SARS-CoV-2 delta variant in African lions (Panthera leo) and humans at Utah's Hogle Zoo, USA, 2021-22.

AIMS: We conducted a One Health investigation to assess the source and transmission dynamics of SARS-CoV-2 infection in African lions (Panthera leo) at Utah's Hogle Zoo in Salt Lake City from October 2021 to February 2022. METHODS AND RESULTS: Following observation of respiratory illness in the lions, zoo staff collected pooled faecal samples and individual nasal swabs from four lions. All specimens tested positive for SARS-CoV-2 by reverse transcription-polymerase chain reaction (RT-PCR). The resulting investigation included: lion observation; RT-PCR testing of lion faeces every 1-7 days; RT-PCR testing of lion respiratory specimens every 2-3 weeks; staff interviews and RT-PCR testing; whole-genome sequencing of viruses from lions and staff; and comparison with existing SARS-CoV-2 human community surveillance sequences. In addition to all five lions, three staff displayed respiratory symptoms. All lions recovered and no hospitalizations or deaths were reported among staff. Three staff reported close contact with the lions in the 10 days before lion illness onset, one of whom developed symptoms and tested positive for SARS-CoV-2 on days 3 and 4, respectively, after lion illness onset. The other two did not report symptoms or test positive. Two staff who did not have close contact with the lions were symptomatic and tested positive on days 5 and 8, respectively, after lion illness onset. We detected SARS-CoV-2 RNA in lion faeces for 33 days and in lion respiratory specimens for 14 weeks after illness onset. The viruses from lions were genetically highly related to those from staff and two contemporaneous surveillance specimens from Salt Lake County; all were delta variants (AY.44). CONCLUSIONS: We did not determine the sources of these infections, although human-to-lion transmission likely occurred. The observed period of respiratory shedding was longer than in previously documented SARS-CoV-2 infections in large felids, indicating the need to further assess duration and potential implications of shedding.

Investigation of SARS-CoV-2 Transmission in The Tabernacle Choir at Temple Square in the Context of Prevention Protocols, Utah, September-November 2021.

Group singing and playing of wind instruments increase COVID-19 transmission risk. After a pause during the initial period of the COVID-19 pandemic, The Tabernacle Choir at Temple Square organization (hereinafter, Choir) resumed musical events in September 2021 with prevention protocols, including required vaccination and pre-event rapid antigen testing. We investigated potential SARS-CoV-2 transmission at Choir events during September 21-November 7, 2021. We interviewed COVID-19-positive members (hereinafter, case-members) and identified members exposed when a case-member attended a Choir event during his or her infectious period. We compared whole genome sequencing results to assess the genetic relatedness of available SARS-CoV-2 specimens obtained from case-members. We identified 30 case-members through pre-event testing (n = 10), self-reported positive test results (n = 18), and a review of Utah's disease surveillance system (n = 2). All 30 case-members reported symptoms; 21 (70%) were women and 23 (77%) received a positive test result by nucleic acid amplification test. No hospitalizations or deaths were reported. We identified 176 test-eligible exposed members from 14 instances of case-members attending events during their infectious periods. All were tested at least once 2 to 14 days after exposure: 74 (42%) by rapid antigen test only (all negative) and 102 (58%) by nucleic acid amplification test (4 positive, 97 negative, and 1 equivocal). Among viral sequences available from 15 case-members, the smallest single-nucleotide polymorphism distance between 2 sequences was 2, and the next-smallest distance was 10. The lack of disease detected in most exposed members suggests that minimal, if any, transmission occurred at Choir events. When community COVID-19 incidence is high, prevention protocols might help limit SARS-CoV-2 transmission during group musical activities.

