Alterations in lipids and minerals in relation to larval trematode infections of Nerita polita marine snails

Marine snails are known to have a high nutritive value, they have a great percent of polyunsaturated fatty acids which are needed for human fitness. Marine snail’s fl esh has a great value in nutrition as a source of lipids, and minerals (Alves et al., 2014). They constitute a major part of the trematode’s life cycle as the first and second intermediate hosts for trematode parasites. These parasites are transmitted to marine fish and marine vertebrates. Trematode parasites have a complicated life cycle as they need the snails as intermediate hosts for transformation into the infective stage i.e. cercariae. In the first intermediate host, sporocysts are consisted and get their nutritional needs through their tegument and develop into rediae containing cercariae inside the snail host. Cercariae have a wide range of shapes and features. They are modifi ed physiologically and behaviorally to find and penetrate their final hosts (Toledo et al., 2014; Hassan et al., 2018a; Al-Solami et al., 2019; Turkistani et al., 2021).

Marine snails comprise a high nutritional value including vitamins, proteins, minerals, and essential fatty acids. Concerning lipids, they play important functions in the energy and structure of biological systems and animal’s survival under physiological stress like low food supply, parasites that need lipid consumption after depletion of the carbohydrates which lead to changes in lipids structure. Lipids are a major constituent in the biological membranes and any change in the metabolic state of the snail as response to stress may influence the number and structure of these lipids (Stuart et al., 1998; Storey, 2002; Giokas et al., 2005; Bandstra et al., 2006; Ab Lah, et al., 2017).

Snail’s mucus was found to be consist of a complicated mixture of proteoglycans, glycosaminoglycans, glycoprotein enzymes, hyaluronic acid, copper peptides, antimicrobial peptides, and metal ions (Smith et al., 2009). It contains primarily allantoin, collagen, elastin, and glycolic acid (Thomas, 2013). Many studies have dealt with the peptide constituent of snail’s mucus-like mucin which contains both Gram-positive and Gram-negative antibacterial activity which stimulates some components of the immune system including barrier repair and inflammatory cell recruitment (Thomas, 2013; Etim et al., 2016). Another study showed that snail mucus is beneficial in bone repairing and teeth regeneration due to its ability to increase the osteopontin expression and stimulate the expression of some inflammatory genes in the cells of the dental pulp (Kantawong et al., 2016). It can also be useful to decline pigmentation scarring and wrinkles. Furthermore, it can assist wound healing and stop its infections (Adikwu & Alozie, 2007; Adikwu & Enebeke, 2007).

Studies dealt with marine host-parasite systems remain scarce. The present study aimed to investigate the effect of larval trematode infections on the content of lipids and minerals in infected snails’ Digestive Gland- Gonad complex (DGG), hemolymph, and Snail-Conditioned Water (SCW) as well as uninfected ones.

Informed consent was obtained from all individuals included in this study. The conducted research is not related to human or animal use

A total of 875 snails of N. polita were collected, of which 4.30 % were found infected with ocellate furcocercus cercaria, Trichobilharzia regenti (Fig. 3) and 16.12 % with xiphidiocercaria cercaria, Litorina saxatilis VII (Fig. 4). As shown in Figure 5, according to the length of snail, the total infection highest (40 %) in 21 – 22.9 mm long snails and lowest (10 %) in 11 – 12.9 mm snails. The infection percentage in the 9 – 10.9 mm length was 14.28 % compared to that in the length of 15 – 16.9 mm (16 %). In the lengths of 17 – 18.9 mm and 19 – 20.9 mm, infection prevalence was 33.34 % and 29.42 % respectively.

Fig. 3

Fig. 4

Fig. 5

The biochemical analysis of triglycerides, cholesterol, and phospholipids in the DGG complex of snails infected with ocellate furcocercus cercaria showed a significant decline in triglycerides (P = 007) and cholesterol (P = 0.02) while phospholipids were insignificantly increased (P = 0.06). In the case of xiphidiocercaria infection, DGG complex showed a significant increase in triglycerides (P = 0.021), cholesterol (P = 0.03) and phospholipids (P = 0.01) compared to uninfected snails (Table 1)

The effect of infection with ocellate furcocercus cercaria and xiphidiocercaria on the hemolymph of Nerita polita showed a significant decrease in triglycerides (P = 0.005 and 0.002, respectively) and cholesterol (P = 0.003 and 0.01, respectively) in hemolymph and an insignificant increase in phospholipids of hemolymph (P = 0.075 and 0.175, respectively) in infected snails compared with non-infected one as shown in (Table 2).

Table 3 shows that the triglycerides (P = 0.012) and cholesterol (P = 0.004) in SCW of Nerita polita, were significantly lower in infection with ocellate furcocercus cercaria than uninfected snails. Whereas, Triglycerides (P = 0.1) and cholesterol (P = 0.375) are insignificantly decreased in xiphidiocercaria infected snails compared with non-infected ones. The phospholipids were insignificantly increased (P = 0.231) in infection with furcocercus infection and significantly (P = 0.031) in xiphidiocercaria infected snails than uninfected.

