes in the same tissues32. Such evidence indicates high diversity of the spore dimension; hence, molecular analysis is essential in the identification of microsporidian species16.
The SSU rRNA sequence has been widely used as a molecular marker to estimate phylogenetic relationships between microsporidia, because it is a highly conserved gene12. However, this gene alone cannot be used to distinguish closely related species. This is a limitation of this gene for a more refined phylogenetic separations between species of this pathogen33,34. However, they can be used in the taxonomic classification at the genus level35,36. In this case, the species is well resolved, even amongst members of the same genus.
The use of microsporidia as a biological control agent offers many properties that make them attractive for pest control applications. They cause a chronic disease, which is debilitating to the host37. In addition, the transmission of microsporidia occurs by one or more several means - ingestion of spores present in the environment, parental transmission to offspring, which facilitates their multiplication in the target population38. This high transmissibility of microsporidia in host population coupled with low lethality is key for their high and long-term prevalence, which could be desirable for augmentative biocontrol strategy in forest pests. However, the method of transmission in G. platensis is currently unknown.
Associated pathogens may also be present in other countries, so there is a need for a more in-depth study aimed at detecting the microsporidium in Australia, native land to Gonipterus, and in other countries with the presence of ESB and other species of Gonipterus. We also need to investigate the host spectrum of this new microsporidiosis to native beetles in Brazil, especially concerning predatory beetles, in order to find out if this pathogen could be lethal or harmless to non-target hosts.
In summary, this data indicates a probable new species of this pathogen, providing support for new studies as well as for the elaboration of control methods to contribute to its integrated management in Brazilian eucalyptus plantations.