This study was conducted in early 2024 in two localities across two departments in southern Benin: Azowlissè in Adjohoun (Ouémé) and Sô-Ava in the Nokoué Lake region (Atlantique). These areas are situated in southern Benin and are characterized by a sub-equatorial climate with high humidity [10] . The region experiences two dry seasons (December to March and mid-July to mid-September), alternating with two rainy seasons (April to mid-July and mid-September to November). The temperatures are typically around 30˚C or higher, with frequent rainfall averaging 1200 mm annually. Human activities in these areas include heavy reliance on water sources for agriculture (market gardening, seasonal and annual crops, and perennial plantations), fisheries, and aquaculture (fish farming ponds).

Bulinus species were collected from natural water retention areas. These sites were selected based on visual observation and preliminary sociological surveys. The survey aimed to gather information from local residents about the aquatic environments they frequently use for their daily needs. In each identified water point, Bulinus snails were collected. The georeferencing of the sampling sites was conducted using a Global Positioning System (GPS). At each site, random sampling was carried out over approximately two hours in various parts of the water, covering areas of varying sizes. The collected Bulinus snails were placed in perforated jars and transported to the Unit of Research in Phylogeography, Genetics, and Evolution (UR-PGE) at the Faculty of Sciences and Technology of the University of Abomey-Calavi (FAST-UAC) for breeding and comprehensive identification, which included additional ecological and morphological studies (shell morphology and determination of parasitic infestation rates for each identified species). The snails were then preserved in 70% alcohol for subsequent molecular analyses.

DNA extraction from Bulinus out of the 293 Bulinus snails collected and morphologically identified, a random selection was made from each species previously confirmed to be infected, to form a sample for molecular analysis. In total, 80 individuals of Bulinus globosus and 10 of Bulinus truncatus were analyzed. To better assess the genetic diversity at each site, two to five snails were randomly selected per locality [11] . The preserved Bulinus snails were transported to the Laboratory of Biology and Molecular Typing in Microbiology at the University of Abomey-Calavi for DNA extraction. The snails were immersed in absolute ethanol for two days, and the procedure was repeated, replacing the ethanol if it became cloudy or yellowish due to mucus secretion. Approximately 20 mg of tissue (excluding the shell) from the whole snail was immersed in 1 mL of absolute ethanol for two days to eliminate any mucus residue. The tissue was then crushed and lightly ground with a pestle in a porcelain mortar before being transferred to a 2 mL Eppendorf tube containing 1 mL of ethanol. After two days, 750 µL of TE buffer were added to the Eppendorf tube to eliminate the remaining ethanol, and the mixture was incubated for 1 hour at room temperature (30˚C) to soften the tissue. Afterwards, the TE buffer was discarded, and distilled water was added to each tube, followed by another 1-hour incubation. The water was then discarded, and 600 µL of preheated Urea-SDS buffer (60˚C) and 10 µL of Proteinase K were added.

The mixture was subjected to mechanical disruption using a silica bead in a grinder for 45 seconds and left in a 60˚C water bath overnight (10 - 14 hours) to lyse the cells. Then, 750 µL of phenol-chloroform-isoamyl alcohol (PCIA) solution (24:1) were added and mixed by inversion for 5 minutes. The mixture was centrifuged at 10,000 rpm at 4˚C for 15 minutes, and the supernatant was carefully transferred to a new 1.5 mL Eppendorf tube, ensuring the white intermediate layer remained intact. The aqueous phase was precipitated with 750 µL of cold absolute ethanol (−20˚C) and 40 µL of 3M sodium acetate. The mixture was gently stirred for a few minutes and incubated at −20˚C for 1 hour. The pellet was recovered by centrifugation at 10,000 rpm at 4˚C for 10 minutes, then washed with 600 µL of 70% ethanol. This step was repeated two or three times, depending on the appearance of the pellet. The tubes were left to dry at room temperature on blotting paper. Finally, the pellet was resuspended in 100 µL of ultrapure water. After the complete dissolution of the pellets, the DNA extracts were quantified using a NanoDrop spectrophotometer after 4 hours to assess the quality and quantity of native DNA. The purity index (I) averaged between 1.8 and 2, confirming satisfactory conditions for DNA extraction.

