Optimal counting limit for fungal spore abundance estimation using Sporormiella as a case study

The use of non-pollen palynomorphs, and among them spores of coprophilous fungi, has become greatly important in palaeoecological studies. Particularly, the genus Sporormiella has been demonstrated to be the most valuable proxy for the presence of wild and domestic herbivores. This genus could also be used to determine livestock density and reconstruct pastoral pressure during the Holocene. Non-standard counting methods have been established to determine coprophilous fungal spore abundance in sediments. Moreover, these analyses are faced with the recurrent problem of setting the minimum counting sum as small as possible to save time. We researched the reliability of Sporormiella concentration estimates based on different counting sums, using low to high count samples. Box-plots indicate that the variability of inferred Sporormiella concentrations decreases progressively with increasing sums. Statistical comparisons show that the means of box-plots became stabilised after the counts have reached 300–350 exotic marker grains. Moreover, a count of 300–350 exotic marker grains is sufficient to produce a Sporormiella concentration estimate, whatever the amount. Finally, we propose that this counting limit is valid for other fungal spores as well.     

Geographic variation in the ecological effects of extinction of Australia's Pleistocene megafauna

Recent studies suggest that extinction of Pleistocene megafauna had large impacts on the structure and functioning of ecosystems, including increased fire and shifts in vegetation state. We argue that the ecological effects of mega‐herbivore extinction are likely to have varied geographically, and might have been reduced in environments of low productivity. We tested this at Caledonia Fen, a cool, high‐elevation site in southeast Australia with a palynological record reaching back approximately 140 ka. The dung fungus Sporormiella indicated that large herbivores were present through most of the early part of the last glacial cycle, but declined abruptly between 50–40 ka and did not recover. This event corresponds with evidence for continent‐wide extinction of Australia's Pleistocene megafauna at that time. An earlier episode of low Sporormiella occurrence coincided with evidence of raised water levels in the fen. Changes in wetland conditions can alter the accumulation of Sporormiella, but there was no such change when Sporormiella counts fell in the period 50–40 ka. We found no evidence that the decline in Sporormiella triggered increased fire or a change in vegetation, which remained a low grass/shrub steppe. This contrasts with a warmer and more humid site, Lynch's Crater in northeast Australia, where decline of dung fungi was followed by increased fire and transition from mixed sclerophyll forest and rainforest to uniform sclerophyll forest. Our results suggest that the magnitude of ecological responses to Pleistocene megafaunal extinction varied geographically, under the control of regional climates.     

Criconemella spp. in Pennsylvania Peach Orchards with Morphological Observations of C. curvata and C. ornata

Criconemella xenoplax and C. curvata, previously associated with decline of peach trees in other parts of the United States, were found in 20 of 25 Pennsylvania peach orchards. Population densities were high in some samples. Morphometrics of juveniles and adult females of Criconemella curvata and C. ornata, are provided. Cuticular crenations were observed on J2 and J3 stages of C. curvata and J2-J4 stages of C. ornata.     

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C_T values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.     

First record of harpacticoid copepods from Lake Tahoe,United States: two new species of Attheyella (Harpacticoida,Canthocamptidae)

Benthic harpacticoids were collected for the first time at Lake Tahoe, California-Nevada, United States. Two species were identified as members of the genus Attheyella Brady, 1880. The genus Attheyella comprises about 150 species within six subgenera, but only twelve species have previously been reported from North American freshwater habitats. The two new species of Attheyella described here have a 3-segmented endopod on P1 and 2-segmented P2–P4 endopods, the distal segment of exopod of P2–P4 has three outer spines, and the P5 has five setae on the exopod and six setae on the baseoendopod. Attheyella (Attheyella) tahoensis sp. n. most closely resembles Attheyella (Attheyella) idahoensis (Marsh, 1903) from Idaho, Montana, and Alaska (United States) and Attheyella (Attheyella) namkungi Kim, Soh & Lee, 2005 from Gosu Cave in South Korea. They differ mainly by the number of setae on the distal endopodal segment of P2–P4. In addition, intraspecific variation has been observed on the caudal rami. Attheyella (Neomrazekiella) tessiae sp. n. is characterized by the extension of P5 baseoendopod, 2-segmented endopod of female P2–P3, and naked third seta of male P5 exopod. The two new species are likely endemic to Lake Tahoe, an isolated alpine lake within the Great Basin watershed in the western United States.     

