Keywords

11.1 Introduction

In the environment, there are many toxicants or stresses having the potential to cause the toxicity at different aspects on nematodes [1,2,3,4,5,6]. For a long time, the researchers in the field of toxicology or environmental science have tried to identify the specific molecular signaling pathways for nematodes in response to certain environmental toxicants or stresses. And then, an important question needs us to further carefully judge or evaluate. That is, are there any specific molecular signals for nematodes in response to certain environmental toxicants or stresses?

In this chapter, we selected three well-described response signals (heavy metal response, heat shock response, and hypoxia response) to discuss this question. We first introduced and discussed the widely accepted molecular signals for the control of these three important responses. Again, we will discuss the potential involvement of these molecular signals in the regulation of toxicity from other environmental toxicants or stresses in nematodes.

11.2 Heavy Metal Response Signaling

11.2.1 Molecular Signaling for Heavy Metal Response

11.2.1.1 Metallothioneins

Metallothioneins are widely considered as the primary player in the detoxification of and protection from toxicity by the heavy metal of cadmium (Cd) [7]. In nematodes, mtl-1 and mtl-2 encode the metallothioneins. A highly sensitive and dose-dependent transcriptional response to Cd, but not Cu or Zn, was observed for both MTL-1 and MTL-2 (Fig. 11.1) [7]. Additionally, Cd exposure induced the sharp induction of both MTL-1::GFP in the pharynx and intestine and MTL-2::GFP in the intestine (Fig. 11.2) [7]. No measurable upregulation of mtl-1 could be detected in mlt-2 mutant nematodes [7], suggesting that these two genes are independent and not synergistic action may be formed.

Fig. 11.1
figure 1

Isoform-specific transcript analysis of mtl-1 and mtl-2 [7]. Utilizing a semiquantitative PCR-based approach, it was possible to assess the dose response to cadmium (a), copper, and zinc (b) and the temporal response to cadmium exposure (c) of mtl-1 and mtl-2 in wild-type nematodes and gk125 the mtl-2 null allele. PCRs were terminated at the half-log phase of amplification and normalized to the invariant ribosomal protein rpa-1

Full size image
Fig. 11.2
figure 2

Promoter-GFP fusions of mtl-1 (a) and mtl-2 (b) after germ line transformation [7]. The background signal (-Cd) was significantly increased by exposure to 10 mM cadmium (+Cd) for a 24 h period. In both isoforms, the primary site of cadmium responsive induction is the intestine, but note that only mtl-1 is expressed constitutively in the lower pharyngeal bulb (right-hand panels)

Full size image

Mutation or RNAi knockdown of mtl-1 or mtl-2 induced a susceptibility to Cd toxicity using body size, generation time, brood size, and lifespan as the toxicity assessment endpoints (Fig. 11.3) [7], which provide the functional evidence to demonstrate the important roles of MTL-1 and MTL-2 in regulating the Cd toxicity in nematodes.

Fig. 11.3
figure 3

Exposure to cadmium slows down development in a dose-responsive manner [7]. This effect is more profound in gk125 and particularly during knockdown of mtl-1, mtl-2, or both by RNAi. All images are representative of 2-day post hatch nematodes. Numbers below the images are average area measurements (G standard error) from four nematodes. Based on a nonparametric Mann–Whitney U-test, identical letters (in brackets) denote that samples are not significantly different (P ≥ 0.05)

Full size image

11.2.1.2 CDR-1

CDR-1 is a predicted 32-kDa, integral membrane protein. cdr-1 is transcribed exclusively in intestinal cells of postembryonic nematodes [8]. In the intestine, the CDR-1 is targeted to the cytoplasmic lysosomes [8]. The cdr-1 transcription was significantly induced by Cd exposure, but not by other examined stressors (heavy metals of Hg, Cu, and Zn, paraquat, hugalone, and heat shock) (Fig. 11.4) [8], suggesting that CDR-1 expression is under the control of Cd exposure in a cell-specific fashion. In addition, RNAi knockdown of cdr-1 also induced a susceptibility to Cd [8, 9].

