Molecular phylogeny supports invalidation of Didelphodiplostomum and Pharyngostomoides (Digenea: Diplostomidae) and reveals a Tylodelphys from mammals.

Alaria, Didelphodiplostomum and Pharyngostomoides are among genera of diplostomid digeneans known to parasitize mammalian definitive hosts. Despite numerous recent molecular phylogenetic studies of diplostomids, limited DNA sequence data is available from diplostomids parasitic in mammals. Herein, we provide the first 28S rDNA and cox1 mtDNA sequences from morphologically identified, adult specimens of Didelphodiplostomum and Pharyngostomoides. Newly generated 28S sequences were used to infer the phylogenetic interrelationships of these two genera among other major lineages of diplostomoideans. The phylogeny based on 28S and a review of morphology clearly suggests that Pharyngostomoides should be considered a junior synonym of Alaria, while Didelphodiplostomum should be considered a junior synonym of Tylodelphys. Pharyngostomoides procyonis (type species), Pharyngostomoides adenocephala and Pharyngostomoides dasyuri were transferred into Alaria as Alaria procyonis comb. nov., Alaria adenocephala comb. nov. and Alaria dasyuri comb. nov.; Didelphodiplostomum variabile (type species) and Didelphodiplostomum nunezae were transferred into Tylodelphys as Tylodelphys variabilis comb. nov. and Tylodelphys nunezae comb. nov. In addition, Alaria ovalis comb. nov. (formerly included in Pharyngostomoides) was restored and transferred into Alaria based on a morphological study of well-fixed, adult specimens and the comparison of cox1 DNA sequences among Alaria spp. The diplostomid genus Parallelorchis was restored based on review of morphology.

PINWORMS ARE ASSOCIATED WITH TAXONOMIC BUT NOT FUNCTIONAL DIFFERENCES IN THE GUT MICROBIOME OF WHITE-THROATED WOODRATS (NEOTOMA ALBIGULA).

Vertebrates rely on their gut microbiome for digestion, and changes to gut microbial communities can impact host health. Past work, primarily in model organisms, has revealed that endoparasites disrupt the gut microbiome. Here, using wild-caught white-throated woodrats (Neotoma albigula), we tested whether naturally acquired parasite infections are associated with different microbiome structure and function. We surveyed wild N. albigula in eastern Utah for gastrointestinal parasites in the spring and fall of 2019, using traditional fecal float methods and testing a PCR-based approach to detect infection. We tested whether the host gut microbiome structure and function differed based on infection with the most prevalent parasite, the pinworm Lamotheoxyuris ackerti. In spring, infected and uninfected animals had significantly different microbiomes, but these differences were not detected in the fall. However, for both sampling periods, infection was associated with differences in particular microbial taxa determined by differential abundance analysis. As N. albigula rely on their microbiomes to digest both fiber and the plant defensive compound oxalate, we compared microbiome function by measuring dry matter digestibility and oxalate intake in infected and uninfected animals. Although we expected infected animals to have reduced fiber degradation and oxalate intake, we found no difference in microbiome function using these assays. This work suggests that parasite effects on the microbiome may be difficult to detect in complex natural systems, and more studies in wild organisms are warranted.

Metabolic Enabling and Detoxification by Mammalian Gut Microbes.

The longstanding interactions between mammals and their symbionts enable thousands of mammal species to consume herbivorous diets. The microbial communities in mammals degrade both plant fiber and toxins. Microbial toxin degradation has been repeatedly documented in domestic ruminants, but similar work in wild mammals is more limited due to constraints on sampling and manipulating the microbial communities in these species. In this review, we briefly describe the toxins commonly encountered in mammalian diets, major classes of biotransformation enzymes in microbes and mammals, and the gut chambers that house symbiotic microbes. We next examine evidence for microbial detoxification in domestic ruminants before providing case studies on microbial toxin degradation in both foregut- and hindgut-fermenting wild mammals. We end by discussing species that may be promising for future investigations, and the advantages and limitations of approaches currently available for studying degradation of toxins by mammalian gut microbes.

