Impact of artemether on some histological and histochemical parameters in Biomphalaria alexandrina

The present study elucidated the molluscicidal properties of the antihelminthic plant derivative, dihydro-artemisinin methyl ether (Artemether) against some histological and histochemical parameters of Biomphalaria alexandrina snails. Results indicated that Artemether has a toxic effect against B. alexandrina snails expressed by LC90 of 32.69 ppm, after 24 h exposure. The rate of infection with Schistosoma mansoni in snails exposed to sublethal concentrations (LC0 and LC10) of Artemether either for 1 or 7 successive days pre-miracidial exposure was significantly lower than that of control group (P < 0.001). The reduction rates of this parameter ranged from 76.9 to 87.3%. Moreover, snails treated with LC25 for 7 days were free from infection (0% infection rate). Moreover, the survival rate in snails exposed to LC25 on 1 and 7 days pre-miracidial exposure were 23.3 and 10%, respectively compared to the 93.3% in control group. Histological investigation of the exposed snails' gland showed that the mature ova lost their normal shape, disappearance of most of the oocytes, spermatogenesis and connective tissues. Moreover, complete destruction of gametogenic cells and severe damage of hermaphrodite gland tissues were clear when the exposure period increased. Haemocytes' morphology showed obvious abnormalities as decrease in their number, activation, and irregular aggregation in exposed snails were recorded. Also, snails’ exposure to LC25 of Artemether demonstrated a decreased glycogen content in hermaphrodite gland tissues while it increased in the haemolymph. In conclusion, the use of sublethal concentration of Artemether showed remarkable molluscicidal activities against B. alexandrina snails which could be a promising and safe biological agent to control schistosomiasis disease.


INTRODUCTION
In the tropics and subtropics, schistosomiasis is the second most important parasitic disease after malaria in terms of prevalence, public health and socio-economic importance (Chitsulo et al., 2004;Steinmann et al., 2006).Freshwater snails of the genus Biomphalaria plays a major role as intermediate hosts of Schistosoma mansoni is the etiological agent of intestinal schistosomiasis while Biomphalaria spp.controled by molluscicides is one of the main strategies to reduce the snail population in infected areas.There are few effective molluscicides commercially available.Natural products are considered as potentially useful and safe molluscicides (Miyasato et al., 2012).
There is a great interest in the use of molluscicides of plant origin by local communities in self-supporting system of schistosomiasis control program.Such molluscicides seem to be less expensive, readily available, rapidly biodegradable and probably easily applicable with simple techniques appropriate to developing countries (Ibrahim et al., 2004).Plant molluscicides have been regarded as possible alternatives to the costly and environmentally hazardous molluscicides currently available (Oliveira-Filho et al., 2010).Also, there is a continuous need to search for new plant species with ideal molluscicidal properties (Tantawy et al., 2004;Bakry and Hamdi, 2007).Many investigators have studied the molluscicidal activity of certain plants and their extracts (El-Emam et al., 1990;Shalaby and Abdel-Hamid, 1999;Mossalem, 2003;Bakry et al., 2007;Abdel-Hamid, 2008;Hasheesh et al., 2011).
A more successful approach to chemoprophylaxis has been to exploit a methyl ether plant derivative; Artemether which was first synthesized and used as an antimalarial drug in China (Hien and White, 1993).The anti-malarial drug Artemether was derived and developed from the plant Artemisia which was known since ancient time for its medicinal importance.Artemether was discovered to also have anti-schistosomal properties and had no drug-related adverse effects, and significantly reduced the incidence and intensity of schistosome infections, including those of S. mansoni, Schistosoma japonicum and Schistosoma haematobium (Xiao et al., 2000a(Xiao et al., , 2002;;Hou et al., 2008).Because Artemether selectively targets the larval migratory stages of the parasite, known as schistosomulae, it blocks the development of ovipositing adult schistosome worm pairs in the vasculature (Xiao et al., 2000).The plant Artemisia was proved to have a molluscicidal and miracidicidal activity (Mansour et al., 2002;Hafez et al., 2007).
In order to promote energy production gastropods categorize primarily carbohydrates, which are stored in certain tissues as glycogen and transported in the haemolymph as glucose (Livingstone and Zwaan, 1983).The molluscicides greatly affect the metabolic activities of the snail intermediate hosts (Rawi et al., 1995).
The aim of this work is to study the molluscicidal effect of the antihelminthic plant derivative Artemether on some histological and histochemical parameters of Biomphalaria alexandrina and its susceptibility to infection with S. mansoni, as a possible safe method of schistosomiasis control.

