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

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. 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. 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. 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.


Background
Angiostrongylus cantonensis is considered the primary causative pathogen of human eosinophilic meningitis and meningoencephalitis in China, Japan, Southeast Asia and the Pacific Islands [1][2][3]. The final host of this parasite is rat; it lives in the rat pulmonary artery where it develops to sexual maturity [4,5]. Human, as an atypical host, mainly acquires this parasite by consuming raw terrestrial freshwater snails, such as the golden apple/channeled apple snail Pomacea canaliculata, in which the infective third-stage larvae (iL3) reside. After passage to the human small intestine, iL3 will infect the central nervous system through the bloodstream and develop into fifth-stage larvae (L5), causing eosinophilic meningitis [6][7][8][9][10].
High percentage of eosinophils, recruited from the circulation into the central nervous system [7], as the primary pathological change in eosinophilic meningitis, is thought to contribute to tissue inflammatory responses and host defense in helminthic infections [11]. High level of the enzyme complex is expressed and released in the granule matrix of eosinophils, such as eosinophil peroxidase [12,13], which is associated with helminthic killing, using the peroxidative oxidation and hydrogen peroxide (H 2 O 2 ) generated by dismutation of superoxide produced during respiratory burst [14][15][16]. In contrast, helminthic worms, parasitizing with a high level of eosinophils, have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival [11]. Therefore, A. cantonensis L5, residing in human cerebrospinal fluid (CSF) with a high percentage of eosinophils, may act to resist the oxidative stress damage from eosinophils.
Recently, we have demonstrated that downregulated Acan-RPS-30 in A. cantonensis L5 plays a defensive role in worm survival against damage due to oxidative stress by inhibiting apoptosis by regulating ced-3 downregulation [17].
Acan-Gal-1, as the homologous protein of human Galectin-9 and Caenorhabditis elegans LEC-1, belongs to the family of galectins that are glycan-binding proteins distributed among animals and fungi and is defined by its conserved carbohydrate recognition domains (CRDs) and affinity for β-galactoside structures [18][19][20]. Galectins are secreted by cells via an unconventional mechanism [21,22] and function in various biological phenomena, such as development, immunity and tumorigenesis via recognition of cell surface or extracellular glycoconjugates [22]. Parasite galectins have a sequence and structure similar to those of mammalian homologs and are presumed to participate in host-parasite interactions [23,24]. Our previous studies showed that upregulated Acan-Gal-1 in A. cantonensis L5 induced the apoptosis of macrophages by binding to Annexin A2 and activating the JNK apoptotic signaling pathway [24]. However, galectins are also found intracellularly and are involved in RNA splicing, cell growth, apoptosis and other functions [25,26]. In the nematode C. elegans, 11 galectins have been determined, and the endogenous ligands and some of the functions of LEC-6, LEC-8 and LEC-10 have been identified. LEC-6 has a role in growth regulation by affecting the localization and function of its ligands such as F57F4.4 [27,28]; LEC-8 acts against bacterial infection by binding to glycolipids [29], and LEC-10 functions against oxidative stress by binding to glycoproteins [27,30]. LEC-1, the homolog of Acan-Gal-1, is a tandem repeat-type galectin, and its two CRDs have different sugar-binding properties [31]. As LEC-1 is mainly localized in the cuticle and pharynx of C. elegans [32], it is thought to have some functions as a component of the durable outer barrier via recognizing and cross-linking glycoconjugates by its two CRDs with different sugar-binding properties and to play a defensive role against damage due to oxidative stress in C. elegans [33]. Therefore, Acan-Gal-1, upregulated in A. cantonensis L5, may have the function of resisting human immune attacks by not only inducing the apoptosis of its host's macrophages extracellularly [24], but also increasing oxidative stress tolerance of the worm itself intracellularly.
In this study, we further explored the intracellular functions of Acan-Gal-1 upregulated in A. cantonensis L5, using the model organism C. elegans as a surrogate, in relation to the lack of effective genetic manipulation in parasitic nematodes, to investigate its role in protecting the worm from its host's immune attacks.

