Molecular insights versus morphological traits: rethinking identification of the closely related Angiostrongylus cantonensis and Angiostrongylus malaysiensis.

BACKGROUND: The closely related Angiostrongylus cantonensis and Angiostrongylus malaysiensis have been reported to coexist in Thailand and share similar hosts and life cycles. Recently, in an angiostrongyliasis outbreak in Thailand, both A. cantonensis and A. malaysiensis were found in the cerebrospinal fluid of affected patients. Morphological similarities, overlapping distribution, shared hosts and habitats, and the close genetics of the two Angiostrongylus species can complicate accurate species identification. Addressing these challenges, this study aims to evaluate whether a correlation between the morphological and genetic identities of A. cantonensis and A. malaysiensis can improve species identification accuracy. METHODS: Angiostrongylus spp. specimens from five zoogeographical regions in Thailand were subjected to morphological and molecular identification using the mitochondrial cytochrome b gene and the nuclear internal transcribed spacer 2 region (ITS2). The morphological characters for males and females were then validated using the species identity obtained from the nuclear ITS2 region. RESULTS: The results revealed that morphological misidentifications between these two closely related species are common due to overlapping morphological characters. Although certain male traits such as body length and width aided species differentiation, female traits were found to be less reliable. Furthermore, hybrid forms (8.2%) were revealed through the ITS2 results, which can further complicate morphological identification. Mito-nuclear discordance was also present in 1.9% of the Angiostrongylus specimens from Thailand, suggesting a complex historical interbreeding between the species. CONCLUSIONS: Based on our findings, we suggest that nuclear ITS2 is a reliable marker for species identification of A. cantonensis and A. malaysiensis, especially in regions where both species coexist. Additionally, the scope and consequences of hybridization between the two closely related Angiostrongylus species should be further investigated in Thailand.

Semperula wallacei (Mollusca, Veronicellidae) um hospedeiro natural recém-descoberto de Angiostrongylus cantonensis (Nematoda, Angiostrongylidae) na Bacia do Pacífico.

Semperula wallacei (Issel, 1874) is a species of terrestrial slug that occurs in southeast China and the Pacific Basin and is the only species of its genus that occurs beyond the Oriental region and to the east of Wallace's line in the Australian region, where it has probably been introduced. In this study, we report for the first time S. wallacei as an intermediate host for Angiostrongylus cantonensis (Chen, 1935) based on histological and molecular analyses of slugs from Tuamasaga, Samoa, deposited at the Medical Malacological Collection (Fiocruz-CMM). DNA was obtained from the deparafinized tissues scraped from specimen slides. Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) targeted to the internal transcribed spacer 2 (ITS2) region were carried out using the restriction enzyme Cla I. The RFLP profile observed for our larval specimen of S. wallacei was identical to the profile previously established for A. cantonensis, demonstrating that S. wallacei can be naturally infected with A. cantonensis and is likely to be an intermediate host for this parasitic nematode species in the field. The potential for geographical range expansion of S. wallacei in the Pacific Basin, its small size, and the general role of veronicellids as crop pests and hosts of nematodes, indicate the significance of S. wallacei as an invasive species in the Pacific Basin. Our work also highlights the importance of biological collections for investigating the environmental impact of invasive species on agriculture, public health, and biodiversity conservation.

[Progress of researches on the role and mechanisms of non - coding RNA in Angiostrongylus cantonensis infection].

Angiostrongylus cantonensis is a food-borne zoonotic parasite, and human infection may cause eosinophilic meningitis. Non-coding RNAs (ncRNAs) may regulate physiological and pathological processes at multiple biological levels; however, there are few studies pertaining to the regulatory role of ncRNAs in A. cantonensis infection. Based on publications retrieved from PubMed, Wanfang Data and CNKI, the regulatory role of ncRNAs in A. cantonensis infections mainly includes immune responses, cell apoptosis and signaling transduction, and ncRNAs may serve as biomarkers for diagnosis of angiostrongyliasis. This review summarizes the main roles of ncRNAs in A. cantonensis infections and the underlying mechanisms, so as to provide insights into diagnosis and treatment of angiostrongyliasis.

