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. 2020 Dec 4;6(12):e05630. doi: 10.1016/j.heliyon.2020.e05630

Usage of pharmaceutical contraceptive drug for controlling Eobania vermiculata snails by baits technique

Mahmoud S Abd El-Atti a,∗, Ashraf SA El-Sayed b, Radwa M Said a
PMCID: PMC7724155  PMID: 33319095

Abstract

The present study focused on evaluating the effects of oral administration of three different concentrations of Yasmin® combined contraceptive pills (estrogen and progesterone) on reproductive hormones levels, histology of the ovotestis and rate of oviposition of E. vermiculata for two months using baits technique. The levels of anti-müllerian hormone (AMH), Follicle stimulating hormone (FSH), Luteinizing hormone (LH), Estradiol (E2), Progesterone(PRG), Thyroid-stimulating hormone (TSH) and Testesterone (T) of treated snails were decreased with increasing the drug concentrations by percentages of -83.3%, -78.9%, - 59.6%,- 98.3 %, - 79.6 %, - 86.7% and 8.2%, respectively. Prolactin (PRL) level was significantly increased (86.9%) compared to control snails after 8 weeks of exposure. Histological investigations on the hermaphrodite glands of snails treated with 909 μg/gm. showed glandular hyperplasia, sloughing of germinal epithelium, acini sizes reduction, suppression of follicular growths, decreased luteinization and vasodilation. Male acini revealed histolytic of spermatogonia and mature sperms. The lowest concentration (303 μg/gm.) caused gradual decrease of the total egg counts that reach 50% at the 8th week of treatment. Higher doses (606 and 909 μg/gm.) resulted in dramatic dwindling of egg numbers and inspiring complete egg cessation at the 7th and 3rd weeks of treatments, respectively. The applications of combined contraceptive drugs as baits give promising results for controlling high population densities of E. vermiculata snails at Sharkia Governorate, Egypt.

Keywords: Contraceptive drug, Oviposition, Hormones, Histopathology, Eobania vermiculata, Food science, Animal science, Agronomy, Pesticide, Zoology

Contraceptive drug; oviposition; Hormones; Histopathology; Eobania vermiculata; Food Science; Animal Science; Agronomy; Pesticide; Zoology.

1. Introduction

Terrestrial gastropods are one of the most prosperous classes of invertebrates inhabiting the terrestrial ecosystems (Arad et al., 1993). They have functional mechanisms versus starvation, desiccation and freezing (Riddle and Miller, 1988; Burla and Gosteli, 1993). Land snails are deliberated as serious economic pests feeding on algae, lichens and grasses in addition to roots, leaves, flowers, fruits and seeds of higher plants (2013; 2015; Lazaridou-Dimitriadou and Kattoulas, 1991; Speiser and Rowell-Rahier, 1993). The helicid chocolate-band Eobania vermiculata (Müller, 1774) snails are native to the Mediterranean region and have been found their way to invade many other countries (Neubert, 1998). They became the most widespread species worldwide excluding southern South America and some Pacific islands (Scott, 1997). This species is characterized by its survival capacities and has been introduced by human activities to numerous countries from the United States to Japan (Ueshima et al., 2004; Ronsmans and Van den Neucker 2016). E. vermiculata forayed many Egyptian agricultural fields (El-Sayed, 2013; El-Sayed et al., 2018; 2019a; c, 2020). Their highest population densities on citrus trees at Sharkia Governorate were detected during spring while the highest vertical distribution on Casuarina trees was noticed during summer (Ismail et al., 2017). Chemical molluscicides degenerated lingeringly in the environment and inspiring critical ecological toxicities (Rug and Ruppel 2000; Gaber et al., 2006). The high expense and resistance exerted against synthetic pesticides encouraged the usage of naturally derived compounds in pests control managements (Ali et al., 2015; Massoud and Habib 2003). Nowadays, many countries have supported the usage of natural plant products and microbial pesticides due to their low mammalian toxicity, low costs and fast biodegradability ( El-Sayed et al., 2020; El-Sayed and Ali, 2020; El-Sayed et al., 2018, 2019a, c; Singh et al., 2000; Genena and Mostafa, 2008; Abd El-Atti et al., 2020). Few studies have been carried out addressing the effect of hormones on reproduction of basommatophoran pulmonates snails and slugs but much fewer studies have done on stylommatophoran land snails (Kruatrachue et al., 1996). The developments of oral contraceptives in 1958 are among the most widely used pharmaceuticals and their combined pills contain active exogenous estrogen and progestin (Pincus et al., 1958). The combination of the two steroids creates a synergistic effect that greatly increases their anti-gonadotropic and ovulation-inhibitory effects (Curtis et al., 2013). Exogenous steroids efficacies are due to a range of effects causing a decrease in FSH and LH, leading to the suppression of follicular activity and ovulation (Baerwald et al., 2004). In primates, estrogen and progestin have been shown to inhibit the growth of pre and medium-sized antral follicles through inhibition of FSH secretion (Koering et al., 1994).