Fig. 11.4
figure 4

Cell-specific expression of cdr-1 [8]. (a, b) C. elegans, strain JF9 (mtIs7, cdr-1/lacZ) was exposed to 100 μM CdCl2 for 24 h and then stained for β-galactosidase activity. (a) Reporter gene activity in the intestinal cells of a young adult and an L3 larva. (b) cdr-1 promoter activity in the intestinal cells of an L1 larva. (c) In situ hybridization of cadmium-treated L1 C. elegans larvae. CDR-1 mRNA was visualized by exposing wild-type nematodes to a digoxigenin-dUTP-labeled CDR-1 antisense probe. (c) shows that the CDR-1 transcript is expressed throughout the intestine of L1. “ph” marks the location of the pharynx in all panels. Nematodes were photographed using Nomarski optics

Full size image

11.2.2 Regulation of Toxicity of Other Environmental Toxicants by MTL-1 and MTL-2

Some engineered nanomaterials (ENMs) have been shown to be toxic for organisms, including the nematodes [10,11,12,13,14,15,16,17]. Among these ENMs, titanium dioxide nanoparticles (TiO2-NPs) and zinc oxide NPs (ZnO-NPs) are two widely used ENMs. In nematodes, mutation of mtl-2 resulted in a susceptibility to the TiO2–NPs toxicity in reducing body length, in reducing brood size, in decreasing locomotion behavior as indicated by the endpoints of head thrash and body bend, and in inducing intestinal autofluorescence and ROS production [18]. In nematodes, pcs-1 encodes a phytochelatin synthase. Additionally, the severe susceptibility to ZnO-NPs toxicity was observed in the triple mutant of mtl-1;mtl-2;pcs-1(zs2) (Fig. 11.5) [19]. Moreover, the mtl-2::GFP expression was significantly increased by exposure to ZnO-NPs in nematodes (Fig. 11.5) [19].

Fig. 11.5
figure 5

Induction of green fluorescence protein (measured as relative fluorescence units) in mtl-2::GFP and pcs-1::GFP transgenic Caenorhabditis elegans in response to ZnO-NPs [19]. Approximately ten worms were observed in each case. Error bars represent the standard error (n = 10). Statistical analysis was performed using the Wilcoxon signed-rank test (p < 0.05). Exposure to ZnO-NPs generates high levels of free radical H2O2 in C. elegans.

Full size image

Nanopolystyrene particles are another important ENM with the wide commercial uses. In nematodes, prolonged exposure to nanopolystyrene particles caused the toxicity on the functions of both primary and secondary targeted organs [20]. It was further found that the intestinal insulin signaling pathway including the daf-16 encoding a FOXO transcriptional factor was involved in the control of toxicity of nanopolystyrene particles [21]. daf-16 has many potential targeted genes during the control of biological processes [22,23,24], and, among them, lys-8, dct-6, nnt-1, ftn-1, pept-1, dod-17, klo-1, ncx-6, cyp-35A3, nhx-2, vha-6, pho-1, gale-1, cyp-34A9, stdh-1, nrf-6, hsp-16.1, hsp-16.2, asah-1, ges-1, cpr-1, acdh-1, vit-5, mtl-2, dod-22, gcy-18, vit-2, lys-7, mtl-1, dct-18, gpb-2, fat-5, cyp-35B1, sod-3, sodh-1, icl-1, fat-7, dod-4, fat-7, asp-3, mdl-1, spp-1, and dct-1 can be expressed in the intestine. Exposure to nanopolystyrene particles (1 μg/L) only significantly increased the transcriptional expressions of sod-3, mtl-1, gpb-2, fat-7, and sodh-1 in wild-type nematodes (Fig. 11.6) [21]. Meanwhile, daf-16 mutation significantly decreased the transcriptional expressions of sod-3, mtl-1, gpb-2, fat-7, and sodh-1 after exposure to nanopolystyrene particles (1 μg/L) (Fig. 11.6) [21]. Among these targeted genes, using VP303 as a genetic tool, it was observed that intestine-specific RNAi knockdown of mtl-1 induced a susceptibility to the toxicity of nanopolystyrene particles in inducing ROS production (Fig. 11.6) [21]. These results suggest that mtl-1 acts as a targeted gene of daf-16 in the regulation of toxicity of nanopolystyrene particles.

