Molecular characterization of a novel Spiruromorpha species in wild Chinese pangolin by mitogenome sequence analysis.

Pangolins are susceptible to a variety of gastrointestinal nematodes due to their burrowing lifestyle and feeding habits, and few parasitic nematodes have been reported. Here, a Chinese pangolin with old wounds on its leg and tail was rescued from the Heyuan City, Guangdong Province. The cox1 and SSU rRNA of the worms from the intestine of the Chinese pangolin had the highest sequence identity of 89.58% and 97.95% to the species in the infraorder Spiruromorpha. The complete mitogenome of the worm was further assembled by next-generation sequencing, with a size of 13,708 bp and a GC content of 25.6%. The worm mitogenome had the highest sequence identity of 78.56% to that of Spirocerca lupi, sharing the same gene arrangement with S. lupi and some species in other families under Spiruromorpha. However, the mitogenome between the worm and S. lupi showed differences in codon usage of PCGs, sequences of NCR, and tRNA secondary structures. Phylogenetic analysis showed that the worm mitogenome was clustered with S. lupi in the family Thelaziidae to form a separate branch. However, it is still difficult to identify the worm in the family Thelaziidae because the species in the family Thelaziidae are confused, specifically S. lupi and Thelazia callipaeda in the family Thelaziidae were separated and grouped with species from other families. Thus, the parasitic nematode from the Chinese pangolin may be a novel species in Spiruromorpha and closely related to S. lupi. This study enriches the data on gastrointestinal nematodes in the Chinese pangolin.

CEBPα/miR-101b-3p promotes meningoencephalitis in mice infected with Angiostrongylus cantonensis by promoting microglial pyroptosis.

BACKGROUND: Angiostrongylus cantonensis (A. cantonensis) infection can induce acute inflammation, which causes meningoencephalitis and tissue mechanical injury to the brain. Parasite infection-induced microRNAs play important roles in anti-parasite immunity in non-permissive hosts. miR-101b-3p is highly expressed after A. cantonensis infection; however, the role of miR-101b-3p and the transcription regulation of miR-101b-3p in A. cantonensis infection remain poorly characterized. RESULTS: In the present study, we found that miR-101b-3p inhibition alleviated inflammation infiltration and pyroptosis in A. cantonensis infection. In addition, we found that CCAAT/enhancer-binding protein alpha (CEBPα) directly bound to the - 6-k to - 3.5-k region upstream of miR-101b, and CEBPα activated miR-101b-3p expression in microglia. These data suggest the existence of a novel CEBPα/miR-101b-3p/pyroptosis pathway in A. cantonensis infection. Further investigation verified that CEBPα promotes pyroptosis by activating miR-101b-3p expression in microglia, and microglial pyroptosis further promoted inflammation. CONCLUSIONS: Our results suggest that a CEBPα/miR-101b-3p/pyroptosis pathway may contribute to A. cantonensis infection-induced inflammation and highlight the pro-inflammatory effect of miR-101b-3p. Video Abstract.

Genus-level evolutionary relationships of FAR proteins reflect the diversity of lifestyles of free-living and parasitic nematodes.