Snails
B. alexandrina snails were collected from different water courses at Giza Governorate, Egypt, during spring, 2012, and transferred in plastic bags to the laboratory.Snails were reared in de-chlorinated water (25°C ± 1) (Liang et al., 1987) at Medical Malacology Laboratory, Theodor Bilharz Research Institute (TBRI), Imbaba, Giza, Egypt.Healthy snails free from trematode infections were used in the experimental testes.

Molluscicidal activity
A stock solution of 1000 ppm was prepared from Artemether powder on the basis of w/v using de-chlorinated water (pH 7.0 to 7.5).A series of concentrations was prepared on the basis of volume/volume (World Health Organization, 1965).Three replicates were used, each of ten snails (6 to 8 mm/L, for each concentration.Exposure and recovery periods were 24 h each; at 25 ± 1°C.For each test, 3 replicates of control snails were maintained under the same experimental conditions in de-chlorinated water.The effectiveness of Artemether has been expressed as LC50 and LC90 (Litchfield and Wilcoxon, 1949).The sublethal concentrations were calculated through a computer program (IPM SPSS Statistics pro-  Finney, 1971).

Effect of Artemether on infection of B. alexandrina snails with S. mansoni
Biomphalaria snails (6 to 8 mm) were exposed to miracidia after one and seven successive days of exposure to sublethal concentrations (LC0, LC10 and LC25) of Artemether.Snails were exposed to miracidia in mass (10 fresh hatched miracidia/snail) for 24 h under ceiling illumination.For each concentration of each group, three replicates (each replicate of 10 snails/L in glass container) were prepared.After that, snails were transferred to clean de-chlorinated water (25 ± 1°C) and daily fed with oven dried lettuce leaves throughout the pre-patent and patent periods (Massoud et al., 1973).A control group of three replicates, each 10 snails/L was exposed to miracidia concurrently with the experimental snails and treated similarly till cercarial emergence.Dead snails were removed daily and surviving snails were individually examined once weekly for cercarial shedding 24 days post-miracidial exposure.The number of snails survived at the first shedding and the number of infected snails were calculated (Youssef, 2010).The survival and infection rates were compared with that of control using CHITEST of EXCEL program, Microsoft Office 2007 for Windows.

Histological and histochemical tests
For each group, three replicates (each of twenty B. alexandrina snails, 6 to 8 mm) were exposed to LC25 of Artemether continuously for 1, 7, 14 and 21 days.Another group of snails was maintained in de-chlorinated water under the same laboratory conditions as control.The haemolymph of about 10 snails of each group was collected according to Michelson (1966).Three snails from each group were crushed gently and the hermaphrodite gland was removed, fixed in Bouin's fluid for 5 h and then transferred to 70% alcohol.Further procedures were followed including dehydration in 100% alcohol, clearing in xylol and paraffin embedding.Five μm paraffin-embedded sections were stained with hematoxylin and eosin for general histological examination (Mohamed and Saad, 1990).Blood film slides were also stained with hematoxylin and eosin.To demonstrate the presence of glycogen, other sections of the hermaphrodite gland and blood film slides were stained with periodic Schiff technique (PAS) (Sheehan and Hrapchak, 1980).Stained slides were examined under light microscope and photos were taken and investigated.

Statistical analysis
Student's t-test and chi-square test (Petrie and Sabin, 2000) were used in comparing the means and rates of experimental and control groups statistically.