Fig. 11.6
figure 6

Identification of targeted genes for daf-16 in the regulation of response to nanopolystyrene particles [21]. (a) Effects of exposure to nanopolystyrene particles on gene expression in wild-type nematodes. (b) Effects of daf-16 mutation on expression of intestinal targeted genes of daf-16 after exposure to nanopolystyrene particles. (c) Effect of intestine-specific RNAi knockdown of sod-3, mtl-1, gpb-2, fat-7, or sodh-1 on induction of ROS production in nematodes exposed to nanopolystyrene particles. Nanopolystyrene exposure concentration was 1 μg/L. Nanopolystyrene exposure was performed from L1-larvae to adult day 3. Bars represent means ± SD. ** P < 0.01 vs control (if not specially indicated)

Full size image

11.3 Heat Shock Response Signaling

11.3.1 Molecular Signaling for Heat Shock Response

11.3.1.1 Heat Shock Proteins

Early in 1983, it was reported that treatment of the nematodes with elevated temperatures could induce the synthesis of the heat shock proteins [25]. In contrast, synthesis of most other proteins present before the heat shock was suppressed [25]. Additionally, the dauer larvae possess little translatable mRNA but, upon the heat shock, synthesizes a set of messages corresponding to the heat shock proteins [25].

11.3.1.2 HSF-1

hsf-1 encodes a heat shock factor. HSF-1 is a master transcriptional regulator of stress-inducible gene expression, as well as protein-folding homeostasis [26]. hsf-1 was required for both the longevity control and the temperature-induced dauer larvae formation in nematodes [26, 27]. Heat shock-induced expression of hsp-16.2 was virtually eliminated in hsf-1 mutant nematodes (Fig. 11.7) [27], suggesting the potential role of HSF-1 as an inducer of heat shock response.

Fig. 11.7
figure 7

Decreased levels of hsp-16.2 mRNA in suppressor mutants, as measured by quantitative real-time PCR [27]. Input levels for each sample were normalized with control PCR reactions using 18S rRNA

Full size image

11.3.2 Regulation of Toxicity of Other Environmental Toxicants by Heat Shock Proteins

In nematodes, heat shock treatment induced the significant increase in HSP-16.1::GFP, HSP-16.2::GFP, HSP-16.41::GFP, and HSP-16.48::GFP (Fig. 11.8) [28]. Meanwhile, hypoxia treatment also induced the significant increase in HSP-16.1::GFP and HSP-16.2::GFP, and ethanol treatment induced the significant increase in HSP-16.1::GFP, HSP-16.2::GFP, HSP-16.41::GFP, and HSP-16.48::GFP (Fig. 11.8) [28].

Fig. 11.8
figure 8

The hypoxia responses of the hsp-16 genes in C. elegans are distinct [28]. (a) The expression of HSP-16.1 is induced by several types of stress. The first row represents the basal Fig. 11.8 (continued) expression of HSP16-1. HSP16-1 is minimally expressed without stress. The second row represents the result of exposure to 7% ethanol for 30 min. The third row represents the result of exposure to 1 mM of sodium azide for 30 min. The forth row represents the result of exposure to 200 mM of NaCl solution for 30 min, and the last row represents the result of starvation for 12 h. The figures of the first and third column are Nomarski images, and the second and fourth are GFP fluorescence driven by the hsp-16-1 promoter. HSP-16.1 was more strongly induced by starvation than by other stresses and appeared as punctuated spots. The scale bar represents 50 mm. (b) Hypoxia response of the hsp-16 genes in a hypoxia chamber. Each column represents the expression of hsp-16.1, hsp-16.2, hsp-16.41, and hsp-16.48, respectively. Upper panels are control animals and lower panels, the animals incubated in the hypoxia chamber. Only hsp-16.1 and hsp-16.2 responded to hypoxia. (c) Hypoxia response of the hsp-16 genes in the animals soaked in the M9 buffer. Each column represents the expression of hsp-16.1, hsp-16.2, hsp-16.41, and hsp-16.48, respectively. All four genes are responsive to heat shock and ethanol stress, but only hsp-16.1 and hsp-16.2 responded to hypoxia. We observed more than 30 animals from at least two independent transgenic lines for each gene and obtained similar results shown in this figure. This figure shows representative animals. The scale bars in (b, c) represent 0.2 mm

