Rate Of Horizontal Transfer Research Articles

Characterization of blaTEM and blaCTX-M beta-lactam resistance genes in chronic rhinosinusitis

BackgroundChronic sinusitis is one of the most challenging health problems of contemporary society. Although several treatment methods have been defined, a comprehensive understanding of the underlying causes (e.g., antibiotic resistance) is still elusive. The aim of this study was to characterize two of the main extended-spectrum beta-lactamase genes—i.e., blaTEM and blaCTX-M genes—and investigate antimicrobial resistance in bacteria isolated from chronic sinusitis. Samples from 70 chronic sinusitis patients and 20 healthy individuals (controls) were analyzed for the presence of blaTEM and blaCTX-M resistance genes using the polymerase chain reaction (PCR) test, followed by gene sequence analysis.ResultsPhenotypic and genotypic beta-lactam resistance was observed in 58.7% and 61.54% of the gram-negative isolates, respectively, with 38.46% carrying the blaTEM gene and 34.62% harboring the blaCTX-M gene. Sequencing data indicated high heterogeneity in blaCTX-M genes (69–100% similarity to reported sequences) and lower heterogeneity in blaTEM genes (93–99%).ConclusionBroad-spectrum beta-lactam resistance is a major pathogenesis factor in chronic rhinosinusitis, and careful consideration is required for antimicrobial therapy. High blaCTX-M heterogeneity could mean high horizontal transfer rate of this gene and warrant a surveillance program.

Evidence of cospeciation between termites and their gut bacteria on a geological time scale.

Termites host diverse communities of gut microbes, including many bacterial lineages only found in this habitat. The bacteria endemic to termite guts are transmitted via two routes: a vertical route from parent colonies to daughter colonies and a horizontal route between colonies sometimes belonging to different termite species. The relative importance of both transmission routes in shaping the gut microbiota of termites remains unknown. Using bacterial marker genes derived from the gut metagenomes of 197 termites and one Cryptocercus cockroach, we show that bacteria endemic to termite guts are mostly transferred vertically. We identified 18 lineages of gut bacteria showing cophylogenetic patterns with termites over tens of millions of years. Horizontal transfer rates estimated for 16 bacterial lineages were within the range of those estimated for 15 mitochondrial genes, suggesting that horizontal transfers are uncommon and vertical transfers are the dominant transmission route in these lineages. Some of these associations probably date back more than 150 million years and are an order of magnitude older than the cophylogenetic patterns between mammalian hosts and their gut bacteria. Our results suggest that termites have cospeciated with their gut bacteria since first appearing in the geological record.

Empirical evidence that complexity limits horizontal gene transfer.

Horizontal gene transfer (HGT) is a major contributor to bacterial genome evolution, generating phenotypic diversity, driving the expansion of protein families, and facilitating the evolution of new phenotypes, new metabolic pathways, and new species. Comparative studies of gene gain in bacteria suggest that the frequency with which individual genes successfully undergo HGT varies considerably and may be associated with the number of protein-protein interactions in which the gene participates, i.e., its connectivity. Two non-exclusive hypotheses have emerged to explain why transferability should decrease with connectivity: the Complexity Hypothesis (Jain, Rivera, & Lake, 1999) and the Balance Hypothesis (Papp, Pál, & Hurst, 2003). These hypotheses predict that the functional costs of HGT arise from a failure of divergent homologues to make normal protein-protein interactions or from gene mis-expression, respectively. Here we describe genome-wide assessments of these hypotheses in which we used 74 existing prokaryotic whole genome shotgun libraries to estimate rates of horizontal transfer of genes from taxonomically diverse prokaryotic donors into E. coli. We show that 1) transferability declines as connectivity increases, 2) transferability declines as the divergence between donor and recipient orthologs increases, and that 3) the magnitude of this negative effect of divergence on transferability increases with connectivity. These effects are particularly robust among the translational proteins, which span the widest range of connectivities. Whereas the Complexity Hypotheses explains all three of these observations, the Balance Hypothesis explains only the first one.

