Microsporidian Infection in Mosquitoes (Culicidae) Is Associated with Gut Microbiome Composition and Predicted Gut Microbiome Functional Content

The animal gut microbiota consist of many different microorganisms, mainly bacteria, but archaea, fungi, protozoans, and viruses may also be present. This complex and dynamic community of microorganisms may change during parasitic infection. In the present study, we investigated the effect of the presence of microsporidians on the composition of the mosquito gut microbiota and linked some microbiome taxa and functionalities to infections caused by these parasites. We characterised bacterial communities of 188 mosquito females, of which 108 were positive for microsporidian DNA. To assess how bacterial communities change during microsporidian infection, microbiome structures were identified using 16S rRNA microbial profiling. In total, we identified 46 families and four higher taxa, of which Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae and Pseudomonadaceae were the most abundant mosquito-associated bacterial families. Our data suggest that the mosquito gut microbial composition varies among host species. In addition, we found a correlation between the microbiome composition and the presence of microsporidians. The prediction of metagenome functional content from the 16S rRNA gene sequencing suggests that microsporidian infection is characterised by some bacterial species capable of specific metabolic functions, especially the biosynthesis of ansamycins and vancomycin antibiotics and the pentose phosphate pathway. Moreover, we detected a positive correlation between the presence of microsporidian DNA and bacteria belonging to Spiroplasmataceae and Leuconostocaceae, each represented by a single species, Spiroplasma sp. PL03 and Weissella cf. viridescens, respectively. Additionally, W. cf. viridescens was observed only in microsporidian-infected mosquitoes. More extensive research, including intensive and varied host sampling, as well as determination of metabolic activities based on quantitative methods, should be carried out to confirm our results.

Microsporidian and fungal infections in Lepidoptera and Orthoptera in Bulgaria

Fourteen hundred and sixty three larvae of 10 lepidopteran species collected from trees and bushes in the spring and summer of 2017, 2018 and 2019 from 5 localities in Northwest and South Bulgaria were investigated for presence of microsporidian and fungal pathogens. Also, 77 grasshopper individuals of Poecilimon thoracicus (Orthoptera, Tettiigoniidae) collected from various shrubs and perennial plants in the spring and summer of 2017 were examined. Conducted microscopic analyses showed the presence of microsporidian infection caused by Endoreticulatis poecilimonae in P. thoracicus and fungal infection in the larvae of mottled umber, Erannis defoliaria caused by Entomophaga auliciae. The studies showed that the average infection rate with the microsporidium E. poecilimonae was 57.1%. The prevalence of the fungus Entomophaga auliciae was 100% during the observed epizootic. E. auliciae is an efficient fungal pathogen which causes strong epizootics and can be used as classical or augmentation biological agent.

Microsporidia Interact with Host Cell Mitochondria via Voltage-Dependent Anion Channels Using Sporoplasm Surface Protein 1

ABSTRACT Microsporidia are opportunistic intracellular pathogens that can infect a wide variety of hosts ranging from invertebrates to vertebrates. During invasion, the microsporidian polar tube pushes into the host cell, creating a protective microenvironment, the invasion synapse, into which the sporoplasm extrudes. Within the synapse, the sporoplasm then invades the host cell, forming a parasitophorous vacuole (PV). Using a proteomic approach, we identified Encephalitozoon hellem sporoplasm surface protein 1 (EhSSP1), which localized to the surface of extruded sporoplasms. EhSSP1 was also found to interact with polar tube protein 4 (PTP4). Recombinant EhSSP1 (rEhSSP1) bound to human foreskin fibroblasts, and both anti-EhSSP1 and rEhSSP1 caused decreased levels of host cell invasion, suggesting that interaction of SSP1 with the host cell was involved in invasion. Coimmunoprecipitation (Co-IP) followed by proteomic analysis identified host cell voltage-dependent anion channels (VDACs) as EhSSP1 interacting proteins. Yeast two-hybrid assays demonstrated that EhSSP1 was able to interact with VDAC1, VDAC2, and VDAC3. rEhSSP1 colocalized with the host mitochondria which were associated with microsporidian PVs in infected cells. Transmission electron microscopy revealed that the outer mitochondrial membrane interacted with meronts and the PV membrane, mitochondria clustered around meronts, and the VDACs were concentrated at the interface of mitochondria and parasite. Knockdown of VDAC1, VDAC2, and VDAC3 in host cells resulted in significant decreases in the number and size of the PVs and a decrease in mitochondrial PV association. The interaction of EhSSP1 with VDAC probably plays an important part in energy acquisition by microsporidia via its role in the association of mitochondria with the PV. IMPORTANCE Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse.

First Identification of Nosema Ceranae (Microsporidia) Infecting Apis Mellifera in Venezuela

Nosema ceranae is a pathogen of Apis mellifera, which seems to have jumped from its original host Asiatic honey bee Apis ceranae. Nosemosis which affects the honey bee Apis mellifera is caused by two parasitic fungi described as etiologic agents of the disease. Nosema apis was the only microsporidian infection identified in A. mellifera until N. ceranae in Taiwan and Europe. Nosema spp. positive samples of adult worker bees from the Venezuelean state of Lara were determined through light microscopy of spores. Samples were then tested to determine Nosema species (N.apis/N.ceranae) using previously reported PCR primers for the 16S rRNA gene. A multiplex PCR assay was used to differentiate both N. apis and N. ceranae species. Only N. ceranae was found in the analyzed samples and the percentage of infected foragers fluctuated between 18% and 60%.

Evaluation of the Effectiveness of Selected Treatments of Nosema Spp. Infection by the Hemocytometric Method and Duplex Pcr

Recent years have witnessed an increase in the mortality of honey bees in many regions of the world. The observed decrease in the bee population results from a combination of factors, and microsporidian parasites Nosema apis and N. ceranae are among the main contributors. Those parasites cause a microsporidian infection that shortens the lifespan of bees and reduces the productivity of bee colonies. The aim of this study was to evaluate the effectiveness of Nozevit, Api Herb and ApiX (acetylsalicylic acid + Artemisia absinthium L. extract) in the control of infections caused by Nosema spp. in a field experiment. Two groups of worker bees were evaluated - hive bees and forager bees returning to the hive. The effect of the analyzed therapies on the number of spores and the microsporidia species were analyzed by the hemocytometric method and duplex PCR. A statistical analysis revealed that the applied treatments had reduced the number of spores by 31.15% on average. In hive bees, Nosema spp. infection was most effectively reduced by Nozevit (67.85%) and ApiX (63.36%). Coinfections (N. ceranae and N. apis) were affirmed in all bee samples before treatments. However, after the treatments, single infection of N. apis and N. ceranae were detected. The tested treatments were more effective in the control of N. apis than N. ceranae.