Full size image

Moreover, it was found that mutation of hsp-16.48 could further result in a susceptibility to the TiO2-NPs toxicity in reducing body length, in reducing brood size, in decreasing locomotion behavior, and in inducing intestinal autofluorescence and ROS production [18]. Similarly, mutation of hsp-16.48 caused the susceptibility to graphene oxide (GO) toxicity in reducing brood size, in decreasing locomotion behavior, and in inducing intestinal ROS production, and the enhanced accumulation of GO in the body of nematodes (Fig. 11.9) [29]. In addition, mutation of hsp-70 or hsp-3 increased the susceptibility of nematodes to Mn toxicity in reducing lifespan, and mutation of hsp-70 enhanced neurotoxicity of Mn in inducing the degradation of dopaminergic neurons in nematodes [30].

Fig. 11.9
figure 9

Distributions of GO-Rho B in wild-type and mutant nematodes [29]. (a) Pictures showing the distributions of GO-Rho B in wild-type and mutant nematodes. (b) Comparison of relative fluorescence of GO-Rho B in the intestine between wild-type and mutant nematodes. (c) Comparison of relative fluorescence of GO-Rho B in the pharynx between wild-type and mutant nematodes. (d) Comparison of relative fluorescence of GO-Rho B in the spermatheca between wild-type and mutant nematodes. The arrowheads indicate the pharynx. The intestine (**) and the spermatheca (*) are also indicated. GO exposure was performed from L1-larvae to young adult. The exposure concentration of GO was 100 mg/L. Bars represent means ± SEM. **P < 0.01 vs. N2

Full size image

11.3.3 Regulation of Toxicity of Other Environmental Toxicants by HSF-1

Environmental pathogen infection can potentially cause the toxicity as different aspects on organisms, including the nematodes [31,32,33,34,35,36,37,38,39]. It was observed that RNAi knockdown of hsf-1 induced a susceptibility to P. aeruginosa PA14 infection in reducing the lifespan, whereas overexpression of HSF-1 induced a resistance to P. aeruginosa PA14 infection in reducing the lifespan (Fig. 11.10) [40]. The susceptibility of hsf-1 mutant nematodes to P. aeruginosa PA14 infection in reducing the lifespan could be further enhanced by RNAi knockdown of pmk-1 encoding a p38 MAPK in p38 MAPK signaling pathway (Fig. 11.10) [40].

Fig. 11.10
figure 10

HSF is required for C. elegans immunity to P. aeruginosa [40]. (a) Wild-type worms grown on E. coli carrying a vector control were untreated or HS-treated and exposed to P. aeruginosa (P < 0.03). In addition, wild-type animals grown on E. coli expressing hsf-1 double-stranded RNA were untreated or HS-treated and exposed to P. aeruginosa (P = 0.2660). hsf-1 RNAi animals were more susceptible to P. aeruginosa compared with vector RNAi controls (P < 0.005). (b) Wild-type or hsf-1(sy441) animals were grown on E. coli carrying a vector control and exposed to P. aeruginosa (P < 0.0001). (c) Wild-type animals grown on vector control or hsf-1 double-stranded RNA were exposed to P. aeruginosa at 20 °C (P < 0.0001). (d) Wild-type or CF1824 (hsf-1 overexpression) animals were exposed to P. aeruginosa (P < 0.001). (e) Wild-type animals grown on E. coli carrying a vector control or expressing pmk-1 double-stranded RNA were exposed to P. aeruginosa (P < 0.0001). (F) hsf-1(sy441) animals grown on E. coli carrying a vector control or expressing pmk-1 double-stranded RNA were exposed to P. aeruginosa (P = 0.0023). For each condition, 80–200 animals were used

Full size image

In nematodes, it was further observed that RNAi knockdown of hsf-1 could suppress the resistance of daf-2 mutant nematodes to pathogen infection (Fig. 11.11) [40], suggesting that HSF-1 may act downstream of insulin signaling pathway to regulate the innate immune response to pathogen infection in nematodes. That is, the increased temperature activated HSF-1 will enhance the innate immunity, and this HSF-1 defense response is mediated by the further induction of a system of chaperones.