Host-specific plasmid evolution explains the variable spread of clinical antibiotic-resistance plasmids

Antibiotic resistance encoded on plasmids is a pressing global health problem. Predicting which plasmids spread in the long term remains very challenging, even though some key parameters influencing plasmid stability have been identified, such as plasmid growth costs and horizontal transfer rates. Here, we show these parameters evolve in a strain-specific way among clinical plasmids and bacteria, and this occurs rapidly enough to alter the relative likelihoods of different bacterium-plasmid combinations spreading. We used experiments with Escherichia coli and antibiotic-resistance plasmids isolated from patients, paired with a mathematical model, to track long-term plasmid stability (beyond antibiotic exposure). Explaining variable stability across six bacterium-plasmid combinations required accounting for evolutionary changes in plasmid stability traits, whereas initial variation of these parameters was a relatively poor predictor of long-term outcomes. Evolutionary trajectories were specific to particular bacterium-plasmid combinations, as evidenced by genome sequencing and genetic manipulation. This revealed epistatic (here, strain-dependent) effects of key genetic changes affecting horizontal plasmid transfer. Several genetic changes involved mobile elements and pathogenicity islands. Rapid strain-specific evolution can thus outweigh ancestral phenotypes as a predictor of plasmid stability. Accounting for strain-specific plasmid evolution in natural populations could improve our ability to anticipate and manage successful bacterium-plasmid combinations.

A mathematical model of plasmid-carried antibiotic resistance transmission in two types of cells

Abstract Antibiotic resistance is a significant public health problem. When resistance genes are being carried on plasmids, the spread can be greatly accelerated. In this paper, the transmission of antibiotic resistance in two types of cells is discussed. A mathematical model is established to describe the dynamics of the transmission of plasmids. The effects of different parameters on the stable solution and sensitivity analysis are studied by numerical simulation. The conclusions show that the concentration of antibiotics must reach a certain level to kill the pathogenic bacteria. If the concentration of antibiotics is not high to a certain extent, the treatment becomes ineffective. If the cost of cells carried on plasmids and the rate of resistance plasmids segregation too high, the drug-resistant cells will gradually die out in the system. The rate of horizontal transfer of resistance plasmids is directly related to the spread of drug resistance. With the increase in the horizontal transfer rate of resistance plasmids, cells in the body gradually turn into cells with antibiotic resistance, which causes substantial difficulties in the treatment of diseases.

Are ecological communities the seat of endosymbiont horizontal transfer and diversification? A case study with soil arthropod community.

Maternally inherited endosymbionts of arthropods are one of the most abundant and diverse group of bacteria. These bacterial endosymbionts also show extensive horizontal transfer to taxonomically unrelated hosts and widespread recombination in their genomes. Such horizontal transfers can be enhanced when different arthropod hosts come in contact like in an ecological community. Higher rates of horizontal transfer can also increase the probability of recombination between endosymbionts, as they now share the same host cytoplasm. However, reports of community‐wide endosymbiont data are rare as most studies choose few host taxa and specific ecological interactions among the hosts. To better understand endosymbiont spread within host populations, we investigated the incidence, diversity, extent of horizontal transfer, and recombination of three endosymbionts (Wolbachia, Cardinium, and Arsenophonus) in a specific soil arthropod community. Wolbachia strains were characterized with MLST genes whereas 16S rRNA gene was used for Cardinium and Arsenophonus. Among 3,509 individual host arthropods, belonging to 390 morphospecies, 12.05% were infected with Wolbachia, 2.82% with Cardinium and 2.05% with Arsenophonus. Phylogenetic incongruence between host and endosymbiont indicated extensive horizontal transfer of endosymbionts within this community. Three cases of recombination between Wolbachia supergroups and eight incidences of within‐supergroup recombination were also found. Statistical tests of similarity indicated supergroup A Wolbachia and Cardinium show a pattern consistent with extensive horizontal transfer within the community but not for supergroup B Wolbachia and Arsenophonus. We highlight the importance of extensive community‐wide studies for a better understanding of the spread of endosymbionts across global arthropod communities.

Evolutionary mechanisms that determine which bacterial genes are carried on plasmids.

