Ben Hanelt

Detection of Schistosoma mansoni in Biomphalaria using nested PCR

A nested polymerase chain reaction (PCR) protocol was developed for detecting the presence of Schistosoma mansoni sporocysts in intermediate host snails of the genus Biomphalaria. To accomplish this, rDNA genes encoding the 18S rRNA of S. mansoni and Biomphalaria alexandrina from Egypt were sequenced, as were 18S-encoding genes of the 13-16-R1 and Salvador strains of Biomphalaria glabrata. Based on a comparison of host and parasite sequences, a nested set of PCR primers was designed to allow specific amplification of portions of S. mansoni 18S rDNA. These primers allowed detection of as little as 10 fg of S. mansoni DNA diluted in 100 ng of snail DNA and did not allow amplification of snail 18S sequences. Using nested PCR, the presence of a single S. mansoni sporocyst within an adult snail could be detected at 1 day postexposure. In DNA samples extracted from each of 74 snails of the M-line strain of B. glabrata exposed to from 1 to 10 S. mansoni miracidia for intervals ranging from 1 to 44 days, use of the outside primer pair alone detected the parasites presence in 51% of the snails, whereas the sequential use of outside and nested primer pairs detected parasites in 92% of the snails. This approach has utility in determining if snails in endemic areas bear prepatent or inactive infections and in assessing the degree of compatibility between local snail and schistosome populations. It will also facilitate studies of resistance of snails to infection.

The life cycle of a horsehair worm, Gordius robustus (Nematomorpha: Gordioidea)

Aspects of the life cycle of the nematomorph Gordius robustus were investigated. Gordius robustus larvae fed to Tenebrio molitor (Coleoptera: Tenebrionidae) readily penetrated and subsequently encysted in the posterior portion of the midgut wall. Parasite development was then arrested in each infected beetle. Upon feeding these cysts to laboratory-reared Gryllus firmus, worm larvae developed to adults in the cricket hemocoel. In an additional experiment, G. robustus larvae fed to G. firmus did not develop to adults. These findings are in contrast to other studies that found direct infection to be an alternative mode of infection. This experiment is the first employing a laboratory-maintained stock of hosts to rear nematomorph worms.

Redescription and compilation of Nearctic freshwater Nematomorpha (Gordiida), with the description of two new species

Abstract We compiled information on freshwater Nematomorpha (horsehair worms) from North America including new records of more than 100 specimens from 66 new locations and the description of two new species. From the 30 species names that have been used for Nearctic nematomorphs, 15 are regarded as valid species. The descriptions of most of these species were supplemented with new material and documented by light and scanning electron microscopy. Three species, Gordius agassizi, Gordius fasciatus and Gordius hawaiiensis are synonymized with Gordius robustus. Gordius leidyi is not a valid species, but its assignment is uncertain. Gordius tenuis is not a nematomorph, but a mermithid nematode. Parachordodes densareolatus is a questionable species and might belong to Gordionus. New combinations are given for three species. The two new species described here raise the number of gordiids known from the Nearctic region to 17. A taxonomic key and a geographic list of species distributions are given in appendices. Species novae: Gordionus sinepilosus (British Columbia, Canada), Pseudochordodes texanus (Texas, U.S.A.).

PHYSID SNAILS AS SENTINELS OF FRESHWATER NEMATOMORPHS

Freshwater nematomorphs, or gordiids, are parasitic as larvae, but free-living in aquatic environments as adults. Studies based on the collection of adults have reported gordiids to be widespread, but discontinuous in distribution. However, a relatively short adult life span and unknown life history make the detection of adults difficult. An alternative approach to investigate gordiid distribution is to use cysts. Of all paratenic hosts, snails were chosen because they lacked internal defense reactions to the cysts and become easily infected. Here, it is reported that the occurrence of gordiids on the basis of the cyst stage is much more common than previously reported, thus altering the perception of how common these worms are. Using this modified survey procedure, gordiid cysts were found at 70% of sites examined, in an area where extensive sampling over 3 yr yielded adults only at a single site. Of 1,000 snails dissected, 395 were infected with gordiids (intensity range: 1–465). Furthermore, different types of human-modified landscapes did not affect gordiid distribution, suggesting that as urban and suburban areas sprawl, human encounters or pseudoparasitism with nematomorphs may increase. The results of this study indicate that use of organismal-specific sampling techniques can be critical in studies of parasite distribution and biodiversity.