Propagation of A. cantonensis and C. elegans
Angiostrongylus cantonensis ZJ strain was maintained and propagated in Wenzhou Medical University, China, by cycling through Pomacea canaliculata and Sprague-Dawley (SD) rats as described previously [8]. Intermediate hosts P. canaliculata were infected with A. cantonensis L1 by feeding on rat feces. L3 were collected at 20 days p.i. Infected snails were shelled and crushed. The intestines and other organs were removed, and the remaining tissue was homogenized. The homogenates were filtered through a 40-mesh sieve, deposited for 5 min at 4 °C and precipitated 2-3 times at room temperature. The sediments were removed, and L3 number and viability were determined by direct observation under a light microscope. Three-week-old Sprague-Dawley (SD) rats [weight 100-120 g, grade clean, Certificate SYXK (zhe2015-0009)], supplied by the Laboratory Animal Center of Wenzhou Medical University, were orally infected with 50 L3/rat. The rats were housed in polypropylene cages with free access to food and water and then killed by anesthesia at 25 days and 45 days p.i., respectively. The L3 worms were collected from the intermediate host P. canaliculata and L5 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/mouse. 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 PBS buffer and stored at − 80 °C. These rats were not used for any other part of the study.

Isolation, purification, treatment and storage of nucleic acids
Total RNA was extracted from worms at different developmental stages employing Trizol reagent (Invitrogen, USA), followed by treatment with 2 U of deoxyribonuclease I (DNase I; Takara Biotechnology Co., Ltd, Japan).
First-strand cDNA was obtained using the Murine leukemia virus reverse transcriptase (M-MLV RTase cDNA Synthesis Kit; Takara Biotechnology Co., Ltd, Japan). RNA samples were stored at − 80 °C.

Quantitative real-time PCR (qRT-PCR) analysis
qRT-PCR was performed to determine the abundance of Acan-gal-1 transcripts in different developmental stages (L3, L5 female, L5 male, adult female, adult male) of A. cantonensis.
Gene expression levels were determined by qRT-PCR using SYBR ® Green PCR Master Mix and a 7500 Real-Time PCR System (Applied Biosystems, USA). Relative gene expression was compared with 18S ribosomal RNA gene (GenBank: AY295804) as an internal loading control. The target genes and primers used are listed in Additional file 1: Table S1. Statistical analysis was conducted using a one-way ANOVA; P < 0.05 was set as the criterion for significance.
To perform cross-species expression of Acan-Gal-1 in C. elegans strains, cDNA sequence was amplified and cloned into pPD95.77 using the promoter of Ce-lec-1 to construct plasmid pCe-lec-1::Acan-gal-1::rfp. All primers used are listed in Additional file 1: Table S1.
All plasmid constructs were confirmed by sequencing. Recombinant plasmids were each microinjected into the gonads of young adult C. elegans hermaphrodites as described [6] together with plasmid pRF4 containing a dominant mutant allele of rol-6 gene, each at a final concentration of 50 μg/ml in the same mixture, using pPD95.77 (pCe-lec-1::rfp) and pRF4 plasmid mixture as a control. Plasmid pRF4 was included in all transformations as a behavioral marker, and transgenic worms showing the roller phenotype were selected. F2 and subsequent generations were analyzed and selected to examine the expression patterns of GFP or RFP using a fluorescent microscope (Olympus IX71). A minimum of three independent lines expressing each transgene was evaluated.

Oxidative stress assay
The oxidative stress assay was performed as described previously [30]. Briefly, adult hermaphrodites (30 worms/ group) were transferred to a 96-well plate containing 3 mM H 2 O 2 in M9 buffer (M9 buffer: 3 g/l KH 2 PO 4 , 6 g/l Na 2 HPO 4 , 5 g/l NaCl and 0.12 g/l MgSO 4 ). 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 using an unpaired two-tailed t-test.