BACKGROUND: Nematodes are a widespread and diverse group comprising free-living and parasitic species, some of which have major detrimental effects on crops, animals, and human health. Genomic comparisons of nematodes may help reveal the genetic bases for the evolution of parasitic lifestyles. Fatty acid and retinol-binding proteins (FARs) are thought to be unique to nematodes and play essential roles in their development, reproduction, infection, and possibly parasitism through promoting the uptake, transport, and distribution of lipid and retinol. However, the evolution of FAR family proteins across the phylum Nematoda remains elusive. RESULTS: We report here the evolutionary relationship of the FAR gene family across nematodes. No FAR was found in Trichocephalida species and Romanomermis culicivorax from Clade I, and FAR could be found in species from Clades III, IV, and V. FAR proteins are conserved in Clade III species and separated into three clusters. Tandem duplications and high divergence events lead to variable richness and low homology of FARs in Steinernema of Clade IVa, Strongyloides of Clade IVb, and intestinal parasitic nematodes from Clades Vc and Ve. Moreover, different richness and sequence variations of FARs in pine wood, root-knot, stem, and cyst nematodes might be determined by reproduction mode or parasitism. However, murine lungworm Angiostrongylus and bovine lungworm Dictyocaulus viviparus from Clade Vd have only 3-4 orthologs of FAR. RNA-seq data showed that far genes, especially far-1 and far-2, were highly expressed in most nematodes. Angiostrongylus cantonensis FAR-1 and FAR-3 have low sequence homology and distinct ligand-binding properties, leading to differences in the cavity volume of proteins. These data indicate that FAR proteins diverged early and experienced low selective pressure to form genus-level diversity. The far genes are present in endophyte or root-colonized bacteria of Streptomyces, Kitasatospora sp., Bacillus subtilis, and Lysobacter, suggesting that bacterial far genes might be derived from plant-parasitic nematodes by horizontal gene transfer. CONCLUSIONS: Data from these comparative analyses have provided insights into genus-level diversity of FAR proteins in the phylum Nematoda. FAR diversification provides a glimpse into the complicated evolution history across free-living and parasitic nematodes.

Speciation and adaptive evolution reshape antioxidant enzymatic system diversity across the phylum Nematoda.

BACKGROUND: Nematodes have evolved to survive in diverse ecological niches and can be a serious burden on agricultural economy, veterinary medicine, and public health. Antioxidant enzymes in parasitic nematodes play a critical role in defending against host oxidative stress. However, the features of the evolution of antioxidant enzymes in the phylum Nematoda remain elusive. RESULTS: Here, we systematically investigated the evolution and gene expression of antioxidant enzymes in the genomes of 59 nematodes and transcriptomes of 20 nematodes. Catalase has been independently lost in several orders, suggesting that it is unnecessary for some nematodes. Unlike in mammals, phospholipid hydroperoxide glutathione peroxidase is widely distributed in nematodes, among which it has evolved independently. We found that superoxide dismutase (SOD) has been present throughout nematode evolutionary process, and the extracellular isoform (SOD3) is diverged from the corresponding enzyme in mammals and has undergone duplication and differentiation in several nematodes. Moreover, the evolution of intracellular and extracellular SOD isoforms in filaria strongly indicates that extracellular SOD3 originated from intracellular SOD1 and underwent rapid evolution to form the diversity of extracellular SOD3. We identify a novel putative metal-independent extracellular SOD presenting independently in Steinernema and Strongyloididae lineage that featured a high expression level in Strongyloides larvae. Sequence divergence of SOD3 between parasitic nematodes and their closest free-living nematode, the specifically high expression in the parasitic female stage, and presence in excretory-secretory proteome of Strongyloides suggest that SOD3 may be related with parasitism. CONCLUSIONS: This study advances our understanding of the complex evolution of antioxidant enzymes across Nematoda and provides targets for controlling parasitic nematode diseases.

A novel strategy to improve the thermostability of Penicillium camembertii mono- and di-acylglycerol lipase.

Penicillium camembertii (PCL), a mono- and di-acylglycerol lipase (DGL), has the vital potential in the oil chemistry for food industry. However, known DGLs are mesophilic enzymes which restricts its application in the industry. To improve thermostability of PCL, we used amino acid substitution by comparison of amino acids compositions of PCL and protein sequences from typical thermophilic bacteria. Then, some conservative residues around active center were avoided to mutate according to homologous alignment analyses. Furthermore, the list was narrowed down to 28 candidate mutational sites of PCL by analyzing the hydrophobic interaction of amino acids in the structure. And among them only the mutant PCL-D25R had formed an additional salt bridge between R25-D32 and increased more hydrogen bonds interaction. Therefore, mutant PCL-D25R were constructed and expressed. Thermal inactivation assay showed that the half-life of mutant PCL-D25R at 45 °C increased 4-fold compared to that of PCL-WT. Melting temperature of mutant PCL-D25R increased to 49.5 °C from 46.5 °C by fluorescence-based thermal stability assay. This study provides a valuable strategy for engineering DGL thermostability.