Two new species and temporal changes in the prevalence of eimerians in a free-living population of Townsend's ground squirrels (Spermophilus townsendii) in Idaho.

More than 1,180 fecal samples were collected from 253 juvenile and 384 adult Townsends ground squirrels (Spermophilus townsendii) at the Snake River Birds of Prey Area near Boise, Idaho, from February to June 1992. Oocysts of 7 eimerians were observed. Five are new host records (Eimeria beecheyi, Eimeria bilamellata, Eimeria callospermophili, Eimeria lateralis, and Eimeria morainensis), 2 species are described here as new, and new structural information on E. morainensis is added. Sporulated oocysts of Eimeria adaensis n. sp. are ovoidal, 19.6 x 22.7 (16-22 x 18-26) microns with sporocysts ellipsoidal 7.2 x 11.9 (6-10 x 9-15) microns. No micropyle or oocyst residuum, but polar bodies, Stieda bodies, and sporocyst residua are present. Sporulated oocysts of Eimeria pseudospermophili n. sp. are ovoidal, 24.7 x 28.5 (21-27 x 25-32) microns with sporocysts ellipsoidal 8.8 x 14.2 (8-10 x 12-17) microns. Micropyle and oocyst residuum are absent, but polar bodies and Stieda bodies are present. Sporulated oocysts of E. morainensis are more variable in size and shape than originally described and contain 2 distinctly different residua not previously described. Temporal changes in the prevalence of eimerians of all 7 species combined in adult squirrels showed significant decline (r2 = 0.79, P < 0.001). We hypothesize that this decline was due to drought during the period of squirrel activity. No significant temporal change in the prevalence of eimerians in juvenile squirrels was observed. The prevalence of E. callospermophili, E. adaensis, and all eimerians combined in adults was significantly greater than in juveniles.(ABSTRACT TRUNCATED AT 250 WORDS)

LIFE CYCLE AND PARATENESIS OF AMERICAN GORDIIDS (NEMATOMORPHA: GORDIIDA)

To complete their life cycle, gordiids must make a transition from aquatic to terrestrial environments. However, epibenthic aquatic larvae and their terrestrial definitive hosts do not overlap in habitat. This has led many investigators to suggest that infections are acquired through the ingestion of insects, which become infected as aquatic larvae with gordiid cysts and subsequently carry gordiids to land. This proposed life cycle was experimentally tested using 3 common American species of gordiids: Gordius robustus, Paragordius varius, and Chordodes morgani. Cysts of all 3 species survived the metamorphosis of Tanytarsus sp., a midge. Subsequent infection trials of definitive hosts with cysts from imagos show that cysts surviving the metamorphosis of insects remained viable and free of host internal defense reaction. Data from naturally infected mayflies, Callibaetus sp., show that encystment and survival of gordiids within aquatic insects occur in nature. Paratenesis between paratenic hosts was also shown to be possible in these 3 species. This latter finding appears to indicate that cysts formed in spurious paratenic hosts may not be lost but may eventually transfer to normal paratenic hosts.

New Records of Nematomorph Parasites (Nematomorpha: Gordiida) of Ground Beetles (Coleoptera: Carabidae) and Camel Crickets (Orthoptera: Rhaphidophoridae) In Washington State

Abstract: From 1998 to 2003, beetles and crickets infected with hairworms were collected from 4 localities within the Hanford Nuclear Site and the Hanford Reach National Monument, located in a shrub-steppe region of Washington State along the Columbia River. Infected hosts comprised 6 species of carabid beetles within 5 genera and 2 camel crickets within 1 genus; all are newly documented insect–nematomorph associations. A large proportion of the infected hosts (48%) were collected from a single site during a single collecting period. Of the 38 infected hosts, 32 contained a single worm, 4 hosts contained 2 worms, and 2 hosts contained 3 worms. Five of the hosts with multiple infections contained at least 1 male and 1 female worm. Camel crickets were infected with Neochordodes occidentalis while carabids were infected with an undescribed species of Gordionus. As the majority of hairworms are collected in the post-parasitic adult phase, host data and hairworm–arthropod associations remain poorly documented and our work adds new data to this area of nematomorph biology.