Successes and limitations of quantitative diet metabarcoding in a small, herbivorous mammal.

DNA metabarcoding is widely used to determine wild animal diets, but whether this technique provides accurate, quantitative measurements is still under debate. To test our ability to accurately estimate the abundance of dietary items using metabarcoding, we fed wild-caught desert woodrats (Neotoma lepida) diets consisting of constant amounts of juniper (Juniperus osteosperma, 15%) and varying amounts of creosote (Larrea tridentata, 1%-60%), cactus (Opuntia sp., 0%-100%) and commercial chow (0%-85%). Using metabarcoding, we compared the representation of items in the original diet samples to that in the faecal samples to test the sensitivity and accuracy of diet metabarcoding, the performance of different bioinformatic pipelines and our ability to correct sequence counts. Metabarcoding, using standard trnL primers, detected creosote, juniper and chow. Different pipelines for assigning taxonomy performed similarly. While creosote was detectable at dietary proportions as low as 1%, we failed to detect cactus in most samples, probably due to a primer mismatch. Creosote read counts increased as its proportion in the diet increased, and we could differentiate when creosote was a minor and major component of the diet. However, we found that estimates of juniper and creosote varied. Using previously suggested methods to correct these errors did not improve accuracy estimates of creosote, but did reduce error for juniper and chow. Our results indicate that metabarcoding can provide quantitative information on dietary composition, but may be limited. We suggest that researchers use caution when quantitatively interpreting diet metabarcoding results unless they first experimentally determine the extent of possible biases.

Molecular phylogenetic analysis of Neodiplostomum and Fibricola (Digenea, Diplostomidae) does not support host-based systematics.

Fibricola and Neodiplostomum are diplostomid genera with very similar morphology that are currently separated based on their definitive hosts. Fibricola spp. are normally found in mammals, while Neodiplostomum spp. typically parasitize birds. Previously, no DNA sequence data was available for any member of Fibricola. We generated nuclear ribosomal and mtDNA sequences of Fibricola cratera (type-species), Fibricola lucidum and 6 species of Neodiplostomum. DNA sequences were used to examine phylogenetic interrelationships among Fibricola and Neodiplostomum and re-evaluate their systematics. Molecular phylogenies and morphological study suggest that Fibricola should be considered a junior synonym of Neodiplostomum. Therefore, we synonymize the two genera and transfer all members of Fibricola into Neodiplostomum. Specimens morphologically identified as Neodiplostomum cratera belonged to 3 distinct phylogenetic clades based on mitochondrial data. One of those clades also included sequences of specimens identified morphologically as Neodiplostomum lucidum. Further study is necessary to resolve the situation regarding the morphology of N. cratera. Our results demonstrated that some DNA sequences of N. americanum available in GenBank originate from misidentified Neodiplostomum banghami. Molecular phylogentic data revealed at least 2 independent host-switching events between avian and mammalian hosts in the evolutionary history of Neodiplostomum; however, the directionality of these host-switching events remains unclear.

Wild herbivorous mammals (genus Neotoma) host a diverse but transient assemblage of fungi.

Fungi are often overlooked in microbiome research and, as a result, little is known about the mammalian mycobiome. Although frequently detected in vertebrate guts and known to contribute to digestion in some herbivores, whether these eukaryotes are a persistent part of the mammalian gut microbiome remains contentious. To address this question, we sampled fungi from wild woodrats (Neotoma spp.) collected from 25 populations across the southwestern United States. For each animal, we collected a fecal sample in the wild, and then re-sampled the same individual after a month in captivity on a controlled diet. We characterized and quantified fungi using three techniques: ITS metabarcoding, shotgun metagenomics and qPCR. Wild individuals contained diverse fungal assemblages dominated by plant pathogens, widespread molds, and coprophilous taxa primarily in Ascomycota and Mucoromycota. Fungal abundance, diversity and composition differed between individuals, and was primarily influenced by animal geographic origin. Fungal abundance and diversity significantly declined in captivity, indicating that most fungi in wild hosts came from diet and environmental exposure. While this suggests that these mammals lack a persistent gut mycobiome, natural fungal exposure may still impact fungal dispersal and animal health.