Changes in erythrocyte polyunsaturated fatty acids and plasma eicosanoids level in patients with asthma.

BACKGROUND: To investigate the changes of polyunsaturated fatty acids (PUFAs) and their downstream eicosanoids in patients with asthma, the levels of erythrocyte membrane lipids and plasma lipid metabolites were examined. METHODS: Erythrocyte membrane lipids were extracted and esterificated, and then fatty acid compositions were determined by gas chromatography. The concentrations of six eicosanoids of PGE2, TXA2, LTB4, PGE1, 6-k-PGF1α and PGF2α in plasma were measured by ELISA. RESULTS: The results showed that the contents of erythrocyte membrane fatty acids in patients with asthma were mainly composed of C16:0, C18:0, C18:1, C18:2(n-6), and C20:4(n-6). The ratio n-6/n-3 PUFAs in patients and health persons were (4.42 ± 1.33):1 and (3.21 ± 0.79):1 (p < 0.01), showing statistically significant differences. ELISA results showed that the levels of plasma PGE2, TXB2, and PGE1 in patients were higher than health persons; and the levels of eicosanoids of PGF2α and 6-k-PGF1α were significantly lower in patient group than healthy group (p < 0.05), but LTB4 was no obvious difference (p = 0.09). Increased ratio of n-6/n-3 PUFAs is consistent to the increased levels of pro-inflammatory PGE2 and TXB2 and anti-inflammatory PGE1 originated from C20:4(n-6) and C18:2(n-6), indicating that increased ratio of n-6/n-3 PUFAs and eicosanoids from n-6 PUFAs might promote the progress of airway inflammation of asthma. CONCLUSION: Changes of erythrocyte fatty acids, n-6/n-3 PUFAs ratio and the levels of plasma PGE2, TXB2, and PGE1 in patients with asthma were relevant to airway inflammation in some extent. Therefore, it could be proposed that increase of n-3/n-6 PUFAs ratio by diet supplementation of n-3 PUFAs might effectively improve airway inflammation in asthma.

Angiostrongylus cantonensis: An optimized cultivation of this parasitic nematode under laboratory conditions.

Angiostrongylus cantonensis (A. cantonensis), a parasitic nematode, is the important neurotropic pathogen which causes human angiostrongyliasis. It has a complex life-cycle and severe parasite-host interaction in contrast to free-living nematode. Establishment of a well-suited life-cycle and in vitro cultivation of A. cantonensis in the laboratory will be one of the key techniques to elucidate the mechanism of parasite-host interaction. However, the low survival and growth rate of worms is still to be the problem. We optimized the known life-cycle of A. cantonensis in the laboratory, showing that small in size, easy to breed, and high compatibility of Biomphalaria straminea precede the common snails as an intermediate host of A. cantonensis. Furthermore, the egg hatching rate in Ham's F-12 medium reached approximately 80% using the eggs of mature female adult worms. We also demonstrated that the survival of larvae could be sustained for more than 30 days by in vitro cultivation of L1 larvae in DMEM with mixed antibiotics (100 units/mL of penicillin G potassium, 50 µg/mL of streptomycin sulfate, and 0.5 µg/mL of amphotericin B) and L3, L4, and L5 larvae in Waymouth's medium with 20% fetal calf serum and mixed antibiotics. Infective L1 and L3 larvae kept high infective rate to the snail and rat after cultivation in these media, respectively. It will provide the basis for studying on genetic manipulations for functional genes, new drug screening, and the mechanism of parasite-host interaction of parasitic nematodes.

Neuronal Apoptosis: Pathological Basis of Behavioral Dysfunctions Induced by Angiostrongylus cantonensis in Rodents Model.

