Isolation of Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea) from the feces of Ambystoma annulatum (Ringed Salamander) in Northwest Arkansas.

The salamander, Ambystoma annulatum, is considered a "species of special concern" in the state of Arkansas, USA, due to its limited geographic range, specialized habitat requirements and low population size. Although metazoan parasites have been documented in this salamander species, neither its native protists nor microbiome have yet been evaluated. This is likely due to the elusive nature and under-sampling of the animal. Here, we initiate the cataloguing of microbial associates with the identification of a new heterlobosean species, Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea), isolated from feces of an adult A. annulatum.

Helminth and leech community structure in tadpoles and caudatan larvae of two amphibian species from Western Nebraska.

Currently no comparative studies exist on helminth and leech community structure among sympatric anuran tadpoles and salamander larvae. During June-August 2007-2009, we examined 50 bullfrog tadpoles, Rana catesbeiana , 50 barred tiger salamander larvae, Ambystoma mavortium , and 3 species of snails from Nevens Pond, Keith County, Nebraska for helminth and leech infections. The helminth and leech compound community of this larval amphibian assemblage consisted of at least 7 species, 4 in bullfrog tadpoles and 4 in barred tiger salamander larvae. Bullfrog tadpoles were infected with 2 species of nematodes ( Gyrinicola batrachiensis and Spiroxys sp.) and 2 types of metacercariae ( Telorchis sp. and echinostomatids), whereas barred tiger salamander larva were infected with 1 species of leech ( Placobdella picta ), 2 species of adult trematodes ( Telorchis corti and Halipegus sp.), and 1 species of an unidentified metacercaria. The component community of bullfrog tadpoles was dominated by helminths acquired through active penetration, or incidentally ingested through respiratory currents, or both, whereas the component community of larval salamanders was dominated by helminths acquired through ingestion of intermediate hosts (χ²  =  3,455.00, P < 0.00001). Differences in amphibian larval developmental time (2-3 yr for bullfrog tadpoles versus 2-5 mo for salamander larvae), the ephemeral nature of intermediate hosts in Nevens Pond, and the ability of bullfrog tadpole to eliminate echinostome infections had significant effects on mean helminth species richness among amphibian species and years (t  =  12.31, P < 0.0001; t  =  2.09, P  =  0.04). Differences in herbivorous and carnivorous diet and time to metamorphosis among bullfrog tadpoles and barred tiger salamander larvae were important factors in structuring helminth communities among the larval stages of these 2 sympatric amphibian species, whereas size was important in structuring helminth and leech communities in larval salamanders, but not in bullfrog tadpoles.

Scoliosis in a tiger salamander (Ambystoma tigrinum) associated with encysted digenetic trematodes of the genus Clinostomum.

A group of 202 tiger salamanders (Ambystoma tigrinum) was brought into captivity due to habitat destruction. Half of these animals died, over two mo, showing generalized hemorrhages compatible with an infectious disease, but an etiologic agent was not determined. Encysted metacercarial stages of trematodes within the skeletal musculature, coelomic cavity, and subcutaneous space were additional necropsy findings. One salamander had scoliosis and multiple skin nodules. A radiograph showed no skeletal abnormality to explain the scoliosis; however, numerous round nodules were more radiodense than the surrounding tissue. A presumptive diagnosis of generalized trematodiasis was made, yet the salamander did not improve after a course of praziquantel and subsequently died. Necropsy revealed massive, encysted trematode infection. Histologic examination revealed marked multifocal intramuscular, subcutaneous, and coelomic trematodiasis with associated necrosis and inflammation. Based on gross morphology, the trematode was identified as a member of the genus Clinostomum.

The role of damselflies (Odonata: Zygoptera) as paratenic hosts in the transmission of Halipegus eccentricus (Digenea: Hemiuridae) to anurans.