RESULTS
This experiment was planned to elucidate the molluscicidal properties of Artemether against adult B. alexandrina snails after 24 h of exposure followed by another of recovery.It was noticed that LC 50 and LC 90 values of Artemether were 21.06 and 32.69 ppm, respectively (Table 1).Table 2 indicated that survival rate, at first cercarial shedding, of Biomphalaria snails exposed for 1 and 7 days to sublethal concentrations (LC 0, LC 10 and LC 25 ) of Artemether decreased compared to that of control.Thus, the survival rates of 7 days groups were 60, 33.3 and 10% compared to that of control one 93.3%,(P < 0.0001).
The infection rates of survived snails in the groups exposed to LC 0 , LC 10 and LC 25 for 1 or 7 days premiracidial exposure were significantly (P < 0.001) less than that of control group (Table 2).Thus, infection rates of 1 day groups were 18.2, 17.6 and 14.3%, respectively, compared to 78.6% for control group.It is worthy mention that no snail was infected in the group exposed to LC 25 for 7 days pre-miracidial exposure (0%).The Light photomicrograph of the hermaphrodite gland complex of B. alexandrina exposed continuously to LC 25 of Artemether 1, 7, 14 and 21 days was shown in Figure 2. Figure 2A showed that B. alexandrina snails exposed to LC 25 of Artemether 1 day resulted in loose of connective tissues, short and irregular sperms, a slight decomposition in ova and a partial damage in some cell walls of hermaphrodite gland.In Figure 2B, snails exposed to Artemether, 7 days, showed lyses of cells and matrix' components.Also, the mature ova appeared dense and irregular in shape with congeal clear and atrophy.A reduction in the number of sperms was noticed.With the increase of exposure period to 14 days, ova lost their normal shape, the damage increases showing discharge, evacuation and disappearance of most of the components of the gonad cells (Figure 2C).Moreover, complete destruction of gametogenic cells and sever damage of ovotestis gland were clear in Figure 2D when the exposure period increased to 21 days.Also, disappearance of the oocytes, spermatogenesis and connective tissue was noticed.Figure 2E showed transverse section in the hermaphrodite gland of unexposed control snails showing the gonadal components, mature ova and oocytes and spermato-genesis occurs in centripetal form toward the lumen of the acinus expressed as sperms and spermatocys.
It is seen also that the photomicrographs in Figure 3 showed PAS positive reaction for glycogen (blue color) in the gonads' cells in Biomphalaria snails exposed continuously to LC 25 of Artemether compared to unexposed control.Glycogen contents aggregated between hermaphrodite cells in their connective tissue condensed in a large amount in case of groups exposed to Artemether 1 and 7 days (Figure 3A and B) compared to control group in which the glycogen is distributed at the

DISCUSSION
Artemisinin (qinghaosu) is the active principle from the leaves of Artemisia annua L., a plant which is widespread throughout China, and also grows naturally in central Europe.Several derivatives of artemisinin showed improved solubility, chemical stability and enhanced antimalarial activity, one of the most important of which are Artemether.The biological activity of artemisinin and its derivatives is based on its endoperoxide bridge which break down releasing carbon-centered free radicals (Wu et al., 2003).The resulting free radical intermediate may then kill by alkylating and poisoning one or more essential proteins (Meshnick, 2002) in the snail.
The present results cleared that Artemether has a toxic effect against B. alexandrina snails with LC 90 of 32.69 ppm, after 24 h exposure according to WHO (1965) recommendations on plant molluscicides.These findings are supported by those of Marston et al. (1993) and Hafez et al. (2007) who reported that Artemisia spp.had a molluscicidal activity against Biomphalaria snails.Also, El Sayed et al. (2011) recorded that LC 90 values for latex of Cryptostegia grandiflora were 8.65 and 14.03 ppm for B. alexandrina and Biomphalaria galabrata, respectively.Similar results were obtained by Sharaf El Din ( 2006) who verified that LC 90 values for latex aqueous solutions of C. grandiflora were 8.48 and 7.55 ppm for B. alexandrina and Lymnaea natalensis, respectively.
Regarding the histological study, the hermaphrodite gland cells of snails exposed to LC 25 Artemether showed loose of connective tissues, short and irregular sperms and the mature ova appeared dense and irregular in shape with congeal clear and atrophy.Elongation of Artemether exposure period resulted in ova lost their normal shape and the damage increased showing discharge, evacuation and complete destruction of gametogenic cells.This may be attributed to the molluscicidal properties of Artemether as it selectively kills the ova; it precludes their development and thus prevents their normal formation (Xiao et al., 2000).These findings were supported by the study of Mohamed et al. (2004) who concluded that B. alexandrina exposed to Mepiquat chloride (plant growth regulator) caused noticeable changes in the histological architecture of the digestive and ovotestis glands.Hasheesh et al. (2011) attributed the reduction in egg laying of Bulinus truncatus treated with Sesbania sesban to severe histological damages to the snail's hermaphrodite gland cells and evacuations of some of its tubules from various gametogenetic stages.Also, Al-Qormuti (2008) found that exposure of B. alexandrina snails to sublethal concentrations of the herbicides Roundup or Topik, showed complete destruction of gametogenic cells.This also agrees with Henry et al. (1989), Brackenbury (1999) and Bakry (2009) who recorded degeneration and evacuation in the gametogenic stages of the exposed snails.
In similar studies, Rosès et al. (1999) noticed that kidney cells of Physa acuta displayed cell lyses when snails were exposed to atrazine (herbicide) for 10 days, and this effect was not reversed after a decontamination process.Also, Langiano and Martinez (2008) reported that short-term exposure to sub-lethal concentration of Roundup induced several histological alterations in Neotropical fish, Prochilodus lineatus.
In the present study, light microscopic investigation revealed that the three types of haemocytes distributed in the haemolymph of B. alexandrina (granulocytes, hyalinocytes and amoebocyte) were influenced by snail continuous exposure to Artemether.The number of cells decreased by the increase of the exposure time.Furthermore, haemocytes' morphology showed obvious abnormalities and irregular aggregation especially in snails exposed for 21 days continuously to LC 25 Artemether.These results confirmed with the findings of Sharaf El-Din (2003), Martin et al. (2006), Souza and Andrade (2006), Kamel et al. (2006Kamel et al. ( , 2007) ) and Gawish et al. (2008).Also, Kamel et al. (2006Kamel et al. ( , 2007) ) evaluated that exposure of B. alexandrina snails to sublethal concentration of Anagallis arvensis and Calendula micrantha caused a significant reduction in total number of haemocytes in comparison with the control.El Sayed (2006) stated that exposure of B. alexandrina snails to sublethal concentrations (LC 0 , LC 10 , LC 25 ) of the dry powder of the plant Cupressus macrocorpa for three weeks significantly decreased the number of circulating haemocytes.
Elongation of the exposure period to 2 and 3 weeks resulted in reduction in localization of glycogen at the peripheral edges of the gonad tubules in the hermaphrodite gland tissue compared to control in which the glycogen appeared distributed at the epithelium of the gonadal cells and in their connective tissues.On the other hand, the continuous exposure to sublethal concentrations of Artemether increased the glycogen amount distributed inside haemocytes causing increase in their size by increasing the time of exposure, showing irregular big cells compared to control ones.This agrees with Hasheesh et al. (2011) who found that the glycogen in haemolymph of Bulinus truncatus snails exposed to methanol extract of Sesbania sesban plant increased, while it decreased in soft tissues when compared with the control group.The reduction in glycogen content might be explained by an increase in the activated conformation of phosporylase, and to a lesser extent the decreased glucose uptake (Xiao et al., 2000).This may also be attributed to the activity of the tested plants that impedes oxygen consumption of snails, thus inducing anaerobic respiration.To restore its energy requirements, the snail has to increase the rate of glycolysis, thus, bringing out a reduction of the glycogen content and increase glucose level in the haemolymph.This finding was reported by Bakry et al. (2001) using Agave franzosini plant against B. alexandrina, and Bakry (2009) using Furcraea gigantea and Lampranthus spectabilis plants.Other similar studies proved also the elevation in glycogen content in snails exposed to molluscicidal plants (for example, Mantawy and Mahmoud, 2002;Bakry et al., 2002Bakry et al., , 2004Bakry et al., , 2012;;Sun et al., 2012).
It can be concluded that the exposure of B. alexandrina snails to the sublethal concentrations of Artemether resulted in remarkable influence on the hermaphrodite gland and glycogen amount and distribution in snails' tissues that may deteriorate their physiological and biological activities.Moreover, the obvious role of Artemether in reduction of snails' susceptibility to infection with S. mansoni may suggest it as a promising and safe agent in schistosomiasis control.