Angiostrongylus cantonensis invades the central nervous system (CNS) of humans to induce eosinophilic meningitis and meningoencephalitis and leads to persistent headache, cognitive dysfunction, and ataxic gait. Infected mice (nonpermissive host), admittedly, suffer more serious pathological injuries than rats (permissive host). However, the pathological basis of these manifestations is incompletely elucidated. In this study, the behavioral test, histological and immunohistochemical techniques, and analysis of apoptotic gene expression, especially caspase-3, were conducted. The movement and motor coordination were investigated at week 2 post infection (PI) and week 3 PI in mice and rats, respectively. The cognitive impairs could be found in mice at week 2 PI but not in rats. The plaque-like lesion, perivascular cuffing of inflammatory cells, and dilated vessels within the cerebral cortex and hippocampus were more serious in mice than in rats at week 3 PI. Transcriptomic analysis showed activated extrinsic apoptotic pathway through increased expression of TNFR1 and caspase-8 in mice CNS. Immunohistochemical and double-labeling for NeuN and caspase-3 indicated the dramatically increased expression of caspase-3 in neuron of the cerebral cortex and hippocampus in mice but not in rats. Furthermore, western-blotting results showed high expression of cleaved caspase-3 proteins in mice but relatively low expression in rats. Thus, extrinsic apoptotic pathway participated in neuronal apoptosis might be the pathological basis of distinct behavioral dysfunctions in rodents with A. cantonensis infection. It provides the evidences of a primary molecular mechanism for the behavioral dysfunction and paves the ways to clinical diagnosis and therapy for A. cantonensis infection.

Screening and characterization of a thermostable lipase from marine Streptomyces sp. strain W007.

A screening method along with the combination of genome sequence of microorganism, pairwise alignment, and lipase classification was used to search the thermostable lipase. Then, a potential thermostable lipase (named MAS1) from marine Streptomyces sp. strain W007 was expressed in Pichia pastoris X-33, and the biochemical properties were characterized. Lipase MAS1 belongs to the subfamily I.7, and it has 38% identity to the well-characterized Bacillus subtilis thermostable lipases in the subfamily I.4. The purified enzyme was estimated to be 29 kDa. The enzyme showed optimal temperature at 40 °C, and retained more than 80% of initial activity after 1 H incubation at 60 °C, suggesting that MAS1 was a thermostable lipase. MAS1 was an alkaline enzyme with optimal pH value at 7.0 and had stable activity for 12 H of incubation at pH 6.0-9.0. It was stable and retained about 90% of initial activity in the presence of Cu(2+) , Ca(2+) , Ni(2+) , and Mg(2+) , whereas 89.05% of the initial activity was retained when ethylene diamine tetraacetic acid was added. MAS1 showed the tolerance to organic solvents, but was inhibited by various surfactants. MAS1 was verified to be a triglyceride lipase and could hydrolyze triacylglycerol and diacylglycerol. The result represents a good example for researchers to discover thermostable lipase for industrial application.

Association of decreased spermatozoa omega-3 fatty acid levels and increased oxidative DNA damage with varicocele in infertile men: a case control study.

Varicocele is commonly associated with male infertility because it impairs normal sperm morphology and activity. Polyunsaturated fatty acids (PUFA) are important determinants of sperm cell structure and function, but their relationship with varicocele remains unclear. The aim of the present study was to investigate the PUFA composition in spermatozoa of infertile men with varicocele and to evaluate the potential relationship between PUFA and varicocele. This case control study recruited 92 infertile men with varicocele, 99 infertile men without varicocele and 95 fertile male control subjects. Semen morphology and activity parameters were assessed and seminal plasma 8-hydroxy-2-deoxyguanosine (8-OHdG) content was determined by ELISA. Sperm concentrations of omega-3 and omega-6 fatty acids were measured by gas chromatography. Infertile men with varicocele had lower concentrations of omega-3 PUFA, higher omega-6:omega-3 PUFA ratios and greater oxidative DNA damage in spermatozoa compared with infertile men without varicocele and normal subjects. The degree of varicocele and DNA damage was associated with decreased omega-3 PUFA concentrations and semen quality in infertile men with varicocele. The findings suggest that omega-3 PUFA deficiency could be implicated in varicocele-associated infertility, and highlight the need for intervention trials to test the usefulness of omega-3 supplementation in reducing sperm abnormalities in infertile men with varicocele.