Revisiting the Life Cycle of Dung Fungi,Including Sordaria fimicola

Dung fungi, such as Sordaria fimicola, generally reproduce sexually with ascospores discharged from mammalian dung after passage through herbivores. Their life cycle is thought to be obligate to dung, and thus their ascospores in Quaternary sediments have been interpreted as evidence of past mammalian herbivore activity. Reports of dung fungi as endophytes would seem to challenge the view that they are obligate to dung. However, endophyte status is controversial because surface-sterilization protocols could fail to kill dung fungus ascospores stuck to the plant surface. Thus, we first tested the ability of representative isolates of three common genera of dung fungi to affect plant growth and fecundity given that significant effects on plant fitness could not result from ascospores merely stuck to the plant surface. Isolates of S. fimicola, Preussia sp., and Sporormiella sp. reduced growth and fecundity of two of three populations of Bromus tectorum, the host from which they had been isolated. In further work with S. fimicola we showed that inoculations of roots of B. tectorum led to some colonization of aboveground tissues. The same isolate of S. fimicola reproduced sexually on inoculated host plant tissues as well as in dung after passage through sheep, thus demonstrating a facultative rather than an obligate life cycle. Finally, plants inoculated with S. fimicola were not preferred by sheep; preference had been expected if the fungus were obligate to dung. Overall, these findings make us question the assumption that these fungi are obligate to dung.     

Dung fungi as a proxy for megaherbivores: opportunities and limitations for archaeological applications

The use of spores of coprophilous fungi from sedimentary sequences as proxy evidence for large herbivore abundance has garnered pronounced attention and scrutiny over the past three decades. In response to the rapid rate at which new information is being discovered on this topic, this paper presents a brief review of the archaeological applications so far, and outlines opportunities and limitations of using Sporormiella as a proxy for herbivore abundance. Specific archaeological uses of this proxy include understanding megaherbivore extinctions and human land use patterns such as pastoralism and agriculture. We analyse how dung fungal records are formed and review the mycological literature to outline factors affecting spore reproduction and preservation. These include how strongly each commonly used dung fungal taxon relies on dung as a substrate and environmental factors affecting dung fungal reproduction and coprophilous fungi deposition. Certain laboratory preparation techniques adversely affect spore representation on pollen slides. The methods of analysis and quantification of spore records also impact our understanding. We describe good practice to increase precision of analytical methods. Due to limitations imposed by some of these factors, it is possible that an absence of dung fungi from a palaeoecological record does not imply an absence of herbivores. However, consideration of these factors and inclusion of as wide a range of coprophilous spore records as possible increases the reliability of such inferences.

     

Effect of Controlled Cold Storage on Recovery of Rotylenchulus reniformis from Naturally Infested Soil

Rotylenchulus reniformis is rapidly becoming the most economically important pest associated with cotton in the southeastern United States. Incentive programs have been implemented to support sampling of production fields to determine the presence and abundance of R. reniformis. These sampling programs have dramatically increased the number of soils samples submitted to nematology laboratories during autumn. The large numbers of samples overwhelm most labs and require placement in cold storage until extraction. Therefore, the objective of this study was to examine the length of time soils infested with R. reniformis can be stored before nematode extraction without compromising the accuracy of estimates of population densities. A sandy loam and a silty loam were the two cotton production soils used in this study. Rotylenchulus reniformis numbers decreased 61%during the first 180 days of storage in both soils. Rotylenchulus reniformis numbers from the initial sampling through 180 days decreased as a linear function. The decline of R. reniformis numbers during storage was estimated as 0.28% of the population lost daily from the maximum population through 180 days. The diminution of nematode numbers from 180 through 1,080 days in storage continued, but at a slower rate. Numbers of R. reniformis declined to less than 89%, 93%, and 99% of the initial population within 360, 720, and 1,080 days, respectively, of storage. The reduction of R. reniformis numbers over 180 days can be adjusted, allowing a more accurate estimation of R. reniformis levels in soil samples stored at 4 °C.     

A Plant Health Care Program for Brambles in the Pacific Northwest

Pratylenchus and Xiphinema species have been associated with decline and mortality of brambles (Rubus species) in the Pacific Northwest of the United States. These nematodes cause direct feeding damage and (or) transmit viruses that result in poor fruit quality and plant decline. A nematode management program has been developed by the author to minimize chemical use and nematode-induced damage while optimizing fruit production. Nematode management is an integral part of a total plant health care program in which foliar and soil pests, plant stresses, and fertility are managed.     