Benchmark datasets for SARS-CoV-2 surveillance bioinformatics.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has spread globally and is being surveilled with an international genome sequencing effort. Surveillance consists of sample acquisition, library preparation, and whole genome sequencing. This has necessitated a classification scheme detailing Variants of Concern (VOC) and Variants of Interest (VOI), and the rapid expansion of bioinformatics tools for sequence analysis. These bioinformatic tools are means for major actionable results: maintaining quality assurance and checks, defining population structure, performing genomic epidemiology, and inferring lineage to allow reliable and actionable identification and classification. Additionally, the pandemic has required public health laboratories to reach high throughput proficiency in sequencing library preparation and downstream data analysis rapidly. However, both processes can be limited by a lack of a standardized sequence dataset. Methods: We identified six SARS-CoV-2 sequence datasets from recent publications, public databases and internal resources. In addition, we created a method to mine public databases to identify representative genomes for these datasets. Using this novel method, we identified several genomes as either VOI/VOC representatives or non-VOI/VOC representatives. To describe each dataset, we utilized a previously published datasets format, which describes accession information and whole dataset information. Additionally, a script from the same publication has been enhanced to download and verify all data from this study. Results: The benchmark datasets focus on the two most widely used sequencing platforms: long read sequencing data from the Oxford Nanopore Technologies platform and short read sequencing data from the Illumina platform. There are six datasets: three were derived from recent publications; two were derived from data mining public databases to answer common questions not covered by published datasets; one unique dataset representing common sequence failures was obtained by rigorously scrutinizing data that did not pass quality checks. The dataset summary table, data mining script and quality control (QC) values for all sequence data are publicly available on GitHub: https://github.com/CDCgov/datasets-sars-cov-2. Discussion: The datasets presented here were generated to help public health laboratories build sequencing and bioinformatics capacity, benchmark different workflows and pipelines, and calibrate QC thresholds to ensure sequencing quality. Together, improvements in these areas support accurate and timely outbreak investigation and surveillance, providing actionable data for pandemic management. Furthermore, these publicly available and standardized benchmark data will facilitate the development and adjudication of new pipelines.

One Health Investigation of SARS-CoV-2 Infection and Seropositivity among Pets in Households with Confirmed Human COVID-19 Cases-Utah and Wisconsin, 2020.

Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pets during a COVID-19 household transmission investigation. Pets from households with ≥1 person with laboratory-confirmed COVID-19 were eligible for inclusion from April-May 2020. We enrolled 37 dogs and 19 cats from 34 households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dog's fur swabs (2%) tested positive by rRT-PCR at the first sampling. Among 47 pets with serological results, eight (17%) pets (four dogs, four cats) from 6/30 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40-100%) compared to households with no seropositive pet (median 37%; range: 13-100%) (p = 0.01). Thirty-three pets with serologic results had frequent daily contact (≥1 h) with the index patient before the person's COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the index patient after diagnosis and none were seropositive; of the 19 (58%) pets with continued contact, four (21%) were seropositive. Seropositive pets likely acquired infection after contact with people with COVID-19. People with COVID-19 should restrict contact with pets and other animals.

Methods and feasibility study for exome sequencing as a universal second-tier test in newborn screening.

PURPOSE: Newborn screening disorders increasingly require genetic variant analysis as part of second-tier or confirmatory testing. Sanger sequencing and gene-specific next-generation sequencing (NGS)-based tests, the current methods of choice, are costly and lack scalability when expanding to new conditions. We describe a scalable, exome sequencing-based NGS pipeline with a priori analysis restriction that can be universally applied to any NBS disorder. METHODS: De-identified abnormal newborn screening specimens representing severe combined immune deficiency (SCID), cystic fibrosis (CF), VLCAD deficiency, metachromatic leukodystrophy (MLD), and in silico sequence read data sets were used to validate the pipeline. To support interpretation and clinical decision-making within the bioinformatics pipeline, variants from multiple databases were curated and validated. RESULTS: CFTR variant panel analysis correctly identified all variants. Concordance compared with diagnostic testing results for targeted gene analysis was between 78.6% and 100%. Validation of the bioinformatics pipeline with in silico data sets revealed a 100% detection rate. Varying degrees of overlap were observed between ClinVar and other databases ranging from 3% to 65%. Data normalization revealed that 11% of variants across the databases required manual curation. CONCLUSION: This pipeline allows for restriction of analysis to variants within a single gene or multiple genes, and can be readily expanded to full exome analysis if clinically indicated and parental consent is granted.

Infection control response to an outbreak of OXA-23 carbapenemase-producing carbapenem-resistant Acinetobacter baumannii in a skilled nursing facility in Utah.