Ancestral genetic complexity of arachidonic acid metabolism in Metazoa.

Eicosanoids play an important role in inducing complex and crucial physiological processes in animals. Eicosanoid biosynthesis in animals is widely reported; however, eicosanoid production in invertebrate tissue is remarkably different to vertebrates and in certain respects remains elusive. We, for the first time, compared the orthologs involved in arachidonic acid (AA) metabolism in 14 species of invertebrates and 3 species of vertebrates. Based on parsimony, a complex AA-metabolic system may have existed in the common ancestor of the Metazoa, and then expanded and diversified through invertebrate lineages. A primary vertebrate-like AA-metabolic system via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways was further identified in the basal chordate, amphioxus. The expression profiling of AA-metabolic enzymes and lipidomic analysis of eicosanoid production in the tissues of amphioxus supported our supposition. Thus, we proposed that the ancestral complexity of AA-metabolic network diversified with the different lineages of invertebrates, adapting with the diversity of body plans and ecological opportunity, and arriving at the vertebrate-like pattern in the basal chordate, amphioxus.

Diversity and composition of the bacterial community in Amphioxus feces.

Amphioxus is a typical filter feeder animal and is confronted with a complex bacterial community in the seawater of its habitat. It has evolved a strong innate immune system to cope with the external bacterial stimulation, however, the ecological system of the bacterial community in Amphioxus remains unknown. Through massive parallel 16S rRNA gene tag pyrosequencing, the investigation indicated that the composition of wild and lab-cultured Amphioxus fecal bacteria was complex with more than 85,000 sequence tags being assigned to 12/13 phyla. The bacterial diversity between the two fecal samples was similar according to OTU richness of V4 tag, Chao1 index, Shannon index and Rarefaction curves, however, the most prominent bacteria in wild feces were genera Pseudoalteromonas (gamma Proteobacteria) and Arcobacter (epsilon Proteobacteria); the highly abundant bacteria in lab-cultured feces were other groups, including Leisingera, Phaeobacter (alpha Proteobacteria), and Vibrio (gamma Proteobacteria). Such difference indicates the complex fecal bacteria with the potential for multi-stability. The bacteria of habitat with 28 assigned phyla had the higher bacterial diversity and species richness than both fecal bacteria. Shared bacteria between wild feces and its habitat reached to approximately 90% (153/169 genera) and 28% (153/548 genera), respectively. As speculative, the less diversity of both fecal bacteria compared to its habitat partly because Amphioxus lives buried and the feces will ultimately end up in the sediment. Therefore, our study comprehensively investigates the complex bacterial community of Amphioxus and provides evidence for understanding the relationship of this basal chordate with the environment.

Biochemical properties of a new cold-active mono- and diacylglycerol lipase from marine member Janibacter sp. strain HTCC2649.

Mono- and di-acylglycerol lipase has been applied to industrial usage in oil modification for its special substrate selectivity. Until now, the reported mono- and di-acylglycerol lipases from microorganism are limited, and there is no report on the mono- and di-acylglycerol lipase from bacteria. A predicted lipase (named MAJ1) from marine Janibacter sp. strain HTCC2649 was purified and biochemical characterized. MAJ1 was clustered in the family I.7 of esterase/lipase. The optimum activity of the purified MAJ1 occurred at pH 7.0 and 30 °C. The enzyme retained 50% of the optimum activity at 5 °C, indicating that MAJ1 is a cold-active lipase. The enzyme activity was stable in the presence of various metal ions, and inhibited in EDTA. MAJ1 was resistant to detergents. MAJ1 preferentially hydrolyzed mono- and di-acylglycerols, but did not show activity to triacylglycerols of camellia oil substrates. Further, MAJ1 is low homologous to that of the reported fungal diacylglycerol lipases, including Malassezia globosa lipase 1 (SMG1), Penicillium camembertii lipase U-150 (PCL), and Aspergillus oryzae lipase (AOL). Thus, we identified a novel cold-active bacterial lipase with a sn-1/3 preference towards mono- and di-acylglycerides for the first time. Moreover, it has the potential, in oil modification, for special substrate selectivity.