Fig. 11.11
figure 11

HSF-1 is required for immunity to Gram-negative and Gram-positive pathogens [40]. (a) Wild-type worms grown on E. coli carrying a vector control or on E. coli expressing hsf-1 double-stranded RNA were exposed to Y. pestis KIM5 (P < 0.0001). daf-2(e1370) worms grown on E. coli carrying a vector control or on E. coli expressing hsf-1 double-stranded RNA were exposed to Y. pestis KIM5 (P < 0.001). (b) Wild-type worms grown on E. coli carrying a vector control or on E. coli expressing hsf-1 double-stranded RNA were exposed to S. enterica SL1344 (P < 0.0001). daf-2(e1370) worms grown on E. coli carrying a vector control or on E. coli expressing hsf-1 double-stranded RNA were exposed to S. enterica SL1344 (P < 0.0001). (c) Wild-type worms grown on E. coli carrying a vector control or on E. coli expressing hsf-1 double-stranded RNA were exposed to En. faecalis OG1RF (P < 0.0001). daf-2(e1370) worms grown on E. coli carrying a vector control or on E. coli expressing hsf-1 double-stranded RNA also were exposed to En. faecalis OG1RF (P < 0.0001). (d) HSF-1 activated by HS and the DAF-2_DAF-16 pathway enhances C. elegans immunity. HSF-1 mediates protection via induction of Hsp90 and small HSPs in a PMK-1-independent manner

Full size image

11.4 Hypoxia Response Signaling

11.4.1 Molecular Signaling for Hypoxia Response

11.4.1.1 HIF-1 and EGL-9

hif-1 encodes a bHLH-PAS hypoxia-inducible transcription factor. During the response to hypoxia stress, a physical complex is formed, and this complex contains HIF-1 and AHA-1, which are encoded by C. homologs of hypoxia-inducible factor (HIF) α and β subunits, respectively [41]. The expression of HIF-1:GFP could be highly induced by hypoxia and was subsequently reduced upon reoxygenation (Fig. 11.12) [41]. In addition, the subcellular localization of AHA-1 was disrupted in the hif-1 (ia04) mutant nematodes [41], suggesting that HIF-1 acts upstream of AHA-1 to regulate the hypoxia stress in a complex.

Fig. 11.12
figure 12

HIF-1:GFP protein levels are increased by hypoxia [41]. The transgenic C. elegans strain pHJ06 Ex6, which expresses the complete HIF-1 protein fused to GFP, was harvested before (21% O2) and after incubation in 0.1% oxygen (upper) or 1% oxygen (lower) for 8 h. In each experiment, a fraction of the hypoxia-treated worms was reoxygenated for 10 min (ReOx). Worms were lysed by boiling in loading buffer, and equal amounts of total protein were loaded in each lane. The expression of HIF-1:GFP and AHA-1 was assayed by probing the immunoblot with a GFP-specific antibody and the AHA-1-specific mAb 10H8

Full size image

HIF-1 is a critical regulator for the responses to low oxygen levels. The hif-1 mutant nematodes exhibited no adaptation to hypoxia stress, and the majority of hif-1-defective nematodes would die under these conditions (Fig. 11.13) [41, 42]. Additionally, it was found that some HIF-1 target genes could negatively regulate the formation of stress-resistant dauer larvae [42], suggesting the potential involvement of HIF-1 in the regulation of other stresses in nematodes.

Fig. 11.13
figure 13

Viability assays in normoxia (21% oxygen) and hypoxia (0.5% oxygen) [42]. Animals carrying strong loss-of-function mutations in downstream targets of HIF-1 were incubated in 0.5% oxygen (hypoxia) or room air (normoxia) for 24 h and then assayed for successful completion of embryogenesis (a) and survival to adulthood (b). Asterisks indicate that the viability of hypoxia-treated mutants is significantly lower than that of wild-type (WT)

Full size image

A summary for the basics molecular basis for the hypoxia response signaling is provided in Fig. 11.14 [42]. During the control of response to hypoxia stress, EGL-9/PHD modifies the HIF-1, thereby increasing its affinity for VHL-1/E3 ligase to target the HIF-1 for proteasomal degradation (Fig. 11.14) [42]. That is, the proteasomal degradation of HIF-1 is mediated by both the EGL-9 and the VHL-1 (Fig. 11.14) [42]. Meanwhile, the expression of egl-9 could also be regulated by hif-1 mutation [42]. This suggests that the positive regulation of egl-9 transcription by HIF-1 will further help to maintain the EGL-9 activity when oxygen substrate is limiting, and a feedback loop will be activated to attenuate the HIF-1 activation (Fig. 11.14) [42]. In addition, the response to hypoxia is mediated by HIF-1-dependent or by HIF-1-independent pathway(s) (Fig. 11.14) [42]. HIF-1 can further positively regulate the expression of npc-1 and cam-1, regulators of dauer formation [42]. The increased expression in daf-16 is a common feature of both the hypoxia response and of the perturbations in insulin signaling pathway [42].