BACKGROUND: Antibiotic-resistant Acinetobacter species are a growing public health threat, yet are not nationally notifiable, and most states do not mandate reporting. Additionally, there are no standardized methods to detect Acinetobacter species colonization. METHODS: An outbreak of carbapenem-resistant Acinetobacter baumannii (CRAB) was identified at a Utah ventilator unit in a skilled nursing facility. An investigation was conducted to identify transmission modes in order to control spread of CRAB. Culture-based methods were used to identify patient colonization and environmental contamination in the facility. RESULTS: Of the 47 patients screened, OXA-23-producing CRAB were detected in 10 patients (21%), with 7 patients (15%) having been transferred from out-of-state facilities. Of patients who screened positive, 60% did not exhibit any signs or symptoms of active infection by chart review. A total of 38 environmental samples were collected and CRAB was recovered from 37% of those samples. Whole genome sequencing analyses of patient and environmental isolates suggested repeated CRAB introduction into the facility and highlighted the role of shared equipment in transmission. CONCLUSIONS: The investigation demonstrated this ventilated skilled nursing facility was an important reservoir for CRAB in the community and highlights the need for improved surveillance, strengthened infection control and inter-facility communication within and across states.

Sepsis Attributed to Bacterial Contamination of Platelets Associated with a Potential Common Source - Multiple States, 2018.

During May-October 2018, four patients from three states experienced sepsis after transfusion of apheresis platelets contaminated with Acinetobacter calcoaceticus-baumannii complex (ACBC) and Staphylococcus saprophyticus; one patient died. ACBC isolates from patients' blood, transfused platelet residuals, and two environmental samples were closely related by whole genome sequencing. S. saprophyticus isolates from two patients' blood, three transfused platelet residuals, and one hospital environmental sample formed two whole genome sequencing clusters. This whole genome sequencing analysis indicated a potential common source of bacterial contamination; investigation into the contamination source continues. All platelet donations were collected using apheresis cell separator machines and collection sets from the same manufacturer; two of three collection sets were from the same lot. One implicated platelet unit had been treated with pathogen-inactivation technology, and two had tested negative with a rapid bacterial detection device after negative primary culture. Because platelets are usually stored at room temperature, bacteria in contaminated platelet units can proliferate to clinically relevant levels by the time of transfusion. Clinicians should monitor for sepsis after platelet transfusions even after implementation of bacterial contamination mitigation strategies. Recognizing adverse transfusion reactions and reporting to the platelet supplier and hemovigilance systems is crucial for public health practitioners to detect and prevent sepsis associated with contaminated platelets.

Metagenomic sequencing provides insights into microbial detoxification in the guts of small mammalian herbivores (Neotoma spp.).

Microbial detoxification of plant toxins influences the use of plants as food sources by herbivores. Stephen's woodrats (Neotoma stephensi) specialize on juniper, which is defended by oxalate, phenolics and monoterpenes, while closely related N. albigula specialize on cactus, which only contains oxalate. Woodrats maintain two gut chambers harboring dense microbial communities: a foregut chamber proximal to the major site of toxin absorption, and a cecal chamber in their hindgut. We performed several experiments to investigate the location and nature of microbial detoxification in the woodrat gut. First, we measured toxin concentrations across gut chambers of N. stephensi. Compared to food material, oxalate concentrations were immediately lower in the foregut, while concentrations of terpenes remained high in the foregut, and were lowest in the cecal chamber. We conducted metagenomic sequencing of the foregut chambers of both woodrat species and cecal chambers of N. stephensi to compare microbial functions. We found that most genes associated with detoxification were more abundant in the cecal chambers of N. stephensi. However, some genes associated with degradation of oxalate and phenolic compounds were more abundant in the foregut chambers. Thus, microbial detoxification may take place in various chambers depending on the class of chemical compound.

Whole-Genome Sequencing and Bioinformatic Analysis of Isolates from Foodborne Illness Outbreaks of Campylobacter jejuni and Salmonella enterica.