PCR (Polymerase Chain Reaction) was used to obtain numerous copies of a specific DNA segment, called the target DNA. A fragment of approximately 1020 bp from the SSU gene located on the nuclear genome was amplified using primers with the following sequences: 18S Baso3F: 5’-GTGCTCTTCNCTGAGGGTCC-3’ and 18S Baso9R: 5’-TACGGAAGCCTTGTTACGA-3’ (Jorgensen et al. 2011). The PCR program included an initial denaturation at 95˚C for 4 minutes, followed by 35 cycles of denaturation (30 seconds at 94˚C), annealing at 68˚C, and elongation (60 seconds at 72˚C), ending with a final elongation phase at 72˚C for 5 minutes. The reaction mixture consisted of 5 µL of PCR buffer (6X green buffer), 1 µL of 200 µM dNTP, 2.5 µL of each primer at 0.2 µM, 1 µL of 25 mM MgCl₂, 0.1% BSA (Bovine Serum Albumin), 0.2 U of OneTaq polymerase, 3 µL of 10 ng/µL DNA, and sufficient water for a final volume of 25 µL. The amplified products were stored at 4˚C before being digested with the restriction endonucleases HaeIII, DdeI, and HinfI. The digestion mixture was prepared with 5 µg of PCR products, 5 µL of digestion buffer, 1.5 µL of each enzyme, and ultrapure water to a final volume of 25 µL. The digestion mixtures were incubated at 37˚C overnight (approximately 10 hours) for optimal activation, and then inactivated at 65˚C (for DdeI) or 80˚C (for HaeIII and HinfI) for 20 minutes, depending on the characteristics of each restriction enzyme. The resulting fragments, hydrolyzed according to specific cutting sites, were separated by electrophoresis on a 2% agarose gel using 10 µL of digestion products mixed with loading buffer. The migration was carried out under 80V for 45 minutes to 1 hour in 0.5X TBE buffer. A 1.5 µL volume of ethidium bromide (BET) was added to each gel well. After migration, the gel was visualized under UV light using a transilluminator. A 100 bp size marker was used during migration to estimate the approximate size of the DNA fragments generated by digestion and to assess their polymorphism.

A total of 283 Bulinus snails were collected from the two sampling sites during the 2024 recession period ( Table 1 ). Two different species of Bulinus were identified based on previously reported morphological criteria: Bulinus globosus and Bulinus truncatus.

The Kruskal-Wallis non-parametric test indicated a significant difference (P = 0.01) in the number of infested Bulinus between the two localities. Out of the 283 Bulinus snails tested for cercarial shedding, 265 excreted cercariae, leading to an overall infestation rate of 69.19% ± 6.80%. However, this infestation rate varied according to the locality: Sô-Ava: 64.12% ± 4.6% Azowlissè: 76% ± 4.90%. For Bulinus truncatus, 17 out of 23 samples released schistosome cercariae, with an infestation rate of 73.91% ± 10.65%. The infestation rate of B. truncatus also showed variability between localities: Azowlissè: 66.67% ± 33.33%; Sô-Ava: 75.00% ± 11.18%. Furthermore, out of 260 Bulinus globosus samples, 248 shed schistosome cercariae, resulting in an infestation rate of 73.37% ± 2.80%. These infestation rates also showed variability between the localities: for B. truncatus, 66.67% ± 33.33% in Azowlissè and 75.00% ± 11.18% in Sô-Ava; for B. globosus, 76.00% ± 4.90% in Azowlissè and 64.12% ± 4.06% in Sô-Ava. Consequently, the infestation rate of B. globosus is higher in Azowlissè than in Sô-Ava, while B. truncatus shows a higher rate in Sô-Ava compared to Azowlissè. The variability in the infestation rate of B. truncatus suggests a less consistent infection pattern in Sô-Ava than in Azowlissè. These observed differences could be attributed to environmental and ecological factors affecting the infection dynamics of Bulinus species in the two localities. ( Table 2 )

The recorded water temperature in Sô-Ava is 28˚C, slightly higher than the 27.9˚C observed in Azowlissè. The pH values are 7.31 in Sô-Ava and 7.01 in Azowlissè, indicating neutral conditions in both locations. Dissolved oxygen levels are higher in Sô-Ava (6.71 mg/L) compared to Azowlissè (5.06 mg/L). Conductivity is 45 S/m in Sô-Ava but significantly higher at 93 S/m in Azowlissè. Lastly, salinity is 0 ppt in Sô-Ava and 0.01 ppt in Azowlissè. ( Table 3 )

The generalized linear regression model of the fish family shows that all the parameters evaluated have a significant effect at the 0.1% threshold on the number of Bulinus snails collected and those releasing cercaria ( Table 4 ). The analysis reveals that conductivity and pH are negatively correlated with the number of Bulinus snails collected, while there is a positive relationship between the other parameters and the number of Bulinus snails. Analysis of the same tables shows that conductivity and pH are negatively correlated with the number of Bulinus snails infested, while there is a positive relationship between the other parameters and the number of Bulinus snails infested.

*Significant at the 5% level; ** Significant at the 1% level; *** Significant at the 0.1% level.

