otb: an Automated HiC/HiFi Pipeline Assembles the Prosapia bicincta Genome.

The implementation of a new genomic assembly pipeline named only the best (otb) has effectively addressed various challenges associated with data management during the development and storage of genome assemblies. otb, which incorporates a comprehensive pipeline involving a setup layer, quality checks, templating, and the integration of Nextflow and Singularity. The primary objective of otb is to streamline the process of creating a HiFi/HiC genome, aiming to minimize the manual intervention required in the genome assembly process. The Two-lined spittlebug, (Prosapia bicincta, Hemiptera: Cercopidae), a true bug insect herbivore, serves as a practical test case for evaluating otb. The two-lined spittlebug is both a crucial agricultural pest and a genomically understudied insect belonging to the order Hemiptera. This insect is a significant threat to grasslands and pastures, leading to plant wilting and phytotoxemia when infested. Its presence in tropical and subtropical regions around the world poses a long-term threat to the composition of plant communities in grassland landscapes, impacting rangelands, and posing a substantial risk to cattle production.

Impact of Larval Food Source on the Stability of the Bactrocera dorsalis Microbiome.

Bacterial symbionts are crucial to the biology of Bactrocera dorsalis. With larval diet (fruit host) being a key factor that determines microbiome composition and with B. dorsalis using more than 400 fruits as hosts, it is unclear if certain bacterial symbionts are preserved and are passed on to B. dorsalis progenies despite changes in larval diet. Here, we conducted a fly rearing experiment to characterize diet-induced changes in the microbiome of female B. dorsalis. In order to explicitly investigate the impacts of larval diet on the microbiome, including potential stable bacterial constituents of B. dorsalis, we performed 16S rRNA sequencing on the gut tissues of teneral female flies reared from four different host fruits (guava, mango, papaya, and rose apple) infested using a single cohort of wild B. dorsalis that emerged from tropical almond (mother flies). Although B. dorsalis-associated microbiota were predominantly shaped by the larval diet, some major bacterial species from the mother flies were retained in progenies raised on different larval diets. With some variation, Klebsiella (ASV 1 and 2), Morganella (ASV 3), and Providencia (ASV 6) were the major bacterial symbionts that were stable and made up 0.1-80% of the gut and ovipositor microbiome of female teneral flies reared on different host fruits. Our results suggest that certain groups of bacteria are stably associated with female B. dorsalis across larval diets. These findings provide a basis for unexplored research on symbiotic bacterial function in B. dorsalis and may aid in the development of novel management techniques against this devastating pest of horticultural importance.

Toward an Integrated Understanding of the Lepidoptera Microbiome.

Research over the past 30 years has led to a widespread acceptance that insects establish widespread and diverse associations with microorganisms. More recently, microbiome research has been accelerating in lepidopteran systems, leading to a greater understanding of both endosymbiont and gut microorganisms and how they contribute to integral aspects of the host. Lepidoptera are associated with a robust assemblage of microorganisms, some of which may be stable and routinely detected in larval and adult hosts, while others are ephemeral and transient. Certain microorganisms that populate Lepidoptera can contribute significantly to the hosts' performance and fitness, while others are inconsequential. We emphasize the context-dependent nature of the interactions between players. While our review discusses the contemporary literature, there are major avenues yet to be explored to determine both the fundamental aspects of host-microbe interactions and potential applications for the lepidopteran microbiome; we describe these avenues after our synthesis.

Gut bacterial population and community dynamics following adult emergence in pest tephritid fruit flies.

Gut microbiota are important contributors to insect success. Host-microbe interactions are dynamic and can change as hosts age and/or encounter different environments. A turning point in these relationships the transition from immature to adult life stages, particularly for holometabolous insects where there is radical restructuring of the gut. Improved knowledge of population and community dynamics of gut microbiomes upon adult emergence inform drivers of community assembly and physiological aspects of host-microbe interactions. Here, we evaluated the bacterial communities of the pest tephritid species melon fly (Zeugodacus cucurbitae) and Medditeranean fruit fly (medfly, Ceratitis capitata) associated with the pupae life stage and timepoints immediately following adult eclosion. We used a combination of culturing to determine cultivatable bacterial titers, qPCR to determine 16S-rRNA SSU copy numbers, and 16S V4 sequencing to determine changes in communities. Both culturing and qPCR revealed that fly bacterial populations declined upon adult emergence by 10 to 100-fold followed by recovery within 24 h following eclosion. Titers reached ~ 107 CFUs (~ 108 16S rRNA copies) within a week post-emergence. We also observed concurrent changes in amplicon sequence variance (ASVs), where the ASV composition differed overtime for both melon fly and medfly adults at different timepoints. Medfly, in particular, had different microbiome compositions at each timepoint, indicating greater levels of variation before stabilization. These results demonstrate that tephritid microbiomes experience a period of flux following adult emergence, where both biomass and the makeup of the community undergoes dramatic shifts. The host-microbe dynamics we document suggest plasticity in the community and that there may be specific periods where the tephritid gut microbiome may be pliable to introduce and establish new microbial strains in the host.