The evolutionary pressures that determine the location (chromosomal or plasmid‐borne) of bacterial genes are not fully understood. We investigate these pressures through mathematical modeling in the context of antibiotic resistance, which is often found on plasmids. Our central finding is that gene location is under positive frequency‐dependent selection: the higher the frequency of one form of resistance compared to the other, the higher its relative fitness. This can keep moderately beneficial genes on plasmids, despite occasional plasmid loss. For these genes, positive frequency dependence leads to a priority effect: whichever form is acquired first—through either mutation or horizontal gene transfer—has time to increase in frequency and thus becomes difficult to displace. Higher rates of horizontal transfer of plasmid‐borne than chromosomal genes therefore predict moderately beneficial genes will be found on plasmids. Gene flow between plasmid and chromosome allows chromosomal forms to arise, but positive frequency‐dependent selection prevents these from establishing. Further modeling shows that this effect is particularly pronounced when genes are shared across a large number of species, suggesting that antibiotic resistance genes are often found on plasmids because they are moderately beneficial across many species. We also revisit previous theoretical work—relating to the role of local adaptation in explaining gene location and to plasmid persistence—in light of our findings.

Radium isotope ratios as a tool to characterise nutrient dynamics in a variably stratified temperate fjord

The effect of freshwater discharge on solute vertical mixing and horizontal transfer rates is evaluated in a variably stratified temperate fjord (Killary Harbour, Ireland) using the activity ratios of 224Ra and 223Ra in the water column. With high river discharge (>1.7 × 106 m3 d−1), surface 224Ra/223Ra activity ratios (ARs) decreased from 18.0 ± 7.1 to 8.6 ± 0.9 from the inner to the outer fjord and the system becomes stratified, resulting in clear differences of 224Ra/223Ra ARs between surface and bottom layers. When river discharge was low (<0.6 × 106 m3 d−1), 224Ra/223Ra ARs dipped from 19.6 ± 5.1 to 8.0 ± 1.8 in the inner system before increasing again towards the outlet to the sea up to 11.3 ± 3.6, and the water column was well mixed: the difference in 224Ra/223Ra ARs and salinity between surface and bottom layers was minimal. Longitudinal seaward decreases of surface water 224Ra/223Ra ARs within the inner fjord independently of river discharge suggested that the contribution of benthic-pelagic coupling to surface solute inventories is small in the inner section of the system. Conversely, the increase of 224Ra/223Ra ARs during low discharge conditions indicated that the slower flushing of the system, coupled with a well-mixed water column, facilitated solute transfers from deeper waters to the surface in the outer fjord. The effect of discharge on the residence time of water in the inner fjord was also determined using radium ages: longitudinal water flushing timescales are quickly reduced in the inner part of the bay with increasing discharge. We show that the degree of vertical mixing is an important driver of nutrient ratios in the system. Retention of soluble reactive phosphorus (SRP) and Ra in deep water and a higher molar N:P ratio in surface water were more likely under stratified conditions. Stratification may therefore amplify the effect of variable watershed inputs on the estuarine N:P ratio in Killary Harbour.

Comparative genomics reveals high rates of horizontal transfer and strong purifying selection on rhizobial symbiosis genes.

Horizontal transfer (HT) alters the repertoire of symbiosis genes in rhizobial genomes and may play an important role in the on-going evolution of the rhizobia-legume symbiosis. To gain insight into the extent of HT of symbiosis genes with different functional roles (nodulation, N-fixation, host benefit and rhizobial fitness), we conducted comparative genomic and selection analyses of the full-genome sequences from 27 rhizobial genomes. We find that symbiosis genes experience high rates of HT among rhizobial lineages but also bear signatures of purifying selection (low Ka : Ks). HT and purifying selection appear to be particularly strong in genes involved in initiating the symbiosis (e.g. nodulation) and in genome-wide association candidates for mediating benefits provided to the host. These patterns are consistent with rhizobia adapting to the host environment through the loss and gain of symbiosis genes, but not with host-imposed positive selection driving divergence of symbiosis genes through recurring bouts of positive selection.

