BackgroundEosinophilic meningitis (angiostrongyliasis) caused by Angiostrongylus cantonensis is emerging in mainland China. However, the distribution of A. cantonensis and its intermediate host snails, and the role of two invasive snail species in the emergence of angiostrongyliasis, are not well understood.Methodology/Principal FindingsA national survey pertaining to A. cantonensis was carried out using a grid sampling approach (spatial resolution: 40×40 km). One village per grid cell was randomly selected from a 5% random sample of grid cells located in areas where the presence of the intermediate host snail Pomacea canaliculata had been predicted based on a degree-day model. Potential intermediate hosts of A. cantonensis were collected in the field, restaurants, markets and snail farms, and examined for infection. The infection prevalence among intermediate host snails was estimated, and the prevalence of A. cantonensis within P. canaliculata was displayed on a map, and predicted for non-sampled locations. It was confirmed that P. canaliculata and Achatina fulica were the predominant intermediate hosts of A. cantonensis in China, and these snails were found to be well established in 11 and six provinces, respectively. Infected snails of either species were found in seven provinces, closely matching the endemic area of A. cantonensis. Infected snails were also found in markets and restaurants. Two clusters of A. cantonensis–infected P. canaliculata were predicted in Fujian and Guangxi provinces.Conclusions/SignificanceThe first national survey in China revealed a wide distribution of A. cantonensis and two invasive snail species, indicating that a considerable number of people are at risk of angiostrongyliasis. Health education, rigorous food inspection and surveillance are all needed to prevent recurrent angiostrongyliasis outbreaks.

An infection with Angiostrongylus cantonensis, the main causative agent for human eosinophilic encephalitis, can be acquired through the consumption of the freshwater snail Pomacea canaliculata. This snail also provides a suitable model to study the developmental morphology and behavior of A. cantonensis larvae, facilitated by the snail's distinct lung structure. We used microanatomy for studying the natural appearance and behavior of A. cantonensis larvae while developing within P. canaliculata. The distribution of refractile granules in the larval body and characteristic head structures changed during the developmental cycle. Two well-developed, rod-like structures with expanded knob-like tips at the anterior part were observed under the buccal cavity as early as the late second developmental stage. A "T"-shaped structure at the anterior end and its tenacity distinguished the outer sheath from that shed during the second molting. Early first-stage larvae obtained from fresh rat feces are free moving and characterized by a coiled tail, whereas a mellifluous "Q"-movement was the behavioral trait of third-stage A. cantonensis larvae outside the host tissue. In combination, the distribution of refractive granules, distinct head features, variations in sheaths, and behavioral characteristics can be utilized for differentiation of larval stages, and for distinguishing A. cantonensis larvae from those of other free-living nematodes.

AimTo determine the genetic diversity of invasive snails (Pomacea spp.) that are implicated in crop damage, environmental degradation and human disease, and to determine their distribution pattern in a large part of eastern Asia.Location People's Republic of China (P.R. China). MethodsWe collected Pomacea snails in a national survey using a grid sampling approach. Overall, 544 snails from 54 sites were used for the present investigation. The mitochondrial cox1 gene was amplified and sequenced from all the snails. We determined and classified the haplotypes using network analyses and mapped them within P.R. China. Haplotypes from this study, together with sequences available from GenBank, were used to reveal the global distribution of Pomacea canaliculata and P. insularum. Results We obtained 521 cox1 sequences and identified 24 unique haplotypes. Six haplotypes were commonly found in P.R. China. Two species, P. canaliculata and P. insularum, and one cryptic group were observed. The distribution of the 24 haplotypes across P.R. China shows a mosaic pattern. Globally, only six of 112 haplotypes of P. canaliculata, P. insularum, P. dolioides, P. lineata and P. paludosa are shared between introduced and native snail populations. We found 16 haplotype clusters, five of which occur in mainland P.R. China. Three of the five clusters could be traced back to South America. The remaining two clusters were unique to P.R. China.Main conclusions Phylogenetic analyses indicate that P. canaliculata, P. insularum and two cryptic groups, discovered by the present and previous studies, coexist in the mainland of P.R. China. The mosaic distribution and the high diversity found in the collection sites suggests multiple and secondary introductions. The findings indicate the importance of preventing further intentional introductions and call for appraisal of the risk posed by these snails in vulnerable areas. Discrimination of the ecological impacts of the different species or genotypes will help to develop setting-specific management strategies.