Our goal was to determine the effect of Yasmin® (Estrogen and Progesteron) contraceptive drug on the reproductive hormones, histological structure of the hermaphrodite gland and rate of egg production of E. vermiculata under laboratory conditions using bait technique. This research aimed also to apply an environmentally safe method for decreasing fertilities of these harmful agricultural pests and consequently controlling their high populations on citrus trees at Sharkia Governorate, Egypt.

2. Materials and methods

2.1. Collection and acclimatization of snails

Adult E. vermiculata snails (29 ± 0.4 mm length, 16 ± 0.2 mm width and 2.6 ± 1 gm. weight) were collected from citrus fields at Alrobomaya village, Meniet El-kamh district, Sharkia Governorate, Egypt, during October 2019. The collected specimens were transferred to the laboratory and maintained in glass cages (40 × 30 × 30 cm) filled with moist soil of about 10 cm height and covered with a nylon-mesh lid. They fed on fresh lettuce leaves daily for two weeks. The experimental animals were maintained at 20 °C ± 2 °C and relative humidity 80–90 %.

2.2. Synthetic hormones and baits formulation

Yasmin® is an Egyptian contraceptive drug containing 0.03 mg ethinyl estradiol (Estrogen) and 3 mg drospirenone (Progesterone)/tablet. Baits were formulated by adding 95 g wheat bran to 5 g of sugarcane syrup and the mixture was moistened with small amounts of water. Different baits concentrations (303, 606 and 909 μg/g) were prepared by grinding of one, two and three tablets, respectively with 10 g of the bait.

2.3. Experimental design and egg masses count

The chocolate-band snails were divided into four groups comprising 10 snails for each. The first group left as control group. The remaining three groups were fed on baits containing the tested drug concentrations for two months and three replicates were applied for each group. During oviposition, snails were not disturbed. The soil was inspected with the aid of hand lens for eggs masses and the eggs were counted and removed weekly for eight successive weeks. Oviposition was observed from mid of November 2019 to mid of January 2020.

2.4. Hormonal analysis

Soft tissues of control and treated with different doses of contraceptive drug for 8 weeks were dissected out and homogenized in Phosphate-buffered saline. The homogenates were centrifuged at 8000 rpm at 5 °C and the supernatants were stored at - 80 °C. Levels of anti-müllerian hormone (AMH), Follicle stimulating hormone (FSH), Luteinizing hormone (LH), Prolactin (PRL), Testesterone (T), Estradiol (E2) and Thyroid-stimulating hormone (TSH) were measured using kits, two-immunological step sandwich type assay (Immunotech version; Beckman Coulter, Marseille, France). Progesterone (PRG) hormone was analyzed by ELISA technique.

2.5. Histological investigations

The hermaphrodite glands of both control and treated snails were dissected out and fixed in alcoholic Bouin. Specimens were dehydrated in ascending series of ethanol, cleared in Xylene for 20 min and embedded in paraffin wax. Sections (4–5 μ m thick) were cut, mounted, and stained with Hematoxylin and Eosin.

2.6. Statistical analysis

All data were performed using the Statistical Package for the Social Sciences software (version 20, IBM SPSS, Armonk, NY, USA). Two-way ANOVA was applied to examine the effect of different concentration of contraceptive drug and exposure time on the total egg count. Comparisons of the effects of different drug concentrations on hormonal levels were performed using one-way analysis of variance (ANOVA). Duncan test was used to calculate significant differences (P < 0.05) between tested treatments. Variables were presented as mean ± S.E.