RNAi feeding experiments
RNAi experiments were performed with feeding worms, E. coli strain HT115 (DE3), transformed with either the empty vector L4440 as the control or lec-1gene-targeting constructs from the C. elegans Ahringer RNAi Collection [36]. To generate lec-1-specific RNAi vectors, lec-1 partial cDNA was cloned into 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 [17]. Briefly, overnight bacterial culture in LB supplemented with ampicillin (100 mg/ml) at 37 °C was seeded onto NGM plates containing IPTG (1 mM) and ampicillin (100 mg/ml) and incubated overnight at room temperature to induce double-stranded RNA production. Gravid adult ced-3 mutant worms and fat-6;fat-7 double-mutant worms were allowed to lay eggs overnight on the RNAi plates, and adult worms were picked off. Embryos were placed on RNAi plates and incubated at 20 °C until adulthood to score phenotypes.

Lipid staining and quantitation
Lipid staining was performed using Oil Red O dyeing solution as described previously [37]. Worms were washed off the NGM or RNAi plates and incubated in PBS (phosphate-buffered saline) buffer for 30 min on a shaker at room temperature. The worms were then fixed in Modified Ruvkun's Witches Brew (MRWB) buffer containing 1% paraformaldehyde. After three rounds of freezing/thawing, the worms were dehydrated in 60% isopropanol followed by addition of saturated Oil Red O (Sigma, USA) solution. Fixed worms were incubated overnight on a shaker at room temperature, mounted on slides and viewed using a microscope with differential interference contrast optics (Nikon, Japan).
For quantification of Oil Red O staining, using ImageJ image processing software, we separated out each color image into its Red, Green and Blue (RGB) channel components. As it has been reported that Oil Red O absorbs light at 510 nm, we used the green channel for further analysis [38]. We measured the average pixel intensity for a 40-pixel radius immediately behind the pharynx of each animal. In addition, we measured a 40-pixel radius of the background, which was later subtracted from the values obtained from the staining. A minimum of nine animals was measured for each strain, and we repeated the experiments two additional times. Significance was determined by Student's t-test.

Structural characterization of Acan-Gal-1
To characterize the structure of Acan-Gal-1, amino acid sequence alignment and structural analysis were performed. Full-length Acan-Gal-1 was composed of 285 amino acids, containing N-terminal carbohydrate recognition domain (CRD) (NCRD, residues 1-150) and C-terminal CRD (CCRD, residues 158-285) in the manner of tandem repeat, and a short linker (residues 151-157) held them together (Fig. 1a). The amino acid sequence was aligned with Ce-Lec-1 (Fig. 1b). The results showed that Acan-Gal-1 had a similarity of 84.4% to Ce-Lec-1. The conserved motifs HXXXR and WGXEER, involved with carbohydrate binding sites [35], were located in both NCRD and CCRD. The charged Arg 69 /Arg 203 and Glu 88 /Glu 222 were conserved in the motifs, which are critical amino acids for recognizing carbohydrate binding and affect protein folding and structure [35]. The linker "GKYYPVP, " shorter than other tandem repeattype galectins such as galectin-9, which are flexible and susceptible to proteolysis [20,39,40], may indicate the structural stability of Acan-Gal-1. Structural analysis from homology models revealed that the NCRD of Acan-Gal-1 possessed 11 β-sheets and a small α-helix located between β9 and β10, whereas the CCRD contained 10 β-sheets and no α-helix (Fig. 1b, c). The conserved motifs HXXXR and WGXEER, the carbohydrate binding sites, were located in the concave surface surrounded by β4 and β6 of NCRD, and those were also located in the surface formed by β4′ and the loop between β5′ and β6′ of CCRD (Fig. 1c).

