Global amphibian declines are linked with the presence of specific, highly virulent genotypes of the emerging fungal disease chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) known as the global panzootic lineage (Bd-GPL). The global trade in amphibians for human consumption is suspected to have facilitated emergence of the disease, but evidence to support this is largely lacking. Here, we investigated the role the Lithobates catesbeianus (North American bullfrog) trade in spreading Bd genotypes by comparing strains associated with L. catesbeianus to a global panel using 36 sequenced loci from multiple chromosomal regions. Most bullfrogs were infected with Bd-GPL genotypes, but we also detected novel, highly divergent Bd genotypes (Bd-Brazil) from a live bullfrog in a US market and from native Brazilian anurans in the Atlantic Forest where bullfrogs are widely farmed. Sexual reproduction was also detected for the first time in Bd in the form of a hybrid genotype between the Bd-GPL and Bd-Brazil lineages in the Atlantic Forest. Despite the demonstration that ribosomal RNA types in Bd fail to undergo concerted evolution (over 20 sequence types may be found in a single strain), the Bd-GPL and Bd-Brazil lineages form largely separate clusters of related internal transcribed spacer (ITS) RNA sequences. Using ITS sequences, we then demonstrate the presence of Bd-Brazil in Japan, primarily on invasive L. catesbeianus. The finding that Bd is capable of sexual reproduction between panzootic and endemic genotypes emphasizes the risk of international wildlife trade as a source of additional Bd epizootics owing to hybridization.

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Global trade in the North American bullfrog Rana catesbeiana has been implicated in the introduction and spread of the amphibian pathogen Batrachochytrium dendrobatidis. Large-scale production of the North American bullfrog for human consumption has been ongoing since the early part of the 20th century, particularly in Brazil, a pioneer in bullfrog farming following its introduction in the 1930s. In order to determine whether bullfrogs introduced to Brazil and farmed for the food trade serve as reservoirs for amphibian disease, we tested individuals located in the Brazilian states of Sa˜o Paulo and Par´a to determine the prevalence of B. dendrobatidis infections. We were able to confirm B. dendrobatidis on five farms (78.5% infection prevalence overall) by PCR. Isolates were obtained from three of the five farms and genotyped at 17 loci using multilocus sequence typing. Five isolates from the Brazilian farms were genotypically similar to each other as well as to isolates from Central and South America. Isolates from farmed bullfrogs were more similar to isolates from introduced populations in Venezuela than those from the native range of the bullfrog in eastern North America. These results could have important implications for the origin and spread of B. dendrobatidis in Brazil, its neighboring regions and the conservation of native amphibian fauna. They may also suggest a potential for prior recombination within B. dendrobatidis in bullfrogs, which may have implications for the recent emergence of this pathogen.

Abstract. Haemogregarina bigemina Laveran et Mesnil, 1901 was examined in marine fishes and the gnathiid isopod, Gnathia africana Barnard, 1914 in South Africa. Its development in fishes was similar to that described previously for this species. Gnathiids taken from fishes with H. bigemina, and prepared sequentially over 28 days post feeding (d.p.f.), contained stages of syzygy, immature and mature oocysts, sporozoites and merozoites of at least three types. Sporozoites, often five in number, formed from each oocyst from 9 d.p.f. First-generation merozoites appeared in small numbers at 11 d.p.f., arising from small, rounded meronts. Mature, second-generation merozoites appeared in large clusters within gut tissue at 18 d.p.f. They were presumed to arise from fan-shaped meronts, first observed at 11 d.p.f. Third-generation merozoites were the shortest, and resulted from binary fission of meronts, derived from second-generation merozoites. Gnathiids taken from sponges within rock pools contained only gamonts and immature oocysts. It is concluded that the development of H. bigemina in its arthropod host illustrates an affinity with Hemolivia and one species of Hepatozoon. However, the absence of sporokinetes and sporocysts also distances it from these genera, and from Karyolysus. Furthermore, H. bigemina produces fewer sporozoites than Cyrilia and Desseria, although, as in Desseria, Haemogregarina (sensu stricto) and Babesiosoma, post-sporogonic production of merozoites occurs in the invertebrate host. The presence of intraerythrocytic binary fission in its fish host means that H. bigemina is not a Desseria. Overall it most closely resembles Haemogregarina (sensu stricto) in its development, although the match is not exact.

Background information. Nitric oxide (NO) is an important molecule in innate immune responses. In molluscs NO is produced by mobile defence cells called haemocytes; however, the molecular mechanisms that regulate NO production in these cells is poorly understood. The present study focused on the role of cell signalling pathways in NO production by primary haemocytes from the snail Lymnaea stagnalis.Results. When haemocytes were challenged with PMA (10 µM) or the β-1,3-glucan laminarin (10 mg/ml), an 8-fold and 4-fold increase in NO production were observed after 60 min respectively. Moreover, the NOS (NO synthase) inhibitors L-NAME (N G -nitro-L-arginine methyl ester) and L-NMMA (N G -monomethyl-L-arginine) were found to block laminarin-and PMA-induced NO synthesis. Treatment of haemocytes with PMA or laminarin also increased the phosphorylation (activation) status of PKC (protein kinase C). When haemocytes were preincubated with PKC inhibitors (calphostin C or GF109203X) or inhibitors of the ERK (extracellular-signal-regulated kinase) pathway (PD98059 or U0126) prior to challenge, significant reductions in PKC and ERK phosphorylation and NO production were observed following exposure to laminarin or PMA. The greatest effect on NO production was seen with GF109203X and U0126, with PMA-induced NO production inhibited by 94% and 87% and laminarin-induced NO production by 50% and 91% respectively.Conclusions. These data suggest that ERK and PKC comprise part of the signalling machinery that regulates NOS activation and subsequent production of NO in molluscan haemocytes. To our knowledge, this is the first report that shows a role for these signalling proteins in the generation of NO in invertebrate defence cells.

Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.