A combined amplicon approach to nematode polyparasitism occurring in captive wild animals in southern China.

BACKGROUND: Gastrointestinal tract (GIT) nematodes prefer to live in the intestines of wild animals, causing damage and even death, and posing a zoonotic risk. The polyparasitism of GIT nematodes results in the complex dynamics of the nematode communities that occur naturally in wild animals. However, the nematode community in captive wild animals is poorly understood. METHODS: We combined  microscopic examination and amplicon sequencing for community diversity. RESULTS: We characterized GIT nematode assemblages to one order, one family, four genera, and ten species, in 512 fecal samples of 121 species from captive wild animals in southern China. The positive rate of GIT nematodes was 20.7% (106/512), including 42.3% (11/26) in reptiles, 26.5% (39/147) in herbivores, 25.0% (25/100) in non-human primates, 20.0% (5/25) in omnivores, 12.2% (9/74) in carnivores, and 12.1% (17/140) in avians. The dominant nematodes were Haemonchus contortus in herbivores and Trichuris species in primates. The nematode communities of arboreal primates differed from their terrestrial counterparts, reflecting both host phylogeny and ecological constraints. Soil-transmitted Strongyloides species were widespread throughout the herbivore, primate, avian, and carnivore communities, and tended to infect omnivorous primates and terrestrial herbivores. In addition, new Trichuris and Heterakis species were found in the nematode communities of captive porcupines and peafowls. CONCLUSION: This study highlights the variation in the composition of the GIT nematode community and strengthens the attention to the harms induced by zoonotic nematodes and co-infective nematodes with low species richness.

Effect of low­frequency repetitive transcranial magnetic stimulation on cognitive function in rats with medial temporal lobe epilepsy.

Epilepsy, especially the medial temporal lobe epilepsy (TLE), can result in cognitive impairment. Low­frequency repetitive magnetic stimulation (rTMS) has been verified to suppress neural excitability and reduce seizures. Given its potential in modifying cortical activity, we aimed to investigate its impact on cognitive function in the context of epilepsy, a condition where the use of rTMS has not been extensively explored. However, the influence on cognitive function has not yet been investigated. Therefore, this study aimed to investigate the effects of low­frequency rTMS on cognitive improvement in epileptic rats. Rats used in this study were randomly divided into five groups: the sham group, the epilepsy group, and three epilepsy groups treated with rTMS at different frequencies. Each group underwent the Morris water maze test to investigate hippocampus­dependent episodic memory, to evaluate their cognitive performance. Further assessments included patch clamp and western blot techniques to estimate the synaptic function in the hippocampus. Comparison between groups showed that low­frequency rTMS significantly reduced spontaneous recurrent seizures and improved spatial learning and memory impairment in epileptic rats. Additionally, rTMS remodeled the synaptic plasticity affected by seizures and notably enhanced the expression of AMPAR and synaptophysin. Low­frequency rTMS can antagonize the cognitive impairment caused by TLE, and promote synaptic connections.

Heteroplasmic mitochondrial genomes of a Raillietina tapeworm in wild Pangolin.