Hotter days, stronger immunity? Exploring the impact of rising temperatures on insect gut health and microbial relationships.

Climate change can generate cascading effects on animals through compounding stressors. As ectotherms, insects are particularly susceptible to variation in temperature and extreme events. How insects respond to temperature often occurs with respect to their environment, and a pertinent question involves how thermal stress integrates with insect capabilities to resolve interactions with gut microorganisms (microbiome and gut pathogens). We explore the impact of elevated temperatures and the impact of the host physiological response influencing immune system regulation and the gut microbiome. We summarize the literature involving how elevated temperature extremes impact insect gut immune systems, and how in turn that alters potential interactions with the gut microbiome and potential pathogens. Temperature effects on immunity are complex, and ultimate effects on microbial components can vary by system. Moreover, there are multiple questions yet to explore in how insects contend with simultaneous abiotic stressors and potential trade-offs in their response to opportunistic microbiota.

Tomato Chemical Defenses Intensify Corn Earworm (Helicoverpa zea) Mortality from Opportunistic Bacterial Pathogens.

Insect herbivores face multiple challenges to their ability to grow and reproduce. Plants can produce a series of defenses that disrupt and damage the herbivore digestive system, which are heightened upon injury by insect feeding. Additionally, insects face threats from virulent microorganisms that can incur their own set of potential costs to hosts. Microorganisms that invade through the digestive system may function in concert with defenses generated by plants, creating combined assailments on host insects. In our study, we evaluated how tomato defenses interact with an enteric bacterial isolate, Serratia marcescens, in the corn earworm (Helicoverpa zea). We performed bioassays using different tomato cultivars that were induced by methyl jasmonate and larvae orally inoculated with a S. marcescens isolate. Untreated corn earworm larval mortality was low on constitutive tomato, while larvae inoculated with S. marcescens exhibited > 50% mortality within 5 days. Induction treatments elevated both control mortality (~ 45%) and in combination with S. marcescens (> 95%). Larvae also died faster when encountering induced defenses and Serratia. Using a tomato mutant, foliar polyphenol oxidase activity likely had stronger impacts on S. marcescens-mediated larval mortality. Induction treatments also elevated the number of bacterial colony-forming units in the hemolymph of larvae inoculated with Serratia. Larval mortality by S. marcescens was low (< 10%) on artificial diets. Our results demonstrate that plant chemical defenses enhance larval mortality from an opportunistic gut microbe. We propose that the combined damage from both the plant and microbial agent overwhelm the herbivore to increase mortality rates and expedite host death.

Head and thorax elevation during cardiopulmonary resuscitation using circulatory adjuncts is associated with improved survival.

BACKGROUND: Survival after out-of-hospital cardiac arrest (OHCA) remains poor. A physiologically distinct cardiopulmonary resuscitation (CPR) strategy consisting of (1) active compression-decompression CPR and/or automated CPR, (2) an impedance threshold device, and (3) automated controlled elevation of the head and thorax (ACE) has been shown to improve neurological survival significantly versus conventional (C) CPR in animal models. This resuscitation device combination, termed ACE-CPR, is now used clinically. OBJECTIVES: To assess the probability of OHCA survival to hospital discharge after ACE-CPR versus C-CPR. METHODS: As part of a prospective registry study, 227 ACE-CPR OHCA patients were enrolled 04/2019-07/2020 from 6 pre-hospital systems in the United States. Individual C-CPR patient data (n = 5196) were obtained from three large published OHCA randomized controlled trials from high-performing pre-hospital systems. The primary study outcome was survival to hospital discharge. Secondary endpoints included return of spontaneous circulation (ROSC) and favorable neurological survival. Propensity-score matching with a 1:4 ratio was performed to account for imbalances in baseline characteristics. RESULTS: Irrespective of initial rhythm, ACE-CPR (n = 222) was associated with higher adjusted odds ratios (OR) of survival to hospital discharge relative to C-CPR (n = 860), when initiated in <11 min (3.28, 95 % confidence interval [CI], 1.55-6.92) and < 18 min (1.88, 95 % CI, 1.03-3.44) after the emergency call, respectively. Rapid use of ACE-CPR was also associated with higher probabilities of ROSC and favorable neurological survival. CONCLUSIONS: Compared with C-CPR controls, rapid initiation of ACE-CPR was associated with a higher likelihood of survival to hospital discharge after OHCA.

