Despite the ecological importance of symbiotic relationships, few studies have explored how microbial communities vary across species complexes or within hybrid zones. We characterized the spatial and temporal variation in microbial communities in Culex pipiens complex and Culex restuans mosquitoes, the most important vectors for West Nile virus in the midwestern United States. Cx. pipiens complex and Cx. restuans mosquitoes were collected monthly from May to September 2023 from three geographic regions (Champaign County, IL, Cook County, IL and Dane County, WI). DNA from individual mosquitoes was sequenced using Illumina NovaSeq amplicon sequencing to identify the mosquito species and characterize their bacterial communities. Microbial composition differed between Cx. pipiens complex and Cx. restuans, driven by high relative abundance of Wolbachia in Cx. pipiens complex compared to Cx. restuans, which also contributed to greater within-species microbiome variability. Despite the limited sample sizes for some forms and hybrids, there were no detectable differences in the bacterial diversity or community composition among the five observed Cx. pipiens forms. Microbial diversity also varied regionally and over the sampling season, increasing later in the season. These findings demonstrate a microbial convergence within Cx. pipiens complex and reveal that spatiotemporal factors influence acquisition of environmentally acquired microbes highlighting the dynamic nature of mosquito-microbe interactions.

Invertebrates possess an innate immune system that acts non-specifically against pathogens and is regulated by the circadian clock. Using the host-parasite system, Daphnia magna and its bacterial parasite Pasteuria ramosa, we investigated day-night differences in susceptibility. In an infection experiment where hosts were exposed to spores either during the day or night, infection was slightly higher during daytime exposure and in animals treated with exogenous melatonin. Daphnia exhibited rhythmic expression of five immune genes, with low daytime expression and a pronounced, synchronized peak immediately after the transition from day to night. This timing aligns with the documented increase in daytime susceptibility, which may benefit Pasteuria as encounter rates rise when Daphnia forage in sediment during diel vertical migration. Melatonin exposure altered immune gene expression and increased susceptibility both day and night. Melatonin can act as an immune suppressor and may also influence parasite spore maturation. Disruption of circadian rhythms and melatonin signaling by anthropogenic stressors alters the infection dynamics in this freshwater keystone organism, with consequences for population stability, ecosystem functioning, and the conservation of freshwater biodiversity. Our results spotlight the mechanisms underlying infection risk in host-pathogen systems, highlighting the importance of circadian regulation for disease dynamics in freshwater ecosystems.

By changing the behavior of infected hosts, pathogens can shape the outcome of both disease transmission and other interspecific interactions within the food web. This change in behavior is most often recognized in trophically-transmitted parasites, where changing the behavior of an intermediate host can increase the rate at which the parasite is transmitted to the next host. However, behavioral changes also occur in terminal hosts. In a laboratory experiment, we investigated if infection by the ascomycete yeast Australozyma monospora (formerly Metschnikowia bicuspidata) and/or the time since infection changes the swimming behavior over time of its freshwater cladoceran host, Daphnia dentifera. Infection influenced the net distance and total distance traveled as well as the swimming speed and mean depth of the Daphnia. Uninfected individuals traveled further, both in terms of net distance and total distance, and they also swam faster. The influence of time since infection was more nuanced. Swimming speed, net distance, and total distance traveled varied with time, but mean depth did not. Though we found no main effect of time since infection on mean swimming depth, time did interact with infection status to influence mean depth. A better understanding of how pathogens may alter the movement and habitat selection of infected hosts can help to inform predictive models of disease spread in lakes that are ever-changing due to increased anthropogenic stressors.

We explored variation in infection outcomes and processes explaining that variation in multiple taxa of Daphnia exposed to the pathogen, Australozyma monospora (formerly Metschnikowia bicuspidata). Three laboratory assays were conducted to explore (1) variation in likelihood of infection and pathogen load, (2) host encounter and defense traits, and the influence of host maturity on these traits, and (3) the fitness costs of preventing infection. We found considerable among-taxon variation in susceptibility as well as the pathogen’s ability to reproduce following successful infection. Notably, in both the most susceptible (D. dentifera) and least susceptible (D. pulicaria) host species, we discovered that host barriers and immune responses, but not encounter rates, play an important role in shaping infection outcomes. Although differences in body size could underlie variation in barriers and immune responses between the two taxa, they could not explain infection differences across the broader set of host taxa. Finally, we found that the least susceptible taxon did not exhibit a cost of resistance, whereas the most susceptible taxon-suffered fitness declines when preventing infection. These results underscore the complexity of taxon-level differences in host–pathogen interactions and provide a framework for understanding how variation among and within taxa can shape disease outbreaks.