The two rodent intra-arterial nematodes, Angiostrongylus cantonensis and Angiostrongylus costaricensis, can cause human ill-health. The present study aimed to characterize and compare the mitochondrial (mt) genomes of these two species, and clarify their phylogenetic relationship and the position in the phylum Nematoda. The complete mt genomes of A. cantonensis and A. costaricensis are 13,497 and 13,585 bp in length, respectively. Hence, they are the smallest in the class of Chromadorea characterized thus far. Like many nematode species in the class of Chromadorea, they encode 12 proteins, 22 transfer RNAs, and two ribosomal RNAs. All genes are located on the same strand. Nucleotide identity of the two mt genomes is 81.6%, ranging from 77.7% to 87.1% in individual gene pairs. Our mt genome-wide analysis identified three major gene arrangement patterns (II-1, II-2, and II-3) from 48 nematode mt genomes. Both patterns II-1 and II-2 are distinct from pattern II-3, which covers the Spirurida, supporting a closer relationship between Ascaridida and Strongylida rather than Spirurida. Thymine (T) was highly concentrated on coding strands in Chromadorea, but balanced between the two strands in Enoplea, probably due to the gene arrangement pattern. Interestingly, the gene arrangement pattern of mt genomes and phylogenetic analysis based on concatenated amino acids indicated a closer relationship between the order Ascaridida and Rhabditida rather than Spirurida as indicated in previous studies. These discrepancies call for new research, reassessing the position of the order of Ascaridida in the phylogenetic tree. Once consolidated, the findings are important for population genetic studies and target identification.

Angiostrongyliasis cantonensis, clinically presented as eosinophilic meningitis, is a snail-borne parasitic disease. We studied the effects of different temperatures on the larval development of Angiostrongylus cantonensis in the freshwater snail Pomacea canaliculata. Six groups of snails were infected and each group was cultured under different temperature conditions. At predefined intervals, four snails from each group were dissected to examine the larval development. The development-time curve of each group was drawn according to the fraction of third-stage larvae present. The developmental time was defined as the time needed until 50% of the first-stage larvae developed into third-stage larvae. A linear regression model was established based on the time (D; in days) and the corresponding temperature (T; in degrees Celsius): DT = 15.04 x D + 262.53. The threshold temperature for larval development was 15.04 degrees C and the thermal constant was 262.53 degree-days. These parameters could be helpful in estimating the number of parasite generations in a year and the impact of climate change on the distribution of A. cantonensis.

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Abstract. The human lung fluke Paragonimus is transmitted by gastropod taxa of two superfamilies: Ceritheoidea and Rissooidea. The question whether or not Paragonimus shows the same specificity of host-parasite coevolved relationship as the human blood fluke Schistosoma was inspired by the finding of two sympatric snail species as hosts for Paragonimus skrjabini in Fujian Province, China: Gammatricula and Erhaia. The former species can clearly be classified as Pomatiopsidae: Triculinae. The latter has previously been classified as Pomatiopsidae: Pomatiopsinae. However, this classification based on anatomical characteristics is uncertain. In order to obtain a robust phylogenetic hypothesis for Erhaia, we have studied three gene fragments from this taxon as well as from twelve related taxa. The data show that the species involved represent four families: Pomatiopsidae, Hydrobiidae, Cochliopidae (here raised to family status), and Amnicolidae. Erhaia fits securely into the Amnicolidae. This indicates that P. skrjabini has not coevolved with snail lineages. However, P. skrjabini has so far only been reported from rissooidean snails, whereas members of the Paragonimus westermani complex have only been found in ceritheoidean snails. The implication is that there is a host specificity on the superfamily level. However, Asian freshwater species of the Ceritheoidea and Rissooidea usually are not sympatric and often prefer different habitats. It is therefore possible that ecological niche partitioning plays the primary role for Paragonimus evolution.

Hepatocellular carcinoma (HCC) is the fourth largest cause of cancer-related deaths worldwide with limited therapeutic interventions. Renewed interest in natural products as drug leads has resulted in a paradigm shift toward the rapid screening of medicinal plants for the discovery of new chemical entities. Rotundic acid (RA), a plant-derived triterpenoid, has been anecdotally reported to possess anti-inflammatory and cardio-protective abilities. The present study highlights the anti-cancer efficacy of RA on HCC in vitro and in vivo . The inhibitory effects of RA on HCC cell viability was determined by MTT. Soft agar colony formation and clonogenic assays also showed that RA inhibited HCC cell proliferation. Flow cytometry, confocal, and western blot results further indicated that RA induced cell cycle arrest, DNA damage, and apoptosis by modulating the AKT/mTOR and MAPK pathways. Besides the suppression of migration and invasion, tube formation and VEGF-ELISA revealed the anti-angiogenic abilities of RA on HCC. Moreover, RA also inhibited tumor growth in a HepG2 xenograft mouse model. To our best knowledge, this is the first extensive study of the anticancer activity of RA on HCC. The results demonstrate that RA could be a potential drug candidate for HCC treatment.