Toxin tolerance across landscapes: Ecological exposure not a prerequisite.

Little is known about the tolerances of mammalian herbivores to plant specialized metabolites across landscapes.We investigated the tolerances of two species of herbivorous woodrats, Neotoma lepida (desert woodrat) and Neotoma bryanti (Bryant's woodrat) to creosote bush (Larrea tridentata), a widely distributed shrub with a highly toxic resin. Woodrats were sampled from 13 locations both with and without creosote bush across a 900 km transect in the US southwest. We tested whether these woodrat populations consume creosote bush using plant metabarcoding of feces and quantified their tolerance to creosote bush through feeding trials using chow amended with creosote resin.Toxin tolerance was analyzed in the context of population structure across collection sites with microsatellite analyses. Genetic differentiation among woodrats collected from different locations was minimal within either species. Tolerance differed substantially between the two species, with N. lepida persisting 20% longer than N. bryanti in feeding trials with creosote resin. Furthermore, in both species, tolerance to creosote resin was similar among woodrats near or within creosote bush habitat. In both species, woodrats collected greater than 25 km from creosote had markedly lower tolerances to creosote resin compared to animals from within the range of creosote bush.The results imply that mammalian herbivores are adapted to the specialized metabolites of plants in their diet, and that this tolerance can extend several kilometers outside of the range of dietary items. That is, direct ecological exposure to the specialized chemistry of particular plant species is not a prerequisite for tolerance to these compounds. These findings lay the groundwork for additional studies to investigate the genetic mechanisms underlying toxin tolerance and to identify how these mechanisms are maintained across landscape-level scales in mammalian herbivores.

Microbiome stability and structure is governed by host phylogeny over diet and geography in woodrats (Neotoma spp.).

The microbiome is critical for host survival and fitness, but gaps remain in our understanding of how this symbiotic community is structured. Despite evidence that related hosts often harbor similar bacterial communities, it is unclear whether this pattern is due to genetic similarities between hosts or to common ecological selection pressures. Here, using herbivorous rodents in the genus Neotoma, we quantify how geography, diet, and host genetics, alongside neutral processes, influence microbiome structure and stability under natural and captive conditions. Using bacterial and plant metabarcoding, we first characterized dietary and microbiome compositions for animals from 25 populations, representing seven species from 19 sites across the southwestern United States. We then brought wild animals into captivity, reducing the influence of environmental variation. In nature, geography, diet, and phylogeny collectively explained ∼50% of observed microbiome variation. Diet and microbiome diversity were correlated, with different toxin-enriched diets selecting for distinct microbial symbionts. Although diet and geography influenced natural microbiome structure, the effects of host phylogeny were stronger for both wild and captive animals. In captivity, gut microbiomes were altered; however, responses were species specific, indicating again that host genetic background is the most significant predictor of microbiome composition and stability. In captivity, diet effects declined and the effects of host genetic similarity increased. By bridging a critical divide between studies in wild and captive animals, this work underscores the extent to which genetics shape microbiome structure and stability in closely related hosts.

The ecological consequences of a pandemic.

The COVID-19 pandemic has altered human behaviour in profound ways, prompting some to question whether the associated economic and social impacts might outweigh disease impacts. This fits into a burgeoning ecological paradigm suggesting that for both predator-prey and parasite-host interactions, non-consumptive effects (avoidance) can be orders of magnitude stronger than consumptive effects (sickness and death). Just as avoidance of predators and parasites imposes substantial costs on prey and hosts, altered behaviour to reduce the transmission of COVID-19 has impacted human fitness and wellbeing. But the effects of infectious disease avoidance do not stop there; non-consumptive effects of predators and parasites often trigger cascading indirect effects in natural systems. Similarly, shifts in human behaviour due to COVID-19 have triggered myriad indirect effects on species and the environment, which can be positive, negative or neutral. We urge researchers to recognize that the environmental impacts associated with lockdowns are indirect effects of the virus. In short, the global response to COVID-19 suggests that the non-consumptive effects of a pathogen, and resulting indirect effects, can be profound.

