What type of fungal genus grow on insects

At first, he thought that the cicadas had picked up a fungal infection, but he found the same cells in every species that lacked Hodgkinia. These insects had clearly adopted some kind of fungus and turned it into an endosymbiont that replaced the missing bacterium.

Cordyceps fungi excel at infecting and killing insects. One particular species, Ophiocordyceps unilateralis , has become famous for its ability to turn ants into zombies.

Then it compels the ant to climb a plant stem, and clamp its jaws on the underside of a leaf. In this way, the zombie fungus can claim an entire colony. The fungi that Matsuura discovered in the cicadas are all close relatives of this ant-killing species—all part of the same Ophiocordyceps genus. And that, to put it bluntly, is extraordinary. Their hosts almost always end up dead, with spore-tipped stalks erupting from their corpses. And yet Matsuura showed that cicadas have domesticated Ophiocordyceps , turning it into an essential part of their own bodies.

Many beneficial microbes evolve from parasitic ancestors, and the divide between these two lifestyles is more of a continuum. Cicadas certainly encounter a lot of fungi. They spend most of their lives underground, and are constantly surrounded by fungi that live in soil. These include several species of Ophiocordyceps that specialize in parasitizing cicadas and nothing else. These strains came to coexist with their hosts. Perhaps their presence conferred some kind of benefit, like resistance to viral infections.

And perhaps they might even have saved some of the cicadas from extinction. Remember Hodgkinia , the bacterial symbiont that cicadas rely on? He has shown that this single microbe tends to split into what are effectively several daughter species. A single cicada might have dozens of these daughter microbes. This chaotic mess leaves the cicada in a precarious position.

To define whether the microbiota of FGI exhibits a taxonomic and functional configuration characteristic of this environment, we compared these communities to several hosts ranging diverse diets e. For expanding the geographic distribution of FGI microbiota that are publicly available, we performed shotgun metagenome sequencing for microbial communities associated with fungus gardens of the attine ants Mycocepurus goeldii and Atta sexdens rubropilosa , both species widely distributed in Brazil 40 , 41 , which were grouped to a dataset from a previous study with FGI The FGI microbiota functional profile exhibit similarities with the gut microbiota of both herbivorous and omnivorous hosts, though some differentially abundant features codified by the FGI microbiota suggest these communities occupying microhabitats that could be characteristic of fungiculture.

By suggesting the microbiota as functionally adapted to fungiculture environment, our findings reinforce the bacterial community as a structured and metabolically important feature of FGI ecosystems, possibly composing an essential part of FGI ecology.

Microbiota composition at class level. Hosts are depicted according to their phylogenetic relationship and diet detailed in Supplementary Table S1. Microbiota composition and similarity were estimated based on the normalized abundance of protein coding sequences taxonomically assigned at class level. Pencil drawings by Mariana O. We seek to expand the geographic distribution of the microbiota associated with FGI that are already available. Thus, we shotgun sequenced the metagenomes from fungus gardens of the lower attine M.

Sequencing of the bacterial community obtained from M. The bacterial community from At. Reads of each library were assembled into metagenomes consisting of — Mbp of sequence data. Assembled contigs comprised good quality and length sequences Supplementary Table S2. Fungus garden metagenomes from M.

Gammaproteobacteria and Bacteroidia are similarly abundant for the microbiota of M. At genera level, Pseudomonas , Dysgonomonas, Bacteroides, Enterobacter, Parabacteroides, Prevotella, Comamonas, and Burkholderia are amongst the most abundant taxa in the bacterial community of M. On the other hand, the microbiota of At.

Bacterial genera abundant in At. Most abundant bacterial genera of the microbiota associated with Mycocepurus goeldii , Atta sexdens rubropilosa, and other FGI. Sequences taxonomically assigned to the most abundant bacterial genera classified by COG functional categories for the microbiota of a Mycocepurus goeldii and b Atta sexdens rubropilosa.

The data presented in this figure also feature in the masters dissertation of M. When comparing the microbiota taxonomic composition between hosts with different diets and differing in phylogenetic distribution, UPGMA-clustering indicates the microbiota of FGI clustering separately from other hosts Fig. Exceptions to this pattern may be observed in the microbiota composition of M.

For instance, while the relative abundance of Gammaproteobacteria is higher in the FGI microbiota, the relative abundance of Clostridia and Spirochaetia is higher in the gut microbiota of herbivorous insects Supplementary Fig.

