The population genetics of the causative agent of snake fungal disease indicate recent introductions to the USA.

Snake fungal disease (SFD; ophidiomycosis), caused by the pathogen Ophidiomyces ophiodiicola (Oo), has been documented in wild snakes in North America and Eurasia, and is considered an emerging disease in the eastern United States of America. However, a lack of historical disease data has made it challenging to determine whether Oo is a recent arrival to the USA or whether SFD emergence is due to other factors. Here, we examined the genomes of 82 Oo strains to determine the pathogen's history in the eastern USA. Oo strains from the USA formed a clade (Clade II) distinct from European strains (Clade I), and molecular dating indicated that these clades diverged too recently (approximately 2,000 years ago) for transcontinental dispersal of Oo to have occurred via natural snake movements across Beringia. A lack of nonrecombinant intermediates between clonal lineages in Clade II indicates that Oo has actually been introduced multiple times to North America from an unsampled source population, and molecular dating indicates that several of these introductions occurred within the last few hundred years. Molecular dating also indicated that the most common Clade II clonal lineages have expanded recently in the USA, with time of most recent common ancestor mean estimates ranging from 1985 to 2007 CE. The presence of Clade II in captive snakes worldwide demonstrates a potential mechanism of introduction and highlights that additional incursions are likely unless action is taken to reduce the risk of pathogen translocation and spillover into wild snake populations.

Predator preferences shape the diets of arthropodivorous bats more than quantitative local prey abundance.

Although most predators are generalists, the majority of studies on the association between prey availability and prey consumption have focused on specialist predators. To investigate the role of highly generalist predators in a complex food web, we measured the relationships between prey consumption and prey availability in two common arthropodivorous bats. Specifically, we used high-throughput amplicon sequencing coupled with a known mock community to characterize seasonal changes in little brown and big brown bat diets. We then linked spatiotemporal variation in prey consumption with quantitative prey availability estimated from intensive prey community sampling. We found that although quantitative prey availability fluctuated substantially over space and time, the most commonly consumed prey items were consistently detected in bat diets independently of their respective abundance. Positive relationships between prey abundance and probability of consumption were found only among prey groups that were less frequently detected in bat diets. While the probability of prey consumption was largely unrelated to abundance, the community structure of prey detected in bat diets was influenced by the local or regional abundance of prey. Observed patterns suggest that while little brown and big brown bats maintain preferences for particular prey independently of quantitative prey availability, total dietary composition may reflect some degree of opportunistic foraging. Overall, our findings suggest that generalist predators can display strong prey preferences that persist despite quantitative changes in prey availability.

The sexual spore pigment asperthecin is required for normal ascospore production and protection from UV light in Aspergillus nidulans.

Many fungi develop both asexual and sexual spores that serve as propagules for dissemination and/or recombination of genetic traits. Asexual spores are often heavily pigmented and this pigmentation provides protection from UV light. However, little is known about any purpose pigmentation that may serve for sexual spores. The model Ascomycete Aspergillus nidulans produces both green pigmented asexual spores (conidia) and red pigmented sexual spores (ascospores). Here we find that the previously characterized red pigment, asperthecin, is the A. nidulans ascospore pigment. The asperthecin biosynthetic gene cluster is composed of three genes: aptA, aptB, and aptC, where deletion of either aptA (encoding a polyketide synthase) or aptB (encoding a thioesterase) yields small, mishappen hyaline ascospores; while deletion of aptC (encoding a monooxygenase) yields morphologically normal but purple ascospores. ∆aptA and ∆aptB but not ∆aptC or wild type ascospores are extremely sensitive to UV light. We find that two historical ascospore color mutants, clA6 and clB1, possess mutations in aptA and aptB sequences, respectively.

Drivers of genetic diversity in secondary metabolic gene clusters within a fungal species.

