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

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. 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. 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. 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.


Background
Angiostrongylus cantonensis is a human zoonotic pathogen that may cause eosinophilic meningitis [1]. Several different hosts are required to complete the life-cycle of A. cantonensis. Humans are an atypical host are are mainly be infected through accidental ingestion of undercooked intermediate hosts, such as the golden apple/ channeled apple snail Pomacea canaliculata in which the infective third-stage larvae (iL3) resides [2]. After passage to the small intestine, iL3 will penetrate the blood-brain barrier, subsequently infecting the central nervous system where it will develop into the fifth-stage larvae (L5) and cause angiostrongyliasis with neurological symptoms [3][4][5][6].
Eosinophils, recruited from the circulation into the central nervous system [3], are robust producers of extracellular superoxide due to expression of high levels of the enzyme complex that generates superoxide [7], thereby contributing to tissue inflammatory responses and host defense in helminthic infections [8]. Eosinophil peroxidase (EPO), resident in the granule matrix of eosinophils, would be released in this response [9,10]. EPO is associated with the killing of helminths through peroxidative oxidation and the hydrogen peroxide (H 2 O 2 ) generated by dismutation of the superoxide produced during respiratory bursts [11][12][13]. In contrast, residing in a host with high levels of eosinophils, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses to facilitate their survival in that host [8]. Therefore, A. cantonensis L5 that reside in the cerebrospinal fluid together with eosinophils may be resistant to damage by oxidative stress. In a previous study, we showed that the expression level of A. cantonensis ribosomal protein 30 (Acan-RPS-30) was lower in L5 than in iL3, based on the proteomic analysis of different developmental stages using two-dimensional difference gel electrophoresis [4].
Acan-rps-30 is a homologous gene of human Fau [14] (FBR-MuSV associated ubiquitously expressed gene), which was originally isolated from a radiation-induced osteosarcoma [15]. Fau is inversely inserted as the fox sequence in FBR-MuSV [16,17], and expression of fox enhances the transformation of FBR-MuSV, presumably by inactivating Fau expression [18,19]. Fau may play an important role in inhibiting tumorigenesis, based on results showing that it is downregulated in both breast cancer [20] and ovarian cancer [21]. Fau also regulates apoptosis in human T-cell lines and HEK293/17 cells [20]. A sequence antisense to Fau is able to decrease apoptosis induced by dexamethasone, ultraviolet light or cisplatin in W7.2c cells [19]. In the parasitic nematode Haemonchus contortus, RPS-30 can regulate the fourth-stage larval diapauses [22]. Fau encodes an ubiquitin-like protein (UBiL) fused to ribosomal protein S30 (S30) as a carboxy-terminal extension [14]. These two products are thought to result from post-translational cleavage [23]. Human Fau-UBiL has 37% amino acid sequence similarity to ubiqutin and contains the C-terminal Gly-Gly dipeptide motif that participates in isopeptide bond formation between the ubiquitin and lysine of target proteins [14]. However, a lack of internal lysine residues, which are sites of poly-ubiquitin chain formation, indicates that the biological function of UBiL is different from that of ubiquitin [23]. The identification of UBiL covalently bound to Bcl-G, a member of the Bcl-2 family of apoptosis control proteins [24], suggests a pro-apoptotic regulatory role for Fau, mediated via Bcl-G [19,23].
Apoptosis is closely related to oxidative stress in many cell lines, both mammalian and the model organism Caenorhabditis elegans [25,26]. Therefore, in this study, our aim was to determine the structures and functions of Acan-RPS-30 in A. cantonensis L5 in order to investigate its role in regulating oxidative stress resistance. L3 were determined by direct observation under a light microscope. Three-week-old SD rats (weight 100-120 g, grade clean, certificate SYXK [ZHE] 2005-0061), supplied by the Laboratory Animal Center of Wenzhou Medical University, were orally infected with 50 L3 per rat. The rats were housed in polypropylene cages with free access to food and water and then sacrificed by anesthesia at 25 days and 45 days post-infection, respectively. The L3 worms were collected from the intermediate hosts P. canaliculata; the L5 were harvested from the brains of mice (C57BL/6J [B6], Certificate SYXK [zhe2015-0009]) (non-permissive host same as humans), which were orally infected with 30 L3 per mouse; the adult worms were collected from the blood vessels of the hearts and lungs. Individuals of different sexes were separated using morphological criteria: females are usually longer and thinner than males, and males exhibit typical copulatory bursa. L3, L5 and adults were washed three times with 0.01 mol/l phosphate buffered saline and stored at − 80 °C. These rats were not used for any other part of the study.
Caenorhabditis elegans strains N2, rps-30 (tm6034/ nt1) and ced-3 (ok2734) were maintained on Nematode Growth Media agar plates at 15 °C, as described previously [27]. Worms were fed Escherichia coli strain OP50 unless otherwise stated. The mutant strain ced-3 (ok2734) was obtained from the Caenorhabditis Genetic Center (CGC) of the University of Minnesota (Minneapolis, MN, USA). The mutant strain rps-30 (tm6034/nt1) was originally provided by Shohei Mitani of Tokyo Women's Medical University School of Medicine (Tokyo, Japan). The gene rsp-30 is essential for the survival of the worms, and if the gene is deleted, worms are sterile. Therefore, the mutant strain tm6034/nt1 was used to produce trans-heterozygous animals using a translocational balancer (nT1) that has fluorescent marker: fluorescence-positive animals carry nT1 but animals without nT1 are considered to be homozygous for the mutation.