The recent introduction of Angiostrongylus cantonensis and its intermediate host Achatina fulica into Guadeloupe detected by phylogenetic analyses.

BACKGROUND: Angiostrongylus cantonensis (rat lungworm) is the main pathogen responsible for eosinophilic meningitis in humans. One of its intermediate snail hosts, Achatina fulica, was already present in many countries around the world before it appeared in the West Indies in the late 1980s. In the French territories in the Caribbean and northern South America, the first cases of human neuroangiostrongyliasis were reported in Martinique, Guadeloupe and French Guiana in 2002, 2013 and 2017, respectively. In order to better characterize angiostrongyliasis in Guadeloupe, particularly its geographical origin and route of introduction, we undertook molecular characterization of adult worms of Angiostrongylus cantonensis and its intermediate host Achatina fulica. METHODS: Genomic DNA of adult Angiostrongylus cantonensis and Achatina fulica was extracted and amplified by polymerase chain reaction (PCR) targeting the mitochondrial genes cytochrome B and C for A. cantonensis and 16S ribosomal RNA for A. fulica. The PCR products were sequenced and studied by phylogenetic analysis. RESULTS: Cytochrome B and cytochrome C molecular markers indicate a monophyletic lineage of A. cantonensis adult worms in Guadeloupe. Two sequences of A. fulica were identified. CONCLUSIONS: These results confirm the recent introduction of both Angiostrongylus cantonensis and Achatina fulica into Guadeloupe. Achatina fulica in Guadeloupe shares a common origin with those in Barbados and New Caledonia, while Angiostrongylus cantonensis in Guadeloupe shares a common origin with those in Brazil, Hawaii and Japan.

Identification and genetic characterization of Angiostrongylus cantonensis isolated from the human eye.

Angiostrongylus cantonensis, or the rat lungworm, is the causative agent of human angiostrongyliasis associated with eosinophilic meningitis or meningoencephalitis. Additionally, this nematode can cause ocular angiostrongyliasis, though this is rare. The worm can cause permanent damage to the affected eye and sometimes even blindness. Genetic characterization of the worm from clinical samples is limited. In the present study, we investigated the genetics of A. cantonensis recovered from a patient's eye in Thailand. We sequenced two mitochondrial genes (cytochrome c oxidase subunit I, or COI, and cytochrome b, or cytb) and nuclear gene regions (66-kDa protein and internal transcribed spacer 2, or ITS2) from a fifth-stage larva of Angiostrongylus sample that was surgically removed from the human eye. All sequences of the selected nucleotide regions were highly similar (98-100%) to the sequences of A. cantonensis in the GenBank database. The maximum likelihood and neighbor-joining trees of the COI gene indicated that A. cantonensis was closely related to the AC4 haplotype, whereas the cytb and 66-kDa protein genes were closely clustered with the AC6 and Ac66-1 haplotypes, respectively. In addition, the phylogeny of the concatenated nucleotide datasets of the COI and cytb revealed that the worm was closely related to the Thai strain and strains from other countries. This study confirms the identification and genetic variation of the fifth-stage larvae of A. cantonensis recovered from a patient's eye in Thailand. Our findings are important for future research on the genetic variation of A. cantonensis that causes human angiostrongyliasis.

Angie-LAMP for diagnosis of human eosinophilic meningitis using dog as proxy: A LAMP assay for Angiostrongylus cantonensis DNA in cerebrospinal fluid.

BACKGROUND: Angiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and a rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the other neurotropic Angiostrongylus species, which is native to Australia. METHODOLOGY/PRINCIPAL FINDINGS: Cerebrospinal fluid (CSF) from dogs with a presumptive diagnosis of A. cantonensis infection (2020-2022) were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR targeting AcanR3390. A newly designed LAMP assay targeting the same gene target was directly compared to the reference ultrasensitive qPCR in a diagnostic laboratory setting to determine the presence of A. cantonensis DNA to diagnose CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa = 0.81, 95%CI 0.69-0.92; n = 93) and rapid single-step lysis of archived CSF samples (Kappa = 0.77, 95%CI 0.59-0.94; n = 52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1. CONCLUSIONS/SIGNIFICANCE: The Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in canine CSF and performs comparably to a laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension, to humans.

