Published On: Tue, Apr 16th, 2019

Nematicidal actions of the marigold exudate {alpha}-terthienyl: oxidative stress-inducing compound penetrates nematode hypodermis [RESEARCH ARTICLE]


α-terthienyl is photoactivated generator of singlet oxygen and superoxide anion radicals (Huang et al., 2017; Nivsarkar et al., 2001). Mosquito larval mortality was 100% after treatment with naturally extracted or commercially synthesized α-terthienyl at concentrations of 33 ppb (≈0.13 µM) and ultraviolet excitation (366 nm) (Nivsarkar et al., 2001). No difference was observed between light-excited and non-treated α-terthienyl against the cyst nematode H. zeae; mortality of H. zeae was 100% when treated with commercially available α-terthienyl at concentrations of 0.125% (≈5 mM) for 24 h (Faizi et al., 2011). Our experiments clearly indicated that photoactivated α-terthienyl shows much greater toxicity against C. elegans young adults, dauer larvae, and M. incognita J2s than that without photoactivation. Since α-terthienyl is expected to exert nematicidal action in the soil, we investigated in detail the action of the chemical without photoactivation.

SODs are enzymes that detoxify superoxide (O2) radicals into oxygen (O2) and hydrogen peroxide (H2O2), followed by the detoxification of H2O2 to H2O and O2 by CTLs. Phase II enzymes catalyze reactions that conjugate critical reducing agents with reactive xenobiotics and/or oxidative biomolecules metabolized by Phase I enzymes (An and Blackwell, 2003). Biological defense systems against such harmful molecules are broadly conserved in animals. In mammals, NF-E2-related factor 2 (Nrf2) is a central transcription factor involved in the transcriptional activation of many genes encoding Phase II enzymes via the antioxidant response element (Nguyen et al., 2003). Functional similarity of C. elegans SKN-1 to mammalian Nrf has been reported (An and Blackwell, 2003). In C. elegans, SKN-1 is a transcription factor required for response to oxidative stresses; SKN-1 activation induces the expression of ctl, sod, and at least nine gst genes (Park et al., 2009). The SKN-1 binding site is present in the promoter regions of many Phase II detoxification enzymes and also that of ctl and sod (An and Blackwell, 2003; Lindblom and Dodd, 2006). Steady-state mRNA levels of the gst-4 increased 40-fold, and the sod-1 and -3 increased twofold in the C. elegans homogeneous larval population in response to exposure to an oxidative stress inducer paraquat (Tawe et al., 1998).

Nuclear accumulation of SKN-1 is repressed by WDR-23 which is expressed in intestinal-, hypodermal- and neuronal-cell nuclei and interacts with DDB-1 and SKN-1 (Choe et al., 2009). Loss of function of WDR-23 causes constitutive transcription of Phase II detoxification genes, accumulation of SKN-1 in the intestinal nuclei, and elevation of SKN-1 protein levels (Choe et al., 2009). skn-1 RNAi prevented GST-4 expression induced by the strong GST inducer acrylamide except for that in the pharynx and body wall muscle (Hasegawa et al., 2008). In addition, the upregulations of sod-1 and ctl-2 were inhibited 80% and 100%, respectively, by skn-1 RNAi (Park et al., 2009). Since GST-4 and SOD-1 were induced in the C. elegans hypodermis after treatment with α-terthienyl (Figs 3 and 4), this chemical quickly permeates the nematode cuticle and acts as an oxidative stressor. If α-terthienyl is taken by mouth, expression of these detoxifying enzymes should also be observed in the pharyngeal muscle and intestine, as observed in the transgenics treated with acrylamide (Figs 4 and 5; Fig. S1). We confirmed that the susceptibility of nematodes to α-terthienyl increased when expression of GST-4 and SOD-1 was suppressed by skn-1 RNAi. Furthermore, we found that knocking down of wdr-23 induced GST-4 and SOD-1 expression and conferred resistance against α-terthienyl. CTL-1 is constitutively expressed in the nematode intestine, but its expression was not induced when transgenic KHA166 (ctl-1::gfp) was treated with α-terthienyl. We also confirmed that CTL-1 was not affected neither by the RNAi of either skn-1 or wdr-23 (data not shown). This result supports the hypothesis that the defense system against α-terthienyl is largely dependent on the SKN-1/WDR-23.

Furthermore, we found that dauer larvae exhibited much greater sensitivity to α-terthienyl (Figs 1 and 2). C. elegans dauer larvae are morphologically and metabolically specialized to be able to survive harsh conditions (Cassada and Russell, 1975; Erkut and Kurzchalia, 2015). Vanfleteren and De Vreese (1995) found that SOD and CTL activity was elevated in dauer larvae, which is in contrast to our results. Although constitutive expression of these enzymes is more frequent in the dauer stage than in the propagative stage, their inducible responses against xenobiotics might be stagnant. We did not observe induction of GST-4 and SOD-1 expression in dauer larvae of the transgenics after α-terthienyl treatment (data not shown). From these results, we concluded that α-terthienyl is an oxidative stress-inducing chemical that effectively penetrates the nematode hypodermis even in the dauer/infectious larvae and exerts nematicidal activity.

Other plants with nematicidal properties have been reported and these are believed to release nematicidal compounds when incorporated into the soil. 1,2-dehydropyrrolizidine alkaloids (PAs) present in Crotalaria are used in nematode control (Ntalli and Caboni, 2012). One of the main allelochemical compounds of monocrotaline has harmful effects on livestock and humans, damaging the central nervous system (Ntalli and Caboni, 2012). A new active compound 6′-methyl-fungichromin (fungichromin B) extracted from the potent nematicidal bacteria Streptomyces albogriseolus HA10002 could also be a promising candidate as a natural microorganism-based product (Zeng et al., 2013). These practicalities could also be tested on the model nematode C. elegans.

In this model system we show that α-terthienyl (1) has nematicidal activity even without photoactivation, (2) is more effective against dauer larvae than adults, (3) penetrates the nematode hypodermis and exerts effects, and (4) acts through oxidative stress. This substance would be effective even in soils with limited or no light, and exerts its nematicidal actions against non-feeding larvae by penetrating their cuticle, suggesting high potential for practicable use as an agricultural nematode control agent.

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