BACKGROUND: Raillietina species belong to the family Davaineidae, which parasitizes in a wide variety of mammals and birds, causing stunted growth, lethargy, emaciation, and digestive tract obstruction. However, only a limited number of Raillietina species have been identified in wild animals. METHODS: We analyzed and annotated the complete mitochondrial (mt) genome of a worm from the intestine of a wild pangolin using Illumina sequencing of whole genomic DNA. RESULTS: These findings showed the presence of two mtDNA sequences in Raillietina sp., designated as mt1 and mt2, with the lengths of 14,331 bp and 14,341 bp, respectively. Both the mts genomes of Raillietina sp. comprised 36 genes, containing 12 protein-coding genes (PCGs), 2 ribosomal RNAs, and 22 transfer RNAs. Gene arrangements of both mt genomes of Raillietina sp. were similar to those of most flatworms, except for taeniids, which shift positions between tRNAL1 and tRNAS2 genes. Twenty of 22 tRNA secondary structures of Raillietina sp. had a typical cloverleaf structure similar to Raillietina tetragona. Sequence differences between the mt1 and mt2 genomes were 4.4%, and this difference arises from the mtDNA heteroplasmic mutations. Moreover, heteroplasmic mtDNA mutations were detected in PCGs, tRNAs, rRNAs, NCRs, and intergenes, but the highest proportion of heteroplasmy of 79.0% was detected in PCGs, indicating the occurrence of mtDNA heteroplasmy in Raillietina sp. To our knowledge, this is the first report of mtDNA heteroplasmy in tapeworm parasites. Phylogenetic analyses of 18S rRNA, ITS2, and 12 PCG sequences demonstrated that the worm was clustered with other Raillietina species in the Davaneidae family. CONCLUSIONS: We found a novel Raillietina species in wild pangolin with the existence of mitochondrial DNA heteroplasmy. Thus, these findings provide insights into the heterogeneity of the mt genome in parasitic cestodes, and mt genome data contributes to the understanding of pangolin-parasitic cestodes in terms of their molecular biology, epidemiology, diagnosis, and taxonomy.

Molecular detection of a novel Ancylostoma sp. by whole mtDNA sequence from pangolin Manis javanica.

BACKGROUND: Ancylostoma species are hematophagous parasites that cause chronic hemorrhage in various animals and humans. Pangolins, also known as scaly anteaters, are mammals that live in soil environments where they are readily exposed to soil-borne parasitic nematodes. However, only a limited number of helminth species have been identified in this animal host so far. METHODS: Ancylostoma sp. was isolated from a wild pangolin, and the complete mitochondrial (mt) genome of Ancylostoma sp. was obtained by Illumina sequencing of total genomic DNA. RESULTS: The circular complete mt genome that was assembled had a total length of 13,757 bp and comprised 12 protein-coding genes (PCGs), 22 transfer ribosomal RNAs, two ribosomal RNAs (rRNAs), two non-coding regions and one AT-rich region, but lacked the gene coding for ATPase subunit 8 (atp8). The overall AT content of the mt genome of Ancylostoma sp. was 76%, which is similar to that of other nematodes. The PCGs used two start codons (ATT and TTG) and three stop codons (TAA, TAG, and T). The nucleotide identity of the 12 PCGs ranged from 83.1% to 89.7% and had the highest sequence identity with Ancylostoma caninum among species in the Ancylostomatidae family. Also, the pangolin-derived Ancylostoma sp. lacked repeat sequences in the non-coding regions and in the unique sequence of the short non-coding regions, which differentiated it from other Ancylostoma species. In addition, phylogenetic analyses of 18S rRNA and mtDNA sequences revealed that the Ancylostoma sp. was positioned in a separate branch in the subfamily Ancylostomatinae along with other Ancylostoma species. CONCLUSIONS: The Ancylostoma sp. isolated from a pangolin in this study was identified as a possible new Ancylostoma species. The identification of this Ancylostoma sp. from pangolin enriches our knowledge of the species in the Ancylostomatidae family and provides information that will lead to a better understanding of the taxonomy, diagnostics, and biology of hookworms.

Gut Microbiota Modulates Intestinal Pathological Injury in Schistosoma japonicum-Infected Mice.

