Three new species of Cosmocerca Diesing, 1861 (Nematoda: Cosmocercidae) parasitising frogs Cacosternum boettgeri Boulenger, 1882, Kassina senegalensis Dumeril and Bibron, 1841 and Phrynomantis bifasciatus Smith, 1847 from South Africa.

Cosmocercid nematodes have been documented with much criticism due to the numerous inaccurate descriptions, redescriptions and synonymisation of found species. This is due to indistinguishable characters of females and the lack of male specimens found. Consequently, the species C. ornata is the most commonly found species worldwide and the only species of the genus reported in South Africa. In the present study, we found Cosmocerca in three different amphibian species, namely Cacosternum boettgeri, Kassina senegalensis and Phrynomantis bifasciatus. Based on differences in the shape of the gubernaculum and number of papillae, the found nematodes were assigned to three new species namely C. daly n. sp., C. monicae n. sp. and C. makhadoensis n. sp. Descriptions of species are followed by pairwise and phylogenetic analysis of partial ITS-28S sequences. All three species were found only in their host types from distant localities. Therefore, we hypothesise that host specificity of Cosmocerca from South African amphibians might be rather high and that the presence of C. ornata throughout South Africa is rather doubtful.

Taxonomy of the super-cryptic Hyperolius nasutus group of long reed frogs of Africa (Anura: Hyperoliidae), with descriptions of six new species.

Specimens from across the range of the Hyperolius nasutus species group were sequenced for two mitochondrial genes and one nuclear gene. Advertisement calls were recorded from the same specimens where possible, and morphological characters were compared. Bayesian inference and maximum likelihood produced a tree indicating 16 clades. The clades show little or no overlap in combinations of 16S sequence difference, shared tyr haplotypes, advertisement call parameters, snout profiles and webbing. On the basis of these data we recognise H. acuticeps, H. adspersus, H. benguellensis, H. dartevellei, H. igbettensis, H. nasutus, H. nasicus, H. poweri, H. viridis and describe six new species: Hyperolius friedemanni sp. nov. Mercurio & Rödel, Hyperolius howelli sp. nov. Du Preez & Channing, Hyperolius inyangae sp. nov. Channing, Hyperolious jacobseni sp. nov. Channing, Hyperolius rwandae sp. nov. Dehling, Sinsch, R6del & Channing, and Hyperolius lupiroensis sp. nov. Channing. Hyperolius lamottei is confirmed to be outside the H. nasutus group clade. Hyperolius granulatus, H. oxyrhynchus, H. punctulatus and H. sagitta are assigned as junior synonyms. As our results are based on a small number of specimens, these hypotheses await testing with larger sample sizes and more characters. A species distribution model suggests where outlier populations might be found.

Polystoma nacialtuneli n. sp. (Monogenea: Polystomatidae) from the eastern spadefoot, Pelobates syriacus (Pelobatidae) in Turkey.

Polystoma nacialtuneli n. sp. is described from the urinary bladder of the eastern spadefoot, Pelobates syriacus from Turkey. This is the fifth polystome species known from Turkey and the third species in Pelobates. We show that this new parasite species can be distinguished from other polystome species in the area by a combination of characteristics, including parasite size and the shape and size of the hamuli. Polystoma pelobatis from Pelobates cultripes has a pair of well-developed hamuli, while P. fuscus from Pe. fuscus characteristically has a pair of underdeveloped hamuli barely larger than the marginal hooklets. Polystoma nacialtuneli n. sp. has well-developed hamuli that vary significantly in shape. Phylogenetic relationships of P. nacialtuneli n. sp. within Polystoma, supplemented with molecular divergences estimated from internal transcribed spacer 1 (ITS1) sequences, indicate that they are well separated from their closest relatives, i.e. P. fuscus and P. pelobatis from Pe. fuscus and Pe. cultripes, respectively.

Invasive species threat: parasite phylogenetics reveals patterns and processes of host-switching between non-native and native captive freshwater turtles.

One of the major threats to biodiversity involves biological invasions with direct consequences on the stability of ecosystems. In this context, the role of parasites is not negligible as it may enhance the success of invaders. The red-eared slider, Trachemys scripta elegans, has been globally considered among the worst invasive species. Since its introduction through the pet trade, T. s. elegans is now widespread and represents a threat for indigenous species. Because T. s. elegans coexists with Emys orbicularis and Mauremys leprosa in Europe, it has been suggested it may compete with the native turtle species and transmit pathogens. We examined parasite transfer from American captive to the two native species that co-exist in artificial pools of a Turtle Farm in France. As model parasite species we used platyhelminth worms of the family Polystomatidae (Monogenea) because polystomes have been described from American turtles in their native range. Phylogenetic relationships among polystomes parasitizing chelonian host species that are geographically widespread show patterns of diversification more complex than expected. Using DNA barcoding to identify species from adult and/or polystome eggs, several cases of host switching from exotic to indigenous individuals were illustrated, corroborating that parasite transmission is important when considering the pet trade and in reintroduction programmes to reinforce wild populations of indigenous species.

