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.

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.