Host-Specific larval lepidopteran mortality to pathogenic Serratia mediated by poor diet.

Insect guts often harbor an abundance of bacteria. Many of these members are commensal, but some may emerge as opportunistic pathogens when the host is under stress. In this study, we evaluated how dietary nutritional concentration mediates a shift from commensal to pathogenic, and if host species influences those interactions. We used the lepidopterans (Noctuidae) fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), and corn earworm (Helicoverpa zea) as hosts and a Serratia strain initially isolated from healthy fall armyworm. Diet concentration was altered by bulk reduction in nutritional content with dilution using cellulose. Our experiments revealed that low nutrient diet increased mortality from Serratia for beet armyworm and corn earworm. However, for fall armyworm, little mortality was observed in any of the diet combinations. Dietary nutrition and oral inoculation with Serratia did not change the expression of two antimicrobial peptides in fall and beet armyworm, suggesting that other mechanisms that mediate mortality were involved. Our results have implications for how pathogens may persist as commensals in the digestive tract of insects. These findings also suggest that diet plays a very important role in the switch from commensal to pathogen. Finally, our data indicate that the host response to changing conditions is critical in determining if a pathogen may overtake its host and that these three lepidopteran species have different responses to opportunistic enteric pathogens.

Opposing Growth Responses of Lepidopteran Larvae to the Establishment of Gut Microbiota.

Gut microbiota can have diverse impacts on hosts, the nature of which often depend on the circumstances. For insect gut microbes, the quality and nature of host diets can be a significant force in swinging the pendulum from inconsequential to functionally important. In our study, we addressed whether beneficial microbes in one species impart similar functions to related species under identical conditions. Using fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), and other noctuid hosts, we implemented an axenic rearing strategy and manipulated gut bacterial populations and dietary conditions. Our results revealed that some gut Enterococcus and Enterobacter isolates can facilitate utilization of a poor diet substrate by fall armyworm, but this was not the case for other more optimized diets. While Enterococcus provided benefits to fall armyworm, it was decidedly antagonistic to beet armyworm (Spodoptera exigua) under identical conditions. Unique isolates and bacterial introductions at early growth stages were critical to how both larval hosts performed. Our results provide robust evidence of the roles in which bacteria support lepidopteran larval growth, but also indicate that the directionality of these relationships can differ among congener hosts. IMPORTANCE Insects have intimate relationships with gut microbiota, where bacteria can contribute important functions to their invertebrate hosts. Lepidopterans are important insect pests, but how they engage with their gut bacteria and how that translates to impacts on the host are lacking. Here we demonstrate the facultative nature of gut microbiota in lepidopteran larvae and the importance of diet in driving mutualistic or antagonistic relationships. Using multiple lepidopteran species, we uncover that the same bacteria that can facilitate exploitation of a challenging diet in one host severely diminishes larval performance of another larval species. Additionally, we demonstrate the beneficial functions of gut microbiota on the hosts are not limited to one lineage, but rather multiple isolates can facilitate the exploitation of a suboptimal diet. Our results illuminate the context-dependent nature of the gut microbiomes in invertebrates, and how host-specific microbial engagement can produce dramatically different interactions.

CDC field triage criteria accurately predicts outcomes in high impact trauma.