Anthropogenic changes can drive rapid evolution in wild populations, but the role of phenotypic plasticity in such scenarios remains unclear. This uncertainty can affect applications like the design of resistance management approaches. In the case of insecticide resistance in mosquitoes, however, little is known regarding how environmental conditions, genetic variation, and their interactions jointly shape resistance phenotypes. To address this, we employed a full-sibling design to investigate the effects of larval food availability on adult broad-sense heritability and phenotypic plasticity in resistance to permethrin. Two experiments measured resistance levels in West Nile virus vectors (laboratory colony of Culex pipiens and two field populations of Culex restuans) using CDC bottle bioassays, and the time until death was tracked. Wing lengths were measured to assess if there is a relationship between body size and permethrin resistance. Based on likelihood ratio tests, the broad-sense heritability values for resistance were significant. There was substantial variance and phenotypic plasticity in both Cx. restuans field populations, while the laboratory colony of Cx. pipiens exhibited less variation. Larval food availability significantly affected resistance, but the sign of the effect varied across populations from different geographic regions, highlighting the importance of genotype by environmental interactions in this system. Our results offer valuable insights into the potential for insecticide resistance to evolve in mosquito populations and have important implications for how resistance in vectors can be assessed. We suggest changes to improve the current methodology for insecticide resistance testing and recommend that population-specific data should inform vector control schemes.

Non-consumptive effects of predators upon their prey can alter disease dynamics by shifting prey behavior and physiology. In the Chaoborus-Daphnia-Australozyma (formerly Metschnikowia) predator-prey-host-parasite system, Chaoborus cues increase the prevalence of Australozyma infection in Daphnia, but the mechanism of this effect is unknown. This study investigated two potential mechanisms for the non-consumptive effect on infection. It has been hypothesized, but not directly tested, that Daphnia encounter more Australozyma spores on exposure to Chaoborus cues as a result of increased feeding rate. We also hypothesized that the production of morphological anti-predator defenses imposes a resource allocation trade-off with immune function. We tested both hypotheses by rearing Daphnia dentifera factorially in the presence/absence of Chaoborus kairomone and the presence/absence of Australozyma spores, measuring infection, morphology, reproduction and feeding rate. Our results did not support either hypothesis. Feeding rate did not increase in response to kairomone exposure, nor was it predictive of infection. Exposure to Australozyma did not significantly decrease anti-predator morphology, nor was morphology predictive of infection. While the mechanism of the effect remains unknown, we have tested and provided evidence against two likely hypotheses as to its functioning.

In both human and wildlife disease systems, temporal shifts in host immunity may shape the timing and severity of epidemics. Yet, immune responses, as well as seasonal patterns in their expression, are difficult to measure. Rather, field studies collect phenomenological data on infection outcomes. Pairing epidemic data of multiple outbreaks with models that directly parameterize immune metrics can be a powerful approach for exploring the role of time-varying immunity on disease. Field data can be used to determine how well a parameterized model can reproduce trends and differences observed among outbreaks. Previous work in the Daphnia dentifera-Metschnikowia bicuspidata focal host-fungal pathogen disease system has not taken full advantage of coupling patterns in nature with mechanisms predicted by theory. Here, we study a mathematical model accounting for host immunity in the form of resistance to and recovery from M. bicuspidata infections and temporal variation in key aspects of the system’s epidemiology and ecology. Specifically, host population birth, predation and transmission rates, the fraction of recovering hosts, as well as the fungal spore yield were allowed to vary within the epidemic season. Modifying the system’s carrying capacity produces good correspondence between observed and model-estimated densities. Adjusting the transmission rate, spore yield, and the fraction of recovering hosts, captures the timing of disease outbreaks, as well as other qualitative features of outbreaks, such as the disparity between the prevalence of early- and late-stage infections. Our findings suggest that host immunological parameters are an important within-host constraint on disease dynamics.