Figure 3 .
Figure 3.Light photomicrographs of T.S (Transverse section) in the hermaphrodite gland of Biomphalaria alexandrina exposed continuously to LC25 of Artemether 1, 7, 14 and 21 days (A, B, C and D) showing reaction of glycogen contents.E: T.S section in the hermaphrodite gland of unexposed control snails.(PAS stain) ×400.
Sharaf El-Din et al. (2001),Mostafa and Tantawy (2000),Gawish et al. (2008),Mostafa et al. (2005),Bakry et al. (2007), Abdel-Hamid (2008) andHasheesh et al. (2011).Exposure of B. alexandrina snails to sublethal concentrations (LC 0 , LC 10 and LC 25 ) of Artemether either for 1 or 7 days pre-miracidial exposure significantly reduced their infection rates with S. mansoni.These results agree with that of El Sayed et al. (2011) who found that exposure of B. alexandrina and B. glabrata snails to sublethal concentration of C. grandiflora 3 days pre-miracidial exposure led to a significant reduction in the infection rate with S. mansoni by 55.47 and 58.9%, respectively.Also, Mahmoud et al. (2011) recorded a reduction in infection rate of B. alexandrina snails exposed to LC 10 of Datura stramonium and Sesbania sesban during S. mansoni miracidial exposure by 41.7

Table 1 .
Molluscicidal activity of Artemether against adult B. alexandrina snails after 24 h exposure.

Table 2 .
The survival and infection rates of B. alexandrina snails with S. mansoni after exposure to Artemether for one and seven days.