Do cestode parasites affect As toxicity in Artemia?

This study tested the effect of parasites on Artemia sensitivity to As, and explored the physiological crosstalk between the parasite and the host, measuring oxidative stress and lipid content in infected and uninfected Artemia. Our study provides the first empirical evidence that parasites can increase resistance to metal or metalloid pollution, rather than decrease it. It is also the first study to consider the influence of temperature change on parasite-pollutant interactions. In three separate acute toxicity experiments, Artemia infected with cestodes consistently showed lower sensitivity to As than uninfected individuals. The higher sensitivity of infected Artemia in April suggests that multiple infections may reduce the benefits of cestode infection to host resistance.

Our results contradict the pre-existing view that pollution and parasites are stressors that both have negative effects on the health of free living organisms. This view was based on previous field and laboratory investigations (including chronic and acute exposure to a wide variety of toxicants, in vertebrates and invertebrates, intermediate and definitive hosts, and in several groups of parasites [43,44,45].

The results of oxidative stress analysis provide a mechanistic explanation for our findings. Infected individuals exhibited much higher levels of CAT and GR, reflecting a superior ability to combat the effects of exposure to pollutants with oxidative potential, such as As. The particularly high levels of CAT in infected individuals (nearly double that of uninfected Artemia) is related to the increased levels of hemoglobins in F. liguloides-infected Artemia compared with uninfected individuals (Fig 2C and 2D). CAT is a haematin protein complex with four porphyrin heme groups that allow the enzyme to react with hydrogen peroxide. There is a close linear relationship between CAT activity and hemoglobin concentration in human blood [46]. CAT also has one of the highest rates of all enzymes; one CAT molecule is able to catalyse the conversion of 5 million molecules of hydrogen peroxide per second to water and oxygen. Thus, even if the level of SOD was lower in infected individuals, the control mechanisms via CAT and GR seem to be sufficient to avoid the establishment of an oxidative stress condition, as indicated by the lack of changes in TBARS between infected and uninfected individuals. TBARS is a by-product of lipid peroxidation, so this result indicates that parasites are not inducing damage by oxidative stress. Our results conflict with what most studies have shown up to now (but see for example Marcogliese et al 2005 [6]). Infection by parasites and pathogens of a wide range of taxa are generally associated with the inhibition or weakening of the host antioxidant system, and the concomitant increase in TBARS [47,48,49].

This enhancement of the anti-oxidative defense mechanisms is probably connected with the trophic transmission mode of cestodes that infect Artemia, which means they require “healthy” hosts in order to increase transmission success (through predation by the definitive host). Decrease in antioxidant status enhances short-term survival prospects [50], so potentiating it may be part of the transmission strategy of the parasite to increase longevity and probability of transmission (as in the “parasite manipulation hypothesis” [51]).

We also found that there were more lipid droplets in infected individuals, which is consistent with previous work indicating that F. liguloides increases total lipid levels [52] and that C. podicipina increases triglyceride levels in Artemia [26] (Fig 2B). Lipids have a high heavy metal binding capacity, and lipid content has a significant effect on the accumulation of As in other organisms [53]. Neutral lipids such as those in lipid droplets can protect organisms against pollutants, sequestering them away from sensitive target sites [54,55]—a principle known as survival of the fattest [41]. Although many studies support this principle, none have addressed parasite-mediated effects. The only previous study to suggest that parasites can increase host survival under polluted conditions through a lipid-related effect was on the freshwater clam Pisidium amnicum [56]. Clams infected by digenean trematode larvae are less sensitive to pentachlorophenol, perhaps because the high lipid content of the parasite changes the internal distribution of the toxicant. Pentachlorophenol is moderately lipophilic so is expected to accumulate in adipose tissue, whereas the target sites for toxicity are the mitochondria [56]. In parasitized Artemia, additional lipids accumulate in the host, not in the parasite as in the case of Pisidium.

Lipid droplets in infected Artemia are associated with carotenoids (see Fig 2B) and this, together with hemoglobins induced by parasites, largely explains the colour change that allowed us to separate infected individuals with the naked eye (Fig 1). Both F. liguloides and C. podicipina increase the concentration of carotenoids in infected Artemia [52]. In contrast, carotenoid concentrations in other animals are often negatively correlated with parasite load [57] and with pollutants [58]. Carotenoids are potent lipid-soluble antioxidants [59] and are able to inhibit lipid peroxidation in liposomes [60]. The accumulation of carotenoids in infected Artemia is also considered a parasite strategy to increase the probability of being predated by birds (the final host) by increasing visibility [61] and enhancing nutritive value [26]. Carotenoids provide protection against oxidative stress in many free living organisms [62,63,64] so they may also increase longevity in infected Artemia. Given that oxidative stress is a common marker of toxicity, not only for As in plants, invertebrates and vertebrates [65,66,67] but also for heavy metals (e.g. lead, cadmium and mercury [68]), cestode parasites may protect Artemia against a broader range of pollutants.

Unlike carotenoids, a positive effect of parasites on host lipid content is common in nature, e.g. in acanthocephalans infecting gammarids [69] or trematodes infecting bivalves [70]. Therefore, our finding regarding increased resistance to As in the presence of parasites may not be an isolated case, and more studies are needed to evaluate how frequently this occurs in nature.

The differences in sensitivity to As observed in infected Artemia collected on different dates have several possible explanations, including a negative effect of the generally higher infection levels, or a higher pathogenicity of C. podicipina which was absent in May. Alternatively, it could be related to seasonal changes in the ages of the parasites or their hosts, or changes in the lipid or carotenoid levels in the host. Increased heavy metal accumulation with age in cysticercoids has been shown in other cestodes [71,72]. Previous studies of the interactions between parasites and pollutants on toxicity have focused on individual parasite species (but see Gismondi et al. [7]). In nature, co-infections of different parasites are extremely common, and our study illustrates the need to consider the effects of co-infections in environmental parasitology.