A vast literature explores a model system that consists of a prey crustacean, the water flea Daphnia spp., and an obligately pathogenic yeast that has been referred to as Metschnikowia bicuspidata and thought to represent the material used by Metschnikoff in his study of innate immunity. Typification of species bearing that name and indeed the whole genus has been problematic as regards yeasts that only grow or form aciculate ascospores in hospite. The neotype of M. bicuspidata, unlike the Daphnia parasite, is easily cultured on a variety of laboratory media, although it too can cause serious infections in a variety of mostly aquatic animals. It has become evident that the Daphnia parasite studied by Metschnikoff or current workers is not closely related to M. bicuspidata as currently understood. Analysis of whole genome DNA extracted from the yeast repeatedly found in infected Daphnia specimens shows that it belongs to the recently circumscribed genus Australozyma. The yeast is described here as Australozyma monospora sp. nov. The species, although haplontic and heterothallic, forms single-spored asci without mating. It also appears that all species in the genus are restricted to asexual reproduction, which may explain their rare status. The holotype is MICH 346683. The name is registered in Mycobank under the number MB 859667.

The levels of the hormone melatonin fluctuate daily, with higher concentrations often found at night. These fluctuations likely influence multiple aspects of physiology, including the immune response. We demonstrated that the addition of exogenous melatonin increased the proportion of the freshwater zooplankton Daphnia dentifera that became infected by the fungal pathogen Metschnikowia bicuspidata , during the day but not at night. To determine the stage of this host–pathogen interaction at which melatonin may increase susceptibility, we conducted a series of laboratory experiments in which we raised Daphnia in the presence and absence of exogenous melatonin. To complete its life cycle, Metschnikowia must encounter a foraging host, overcome the host's barrier resistance (gut wall), and evade the host's immune response (internal clearance). We quantified encounter rate by measuring the gut passage time and the number of spores that entered the gut. We also measured the number of spores that successfully entered the body cavity (barrier resistance) and the hemocyte response to spores entering the body cavity (one metric of internal clearance). Finally, we quantified the effect of exogenous melatonin on triggering molting. The addition of exogenous melatonin lengthened gut passage time and decreased the number of spores present in the gut. We found no effect of melatonin on the percentage of gut spores successfully entering the host's body cavity, nor on the hemocyte response. Melatonin is known to influence the timing of molting and hosts that molted during exposure were more likely to become infected, likely due to a decrease in barrier resistance. In a fully factorial experiment, there was a high death rate, low infection rate, and therefore no discernible effect of melatonin on molting, nor molting or melatonin on infection. Our results provide insight into the stages of infection where melatonin does and does not have significant effects.

Immune responses can be energetically expensive and subject to trade-offs. Prior work on the freshwater zooplankton, Ceriodaphnia cornuta, demonstrated an association between eye size and infection, leading to questions about whether investment in eyes trades off against investment in immunity. We used the crustacean host, Daphnia dentifera, and its fungal parasite, Metschnikowia bicuspidata, to investigate the relationships between eye size, parasite resistance and infection. In the field, we found a negative correlation between size-corrected eye area (SCEA) and Metschnikowia infection, suggesting that either SCEA decreases infection (thereby indicating resistance) or that infection decreases SCEA. Controlled laboratory experiments reinforced the latter result: exposure to the fungal parasite decreased a host's SCEA, regardless of the parasite dose or host genotype. We also uncovered significant plasticity in this trait-both host age and resource level increased SCEA. Identifying causality in physiological correlations is challenging. Our results suggest that negative associations between parasitism and energetically-expensive traits can arise through plasticity.