Filamentous fungi produce a diverse array of secondary metabolites (SMs) critical for defense, virulence, and communication. The metabolic pathways that produce SMs are found in contiguous gene clusters in fungal genomes, an atypical arrangement for metabolic pathways in other eukaryotes. Comparative studies of filamentous fungal species have shown that SM gene clusters are often either highly divergent or uniquely present in one or a handful of species, hampering efforts to determine the genetic basis and evolutionary drivers of SM gene cluster divergence. Here, we examined SM variation in 66 cosmopolitan strains of a single species, the opportunistic human pathogen Aspergillus fumigatus. Investigation of genome-wide within-species variation revealed 5 general types of variation in SM gene clusters: nonfunctional gene polymorphisms; gene gain and loss polymorphisms; whole cluster gain and loss polymorphisms; allelic polymorphisms, in which different alleles corresponded to distinct, nonhomologous clusters; and location polymorphisms, in which a cluster was found to differ in its genomic location across strains. These polymorphisms affect the function of representative A. fumigatus SM gene clusters, such as those involved in the production of gliotoxin, fumigaclavine, and helvolic acid as well as the function of clusters with undefined products. In addition to enabling the identification of polymorphisms, the detection of which requires extensive genome-wide synteny conservation (e.g., mobile gene clusters and nonhomologous cluster alleles), our approach also implicated multiple underlying genetic drivers, including point mutations, recombination, and genomic deletion and insertion events as well as horizontal gene transfer from distant fungi. Finally, most of the variants that we uncover within A. fumigatus have been previously hypothesized to contribute to SM gene cluster diversity across entire fungal classes and phyla. We suggest that the drivers of genetic diversity operating within a fungal species shown here are sufficient to explain SM cluster macroevolutionary patterns.

Relationships among wood-boring beetles, fungi, and the decomposition of forest biomass.

A prevailing paradigm in forest ecology is that wood-boring beetles facilitate wood decay and carbon cycling, but empirical tests have yielded mixed results. We experimentally determined the effects of wood borers on fungal community assembly and wood decay within pine trunks in the southeastern United States. Pine trunks were made either beetle-accessible or inaccessible. Fungal communities were compared using culturing and high-throughput amplicon sequencing (HTAS) of DNA and RNA. Prior to beetle infestation, living pines had diverse fungal endophyte communities. Endophytes were displaced by beetle-associated fungi in beetle-accessible trees, whereas some endophytes persisted as saprotrophs in beetle-excluded trees. Beetles increased fungal diversity several fold. Over forty taxa of Ascomycota were significantly associated with beetles, but beetles were not consistently associated with any known wood-decaying fungi. Instead, increasing ambrosia beetle infestations caused reduced decay, consistent with previous in vitro experiments that showed beetle-associated fungi reduce decay rates by competing with decay fungi. No effect of bark-inhabiting beetles on decay was detected. Platypodines carried significantly more fungal taxa than scolytines. Molecular results were validated by synthetic and biological mock communities and were consistent across methodologies. RNA sequencing confirmed that beetle-associated fungi were biologically active in the wood. Metabarcode sequencing of the LSU/28S marker recovered important fungal symbionts that were missed by ITS2, though community-level effects were similar between markers. In contrast to the current paradigm, our results indicate ambrosia beetles introduce diverse fungal communities that do not extensively decay wood, but instead reduce decay rates by competing with wood decay fungi.

A scalable platform to identify fungal secondary metabolites and their gene clusters.

The genomes of filamentous fungi contain up to 90 biosynthetic gene clusters (BGCs) encoding diverse secondary metabolites-an enormous reservoir of untapped chemical potential. However, the recalcitrant genetics, cryptic expression, and unculturability of these fungi prevent scientists from systematically exploiting these gene clusters and harvesting their products. As heterologous expression of fungal BGCs is largely limited to the expression of single or partial clusters, we established a scalable process for the expression of large numbers of full-length gene clusters, called FAC-MS. Using fungal artificial chromosomes (FACs) and metabolomic scoring (MS), we screened 56 secondary metabolite BGCs from diverse fungal species for expression in Aspergillus nidulans. We discovered 15 new metabolites and assigned them with confidence to their BGCs. Using the FAC-MS platform, we extensively characterized a new macrolactone, valactamide A, and its hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS). The ability to regularize access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Selenate sensitivity of a laeA mutant is restored by overexpression of the bZIP protein MetR in Aspergillus fumigatus.