The application of metagenomic next-generation sequencing for Angiostrongylus eosinophilic meningitis in a pediatric patient: A case report.

Background: Angiostrongylus eosinophilic meningitis (AEM) is a rare yet emerging disease caused by Angiostrongylus cantonensis infection. Its atypical symptoms may delay the diagnosis and cause fatal outcomes, especially in the early stages of infection and among children. Case presentation: Here we reported the use of metagenomic next-generation sequencing (mNGS) to facilitate the diagnosis and treatment of an 8-year-old boy with severe A. cantonensis infection. The mNGS tests consistently identified the infection of A. cantonensis prior to the detection by the immunologic method and confirmed it as AEM. Owing to the multidisciplinary team (MDT)-administrated treatments and close disease monitoring based on regular clinical tests and sequential mNGS tests, the patients eventually fully recovered from severe infectious conditions. Conclusion: This case demonstrated the advantages of mNGS for early diagnosis of AEM in pediatric patients, highlighting its application for pan-pathogen detection, as well as disease monitoring for severe A. cantonensis infection.

Autochthonous Angiostrongylus cantonensis Lungworms in Urban Rats, Valencia, Spain, 2021.

To determine the role of rats as potential reservoirs of zoonotic parasites, we examined rats trapped in urban sewers of Valencia, Spain, in 2021. Morphologic and molecular identification and sequencing identified autochthonous Angiostrongylus cantonensis nematodes, the most common cause of human eosinophilic meningitis, in pulmonary arteries of Rattus norvegicus and R. rattus rats.

[First report of invasive Pomacea snails in Shandong Province].

OBJECTIVE: To characterize the species of invasive Pomacea snails that were discovered for the first time in Shandong Province. METHODS: Pomacea snails samples were collected in the field of Jining City, Shandong Province on October 2021 for morphological identification. Pomacea snails were randomly sampled and genomic DNA was extracted from foot muscle tissues of Pomacea snails for multiplex PCR amplification. The PCR amplification product was sequenced. Then, the sequence was aligned and a phylogenetic tree was created using the software MegAlign 7.1.0. In addition, Angiostongylus cantonensis infection was detected in Pomacea snails with the lung microscopy. RESULTS: A total of 104 living Pomacea snails were collected, and all were characterized as Pomacea spp. based on morphological features. Of 12 randomly selected adult Pomacea snails, multiplex PCR assay and sequencing identified eleven snails as P. canaliculata and one as P. maculata. No A. cantonensis infection was detected in 104 Pomacea snails. CONCLUSIONS: This is the first report of invasive Pomacea snails in Shandong Province, where P. canaliculata and P. maculata are found.

A Rare Etiology for Ascending Paralysis in an Infant.

An 11-month-old male infant with ascending paralysis had an unremarkable initial cerebrospinal fluid (CSF) analysis and imaging. Progressive neurological symptoms resulted in repeated CSF sampling, microscopy, and plasma microbial cell-free DNA next-generation sequencing analysis, that in combination with epidemiology, confirmed the diagnosis.

Angiostrongylus cantonensis Nematode Invasion Pathway, Mallorca, Spain.

Neural angiostrongyliasis is an emerging zoonosis caused by the rat lungworm, Angiostrongylus cantonensis. In humans, infection with this nematode often results in eosinophilic meningitis and other severe disorders of the central nervous system. Europe was deemed a nonendemic region until 2018, when A. cantonensis worms were detected on the Mediterranean island of Mallorca, Spain, a tourism hotspot. Since that time, a sentinel surveillance system and a molecular approach have been used to follow the invasion path of the rat lungworm on the island. A. cantonensis worms have been found in animals from 8 locations on the island over 3 consecutive years. Our preliminary results show a recognizable pattern of clinical signs in infected hedgehogs and a single mitochondrial haplotype circulating in Mallorca. We present strong evidence confirming that the rat lungworm has successfully established and colonized an island in Europe and discuss observations and possible strategies for its early detection across continental Europe.

