First Record of A New Microsporidium Pathogenic to the Eucalyptus Snout Beetle in Brazil

Microsporidia are naturally occurring fungal-related parasites that can infect nearly all animal hosts, but their biocontrol potential of insect pests is routinely overlooked in agriculture and forestry. This research brings the rst report describing the natural occurrence of a microsporidium causing disease in eld-collected populations of the invasive eucalyptus snout beetle, Gonipterus platensis (Coleoptera: Curculionidae), a major destructive pest of eucalyptus plantations in Brazil. Adult beetles were collected during eld surveys in commercial eucalyptus plantations in southern Brazil to be examined and dissected with typical symptoms to verify presence of microsporidian spores in haemolymph. From 14 plantations in different sites, the natural infection occurrence in these populations ranged from 0 to 65%, while a lab colony exhibited an infection incidence of 70%. Spore density in haemolymph of symptomatic insects averaged 2.1(±0.4) × 10 7 spores/mL. Symptoms in infected adults were identied by abnormal abdomen with malformation of the second pair of wings, impairing their ight activity. Electron transmission microscopy of the pathogen showed morphological features similar to species belonging to the genus Nosema or Vairimorpha. Phylogenetic analysis of the full-length small subunit ribosomal RNA gene suggests this pathogen’s placement in the genus Vairimorpha, but with a sequence identity of ~94% with the nearest neighbours. The low level of sequence identity suggests this pathogen may represent a novel taxon in the genus and further requires whole genome sequencing for denitive taxonomic resolution. These ndings provide insights on the natural occurrence of this novel pathogen for potential use in biocontrol of this invasive pest in Eucalyptus plantations in Brazil, and prompts the design of conservative or augmentative strategies for the establishment of this microsporidium in eld populations for sustainable management of eucalyptus snout beetles.


Introduction
Various environmental, biological and genetic factors can in uence performance and tness during an insect's life cycle. A relevant factor is the occurrence of pathogens such as bacteria, viruses, fungi, and parasites that are responsible for about 80% of the diseases in insect populations 1 . Recent research has highlighted several cases of success in insect invasions facilitated by microorganismsc 2,3,4,5,6 , including some microsporidia.
Microsporidia have been reported to cause substantial deleterious effects on host tness in other host insects. These effects include malformations in infected pupae, increased larval mortality, developmental delay of immatures, reduced fertility and longevity of adults, and increased susceptibility to stress conditions 7 . These stress factors cause biological changes in the host insect and may be associated with the failure of its parasitism 1 . As microsporidian pathogens generally display e cient transmission mechanisms and moderate virulence, these traits may make them more effective agents in establishing enzootics in host population 8 , as evidenced by the use of a microsporidium to control locusts 9 . In this context, microsporidian entomopathogens hold a great potential as long-term biocontrol agents of numerous arthropod pests, but their natural incidence and pathogenicity in populations of forest pests have been underexplored 10 .
In the past, microsporidia were considered to be spore-forming protozoa, but in the light of modern taxonomy, this group was relocated to in or near the Fungi kingdom and are now called non-agellate, single-celled fungi, and obligate intracellular parasites 11 . Several studies suggest a new classi cation for microsporidia within or near the fungi group, and the majority of entomopathogenic microsporidia belong to the genus Nosema, with more than 150 insect host species described in 12 insect orders, notably Lepidoptera, Hymenoptera, Diptera, Orthoptera and Coleoptera are among them 12 .
Microsporidia generally cause sublethal and chronic deleterious effects in infected hosts and, as a result, are a serious problem in insects that are massively bred in laboratories and bio-factories 1,7 . Spores are generally small and most entomopathogenic microsporidia are approximately 2 to 6 µm in length. Spores can have different morphologies, including rounded, oval, or pyriform, and less frequently reniform, long ovals to almost tubular shape and refringent appearance when examined under phase contrast microscopy7.
The Eucalyptus snout beetle (ESB), Gonipterus platensis Marelli 1927, also known as the eucalyptus beetle, is currently the primary coleoptereus pest in commercial eucalyptus forestry in Brazil. ESB is native to Australia, have a high destructive potential as adults and larvae feed mainly on young leaves 13 , and are distributed across the South and Southeast of Brazil 14 . In a study carried out in Portugal, defoliation by G. platensis resulted in wood losses of 648 million euros in the last twenty years 15 . This demonstrates the need to better understand the insect in order to have better pest management and avoid losses.
Despite microsporidiosis being reported in a variety of different insect populations, there are no previous studies reporting the presence of this pathogen in populations of G. platensis in the literature. Recently, microsporidiosis symptoms observed in infected adult beetles included typical morphological malformations comprising an abnormal abdomen with the second pair of wings displaced, which impairs ight activity in a laboratory colony. This promptly motivated us to identify the pathogen and determine its prevalence in natural populations. Identifying the pathogen would also allow us to evaluate the potential of the organism to serve as a biological control agent for this pest in forest plantations. The knowledge of the possible microsporidium associated with G. platensis is of great importance to biological control programs attempting to control this pest and to prevent the pathogen from infecting laboratory colonies of this insect.