Circulation of Tick-Borne Spirochetes in Tick and Small Mammal Communities in Santa Barbara County, California, USA.

A diversity of Borrelia burgdorferi sensu lato (Johnson, Schmid, Hyde, Steigerwalt & Brenner) (Spirochaetales: Spirochaetaceae) genomospecies, including the Lyme disease agent, Borrelia burgdorferi sensu stricto (s.s.), have been identified in the western United States. However, enzootic transmission of B. burgdorferi s.l. in small mammals and ticks is poorly characterized throughout much of the region. Here we report prevalence of B. burgdorferi s.l. in small mammal and tick communities in the understudied region of southern California. We found B. burgdorferi s.l. in 1.5% of Ixodes species ticks and 3.6% of small mammals. Infection was uncommon (~0.3%) in Ixodes pacificus Cooley and Kohls (Acari: Ixodidae), the primary vector of the Lyme disease agent to humans in western North America, but a diversity of spirochetes-including Borrelia bissettiae, Borrelia californiensis, Borrelia americana, and B. burgdorferi s.s.-were identified circulating in Ixodes species ticks and their small mammal hosts. Infection with B. burgdorferi s.l. is more common in coastal habitats, where a greater diversity of Ixodes species ticks are found feeding on small mammal hosts (four species when compared with only I. pacificus in other sampled habitats). This provides some preliminary evidence that in southern California, wetter coastal areas might be more favorable for enzootic transmission than hotter and drier climates. Infection patterns confirm that human transmission risk of B. burgdorferi s.s. is low in this region. However, given evidence for local maintenance of B. burgdorferi s.l., more studies of enzootic transmission may be warranted, particularly in understudied regions where the tick vector of B. burgdorferi s.s. occurs.

The secret social lives of African crested rats, Lophiomys imhausi.

The crested rat, Lophiomys imhausi, is the only mammal known to sequester plant toxins. Found in eastern Africa, this large rodent is thought to defend against predation by coating specialized hairs along its sides with cardenolide toxins from the poison arrow tree, Acokanthera schimperi. To better understand the ecology of this unusual poisonous mammal, we used camera traps, livetrapping, and captive behavioral observations, to study L. imhausi in central Kenya. Although crested rats were rarely detected with camera traps, 25 individuals were caught in live traps, with estimated densities of up to 15 rats/km2 at one of nine trapping sites. Trapping records and behavioral observations suggest that L. imhausi live in male-female pairs, with juveniles that might exhibit delayed dispersal. We observed chewing of A. schimperi and/or anointing in 10 of 22 individuals, confirming the previous poison sequestration observation. We monitored crested rat activity using cameras and found that chewing on A. schimperi and cardenolide exposure had no effect on feeding, movement, or total activity. One crested rat also fed on milkweed (Gomphocarpus physocarpus; Gentaniales: Apocynaceae), but did not anoint with this cardenolide containing plant. This observation, combined with L. imhausi's selective use of A. schimperi, suggests the potential for use of alternative poison sources. This research provides novel insight into the ecology of L. imhausi, while also suggesting that more field observations, feeding trials, and chemical analyses are needed to understand their behavior and physiology. Furthermore, their complex social interactions, slow life history, and fragmented populations suggest that L. imhausi could be at risk of decline.

Infection at an ecotone: cross-system foraging increases satellite parasites but decreases core parasites in raccoons.

Ecotones can increase free-living species richness, but little is known about how parasites respond to ecotones. Here, we use parasite communities in raccoons (Procyon lotor) to test the hypothesis that parasite communities can be divided into core and satellite species, each with fundamentally different responses to ecotones. We used published parasite surveys to classify parasites as common core or rare satellite species, and then surveyed raccoons in coastal California to examine how proximity to two aquatic ecotones altered parasite communities. Raccoons near ecotones had more satellite and fewer core parasite species. Specifically, the marine ecotone increased parasite diversity by adding satellite species to a persistent core community, whereas the freshwater ecotone shifted the community from core to satellite species without a net change in parasite richness. We hypothesize that increased parasite richness at the marine ecotone resulted from increased diet diversity, but that raccoons were sinks for some parasites. Increased exposure to rare parasites at ecotones has implications for wildlife health and provides insight into observed associations between ecotones and emerging disease.