2.7. Ethical approval

All the studies involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

3. Results and discussion

3.1. Hormonal levels of treated snails

Levels of tested hormones of treated snails with three different concentrations of Yasmin® (Estrogen and Progesterone) contraceptive drug were decreased significantly with increasing drug concentrations, while Prolactin levels were significantly increased compared to control (Table 1). Percentage of decreases of anti-müllerian hormone (AMH), Follicle stimulating hormone (FSH), Luteinizing hormone (LH), Estradiol (E2), Progesterone(PRG), Thyroid-stimulating hormone (TSH) and Testesterone (T) of treated snails were decreased at the end of the 8th week by -83.3%, -78.9%, - 59.6%,- 98.3 %, - 79.6 %,- 86.7% and -8.2%, respectively compared to control.Prolactin (PRL) levels were increased significantly (86.9%) compared to control (Figure 1).

Table 1.

Mean values of reproductive hormones levels of E. vermiculata exposed to different concentrations of contraceptive drug for 8 weeks.

Hormones Drug concentrations
Control 303 (μg/g) 606 (μg/g) 909 (μg/g) F-value P-value
AMH (ng/ml) 3.71 ± 0.41a 2.42 ± 0.81b 1.2 ± 0.2c 0.6 ± 0.1c 59.26 0.001
FSH (mIU/ml) 1.42 ± 0.21a 0.68 ± 0.25b 0.48 ± 0.06b 0.30 ± 0.02b 13.69 0.014
LH (mIU/ml) 1.51 ± 0.29a 0.95 ± 0.07b 0.77 ± 0.06b 0.61 ± 1.32c 12.55 0.02
PRL (ng/ml) 1.69 ± 0.31a 2 ± 0.26a 2.44 ± 0.07ab 3.16 ± 0.16b 8.39 0.03
T (ng/dl) 8.50 ± 0.00a 8.30 ± 0.2a 8.20 ± 0.3a 7.8 ± 0.75a 0.58 0.66
E2 (Pg/ml) 1905.5 ± 12.2a 1328.2 ± 30b 1030 ± 19.1c 32.70 ± 5.97d 1701.3 0.00
PRG (ng/dl) 2.50 ± 0.04a 1.80 ± 0.21b 1.02 ± 0.12c 0.51 ± 0.1d 45.72 0.001
TSH (mU/ml) 0.15 ± 0.01a 0.09 ± 0.01ab 0.07 ± 0.01bc 0.02 ± 0.0c 8.03 0.04
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Variables were presented as mean ± S.E. Mean values with the same alphabetical superscripts are statistically insignificant. Values are statistically significant at P < 0.05.

Figure 1.

Figure 1

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Changes (%) of reproductive hormones levels in E. vermiculata treated with different concentrations of contraceptive drug for 8 weeks.

The endocrine role of the dorsal bodies in pulmonate snails was firstly reported by Lever (1958). Gonadal hormones of land snails control gametogenesis through a group of neuroendocrine mechanisms (Takeda, 1989). Synthetic steroids (estrogens and progestogens) are potent endocrine cripples altering endogenous hormone levels through interfering with the fabrication processes of hormones and this, in turn, alters various biochemical processes and disturbs reproduction and development in freshwater snails (Diaz-Cruz et al., 2003a). Anti-Müllerian hormone (AMH) is the best endocrine marker for detecting used to evaluate ovarian reserve and the age-related decrease of the ovarian pool in females (Grynnerup et al., 2012). In the present study, AMH levels were decreased significantly in treated snails and this decrease may be due to dramatic decrease in egg reserves within their hermaphroditic acini. Prolonged treatment with contraceptives drug caused a reduction of granulosa cell mass and this resulted in a significant decrease in AMH secretion (Sanna et al., 2013).