Morphological characterization and spore density of microsporidium
Field-collected G. platensis adult beetles were observed with visible symptoms of microsporidiosis disease, as they exhibited notable malformations characterized by spread pair of wings with abnormal and shrivelled abdomen, and the membranous pair of wings completely extended from the elytra (Fig. 1A). In this study, oval spores were identi ed as resistance structures of intracellular parasites isolated from the body content of G. platensis adults. In slides prepared with the beetle's body content, a large amount of spores of the pathogen was easily and clearly observed, which was also used as a quick and easy diagnosis parameter to con rm microsporidiosis in eld-collected beetles from different localities, as described later in this study (Fig. 1B). Microscopy is an inexpensive and routine technique for the conformation of microsporidiosis, but it is not an accurate method for species identi cation since the morphological structures of some pathogens are similar 16 .
Light microscopy revealed that fresh Vairimorpha sp. spore were generally elongated ovoid or oval shapes. Spore dimensions (n = 10) averaged 2.07 (± 0.136) µm in length and 1.20 (± 0.066) µm in width, respectively (Fig. 1B). Ultrastructure of microsporidian spores were examined under transmission microscopy and revealed spore wall consisted of an electron-dense exospore. The coiled region of the polar tube comprised 8 turns, and the diplokaryotic nuclei were slightly separated from each other (Fig. 1C). However, this does not discard that this pathogen also forms uninucleate spores as well. All the above-mentioned cellular features corresponded to the basic characteristics found in the genus Vairimorpha. Microsporidian spores from 10 beetles with similar size and weight were collected in Eucalyptus plantation located at São Jerônimo da Serra, SP, Brazil, and presented a sex ratio of 1:1. As result, the averaged of spore load of symptomatic beetles was determined at 2.15 (± 0.402) × 10 7 spores per adult with (Fig. 1D).

Molecular identi cation and phylogenetic construction
Sequencing of the SSU rRNA gene was performed to con rm the identi cation of the pathogen found infecting populations of G. platensis. The PCR results con rmed the isolate was closely related to Microsporidia species, with the highest sequence identity (98%) to a sequence submitted to Genbank as Microsporidia sp. MB-2008 (GenBank accession no. EU589246). A phylogenetic analysis of the strain and closely related strains of Microsporidia species was conducted (Fig. 2). The closest phylogenetic neighbor of the isolate found in G. platensis was found to be Microsporidia sp. MB-2008, which was isolated from another weevil, Otiothynchus sulcatura (Coleoptera: Curculionidae), followed by Vairimorpha apis, isolated from Apis cerana (Hymenoptera: Apidae). Based on the recent formal rede nition of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae), the strain belongs to the Vairmorpha genus. Based on these ndings, the name Vairimorpha curculionidae is proposed, when the species is formally circumscribed in the future.

Prevalence of microsporidiosis in different eld populations of Gonipterus spp.
To determine the presence of microsporidium in other populations of the host insect, collections were made at 13 different sampling points distributed in three states as shown in the Fig. 3A. Identi cation of infected adult beetles was based on visual diagnosis of the microsporidiosis symptopms and by checking for spores in hemolymph. Positive results were obtained for microsporidium in most surveyed eld sites, including beetles sampled from a mass reared insect colony up to the 2nd generation in Botucatu-SP. There was a signi cant variation in natural infection caused by microsporidium across different regions or sites of collection in South and Southeast Brazil (χ 2 = 62.87, df = 13, P < 0.0001). The prevalence of natural infection in these eld-collected beetle populations sampled at different locations under eld conditions ranged from 0 to 65%, but the highest pathogen incidence was observed in the insect colony maintained at the laboratory corresponding to 70% infection in adults (Fig. 3B).
Interestingly, it was the rst time that we found microsporidium infecting Gonipterus pulverulentus (26.7% infection), despite the lower frequency of this insect species compared to G. platensis throughout Brazil.

Discussion
The present work is the rst to describe the occurrence of microsporidiosis in an insect colony from the second generation and in eld populations of eucalyptus snout beetles collected in commercial Eucalyptus plantations, with the disease being con rmed in two species present in Brazil, G. platensis and G. pulverulentus (Coleoptera: Curculionidae). Microsporidian infections have been reported in other Curculionidae 17,18 as well as other coleopterans 19,20,21,22, 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 eld 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 year 23,24 . There are several reports of these microorganisms infecting lepidopterans 25 , hymenopterans 26 and orthopterans 27 , 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 infections 28 .
Microsporidiosis is considered an important problem in the life cycle of insects 29 because of the reduction in pupal size, number and viability, along with a longer duration of the pupal stage 30 . 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 y 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) × 10 7 spores in symptomatic insects. This is similar to other microsporidia infections, Nosema cerane in honey bees yielded 1.15 × 10 7 spores/bee at 18 days post-inoculation 31 . However, diet had a considerable effect on the spore load observed in honey bees 31 . This difference illustrates the speci c 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 identi cation to species di cult. Therefore, other methods are needed to con rm identi cation. Classi cation based on spore morphology can be di cult 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 tissues 32 . Such evidence indicates high diversity of the spore dimension; hence, molecular analysis is essential in the identi cation of microsporidian species 16 .
The SSU rRNA sequence has been widely used as a molecular marker to estimate phylogenetic relationships between microsporidia, because it is a highly conserved gene 12 . However, this gene alone cannot be used to distinguish closely related species. This is a limitation of this gene for a more re ned phylogenetic separations between species of this pathogen 33,34 . However, they can be used in the taxonomic classi cation at the genus level 35,36 . 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 host 37 . 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 population 38 . 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 nd 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.