Expression patterns of Acan-Gal-1
To determine the relative abundance of Acan-gal-1 transcript in different developmental stages (L3, L5 and adult) and genders [females (F) and males (M)] of the life cycle of A. cantonensis, qRT-PCR was performed with the 18S ribosomal RNA gene as an internal loading control. The results showed that Acan-gal-1 was transcribed in larval and adult developmental stages examined in different levels ( Fig. 2; Additional file 2: Table S2). The expressions of Acan-gal-1 were greatly upregulated in both L5 and adult compared with those in L3, whereas the expression levels were not significantly changed among L5, adult and different genders. This might indicate the important roles of Acan-Gal-1 in L5 and adults, which reside in mammals, such as humans and rats, where a full immune system exists.
Because of the lack of functional genetic and in vitro culture methods, it is not possible to detect the functions of Acan-Gal-1 directly in A. cantonensis. Here, 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 [41][42][43], was used to investigate the anatomical expression patterns of Acan-Gal-1.
Wild-type C. elegans (N2 strain) were transformed with the construct pAcan-gal-1::gfp and pCe-lec-1::rfp, respectively. The results showed that GFP under the promoter pAcan-gal-1 was only expressed in pharyngeal neurons of C. elegans (Fig. 3a-c), in contrast to the situation in worms expressing pCe-lec-1::rfp, where RFP was mainly localized in cuticle and less in  residues 1-285). b Alignment of amino acid sequences of Acan-Gal-1 from A. cantonensis with that from C. elegans and the secondary structures. The accession number of Ce-Lec-1 sequence available from the current database is: NP_001370038.1. Identical and similar residues are shown in black and gray blocks, respectively. Secondary elements are shown by coil (α-helix) and arrows (green indicates β-sheets of NCRD and blue indicates those of CCRD). The short linker between NCRD and CCRD is indicated by a red line. The conserved motifs HXXXR and WGXEER are indicated by blue boxes. The critical amino acids for recognizing carbohydrate binding are indicated by red stars. c Predicted tertiary structure of Acan-Gal-1, HXXXR motif and WGXEER motif in NCRD and CCRD, respectively intestine, nervous system and pharynx (Fig. 3d-f ). This result was in agreement with the previous report [32].

Cross-species expressions of Acan-Gal-1 in C. elegans worms
The different activities of pAcan-gal-1 and pCe-lec-1 might be due to the heterologous expression, with low promoter sequence similarity (data not shown).
Therefore, pCe-lec-1 was used as the promoter in this research on the functions of Acan-Gal-1 in C. elegans.
Morphological changes were further detected under a light microscope, and we found the level of lipid storage in lec-1 mutant worms expressing pCe-lec-1::Acan-gal-1::rfp was lower (Fig. 4d) than that in lec-1 mutant worms expressing pCe-lec-1::rfp (Fig. 4g). This might suggest the function of Acan-Gal-1 in reducing lipid deposition in C. elegans.

Functional role of Acan-Gal-1 in lipid storage
To explore the function of Acan-Gal-1 in reducing lipid deposition, Oil Red O fat staining was performed in C. elegans. The results showed that lec-1 mutant worms stored significantly more lipid than N2 worms, and this lipid accumulation phenotype could be rescued by expressing pCe-lec-1::Acan-gal-1::rfp in lec-1 mutant worms. The N2 worms expressing pCe-lec-1::Acangal-1::rfp exhibited much less lipid storage than the N2 worms expressing pCe-lec-1::rfp ( Fig. 5; Additional file 2: Table S2). This might suggest the function of Acan-Gal-1 in reducing lipid deposition in C. elegans. Data shown are mean ± SEM derived from three technical replicates with two biological replicates. Relative transcription of the Acan-gal-1 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 ANOVA. *P < 0.05; **P < 0.01

