APOLOCYSTIS BOSANQUETI N. SP. (APICOMPLEXA: EUGREGARINORIDA) FROM THE INVASIVE EARTHWORM AMYNTHAS AGRESTIS (ANNELIDA: MEGASCOLECIDAE), WITH SIGNIFICANCE FOR THE MONOPHYLY OF THE FAMILY MONOCYSTIDAE.

Apolocystis bosanqueti n. sp., a parasite of an important invasive earthworm in North America, Amynthas agrestis, is described from a site in northern Vermont. The earthworm host follows an annual life cycle in Vermont, so the entire life cycle of the parasite can be observed in 7 mo. In spring, the parasites were first seen in juvenile worms as paired gamonts (suggesting precocious association). These paired gamonts mature into gametocytes that form an opaque structure, with a thick gelatinous envelope (epicyst), that becomes full of zygotes. The resulting gametocyst becomes packed with ∼105 fusiform oocysts. The mature orbicular gametocysts are large (∼1 mm in diameter) and visible to the naked eye through the body wall of the host's anterior segments. The new species most resembles Apolocystis herculea described from many lumbricid earthworm species in Europe but differs from that parasite because Ap. herculea infects the intestinal wall in the posterior of the host rather than the anterior segments. A survey of 9 other earthworm species sympatric with Am. agrestis revealed that only Amynthas tokioensis, also an invasive species, was infected with Ap. bosanqueti, albeit much less commonly. Diagnosis for the family Monocystidae is problematic because cardinal characters are lacking, and the commonly cited character, a trophozoite with no anterior differentiation, is violated in most genera placed in the family. For the first time, a molecular phylogeny is presented that includes 3 genera of monocystids with diverse cell morphology (including the new species) and supports the monophyly of the family. The only morphological character that may be used to diagnose the Monocystidae is the morphology of oocysts, which are fusiform with extended terminal tips. A comparison of oocysts from 7 parasites recovered from local earthworms, including from 3 monocystid species in the phylogeny, confirms the utility of this diagnostic trait. The 2 hosts of the new species were most likely introduced from Japan, so the range of Apolocystis likely extends into East Asia.

Effect of post-production vermicompost and thermophilic compost blending on nutrient availability.

Composting is a common waste management strategy for recycling nutrients from organic household or agricultural wastes. However, thermophilic (e.g. windrow) composting and vermicomposting (using earthworms) produce different nutrient and enzyme profiles. Vermicompost is purported to have greater fertility benefits, but is also more expensive than thermophilic compost. The objective of this study was to examine a novel approach to designing organic fertility amendments by blending mature vermicompost and thermophilic compost. To examine the effect of blending, vermicompost was admixed to thermophilic compost at 20, 50 and 70 % by mass, with and without the addition of coir (cocopeat). Electric conductivity, water-extractable, immediately available N, P and K were measured. Vermicompost and coir synergistically affected the availability of these nutrients. Synergistic effects were between 15 and 40 % for total inorganic N in blends with coir. Without coir, synergism occurred only at vermicompost additions ≥50 %. Synergism for available P and K was present in all blends and ranged from 10% to 35%. Electrical conductivity measurements suggest that blending affected compost within three days of starting the incubation. The activity of five of seven measured enzymes were linearly and positively related to the fraction of vermicompost in the blend. Blending mature composts with differing properties may be another tool, in addition to adjusting feedstock and process parameters, to affect positively the fertility properties of composts.

Genetic population structure and reproductive system of two invasive Asian earthworms, Amynthas tokioensis and Amynthas agrestis.

The invasive Asian earthworms, Amynthas tokioensis and A. agrestis, have been successful in entering North American forests in recent decades, with significant damage to both soils and above-ground environments. This success could be driven in part by a polyploid genetic system and parthenogenetic reproduction, often suggested as benefits for invasive species. Therefore, we assessed the genetic population structure, genetic diversity, and reproductive system of both species using morphological traits and panels of microsatellite markers. A total of 216 A. tokioensis and 196 A. agrestis from six sites in Vermont USA were analyzed. Although all worms were morphologically hermaphroditic, all the A. agrestis lacked the male pore (the structure allowing pass of sperm between individuals), and only 19% of the A. tokioensis possessed the male pore. All A. tokioensis earthworms were triploid (scored for three alleles for at least 1 locus, and usually several), and A. agrestis was a mix of triploid and diploid individuals. Notable was the high proportion (80%) of A. agrestis earthworms that were diploid at one site. There was clearly clonal reproduction, with identical seven- locus genotypes observed for earthworms from each site, with as many as 45 individuals with the identical genotype at one site. However, the earthworms were also genetically diverse, with 14 genotypes observed for A. tokioensis and 54 for A. agrestis, and with many singleton genotypes (a single individual). Most genotypes (71% for A. tokioensis and 92% for A. agrestis) were found at a single site. The greatest number of genotypes was found at a commercial nursery where fully 23/26 A. agrestis earthworms were singleton genotypes. As expected for the pattern of private clone alleles at sites, several measures of geographic genetic differentiation were positive, and as expected for triploid systems, an AMOVA analysis showed high within-individual genetic diversity. The paradox of clear clonal reproduction, but with a great number of genotypes for each species, and the mix of triploid and diploid individuals could be explained if the worms have been sexually reproductive, with the switch to the uniparental system only recently (or even if sexual reproduction is episodic). Last, a large number of microsatellite loci were recovered for each species and there sequence and suggested PCR primers are provided for free use by other researchers.