Halipegus eccentricus is a common hemiurid trematode in the eustachian tubes of North America frogs. However, the life cycle of this species has never been completely elucidated. Studies on H. eccentricus suggest that it has a 3-host life cycle. Here, we show through fieldwork and host specificity experimental infections that the life cycle of Halipegus eccentricus utilizes 4 hosts. Metamorphosed anurans become infected with H. eccentricus by feeding on infected damselflies; worms reside in the stomach of anurans, migrate to the eustachian tubes within 32-39 days post-exposure (DPE), and release eggs 50-60 DPE. Cystophorous cercariae develop in Physa gyrina snails within 32-35 DPE, infect ostracod ( Cypridopsis sp.) second intermediate hosts, and develop to metacercariae. Fifteen- to 19-day-old metacercariae from ostracods are infective to both damselfly larvae and metamorphosed anurans. Field surveys of damselflies and tadpoles, along with laboratory exposure of damselfly larvae, metamorphosed anurans, and tadpoles with infected ostracods, indicated that only metamorphosed anurans and damselflies become infected with H. eccentricus , whereas field-collected tadpoles and laboratory-exposed tadpoles were never infected with H. eccentricus . Because little morphological change occurred in the metacercaria stage of H. eccentricus between the ostracod second intermediate host and damselfly host, and metamorphosed anurans became infected with H. eccentricus metacercariae recovered from both host groups, we suggest that odonates serve as paratenic hosts in this life cycle. Additionally, our field work and experimental infections provide data on the use of odonates as the route of infection by another North American Halipegus sp. that matures in the stomach of frogs. Our data indicate that when the life cycles are known, the use of odonates as the route of infection to anurans is common in the life cycles of Halipegus spp., and all species exhibit remarkable infection site fidelity in their amphibian hosts.

A New Species of Eimeria (Apicomplexa: Eimeriidae) from the marbled salamander, Ambystoma opacum (Caudata: Ambystomatidae), from northern Louisiana.

Between December 2002 and June 2004, 10 marbled salamanders, Ambystoma opacum, were examined for coccidian parasites. Salamanders were collected in Bradley (n = 2), Little River (n = 1), Miller (n = 1), and Sevier (n = 1) Counties, Arkansas; Webster Parish, Louisiana (n = 2); and Bowie (n = 1) and Nacogdoches (n = 2) Counties, Texas. Two of 10 (20%) A. opacum from Louisiana harbored an undescribed species of Eimeria. Oocysts of Eimeria trauthi n. sp. were ellipsoidal, 36.6 x 33.1 (33-40 x 29-37) microm, with a thin, single-layered wall; shape index 1.1. Polar granule(s) and micropyle were absent. Oocyst residuum was composed of hundreds of loosely packed homogenous granules of various sizes enclosing a vacuole. Sporocysts were elongate-ellipsoidal, 20.8 x 8.1 (19-22 x 7-9) microm; shape index 2.6. Sporocyst residuum was spherical and composed of a cluster of granules often membrane-bound. This is the first time a coccidium has been reported from an amphibian species in Louisiana and the second time a coccidium has been described from this salamander host. In addition, the following 26 salamanders from various counties in Arkansas, Oklahoma, and Texas were surveyed during the study period and were negative for coccidia: Ambystomatidae, 4 spotted salamanders (Ambystoma maculatum) and 7 mole salamanders (Ambystoma talpoideum); Cryptobranchidae, 4 Ozark hellbenders (Cryptobranchus alleganiensis bishopi); Plethodontidae, 6 spotted dusky salamanders (Desmognathus conanti) and 3 many-ribbed salamanders (Eurycea multiplicata multiplicata); and Salamandridae, 2 central newts (Notophthalmus viridescens louisianensis).

Infection of spotted salamanders (Ambystoma maculatum) with Ichthyophonus-like organisms in Virginia.

Ichthyophonus-like organisms were found in two free-ranging adult spotted salamanders (Ambystoma maculatum) captured within two different vernal ponds in the Virginia Commonwealth University Rice Center for Environmental Life Sciences in Charles City County, Virginia. Histopathologic examination of necropsied specimens revealed large spores, often enclosed by granulomas. These enclosed spores resembled those caused by the fish pathogen Ichthyophonus hoeferi. One salamander displayed an externally visible large swelling beneath the jaws. The other lacked macroscopic abnormalities, but histologic sections of ventral muscle revealed early-stage Ichthyophonus-like organisms and minimal granulomatous reactions. This is the first report of Ichthyophonus-like infection of Ambystoma maculatum in Virginia.