Cross-Institute Evaluations of Inhibitor-Resistant PCR Reagents for Direct Testing of Aerosol and Blood Samples Containing Biological Warfare Agent DNA

Rapid pathogen detection is crucial for the timely introduction of therapeutics. Two groups (one in the United Kingdom and one in the United States) independently evaluated inhibitor-resistant PCR reagents for the direct testing of substrates. In the United Kingdom, a multiplexed Bacillus anthracis (target) and Bacillus subtilis (internal-control) PCR was used to evaluate 4 reagents against 5 PCR inhibitors and down-selected the TaqMan Fast Virus 1-Step master mix (Life Technologies Inc.). In the United States, four real-time PCR assays (targeting B. anthracis, Brucella melitensis, Venezuelan equine encephalitis virus [VEEV], and Orthopoxvirus spp.) were used to evaluate 5 reagents (plus the Fast Virus master mix) against buffer, blood, and soil samples and down-selected the KAPA Blood Direct master mix (KAPA Biosystems Inc.) with added Platinum Taq (Life Technologies). The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples—which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR.     

Esterase Allozymes of Soybean Cyst Nematode,Heterodera glycines,from China,Japan, and the United States

Individual females from 19 populations of Heterodera glycines from China, Japan, and the United States were analyzed for esterase allozyme polymorphism. Eight esterase electrophoretic phenotypes were resolved. Four putative loci, est-1, est-2, est-3, and est-4, were identified, having one, one, two, and one allele, respectively. The four loci expressed six genotypes in the four Chinese populations. Loci est-2, est-3, and est-4 were identified in five Japanese populations and expressed five genotypes, whereas only loci est-2 and est-3 were identified in 10 populations from the United States and expressed four genotypes. Putative alleles at each locus were defined as characters for data analysis. Phylogenetic analysis using parsimony (PAUP) was utilized to determine relationships among the 19 populations. More loci and alleles in populations from China and phylogenetic similarities among populations from Japan and the United States are consistent with a founder effect resulting from dissemination of progenitor H. glycines from China to Japan and subsequent introductions of founder populations from Japan to the United States.     

Cultivable bacterial composition and BIOLOG catabolic diversity of biofilm communities developed on Phragmites australis

Biofilm samples formed on submerged young and old stems of reed, Phragmites australis (Cav.) Trin ex Steudel were taken during summer at different sites of Lake Velencei, Hungary. BIOLOG GN microplates were used to analyze the patterns of sole carbon source utilizations by microbial communities. From the carbon sources, carbohydrates and amino acids were preferred by all microbial communities. In the case of the old reed stem samples, higher number of carbohydrates, carboxylic acids and polymers were used than in young samples. Biofilm bacterial communities from the old reed samples of the nature conservation area of the lake used the highest number of (≥50% of the available) substrates. In principal component analysis (PCA), the metabolic potential of the microbial communities from the middle open water region of the lake showed the smallest variability. The variability within metabolic potential of the reed stem microbial communities from a given sampling site was the largest in the case of samples originating from the western, reed-covered nature conservation area. A total of 251 bacterial isolates obtained after serial dilutions and plating onto different media were characterized by traditional phenotypic tests. The strains showed high activities mainly in the hydrolysis of certain biopolymers (gelatine and casein). PCA was used to evaluate the phenotypic variability of strain groups of different sampling sites. The two open water regions were similar to each other, and separated from the western reed covered part of the lake. Similarly to the BIOLOG community-level physiological profiles, strain groups of the young and old reed stem samples originating from the nature conservation area had the largest metabolic potential. On the basis of 16S rDNA sequence analysis, 23 representative strains with different ARDRA patterns were identified. The cultivation-based investigations of bacterial diversity showed characteristic differences in the number of identified taxa in connection with the sampling sites. No characteristic differences could be observed according to medium or sample type (young, first year and more than 1-year old stems) among the identified species. 16S rDNA sequence comparisons resulted in the identification of the genera Aureobacterium, Arthrobacter, Kocuria, Microbacterium, Micrococcus, Rhodococcus, Bacillus, Marinibacillus, Rhodobacter, Defluvibacter, Pseudomonas, Klebsiella, Serratia and Aeromonas. The results of the cultivation-based and BIOLOG investigations revealed characteristic differences in the bacterial community composition and activities of the open water region and the reed covered nature conservation part of the lake.     