Isolation, purification, treatment and storage of nucleic acids
Total genomic DNA was extracted from A. cantonensis ZJ strain adult worms using a small-scale genomic DNA extraction Kit (Takara Biotechnology Co. Ltd., Kusatsu, Shiga, Japan). Total RNA was extracted from worms at different developmental stages employing TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by treatment with 2 U of DNase I (Takara Biotechnology Co., Ltd.). First-strand cDNA was obtained using the M-MLV RTase cDNA Synthesis kit (Takara Biotechnology Co., Ltd.). Both DNA and RNA samples were stored at − 80 °C until used.

Isolation of full-length cDNA and genomic DNA encoding Acan-rps-30 from A. cantonensis
Using two degenerate primers, rps-30DF and rps-30DR (Additional file 1: Table S1), designed on the basis of a relatively conserved S30 domain, with reference to the C. elegans gene (NC_003283.11) and Homo sapiens gene (NC_000011.10), a portion of Acan-rps-30 was amplified by PCR from cDNA synthesized from total RNA extracted from adult worms. PCR products were cloned into the pMD18-T vector (Takara Biotechnology Co., Ltd.) and sequenced. Based on the available sequence information, gene-specific primer pairs (Additional file 1: Table S1) were then designed. Using 5′-and 3′-rapid amplification of cDNA ends (RACE) method (Takara Biotechnology Co., Ltd.), we obtained two partially overlapping cDNA fragments. The products were cloned into the pMD18-T vector and sequenced. Based on these sequences, we designed additional primers (Additional file 1: Table S1) to amplify the full-length Acan-rps-30.
Full-length genomic DNA of Acan-rps-30 from the ZJ strain of A. cantonensis was obtained using a Genome Walking kit (Takara Biotechnology Co., Ltd.), using primers designed based on the acquired cDNA sequence (Additional file 1: Table S1), following the manufacturer's instructions. The third-round PCR products were cloned into a pMD18-T vector and sequenced.

Bioinformatics analysis
A sequence alignment between Acan-RPS-30, Hs-RPS-30 (NP_001988.1) and Ce-RPS-30 (NP_505007.1) was generated using Clustal Omega. Homology models were built by SWISS-MODEL using H. sapiens ribosome (Protein Data Bank codes 5LKS and 2L7R) as templates. Three-dimensional structural analysis was performed using the PyMOL program. All calculations were carried out under default conditions. The amino acid sequence inferred for Acan-RPS-30 and seven other selected homologous sequences were subjected to phylogenetic analyses. The phylogenetic analysis was conducted using the neighbor-joining (NJ) and maximum parsimony (MP) methods, respectively, based on the Jones-Taylor-Thornton (JTT) model [28]. Confidence limits were assessed using a bootstrap procedure with 1000 pseudo-replicates for NJ and MP trees, and other settings were obtained using the default values in MEGA v.5.0. A 50% cut-off value was implemented for the consensus tree.