Helminth communities in five species of sympatric amphibians from three adjacent ephemeral ponds in southeastern Wisconsin.

Representatives of 5 amphibian species (313 individuals), including eastern American toads (Bufo americanus), wood frogs (Rana sylvatica), spring peepers (Pseudacris crucifer), blue-spotted salamanders (Ambystoma laterale), and central newts (Notophthalmus viridescens louisianensis), were collected from 3 ephemeral ponds during spring 1994, and they were inspected for helminth parasites. The component communities of anurans were more diverse than those of caudates. Infracommunities of all host species were isolationist and depauperate, due mostly to host ectothermy and low vagility. Toad infracommunities were dominated by skin-penetrating nematodes, and they had the highest values of mean total parasite abundance, mean species richness, and overall prevalence. This was likely due to their greater vagility compared with other host species. Infracommunities of wood frogs and blue-spotted salamanders had intermediate values for these measures of parasitism, whereas spring peeper and newt infracommunities had the lowest values. In addition to relative vagility, feeding habits and habitat preference were likely important in helminth community structure. Body size also seemed to play a role because mean wet weight of host species followed the same general trend as values of parasitism. However, effects of size were variable within host species and difficult to separate from other aspects of host ecology.

Adding infection to injury: synergistic effects of predation and parasitism on amphibian malformations.

We explored the importance of interactions between parasite infection and predation in driving an emerging phenomenon of conservation importance: amphibian limb malformations. We suggest that injury resulting from intraspecific predation in combination with trematode infection contributes to the frequency and severity of malformations in salamanders. By integrating field surveys and experiments, we evaluated the individual and combined effects of conspecific attack and parasite (Ribeiroia ondatrae) infection on limb development of long-toed salamanders (Ambystoma macrodactylum). In the absence of Ribeiroia, abnormalities involved missing digits, feet, or limbs and were similar to those produced by cannibalistic attack in experimental trials. At field sites that supported Ribeiroia, malformations were dominated by extra limbs and digits. Correspondingly, laboratory exposure of larval salamanders to Ribeiroia cercariae over a 30-day period induced high frequencies of malformations, including extra digits, extra limbs, cutaneous fusion, and micromelia. However, salamander limbs exposed to both injury and infection exhibited 3-5 times more abnormalities than those exposed to either factor alone. Infection also caused significant delays in limb regeneration and time-to-metamorphosis. Taken together, these results help to explain malformation patterns observed in natural salamander populations while emphasizing the importance of interactions between parasitism and predation in driving disease.

Metacercariae of Clinostomum attenuatum in Ambystoma tigrinum mavortium, Bufo cognatus and Spea multiplicata from west Texas.

Tissues from barred tiger salamanders (Ambystoma tigrinum mavortium), Great Plains toads (Bufo cognatus) and New Mexico spadefoots (Spea multiplicata) collected from 16 playa wetlands in Texas during 1999 and 2000 were examined by light microscopy. Digenean cysts were primarily distributed subcutaneously throughout the specimens and occasionally coelomic invasion was noted. The parasites within the cysts were 1.5-2 mm in diameter, with a thin (c. 10 microm wide) eosinophilic-staining tegument, two suckers (oral and ventral), posteriorly located primordial genitalia and paired digestive caeca. These digeneans were identified as the metacercariae of Clinostomum attenuatum. This is the first record of Clinostomum attenuatum in these amphibian species.

Helminth community structure of sympatric eastern American toad, Bufo americanus americanus, northern leopard frog, Rana pipiens, and blue-spotted salamander, Ambystoma laterale, from southeastern Wisconsin.