The assembly of host-associated microbial communities is influenced by multiple factors, but the effect of microbiomes on host phenotypes is often not well understood. To address questions of food-web effects on host microbiome assembly, we manipulated the resource environment (grass only [G] vs. grass + nutrients [GN]), competition type (intra- vs. inter-specific) and density (high vs. low) for Culex restuans mosquito larvae. We predicted the microbial communities in fourth-instar larvae would differ between these environmental treatments and that these treatments would translate into differences in the adult phenotype. Resource environment and density influenced the larval microbiome. In addition, the larval microbiome exhibited notable differences compared to the free-living microbial communities. Resource-driven differences in the larval samples can be attributed to Arcobacteraceae being more abundant in larvae reared in the GN treatments relative to those reared in the G treatments and Comamonadaceae being more abundant in the G treatment. Although significant, the difference in community structure between density treatments was difficult to discern. This appears to be driven by Weeksellaceae only being abundant in the high-density, interspecific, GN treatment. Rearing larvae to adulthood under severe food limitation resulted in low survival (<25%) in both resource environments. Approximately 60% of survivors to adulthood were male. Larvae reared in the intraspecific, G treatment had the shortest development time to adulthood and emerged as the smallest adults. These results demonstrate how environmental variation can significantly alter the alpha and beta diversity of free-living microbes, which in turn can significantly affect host phenotype and critical life history traits, such as development time, size at adulthood, and survival. These findings highlight the importance of considering environmental influences on microbiome diversity to understand and predict host outcomes, offering valuable insights for diverse applications in fields such as ecology, public health, and agriculture.

Dormant propagules can provide a rapid colonization source for temporary aquatic habitats and set the trajectory for community dynamics, yet the egg banks of stormwater management systems have received little attention. We asked which species hatched from the sediment of drainage ditches in Champaign County, IL, and found bdelloid rotifers and ostracods (Heterocypris incongruens) to be the most common taxa. These sites also are colonized by mosquitoes, and we established laboratory experiments to examine interspecific interactions between common co-occurring taxa. Culex restuans larvae were reared in the presence or absence of H. incongruens at two intra- and interspecific densities (20 or 40 total individuals) and their survivorship to adulthood, development time to adulthood, adult body size, and sex ratio were determined. Survival for Cx. restuans was significantly lower at high larval density than at low larval density in both treatments. Culex restuans larvae reared in the presence of H. incongruens had a shorter development time to adulthood and emerged as larger adults compared to those reared in the absence of H. incongruens. The sex ratios in the H. incongruens treatments were female-biased whereas those in the Culex-only treatments were male-biased. These differences may have epidemiological implications, as only female mosquitoes serve as disease vectors. Our results emphasize the importance of understanding interspecific interactions in influencing larval mosquito development traits.

The healthy herds hypothesis proposes that predators can reduce parasite prevalence and thereby increase the density of their prey. However, evidence for such predator-driven reductions in the prevalence of prey remains mixed. Furthermore, even less evidence supports increases in prey density during epidemics. Here, we used a planktonic predator-prey-parasite system to experimentally test the healthy herds hypothesis. We manipulated density of a predator (the phantom midge, Chaoborus punctipennis) and parasitism (the virulent fungus Metschnikowia bicuspidata) in experimental assemblages. Because we know natural populations of the prey (Daphnia dentifera) vary in susceptibility to both predator and parasite, we stocked experimental populations with nine genotypes spanning a broad range of susceptibility to both enemies. Predation significantly reduced infection prevalence, eliminating infection at the highest predation level. However, lower parasitism did not increase densities of prey; instead, prey density decreased substantially at the highest predation levels (a major density cost of healthy herds predation). This density result was predicted by a model parameterized for this system. The model specifies three conditions for predation to increase prey density during epidemics: (i) predators selectively feed on infected prey, (ii) consumed infected prey release fewer infectious propagules than unconsumed prey, and (iii) sufficiently low infection prevalence. While the system satisfied the first two conditions, prevalence remained too high to see an increase in prey density with predation. Low prey densities caused by high predation drove increases in algal resources of the prey, fueling greater reproduction, indicating that consumer-resource interactions can complicate predator-prey-parasite dynamics. Overall, in our experiment, predation reduced the prevalence of a virulent parasite but, at the highest levels, also reduced prey density. Hence, while healthy herds predation is possible under some conditions, our empirical results make it clear that the manipulation of predators to reduce parasite prevalence may harm prey density.