Functional role of Acan-Gal-1 in resisting oxidative stress
LEC-1 plays a defensive role against damage due to oxidative stress in C. elegans, and lec-1 (tm1345) worms show susceptibility to H 2 O 2 [33]. To investigate the role of Acan-Gal-1 in regulating oxidative stress resistance, we performed oxidative stress assays using H 2 O 2 . We found that the incidence of rapid death among lec-1 deletion mutants was significantly higher than that among the N2 worms, and this oxidative stress susceptibility phenotype could be rescued by expressing pCe-lec-1::Acan-gal-1::rfp in lec-1 mutant worms. The N2 worms expressing pCelec-1::Acan-gal-1::rfp were significantly more resistant to H 2 O 2 than the N2 worms expressing pCe-lec-1::rfp ( Fig. 6a; Additional file 2: Table S2). This might suggest the regulating role of Acan-Gal-1 in increasing oxidative stress tolerance.
As we have demonstrated that downregulated Acan-RPS-30 in A. cantonensis L5 can resist oxidative stress damage by inhibiting worm apoptosis [17], and oxidative stress is thought to be one of the major factors that promote apoptosis [44], we next determined whether Acan-Gal-1 increased oxidative stress tolerance via inhibiting worm apoptosis. Then, the expression levels of apoptosis genes were detected in C. elegans. The results showed that all the apoptosis genes were not significantly changed in lec-1 mutant worms expressing pCe-lec-1::Acan-gal-1::rfp compared with those in lec-1 mutant worms expressing pCe-lec-1::rfp (Fig. 6b). This indicated that expressing Acan-Gal-1 could not regulate apoptosis genes in transcriptional level.
ced-3 is the final executive gene in the core apoptosis pathway in C. elegans, and apoptosis is inhibited in ced-3 mutant worms [45]. Therefore, oxidative stress assays were further performed in ced-3 mutant worms to detect the effects of expressing Acan-Gal-1 on the oxidative stress damage. The results showed that the incidence of rapid death among ced-3 mutant worms expressing pCelec-1::Acan-gal-1::rfp was significantly lower than that among ced-3 mutant worms expressing pCe-lec-1::rfp ( Fig. 6c; Additional file 2: Table S2), whereas lec-1 RNAi made ced-3 mutant worms greatly more susceptibility to oxidative stress ( Fig. 6d; Additional file 2: Table S2). This indicated that Acan-Gal-1 regulated the oxidative stress resistance was not via apoptosis in C. elegans. Fatty acid metabolism is involved in the oxidative stress resistance in C. elegans, and reduction of fat storage makes worms strongly resistant to oxidative stress [46,47]. We next investigated whether Acan-Gal-1 increased oxidative stress tolerance via reducing lipid deposition in C. elegans. fat-6;fat-7 double-mutant worm was selected because fat-6 and fat-7 genes encode stearoyl-CoA desaturases, which are key lipogenic enzymes, and fat-6;fat-7 double-mutant worms have decreased fat storage [48]. Then, oxidative stress assays were performed to determine the effects of expressing Acan-Gal-1 on the oxidative stress damage in fat-6; fat-7 double-mutant worms. The results showed that the incidence of rapid death was not significantly changed among fat-6;fat-7 doublemutant worms expressing pCe-lec-1::Acan-gal-1::rfp compared with that among fat-6;fat-7 double-mutant worms expressing pCe-lec-1::rfp (Fig. 6e). The susceptibility to oxidative stress was not influenced by lec-1 RNAi in fat-6;fat-7 double-mutant worms (Fig. 6e). Expression of either Acan-Gal-1 or lec-1 RNAi had no significant effect on oxidative stress resistance in fat-6;fat-7 doublemutant worms, where fat storage is reduced dramatically. This might indicate the function of Acan-Gal-1 in increasing oxidative stress tolerance was related to fat storage reduction in C. elegans.