Glyphosate escalates horizontal transfer of conjugative plasmid harboring antibiotic resistance genes

ABSTRACT Glyphosate has been frequently detected in water environments because of the wide use for controlling weed in farm lands and urban areas. Presently, the focus of the majority of studies is placed on the toxicity of glyphosate on humans and animals. However, the effects of glyphosate on horizontal transfer of conjugative plasmid carrying antibiotic resistance gene (ARG) are largely unknown. Here, we explored the ability and potential mechanism of glyphosate for accelerating horizontal transfer of conjugative plasmid-mediated ARG. The results showed that glyphosate can effectively boost horizontal transfer rate of conjugative plasmid carrying ARG. The possible mechanism analysis demonstrated that over-production of reactive oxygen species and reactive nitrogen species effectively regulated expression levels of bacterial outer membrane protein and conjugative transfer-related genes, thereby resulting into elevated horizontal transfer rate of plasmid-mediated ARG. In conclusion, this study casts new understanding into the biological effects of glyphosate on ARG.

Global survey of mobile DNA horizontal transfer in arthropods reveals Lepidoptera as a prime hotspot.

More than any other genome components, Transposable Elements (TEs) have the capacity to move across species barriers through Horizontal Transfer (HT), with substantial evolutionary consequences. Previous large-scale surveys, based on full-genomes comparisons, have revealed the transposition mode as an important predictor of HT rates variation across TE superfamilies. However, host biology could represent another major explanatory factor, one that needs to be investigated through extensive taxonomic sampling. Here we test this hypothesis using a field collection of 460 arthropod species from Tahiti and surrounding islands. Through targeted massive parallel sequencing, we uncover patterns of HT in three widely-distributed TE superfamilies with contrasted modes of transposition. In line with earlier findings, the DNA transposons under study (TC1-Mariner) were found to transfer horizontally at the highest frequency, closely followed by the LTR superfamily (Copia), in contrast with the non-LTR superfamily (Jockey), that mostly diversifies through vertical inheritance and persists longer within genomes. Strikingly, across all superfamilies, we observe a marked excess of HTs in Lepidoptera, an insect order that also commonly hosts baculoviruses, known for their ability to transport host TEs. These results turn the spotlight on baculoviruses as major potential vectors of TEs in arthropods, and further emphasize the importance of non-vertical TE inheritance in genome evolution.

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs) that are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here we show that AMP- and antibiotic-resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota with implications for disease risks.

Function-related replacement of bacterial siderophore pathways

Bacterial genomes are rife with orphan biosynthetic gene clusters (BGCs) associated with secondary metabolism of unrealized natural product molecules. Often up to a tenth of the genome is predicted to code for the biosynthesis of diverse metabolites with mostly unknown structures and functions. This phenomenal diversity of BGCs coupled with their high rates of horizontal transfer raise questions about whether they are really active and beneficial, whether they are neutral and confer no advantage, or whether they are carried in genomes because they are parasitic or addictive. We previously reported that Salinispora bacteria broadly use the desferrioxamine family of siderophores for iron acquisition. Herein we describe a new and unrelated group of peptidic siderophores called salinichelins from a restricted number of Salinispora strains in which the desferrioxamine biosynthesis genes have been lost. We have reconstructed the evolutionary history of these two different siderophore families and show that the acquisition and retention of the new salinichelin siderophores co-occurs with the loss of the more ancient desferrioxamine pathway. This identical event occurred at least three times independently during the evolution of the genus. We surmise that certain BGCs may be extraneous because of their functional redundancy and demonstrate that the relative evolutionary pace of natural pathway replacement shows high selective pressure against retention of functionally superfluous gene clusters.

Mathematical model for the transport of fluoroquinolone and its resistant bacteria in aquatic environment.