Follicle stimulating hormone (FSH) promotes the follicular growth in females and the development of primordial germ cells in males (Ulloa-Aguirre et al., 2018).Luteinizing hormone (LH) controls the ovulation and the production of both estrogen and progesterone from the ovary (Balasch et al., 2001; Kumar and Sait, 2011). FSH and LH regulate gonadal function and fertility (Osunkeye et al., 2019). The present study showed that FSH and LH levels were significantly decreased in treated E. vermiculata for 28 days. Synthetic hormones may decrease gonadotropin releasing hormones and consequently inhibiting FSH and LH secretion. The progestogen negative feedback and lack of estrogen positive feedback on LH secretion may stop the mid-cycle of LH flow and prevent ovulation (Baird and Glasier., 1993). Contrarily, exogenous administration of gonadotropin, Progesterone and 17β-oestradiol increased levels of gene expression of FSH receptors while LH receptors were not affected in all organ of treated Archachatina marginata snail (Osunkeye et al., 2019).

Estrogen is secreted from the ovaries and adrenal gland promotes the growth of the egg follicle (Diaz-Cruz et al., 2003b). Estradiol (E2) is a type of estrogen and its elevated levels may result in loss of sex drive while its low levels resulted in weight gain and cardiovascular disease (Schulster et al., 2016). The present results revealed that E2 levels of treated snails are decreased. Similarly, Omran and Salama (2016) found that E2 level decreased in gonadal tissue of Biomphalaria alexandrina snails treated with sublethal concentrations of atrazine and glyphosate herbicides for 3 weeks. In accordance with this finding, Ibrahim and Sayed (2019) found that the level of E2 hormone were decreased significantly in the gonad tissue of the snail Biomphalaria alexandrina after exposure to sub-lethal concentrations of oxyfluorfen and attributed this decrease to the disturbance in the reproductive hormones. Progesterone (PRG) is a steroid sex hormone involved in embryogenesis of humans and other species and plays an important role in brain functions (Baulieu and Schumacher, 2000; Li et al., 2012). Progesterone levels of treated snails were decreased and this may be due to inhibition of the endocrine ovarian axis (Baird and Glasier., 1993). Testosterone (T) plays a key role in the development of male reproductive tissues (Bassil et al., 2009). Insufficient levels of testosterone in men may lead to abnormalities including frailty of bones (Tuck and Francis, 2009). Increasing levels of testosterone through a negative feedback inhibit the release of FSH and LH, respectively (Swerdloff et al., 1992). Testosterone levels were slightly decreased in treated E. vermiculata snails and this may be due to the inhibitory effects of the drug on the ovarian and adrenal androgens synthesis and/or increasing levels of sex hormones-binding globulin (Zimmerman et al., 2014). This finding is in agreement with Ibrahim and Sayed (2019) who found that the level of T hormone was decreased significantly in the gonad tissue of B. alexandrina treated with sub-lethal concentrations of some herbicides. Atrazine and glyphosate are toxic to Sertoli cells in mature rat and induced apoptosis at higher doses in germ cells (Clair et al., 2012). These results may suggest that oral contraceptive pills had side effects on male fertility by damaging developmental stages of spermatogenesis and cells which are responsible for producing testosterone hormone (Wafeeq, 2013). Thyroid-stimulating hormone (TSH) is a pituitary hormone that stimulates the thyroid gland to produce thyroxine (T4), and then triiodothyronine (T3) which promotes the metabolism of body tissues (Magner, 2014). It promotes growth and the maturation of bones and the central nervous system, increase carbohydrates, lipids and protein metabolism (Delitala et al., 2019).The decrease TSH in E. vermiculate may be due to disrupting effect of synthetic combined contraceptive drug. Prolactin (PRL), stimulates the mammary glands to produce milk which normally starts when levels of progesterone fall by the end of pregnancy and plays an important role in maternal behavior (Lucas et al., 1998). Elevated levels of prolactin decrease the levels of sex hormones — estrogen in women and testosterone in men (Freeman et al., 2000). Elevated prolactin secretion can suppress the secretion of FSH and GnRH, leading to hypogonadism and sometimes causing erectile dysfunction (Banerjee et al., 2004). There is no significant differences among hormones of Archachatina marginata sails at dormancy period except progesterone and testosterone and the mean concentrations of the hormones were found to be significantly influenced by the reproductive phases and aestivation (Okhale et al., 2018).

3.2. Histological investigations

3.2.1. Normal hermaphrodite gland of E. vermiculata

The hermaphrodite gland (ovotestis) of E. vermiculata is embedded in the digestive gland and consists of many ovoid acini each is bounded by interacinar connective tissue supplied with blood vessels and containing some interstitial cells. It composed of many ovoid acini and each acinus is bordered by germinal epithelial layer and many Sertoli cells in between. Their lumens embrace developmental stages of spermatogenesis and oogenesis (Figures 2, 3, 4, and 5).