Whole-genome sequencing (WGS) via next-generation sequencing (NGS) technologies is a powerful tool for determining the relatedness of bacterial isolates in foodborne illness detection and outbreak investigations. WGS has been applied to national outbreaks (for example, Listeria monocytogenes); however, WGS has rarely been used in smaller local outbreaks. The current study demonstrates the superior resolution of genetic and evolutionary relatedness generated by WGS data analysis, compared to pulsed-field gel electrophoresis (PFGE). The current study retrospectively applies WGS and a reference-free bioinformatic analysis to a Utah-specific outbreak of Campylobacter jejuni associated with raw milk and to a national multistate outbreak of Salmonella enterica subsp. enterica serovar Typhimurium associated with rotisserie chicken, both of which were characterized previously by PFGE. Together, these analyses demonstrate how a reference-free WGS workflow is not reliant on determination of a reference sequence, like WGS workflows that are based on single-nucleotide polymorphisms, or the need for curated allele databases, like multilocus sequence typing workflows.

Fatal Sepsis Associated with Bacterial Contamination of Platelets - Utah and California, August 2017.

During August 2017, two separate clusters of platelet transfusion-associated bacterial sepsis were reported in Utah and California. In Utah, two patients died after platelet transfusions from the same donation. Clostridium perfringens isolates from one patient's blood, the other patient's platelet bag, and donor skin swabs were highly related by whole genome sequencing (WGS). In California, one patient died after platelet transfusion; Klebsiella pneumoniae isolates from the patient's blood and platelet bag residuals and a nontransfused platelet unit were matched using WGS. Investigation revealed no deviations in blood supplier or hospital procedures. Findings in this report highlight that even when following current procedures, the risk for transfusion-related infection and fatality persists, making additional interventions necessary. Clinicians need to be vigilant in monitoring for platelet-transmitted bacterial infections and report adverse reactions to blood suppliers and hemovigilance systems. Blood suppliers and hospitals could consider additional evidence-based bacterial contamination risk mitigation strategies, including pathogen inactivation, rapid detection devices, and modified screening of bacterial culture protocols.

Patterns of host gene expression associated with harboring a foregut microbial community.

BACKGROUND: Harboring foregut microbial communities is considered a key innovation that allows herbivorous mammals to colonize new ecological niches. However, the functions of these chambers have only been well studied at the molecular level in ruminants. Here, we investigate gene expression in the foregut chamber of herbivorous rodents and ask whether these gene expression patterns are consistent with results in ruminants. We compared gene expression in foregut tissues of two rodent species: Stephen's woodrat (Neotoma stephensi), which harbors a dense foregut microbial community, and the lab rat (Rattus norvegicus), which lacks such a community. RESULTS: We found that woodrats have higher abundances of transcripts associated with smooth muscle processes, specifically a higher expression of the smoothelin-like 1 gene, which may assist in contractile properties of this tissue to retain food material in the foregut chamber. The expression of genes associated with keratinization and cornification exhibited a complex pattern of differences between the two species, suggesting distinct molecular mechanisms. Lab rats exhibited higher abundances of transcripts associated with immune function, likely to inhibit microbial growth in the foregut of this species. CONCLUSIONS: Some of our results were consistent with previous findings in ruminants (high expression of facilitative glucose transporters, lower expression of B4galnt2), suggestive of possible convergent evolution, while other results were unclear, and perhaps represent novel host-microbe interactions in rodents. Overall, our results suggest that harboring a foregut microbiota is associated with changes to the functions and host-microbe interactions of the foregut tissues.

Bioinformatic Analyses of Whole-Genome Sequence Data in a Public Health Laboratory.

The ability to generate high-quality sequence data in a public health laboratory enables the identification of pathogenic strains, the determination of relatedness among outbreak strains, and the analysis of genetic information regarding virulence and antimicrobial-resistance genes. However, the analysis of whole-genome sequence data depends on bioinformatic analysis tools and processes. Many public health laboratories do not have the bioinformatic capabilities to analyze the data generated from sequencing and therefore are unable to take full advantage of the power of whole-genome sequencing. The goal of this perspective is to provide a guide for laboratories to understand the bioinformatic analyses that are needed to interpret whole-genome sequence data and how these in silico analyses can be implemented in a public health laboratory setting easily, affordably, and, in some cases, without the need for intensive computing resources and infrastructure.

The draft genome sequence and annotation of the desert woodrat Neotoma lepida.