The present work discussed the influence of infection by trematode larvae on the profile of triglyceride, cholesterol, phospholipids, and some minerals in Nerita polita snails over one year. The infection by ocellate furcocercus cercaria and xiphidiocercaria resulted in a significant reduction in triglyceride levels (P =0.005 and P =0.002) respectively) in the hemolymph of Nerita polita. These results are following Pinheiro et al. (2004); Pinheiro et al. (2005); Pinheiro et al. (2009); Tunholi-Alves et al. (2011) who suggested the use of triglycerides as a source of energy by the developing larvae during their asexual multiplication when the miracidium develops into a sporocyst, that develops many cercariae. The development of trematode larval stages inside the snail hosts consume a great amount of lipids to build their new organs and membranes.

Invasion of snail intermediate hosts by trematodes activates several metabolic interactions, needed for the development and multiplication of the parasite. This may consume the energy reserves of the host, primarily polysaccharides causing the snails to utilize other substances like proteins and lipids to maintain the essential needs for the parasite development (Becker, 1980; Pinheiro et al., 2009; Tunholi et al., 2011; Tunholi-Alves et al., 2012; Tunholi-Alves et al., 2013; Alves et al., 2014).

TunholiI-Alves et al. (2011) presented similar results in their study on the infection of Biomphalaria glabrata by Echinostoma paraensei. They found a decrease in the concentration of the triglycerides in the hemolymph of B. glabrata as an outcome of the process of the asexual reproduction of the parasite indicating that triglycerides are important for the parasite’s development. They indicated that there are enzymes in trematodes responsible for lipid metabolism, particularly non-specific lipases and esterase confirming the usefulness of triglycerides as an energy source for the parasite.

Infection by ocellate furcocercus cercaria and xiphidiocercaria both resulted in a significant decrease in triglyceride and cholesterol levels in the DGG complex of the infected N. polita. However, in the SCW the significant decrease was noted only in the triglyceride’s levels in infection by ocellate furcocercus cercaria. Variations occurring in the late stage of the parasite’s multiplication indicated that this stage has the maximum competition for nutrients with the host. Consequently, triglycerides are primarily hydrolyzed into free fatty acids and then oxidized in the mitochondrial matrix producing the energy required to meet the metabolic needs and minimizing the harmful effects of the infection (Tripathi & Singh, 2002; Humiczewska & Rajski, 2005).

Bandstra, et al. (2006); Fried, et al. (1989) observed a significant reduction in the triglyceride fraction in the DGG complex of Biomphalaria glabrata infected by Echinostoma caproni. Cline et al. (2000) deduced significantly lower levels of triacylglycerol in the DGG complex of Cerithidia californica snails infected with three species of tritrud larvae: Euhaplorchis californiensis, Cloacitrema michiganensis, and Mesostephanus appendiculatus.

In our study alterations in the amount of cholesterol in the DGG complex and hemolymph were observed in the Nerita polita snails infected by ocellate furcocercus and xiphidiocercaria. Marsit et al. (2000) suggested that this lipid is included in the constituents of the parasite larval stages, during developmental mitotic activity related to miracidium conversion to sporocyst and its following development leading to fast cell and tissue construction in the larvae. Free sterol, primarily cholesterol has been reported as the major constituent of the trematode larval stages. The resulting rise in cholesterol may be due to two mechanisms: first is the increase in biosynthesis of cholesterol, and second is the remodeling of cell membranes. The latter is accompanied by the liberation of its cholesterol molecules which causes membrane fluidity and raises the rate of metabolic processes including membrane proteins like those working in the transport chain of electrons in the mitochondria and altering the cell permeability (Narayanan & Venkateswarara, 1980). It appears that cholesterol levels are maintained at a very tight range and when alteration occurs, there is need to return rapidly to the homeostatic level (Lustrino et al., 2010).

In the present study, there was a significant reduction in cholesterol levels, in the DGG complex of N. polita snails infected with ocellate furcocercus cercaria as compared to higher cholesterol levels associated with xiphidiocercaria infection. The infection by ocellate furcocercus cercaria and xiphidiocercaria also resulted in a significant decrease in triglyceride levels, in the DGG complex of the infected snails. In the SCW of snails infected by ocellate furcocercus cercaria and xiphidiocercaria there was a significant decrease in the triglycerides. Variations in levels associated with the late stage of parasites’ multiplication indicated that this stage has the maximum competition for nutrients with the host. Consequently, triglycerides are primarily hydrolyzed into free fatty acids and then oxidized in the mitochondrial matrix producing the energy required to meet the metabolic needs and minimizing the harmful effects of the infection (Tripathi & Singh, 2002; Humiczewska & Rajski, 2005).