Figure 2.

Figure 2

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Photomicrographs showing sections in the hermaphrodite gland of control E. vermiculata. Hermaphroditic acini containing different developmental stages of oogenesis and spermatogenesis. H&E X 100. BV: blood vessel; CL: corpus luteium; CT: interacinar connective tissue; FC: Follicular cells; GE: germinal epithelium; HA: hermaphroditic acini; MO: mature oocyte; OO: oogonia; PS: primary spermatocytes; SD: spermatids; SO: secondary oocyte; SP: mature sperms; SR: Sertoli cell.

Figure 3.

Figure 3

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Photomicrographs showing sections in the hermaphrodite gland of control E. vermiculata. Mature oocyte surrounded with Graffian follicle. H&E X 400. BV: blood vessel; CL: corpus luteium; CT: interacinar connective tissue; FC: Follicular cells; GE: germinal epithelium; HA: hermaphroditic acini; MO: mature oocyte; OO: oogonia; PS: primary spermatocytes; SD: spermatids; SO: secondary oocyte; SP: mature sperms; SR: Sertoli cell.

Figure 4.

Figure 4

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Photomicrographs showing sections in the hermaphrodite gland of control E. vermiculata. Corpus luteium lifted after releasing of mature oocyte. H&E X 400. BV: blood vessel; CL: corpus luteium; CT: interacinar connective tissue; FC: Follicular cells; GE: germinal epithelium; HA: hermaphroditic acini; MO: mature oocyte; OO: oogonia; PS: primary spermatocytes; SD: spermatids; SO: secondary oocyte; SP: mature sperms; SR: Sertoli cell.

Figure 5.

Figure 5

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Photomicrographs showing sections in the hermaphrodite gland of control E. vermiculata. Hermaphroditic acini containing developmental stages of sperms. H&E X 400. BV: blood vessel; CL: corpus luteium; CT: interacinar connective tissue; FC: Follicular cells; GE: germinal epithelium; HA: hermaphroditic acini; MO: mature oocyte; OO: oogonia; PS: primary spermatocytes; SD: spermatids; SO: secondary oocyte; SP: mature sperms; SR: Sertoli cell.

3.2.2. Histopathological alterations in treated snails

Histopathological investigations on the hermaphrodite glands of snails treated with 909 μg/gm showed glandular hyperplasia, sloughing of germinal epithelial and with suppression of follicular growths (Figures 6 and 7). Cells of digestive gland tubules suffered from severe vacoulation (Figure 7). Size reduction associated with decreased luteinization and dilation of blood vessels of hermaphroditic acini were observed (Figure 8). Male acini showed histolytic developmental stages of spermatogenesis and mature sperms (Figure 9).

Figure 6.

Figure 6

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Photomicrographs showing sections in the hermaphrodite gland of E. vermiculata treated snails treated with 909 μg/g contraceptive pills for 8 weeks. Hermaphroditic acini showing glandular hyperplasia, decreased luteinization, dilation of blood vessels and oocytes deformations H&E X 100. BL: dilated blood vessel; CT: interacinai connective tissue; DO: Deformed oocytes; DS: Deformed spermatozoa; DT: Vacuolated digestive tubules cells; GE: Sloughed germinal epithelium; GH: Glandular hyperplasia; HA: hermaphroditic acini; OO: oogonia; PS: primary spermatocytes; SO: Secondary oocyte; SR: Sertoli cell; SS: Secondary spermatocytes.

Figure 7.

Figure 7

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Photomicrographs showing sections in the hermaphrodite gland of E. vermiculata treated snails treated with 909 μg/g contraceptive pills for 8 weeks. Sloughing of germinal epithelial tissue and many immature and deformed oocytes and vacuolation of digestive tubular cells. H&E X 400. BL: dilated blood vessel; CT: interacinai connective tissue; DO: Deformed oocytes; DS: Deformed spermatozoa; DT: Vacuolated digestive tubules cells; GE: Sloughed germinal epithelium; GH: Glandular hyperplasia; HA: hermaphroditic acini; OO: oogonia; PS: primary spermatocytes; SO: Secondary oocyte; SR: Sertoli cell; SS: Secondary spermatocytes.