We present the de novo draft genome sequence for a vertebrate mammalian herbivore, the desert woodrat (Neotoma lepida). This species is of ecological and evolutionary interest with respect to ingestion, microbial detoxification and hepatic metabolism of toxic plant secondary compounds from the highly toxic creosote bush (Larrea tridentata) and the juniper shrub (Juniperus monosperma). The draft genome sequence and annotation have been deposited at GenBank under the accession LZPO01000000.

Microbial Community Transplant Results in Increased and Long-Term Oxalate Degradation.

Gut microbes are essential for the degradation of dietary oxalate, and this function may play a role in decreasing the incidence of kidney stones. However, many oxalate-degrading bacteria are susceptible to antibiotics and the use of oxalate-degrading probiotics has only led to an ephemeral reduction in urinary oxalate. The objective of the current study was to determine the efficacy of using whole-community microbial transplants from a wild mammalian herbivore, Neotoma albigula, to increase oxalate degradation over the long term in the laboratory rat, Rattus norvegicus. We quantified the change in total oxalate degradation in lab rats immediately after microbial transplants and at 2- and 9-month intervals following microbial transplants. Additionally, we tracked the fecal microbiota of the lab rats, with and without microbial transplants, using high-throughput Illumina sequencing of a hyper-variable region of the 16S rRNA gene. Microbial transplants resulted in a significant increase in oxalate degradation, an effect that persisted 9 months after the initial transplants. Functional persistence was corroborated by the transfer, and persistence of a group of bacteria previously correlated with oxalate consumption in N. albigula, including an anaerobic bacterium from the genus Oxalobacter known for its ability to use oxalate as a sole carbon source. The results of this study indicate that whole-community microbial transplants are an effective means for the persistent colonization of oxalate-degrading bacteria in the mammalian gut.

Draft Genome Sequence of an Oxalate-Degrading Strain of Clostridium sporogenes from the Gastrointestinal Tract of the White-Throated Woodrat (Neotoma albigula).

The gastrointestinal tract of the white-throated woodrat Neotoma albigula harbors a diverse microbial population that functions in the degradation of ingested plant secondary compounds. Here, we present the draft genome sequence and annotation of Clostridium sporogenes strain 8-O, a novel oxalate-degrading bacterium isolated from the feces of N. albigula.

Effect of Dietary Oxalate on the Gut Microbiota of the Mammalian Herbivore Neotoma albigula.

Diet is one of the primary drivers that sculpts the form and function of the mammalian gut microbiota. However, the enormous taxonomic and metabolic diversity held within the gut microbiota makes it difficult to isolate specific diet-microbe interactions. The objective of the current study was to elucidate interactions between the gut microbiota of the mammalian herbivore Neotoma albigula and dietary oxalate, a plant secondary compound (PSC) degraded exclusively by the gut microbiota. We quantified oxalate degradation in N. albigula fed increasing amounts of oxalate over time and tracked the response of the fecal microbiota using high-throughput sequencing. The amount of oxalate degraded in vivo was linearly correlated with the amount of oxalate consumed. The addition of dietary oxalate was found to impact microbial species diversity by increasing the representation of certain taxa, some of which are known to be capable of degrading oxalate (e.g., Oxalobacter spp.). Furthermore, the relative abundances of 117 operational taxonomic units (OTU) exhibited a significant correlation with oxalate consumption. The results of this study indicate that dietary oxalate induces complex interactions within the gut microbiota that include an increase in the relative abundance of a community of bacteria that may contribute either directly or indirectly to oxalate degradation in mammalian herbivores.

Phenotypic characterization of Sodalis praecaptivus sp. nov., a close non-insect-associated member of the Sodalis-allied lineage of insect endosymbionts.

A Gram-stain-negative bacterium, isolated from a human wound was previously found to share an unprecedentedly close relationship with Sodalis glossinidius and other members of the Sodalis-allied clade of insect symbionts. This relationship was inferred from sequence analysis of the 16S rRNA gene and genomic comparisons and suggested the strain belonged to a novel species. Biochemical and genetic analyses supported this suggestion and demonstrated that the organism has a wide repertoire of metabolic properties, which is consistent with the presence of a relatively large gene inventory. Among members of the Sodalis-allied clade, this is the first representative that has sufficient metabolic capabilities to sustain growth in minimal media. On the basis of the results of this study, we propose that this organism be classified as a representative of a novel species, Sodalis praecaptivus sp. nov. (type strain HS(T) = DSM 27494(T) = ATCC BAA-2554(T)).