Quantitative real-time PCR analysis
Quantitative real-time PCR (qRT-PCR) was performed to determine the abundance of Acan-rps-30 transcripts in different developmental stages (L3, L5 female, L5 male, adult female, adult male) of A. cantonensis.
Gene expression levels were determined by RT-PCR using the SYBR ® Green PCR Master Mix and a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative gene expression was compared with the 18S ribosomal RNA gene (GenBank: AY295804) as an internal loading control. The target genes and the primers used are listed in Additional file 1: Table S1. Statistical analysis was conducted using a one-way analysis of variance, with P < 0.05 set as the criterion for significance.

RNA interference feeding experiments
To generate ced-3-specific RNA interference (RNAi) vectors, ced-3 cDNAs was cloned into the the L4440 vector. Plasmids were transformed into E. coli strain HT115. Primers used for PCR analysis are listed in Additional file 1: Table S1. RNAi plates and media were prepared according to Kwon et al. [29]. Gravid adults of C. elegans were allowed to lay eggs overnight on the RNAi plates and adult worms were picked off. Escherichia coli containing the empty vector were used on separate plates as negative controls.
Recombinant plasmids were each microinjected into the gonad of young, adult C. elegans hermaphrodites as described previously [2,30], together with plasmid pRF4 containing a dominant mutant allele of the rol-6 gene, each at a final concentration of 50 μg/ml in the same mixture, using the pPD95.77 (pCe-rps30::gfp) and pRF4 plasmid mixture as a control. The F2 and subsequent generations with a roller phenotype were analyzed and selected to examine the expression patterns of green fluorescent protein (GFP) or red fluorescent protein (RFP), using a fluorescent microscope (Olympus model IX71; Olympus Corp., Tokyo, Japan). A minimum of three independent lines expressing each transgene were evaluated.

Oxidative stress assay
The oxidative stress assay was performed as described previously [31]. Briefly, adult hermaphrodites (30 worms/ group) were transferred to a 96-well plate containing M9 buffer with 3 mM H 2 O 2 . After incubation at 20 °C for the specified durations, the number of dead worms was determined. Worms were scored as dead when they no longer responded with movement to light prodding of the head. Three (H 2 O 2 ) independent experiments were performed. Statistical analysis was performed with Microsoft Excel 2010 software (Microsoft Corp., Redmond, WA, USA) using an unpaired two-tailed t-test.

Structural characterisation of Acan-RPS-30
The complete cDNAs of Acan-rps-30 was isolated by RACE from A. cantonensis. Acan-rps-30 cDNA was 1209 bp in length, including an open reading frame (ORF) of 393 bp (including stop codon), a 5'-untranslated region (UTR) of 190 bp, and a 3′-UTR of 626 bp (Fig. 2a). The 5′-UTR harbored the consecutive pyrimidines (TTT CTT TTC), which are commonly found at the 5′ end of eukaryotic ribosomal protein mRNAs [17] and which may play a role in regulating translation [32]. The 3′-UTR contained the hexamer AAT AAA (positions, 612 bp downstream of the TAA). The complete Acan-rps-30 gene, isolated by Genome Walking from genomic DNA of A. cantonensis, was 2967 bp in length, consisting of four exons and three introns (Fig. 2a).
To characterize the structure of Acan-RPS-S30, sequence alignment and structural analysis were performed. The cDNA of Acan-rps-30 encoded predicted proteins of 130 amino acids (Fig. 2b), which contained the potential cleavage sites (Gly-Gly) of the fusion protein (UBiL-ribosome protein S30 [S30]). The amino acids sequence was aligned with those from Homo sapiens (Hs-RPS-30) and Caenorhabditis elegans (Ce-RPS-30) (Fig. 2b). The results showed that the C-terminal S30 domains were conserved (Acan-RPS-S30 vs Ce-RPS-S30 and Hs-RPS-30, with 87.9 and 77.6% similarity, The S30 domain contained a nuclear location signal (NLS), KQEKKKKKK, with which RPS-30 can go into the nucleus and involve itself in the small subunit assembly of ribosome. Structural analysis from homology models revealed that the UBiL region possessed three β-sheets and two α-helixes (Fig. 2c), and the S30 region contained two α-helixes (Fig. 2d). The UBiL region did not harbor the K48 and K63 residues, sites of poly-ubiquitin chain formation, consistent with the orthologues from other species, indicating different functions [23], although the structure of UBiL was similar to that of ubiquitin.