S7 , exhibiting low taxa richness Supplementary Fig. S7A and diversity Supplementary Fig. S7B , higher dominance Supplementary Fig. S7C , and low evenness Supplementary Fig. Marine communities both the microbiota associated with corals and gutless worms and herbivorous insects particularly the Termitidae termites gut microbiota present the highest taxa richness, diversity and evenness, as well as the lower dominance Supplementary Fig.

Even that taxonomic similarities within the FGI microbiota group are observed in higher hierarchical levels, their microbiota have particularities regarding genera-level composition. Pantoea, Serratia, and Rahnella are the most abundant genera in M.

The third cluster contains the microbiota of M. Overall, the microbiota of FGI have a distinctive composition when compared to other hosts, being dominated by Gammaproteobacteria at class level and by Pseudomonas at genera level, showing low diversity and high dominance. The microbiota of FGI also group separately from the microbiota of other hosts by alignment-free k- mer based approach for metagenome clustering Fig. S8 and S9. A similar pattern occurs in the cluster comprising the microbiota associated with the omnivorous Panchlora sp.

Heatmaps constructed based on the normalized abundance of CAZy sequences taxonomically assigned. Herbivorous insects clustered in four main groups Fig. S9 : I Cluster encompassing the microbiota of the initial segment of Nasutitermes corniger and Cubitermes ugandensis termites gut, as well as the microbiota of adult Veturius sinuatocollis beetles, that does not present a particular CAZy-codifying microbiota. Herbivorous vertebrates also clustered separately, forming three main groups Fig. S9 : I Macropus eugenii and Ovis aries gut cluster, for which Firmicutes Bacilli and Clostridia and Bacteroidetes are the most abundant groups.

Two general patterns were observed for the taxonomically assigned CAZy sequences of gut microbiota of omnivorous vertebrates Fig. First, the gut microbiota of Canis lupus familiaris and Rattus sp. Second, the gut microbiota of Lemur catta that presents higher abundance of Firmicutes Bacilli and Clostridia , Bacteroidetes, and Gammaproteobacteria Enterobacteria , and clustered with the Primates group.

The marine bacterial communities have low relative abundance of CAZy-annotated sequences, not presenting a particular CAZy-codifying microbiota. In general, when comparing hosts with different diet and lifestyle, the CAZy-codifier community dominated by Gammaproteobacteria seems to be a characteristic feature of the microbiota associated with FGI. S10 ; amino acid pathways related to tyrosine, glutathione, arginine and proline, phenylalanine, tryptophan, valine, leucine and isoleucine metabolism Supplementary Fig.

S11 ; energy pathways related to sulfur and nitrogen metabolism Supplementary Fig. S12 ; glycan pathways related to lipopolysaccharide biosynthesis Supplementary Fig. S13 ; lipid pathways related to fatty acid degradation and biosynthesis of unsaturated fatty acids Fig.

S14 ; cofactors and vitamins pathways related to biotin metabolism and terpenoid-quinone biosynthesis Supplementary Fig. S15 ; terpenoids and polyketides pathways related to geraniol, limonene and pinene degradation, and biosynthesis of siderophore nonribosomal peptides Supplementary Fig. S16 ; secondary metabolism pathways related to tropane, piperidine, pyridine alkaloid, and isoquinoline alkaloid biosynthesis Supplementary Fig.

S17 ; and xenobiotics pathways related to benzoate degradation Supplementary Fig. Box plots calculated based on the normalized abundance of the CAZy families abundantly codified by the microbiota of FGI. In summary, though having some functional overlapping with the gut microbiota of herbivorous and omnivorous hosts, the FGI microbiota differentially codify functions in pathways related to lignocellulose breakdown, detoxification of plant secondary metabolites, metabolism of simple sugars, fungal cell wall deconstruction, biofilm formation, antimicrobials biosynthesis, and diverse nutrient cycling routes Fig.

Possible metabolic roles for these functions were speculated according to the literature Supplementary Table S4. Diverse studies suggest these functions participating in plant biomass metabolism, biofilm formation, fungal biomass metabolism, general nutrition, and antimicrobials biosynthesis. Ant gardens, termite combs, and beetle galleries depicted at the center exhibit characteristic structures deriving from the metabolism of plant biomass.