Development of antibiotic resistance in environmental bacteria is a direct threat to public health. Therefore, it becomes necessary to understand the fate and transport of antibiotic and its resistant bacteria. This paper presents a mathematical model for spatial and temporal transport of fluoroquinolone and its resistant bacteria in the aquatic environment of the river. The model includes state variables for organic matter, fluoroquinolone, heavy metals, and susceptible and resistant bacteria in the water column and sediment bed. Resistant gene is the factor which makes bacteria resistant to a particular antibiotic and is majorly carried on plasmids. Plasmid-mediated resistance genes are transferable between different bacterial species through conjugation (horizontal resistance transfer). This model includes plasmid dynamics between susceptible and resistant bacteria by considering the rate of horizontal resistance gene transfer among bacteria and the rate of losing resistance (segregation). The model describes processes which comprise of advection, dispersion, degradation, adsorption, diffusion, settling, resuspension, microbial growth, segregation, and transfer of resistance genes. The mathematical equations were solved by using numerical methods (implicit-explicit scheme) with appropriate boundary conditions. The development of the present model was motivated by the fact that the Musi River is heavily impacted by antibiotic pollution which led to the development of antibiotic resistance in its aquatic environment. The model was simulated for hypothetical pollution scenarios to predict the future conditions under various pollution management alternatives. The simulation results of the model for different cases show that the concentration of antibiotic, the concentration of organic matter, segregation rate, and horizontal transfer rate are the governing factors in the variation of population density of resistant bacteria. The treatment of effluents for antibiotics might be costly for the bulk drug manufacturing industries, but the guidelines can be made to reduce the organic matter which can limit the growth rate of microbes and reduce the total microbial population in the river. The reduction in antibiotic concentration can reduce the selection pressure on bacteria and can limit the population of resistant culture and its influence zone in the river stretch; however, complete removal of antibiotics may not result in complete elimination of antibiotic-resistant bacteria.

Why so rare if so essentiel: the determinants of the sparse distribution of CRISPR-Cas systems in bacterial genomes

CRISPR-Cas (Cluster of Regularly Interspaced Short Palindromic Repeats) systems confer bacteria and archaea an adaptative immunity against phages and other invading genetic elements playing an important role in bacterial evolution. However, despite the protection they generate and high rate of horizontal transfer, less than 50% of bacterial genomes harbor a CRISPR-Cas system. As a comparison, 90% of archaea encode a CRISPR-Cas system and a bacterial genome codes for two restriction modification systems on average. This review describes CRISPR-Cas systems distribution in bacterial genomes and then details the different hypotheses put forward to explain the relative scarcity of CRISPR-Cas systems. More specifically, phage escape mechanisms, ecological factors such as phage diversity and abundance and intrinsic costs, such as maintenance or autoimmunity, are discussed. Overall, a better understanding of the downsides of encoding CRISPR-Cas systems is essential to explain their evolutionary dynamics and their relative success in different environments and clades.

Inevitability of Genetic Parasites.

Almost all cellular life forms are hosts to diverse genetic parasites with various levels of autonomy including plasmids, transposons and viruses. Theoretical modeling of the evolution of primordial replicators indicates that parasites (cheaters) necessarily evolve in such systems and can be kept at bay primarily via compartmentalization. Given the (near) ubiquity, abundance and diversity of genetic parasites, the question becomes pertinent: are such parasites intrinsic to life? At least in prokaryotes, the persistence of parasites is linked to the rate of horizontal gene transfer (HGT). We mathematically derive the threshold value of the minimal transfer rate required for selfish element persistence, depending on the element duplication and loss rates as well as the cost to the host. Estimation of the characteristic gene duplication, loss and transfer rates for transposons, plasmids and virus-related elements in multiple groups of diverse bacteria and archaea indicates that most of these rates are compatible with the long term persistence of parasites. Notably, a small but non-zero rate of HGT is also required for the persistence of non-parasitic genes. We hypothesize that cells cannot tune their horizontal transfer rates to be below the threshold required for parasite persistence without experiencing highly detrimental side-effects. As a lower boundary to the minimum DNA transfer rate that a cell can withstand, we consider the process of genome degradation and mutational meltdown of populations through Muller’s ratchet. A numerical assessment of this hypothesis suggests that microbial populations cannot purge parasites while escaping Muller’s ratchet. Thus, genetic parasites appear to be virtually inevitable in cellular organisms.