Angiostrongylus cantonensis in a Red Ruffed Lemur at a Zoo, Louisiana, USA.

A red ruffed lemur (Varecia rubra) from a zoo in Louisiana, USA, was euthanized for worsening paresis. Brain and spinal cord histology identified eosinophilic meningoencephalomyelitis with intralesional adult Angiostrongylus sp. nematodes. PCR and sequencing confirmed A. cantonensis infection, indicating this parasite constitutes an emerging zoonosis in the southeastern United States.

Upregulated galectin-1 in Angiostrongylus cantonensis L5 reduces body fat and increases oxidative stress tolerance.

BACKGROUND: Angiostrongylus cantonensis L5, parasitizing human cerebrospinal fluid, causes eosinophilic meningitis, which is attributed to tissue inflammatory responses caused primarily by the high percentage of eosinophils. Eosinophils are also involved in killing helminths, using the peroxidative oxidation and hydrogen peroxide (H2O2) generated by dismutation of superoxide produced during respiratory burst. In contrast, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival. In previous study, we demonstrated the extracellular function of Acan-Gal-1 in inducing the apoptosis of macrophages. Here, the intracellular functions of Acan-Gal-1 were investigated, aiming to further reveal the mechanism involved in A. cantonensis L5 worms surviving inflammatory responses in the human central nervous system. METHODS: In this study, a model organism, Caenorhabditis elegans, was used as a surrogate to investigate the intracellular functions of Acan-Gal-1 in protecting the worm from its host's immune attacks. First, structural characterization of Acan-Gal-1 was analyzed using bioinformatics; second, qRT-PCR was used to monitor the stage specificity of Acan-gal-1 expression in A. cantonensis. Microinjections were performed to detect the tissue specificity of lec-1 expression, the homolog of Acan-gal-1 in C. elegans. Third, microinjection was performed to develop Acan-gal-1::rfp transgenic worms. Then, oxidative stress assay and Oil Red O fat staining were used to determine the functions of Acan-Gal-1 in C. elegans. RESULTS: The results of detecting the stage specificity of Acan-gal-1 expression showed that Acan-Gal-1 was upregulated in both L5 and adult worms. Detection of the tissue specificity showed that the homolog of Acan-gal-1 in C. elegans, lec-1 was expressed ubiquitously and mainly localized in cuticle. Investigating the intracellular functions of Acan-Gal-1 in the surrogate C. elegans showed that N2 worms expressing pCe-lec-1::Acan-gal-1::rfp, with lipid deposition reduced, were significantly resistant to oxidative stress; lec-1 mutant worms, where lipid deposition increased, showed susceptible to oxidative stress, and this phenotype could be rescued by expressing pCe-lec-1::Acan-gal-1::rfp. Expressing pCe-lec-1::Acan-gal-1::rfp or lec-1 RNAi in fat-6;fat-7 double-mutant worms, where fat stores were reduced, had no significant effect on the oxidative stress tolerance. CONCLUSION: In C. elegans worms, upregulated Acan-Gal-1 plays a defensive role against damage due to oxidative stress for worm survival by reducing fat deposition. This might indicate the mechanism by which A. cantonensis L5 worms, with upregulated Acan-Gal-1, survive the immune attack of eosinophils in the human central nervous system.

Rat Lungworm (Angiostrongylus cantonensis) in the Invasive Cuban Treefrog (Osteopilus septentrionalis) in Central Florida, USA.

Cuban treefrogs, Osteopilus septentrionalis, were grossly examined for parasites and parasite species confirmed by PCR. Angiostrongylus cantonensis larvae were recovered from the hind leg muscle of O. septentrionalis. This is the first report of the zoonotic rat lungworm in the Cuban treefrog and new geographic location (Volusia County) in Florida, US.

Comparative proteomics suggests the mode of action of a novel molluscicide against the invasive apple snail Pomacea canaliculata, intermediate host of Angiostrongylus cantonensis.