Southern California and range-wide raccoon gastrointestinal helminth database.

Local and global measurements of parasite prevalence and abundance are critical for understanding the dynamics that underlie the diversity, distribution, and evolution of infectious diseases. Here, we present a data set of gut helminths found in (1) raccoons throughout their range, based on primary literature from 1925-2017 and (2) raccoons in Santa Barbara County, California, USA surveyed from 2012 to 2015. The range-wide data set has 1,256 parasite entries from 217 literature sources across three continents and 32 states in the USA. This data set includes a list of all recorded raccoon gut helminths (n = 100) and their presence and prevalence in surveyed raccoon populations. The Santa Barbara data set includes gut helminth data from 182 raccoons from one Southern California County. In addition to the presence and abundance data for 13 parasite species, this data set includes measurements of 7,465 individual raccoon roundworms (Baylisascaris procyonis). For both range-wide and Santa Barbara data sets, we include information on parasite site of infection in host, sampling method, and sample size. We also provide geographic coordinates for infected raccoon populations (range-wide database) and individuals (Santa Barbara). In the associated metadata, we include sampling methods and summary figures for both the range-wide and Santa Barbara raccoon gut helminth records. There are no copyright or proprietary restrictions for research and/or teaching purposes. S. B. Weinstein and J. C. Van Wert contributed equally to this manuscript and are shared first authors.

Acanthocephalan Parasites of the Oarfish, Regalecus russelii (Regalecidae), With A Description of A New Species of Gymnorhadinorhynchus (Acanthocephala: Gymnorhadinorhynchidae).

Oarfish are rarely seen and seldom studied, which makes their parasite fauna even more enigmatic. Necropsy of 12 oarfish, Regalecus russelii (Regalecidae) (Cuvier, 1816), from Japan yielded 2 species of acanthocephalans. One species was found in 2 oarfish and a total of 76 specimens was collected, but only a single, immature specimen of the second species was found. The former represents an undescribed species from the order Echinorhynchida and is described here. Morphological and phylogenetic analyses of the small subunit ( SSU) rDNA place this species in the family Gymnorhadinorhynchidae, and genus Gymnorhadinorhynchus which is characterized by a cylindrical proboscis with longitudinal rows of hooks, basal circle of enlarged hooks, asymmetry of hook shape, 4 cement glands, and a spineless trunk. Diagnostic characters of this species within the genus are the number of longitudinal rows of hooks (14), smaller body size (males: 4.8-6.6 mm and females: 5.3-6.3 mm) and a number of molecular autapomorphies including a number of long insertions in both the SSU and large subunit rDNA (LSU). A single immature female of Bolbosoma sp. (Palaeacanthocephala: Plagiorhynchidae) was also found with its anterior end embedded in the mucosa of the pyloric ceca. The characters of this specimen are not consistent with any other known species of Bolbosoma; however, because only 1 immature specimen with a partially invaginated proboscis was recovered, it was not designated as a new species.

Detection and Evaluation of Antibody Response to a Baylisascaris-Specific Antigen in Rodent Hosts with the Use of Western Blotting and Elisa.