Evolutionary relationship of Acan-RPS-30 with RPS-30 orthologues from other nematode species
To determine the evolutionary relationship between A. cantonensis and other nematodes, the predicted amino acid sequence of Acan-RPS-30 was aligned with orthologues from other nematodes and subjected to phylogenetic analyses (Fig. 3). Acan-RPS-30 clustered closely with Dv-RPS-30 from Dictyocaulus viviparus, with a similarity of 89.2%. Cladistic analysis showed that the RPS-30 homologues selected from seven parasitic nematodes were mainly grouped into two clades. Haemonchus contortus, Necator americanus, D. viviparus and A. cantonensis were in clade V; Wuchereria bancrofti, Brugia malayi and Loa loa were in clade III. This result is in agreement with a modern phylogenetic analysis of nematodes [33]. When sequences from the S30 regions only were analyzed, bootstrapping did not support the clusters (data not shown), possibly indicating that the divergences of the UBiL regions are likely related to species specificity.

The expression patterns of Acan-rps-30
To determine the relative abundance of the Acan-rps-30 transcript in different developmental stages (L3, L5 and adult) and genders (females [F] and males [M]) of the lifecycle of A. cantonensis, qRT-PCR was performed with the 18S ribosomal RNA gene as an internal loading control. The results showed that Acan-rps-30 was transcribed in both the larval and adult developmental stages examined at different levels ( Fig. 4; Additional file 2: Table S2). The expressions of Acan-rps-30 were significantly downregulated in both A. cantonensis L5 and adults, compared with that in L3; furthermore, the expression level in L5 was much lower than that in the adult, possibly indicating the important roles of Acan-RPS-30 in different developmental stages (L3, L5 and adult) residing in different hosts.
Due to the lack of functional genetic and in vitro culture methods, we were unable to detect the functions of Acan-RPS-30 directly in A. cantonensis. In the present study, we used C. elegans, proposed by numerous authors as a general model for many aspects of basic molecular, cellular and developmental biology in the less tractable parasitic nematodes [33][34][35], to investigate the anatomical expression patterns of Acanrps-30 in order to examine the closed evolutionary relationship between A. cantonensis and C. elegans, both of which belong to clade V according to cladistic analysis [33]. Wild-type C. elegans (N2 strain) were transformed with the construct pAcan-rps-30::gfp and pCe-rps30::gfp, respectively (Fig. 1a). Plasmid pRF4 was included in all transformations as a behavioral marker. Transgenic worms showing the roller phenotype were selected. The results showed that GFP under the promoter pAcan-rps-30 was only expressed in the intestine of C. elegans, mainly in the anterior end ( Fig. 5a-c), which is the major tissue for lifespan regulation in C. elegans [36]. This is in contrast to the situation in the worms expressing pCe-rps-30::gfp, where GFP was expressed in almost all cells, including those of the intestine, nervous system, pharynx and muscle (Fig. 5d-f ). The different activity of pAcanrps-30 and pCe-rps-30 may be due to heterologous expression, with low promoter sequences similarity (data not shown). Therefore, pCe-rps-30 was used as the promoter in subsequent research on the functions of Acan-RPS-30 in C. elegans.