BACKGROUND: The precision of emergency medical services (EMS) triage criteria dictates whether an injured patient receives appropriate care. The trauma triage protocol is a decision scheme that groups patients into triage categories of major, moderate and minor. We hypothesized that there is a difference between trauma triage category and injury severity score (ISS). METHODS: This retrospective, observational study was conducted to investigate a difference between trauma triage category and ISS. Bivariate analysis was used to test for differences between the subgroup means. The differences between the group means on each measure were analyzed for direction and statistical significance using ANOVA for continuous variables and chi square tests for categorical variables. Logistic and linear regressions were performed to evaluate factors predicting mortality, ICU length of stay. RESULTS: With respect to trauma triage category, our findings indicate that minor and moderate triage categories are similar with respect to ISS, GCS, ICU LOS, hospital LOS, and mortality. However, after excluding for low impact injuries (falls), differences between the minor and moderate categories were evident when comparing to ISS, GCS, ICU LOS, and hospital LOS. Additionally, after excluding for low impact injures, ISS, ICU LOS, and hospital stay were found to correlate well with trauma triage category. CONCLUSIONS: In this retrospective, observational study significant differences were not seen when comparing ISS with the trauma triage categories of moderate and minor during our initial analysis. However, a difference was found after excluding for low impact injuries. These findings suggest that CDC criteria accurately predicts outcomes in high impact trauma.

Enterococcal symbionts of caterpillars facilitate the utilization of a suboptimal diet.

Bacterial gut symbionts of insect herbivores can impact their host through different mechanisms. However, in most lepidopteran systems we lack experimental examples to explain how specific members of the gut bacterial community influence their host. We used fall armyworm (Spodoptera frugiperda) as a model system to address this objective. We implemented axenic and gnotobiotic techniques using two semi-artificial diets with pinto bean and wheat germ-based components. Following an initial screen of bacterial isolates representing different genera, larvae inoculated with Enterococcus FAW 2-1 exhibited increased body mass on the pinto bean diet, but not on the wheat germ diet. We conducted a systematic bioassay screening of Enterococcus isolated from fall armyworm, revealing they had divergent effects on the hosts' usage pinto bean diet, even among phylogenetically similar isolates. Dilution of the pinto bean diet revealed that larvae performed better on less-concentrated diets, suggesting the presence of a potential toxin. Collectively, these results demonstrate that some gut microorganisms of lepidopterans can benefit the host, but the dietary context is key towards understanding the direction of the response and magnitude of the effect. We provide evidence that gut microorganisms may play a wider role in mediating feeding breadth in lepidopteran pests, but overall impacts could be related to the environmental stress and the metabolic potentials of the microorganisms inhabiting the gut.

Concerted impacts of antiherbivore defenses and opportunistic Serratia pathogens on the fall armyworm (Spodoptera frugiperda).

Insects frequently confront different microbial assemblages. Bacteria inhabiting an insect gut are often commensal, but some can become pathogenic when the insect is compromised from different stressors. Herbivores are often confronted by various forms of plant resistance, but how defenses generate opportunistic microbial infections from residents in the gut are not well understood. In this study, we evaluated the pathogenic tendencies of Serratia isolated from the digestive system of healthy fall armyworm larvae (Spodoptera frugiperda) and how it interfaces with plant defenses. We initially selected Serratia strains that varied in their direct expression of virulence factors. Inoculation of the different isolates into the fall armyworm body cavity indicated differing levels of pathogenicity, with some strains exhibiting no effects while others causing mortality 24 h after injection. Oral inoculations of pathogens on larvae provided artificial diets caused marginal (< 7%) mortality. However, when insects were provided different maize genotypes, mortality from Serratia increased and was higher on plants exhibiting elevated levels of herbivore resistance (< 50% mortality). Maize defenses facilitated an initial invasion of pathogenic Serratia into the larval hemocoel¸ which was capable of overcoming insect antimicrobial defenses. Tomato and soybean further indicated elevated mortality due to Serratia compared to artificial diets and differences between plant genotypes. Our results indicate plants can facilitate the incipient emergence of pathobionts within gut of fall armyworm. The ability of resident gut bacteria to switch from a commensal to pathogenic lifestyle has significant ramifications for the host and is likely a broader phenomenon in multitrophic interactions facilitated by plant defenses.

Faster time to automated elevation of the head and thorax during cardiopulmonary resuscitation increases the probability of return of spontaneous circulation.