Fungal spores record Iroquoian and Canadian agriculture in 2nd millennium a.d. sediment of Crawford Lake, Ontario, Canada

Pollen analysis identified domestic Zea mays (corn, maize), Phaseolus vulgaris (kidney bean), Helianthus annuus (sunflower) and Cucurbita pepo (squash) accompanied by spores of fungi, many of which cause crop diseases, such as Ustilago maydis (=U. zeae, corn smut), Puccinia sorghi (corn rust), Uromyces appendiculatus (bean rust) and Puccinia graminis (wheat rust). Spores were most abundant in two intervals, ca. a.d. 1300 to ca. 1500 when prehistoric Iroquoian agriculture flourished near the lake and since 1830, when historic Triticum aestivum (wheat) agriculture began. In addition to dispersal by wind, Branta canadensis (Canada goose) also transported fungal spores to the lake via their dung. Spores of fungi that parasitize crop plants may be more abundant than pollen of their host plants and therefore spores may more readily indicate ancient agriculture.     

Effects of a Commercial Formulation of Paecilomyces lilacinus Strain 251 on Overseeded Bermudagrass Infested with Belonolaimus longicaudatus

Belonolaimus longicaudatus is an important parasite of both warm-season bermudagrass and winter overseed grasses used on golf courses in the southeastern United States. Field trials were conducted to study the effects of a commercial formulation of Paecilomyces lilacinus strain 251 applied to overseed grasses during the winter and early spring on population density of B. longicaudatus and bermudagrass health in late spring after bermudagrass broke dormancy. These studies found that P. lilacinus reduced numbers of B. longicaudatus in most cases, but not below damaging levels. Multiple applications of 1 × 10¹⁰ spores/m² were generally more effective than 2 × 10¹⁰ spores/m² in reducing nematode numbers and improving turf roots. These results indicate that application of this formulation of P. lilacinus strain 251 to overseeded turf in the spring may be a useful integrated pest management tool for B. longicaudatus on bermudagrass, but is not sufficient as a stand-alone nematode management tactic.     

Complex Population Structure of Borrelia burgdorferi in Southeastern and South Central Canada as Revealed by Phylogeographic Analysis

Lyme disease, caused by the bacterium Borrelia burgdorferi sensu stricto, is an emerging zoonotic disease in Canada and is vectored by the blacklegged tick, Ixodes scapularis. Here we used Bayesian analyses of sequence types (STs), determined by multilocus sequence typing (MLST), to investigate the phylogeography of B. burgdorferi populations in southern Canada and the United States by analyzing MLST data from 564 B. burgdorferi-positive samples collected during surveillance. A total of 107 Canadian samples from field sites were characterized as part of this study, and these data were combined with existing MLST data for samples from the United States and Canada. Only 17% of STs were common between both countries, while 49% occurred only in the United States, and 34% occurred only in Canada. However, STs in southeastern Ontario and southwestern Quebec were typically identical to those in the northeastern United States, suggesting a recent introduction into this region from the United States. In contrast, STs in other locations in Canada (the Maritimes; Long Point, Ontario; and southeastern Manitoba) were frequently unique to those locations but were putative descendants of STs previously found in the United States. The picture in Canada is consistent with relatively recent introductions from multiple refugial populations in the United States. These data thus point to a geographic pattern of populations of B. burgdorferi in North America that may be more complex than simply comprising northeastern, midwestern, and Californian groups. We speculate that this reflects the complex ecology and spatial distribution of key reservoir hosts.     

Ultrastructure and molecular characterization of a microsporidium, Tubulinosema hippodamiae, from the convergent lady beetle, Hippodamia convergens Guérin-Méneville