1) Case of the HinfI Endonuclease

2) Case of the HaeIII Endonuclease

3) Case of the DdeI Endonuclease

1) Cases of the endonucleases HinfI, HaeIII, and DdeI

PCR amplification performed on the species of Bulinus, with highly intact indices on agarose gels, was subsequently subjected to RFLP analysis ( Figures 1-4 ). The species Bulinus globosus produced the same profiles in Sô-Ava and Azowlissè, regardless of the endonuclease used. Similarly, the species Bulinus truncatus also produced the same profiles in Sô-Ava and Azowlissè, irrespective of the endonuclease employed. Typical bands of the Bulinus species were obtained from 13 snails in Nigeria [12] . Consistent with these published RFLP reference profiles from Nigeria, which clearly identified B. truncatus and B. globosus, the profiles obtained in Sô-Ava and Azowlissè can be identified as Bulinus globosus and Bulinus truncatus using the aforementioned RFLP reference profiles.

The results of this study demonstrated a heterogeneous distribution of the species Bulinus globosus and Bulinus truncatus, as well as infestation rates in the two localities in Benin studied. The species Bulinus truncatus and Bulinus globosus each exhibit distinct ecological structures. These species are unevenly distributed across the collection sites. The causes of this dissimilar distribution can be traced to both abiotic and biotic factors. This difference may be explained by the density of water hyacinths and the physicochemical parameters of the water (temperature, salinity, turbidity, dissolved oxygen) [13] . The pH and conductivity correlated negatively with the number of collected bulins and the number of infested bulins. The slightly basic pH obtained respectively in Sô-Ava and Azowlissè (7.01 and 7.31) confirms the work carried out by [14] in the waters of the Senegal River which showed that the pH values measured in the waters of the Senegal River are not significant and have no influence on the life of these gastropods. The data obtained during this study comply with the standard recommended by the WHO (6.5 < pH < 8.5). The electrical conductivity (EC) which is a measure of the capacity of an aqueous solution to conduct electric current shows less significant variations for the localities Sô-Ava and Azowlissè (45 and 93 mS/cm). These low contents obtained in Sô-Ava (45 mS/cm) and Azowlissè (93 mS/cm) would be due to the low inputs of organic matter in these waters, leading to very low mineralization. Our results are consistent with those of [15] who showed that conductivity contents (74 and 77.4 mS/cm) obtained in the Ivory Coast on the dams of Lake Taabo did not achieve a significant correlation with the populations of these gastropods because of its low values. The temperatures recorded in Sô-Ava and Azowlissè could be explained by the fact that the freshwater ecosystem found in these environments is less penetrated by light due to its highly developed eutrophication which would have an impact on the multiplication of the number of bulins, hence an increase in the number of bulins and the infestation rate of these bulins. The measured salinity correlated positively with the harvested molluscs and the number of infested molluscs; This could be due to its very low content. The very low salt contents indicate that the watercourses of the two localities Sô-Ava and Azowlissè are freshwater ecosystems. This absence of salinity would favor the proliferation of these aquatic species, more specifically water hyacinths (Eichhornia crassipes) which is very high in Sô-Ava and Azowlissè which, by developing, would prevent the penetration of solar rays into the water and contribute to the depletion of dissolved oxygen (O₂) [16] . These bulins are often stuck to water hyacinths which are their host plant species. The positive influence of oxygen measurements on molluscs shows that the latter record optimal values favoring life in the habitat of these gastropods. Environmental factors influencing the distribution of snails are often overlooked, even though they can vary significantly from one site to another and across ecological zones, even over short distances [17] . It should be noted that the environments of these two localities better meet the ecological requirements of these two species of Bulinus, and that there is a nearly constant communication established between them, facilitating a continuous exchange of organisms and physicochemical elements. The predominance of Bulinus globosus and Bulinus truncatus in Sô-Ava (Lake Nokoué) compared to Azowlissè (Ouémé River) can be attributed to the low marine current and the shallow gradient of the lake, although dissolved oxygen levels and salinity are not negligible factors. The results of the infestation rates in this study clearly indicate that Sô-Ava and Azowlissè are two localities that exhibit significant infestation rates.

The molecular characterization study of Bulinus using endonucleases (HaeIII, HinfI, and DdeI) with the molecular marker SSU through PCR-RFLP amplification showed specific cuts for each locality and each species of Bulinus, releasing nearly the same fragment size per enzyme for all individuals of the Bulinus populations. Regardless of the endonucleases used, Sô-Ava and Azowlissè revealed the same profiles for each species of Bulinus. Our results demonstrating the different profiles of Bulinus globosus and Bulinus truncatus obtained in this study at Sô-Ava and Azowlissè with the endonucleases (HaeIII, HinfI, and DdeI) on the SSU gene in southern Benin are consistent with the work conducted in Nigeria [18] - [20] in Kenya, who confirmed that these Bulinus species obtained were indeed Bulinus globosus and Bulinus truncatus, among other species. The observed data indicate that stable genetic markers are present for B. globosus and B. truncatus in Benin. The evolution of the SSU molecular marker is rapid enough to assist in species identification. Accurate identification of the host and parasite would not only clarify species diversity but also facilitate the development of strategies to significantly reduce transmission and find means to combat this pathology.