Pencil drawings by Mariana Barcoto. Besides obtaining nutrients through a symbiotic association with fungi, FGI are associated with a bacterial community physiologically important for the insect-host lifestyle 17 , 27 , 28 , Even though FGI differ regarding geographic distribution, evolutionary history, and fungal taxa maintained as crops 9 , marked similarities in microbiota taxonomic composition at higher hierarchical levels e.

At class level, FGI colonies and galleries seem to assemble a microbiota particular to these environments, having the Gammaproteobacteria as the most abundant group Fig. S1 and low class-diversity Supplementary Fig. At genera level, despite particularities regarding the relative abundance of specific genera, Pseudomonas, Pantoea, Klebsiella, Enterobacter, and Serratia are relatively abundant for fungus-growing ants, termites, and beetles Fig.

The microbiota of M. However, based on the small amount of metagenomic data available for lower attine ants, we are not able to determine whether the taxonomic composition of M. Even so, considering the diversity of lower attine ants 11 , it is also possible that different ant species could host taxonomically diverse microbiota that would include different dominant taxa.

Therefore, having a particular taxonomic composition could not be an exclusivity of the M. Gammaproteobacteria-enriched communities of FGI codify for diverse carbohydrate-active enzymes Fig. Some of these features overlap with herbivorous and omnivorous hosts, indicating functional similarities with these environments at a certain extent Fig. Together, abundantly codified CAZy families and KEGG pathways may reflect functions important for the FGI microbiota metabolism, suggesting the community participating in lignocellulose breakdown, detoxification of plant secondary metabolites, metabolism of simple sugars, fungal cell wall deconstruction, biofilm formation, antimicrobials biosynthesis, and diverse nutrient cycling routes Fig.

Fungus gardens, combs, and galleries are considered to act as aerobic external guts, metabolizing recalcitrant plant biomass into simpler carbohydrates 21 , 22 , 23 , 24 , that become available to the insect host 17 , 20 , 44 , 45 , Because oxygen is required for lignin breakdown 49 , aerobic conditions could favor lignin depolimerization by microorganisms codifying ligninolytic enzymes.

The FGI microbiota has been suggested as part of plant biomass metabolism 17 , 20 , 27 , 35 , 43 , though the mechanisms and pathways for this integration remain to be further explored. By potentially metabolizing complex plant components and degrading toxic compounds Fig.

Fungiculture environments could also favor groups of microorganisms degrading plant fibers via pathways alternative to those commonly codified by herbivorous gut microbiota for instance, those able to metabolize lignocellulose in aerobic conditions that are not found in the gut; Figs.

Independent of the taxonomic composition, these microbial communities would have similar functional groups Fig. Plant cell wall deconstruction in FGI symbiosis could sustain complimentary roles of the fungal symbiont and the associated microbiota, resulting in a multipartite metabolism of lignocellulose.

By assembling the plant biomass degradation in tandem, the fungal-microbiota association could efficiently metabolize lignocellulose even whether none of the organisms codify the complete enzymatic pathway 50 , 60 , Also crucial for maintaining a healthy fungiculture is detoxifying plant secondary compounds, as several of these metabolites specially terpenoids are harmful for both the insect and fungal symbiont 62 , The FGI microbiota may have an important role in detoxifying plant metabolites 43 , which could select microbial members able to metabolize these toxic compounds, influencing the microbiota composition.

Members of the FGI core microbiota including Pseudomonas, Rahnella, Serratia, Burkholderia 43 , 64 , and Stenotrophomonas 65 , are reported to detoxify plant compounds, which could also be accomplished in the fungiculture environment.

Differentially abundant functions related to biofilm formation Fig. Communities embedded in biofilm matrix optimize lignocellulose breakdown by retaining and accumulating degradative enzymes and depolymerization products, allowing the attachment to the plant substrate and permanence at the hydrolysis site, supporting syntrophic associations between microorganisms and thus forming trophic chains required for degradation of plant polymers 51 , 66 , 67 , Host-associated biofilm-forming communities not only detoxify plant secondary compounds through sorption of toxins into the matrix 53 , but also retain nutrients and metabolic products that become available for assimilation by the community and the host 51 , 66 , Nutritional support to the fungal symbiont has already been suggested as a role of the FGI microbiota 26 , 27 , 28 , which could involve pathways related to nitrogen, sulfur, amino acids, lipids, and vitamins metabolism Fig.