Polystoma vernoni n. sp. (Monogenea: Polystomatidae) from the sharp nosed grass frog Ptychadena oxyrhynchus (Smith, 1849) in South Africa.

Polystoma vernoni (Polystomatidae) is a new species in the urinary bladder of Ptychadena oxyrhynchus in KwaZulu-Natal province, South Africa. Ptychadena is a true African species and the 49 currently known species in Africa harbour 11 polystome parasites. Ptychadena oxyrhynchus has an extensive distribution through Africa and harbours Polystoma prudhoei in West Africa. This new parasite differs significantly from P. prudhoei. The new species is distinguished by its long body relative to its haptor length, its unique shape and its ratio of marginal hooklets. In a sample of 13 frogs, 53.8% were infected with up to 18 parasites per host (mean intensity 4.86).

Polystoma okomuensis n. sp. (Monogenea: Polystomatidae) from Boulenger's striped frog, Phlyctimantis boulengeri (Perret, 1986) in Nigeria.

Polystoma okomuensis is described as a new species of the Polystomatidae parasitic in the urinary bladder of Boulenger's striped frog Phlyctimantis boulengeri in the Okomu National Park, Edo State, Nigeria. Although other African polystomes have been reported from Nigeria, this is the first to be described from the country and the first from Phlyctimantis. It is distinguished from other African Polystoma species by a combination of characters, including the body size, size and shape of the hamuli, size and shape of marginal hooklets and intestinal location. Phlyctimantis boulengeri was found to be infected in two of four seasonal lakes where specimens were caught with prevalences and mean intensities ranging from 14.3 to 22% and 1.0 to 1.5, respectively. Of the total number of 45 frogs examined, the prevalence was 15.6% and mean intensity 1.4.

Sediment TCDD-EQs and EROD and MROD activities in Ranid frogs from agricultural and nonagricultural sites in Michigan (USA).

In vitro studies have demonstrated atrazine-mediated induction of 7-ethoxyresorufin O-deethylase (EROD) activity. EROD is an enzyme active in the metabolism of many compounds, including many xenobiotics. These studies have suggested that atrazine may affect reproductive function by altering steroid metabolism. The goal of this study was to determine whether relationships could be detected between measured atrazine concentrations in surface waters and the liver-somatic index (LSI) and EROD and 7-methoxyresorufin O-deethylase (MROD) activities in the livers of ranid frogs. In addition, sediment dioxin toxic equivalents (TCDD-EQs) were determined using the H4IIE-luc cell bioassay. Adult and juvenile green frogs (Rana clamitans), bullfrogs (R. catesbeiana), and Northern leopard frogs (R. pipiens) were collected from areas with extensive corn cultivation and areas where there was little agricultural activity in south central Michigan in the summer of 2003. Atrazine concentrations at nonagricultural sites ranged from less than the limit of quantification (0.17 microg atrazine/L) to 0.23 microg atrazine/L and did not exceed 1.2 microg atrazine/L at agricultural sites. Sediment TCDD-EQs were measurable only at one agricultural site. Of the measured parameters, only LSI values in adult male frogs differed significantly between agricultural and nonagricultural sites, with greater values observed at agricultural sites. In green frogs, EROD and MROD activities were measurable in both adult and juvenile frogs and were similar among sites. Median EROD activities ranged from 13 to 21 pmol/min/mg protein in adult male green frogs and from 5 to 13 pmol/min/mg protein in adult female green frogs. Juvenile frogs had greater EROD and MROD activities than adult frogs. Bullfrogs and leopard frogs had greater activities than did green frogs. Atrazine concentrations were significantly and negatively correlated with MROD activity in adult male green frogs (Spearman R = -0.800). LSI and EROD and MROD activities of adult female or juvenile green frogs were not significantly correlated with atrazine concentrations. These results suggest that atrazine does not appear to have a consistent association with EROD or MROD activities in wild-caught green frogs.

Plasma steroid hormone concentrations, aromatase activities and GSI in ranid frogs collected from agricultural and non-agricultural sites in Michigan (USA).