Natural Selection for Operons Depends on Genome Size

In prokaryotes, genome size is associated with metabolic versatility, regulatory complexity, effective population size, and horizontal transfer rates. We therefore analyzed the covariation of genome size and operon conservation to assess the evolutionary models of operon formation and maintenance. In agreement with previous results, intraoperonic pairs of essential and of highly expressed genes are more conserved. Interestingly, intraoperonic pairs of genes are also more conserved when they encode proteins at similar cell concentrations, suggesting a role of cotranscription in diminishing the cost of waste and shortfall in gene expression. Larger genomes have fewer and smaller operons that are also less conserved. Importantly, lower conservation in larger genomes was observed for all classes of operons in terms of gene expression, essentiality, and balanced protein concentration. We reached very similar conclusions in independent analyses of three major bacterial clades (α- and β-Proteobacteria and Firmicutes). Operon conservation is inversely correlated to the abundance of transcription factors in the genome when controlled for genome size. This suggests a negative association between the complexity of genetic networks and operon conservation. These results show that genome size and/or its proxies are key determinants of the intensity of natural selection for operon organization. Our data fit better the evolutionary models based on the advantage of coregulation than those based on genetic linkage or stochastic gene expression. We suggest that larger genomes with highly complex genetic networks and many transcription factors endure weaker selection for operons than smaller genomes with fewer alternative tools for genetic regulation.

Fixation probability of mobile genetic elements such as plasmids

Mobile genetic elements such as plasmids are increasingly becoming thought of as evolutionarily important. Being horizontally transmissible is generally assumed to be beneficial for a gene. Using several simple modelling approaches we show that in fact being horizontally transferable is just as important for fixation as being beneficial to the host, in line with other results. We find fixation probability is approximately 2(s+β), where s is the increased (vertical) fitness provided by the gene, and β the rate of horizontal transfer when rare. This result comes about because when the gene is rare, almost all individuals in the population are possible recipients of horizontal transfer. The ability to horizontally transfer could thus cause a deleterious gene to become fixed in a population even without hitchhiking. Our findings provide further evidence for the importance and ubiquity of mobile genetic elements, particularly in microorganisms.

The interconnection between biofilm formation and horizontal gene transfer

Recent research has revealed that horizontal gene transfer and biofilm formation are connected processes. Although published research investigating this interconnectedness is still limited, we will review this subject in order to highlight the potential of these observations because of their believed importance in the understanding of the adaptation and subsequent evolution of social traits in bacteria. Here, we discuss current evidence for such interconnectedness centred on plasmids. Horizontal transfer rates are typically higher in biofilm communities compared with those in planktonic states. Biofilms, furthermore, promote plasmid stability and may enhance the host range of mobile genetic elements that are transferred horizontally. Plasmids, on the other hand, are very well suited to promote the evolution of social traits such as biofilm formation. This, essentially, transpires because plasmids are independent replicons that enhance their own success by promoting inter-bacterial interactions. They typically also carry genes that heighten their hosts' direct fitness. Furthermore, current research shows that the so-called mafia traits encoded on mobile genetic elements can enforce bacteria to maintain stable social interactions. It also indicates that horizontal gene transfer ultimately enhances the relatedness of bacteria carrying the mobile genetic elements of the same origin. The perspective of this review extends to an overall interconnectedness between horizontal gene transfer, mobile genetic elements and social evolution of bacteria.

Prevalence, transmission and mortality associated with Nosema fumiferanae infections in field populations of spruce budworm Choristoneura fumiferana

The prevalence, intensity and transmission of Nosema fumiferanae (Thomson) (Microsporidae) infections and potential impacts on the survival of field populations of spruce budworm Choristoneura fumiferana (Clem.) were examined in three plots in New Brunswick, Canada, from 1983 to 1992. The highest prevalence of N. fumiferanae infection in post‐hibernation second‐instar larvae occurred in the plot where prevalence in female pupae was the highest in the previous generation, suggesting higher rates of vertical transmission. There was little change in the prevalence of N. fumiferanae infections between the second and sixth instars in the later generations. In the two other plots, N. fumiferanae prevalence increased by approximately 25% from the second to sixth larval stadia. Coincident with the changes in N. fumiferanae prevalence were substantial declines in the populations of spruce budworms, making it difficult to determine rates of horizontal transfer of the disease. In all plots and in all years, there were progressive increases in the intensity of N. fumiferanae infections (spore loads/individual) from the second to sixth instars and pupae. Annual spruce budworm mortality associated with N. fumiferanae was ≤15% of all mortality in reared specimens and was positively correlated with but generally less than 30% of annual N. fumiferanae prevalence.