OBJECTIVES: Resuscitation in the Head Up position improves outcomes in animals treated with active compression decompression cardiopulmonary resuscitation and an impedance threshold device (ACD + ITD CPR).We assessed impact of time to deployment of an automated Head Up position (AHUP) based bundle of care after out-of-hospital cardiac arrest on return of spontaneous circulation (ROSC). METHODS: Observational data were analyzed from a patient registry. Patients received treatment with 1) ACD + and/or automated CPR 2) an ITD and 3) an AHUP device. Probability of ROSC (ROSCprob) from the 9-1-1 call to AHUP device placement was assessed with a restricted cubic spline model and linear regression. RESULTS: Of 11 sites, 6 recorded the interval from 9-1-1 to AHUP device (n = 227). ROSCprobfor all rhythms was 34%(77/227). Median age (range) was 66 years (19-101) and 68% men. TheROSCprobfor shockable rhythms was 47%(18/38). Minutes from 9-1-1 to AHUP device (median, range) varied between sites: 1) 6.4(4,15), 2) 8.0(5,19), 3) 9.9(4, 12), 4) 14.1(6, 36), 5) 15.9(6, 34), 6) 19.0(8, 38),(p = 0.0001).ROSCprobalso varied; 1) 55.1%(16/29), 2) 60%(3/5), 3) 50%(3/6), 4) 22.7%(17/75), 5) 26.4%(9/34), and 6) 37.1%(29/78), (p = 0.019). For all rhythms between 4 and 12 min (n = 85),ROSCprobdeclined 5.6% for every minute elapsed (p = 0.024). For shockable rhythms, between 6 and 15 min (n = 23),ROSCprobdeclined 9.0% for every minute elapsed (p = 0.006). CONCLUSIONS: Faster time to deployment of an AHUP based bundle of care is associated with higher incidence of ROSC. This must be considered when evaluating and implementing this bundle.

Parasitoid Causes Cascading Effects on Plant-Induced Defenses Mediated Through the Gut Bacteria of Host Caterpillars.

Koinobiont endoparasitoid wasps whose larvae develop inside a host insect alter several important facets of host physiology, potentially causing cascading effects across multiple trophic levels. For instance, the hijacking of the host immune responses may have effects on how insects interact with host plants and microbial associates. However, the parasitoid regulation of insect-plant-microbiome interactions is still understudied. In this study, we used the fall armyworm (FAW), Spodoptera frugiperda, and the braconid parasitoid Cotesia marginiventris to evaluate impacts of parasitism on the gut microbiome of FAW larvae, and respective maize plant defense responses. The level of reactive oxygen species and the microbial community in larval gut underwent significant changes in response to parasitism, leading to a significant reduction of Enterococcus, while elevating the relative abundance of Pseudomonas. FAW with parasitism had lower glucose oxidase (GOX) activity in salivary glands and triggered lower defense responses in maize plants. These changes corresponded to effects on plants, as Pseudomonas inoculated larvae had lower activity of salivary GOX and triggered lower defense responses in maize plants. Our results demonstrated that parasitism had cascading effects on microbial associates across trophic levels and also highlighted that insect gut bacteria may contribute to complex interrelationships among parasitoids, herbivores, and plants.

Oviposition preferences, Bt susceptibilities, and tissue feeding of fall armyworm (Lepidoptera: Noctuidae) host strains.

BACKGROUND: The fall armyworm, Spodoptera frugiperda (Smith), is a pest of many economically essential crops across several continents. Documentation of resistance to Bt toxins has caused growing concern in agricultural communities regarding the ability to keep fall armyworm populations below economic thresholds. The existence of two host strains referred to as the 'rice' and 'corn' strains is a complicating and under-researched factor of fall armyworm biology and management. It is essential to characterize the differences between the host strains, as well as their rice/corn hybrid offspring, to elucidate their contributions to field-evolved resistance. RESULTS: Corn was a preferred oviposition host for both rice and corn strain fall armyworm, and a suitable larval host plant for each of the four populations tested. Corn strain females displayed a significant preference towards oviposition on plants that lacked mechanical damage. The rice strain population was generally less tolerant to Cry1F corn tissue than the corn strain and hybrid populations, which performed in a similar way to one another. CONCLUSION: The preference for corn as an ovipositional host may have an impact on resistance management when coupled with differential host strain Bt tolerances, though more studies are needed. Hybrid tolerance to Bt toxins could possibly contribute to the evolution of Bt resistance. This is the first study to compare the larval fitness and survival of rice/corn hybrid fall armyworm to that of pure host strains using a tissue-based approach.