Hippodamia convergens, the convergent lady beetle, is available for aphid control in home gardens and in commercial food production systems throughout the United States and Canada. Beetles received from commercial insectaries for biological control are occasionally infected with a microsporidium. The objective of this study was to describe the pathogen by means of ultrastructure, molecular characterization and tissue pathology. All stages of the microsporidium were in direct contact with the host cell cytoplasm. Early developmental stages were proximal to mature spores and both were observed throughout the tissue sections that were examined. Merogony resulted from binary fission. Early-stage sporoblasts were surrounded by a highly convoluted plasma membrane and contained an electron-dense cytoplasm and diplokaryon. Ovoid to elongated late-stage sporoblasts were surrounded by a relatively complete spore wall. The polar filament, polaroplast, and anchoring disk were readily observed within the cell cytoplasm. Mature spores were typical of terrestrial microsporidia, with a thickened endospore surrounded by a thin exospore. Spores contained well-defined internal structures, including a diplokaryon, lamellar polaroplast and a slightly anisofilar polar filament with 10-14 coils arranged in a single or double row. A prominent indentation was evident at the apical end of the spore wall proximal to the anchoring disk. Aberrant spores were also observed. These had a fully developed endospore and exospore but lacked any discernable internal spore structures, and were, instead, filled with lamellar or vesicular structures. Typical and aberrant spores measured 3.58 ± 0.2 × 2.06 ± 0.2 μm (n = 10) and 3.38 ± 0.8 × 2.13 ± 0.2 μm (n = 10), respectively. Spores were observed in longitudinal muscle surrounding the midgut and within the fat body, Malpighian tubules, pyloric valve epithelium, ventral nerve cord ganglia, muscles and ovaries. The hindgut epithelium was often infected but the connective tissues were rarely invaded. The life cycle and pathology of the microsporidium bears some resemblance to Nosema hippodamiae, the only microsporidium reported from H. convergens by Lipa and Steinhaus in 1959. Molecular characterization of the pathogen genomic DNA revealed that it is 99% similar to Tubulinosema acridophagus and T. ratisbonensis, two pathogens that infect Drosophila melanogaster and 98% similar to T. kingi from D. willistoni. Based on similarities in pathogen ultrastructure and the molecular information gained during this study, we propose that the microsporidium in H. convergens be given the name Tubulinosema hippodamiae.     

Avian Migrants Facilitate Invasions of Neotropical Ticks and Tick-Borne Pathogens into the United States

Migratory birds have the potential to transport exotic vectors and pathogens of human and animal health importance across vast distances. We systematically examined birds that recently migrated to the United States from the Neotropics for ticks. We screened both ticks and birds for tick-borne pathogens, including Rickettsia species and Borrelia burgdorferi. Over two spring seasons (2013 and 2014), 3.56% of birds (n = 3,844) representing 42.35% of the species examined (n = 85) were infested by ticks. Ground-foraging birds with reduced fuel stores were most commonly infested. Eight tick species were identified, including seven in the genus Amblyomma, of which only Amblyomma maculatum/Amblyomma triste is known to be established in the United States. Most ticks on birds (67%) were neotropical species with ranges in Central and South America. Additionally, a single Ixodes genus tick was detected. A total of 29% of the ticks (n = 137) and no avian blood samples (n = 100) were positive for infection with Rickettsia species, including Rickettsia parkeri, an emerging cause of spotted fever in humans in the southern United States, a species in the group of Rickettsia monacensis, and uncharacterized species and endosymbionts of unknown pathogenicity. No avian tick or blood samples tested positive for B. burgdorferi, the etiologic agent of Lyme disease. An extrapolation of our findings suggests that anywhere from 4 to 39 million exotic neotropical ticks are transported to the United States annually on migratory songbirds, with uncertain consequences for human and animal health if the current barriers to their establishment and spread are overcome.     

Identification of Lygus hesperus by DNA barcoding reveals insignificant levels of genetic structure among distant and habitat diverse populations

Background

The western tarnished plant bug Lygus hesperus is an economically important pest that belongs to a complex of morphologically similar species that makes identification problematic. The present study provides evidence for the use of DNA barcodes from populations of L. hesperus from the western United States of America for accurate identification.

Methodology/Principal Findings

This study reports DNA barcodes for 134 individuals of the western tarnished plant bug from alfalfa and strawberry agricultural fields in the western United States of America. Sequence divergence estimates of <3% reveal that morphologically variable individuals presumed to be L. hesperus were accurately identified. Paired estimates of F_st and subsequent estimates of gene flow show that geographically distinct populations of L. hesperus are genetically similar. Therefore, our results support and reinforce the relatively recent (<100 years) migration of the western tarnished plant bug into agricultural habitats across the western United States.

Conclusions/Significance

This study reveals that despite wide host plant usage and phenotypically plastic morphological traits, the commonly recognized western tarnished plant bug belongs to a single species, Lygus hesperus. In addition, no significant genetic structure was found for the geographically diverse populations of western tarnished plant bug used in this study.