The effects of glucose and ascorbic acid on in vitro development of Echinococcus granulosus metacestodes

Echinococcus granulosus-developed metacestodes in the cultured medium are used for the assessment of its susceptibility to different compounds; however, this procedure is time-consuming and risky. In the present study, aspirated protoscoleces from the infected sheep were used to evaluate the effects of glucose, as an energy source, as well as ascorbic acid, as an antioxidant vitamin, on larval development. Protoscoleces were maintained in RPMI1640 culture media containing 10% fetal calf serum, as well as different concentrations of glucose (4, 6, and 8 mg/ml) and ascorbic acid (25, 50, and 100 µg/ml). A culture medium containing 4 mg/ml of glucose was served as the control. Larger cysts were achieved in a shorter time from the medium enriched with 6 mg/ml of glucose (740 ± 20 µm) compared to the control group (420 ± 40 µm). However, in the groups treated with ascorbic acid, the number of cysts was higher in 100 µg/ml (32.5 ± 0.7) compared to the control group (12.5 ± 0.7). Additionally, the mature cysts were achieved on the 7th day of cultivation with 100 µg/ml of ascorbic acid compared to 18 days in the control group.


Introduction
The protoscoleces of Echinococcus granulosus could differentiate in two directions depending on its environmental conditions. The ingested protoscoleces in a dog exposed to the gut environment was differentiated in strobilation to form an adult cestode. On the other hand, in the intermediate hosts, protoscoleces obtained from ruptured cysts escaped into the body cavity, in order to form more hydatid cysts (secondary hydatidosis) (CDC 2019; Schmidt and Roberts 2013). In the cultured medium, both the adult worms and the cysts were obtained from protoscoleces (Smyth and Davies 1974). The in vitro cultivation of E. granulosus metacestodes (protoscoleces or cysts) have been used to study parasite's physiology and biochemistry. In addition, they have been used for the primary assessment of their susceptibility to different compounds (Hemphill et al. 2010;Macpherson and Smyth 1985;Shan et al. 2013;Sharafi et al. 2017).
As the cystic stage of E. granulosus develops slowly, the risk of contamination is high and because of the complication of nutritional conditions, only 1 to 11 percent of protoscoleces develop into small cysts (Elissondo et al. 2017;Elissondo et al. 2004;Rodriguez-Caabeiro and Casado 1988). Thus, in vitro culture conditions were needed to be improved.
It has been shown that tapeworm's metabolism is dependent on glucose. In this regard, protoscoleces of E. granulosus transport glucose via both passive diffusion and the mediated transportation (Jeffs and Arme 1988). It has been shown that the mean concentration of excretory/secretory (E/S) proteins are higher in phosphate-buffered saline (PBS) medium enriched with glucose compared to those of Dulbecco's Modified Eagle's medium (DMEM) and Roswell Park Memorial Institute Medium (RPMI 1640 ) (Haniloo et al. 2011). Many similarities were found between cancer cells and parasites, including multiplying in host organs, resistance to the programmed cell death, escaping from host immunity, and using proteolytic enzymes to reach the suitable tissues. Of note, there are many common drugs between the parasites and cancer cells (Klinkert and Heussler 2006).
Ascorbic acid (vitamin C), which is an antioxidant vitamin, has been shown to play a biphasic role in cancer cells according to dose, time, and expression level of sodium-dependent vitamin C transporter 2 (SVCT-2) (Cho et al. 2018;Park et al. 2004). In low SVCT-2 expressing cell lines, deficient delivery of ascorbic acid was indicated to increase the cancer's proliferation activity. In the E. granulosus genome, three sodium-dependent transporter genes (named as EG_05623, EG_05624, and EG_05625) playing uncharacterized roles exist, which may also play a role in the delivery of acid ascorbic inside the parasite (Zheng et al. 2013) (Sanger Institute 2020).
According to the above-mentioned information, in the current study, the effects of glucose and ascorbic acid on in vitro development of E. granulosus metacestodes were investigated.

Materials and methods
The Protoscoleces were aseptically aspirated from hydatid cysts of livers of sheep that were slaughtered in a local abattoir in Zanjan, Iran. Thereafter, the protoscoleces were passed through two layers of sterile gauze, which were then washed several times with PBS solution (pH 7.2) containing penicillin-streptomycin. Parasite's viability was also assessed by muscular movements using the 0.1% eosin exclusion test. Those samples with more than 90% of living protoscoleces were included to be used in this study.
The effect of glucose on development of E. granulosus metacestodes Approximately, 1800 protoscoleces (180 protoscoleces per milliliter) were incubated in a glucose free RPMI 1640 medium (R1383, Sigma-Aldrich, USA) containing penicillin (100 IU/ml) and streptomycin (100 lg/ml), which was also supplemented with 10% (v/v) fetal bovine serum (FBS) and different concentrations of D-glucose (4, 6, and 8 mg/ml) in 5% CO 2 at 37 AE C. Changes in development, including vesiculation, the appearance of laminated layer, mature cyst formation (with fully developed laminated layer), and number and size of the cysts were all followed using an inverted microscope (MoticÒ, AE31, Spain) in each day for 50 days (Elissondo et al. 2017). The medium was also changed every 3-4 days. To evaluate the effect of the glucose concentration on the development of E. granulosus metacestode, two other groups supplemented with 6 and 8 mg/ml of glucose were selected, as well.
The effect of ascorbic acid on the development of E. granulosus metacestodes As stated in the previous section, RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 4 mg/ml of D-glucose was served as the control group. Thereafter, L-ascorbic acid (Sigma-Aldrich, A4544) with different concentrations (25, 50, and 100 lg/ml) were added to the above-prepared medium, in order to evaluate the effect of ascorbic acid on the development of E. granulosus metacestode. The developmental changes were then evaluated as described earlier. It is noteworthy that the average length of two diameters of all the formed cysts ( Dþd 2 ) was used to calculate the cysts' size. The measurements were done by the microscope using an eyepiece reticle (Fig. 1).