Trapping of Schistosoma japonicum (S. japonicum) eggs in host tissue, mainly in the intestine and liver, causes severe gastrointestinal and hepatic granulomatous immune responses and irreversible fibrosis. Although the gut microbiota plays a central role in regulating pathological responses in several diseases, the effect of the gut microbiota on the pathologenesis progression of schistosomiasis remains largely unknown. In this study, we aimed to investigate the regulatory function of the gut microbiota in schistosomiasis japonica. We found that the depletion of the gut microbiota significantly ameliorated egg granulomas formation and fibrosis in the intestine of infected mice. This role of the gut microbiota in intestinal granuloma formation and fibrosis was reinforced when normal and infected mice were housed together in one cage. Notably, changes in the gut microbiota induced by S. japonicum infection were partly reversible with microbiota transfer in the cohousing experiment. Transfer of the gut microbiota from normal to infected mice attenuated the intestinal pathological responses. Depletion of the gut microbiota by antibiotics, or transfer of the gut microbiota from normal to infected mice decreased the levels of IL-4, IL-5, and IL-13 and promoted the production of cytokines and mRNA levels of IL-10 and TGF-ß in infected mice. Our findings indicated a regulatory effect of the gut microbiota on intestinal pathological injury associated with schistosomiasis japonica in mice, and thus suggested a potential strategy for schistosomiasis treatment.

The potential risk of Schistosoma mansoni transmission by the invasive freshwater snail Biomphalaria straminea in South China.

Schistosomes infect more than 200 million people worldwide, and globally, over 700 million people are at risk of infection. The snail Biomphalaria straminea, as one of the intermediate hosts of Schistosoma mansoni, consecutively invaded Hong Kong in 1973, raising great concern in China. In this study, a malacological survey was conducted over a period of four years, and investigations were performed on the mechanism of susceptibility of B. straminea to S. mansoni. B. straminea was investigated in China from 2014 to 2018. Out of 185 investigated sites, 61 were positive for stages of black B. straminea (BBS), which shows pigmented spots. Twenty of the 61 sites were positive for red B. straminea (RBS), which is partially albino and red colored. Phylogenetic analyses based on cox1 and 18S rRNA sequences demonstrated that both phenotypes were clustered with Brazilian strains. No S. mansoni infections were detected in field-collected snail. However, in laboratory experiments, 4.17% of RBS were susceptible to a Puerto Rican strain of S. mansoni, while BBS was not susceptible. The highest susceptibility rate (70.83%) was observed in the F2 generation of RBS in lab. The density of RBS has increased from south to north and from west to east in Guangdong since 2014. Five tyrosinase tyrosine metabolism genes were upregulated in BBS. Transcriptome comparisons of RBS and BBS showed that ficolin, C1q, MASP-like, and membrane attack complex (MAC)/perforin models of the complement system were significantly upregulated in BBS. Our study demonstrated that B. straminea is widely distributed in Hong Kong and Guangdong Province, which is expanding northwards very rapidly as a consequence of its adaptation to local environments. Our results suggest that B. straminea from South China is susceptible to S. mansoni, implying the high potential for S. mansoni transmission and increased S. mansoni infection risk in China.

Regulatory effect of host miR-101b-3p on parasitism of nematode Angiostrongylus cantonensis via superoxide dismutase 3.

MicroRNA plays a vital role in the regulation of host-parasite interaction. In recent years, genomic and transcriptomic resources have become increasingly available for many helminths, but only a limited number of reports in this area are on the regulatory effects of host microRNAs on parasitic nematodes. In this work, we screened increased expression of host microRNAs after nematode infection from miRNA-seq data and predicted target genes by combined bioinformatics analysis and transcriptional profiling. We elucidated regulatory effects of one host miRNA on nematode infection using miRNA inhibitor and adeno-associated virus (AAV)-based TuD miRNA inhibitor. Using AAV-based TuD miRNA inhibitor, we showed that stable blockade of mmu-miR-101b-3p could alleviate the pathological damages of Angiostrongylus cantonensis, a parasitic nematode. Data from a luciferase report assay showed that mmu-miR-101b-3p targeted the extracellular superoxide dismutase 3 (Acsod3). Increased Acsod3 expression in larvae and alleviated oxidative damages were seen in the groups receiving mmu-miR-101b-3p inhibitor treatment in vitro and AAV-based TuD miRNA inhibitor injection in vivo. Results of this study demonstrate that murine miR-101b-3p inhibits the expression of antioxidant enzyme in A. cantonensis to strengthen host oxidative responses to nematodes. This work expands our knowledge of interspecies regulation of nematode gene expression by of host miRNAs.