Figure 8.

Figure 8

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Photomicrographs showing sections in the hermaphrodite gland of E. vermiculata treated snails treated with 909 μg/g contraceptive pills for 8 weeks. Glandular hyperplasia, degenerated primordial follicles and oocyte deformation H&E X 400. BL: dilated blood vessel; CT: interacinai connective tissue; DO: Deformed oocytes; DS: Deformed spermatozoa; DT: Vacuolated digestive tubules cells; GE: Sloughed germinal epithelium; GH: Glandular hyperplasia; HA: hermaphroditic acini; OO: oogonia; PS: primary spermatocytes; SO: Secondary oocyte; SR: Sertoli cell; SS: Secondary spermatocytes.

Figure 9.

Figure 9

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Photomicrographs showing sections in the hermaphrodite gland of E. vermiculata treated snails treated with 909 μg/g contraceptive pills for 8 weeks. Hermaphroditic acini containing vacuolated and sloughed germinal epithelium histolytic of spermatogonia and mature sperms. H&E X 400. BL: dilated blood vessel; CT: interacinai connective tissue; DO: Deformed oocytes; DS: Deformed spermatozoa; DT: Vacuolated digestive tubules cells; GE: Sloughed germinal epithelium; GH: Glandular hyperplasia; HA: hermaphroditic acini; OO: oogonia; PS: primary spermatocytes; SO: Secondary oocyte; SR: Sertoli cell; SS: Secondary spermatocytes.

Reductions in the follicle-stimulating hormone (FSH), and the luteinizing hormone (LH) observed in this study are believed to suppress ovarian follicular development, and thus prevent ovulation (Tafurt et al., 1980). Similarly, the injection of the gastropods Achatina fulica with vertebrate hormones such as estradiol, testoviron, progesterone and gonadotrophin caused serious histopathological changes in their ovotestis (Kruatrachue et al., 1996). Ovarian follicular development is not completely suppressed during the use of currently available combined oral contraceptives formulations in women (Baerwald et al., 2003). The changes in the ovaries appear to be a result of the suppressive effect of contraceptive steroids on pituitary gonadotropin secretion (Myhre, 1972). In agreement with this study, many histological alterations associated with oral contraceptive usage were detected in the ovaries and blood vessels of women as; glandular hyperplasia, ovarian size reduction suppression of follicle growth and decreased luteinization and fibrosis in blood vessels (Bernard, 1976). Inhibition of gonadotropin levels may result in inhibition of follicle maturation, luteinization as well as consequent reduction in ovarian size. Similarly, non-growing follicles and vacuolated interstitial cells were detected in female rabbits administrated orally with combined pills (Gabri et al., 2010). Contraceptive pills might induce also proliferative changes in liver cells and this may be a result of vascular alterations (Carbia et al., 1970). Light microscopy revealed severe histopathological changes in the pituitary, thyroid and adrenal glands were represented by necrosis, cytoplasmic vacuolationand hyperplasia in Broiler chiken trated with Microgynon contraceptive pills (Layth and Majdy, 2018). Histopathological changes were observed in ovotestis acini of treated snails as damaged and destruction of germinal epithelial layer and reduction of spermatogenesis. Similar results were observed in male mice treated with oral contraceptive pills (Wafeeq, 2013).

3.2.3. Rate of oviposition of E. vermiculata

Mean numbers of eggs/group of treated snails were significantly decreased with increasing the dose of the contraceptive drug and the time of treatments compared to control (Table 2). The lowest tested concentration (303 μg/gm.) caused gradual decrease of the total egg counts that reach 50% at the 8th week of exposure. Higher concentrations of contraceptive Pills (606 and 909 μg/gm.) caused dramatic dwindling of egg numbers and inspiring complete egg cessation at the 7th and 3rd weeks of treatments, respectively (Figure 10).

Table 2.

Mean numbers of eggs of Eobania vermiculata exposed to different doses of contraceptive tablets for 8 weeks.