Bandstra et al. (2006); Fried et al. (1989) observed a significant reduction in the triglyceride fraction in the DGG complex of Biomphalaria glabrata infected by Echinostoma caproni. Cline et al. (2000) using high-performance layer chromatography (HPTLC) analysis showed that triacylglycerol in the DGG of snails Cerithidia californica infected with three species of tritrud larvae; Euhaplorchis californiensis, Cloacitrema michiganensis, and Mesostephanus appendiculatus was significantly less than that of the controls of N.polita snails. This shortage may be due to the consumption of triglycerides by both parasite and host to maintain the energy for metabolism. The increase in cholesterol content is due to the great change in cellular structures of the host especially in the DGG as a result of the severe damage the parasites done. Furthermore, the alteration in lipids profile due to infection indicate the importance of lipid molecules in the snail hosts. Alves et al. (2014) supposed that triglycerides are considered as the first energy resources for the snail and parasite, while cholesterol is included in a manufacturing cell and tissue structures of both organisms.

In our work the results showed an insignificant increase in the phospholipids in the hemolymph of N. polita infected by both ocellate furcocercus and xiphidiocercaria. Phospholipids recorded increase in the SCW was significant only in association with infection by xiphidiocercaria of N.polita. This led Bui et al. (2016) to hypothesize that the level of phosphatidylethanolamine (PE) in B. glabrata is not affected by S. mansoni infection, but by the physiological change of the snail’s body as it ages. To explain the difference in trends of phosphatidylserine serine (PS) level, they propose two explanations: 1) the changes in phosphatidylserine serine (PS) level caused by the parasite outweigh the changes that would otherwise occur naturally as the snail ages, thus skewing the overall trends of phosphatidylserine (PS) level; 2) the parasitism affected the metabolism of the snail, thus causing a difference in the way the snail physiology changes as it ages. They found several differences in phospholipid profile of B. glabrata DGG complex induced by the parasitism.

In a report by Cline et al. (2000) parasitism by three larval trematodes, Euhaplorchis californiensis, Cloacitrema michiganensis, and Mesostephanus appendiculatus resulted in a significant increase in phospholipid and phosphatidylethanolamine content in the DGG complex compared with control levels; whereas infection by both M. appendiculatus and E. californiensis caused a significant increase in phosphatidylcholine content. This clarifies that larval parasitism in the same snail species will induce different lipid profiles in the host, and this can provide chemotaxonomic information to differentiate between larval trematodes from the same snail host.

According to Thompson and Mejia-Scale (1993), the role of lipids as a storage reserve in gastropods is not clear, and the role of lipids in metabolisms of snails and the significance of lipids to developing larvae require further investigation. Schistosoma parasites appear to distort the neutral lipids composition of the snail host, especially during infection. Other studies by Thompson (1987); Fried et al. (1995); Eberemu (2018) also showed that in the presence of parasites there is an initial increase of neutral fat in the infected host cells which is rapidly degraded to fatty acids, as the infection progresses.

The effect of larval trematodes infection on quantities of minerals was studied using different devices. Ong et. al. (2004) using inductively coupled plasma atomic emission spectrometry reported an insignificant decline in the concentrations of Cu, Fe, and K and a significant decrease in the concentration of Zn and Pb in infected snails. Using flame atomic absorption spectrometry, Mostafa (2008) investigated the amount of some metallic ions in the DGG complex and shells of the model Lymnaea stagnalis infected by Fasciola gigantica. He recorded a significant increase in the concentrations of Zn, K, and Cu, an insignificant increase in Fe, and a significant decrease in Pb and Na in infected compared to noninfected snails. Infected pulmonate snails were found to sustain more CaCo3 in their shells than non-infected ones (Ong et al., 2004). Silva et al. (2017) recorded insignificant alteration in the amount of Ca and glucose in the hemolymph of infected and non-infected snails analyzed by an ion-exchange column and high-performance liquid chromatography.

The reasons for metallic ions fluctuation as a result of trematode infections were due to larval stages (rediae, sporocysts, and free cercariae) affection of the ionic balance through influx and outflux of ions from larvae to the snail host. Destruction of the digestive gland cells by larval trematodes probably reduced the storage volume and capacity of elements in the infected snails. The parasites could affect calcium distribution leading to its deposition in the shell and mantle. The hypothesis of hyper-calcification of snail shells induced by trematode larvae should be verified under natural conditions (Evans et al., 2001; Kaufer et al., 2002; Zbikowska, 2003; Ong et al., 2004; Hassan 2008).

Our results of biochemical changes showed a fluctuation in the concentration of lipids and minerals between increase and decrease in snail’s DGG complex, hemolymph, and SCW. The reduction of triglycerides and cholesterol in the SCW may affect the therapeutic value of the studied snails. The nutritive value of the snails is affected by infection through the decrease in some lipids and minerals. Further qualitative studies are recommended.