Biocontrol of invasive pheretimoid earthworms using Beauveria bassiana.

BACKGROUND: Invasive species cause enormous costs of over $120 billion to the U.S. economy. Among biological invasions, the invasion by pheretimoid earthworms has gone relatively unnoticed and their invasion imposes yet unknown damage on USA agriculture and horticulture. The main dispersal is with horticultural goods such as plant material and composts. Pheretimoids affect commercially important hardwood forest. With no chemical agents currently certified for earthworm control nor any best horticultural practices, slowing the invasion is difficult. METHODS: In this study we measured the efficacy of a commercial entomopathogenic fungal isolate of B. bassiana (BotaniGard®) to kill pheretimoid earthworms under greenhouse conditions. Four treatments of B. bassiana were applied: The commercial product as per label, re-cultured commercial B. bassiana, 15 g and 25 g millet grains mycotized with recultured product. In all, three bioassays were conducted in 2 consecutive years with two batches of BotaniGard®. RESULTS: With fresh batches, all B. bassiana treatments with re-cultured product resulted in greater than 70% mortality within 4 weeks. Mortality was less than 60% when BotaniGard® was used as prescribed by the label. When using 1-year old spores (refrigerated at 4 °C), mortality rates for B. bassiana treatments were less than 20% and not significantly different from the controls. However, B. bassiana still affected the earthworms by slowing their development from juvenile to adult stage. CONCLUSION: B. bassiana was effective against pheretimoid earthworms. Overall, mycotized millet grains did not significantly increase mortality over the re-cultured, directly applied B. bassiana spores. More experimentation is needed to find the mode of action of the re-cultured B. bassiana before investigating ways to improve the efficacy of B. bassiana when applied as prescribed on the label.

Potential for Mycorrhizae-Assisted Phytoremediation of Phosphorus for Improved Water Quality.

During this 6th Great Extinction, freshwater quality is imperiled by upland terrestrial practices. Phosphorus, a macronutrient critical for life, can be a concerning contaminant when excessively present in waterways due to its stimulation of algal and cyanobacterial blooms, with consequences for ecosystem functioning, water use, and human and animal health. Landscape patterns from residential, industrial and agricultural practices release phosphorus at alarming rates and concentrations threaten watershed communities. In an effort to reconcile the anthropogenic effects of phosphorus pollution, several strategies are available to land managers. These include source reduction, contamination event prevention and interception. A total of 80% of terrestrial plants host mycorrhizae which facilitate increased phosphorus uptake and thus removal from soil and water. This symbiotic relationship between fungi and plants facilitates a several-fold increase in phosphorus uptake. It is surprising how little this relationship has been encouraged to mitigate phosphorus for water quality improvement. This paper explores how facilitating this symbiosis in different landscape and land-use contexts can help reduce the application of fertility amendments, prevent non-point source leaching and erosion, and intercept remineralized phosphorus before it enters surface water ecosystems. This literature survey offers promising insights into how mycorrhizae can aid ecological restoration to reconcile humans' damage to Earth's freshwater. We also identify areas where research is needed.

Phytoremediation of Heavy Metal-Contaminated Soil by Switchgrass: A Comparative Study Utilizing Different Composts and Coir Fiber on Pollution Remediation, Plant Productivity, and Nutrient Leaching.

We investigated the effects of organic amendments (thermophilic compost, vermicompost, and coconut coir) on the bioavailability of trace heavy metals of Zn, Cd, Pb, Co, and Ni from heavy metal-spiked soils under laboratory conditions. To test switchgrass (Panicum virgatum) as a potential crop for phytoremediation of heavy metal from soil, we investigated whether the addition of organic amendments promoted switchgrass growth, and consequently, uptake of metals. Compost is a valuable soil amendment that supplies nutrients for plant establishment and growth, which is beneficial for phytoremediation. However, excess application of compost can result in nutrient leaching, which has adverse effects on water quality. We tested the nutrient leaching potential of the different organic amendments to identify trade-offs between phytoremediation and water quality. Results showed that the amendments decreased the amount of bioavailable metals in the soils. Organic amendments increased soil pH, electrical conductivity (EC), and soil nutrient status. Switchgrass shoot and root biomass was significantly greater in the amended soils compared to the non-amended control. Amended treatments showed detectable levels of heavy metal uptake in switchgrass shoots, while the control treatment did not produce enough switchgrass biomass to measure uptake. Switchgrass uptake of certain heavy metals, and concentrations of some leachate nutrients significantly differed among the amended treatments. By improving soil properties and plant productivity and reducing heavy metal solubility that can otherwise hamper plant survival, organic amendments can greatly enhance phytoremediation in heavy metal-contaminated soils.