Diagnosis of parasitic diseases that involve tissue-stage larvae is challenging, and serology remains the most effective antemortem test for detecting these infections. Baylisascaris procyonis, the raccoon roundworm, is a zoonotic ascarid. Raccoons are the usual definitive host, and humans may be infected as accidental hosts. More than 150 species of birds and mammals may act as paratenic hosts, and rodents play an important role in the transmission and maintenance of this parasite in nature. Migratory larvae in paratenic host tissues can produce ocular disease and severe to fatal neurologic disease, but not all infected hosts develop signs. A sensitive and specific Western blot (WB) assay based on a recombinant Baylisascaris-specific antigen (rBpRAG-1) has been developed for use in humans. We evaluated the use of this antigen to detect Baylisascaris spp. infections in rodent paratenic hosts. With the use of 4 species of Peromyscus mice ( Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, Peromyscus polionotus) from a previous infection trial, we developed species-adapted WB and ELISA assays and evaluated performance compared to detection of larvae in tissue samples. These assays revealed species-level differences in seroconversion and terminal antibody concentrations, with P. leucopus developing significantly greater antibody concentrations than P. californicus and P. polionotus at all dose levels, and P. maniculatus at the low dose. Some P. californicus and P. polionotus failed to seroconvert despite the recovery of larvae from their tissues. WB and ELISA results were correlated; however, the WB demonstrated higher sensitivity than the ELISA overall (72.2% versus 63.9%, respectively). With the use of experimental samples, specificity was 100% for WB and 94.1% for ELISA. A WB was also used to test Mus and Rattus samples, and although numbers were too limited to evaluate sensitivity and specificity, all animals known to be infected by tissue digestion were WB positive, and all uninfected animals were negative. Finally, the Peromyscus-adapted WB and ELISA were used to test a set of serum samples from wild-trapped P. maniculatus and Rattus rattus. Both assays were generally sensitive, but specificity was equivocal. This emphasizes the challenge of using serology for investigation of wildlife diseases, in which hosts have unknown exposure histories. Nevertheless, serologic methods have utility in the study of Baylisascaris spp. in paratenic hosts, either wild or captive, and have advantageous attributes (non-lethal, high-throughput), but results should be interpreted carefully.

Introduced Rats and an Endemic Roundworm: Does Rattus rattus Contribute to Baylisascaris procyonis Transmission in California?

The introduced black rat, Rattus rattus, occurs throughout the native range of the raccoon roundworm, Baylisascaris procyonis, and might incorporate into its life cycle if rats consume parasite eggs, acquire viable infections, and are eaten by raccoons. Although rats forage at raccoon latrines, their role in B. procyonis transmission remains unknown. Here I tested the potential for rats to amplify B. procyonis transmission in California by surveying wild rodents for B. procyonis and conducting scavenger trials with the use of motion-activated cameras. Rattus rattus were infected with B. procyonis at intensities more than 100 times greater than that of co-occurring native Reithrodontomys megalotis and Peromyscus maniculatus. Rodent carcasses were scavenged by opossums, skunks, and raccoons, suggesting that these rodents, particularly R. rattus, contribute to B. procyonis transmission in this coastal California ecosystem.

Seroprevalence of Baylisascaris procyonis Infection among Humans, Santa Barbara County, California, USA, 2014-2016.

Baylisascaris procyonis (raccoon roundworm) infection is common in raccoons and can cause devastating pathology in other animals, including humans. Limited information is available on the frequency of asymptomatic human infection. We tested 150 adults from California, USA, for B. procyonis antibodies; 11 were seropositive, suggesting that subclinical infection does occur.

Independent origins of parasitism in Animalia.

Nearly half of all animals may have a parasitic lifestyle, yet the number of transitions to parasitism and their potential for species diversification remain unresolved. Based on a comprehensive survey of the animal kingdom, we find that parasitism has independently evolved at least 223 times in just 15 phyla, with the majority of identified independent parasitic groups occurring in the Arthropoda, at or below the level of Family. Metazoan parasitology is dominated by the study of helminthes; however, only 20% of independently derived parasite taxa belong to those groups, with numerous transitions also seen in Mollusca, Rotifera, Annelida and Cnidaria. Parasitism is almost entirely absent from deuterostomes, and although worm-like morphology and host associations are widespread across Animalia, the dual symbiotic and trophic interactions required for parasitism may constrain its evolution from antecedent consumer strategies such as generalist predators and filter feeders. In general, parasitic groups do not differ from their free-living relatives in their potential for speciation. However, the 10 largest parasitic clades contain 90% of described parasitic species, or perhaps 40% of all animal species. Hence, a substantial fraction of animal diversity on the Earth arose following these few transitions to a parasitic trophic strategy.