LaeA is a conserved global regulator of secondary metabolism and development in filamentous fungi. Examination of Aspergillus fumigatus transcriptome data of laeA deletion mutants have been fruitful in identifying genes and molecules contributing to the laeA mutant phenotype. One of the genes significantly down regulated in A. fumigatus ΔlaeA is metR, encoding a bZIP DNA binding protein required for sulfur and methionine metabolism in fungi. LaeA and MetR deletion mutants exhibit several similarities including down regulation of sulfur assimilation and methionine metabolism genes and ability to grow on the toxic sulfur analog, sodium selenate. However, unlike ΔmetR, ΔlaeA strains are able to grow on sulfur, sulfite, and cysteine. To examine if any parameter of the ΔlaeA phenotype is due to decreased metR expression, an over-expression allele (OE::metR) was placed in a ΔlaeA background. The OE::metR allele could not significantly restore expression of MetR regulated genes in ΔlaeA but did restore sensitivity to sodium selenate. In A. nidulans a second bZIP protein, MetZ, also regulates sulfur and methionine metabolism genes. However, addition of an OE::metZ construct to the A. fumigatus ΔlaeA OE::metR strain still was unable to rescue the ΔlaeA phenotype to wildtype with regards gliotoxin synthesis and virulence in a zebrafish aspergillosis model.

Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome.

Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.

FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection.

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.

Caspofungin exposure alters the core septin AspB interactome of Aspergillus fumigatus.

Aspergillus fumigatus, the main etiological agent of invasive aspergillosis, is a leading cause of death in immunocompromised patients. Septins, a conserved family of GTP-binding proteins, serve as scaffolding proteins to recruit enzymes and key regulators to different cellular compartments. Deletion of the A. fumigatus septin aspB increases susceptibility to the echinocandin antifungal caspofungin. However, how AspB mediates this response to caspofungin is unknown. Here, we characterized the AspB interactome under basal conditions and after exposure to a clinically relevant concentration of caspofungin. While A. fumigatus AspB interacted with 334 proteins, including kinases, cell cycle regulators, and cell wall synthesis-related proteins under basal growth conditions, caspofungin exposure altered AspB interactions. A total of 69 of the basal interactants did not interact with AspB after exposure to caspofungin, and 54 new interactants were identified following caspofungin exposure. We generated A. fumigatus deletion strains for 3 proteins (ArpB, Cyp4, and PpoA) that only interacted with AspB following exposure to caspofungin that were previously annotated as induced after exposure to antifungal agents, yet only PpoA was implicated in the response to caspofungin. Taken together, we defined how the septin AspB interactome is altered in the presence of a clinically relevant antifungal.

Secondary metabolism and development is mediated by LlmF control of VeA subcellular localization in Aspergillus nidulans.

Secondary metabolism and development are linked in Aspergillus through the conserved regulatory velvet complex composed of VeA, VelB, and LaeA. The founding member of the velvet complex, VeA, shuttles between the cytoplasm and nucleus in response to alterations in light. Here we describe a new interaction partner of VeA identified through a reverse genetics screen looking for LaeA-like methyltransferases in Aspergillus nidulans. One of the putative LaeA-like methyltransferases identified, LlmF, is a negative regulator of sterigmatocystin production and sexual development. LlmF interacts directly with VeA and the repressive function of LlmF is mediated by influencing the localization of VeA, as over-expression of llmF decreases the nuclear to cytoplasmic ratio of VeA while deletion of llmF results in an increased nuclear accumulation of VeA. We show that the methyltransferase domain of LlmF is required for function; however, LlmF does not directly methylate VeA in vitro. This study identifies a new interaction partner for VeA and highlights the importance of cellular compartmentalization of VeA for regulation of development and secondary metabolism.

Prototype of an intertwined secondary-metabolite supercluster.

The hallmark trait of fungal secondary-metabolite gene clusters is well established, consisting of contiguous enzymatic and often regulatory gene(s) devoted to the production of a metabolite of a specific chemical class. Unexpectedly, we have found a deviation from this motif in a subtelomeric region of Aspergillus fumigatus. This region, under the control of the master regulator of secondary metabolism, LaeA, contains, in its entirety, the genetic machinery for three natural products (fumitremorgin, fumagillin, and pseurotin), where genes for fumagillin and pseurotin are physically intertwined in a single supercluster. Deletions of 29 adjoining genes revealed that fumagillin and pseurotin are coregulated by the supercluster-embedded regulatory gene with biosynthetic genes belonging to one of the two metabolic pathways in a noncontiguous manner. Comparative genomics indicates the fumagillin/pseurotin supercluster is maintained in a rapidly evolving region of diverse fungal genomes. This blended design confounds predictions from established secondary-metabolite cluster search algorithms and provides an expanded view of natural product evolution.

A novel automethylation reaction in the Aspergillus nidulans LaeA protein generates S-methylmethionine.

