The identification of alternative oxidase in intermediate host snails of Schistosoma and its potential role in protecting Oncomelania hupensis against niclosamide-induced stress

Abstract

Background: Snail intermediate hosts are mandatory for the transmission of schistosomiasis, which has to date infected more than 200 million people worldwide. Our previous studies showed that niclosamide treatment caused the inhibition of aerobic respiration and oxidative phosphorylation, and the disruption of energy supply, in one of the intermediate hosts of schistosomiasis, Oncomelania hupensis, which eventually led to the death of the snails. Meanwhile, the terminal oxidase in the mitochondrial respiratory chain, alternative oxidase (AOX), was significantly up-regulated, which was thought to counterbalance the oxidative stress and maintain metabolic homeostasis in the snails. The aims of the present study are to identify the AOXs in several species of snails and investigate the potential activation of O. hupensis AOX (OhAOX) under niclosamide-induced stress, leading to enhanced survival of the snail when exposed to this molluscicide.

Methods: The complete complementary DNA was amplified from the AOXs of O. hupensis and three species of Biomphalaria; the sequence characteristics were analysed and the phylogenetics investigated. The dynamic expression and localisation of the AOX gene and protein in O. hupensis under niclosamide-induced stress were examined. In addition, the expression pattern of genes in the mitochondrial respiratory complex was determined and the production of reactive oxygen species (ROS) calculated. Finally, the molluscicidal effect of niclosamide was compared between snails with and without inhibition of AOX activity.

Results: AOXs containing the invertebrate AOX-specific motif NP-[YF]-XPG-[KQE] were identified from four species of snail, which phylogenetically clustered together into Gastropoda AOXs and further into Mollusca AOXs. After niclosamide treatment, the levels of OhAOX messenger RNA (mRNA) and OhAOX protein in the whole snail were 14.8 and 2.6 times those in untreated snails, respectively, but varied widely among tissues. Meanwhile, the level of cytochrome C reductase mRNA showed a significant decrease in the whole snail, and ROS production showed a significant decrease in the liver plus gonad (liver-gonad) of the snails. At 24 h post-treatment, the mortality of snails treated with 0.06-0.1 mg/L niclosamide and AOX inhibitor was 56.31-76.12% higher than that of snails treated with 0.1 mg/L niclosamide alone.

Conclusions: AOX was found in all the snail intermediate hosts of Schistosoma examined here. AOX was significantly activated in O. hupensis under niclosamide-induced stress, which led to a reduction in oxidative stress in the snail. The inhibition of AOX activity in snails can dramatically enhance the molluscicidal effect of niclosamide. A potential target for the development of an environmentally safe snail control method, which acts by inhibiting the activity of AOX, was identified in this study.

Keywords: Alternative oxidase; Intermediate host; Mitochondrial respiratory chain; Niclosamide; Oncomelania hupensis; Schistosoma.

Fig. 1

Phylogenetic tree (maximum likelihood; ML) of alternative oxidases (AOXs) based on protein sequences. The number beside each node shows the ML support rate (bootstrap value). The four snail AOXs identified in this study are highlighted in red (in the Gastropoda branch)

Fig. 2a–c

Dynamic expression profiles of Oncomelania hupensis AOX (OhAOX) messenger RNA (mRNA) and OhAOX protein in O. hupensis snails under wettable powder of niclosamide (WPN) stress. a Expression profiles of OhAOX in the whole snail, head and foot muscle (head-foot) region, and liver and gonad (liver-gonad) region after WPN treatment. Asterisks indicate significant difference (*P < 0.05, **P < 0.01) between time points and 0 h within the same treatment; underlined asterisks indicate significant difference between WPN- and H₂O-treated snails at the same time point. b Expression of OhAOX protein (upper panel) in the whole snail 0–48 h after WPN and H₂O treatment. The OhAOX mRNA profile calculated using quantitative real-time PCR (qPCR) is shown in the lower panel (and corresponds with the whole snail data in a). c Statistical analysis of OhAOX protein expression in the western blot (b); *P < 0.05, ***P < 0.001. bp Base pairs

Fig. 3a, b

Tissue expression of OhAOX mRNA in O. hupensis after WPN treatment following in situ hybridisation. a Tissue distribution and statistical evaluation of OhAOX mRNA in the muscle of the head-foot region. b Tissue distribution and statistical evaluation of OhAOX mRNA in the liver. The purple-blue area was identified as a positive signal of OhAOX mRNA. Asterisks indicate statistically significant difference (*P < 0.05, **P < 0.01) in the quantity of OhAOX mRNA between the given time point and 0 h under the same treatment. Underlined asterisks indicate the level of significant difference between WPN- and H₂O-treated groups at the same time point. IOD Integrated optical density; for other abbreviations, see Fig. 2

Fig. 4a, b

Tissue distribution of OhAOX protein determined by immunohistochemistry. a Different tissues of the untreated snails. b Head-foot muscle at 48 h after WPN treatment. c Liver at 48 h after WPN treatment. 1 Blank controls, 2 phosphate-buffered saline (PBS) immune serum, 3 anti-OhAOX serum, HE hematoxylin–eosin staining, M muscle, EP epithelium, GT gland tissue, CT connective tissue, T testis, O ovary. The regions stained brown-yellow indicate the distribution of OhAOX protein

Fig. 5

Expression profiles of genes involved in the mitochondrial respiratory complex (MRC), and reactive oxygen species (ROS) production in O. hupensis after WPN treatment. a Dynamic expression profile of genes coding for NADH dehydrogenase (NADH), succinic dehydrogenase (SDH), cytochrome C oxidase (CCO), ATPase, and cytochrome c reductase (CCR) in the whole snail determined using qPCR. b ROS production in the whole snail, head-foot region, and liver-gonad determined using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) method. Asterisk indicates statistically significant difference (*P < 0.05) when the level is compared with that at 0 h for the same tissue. For other abbreviations, see Fig. 2

Fig. 6a, b

Dynamic mortality rate of O. hupensis after WPN treatment with the addition of salicylhydroxamic acid (SHAM). a SHAM added together with WPN at 0 h. b SHAM added at 6 h after WPN treatment. 0.1WPN Niclosamide at 0.1 mg/L, SHAM SHAM at 0.4 mM, 0.1WPN–SHAM niclosamide at 0.1 mg/L and SHAM at 0.4 mM (added at the same time), Methanol methanol at 0.08% (control solvent for SHAM), hash symbol indicates that the corresponding reagent was added at 6 h

Fig. 7

Mortality rate of O. hupensis treated with different concentrations of niclosamide with and without inhibition of OhAOX activity through the addition of SHAM. For abbreviations, see Figs. 2 and 6