Cross-species expressions of Acan-RPS-30 in C. elegans N2 strain and the rps-30 deletion mutant worms
In order to clarify the role of Acan-RPS-30, cross-species expression of Acan-rps-30 in C. elegans was performed. The expressing constructs containing Acan-rps-30::rfp coding sequences driven by Ce-rps-30 promoters (Fig. 1b) were used to transform C. elegans N2 strain and rps-30 deletion mutant strain (tm6034), respectively. In N2 worms transformed with pCe-rps30::Acan-rps-30::rfp, RFP was expressed widely (Fig. 6b, c), consistent with the pCe-rps30::gfp expression pattern (Fig. 5d-f ). In addition, RFP mainly focused on the nucleus for the existence  Data shown are the mean ± standard error of the mean from three technical replicates with two biological replicates. Relative transcription of the Acan-rps-30 gene in each sample was calculated by normalization of the raw data, followed by the determination of abundance relative to the 18S ribosomal RNA gene (GenBank: AY295804), which served as an internal loading control. Statistical analysis was conducted using a one-way analysis of variance. Asterisks indicate statistical difference at *P < 0.05 and **P < 0.01 of a NLS in the S30 region. "Button-like" apoptotic cell corpses arising from developmental apoptosis, which are the gold standard for quantification of apoptosis in C. elegans [25], were seen in the anterior pharynx (Fig. 6a,  d), possibly suggesting the pro-apoptotic effect of Acan-RPS-30, consistent with the pro-apoptotic regulatory role of Hs-RPS-30 [19,23].

Functional role of Acan-RPS-30 in oxidative stress
To investigate the role of Acan-RPS-30 in regulating oxidative stress resistance, we performed oxidative stress assays using H 2 O 2 . We found that the incidence of rapid death among the C. elegans N2 worms expressing pCe-rps30::Acan-rps-30::rfp was significantly higher than that among the N2 worms expressing pCe-rps30::gfp; in addition, the rps-30 deletion mutants (rps-30 −/− ) were significantly more resistant than N2 worms. This oxidative stress resistance phenotype could be rescued and inhibited by expressing pCe-rps30::Acan-rps-30::rfp in rps-30 −/− mutant worms ( Fig. 7a; Additional file 2: Table S2 ). These results may indicate the regulating role of Acan-RPS-30 in promoting susceptibility to oxidative stress.
As oxidative stress is considered to be one of the major factors that promote apoptosis [26], we next detected the expression levels of apoptosis genes in C. elegans. The results showed that all of the apoptosis genes were downregulated in rps-30 −/− mutant worms, with the exception of upregulation of akt-1 (Fig. 7b), which inhibits CEP-1 and decreases DNA damageinduced apoptosis [37]. ced-3 and ced-4, the core apoptosis executive genes [25], were both upregulated in N2 worms expressing pCe-rps30::Acan-rps-30::rfp, whereas ced-9 was downregulated (Fig. 7c); this latter gene encodes the homologous protein to the anti-apoptotic B-cell lymphoma 2 (Bcl-2) family of proteins [38]. These results may indicate the role of Acan-RPS-30 in promoting apoptosis in C. elegans.
To further determine the effect of apoptosis regulated by Acan-RPS-30 on oxidative stress susceptibility, expressing pCe-rps30::Acan-rps-30::rfp was almost the same as that among the ced-3 −/− worms expressing pCe-rps30::gfp; and that the incidence of rapid death among the N2 worms expressing pCe-rps30::Acanrps-30::rfp was significantly higher than that among the N2 worms expressing pCe-rps30::Acan-rps-30::rfp with ced-3 knocked down ( Fig. 7d; Additional file 2: Table S2). These results may suggest that the regulating role of Acan-RPS-30 in promoting susceptibility to oxidative stress plays through CED-3, which is the core executive effector in worm cell apoptosis [25].