Discussion
The parasitic nematode A. cantonensis L5, residing in human cerebrospinal fluid, can cause eosinophilic meningitis, primarily due to eosinophil-inducing tissue inflammatory responses. Eosinophils, as well-equipped immune cells, can be recruited from the circulation into inflammatory sites in response to helminthic infections to play a role in protecting the host against the infection; at the same time, they cause some tissue damage when the inflammatory response is serious. Eosinophil peroxidase, as a cytotoxic granular protein in eosinophils, plays a crucial role in killing helminths [49]. A high level of the enzyme complex that generates superoxide is expressed in eosinophils, and superoxide anions are produced in response to helminth-derived immunomodulating agents. Therefore, eosinophils are robust producers of extracellular superoxide. In contrast, helminthic worms have evolved to attenuate the oxidative stress damage from eosinophils for their survival in hosts, such as inducing apoptosis of eosinophils [50,51] and increasing oxidative stress resistance of the worm itself [17]. We have demonstrated that Acan-Gal-1, upregulated in A. cantonensis L5, which resides in humans with more sophisticated immune systems, could induce apoptosis of macrophages extracellularly when secreted by cells via an unconventional mechanism [24]. Acan-Gal-1 could also function intracellularly [25,26,33]. In this study, we investigated the intracellular functions of Acan-Gal-1in protecting worms from immune attacks in hosts.
Lack of effective genetic manipulation in parasitic nematodes and A. cantonensis L5 in in vitro culture methods makes it impossible to study the in vivo functions of Acan-Gal-1 in A. cantonensis. The free-living nematode C. elegans, which belongs to clade V in the phylogenetic relationship, like A. cantonensis [17,41], has been used widely in scientific research as the model organism, especially for exploring gene functions of parasitic nematodes, such as Haemonchus contortus, Strongyloides stercoralis, etc. [52][53][54]. Therefore, in this study, it was employed as a surrogate to explore the intracellular functions through cross-species expression of Acan-Gal-1.
Here, the expression pattern of Acan-gal-1 promoter was not consistent with that of Ce-lec-1 promoter in C. elegans. This might be because of the low promoter sequence similarity between Acan-gal-1 promoter and Ce-lec-1 promoter. There are many examples of heterologous expression in C. elegans, such as daf-2, daf-16 and rps-30 from Haemonchus contortus [37,52,54], daf-16 from Strongyloides stercoralis, et al. [53] and GFP or RFP mainly expressed in the intestinal tract or nervous system, where heterologous expression easily occurs. For example, in the parasitic nematode Haemonchus contortus, pHc-rps30::gfp is expressed in the distal intestine of C. elegans using cross-species expression, in contrast to pCe-rps30::gfp, expressed ubiquitously. Hc-rps30 is also expressed ubiquitously in H. contortus, consistent with the expression pattern of pCe-rps30::gfp in C. elegans, when it is localized using whole-mount in situ hybridization [37]. The expression pattern of Hc-rps30 in Haemonchus contortus is consistent with that of Ce-rps30 in C. elegans, though cross-species expression of Hc-rps30 is very different. In this study, we tried to determine the expression pattern of Acan-gal-1 in Angiostrongylus catonensis using whole-mount in situ hybridization. Unfortunately, A. cantonensis L5 worms were too big, with thick cuticle and dark color, and the location was not successful. However, in our opinion, the expression pattern of Acan-gal-1 in Angiostrongylus catonensis should be consistent with that of Ce-rps30 in C. elegans, and it was reasonable to express Acan-gal-1 under Fig. 6 Upregulated Acan-Gal-1 plays a defensive role against oxidative stress. a Oxidative stress assays using H 2 O 2 in N2 and lec-1 mutant worms expressing pCe-lec-1::Acan-gal-1::rfp. b Relative expression levels of apoptosis genes in lec-1 mutant worms expressing pCe-lec-1::rfp and in lec-1 mutant worms expressing pCe-lec-1::Acan-gal-1::rfp. c Oxidative stress assays using H 2 O 2 in ced-3 mutant worms expressing pCe-lec-1::Acan-gal-1::rfp or pCe-lec-1::rfp. d Oxidative stress assays using H 2 O 2 in ced-3 mutant worms with lec-1 RNAi. e Oxidative stress assays using H 2 O 2 in fat-6;fat-7 double-mutant worms expressing pCe-lec-1::Acan-gal-1::rfp or with lec-1 RNAi. The worms were counted as described in "Methods" section. The error bars indicate standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001