S11 , S12 , S14 , S Investigating nutrient-based interactions could reveal fungal-microbiota integrated networks for nutrient cycling important for FGI ecosystem functioning.

For instance, bacteria in some plant decomposer communities make nitrogen available to fungi while receiving labile carbon compounds in exchange 70 , 71 , and similar networks could be operating in the fungiculture environment Moreover, functions codified by the FGI microbiota suggest the attachment to fungal cell walls, possibly via biofilm formation Fig.

In the fungiculture scenario, biofilms could mediate fungal-microbiota interactions 70 , 72 including bacterial mycolytic activity, as pathways related to the metabolism of chitin may reflect the populations obtaining nutrients from hyphae 63 , 64 , 65 , 73 , 74 , 75 , 76 , This opens the possibility of populations within the FGI microbiota participating in fungal biomass turnover by consuming fungal nutrients from old and metabolically inactive portions of fungus gardens, combs, and galleries.

Alternatively, bacterial populations could act as commensals throughout the system, obtaining resources from hyphae as carbohydrates, protein, lipids 73 and exudates low molecular weight metabolites , but not leading to harmful interactions It is curious to observe that communities living in ectomycorrhiza mycosphere i.

It also remains to be investigated the likelihood, extent, and metabolic outcomes of interactions occurring among bacterial populations within the FGI microbiota For instance, the abundance of pathways related to antimicrobials biosynthesis Fig. Also insightful would be to analyze the distribution, diversity, and stability of bacterial populations across the gradient of nutrients that derive from plant biomass metabolism by the fungal symbiont 82 , 83 , Overall, features abundantly codified by the FGI microbiota may reflect a multiplicity of microhabitats distinctive of fungiculture, deriving from an assemblage of conditions including the availability of raw plant biomass, simpler carbohydrates and lignin-derivatives resulted from fungal metabolism, fungal biomass, and aerobic environments.

Merging these conditions could result in niches i. Such environmental particularities shaping the microbiota could result in the low class diversity and high dominance observed for this group Supplementary Fig. Our findings highlight the complexity and heterogeneity of the FGI microbiota metabolic pathways, suggesting the microbiota as possibly adapted to the fungiculture environment.

Such perspective emphasizes the need to further investigate FGI ecosystems, not only for their potential to codify for natural products 88 , 89 and biotechnologically important enzymes 90 , but also to unveil the ecological relevance of microbiota-fungal metabolic networks fundamental to FGI evolutionary success. We expanded the dataset of FGI microbiota by sequencing the microbial community from fungus gardens of the lower attine M.

Fungus gardens, ants, and brood from visibly healthy colonies of At. Both At. Top and bottom sections of fungus gardens were sampled from two colonies of At. Because of the smaller size of M. Bacterial fractions were obtained from fungus-gardens through a centrifugation and filtration protocol modified from Suen et al. This mixture was incubated at room temperature for six days for fungus gardens of At. During this period, the fungus garden settled at the bottom of the tubes.

The washing and incubation steps were repeated three times. Then, the several pellets resulting from the same sample were joined. The presence of bacteria in the final pellet was confirmed through bright-field microscopy.

DNA from 0. We empirically verified this adaptation resulting in DNA samples with higher quantity and quality from our bacterial samples. Quality control and preprocessing of reads were carried out in Solexa QA v3. Preprocessing quality was checked in FastQC. Quality-controlled contigs were uploaded to the Integrated Microbial Genomes IMG database for gene identification and annotation through the standard pipeline of IMG Taxonomic classification was further confirmed through two distinct approaches.

Since there is only a single copy of these genes in most of bacterial genomes, their sequences are considered proper for bacterial taxonomic classification 96 , Maximum-likelihood phylogenies were inferred through PhyML 99 , using WAG as substitution model and replicates of non-parametric bootstrap analysis.

Metagenomes from M. Comparisons were based on the relative abundance of protein-coding sequences, i. Relative abundances were multiplied by 10 6 for statistical analysis. Diversity indices were estimated based on the relative abundance of bacterial class using PAST 3. KO annotated sequences were compiled as metabolic pathways, which were subsequently compiled as: carbohydrate metabolism, amino acid metabolism, energy metabolism, lipid metabolism, glycan metabolism, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides, biosynthesis of other secondary metabolites, and xenobiotics biodegradation and metabolism.

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