While vertebrate immune systems are appreciated for their complexity and adaptability, invertebrate immunity is often considered to be less complex. However, immune responses in many invertebrates likely involve sophisticated processes. Interactions between the crustacean host Daphnia dentifera and its fungal pathogen Metschnikowia bicuspidata provide an excellent model for exploring the mechanisms underlying crustacean immunity. To explore the genomic basis of immunity in Daphnia, we used RNA-sequencing technology to quantify differential gene expression between individuals of a single host genotype exposed or unexposed to M. bicuspidata over 24 h. Transcriptomic analyses showed that the number of differentially expressed genes between the control (unexposed) and experimental (exposed) groups increased over time. Gene ontology enrichment analysis revealed that differentially expressed genes were enriched for immune-related molecules and processes, such as cuticle development, prostaglandin, and defense response processes. Our findings provide a suite of immunologically relevant genes and suggest the presence of a rapidly upregulated immune response involving the cuticle in Daphnia. Studies involving gene expression responses to pathogen exposure shine a light on the processes occurring during the course of infection. By leveraging knowledge on the genetic basis for immunity, immune mechanisms can be more thoroughly understood to refine our understanding of disease spread within invertebrate populations.

Theory often predicts that host populations should evolve greater resistance when parasites become abundant. Furthermore, that evolutionary response could ameliorate declines in host populations during epidemics. Here, we argue for an update: when all host genotypes become sufficiently infected, higher parasite abundance can select for lower resistance because its cost exceeds its benefit. We illustrate such a “resistance is futile” outcome with mathematical and empirical approaches. First, we analyzed an eco-evolutionary model of parasites, hosts, and hosts’ resources. We determined eco-evolutionary outcomes for prevalence, host density, and resistance (mathematically, “transmission rate”) along ecological and trait gradients that alter parasite abundance. With high enough parasite abundance, hosts evolve lower resistance, amplifying infection prevalence and decreasing host density. In support of these results, a higher supply of nutrients drove larger epidemics of survival-reducing fungal parasites in a mesocosm experiment. In two-genotype treatments, zooplankton hosts evolved less resistance under high-nutrient conditions than under low-nutrient conditions. Less resistance, in turn, was associated with higher infection prevalence and lower host density. Finally, in an analysis of naturally occurring epidemics, we found a broad, bimodal distribution of epidemic sizes consistent with the resistance is futile prediction of the eco-evolutionary model. Together, the model and experiment, supplemented by the field pattern, support predictions that drivers of high parasite abundance can lead to the evolution of lower resistance. Hence, under certain conditions, the most fit strategy for individual hosts exacerbates prevalence and depresses host populations.

Despite all that is known about Daphnia and their interactions with algal resources, questions remain as to how a changing resource environment influences a host's susceptibility to parasites. Theory and empiricism have demonstrated that increasing resource quantity can positively, negatively, and even non-linearly correlate with susceptibility. The nature of this correlation depends on the complex dynamics between the host's immune traits (which are assumed to be costly) and a parasite's ability to evade that immune system and "steal" resources from the host. We used three separate assays to examine how resources influence host immune responses and infection outcomes in eight genotypes of Daphnia dentifera. We challenged Daphnia with the fungal parasite Metschnikowia bicuspidata at three concentrations of the green algae Ankistrodesmus falcatus. In the first assay, we investigated how this resource gradient influences the number of fungal spores consumed (a measure of encounter with the parasite), host gut penetrability (a measure of resistance to the parasite), and the haemocyte response (a measure of clearance of the parasite). In the second assay, we explored how these traits combined to determine overall sus-ceptibility to infection. Finally, our third assay investigated the potential for tolerance in this system by comparing reproduction among hosts that managed to avoid, resist, or clear infection to those that developed late-stage infec-tions. We found that host immune responses changed non-uniformly with resources: the number of fungal spores consumed decreased with increasing resources, gut penetrability showed no relationship with resources (but was strongly driven by host genotype), and haemocyte counts peaked at intermediate resource levels. Ultimately, over-all susceptibility demonstrated a strong genotype by environment interaction, with some genotypes showing the highest proportion infected in high resource environments, others in low resource environments, and one genotype had the highest proportion infected at the intermediate resource level. In all resource environments, individuals that avoided, resisted, or cleared infection had higher reproduction than those that developed late-stage infections, suggesting that Daphnia hosts use resistance rather than tolerance with this parasite. Our results demonstrate the importance of integrating resource supply with immunological mechanisms and examining those effects across a range of genotypes that differ in their responses to the environment.