The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that can adapt to their environment by producing various secondary metabolites, including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are globally regulated by the LaeA protein. Null mutants of LaeA in all pathogenic fungi examined to date show decreased virulence coupled with reduced secondary metabolism. Although the amino acid sequence of LaeA contains the motifs characteristic of seven-ß-strand methyltransferases, a methyl-accepting substrate of LaeA has not been identified. In this work we did not find a methyl-accepting substrate in Aspergillus nidulans with various assays, including in vivo S-adenosyl-[methyl-(3)H]methionine labeling, targeted in vitro methylation experiments using putative protein substrates, or in vitro methylation assays using whole cell extracts grown under different conditions. However, in each experiment LaeA was shown to self-methylate. Amino acid hydrolysis of radioactively labeled LaeA followed by cation exchange and reverse phase chromatography identified methionine as the modified residue. Point mutations show that the major site of modification of LaeA is on methionine 207. However, in vivo complementation showed that methionine 207 is not required for the biological function of LaeA. LaeA is the first protein to exhibit automethylation at a methionine residue. These findings not only indicate LaeA may perform novel chemistry with S-adenosylmethionine but also provide new insights into the physiological function of LaeA.

Disease-related population declines in bats demonstrate non-exchangeability in generalist predators.

The extent to which persisting species may fill the functional role of extirpated or declining species has profound implications for the structure of biological communities and ecosystem functioning. In North America, arthropodivorous bats are threatened on a continent-wide scale by the spread of white-nose syndrome (WNS), a disease caused by the fungus Pseudogymnoascus destructans. We tested whether bat species that display lower mortality from this disease can partially fill the functional role of other bat species experiencing population declines. Specifically, we performed high-throughput amplicon sequencing of guano from two generalist predators: the little brown bat (Myotis lucifugus) and big brown bat (Eptesicus fuscus). We then compared changes in prey consumption before versus after population declines related to WNS. Dietary niches contracted for both species after large and abrupt declines in little brown bats and smaller declines in big brown bats, but interspecific dietary overlap did not change. Furthermore, the incidence and taxonomic richness of agricultural pest taxa detected in diet samples decreased following bat population declines. Our results suggest that persisting generalist predators do not necessarily expand their dietary niches following population declines in other predators, providing further evidence that the functional roles of different generalist predators are ecologically distinct.

Telomere position effect is regulated by heterochromatin-associated proteins and NkuA in Aspergillus nidulans.

Gene-silencing mechanisms are being shown to be associated with an increasing number of fungal developmental processes. Telomere position effect (TPE) is a eukaryotic phenomenon resulting in gene repression in areas immediately adjacent to telomere caps. Here, TPE is shown to regulate expression of transgenes on the left arm of chromosome III and the right arm of chromosome VI in Aspergillus nidulans. Phenotypes found to be associated with transgene repression included reduction in radial growth and the absence of sexual spores; however, these pleiotropic phenotypes were remedied when cultures were grown on media with appropriate supplementation. Simple radial growth and ascosporogenesis assays provided insights into the mechanism of TPE, including a means to determine its extent. These experiments revealed that the KU70 homologue (NkuA) and the heterochromatin-associated proteins HepA, ClrD and HdaA were partially required for transgene silencing. This study indicates that TPE extends at least 30 kb on chromosome III, suggesting that this phenomenon may be important for gene regulation in subtelomeric regions of A. nidulans.

Involvement of transposon-like elements in penicillin gene cluster regulation.

Subtelomeric secondary metabolite (SM) gene clusters are frequently surrounded by DNA repeats and transposon-like elements. The Aspergillus nidulans penicillin cluster, 30kb from the telomere of chromosome VI, is bordered by such elements. Deletions of penicillin telomere proximal and distal border regions resulted in decreased penicillin production. A 3.7kb distal region called PbIa, consisting of the putative transposable element DNA-2, was examined further where its replacement by a pyrG marker presented a similar phenotype as loss of the global SM regulator LaeA, resulting in a decrease in penicillin gene expression and product formation. In contrast, placement of the pyrG marker on either side of PbIa had no effect on penicillin synthesis. A requirement for PbIa in penicillin production was also apparent in a histone deacetylase mutant, DeltahdaA, enhanced for penicillin production. Trans-complementation of the PbIa element near and within the terrequinone A cluster on chromosome V did not restore penicillin biosynthesis or increase production of terrequinone A. Taken together, this data provides evidence for transposon involvement in SM cluster regulation.

Modification of transcriptional factor ACE3 enhances protein production in Trichoderma reesei in the absence of cellulase gene inducer.