Fig. 11.14
figure 14

Models for hypoxia signaling and response [42]

Full size image

11.4.1.2 Aminoacyl-tRNA Synthetase RRT-1

rrt-1 encodes a arginyl-transfer RNA (tRNA) synthetase, an enzyme essential for protein translation. Mutation of RNAi knockdown of rrt-1 or other genes encoding the aminoacyl-tRNA synthetase could rescue the nematodes from the hypoxia-induced death [43]. More importantly, this hypoxia resistance was inversely correlated with the protein translation rate [43]. The ER unfolded protein response (UPR) induced by hypoxia was required for the resistance of rrt-1 mutant nematodes to the hypoxia stress [43], suggesting that the translational suppression can induce the hypoxia resistance in part by reducing the unfolded protein toxicity in nematodes.

11.4.2 Regulation of Toxicity of Other Environmental Toxicants by HIF-1 and EGL-9

In nematodes, it was found that loss-of-function mutation of hif-1 promoted the resistance of nematodes to exogenous mitochondrial stressor ethidium bromide (EtBr) and suppressed the EtBr-induced ROS production (Fig. 11.15) [44]. Similarly, mutation of hif-1 also induced a resistance to high-glucose diets stress in reducing lifespan in nematodes [45]. During the control of EtBr toxicity, p38 MAPK signaling was identified as an indispensable factor for the survival against mitochondrial stress in hif-1 mutant nematodes [45].

Fig. 11.15
figure 15

hif-1 and djr-1.1 mutants are highly resistant to EtBr [44]. (a) hif-1 animals exposed to EtBr have no effect on fecundity over three generations, while wild-type worms showed a significant decrease in brood size. Brood size of N2 (wild-type), djr-1.1, and hif-1 animals in 25 μg/mL EtBr were counted for three generations: F1, F2, and F3. Relative fecundity is measured as the number of progeny grown to adulthood on EtBr divided by the number of progeny grown to adulthood in the absence of EtBr and would include animals that arrest at the L3 stage. Animals with ruptured vulva were not counted. Asterisk marks denote significant difference of fecundity between P0 (normalized to 1.0) and the following generations for each strain (* denotes p < 0.05 and ** p < 0.01) using one-way ANOVA. (b) Concentration dependence of EtBr. N2, hif-1, vhl-1, and djr-1.1 mutant animals (first generation) were grown on worm plates containing nematode growth media and various concentrations of EtBr (0, 25, 50, and 100 μg/mL). The number of worms that grew to adulthood was determined and plotted. To determine significant differences between strains, the data were subjected to two-way ANOVA comparing groups (different strains) over different concentrations of EtBr. All strains showed a significant difference compared to N2 (***p < 0.001). (c–h) Growth of strains on EtBr. N2, djr-1.1 and hif-1 worms (as labeled) 4 days after egg-laying in plates containing either no EtBr (c, e, g) or 50 μg/mL EtBr (d, f, h)

Full size image

Moreover, it was observed that mutation of hif-1 induced a susceptibility to pore-forming toxins (PFTs) toxicity in reducing the lifespan (Fig. 11.16) [46]. In contrast, the Cry21A PFT resistance was observed in egl-9 mutant nematodes (Fig. 11.16) [46]. In nematodes, the intestinal specific expression of egl-9 was sufficient to rescue the Cry21A PFT resistance [46]. Two of the downstream effectors of this pathway were identified, and they were nuclear receptor NHR-57- and XBP-1-mediated ER UPR signaling during the control of PFTs toxicity in nematodes.