Host density shapes infection risk through two opposing phenomena. First, when infective stages are subdivided among multiple hosts, greater host densities decrease infection risk through ‘safety in numbers’. Hosts, however, represent resources for parasites, and greater host availability also fuels parasite reproduction. Hence, host density increases infection risk through ‘density-dependent transmission’. Theory proposes that these phenomena are not disparate outcomes but occur over different timescales. That is, higher host densities may reduce short-term infection risk, but because they support parasite reproduction, may increase long-term risk. We tested this theory in a zooplankton-disease system with laboratory experiments and field observations. Supporting theory, we found that negative density–risk relationships (safety in numbers) sometimes emerged over short timescales, but these relationships reversed to ‘density-dependent transmission’ within two generations. By allowing parasite numerical responses to play out, time can shift the consequences of host density, from reduced immediate risk to amplified future risk.

Host density shapes infection risk through two opposing phenomena. First, when infective stages are subdivided among multiple hosts, greater host densities decrease infection risk through 'safety in numbers'. Hosts, however, represent resources for parasites, and greater host availability also fuels parasite reproduction. Hence, host density increases infection risk through 'density-dependent transmission'. Theory proposes that these phenomena are not disparate outcomes but occur over different timescales. That is, higher host densities may reduce short-term infection risk, but because they support parasite reproduction, may increase long-term risk. We tested this theory in a zooplankton-disease system with laboratory experiments and field observations. Supporting theory, we found that negative density-risk relationships (safety in numbers) sometimes emerged over short timescales, but these relationships reversed to 'density-dependent transmission' within two generations. By allowing parasite numerical responses to play out, time can shift the consequences of host density, from reduced immediate risk to amplified future risk.

Host density shapes infection risk through two opposing phenomena. First, when infective stages are subdivided among multiple hosts, greater host densities decrease infection risk through ‘safety in numbers’. Hosts, however, represent resources for parasites, and greater host availability also fuels parasite reproduction. Hence, host density increases infection risk through ‘density-dependent transmission’. Theory proposes that these phenomena are not disparate outcomes but occur over different timescales. That is, higher host densities may reduce short-term infection risk, but because they support parasite reproduction, may increase long-term risk. We tested this theory in a zooplankton-disease system with laboratory experiments and field observations. Supporting theory, we found that negative density–risk relationships (safety in numbers) sometimes emerged over short timescales, but these relationships reversed to ‘density-dependent transmission’ within two generations. By allowing parasite numerical responses to play out, time can shift the consequences of host density, from reduced immediate risk to amplified future risk.

Differential predation on species with intraspecific colour variation has been explored in various systems and is often implicated as the driving force behind colour polymorphism maintenance. Here, investigation done on whether predation contributes to the maintenance of extensive colour variation in the Neotropical tortoise beetle, Chelymorpha alternans (Chrysomelidae). Recorded predation rates on different colour pattern phenotypes by three common, generalist invertebrate predators, and identified potential chemical signals of unpalatability. Predaceious mantids (Orthoptera, Mantidae) consumed no beetles, regardless of phenotype, whereas the giant orb‐weaving spider ( Trichonephila clavipes ; Araneidae) consumed all three beetle phenotypes. The carton‐nest ant, Azteca chartifex (Formicidae), displayed differential predation; the rufipennis phenotype of C. alternans was sometimes consumed, the metallic phenotype was never consumed, and the veraguensis phenotype was consumed in the first three encounters and subsequently discarded, suggesting a learned avoidance behaviour. Using gas chromatography–mass spectrometry, it was determined that cuticular hydrocarbon profiles were similar between the metallic and militaris‐a phenotypes. The rufipennis phenotype showed pronounced differences and displayed the greatest among‐individual variation in elytral cuticular profiles. Between‐phenotype variation in chemical cues, and differences in how predators receive those cues, may mediate predator response, and play a role in maintaining colour variation in this species.