Statistical analysis
All the experiments were repeated three times and the obtained data were presented as the mean value ± standard deviation (mean ± SD). The differences between the groups were determined by ANOVA using SPSS (version 22.0) and a p-value less than 0.05 was considered as statistically significant.

Results
Posterior bladder formation or vesiculation of evaginated protoscoleces, as the first signs of evolution, have started by passing 2 to 6 days from the incubation in different culture flasks. After 14 days of incubating in the control group, a fine membrane has started to form the laminated layer and by the day 17, some completely developed cysts (mature cysts) were detected (Fig. 2).
The effect of glucose on development of E. granulosus metacestodes As shown in Table 1, the appearances of laminated layer as well as the formation of mature cysts in the cultured medium supplemented with 6 mg/ml of glucose were observed to be faster than the use of 4 and 8 mg/ml of this sugar (p \ 0.05). Moreover, larger cysts were achieved from the cultured medium with 6 mg/ml of glucose (740 ± 20 lm) compared with the control group (420 ± 40 lm) (p \ 0.05).
The effect of ascorbic acid on the development of E. granulosus metacestodes The appearance of laminated layer and the formation of mature cyst occurred after 5 and 7 days in the cultured media supplemented with 100 lg/ml of ascorbic acid; whereas it took place after about 14 and 18 days in the control group, respectively (Table 2). Although the size of the cysts was not very different between the study groups, the number of the obtained cysts was significantly higher in the groups supplemented with 100 lg/ml of ascorbic acid (32.5 ± 0.7) compared to the control group (12.5 ± 0.7) (p \ 0.05).

Discussion
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 lg/ml of ascorbic acid. 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.
The cysts obtained from the 6 mg/ml glucose culture medium were larger (9 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 and Roberts 2013).
Cultivated E. granulosus protoscoleces in a Connaught Medical Research Laboratories (CMRL-1066) culture medium that was supplemented with fetal calf serum (FCS), glucose (30% in distilled water), and yeast extract. The mature cyst formation occurred between days of 19 to 37 (Gordo and Bandera 1997), while in our study, the mature cyst formation with 6 mg/ml of glucose and 100 lg/ml ascorbic acids on RPMI 1640 was achieved on days 14 and 7 post-incubation, respectively. Elissondo et al. (2004) for the first time reported that the cysts formation from protoscoleces of cattle origin using a M199 culture medium containing 4 mg/ml of glucose (Elissondo et al. 2004). After 14 days of incubation, some laminated layers appeared and on day 20, some cysts with a complete laminated layer were observed.
Moreover, these authors have cultivated protoscoleces from sheep origin (Elissondo et al. 2005); Culture condition was the same as our study except the culture medium (M199 instead of RPMI 1640 ). They were reported that after 14 days of cultivation a laminated layer appeared.
For a long time, these culture conditions were used for study on Echinococcus species in vitro. In 2017, Elissondo et al. designed another study to improve the E. granulosus cultivation process by using of insulin in a M199 medium. As a result, they were reported that a laminated layer appeared after 11 days of culturing, 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 et al. 2017). Of note, insulin is a peptide hormone that can regulate the metabolism of  Â 100 ** All formed cysts were measured, Cysts size measured as diameters mean ( Dþd 2 ), p \ 0.05 carbohydrate and promote the absorption of glucose; therefore, the observed similarity was expected.
In order to investigate the effect of glucose on E. granulosus, other study in 2011 reported that protoscoleces incubated in PBS enriched with glucose produced a higher concentration of E/S proteins compared to protoscoleces incubated in DMEM and RPMI 1640 media during 24 h of culturing. Correspondingly, this indicates the effect of glucose on the protoscoleces' metabolism (Haniloo et al. 2011).
Cestodes lack an alimentary tract and interact with their environment via the tegument. Accordingly, the tegument contains many structural proteins and enzymes (Thompson and Geary 2003). For E. granulosus, some sugar transporters were identified based on the gene database (Zheng et al. 2013;Sanger institute 2020). Glucose absorption in E. granulosus protoscoleces is through both passive diffusion and mediated transport (Jeffs and Arme 1988). Cestodes drive most of their energy from glucose.
The enrichment of protoscoleces environment by optimal concentration of glucose can play an important role in the survival and growth of the organism.
Another problem in E. granulosus cultivation is that it takes a long time to reach the developed cyst. In the present study, vitamin C used to reduce cysts achievement time. As well, mature cysts were achieved in the 7 th day of cultivation with ascorbic acid in100 lg/ml compared to the 18th days in the control group. Vitamin C is one of the watersoluble vitamins with an anti-oxidant role. 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).
There are some studies that have evaluated the relation between plasma level of ascorbic acid and hydatid diseases, but there is no investigation on the role of this vitamin on this parasite.
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).
However, in other study, vitamin C plasma level in sheep naturally infected with hydatid cysts was not affected by infection (Cinar et al. 2018). Further studies are needed to determine the exact effect of ascorbic acid on E. granulosus larval stages.