The triazine herbicide atrazine has been hypothesized to disrupt sexual development in frogs by up-regulating aromatase activity, resulting in greater estradiol (E2) concentrations and causing feminization in males. The goal of this study was to collect native ranid frogs from atrazine-exposed ponds and determine whether relationships exist between measured atrazine concentrations and the gonadosomatic index (GSI), plasma concentrations of testosterone (T), E2 or 11-ketotestosterone (KT), or with aromatase activity. In the summer of 2002 and 2003, adult and juvenile green frogs (Rana clamitans), bullfrogs (R. catesbeiana) and Northern leopard frogs (R. pipiens) were collected from areas with extensive corn cultivation and areas where there was little agricultural activity in south-central Michigan. Atrazine concentrations were below the limit of quantification at non-agricultural sites. Atrazine concentrations did not exceed 2 microg/L at most agricultural sites, but a concentration of 250 microg atrazine/L was measured in one sample from one site in 2002. Plasma steroid concentrations varied among locations. Aromatase activity was measurable in less than 11% of testes in adult males, and in less than 4% of testes in juvenile males. Median aromatase activities in ovaries of adult females ranged from 3 to 245 pmol/h/mg protein, and maximum activities were 2.5-fold greater in juveniles than in adults. Atrazine concentrations were not significantly correlated with any of the parameters measured in this study. These results indicate that atrazine does not up-regulate aromatase in green frogs in the wild, and does not appear to affect plasma steroid hormone concentrations.

Atrazine concentrations, gonadal gross morphology and histology in ranid frogs collected in Michigan agricultural areas.

The triazine herbicide atrazine has been suggested to be a potential disruptor of normal sexual development in male frogs. The goals of this study were to collect native ranid frogs from sites in agricultural and non-agricultural areas and determine whether hypothesised atrazine effects on the gonads could be observed at the gross morphological and histological levels. Juvenile and adult green frogs (Rana clamitans), bullfrogs (R. catesbeiana) and leopard frogs (R. pipiens) were collected in the summers of 2002 and 2003. Atrazine concentrations were below the limit of quantification at non-agricultural sites, and concentrations did not exceed 2 microg/L at most agricultural sites. One concentration greater than 200 microg atrazine/L was measured once at one site in 2002. Hermaphroditic individuals with both male and female gonad tissue in either one or both gonads, were found at a low incidence at both non-agricultural and agricultural sites, and in both adults and juveniles. Testicular oocytes (TO) were found in male frogs at most of the sites, with the greatest incidence occurring in juvenile leopard frogs. TO incidence was not significantly different between agricultural and non-agricultural sites with the exception of juveniles collected in 2003. Atrazine concentrations were not significantly correlated with the incidence of hermaphroditism, but maximum atrazine concentrations were correlated with TO incidence in juvenile frogs in 2003. However, given the lack of a consistent relationship between atrazine concentrations and TO incidence, it is more likely the TOs observed in this study result from natural processes in development rather than atrazine exposure.

Seasonal exposures to triazine and other pesticides in surface waters in the western Highveld corn-production region in South Africa.

The objective of this study was to characterize concentrations of atrazine, terbuthylazine, and other pesticides in amphibian habitats in surface waters of a corn-production area of the western Highveld region (North-West Province) of South Africa. The study was conducted from November 2001 to June 2002, coinciding with the corn-production season. Pesticide residues were measured at regular intervals in surface water from eight ponds, three in a non-corn-growing area (NCGA) and five within the corn-growing area (CGA). Measured atrazine concentrations differed significantly among sites and between samples. In the five CGA sites, the maximum atrazine concentrations measured during the study ranged from 1.2 to 9.3 microg/L. Although no atrazine was recorded as being applied in the catchment of the three NCGA sites, maximum concentrations from 0.39 to 0.84 microg/L were measured during the study, possibly as a result of atmospheric transport. Maximum measured concentrations of terbuthylazine ranged from 1.22 to 2.1 microg/L in the NCGA sites and from 1.04 to 4.1 microg/L in the CGA sites. The source of terbuthylazine in the NCGA sites may have been in use other than in corn. The triazine degradation products, deisopropylatrazine (DIA) and deethylatrazine (DEA) and diaminochlorotriazine (DACT) were also found in water from both the CGA and NCGA sites. Concentrations of DIA were > or = 1 microg/L throughout the season, while DEA concentrations were mostly <0.5 microg/L before planting but increased after planting and application of herbicides to concentrations >2 microg/L in some locations. Concentrations of DACT were highly variable (LOD to 8 microg/L) both before and after planting and application, suggesting that they resulted from historical use of triazines in the area. Other herbicides such as simazine and acetochlor were only detected infrequently and pesticides such as S-metolachlor, cypermethrin, monocrotophos, and terbuphos, known to be used in the CGA, were not detected in any of the samples. Because of dilution by higher than normal rainfall in the study period, these concentrations may not be predictive of those in years of normal rainfall.