Discussion
Eosinophilic meningitis, caused by A. cantonensis L5, is mainly attributed to the eosinophils [39], which contribute to tissue inflammatory responses in helminthic infections [8]. Eosinophil are well-equipped immune cells recruited from the circulation into inflammatory foci [40] that directly recognize helminth-derived immunomodulating agents and function in host defense mechanisms against helminth infection [8]. The cell surface of eosinophils possess a variety of receptors for cell signaling associated with chemotaxis, adhesion, respiratory burst, degranulation, apoptosis or survival [41], all of which may be closely associated with eosinphil-mediated tissue inflammatory responses in helminth infection [8]. Eosinophils primarily contain four main granules: crystalloid granules, primary granules, small granules and secretory vesicles [42]. Cytotoxic granular proteins, including the major basic proteins, EPO, eosinophil cationic protein and eosinophil-derived neurotoxin, reside in the crystalloid granules [9,10]. The functional role of EPO is associated with the killing of helminths killing [43]. EPO catalyzes the peroxidative oxidation of halides and thiocyanate present in the plasma together with H 2 O 2 generated by dismutation of the superoxide produced during respiratory burst [11][12][13]. Eosinophils, the robust producers of extracellular superoxide due to the expression of high levels of the enzyme complex that generates superoxide [7], produce superoxide anions in response to helminth-derived cysteine proteases [44]. However, helminthic worms residing in the host with high level of eosinophils have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival in hosts, such as inducing the apoptosis of eosinophils [45,46] and blocking the chemotactic effects on eosinophils [47]. In this study, we identified Acan-rps- 30  rps-30 −/− mutant worms were resistant to oxidative stress. This observation might indicate the regulating function of Acan-RPS-30 in attenuating eosinophil-mediated immue attack upon L5 worms in the central nervous system of humans by due to a lower expression. In comparison L3 worms, with a higher level of Acan-rps-30, reside in intermediate hosts (e.g. Pomacea canaliculata) in which the immune system is lower than that in mammalians, or the immune attack may be weaker, or even there may be no eosinophil-mediated superoxide attack. Therefore, the higher level of Acan-rps-30 in L3 worms may indicate its multi-function in different developmental stages, such as promoting the development of L3 worms with the S30 region [22]. Furthermore, the expression level in L5 was significantly lower than that in adult, which possesses a thicker cuticle than L5 larva. Alhough adult worms in the blood vessels of the hearts and lungs of rats, in which there is an active immune system, are attacked by superoxide from eosinophils, the thick cuticle may provide some protection [48]. In addition, many other proteins may be differently expressed in the cuticle, such as the homologous gene of lec-1, which plays an important role against damage due to oxidative stress [4,48]. Angiostrong cantonensis is relatively closely related to the model organism C. elegans, with both belonging to clade V [2,33], and the homologous gene of Acan-rps-30 is Ce-rps30 (C26F1.4). Here, we used C. elegans as a surrogate to explore the in vivo functions of the homologous gene Acan-rps-30 for the lack of effective genetic manipulation in parasitic nematode. In C. elegans, apoptosis is characterized by the refractile "button-like" apoptotic corpses that are the result of inefficient engulfment from healthy neighboring cells [49][50][51]. The "button-like" appearance under differential interference contrast (DIC) optics is the gold standard for quantification of apoptosis in C. elegans [25]. In this study, the "button-like" corpses were seen in the anterior pharynx of the transgenic worm expressing pCe-rps30::Acan-rps-30::rfp, indicating that apoptosis was occurring. CED-1 and CED-5 proteins can recognize corpses and are critical to engulfment [49]. The downregulated expression of ced-1 and ced-5 in the transgenic worm expressing pCe-rps30::Acan-rps-30::rfp may contribute to the formation of corpses.
Four genes, comprising the core apoptosis pathway in C. elegans, have been identified [37,52]. egl-1 encodes a proapoptotic BH3-only protein that antagonizes the CED-9 protein [53]. ced-9, which functions upstream of ced-4 to prevent activation of the CED-3 caspase, encodes the homologous protein to the anti-apoptotic B-cell lymphoma 2 (Bcl-2) family of proteins [38]. ced-3 encodes a proteolytic caspase protein that is activated by CED-4, the worm homologue of mammalian apoptotic protease activation factor 1 [54]. Therefore, CED-3 is the core executioner [25]. In the worms expressing pCe-rps30::Acan-rps-30::rfp, the ced-3 was upregulated and the worms exhibited apoptosis and susceptibility to oxidative stress; whereas in the rps-30 −/− mutant worms, the ced-3 was downregulated and the worms exhibited resistance to oxidative stress. This phenotype could be converted with the ced-3 defective mutation and RNAi. Therefore, the function of Acan-RPS-30 in promoting susceptibility to oxidative stress may possibly be conducted through apoptosis by regulating CED-3. In A. cantonensis L5, Acan-RPS-30 was downregulated to enhance the resistance to oxidative stress from eosinophils to ensure wormssurvival in host.