One-hundred twelve amphibians, including 51 blue-spotted salamanders, Ambystoma laterale, 30 eastern American toads, Bufo americanus americanus, and 31 northern leopard frogs, Rana pipiens, were collected during April-October 1996 from Waukesha County, Wisconsin and examined for helminth parasites. The helminth compound community of this amphibian assemblage consisted of at least 10 species: 9 in American toads, 8 in leopard frogs, and 3 in blue-spotted salamanders. American toads shared 7 species with leopard frogs, and 2 species occurred in all 3 host species. Although there was a high degree of helminth species overlap among these sympatric amphibians, statistically significant differences were found among host species and percent of indirect or direct-life cycle parasites of amphibian species individual component communities (chi2 = 1,015, P < 0.001). American toads had a higher relative abundance of nematodes, 59%, than larval cestodes, 31%, and larval and adult trematodes, 10%, whereas leopard frogs had a higher relative abundance of larval cestodes, 71.3%, and larval and adult trematodes, 25.3%, than nematodes 3.4%. This is related to ecological differences in habitat and dietary preferences between these 2 anuran species. Helminth communities of blue-spotted salamanders were depauperate and were dominated by larval trematodes, 94%, and few nematodes, 6%. Low helminth species richness in this host species is related to this salamander's relatively small host body size, smaller gape size, lower vagility, and more fossorial habitat preference than the other 2 anuran species. Adult leopard frogs and toads had significantly higher mean helminth species richness than metamorphs, but there was no significant difference in mean helminth species richness among adult and metamorph blue-spotted salamanders. Considering adult helminths, the low species richness and low vagility of caudatans as compared with anurans suggest that local factors may be more important in structuring caudatan helminth communities of salamanders than of anuran hosts. Helminth species infecting salamanders may be more clumped in their geographic distribution as compared with anurans, and the role of other hosts and their parasites at the compound community level may be important in structuring helminth communities of salamanders.

[Infracommunity of parasite helminths of Ambystoma lernaensis (Caudata: Ambystomatidae) in Lerma, Mexico]. / Infracomunidades de helmintos parásitos de Ambystoma lermaensis (Caudata: Ambystomatidae) en Lerma, México.

The helminth infracommunity structure was analyzed in 48 salamanders (Ambystoma lermaensis) from San Pedro Tlaltizapán, Lerma, Estado de Mexico (June 1997 to March 1999). Richness (2.46 +/- 1.8), abundance (29.5 +/- 42.3), and diversity (0.64 +/- 0.58) levels characterize these communities as depauperate, similar to other amphibian helminth communities worldwide. Apparently, the main forces determining infracommunity structure are ectothermy (as regulator of the ingestion rate), and the opportunistic feeding habits of the hosts, because 80% of the helminth taxa enter the host by ingestion of intermediate hosts.

Differentiation of Mexican species of Haematoloechus looss, 1899 (Digenea: Plagiorchiformes): molecular and morphological evidence.

Molecular evidence is interpreted in the light of morphology to examine the validity of several species of Haematoloechus described as Mexican endemics. Internal transcribed spacers 1 and 2 and 28S ribosomal genes were sequenced for 11 isolates. Phylogenetic analysis of separate partitions and combined databases was conducted. Results were analyzed, in the light of morphological evidence. Haematoloechus macrorchis is proposed as a junior synonym of Haematoloechus longiplexus. Haematoloechus pulcher is a sibling species with Haematoloechus complexus in Lerma wetlands. In Mexico, Haematoloechus medioplexus is distributed along the east coast coinciding with the distribution of Rana berlandieri. The sister species of H. medioplexus is Haematoloechus coloradensis, sharing the distribution of the uterus as a synapomorphic character. Haematoloechus illimis is more closely related to H. medioplexus and H. coloradensis than to H. complexus. It can be distinguished by the distribution of the uterus, lobed ovary, and testes.

Morphology and infectivity of cultivated Trypanosoma ambystomae.

Cultures of Trypanosoma ambystomae were initiated and maintained in hypo-osmotic biphasic blood-agar medium. Epimastigotes and trypomastigotes occurred in culture and although they were similar in size, epimastigotes predominated with about 70% of all stages. Epimastigotes divided by equal binary fission and occasionally formed rosettes. Cultured trypomastigotes did not divide. Cultures isolated 118 days earlier were infective to all 3 laboratory-reared Ambystoma jeffersonianum and to 2 of 3 Ambystoma mexicanum; the latter is a new host record. Trypanosomes were morphometrically similar in both experimental hosts. Specimens from 20-day-old experimental infections were smaller than those from natural infections. Dividing bloodstream forms were detected in experimental infections. Standard culture conditions should be adopted if the morphology of culture forms is to have taxonomic significance.