Exposure Time Drug conc.
Number of eggs/10 snails
Control 303 μg/g 606 μg/g 909 μg/g
1st week 91 ± 4a 80 ± 10a 73 ± 3ab 50 ± 5b
2nd week 83 ± 3a 69 ± 8a 66 ± 4a 38 ± 2b
3rd week 94 ± 14a 73 ± 2b 61 ± 6b 0 ± 0.0c
4th week 94 ± 6a 65 ± 14b 49 ± 11b 0 ± 0.0c
5th week 103 ± 7a 63 ± 7b 44 ± 4b 0 ± 0.0c
6th week 110 ± 20a 52 ± 11b 35 ± 5bc 0 ± 0.0c
7th week 120 ± 10a 50 ± 3b 0 ± 0.0c 0 ± 0.0c
8th week 113 ± 23a 40 ± 12b 0 ± 0.0c 0 ± 0.0c
Two-way ANOVA F-Value P-value
Exposure time 7.59 0.000
Tested concentrations 169.04 0.000
Exposure time x conc. 5.11 0.000
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Variables were presented as mean ± S.E.Mean values with the same alphabetical superscripts are statistically insignificant. Values are statistically significant at P < 0.05.

Figure 10.

Figure 10

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Egg count/10 Eobania vermiculata snails exposed to three different doses of contraceptive drug for 8 weeks.

Egg production in several species of stylommatophoran gastropods is stimulated by mating behavior.

(Bride et al., 1991). The breeding period of land snails depends upon the climate of the geographic region, temperature and humidity (Attia, 2004). In Western Europe the breeding period lasts from April to June while at the Mediterranean multiple copulations and ovipositions within one reproduction period may occur and last from October to December and February to April (Nicolai et al., 2005). Important triggers of egg oviposition in lymnaea stagnalis pond snails were oxygen - rich clear water, water quality and light (Ter Maat et al., 2012; El-Sayed et al., 2020). Oviposition period of E. vermiculata was averaged from 15 to 25 days (Mohamed and Ali 2009). In this study the mean numbers of eggs of treated snails were significantly decreased with increasing the dose of the contraceptive drug and the time of treatments. The combination of the estrogen and progesterone steroids creates a synergistic effect that greatly increases their anti-gonadotropic and ovulation-inhibitory effects (Curtis et al., 2013). Their efficacy may be due to a range of effects causing a decrease in FSH and LH, leading to the suppression of follicular activity and ovulation (Baerwald et al., 2004; El-Sayed et al., 2017). With no folliclular development and the absence of luteinizing hormone flow to release the follicle, prevention of ovulation occurred (Baird and Glasier., 1993). Similarly, Sublethal concentrations of chlorfluazuron (IGR) complete absence of ova in the ovotestis of E. vermiculata (Mubarak, 2016). Contrary, low concentrations of 17-α-methyltestosterone (a synthetic hormone promoting male monosex in fish aquaculture) significantly increased the number of egg mass (Rivero-Wendt et al., 2014). There was a marked increase in the size of the ovotestis number of oocytes in Achatina fulica snails injected with estradiol and progesterone (Kruatrachue et al., 1996). Chitosan and emamectin benzoate caused prevention of egg production in treated E. vermiculata snails (Patel et al., 2016; Kandil et al., 2020). Finally, the squeaky decreases of the tested reproductive hormones levels, malformations of sperms and ova and complete egg cessation at the third week after treatment of snails with contraceptive pills as baits may assure the efficacy of this method in controlling high population densities of E. vermiculata devouring citrus trees at Sharkia Governorate, Egypt.

4. Conclusion

Yasmin® contraceptive drug caused dramatic inhibition of hormones levels, deleterious histopathological effects on the hermaphrodite glands in addition to egg cessation and sperms malformations. The application of contraceptive drugs for the first time was promising in controlling high populations of E. vermiculata snails. This environmentally safe baits technique was efficient control method and ensures survival of non-target organisms harmed by using synthetic molluscicides. Usage of contraceptive drugs in controlling E. vermiculata snails by baits technique was effective, easily applicable, ecofreindly technique and could be used in decreasing numbers of other types of land snails damaging economic crops at Egypt.

Declarations

Author contribution statement

Mahmoud S. Abd El-Atti, Ashraf S.A. El-Sayed: Conceived and designed the experiments.

Radwa M. Said:Performed the experiments.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data included in article.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

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