Phylogenetic Analysis
Phylogenetic analysis was conducted with MEGA X using Maximum Likelihood analysis and the Tamura 3-parameter model with a discrete gamma distribution (5 categories), as this model was found to be the best-t using the maximum likelihood-based model selection algorithm implemented in MEGA X. The partial deletion (90%) option was used, and the level of bootstrap support was calculated from 1000 replicates.

Determination of microsporidian spore density in beetles
Microsporidian spores were isolated from typically symptomatic insects, which were originated from the eld, and maintained at the Laboratory for Biological Control of Forest Pests (LCBPF/UNESP, Botucatu, SP, Brazil). The infected insects were homogenized in nuclease-free water in 0.2-mL microtubes. The suspension was subjected to three series of centrifugation: 2,000 rpm for 10 min followed by 2 cycles at 12,000 rpm for 1 min. After each centrifugation, the supernatant was discarded. The spores accumulated at the bottom of the tube forming a "pellet", which was later resuspended in nuclease-free water (adapted from Solter et al., 2012). This procedure was performed individually for 10 insects, in order to determine the average concentration of spores per beetle (n = 10). The spores from each insect were puri ed and suspended in nuclease-free water and then immediately quanti ed with a Neubauer chamber at 400X magni cation under a phase-contrast microscope (Leica DM 2500, Leica Microsystems, Heerbrugg, Switzerland). At the end of the counts, the microsporidium density per insect was determined.

Phase contrast microscopy
Spore immobilization and photomicrographs were performed according to the methods described in Vávra and Maddox (1976). Fresh spores were visualized from macerated body contents of infected insects after dilution in sterile 0.85% NaCl solution (w/v). A drop of this macerate was transferred to a glass slide and the spores were examined under a phase-contrast microscope (Leica DM 2500, Leica Microsystems, Heerbrugg, Switzerland) at 400X magni cation.

Transmission electron microscopy
The material was prepared at the Center for Electron Microscopy (Biosciences Institute -UNESP). Tissue samples from the digestive tract of G. platensis adults were xed in Karnovsky's solution 41 modi ed (glutaraldehyde 25% paraformoldehyde 8% and 0.2 M monosodium / disodium phosphate buffer solution, pH 7.3). The samples were cut into small fragments of up to 2 mm³ for better xation and incubated for at least 3 h at room temperature. The samples were removed from the xative and washed 3 times for 5 min in 0.1 M phosphate buffer with pH 7.3, followed by immersing the material in osmium tetroxide for 2 h. After, the material was washed 3 times for 10 min in distilled water and immersed in 0.5% uranyl acetate in distilled water for about 2 h, in order to have the block contrast, revealing / highlighting the nucleic acids. Dehydration was accomplished with a series of solution containing an increasing amount of acetone. The series consisted of 10 min in 50% acetone, 2 washes for 10 min in 70% acetone, 3 washes for 15 min in 90% acetone, and nally, 3 washes for 15 min in 100% acetone.
The in ltration was done slowly using a 1:1 mixture of Araldite® resin + 100% acetone and left for approximately 12 h at room temperature. The xed biological material was made in an appropriate form and further placed in an oven at 60 °C for 2 to 3 days. Semi-thin cuts (0.5 µm thick) were made for choosing the region of interest and selected regions were trimmed again to further reduce the block surface allowing ultrathin cuts (60-90 nm) to be made and placed in appropriate grids. The cuts were contrasted with a saturated solution of uranyl acetate in 50% alcohol for about 20 min, followed by lead citrate for 10 min.
The slides were rstly examined under a light microscope (Zeiss Axioscop 2, Germany) to select the materials to be subsequently observed in the electron microscope. The analysis of the material was performed using the transmission electron microscope JEM 1011 (JEOL, Inc., Peabody, MA), operating at 40.60, 80 and 100KV JEO at the Electronic Microscopy Laboratory (ESALQ -USP, Piracicaba, SP, Brazil).

Detection of microsporidiosis in different eld-collected ESB populations from Southern Brazil
In order to con rm the presence of the microsporidian pathogen in different eld populations in from South to Southeast Brazil, insects from three different states were collected in commercial Eucalyptus plantations (Table 1). After collection in the eld, the insects were brought to the laboratory, examined for microsporidiosis symptoms in adult beetles to calculate the percent natural infection, and then stored in a -20 °C freezer prior to dissection and microscopy to con rm the presence of the pathogen.