Angiostrongylus cantonensis is a zoonotic parasitic nematode that is the most common cause of human eosinophilic meningitis. The invasive apple snail Pomacea canaliculata is an important intermediate host of A. cantonensis and contributes to its spread. P. canaliculata control will help prevent its invasion and transmission of A. cantonensis. The new molluscicide PBQ (1-(4-chlorophenyl)-3-(pyridin-3-yl)urea) exhibits great potency against P. canaliculata and has low toxicity against mammals and non-target aquatic organisms. We studied the mode of action of PBQ using TMT-based comparative quantitative proteomics analysis between PBQ-treated and control P. canaliculata snails. A total of 3151 proteins were identified, and 245 of these proteins were significantly differentially expressed with 135 downregulated and 110 upregulated. GO and KEGG enrichment analyses identified GO terms and KEGG pathways involved in de novo purine biosynthesis, ribosome components and translation process were significantly enriched and downregulated. The results indicated that PBQ treatment had substantial effects on the synthesis of genetic material, translation process, and protein synthesis of P. canaliculata and were likely the main cause of snail mortality.

Genetic Characterization and Detection of Angiostrongylus cantonensis by Molecular Approaches.

Angiostrongylus cantonensis constitutes a major etiologic agent of eosinophilic meningoencephalitis. The detection methods for angiostrongyliasis mainly depend on morphology or immunology. A firmer diagnosis could be reached by directly detecting the parasite in the cerebrospinal fluid or through laboratory assays that are specific for Angiostrongylus-induced antibodies or the parasite's DNA. A. cantonensis detection could be carried out by larva release from the tissue upon pepsin digestion. However, the procedure requires live mollusks, which might complicate the analysis of large amounts of samples. Since morphological assays are limited, multiple molecular techniques have been put forward for detecting A. cantonensis, including PCR amplification of targets followed by fragment length or DNA sequence analysis. This allows rapid and accurate identification of A. cantonensis for efficient infection management and epidemiological purposes. In this study, we reviewed the current methods, concepts, and applications of molecular approaches to better understand the genetic characterization, molecular detection methods, and practical application of molecular detection in A. cantonensis.

RPAcan3990: an Ultrasensitive Recombinase Polymerase Assay To Detect Angiostrongylus cantonensis DNA.

Angiostrongylus cantonensis is one of the leading causes of eosinophilic meningitis worldwide. A field-deployable molecular detection method could enhance both environmental surveillance and clinical diagnosis of this emerging pathogen. Accordingly, RPAcan3990, a recombinase polymerase assay (RPA), was developed to target a region predicted to be highly repeated in the A. cantonensis genome. The assay was then adapted to produce a visually interpretable fluorescent readout using an orange camera lens filter and a blue light. Using A. cantonensis genomic DNA, the limit of detection was found to be 1 fg/µl by both fluorometer measurement and visual reading. All clinical samples known to be positive for A. cantonensis from various areas of the globe were positive by RPAcan3990. Cerebrospinal fluid samples from other etiologies of eosinophilic meningitis (i.e., Toxocara sp. and Gnathostoma sp.) were negative in the RPAcan3990 assay. The optimal incubation temperature range for the reaction was between 35°C and 40°C. The assay successfully detected 1 fg/µl of A. cantonensis genomic DNA after incubation at human body temperature (in a shirt pocket). In conclusion, these data suggest RPAcan3990 is potentially a point-of-contact molecular assay capable of sensitively detecting A. cantonensis by producing visually interpretable results with minimal instrumentation.

Host snail species exhibit differential Angiostrongylus cantonensis prevalence and infection intensity across an environmental gradient.