BACKGROUND: Trichoderma reesei is one of the best-known cellulolytic organisms, producing large quantities of a complete set of extracellular cellulases and hemicellulases for the degradation of lignocellulosic substances. Hence, T. reesei is a biotechnically important host and it is used commercially in enzyme production, of both native and foreign origin. Many strategies for producing enzymes in T. reesei rely on the cbh1 and other cellulase gene promoters for high-level expression and these promoters require induction by sophorose, lactose or other inducers for high productivity during manufacturing. RESULTS: We described an approach for producing high levels of secreted proteins by overexpression of a transcription factor ACE3 in T. reesei. We refined the ace3 gene structure and identified specific ACE3 variants that enable production of secreted cellulases and hemicellulases on glucose as a sole carbon source (i.e., in the absence of an inducer). These specific ACE3 variants contain a full-length Zn2Cys6 binuclear cluster domain at the N-terminus and a defined length of truncations at the C-terminus. When expressed at a moderate level in the fungal cells, the ACE3 variants can induce high-level expression of cellulases and hemicellulases on glucose (i.e., in the absence of an inducer), and further improve expression on lactose or glucose/sophorose (i.e., in the presence of an inducer). Finally, we demonstrated that this method is applicable to industrial strains and fermentation conditions, improving protein production both in the absence and in the presence of an inducer. CONCLUSIONS: This study demonstrates that overexpression of ACE3 variants enables a high level of protein production in the absence of an inducer, and boosts protein production in the presence of an inducer. It is an efficient approach to increase protein productivity and to reduce manufacturing costs.

Major histocompatibility complex variation is similar in little brown bats before and after white-nose syndrome outbreak.

White-nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans (Pd), has driven alarming declines in North American hibernating bats, such as little brown bat (Myotis lucifugus). During hibernation, infected little brown bats are able to initiate anti-Pd immune responses, indicating pathogen-mediated selection on the major histocompatibility complex (MHC) genes. However, such immune responses may not be protective as they interrupt torpor, elevate energy costs, and potentially lead to higher mortality rates. To assess whether WNS drives selection on MHC genes, we compared the MHC DRB gene in little brown bats pre- (Wisconsin) and post- (Michigan, New York, Vermont, and Pennsylvania) WNS (detection spanning 2014-2015). We genotyped 131 individuals and found 45 nucleotide alleles (27 amino acid alleles) indicating a maximum of 3 loci (1-5 alleles per individual). We observed high allelic admixture and a lack of genetic differentiation both among sampling sites and between pre- and post-WNS populations, indicating no signal of selection on MHC genes. However, post-WNS populations exhibited decreased allelic richness, reflecting effects from bottleneck and drift following rapid population declines. We propose that mechanisms other than adaptive immunity are more likely driving current persistence of little brown bats in affected regions.

H3K9 methylation regulates growth and development in Aspergillus fumigatus.

In most species, chromatin remodeling mediates critical biological processes ranging from development to disease states. In fungi within the genus Aspergillus, chromatin remodeling may regulate expression of metabolic gene clusters, but other processes regulated by chromatin structure remain to be elucidated. In many eukaryotic species, methylation of lysine 9 of histone 3 (H3K9) is a hallmark of heterochromatin formation and subsequent gene silencing. The sole H3K9 methyltransferase in Schizosaccharomyces pombe is Clr4. We report that disruption of the Clr4 homolog in the pathogenic mold Aspergillus fumigatus (ClrD), which is involved in both mono- and trimethylation of H3K9, results in several growth abnormalities. Developmental defects in DeltaAfclrD include reduction in radial growth, reduction in conidial production, and delayed conidiation after developmental competence mediated by delayed expression of brlA, the master regulator of conidiophore development. Sensitivity of DeltaAfclrD to 6-azauracil suggests that ClrD influences transcriptional processing in A. fumigatus. Despite growth abnormalities, macrophage assays suggest ClrD may be dispensable for host interactions.

An improved method for utilizing high-throughput amplicon sequencing to determine the diets of insectivorous animals.

DNA analysis of predator faeces using high-throughput amplicon sequencing (HTS) enhances our understanding of predator-prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair "ZBJ" to results using the novel primer pair "ANML." To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single-copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre- and post-PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects collected in pitfall traps. In PCR tests, the ANML primer pair amplified 58 of 59 arthropod taxa (98%), whereas ZBJ amplified 24-40 of 59 taxa (41%-68%). Furthermore, in an HTS comparison of field-collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.