Genome degeneration and adaptation in a nascent stage of symbiosis.

Symbiotic associations between animals and microbes are ubiquitous in nature, with an estimated 15% of all insect species harboring intracellular bacterial symbionts. Most bacterial symbionts share many genomic features including small genomes, nucleotide composition bias, high coding density, and a paucity of mobile DNA, consistent with long-term host association. In this study, we focus on the early stages of genome degeneration in a recently derived insect-bacterial mutualistic intracellular association. We present the complete genome sequence and annotation of Sitophilus oryzae primary endosymbiont (SOPE). We also present the finished genome sequence and annotation of strain HS, a close free-living relative of SOPE and other insect symbionts of the Sodalis-allied clade, whose gene inventory is expected to closely resemble the putative ancestor of this group. Structural, functional, and evolutionary analyses indicate that SOPE has undergone extensive adaptation toward an insect-associated lifestyle in a very short time period. The genome of SOPE is large in size when compared with many ancient bacterial symbionts; however, almost half of the protein-coding genes in SOPE are pseudogenes. There is also evidence for relaxed selection on the remaining intact protein-coding genes. Comparative analyses of the whole-genome sequence of strain HS and SOPE highlight numerous genomic rearrangements, duplications, and deletions facilitated by a recent expansion of insertions sequence elements, some of which appear to have catalyzed adaptive changes. Functional metabolic predictions suggest that SOPE has lost the ability to synthesize several essential amino acids and vitamins. Analyses of the bacterial cell envelope and genes encoding secretion systems suggest that these structures and elements have become simplified in the transition to a mutualistic association.

Phylogenetic analysis of symbionts in feather-feeding lice of the genus Columbicola: evidence for repeated symbiont replacements.

BACKGROUND: Many groups of insects have obligate bacterial symbionts that are vertically transmitted. Such associations are typically characterized by the presence of a monophyletic group of bacteria living in a well-defined host clade. In addition the phylogeny of the symbiotic bacteria is typically congruent with that of the host, signifying co-speciation. Here we show that bacteria living in a single genus of feather lice, Columbicola (Insecta: Phthiraptera), present an exception to this typical pattern. RESULTS: The phylogeny of Columbicola spp. symbionts revealed the presence of three candidate clades, with the most species-rich clade having a comb-like topology with very short internodes and long terminal branches. Evolutionary simulations indicate that this topology is characteristic of a process of repeated symbiont replacement over a brief time period. The two remaining candidate clades in our study exhibit high levels of nucleotide substitution, suggesting accelerated molecular evolution due to relaxed purifying selection or smaller effective population size, which is typical of many vertically transmitted insect symbionts. Representatives of the fast-evolving and slow-evolving symbiont lineages exhibit the same localization, migration, and transmission patterns in their hosts, implying direct replacement. CONCLUSIONS: Our findings suggest that repeated, independent symbiont replacements have taken place over the course of the relatively recent radiation of Columbicola spp. These results are compatible with the notion that lice and other insects have the capability to acquire novel symbionts through the domestication of progenitor strains residing in their local environment.

A novel human-infection-derived bacterium provides insights into the evolutionary origins of mutualistic insect-bacterial symbioses.

Despite extensive study, little is known about the origins of the mutualistic bacterial endosymbionts that inhabit approximately 10% of the world's insects. In this study, we characterized a novel opportunistic human pathogen, designated "strain HS," and found that it is a close relative of the insect endosymbiont Sodalis glossinidius. Our results indicate that ancestral relatives of strain HS have served as progenitors for the independent descent of Sodalis-allied endosymbionts found in several insect hosts. Comparative analyses indicate that the gene inventories of the insect endosymbionts were independently derived from a common ancestral template through a combination of irreversible degenerative changes. Our results provide compelling support for the notion that mutualists evolve from pathogenic progenitors. They also elucidate the role of degenerative evolutionary processes in shaping the gene inventories of symbiotic bacteria at a very early stage in these mutualistic associations.