Diverse snail species serve as intermediate hosts of the parasitic nematode Angiostrongylus cantonensis, the etiological agent of human neuroangiostrongyliasis. However, levels of A. cantonensis infection prevalence and intensity vary dramatically among these host species. Factors contributing to this variation are largely unknown. Environmental factors, such as precipitation and temperature, have been correlated with overall A. cantonensis infection levels in a locale, but the influence of environment on infection in individual snail species has not been addressed. We identified levels of A. cantonensis prevalence and intensity in 16 species of snails collected from 29 sites along an environmental gradient on the island of Oahu, Hawaii. The relationship between infection levels of individual species and their environment was evaluated using AIC model selection of Generalized Linear Mixed Models incorporating precipitation, temperature, and vegetation cover at each collection site. Our results indicate that different mechanisms drive parasite prevalence and intensity in the intermediate hosts. Overall, snails from rainy, cool, green sites had higher infection levels than snails from dry, hot sites with less green vegetation. Intensity increased at the same rate along the environmental gradient in all species, though at different levels, while the relation between prevalence and environmental variables depended on species. These results have implications for zoonotic transmission, as human infection is a function of infection in the intermediate hosts, ingestion of which is the main pathway of transmission. The probability of human infection is greater in locations with higher rainfall, lower temperature and more vegetation cover because of higher infection prevalence in the gastropod hosts, but this depends on the host species. Moreover, severity of neuroangiostrongyliasis symptoms is likely to be greater in locations with higher rainfall, lower temperature, and more vegetation because of the higher numbers of infectious larvae (infection intensity) in all infected snail species. This study highlights the variation of infection prevalence and intensity in individual gastropod species, the individualistic nature of interactions between host species and their environment, and the implications for human neuroangiostrongyliasis in different environments.

AcanR3990 qPCR: A Novel, Highly Sensitive, Bioinformatically-Informed Assay to Detect Angiostrongylus cantonensis Infections.

BACKGROUND: Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. METHODS: In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. RESULTS: The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. CONCLUSION: These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

Downregulated RPS-30 in Angiostrongylus cantonensis L5 plays a defensive role against damage due to oxidative stress.

BACKGROUND: Eosinophilic meningitis, caused by fifth-stage larvae of the nematode (roundworm) Angiostrongylus cantonensis, is mainly attributed to the contribution of eosinophils to tissue inflammatory responses in helminthic infections. Eosinophils are associated with the killing of helminths via peroxidative oxidation and hydrogen peroxide generated by the dismutation of superoxide produced during respiratory bursts. In contrast, when residing in the host with high level of eosinophils, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival in the hosts. In a previous study we demonstrated that the expression of the A. cantonensis RPS 30 gene (Acan-rps-30) was significantly downregulated in A. cantonensis L5 roundworms residing in cerebrospinal fluid with a high level of eosinophils. Acan-RPS-30 is a protein homologous to the human Fau protein that plays a pro-apoptotic regulatory role and may function in protecting worms from oxidative stress. METHODS: The isolation and structural characterization of Acan-RPS-30 were performed using rapid amplification of cDNA ends (RACE), genome walking and bioinformatics. Quantitative real-time-PCR and microinjection were used to detect the expression patterns of Acan-rps-30. Feeding RNA interference (RNAi) was used to knockdown the apoptosis gene ced-3. Microinjection was performed to construct transgenic worms. An oxidative stress assay was used to determine the functions of Acan-RPS-30. RESULTS: Our results showed that Acan-RPS-30 consisted of 130 amino acids. It was grouped into clade V with C. elegans in the phylogenetic analysis. It was expressed ubiquitously in worms and was downregulated in both L5 larvae and adult A. cantonensis. Worms expressing pCe-rps30::Acan-rps-30::rfp, with the refractile "button-like" apoptotic corpses, were susceptible to oxidative stress. Apoptosis genes ced-3 and ced-4 were both upregulated in the transgenic worms. The phenotype susceptible to oxidative stress could be converted with a ced-3 defective mutation and RNAi. rps-30-/- mutant worms were resistant to oxidative stress, with ced-3 and ced-4 both downregulated. The oxidative stress-resistant phenotype could be rescued and inhibited by through the expression of pCe-rps30::Acan-rps-30::rfp in rps-3-/- mutant worms. CONCLUSION: In C. elegans worms, downregulated RPS-30 plays a defensive role against damage due to oxidative stress, facilitating worm survival by regulating downregulated ced-3. This observation may indicate the mechanism by which A. cantonensis L5 worms, with downregulated Acan-RPS-30, survive in the central nervous system of humans from the immune response of eosinophils.