Effects of maize (Zea mays) genotypes and microbial sources in shaping fall armyworm (Spodoptera frugiperda) gut bacterial communities.

Plants can have fundamental roles in shaping bacterial communities associated with insect herbivores. For larval lepidopterans (caterpillars), diet has been shown to be a driving force shaping gut microbial communities, where the gut microbiome of insects feeding on different plant species and genotypes can vary in composition and diversity. In this study, we aimed to better understand the roles of plant genotypes, sources of microbiota, and the host gut environment in structuring bacterial communities. We used multiple maize genotypes and fall armyworm (Spodoptera frugiperda) larvae as models to parse these drivers. We performed a series of experiments using axenic larvae that received a mixed microbial community prepared from frass from larvae that consumed field-grown maize. The new larval recipients were then provided different maize genotypes that were gamma-irradiated to minimize bacteria coming from the plant during feeding. For field-collected maize, there were no differences in community structure, but we did observe differences in gut community membership. In the controlled experiment, the microbial inoculation source, plant genotype, and their interactions impacted the membership and structure of gut bacterial communities. Compared to axenic larvae, fall armyworm larvae that received frass inoculum experienced reduced growth. Our results document the role of microbial sources and plant genotypes in contributing to variation in gut bacterial communities in herbivorous larvae. While more research is needed to shed light on the mechanisms driving this variation, these results provide a method for incorporating greater gut bacterial community complexity into laboratory-reared larvae.

Fidelity and Timing of Spotted Lanternfly (Hemiptera: Fulgoridae) Attack Patterns on Ornamental Trees in the Suburban Landscape.

Invasive herbivores can have dramatic impacts in new environments by altering landscape composition, displacing natives, and causing plant decline and mortality. One of the most recent invasive insects in the United States, the spotted lanternfly (Lycorma delicatula), has the potential to cause substantial economic and environmental impacts in agriculture and forestry. Spotted lanternfly exhibits a broad host range, yet reports of late-season movement from the surrounding landscapes onto select tree species in suburban environments have been reported. In this study, we aimed to evaluate the fidelity of spotted lanternfly attack on specific, individual trees within the same species during this movement period. In 2018 and 2019, we observed that individual red (Acer rubrum L. [Sapindales: Sapindaceae]) and silver maple (Acer saccharinum L. [Sapindales: Sapindaceae]) trees were preferentially attacked over other nearby trees of the same species. Foliar elemental composition was a good predictor of spotted lanternfly attack numbers, indicating that individual variation in nutrients may influence spotted lanternfly attraction to and/or retention on maple trees. Our data also confirm reports of late-season movement from surrounding landscapes throughout autumn. Collectively, our results show that spotted lanternfly exhibits some fidelity to particular trees in the landscape during this movement period. While other potential mechanisms also contribute to host plant selection by spotted lanternfly, our data show that host nutritional profiles influence spotted lanternfly infestation of suburban trees at the landscape scale. Our data establish that late-season infestations of suburban trees by spotted lanternfly occurred and that variation in host quality should be further considered in the management of this invasive insect pest.

Complex Relationships at the Intersection of Insect Gut Microbiomes and Plant Defenses.

Insect herbivores have ubiquitous associations with microorganisms that have major effects on how host insects may interact in their environment. Recently, increased attention has been given to how insect gut microbiomes mediate interactions with plants. In this paper, I discuss the ecology and physiology of gut bacteria associated with insect herbivores and how they may shape interactions between insects and their various host plants. I first establish how microbial associations vary between insects with different feeding styles, and how the insect host physiology and ecology can shape stable or transient relationships with gut bacteria. Then, I describe how these relationships factor in with plant nutrition and plant defenses. Within this framework, I suggest that many of the interactions between plants, insects, and the gut microbiome are context-dependent and shaped by the type of defense and the isolates present in the environment. Relationships between insects and plants are not pairwise, but instead highly multipartite, and the interweaving of complex microbial interactions is needed to fully explore the context-dependent aspects of the gut microbiome in many of these systems. I conclude the review by suggesting studies that would help reduce the unsureness of microbial interactions with less-defined herbivore systems and identify how each could provide a path to more robust roles and traits.