In this study, the effects of glucose and ascorbic acid on E.granulosus metacestodes development were examined under in vitro. Larger cysts were achieved from the cultured medium with 6 mg/ml of glucose in a shorter time compared to other groups; however, the highest number of cyst was found in the groups treated with 100 µg/ml of ascorbic acid. As well, mature cysts were achieved in the 7th day of cultivation with ascorbic acid in100 µg/ml compared to the 18th days in the control group. To the best of our knowledge, this is the first report in which the effect of ascorbic acid on E. granulosus metacestodes development was studied under in vitro.
Cestodes lack an alimentary tract, which interact with their environment only via the tegument. Accordingly, the tegument contains many structural proteins and enzymes (Thompson and Geary 2003). Glucose absorption in E.granulosus protoscoleces is through passive diffusion, which is mediated of both Na+-dependent and Na+-independent mechanisms (Jeffs and Arme 1988).
In E. multilocularis, EmGLUT1 was introduced as a simply facilitated glucose transporter that may play an essential role in glucose uptake by these parasites (Kashiide et al. 2018). As well, for E. granulosus, some sugar transporters were identified based on the gene database (Zheng, 2013) (Sanger institute).
In the present study, the cysts obtained from the 6 mg/ml glucose culture medium were larger (×1.5), but these were fewer in number compared with the control group. Accordingly, this could be explained by an adaptation phenomenon through which parasite biomass adjusts to the host’s (environment) capacity (SCHMIDT G.D 2013).
Gordo and Bandera in 1997 cultivated E.granulosus protoscoleces in a CMRL-1066 culture medium that was supplemented with fetal calf serum (FCS), 0.014 ml glucose (30% in distilled water) per ml, and yeast extract. Additionally, in this study, the mature cyst formation occurred between days of 19 to 37(Gordo and Bandera 1997), while in our study, the mature cyst formation with 6mg/ml of glucose and 100 µg/ml ascorbic acids was achieved on days 14 and 7 post- incubation, respectively.
In 2004, Elissondo et al. in their study for the first time reported the cysts formation from protoscoleces of cattle origin using a M199 culture medium containing 4 mg/ml of glucose (Elissondo et al. 2004). As reported, after 14 days of incubation, some laminated layers appeared and on day 20, some cysts with a complete laminated layer were observed, which is almost similar to our results obtained from the control groups (4 mg/ml of glucose in RPMI1640).
Moreover, these authors have cultivated protoscoleces from sheep origin in 2005, and reported that there are no differences between the ovine and bovine during the development process and at the time of cysts’ formation (Elissondo et al. 2005).
To improve the E.granulosus cultivation process, Elissondo et al. designed another study in 2017 by the use of insulin in a M199 cultured medium. As a result, they reported that a laminated layer appeared after 11 days of culturing under in vitro, and on day 14, some cysts with a complete laminated layer were detected. Accordingly, this was in line with our results with 6 mg/ml of glucose (Elissondo 2017). Of note, insulin is a peptide hormone that can regulate the metabolism of carbohydrate and promote the absorption of glucose; therefore, the observed similarity was expected.
Haniloo et al. in their study in 2011 reported that protoscoleces maintained in PBS enriched with glucose produced a higher concentration of E/S protein compared to protoscoleces maintained in DMEM and RPMI1640 media during 24 h of culturing. Correspondingly, this indicates the effect of glucose on the protoscoleces’ metabolism (Haniloo et al. 2011).
Vitamin C, also known as L-ascorbic acid, is one of the water- soluble vitamins. In this regard, most of animals make their own vitamin C, but some others cannot do that. Although it is challenging to investigate the vitamin requirement of parasites, some works are available that measured ascorbic acid levels as well as the maturity and growth of cestodes (Ramalingam et al. 2006).
Hayanjeh in 2014 studied naturally infected sheep with hydatid cysts in comparison with healthy sheep. As a result, he reported that the plasma level of ascorbic acid in sheep infected with hydatid cyst was below the normal range and lesser than that of the control group (Hayajneh 2014).
It was observed that the release of reactive oxygen species (ROS) from the activated phagocytes against infectious agents could be harmful to host cells (Hemila 2017). Vitamin C may protect host cells and also reduce the rate of infection during infection diseases such as echinococcosis. However, Cinar et al. in a study in 2018 reported that vitamin C plasma level in sheep naturally infected with hydatid cysts was not affected by infection (Cinar et al. 2018). However, further studies are needed to determine the exact effect of ascorbic acid on E.granulosus larval stages.