SURVIVAL OF LARVAL AND CYST STAGES OF GORDIIDS (NEMATOMORPHA) AFTER EXPOSURE TO FREEZING

Abstract: Hairworms infect terrestrial arthropods and are 1 of the most understudied groups of parasites. Recently, life cycles of 2 gordiids (Paragordius varius and Paragordius obamai) have been domesticated in the laboratory. We tested the viability of laboratory reared and post-frozen larval and cyst stages of the North American gordiid, P. varius, frozen at −80 C for 7 mo, and the viability of field collected and post-frozen cysts of the African (P. obamai) and North American (P. varius) gordiid frozen at −20 C for 2 mo. All snails exposed to post-frozen or control P. varius larvae became infected with cysts, and there was no significant difference in prevalence or mean intensity of cysts among control or experimental snail groups. As with larvae, no significant differences were observed in prevalence or mean intensity of emerging worms from crickets infected with post-frozen or control P. obamai or P. varius cysts. All female P. obamai and P. varius worms from control and post-frozen cyst infections laid eggs and larvae hatched from some of these eggs. Survival and cyst formation of P. varius larvae exposed to different combinations of drying and/or freezing temperatures indicated that gordiid larvae have the ability to survive drying and freezing, but survival significantly increases during freezing at lower temperatures. The major contribution of our study is the demonstration that gordiid larval and cyst stages can survive freezing temperatures to infect and develop in the next host.

Genotyping Natural Infections of Schistosoma mansoni in Biomphalaria alexandrina From Damietta, Egypt, with Comparisons to Natural Snail Infections From Kenya

Abstract The distribution of Schistosoma genotypes among individuals in snail populations provides insights regarding the dynamics of transmission and compatibility between schistosome and snail hosts. A survey of Biomphalaria alexandrina from Damietta (Nile Delta, Egypt), an area subjected to persistent schistosomiasis control efforts, provided only 17 snails infected with Schistosoma mansoni (6.1% overall prevalence), each shown by microsatellite analysis to have a single genotype infection. By contrast, recent studies of uncontrolled S. mansoni transmission foci in Kenya revealed that 4.3% Biomphalaria pfeifferi and 20–25% Biomphalaria sudanica snails had multiple genotype infections. Compared with the 3 Kenyan populations, the Egyptian population of S. mansoni also showed a lesser degree of genetic variability and was genetically differentiated from them. We suggest that tracking of genotype diversity in infected snails could be further developed to serve as an additional and valuable independent indicator of efficacy of schistosomiasis control in Egypt and elsewhere.

Horsehair Worms (Gordius robustus) in Nests of the Western Bluebird (Sialia mexicana): Evidence for Anti-Predator Avoidance?

Abstract Hairworms (Nematomorpha: Gordiida) are internal parasites that alter the behavior of their terrestrial insect host, forcing it to enter the water to reach its reproductive habitat. After reproduction of the free-living adults, the larvae encyst in aquatic insects and are retained upon metamorphosis of the insect into an adult fly. This paratenic host links the aquatic and terrestrial environments after its consumption by omnivorous or predatory insects. Therefore, hairworms are usually only associated with invertebrates, and few reports discuss hairworm interactions with vertebrate species. Here, we report on the finding of horsehair worms in nests of a cavity-nesting bird species in Los Alamos County, New Mexico. From 2004 to 2008, 7 nests within nest boxes occupied by the western bluebird (Sialia mexicana) contained 8 hairworms that were identified as Gordius robustus. All of the nest boxes with worms were less than 100 m from stagnant or low-flowing streams. The most likely explanation for the presence of the worms in the nests is that worms engaged in anti-predator avoidance after their insect hosts were collected and before digestion by nestling birds.