Partitioning of habitable pore space in earthworm burrows.

Earthworms affect macro-pore structure of soils. However, some studies suggest that earthworm burrow walls and casts themselves differ greatly in structure from surrounding soils, potentially creating habitat for microbivorours nematodes which accelerate the decomposition and C and N mineralization. In this study aggregates were sampled from the burrow walls of the anecic earthworm Lumbricus terrestris and bulk soil (not altered by earthworms) from mesocosm incubated in the lab for 0, 1, 3, 5 and 16 weeks. Pore volumes and pore sizes were measured in triplicate with Mercury Intrusion Porosimetry (MIP). This method is well suited to establish pore size structure in the context of habitat, because it measures the stepwise intrusion of mercury from the outside of the aggregate into ever smaller pores. The progress of mercury into the aggregate interior thus resembles potential paths of a nematode into accessible habitable pore spaces residing in an aggregate. Total specific pore volume, V(s), varied between 0.13 and 0.18 mL/g and increased from 3 to 16 weeks in both burrow and bulk soil. Differences between total V(s) of bulk and burrow samples were not significant on any sampling date. However, differences were significant for pore size fractions at the scale of nematode body diameter.

Mesocosm-scale evaluation of faunal and microbial communities of aerated and unaerated leachfield soil.

Aeration improves the capacity of leachfields to decontaminate and reduce the nutrient load of wastewater. To gain a better understanding of the effects of aeration, we examined the faunal and microbial communities of septic system leachfield soil (0-4 and 4-13 cm) using replicated (n = 3) mesocosms that were actively aerated (AIR) or unaerated (LEACH). Protozoa were 40 to 140 times more abundant in AIR than in LEACH soil. No nematodes were found in LEACH soil, whereas AIR soil contained 5 to 14 x 10(3) nematodes (all bacteriovores) kg(-1). Active microbial biomass was four to five times higher in AIR than LEACH soil. Proteobacteria and actinomycetes/sulfate-reducing bacteria constituted a higher proportion of the community in AIR soil, whereas anaerobic Gram-negative bacteria/firmicutes were more prominent in LEACH soil. Ratios of prokaryotic to eukaryotic phospholipid fatty acids (PLFAs) were higher in LEACH soil, as were membrane stress index values, whereas the starvation index was higher in AIR soil. Community-level physiological profiles showed that 29 and 30 different substrates were used for growth by LEACH and AIR soil microorganisms, respectively. The AIR soil had more microorganisms capable of growing on 10 substrates, whereas growth on two substrates was higher in LEACH soil. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA gene fragments revealed greater diversity of dominant phylotypes in AIR than LEACH soil, with communities separated by treatment. Aerated leachfield soil had a larger and more diverse faunal and microbial community than unaerated soil, possibly due to differences in the type and availability of electron acceptors.

Effects of aeration on water quality from septic system leachfields.

We conducted a pilot-scale study at a research facility in southeastern Connecticut to assess the effects of leachfield aeration on removal of nutrients and pathogens from septic system effluent. Treatments consisted of lysimeters periodically aerated to maintain a headspace O(2) concentration of 0.209 mol mol(-1) (AIR) or vented to an adjacent leachfield trench (LEACH) and were replicated three times. All lysimeters were dosed with effluent from a septic tank for 24 mo at a rate of 12 cm d(-1) and subsequently for 2 mo at 4 cm d(-1). LEACH lysimeters had developed a clogging mat, or biomat, 20 mo before the beginning of our study. The level of aeration in the AIR treatment was held constant regardless of loading rate. No conventional biomat developed in the AIR treatment, whereas a biomat was present in the LEACH lysimeters. The headspace of LEACH lysimeters was considerably depleted in O(2) and enriched in CH(4), CO(2), and H(2)S relative to AIR lysimeters. Drainage water from AIR lysimeters was saturated with O(2) and had significantly lower pH, five-day biological oxygen demand (BOD(5)), and ammonium, and higher levels of nitrate and sulfate than LEACH lysimeters regardless of dosing rate. By contrast, significantly lower levels of total N and fecal coliform bacteria were observed in AIR than in LEACH lysimeters only at the higher dosing rate. No significant differences in total P removal were observed. Our results suggest that aeration may improve the removal of nitrogen, BOD(5), and fecal coliforms in leachfield soil, even in the absence of a biomat.