A redescription of Chabaudus leberrei (Bain & Philippon, 1969) (Nematoda: Seuratoidea) from Xenopus spp. in Swaziland.

Chabaudus leberrei (Bain & Philippon, 1969) is redescribed from the pipid anurans Xenopus muelleri (Peters) and X. laevis laevis (Daudin) (new host records) in northern Swaziland, based on light and scanning electron microscope studies. The six anterior protuberances characteristic of the genus Chabaudus Inglis & Ogden, 1965, are, in C. leberrei, formed by bipartite lamellae associated with the internal margins of the three lips. Intraspecific variation in the number and disposition of male caudal papillae and in the development of the cephalic vesicle is documented.

Sundapolystoma chalconotae n. g., n. sp. (Monogenea: Polystomatidae) from Rana chalconota (Schlegel) of Peninsular Malaysia.

Sundapolystoma chalconotae. n. g., n. sp. (Polystomatidae, Polystomatinae) is proposed for a new polystomatid from the urinary bladder of Rana chalconota (Schlegel) in Peninsular Malaysia. This is the first species of polystomatid to be described from the amphibians of Peninsular Malaysia and the second for the Southeast Asian region. This new genus, as exemplified by S. chalconotae, differs from other polystomatids, and in particular Parapolystoma Ozaki, 1935 (P. bulliense (Johnston, 1912) Ozaki, 1935 and P. johnstoni Pichelin, 1995), in having a tubular uterus and a single diffuse testis. P. crooki Vande Vusse, 1976 is similar to S. chalconotae in having a similar type of uterus and testis, and is re-assigned as Sundapolystoma crooki (Vande Vusse, 1976) n. comb. S. chalconotae differs from S. crooki in having anchors with a longer outer root rather than a longer inner root and 7-8 genital spines compared to 9-13 in S. crooki.

Differentiation of two locally sympatric Protopolystoma (Monogenea: Polystomatidae) species by temperature-dependent larval development and survival.

The developmental response of egg stages to different environmental temperature regimes was studied in Protopolystoma xenopodis and Protopolystoma orientalis (Monogenea: Polystomatidae) isolates from southern Africa. Eggs failed to develop at 10 degrees C, whilst at 15 degrees C only P. xenopodis completed larval development, hatching 49--88 days post-collection. Respective hatching windows were 26--34 (P. xenopodis) and 37--49 (P. orientalis) days at 20 degrees C, and 18--26 and 27--37 days at 25 degrees C. Continuous maintenance at 30 degrees C was lethal for eggs of both species. There were no consistent interspecific differences in the response of egg stages to low and high temperature shocks during early embryonic development.

Neopolystoma liewi sp. n. (Monogenea: Polystomatidae) from the eye of the Malayan box turtle (Cuora amboinensis).

Neopolystoma liewi sp. n. is described from the conjunctival cavity of the Malayan box turtle Cuora amboinensis (Daudin, 1802), in Peninsular Malaysia. This is the first record of Neopolystoma in Malaysia and the fourth polystomatid species described from C. amboinensis. Of the 27 Malayan box turtles examined, 8 were found to be infected. A maximum of 2 parasites per eye and 4 individuals per host was recorded. N. liewi sp. n. differs from all other members of the genus by possessing few and short genital spines and small marginal hooks. The oncomiracidium has 64 ciliated cells arranged symmetrically about the sagittal axis.

Supporting experimental evidence of host specificity among southern African polystomes (Polystomatidae:Monogenea).

Although monogeneans of anurans are generally regarded as host-specific, there is a lack of conclusive experimental evidence. Infection and cross-infection experiments were conducted with oncomiracidia of Polystoma australis and P. marmorati. In a series of experiments, oncomiracidia were given a choice between natural and substitute host tadpoles. Oncomiracidia of P. australis became established in substitute hosts but showed a preference for the natural host, whereas the oncomiracidia of P. marmorati showed a strong and statistically significant preference for the natural host. The results indicated that although the oncomiracidia of southern African polystomes showed a strong preference for their natural hosts, not all parasites exercised the same degree of host specificity.