Fig. 11.16
figure 16

Quantitative response of egl-9 and HIF-1 pathway mutants to Cry PFTs [46]. (a) Schematic illustrating O2-dependent regulation of HIF-1 activity. The O2-dependent prolyl hydroxylation of HIF-1 by EGL-9/PHD increases its affinity to VHL-1, leading to ubiquitylation and destruction. (b, c) Dose-dependent mortality assays were performed using (b) Cry21A spore crystal lysates or (c) purified Cry5B to quantitatively compare sensitivities of wild-type N2, egl-9 mutants, and HIF-1 pathway mutants to PFTs. Each data point shows the mean and standard errors of the mean of results from three independent experiments (three wells per experiment; on average, 180 animals per data point). Statistical differences between mutant strains and N2 are given for each concentration using P values represented by asterisks as follows: * P < 0.05; ** P < 0.01; *** P < 0.001

Full size image

Furthermore, it was found that mutation of hif-1 induced a susceptibility to pathogen infection, whereas mutation of egl-9 induced a resistance to pathogen infection (Fig. 11.17) [47]. Moreover, the HIF-1a was dispensable for the host defense gene induction, and SWAN-1-mediated noncanonical pathway inhibited this HIF-1 induced defense gene repression in nematodes [47].

Fig. 11.17
figure 17

egl-9 inactivation causes enhanced susceptibility to S. aureus-mediated killing [47]. (a) egl-9(sa307) animals exhibited enhanced susceptibility, whereas egl-9(sa307);hif-1(ia4) mutants exhibited near wild-type susceptibility. Survival analysis: egl-9 Kaplan–Meier median survival (MS) = 62 h, time to 50% death by nonlinear regression analysis (LT50) = 48.78 h, number of animals (N) = 142, p < 0.0001 (log-rank test, compared with wild-type); egl-9;hif-1 MS = 68 h, LT50 = 62.10 h, N = 122/2, p = 0.0030 (compared with wild-type). (b) vhl-1(ok161) and hif-1(ia4) animals exhibited near wild-type susceptibility. Survival analysis: wild-type MS = 74 h, LT50 = 67.03 h, N = 117/5; vhl-1 MS = 62 h, LT50 = 61.86 h, N = 118, p < 0.0001 (compared with wild-type); hif-1 MS = 74 h, LT50 = 64.77 h, N = 136, p = 0.0943 (compared with wild-type). (c) egl-9(sa330) animals and (d) egl-9(ok478) animals exhibit wild-type susceptibility. (e) egl-9(n586ts) animals are hypersusceptible to S. aureus. Survival analysis: egl-9(sa307) MS = 43 h, N = 95/1, p < 0.0001 (compared with wild-type); egl-9;(n586ts) MS = 43 h, N = 96/15, p < 0.0001 (compared with wild-type); wild-type MS = 50 h, N = 92/9. (f) Wild-type, egl-9(sa307);crp-1::egl-9 (Intestinal egl-9), and egl-9(sa307);crp-1::gfp (Intestinal gfp) animals show that intestinal expression of EGL-9, but not GFP, rescues the egl-9(sa307) enhanced susceptibility phenotype. Survival analysis: wild-type MS = 70 h, N = 108/7; Intestinal egl-9 MS = 61 h, N = 115/14, p < 0.0001 (compared with wild-type), p < 0.0001 (compared with Intestinal gfp); Intestinal gfp MS = 48 h, N = 102/3, p < 0.0001 (compared with wild-type). Results are representative of two independent trials, performed in triplicate. Animals were subjected to cdc-25 RNAi to prevent reproduction and subsequently transferred to S. aureus killing assay plates

Full size image

11.5 Perspectives

In this chapter, we selected three well-described response signals (heavy metal response, heat shock response, and hypoxia response) to discuss the question on the specificity of molecular signals in response to certain environmental toxicants or stresses in nematodes. Actually, there are still other relevant evidence on the study of UV irradiation, osmotic stress, etc. As introduced and discussed in this chapter, so far the obtained data in nematodes does not support the existence of specific molecular signals in response to certain environmental toxicants or stresses. The molecular basis for the response to heavy metal response, heat shock response, or hypoxia response can also involve in the regulation of toxicity of other environmental toxicants or stresses under certain conditions. However, this also not means that the nematodes share the completely same molecular signaling pathways in response to different environmental toxicants or stresses. The further studies are suggested to focus on the definition of direct or primary molecular signals involved in the regulation of certain environmental toxicants or stresses in nematodes.