MORPHOLOGICAL AND BIOINFORMATICS STUDY FOR RADIX AURICULARIA SNAILS IN FRESHWATER IN BASRAH PROVINCE, IRAQ

Radix auricularia (Lymnaea auricularia) snails are the main intermediate host of Fasciola gigantica in the middle and South of Iraq. Recently, some studies suggest these snails are not present in Iraq, in particular Basrah province. Thus, the current study was aimed to use the bioinformatics analysis in the determination of Radix sp. in Basrah and also aimed to identify morphological characteristics of this species in the AL-Sewaib river, Basrah. Four hundred R. auricularia snails were collected from the AL-Sewaib river during May 2018 to March 2019. The results of the bioinformatics analysis showed according to the mitochondrial cytochrome c oxidase subunit I, the presence of R. auricularia snails in Basrah province and these snails are more closely related to Iranian R. auricularia snails than those for European counties and Russia. This study also displayed the existence of the polymorphism phenomenon in R. auricularia snails collected from the AL-Sewaib river which there were six different forms (A, B, C, D, E and F). These forms had variations in the colour and form of shells along with a variation in the number of eggs (30-56) per an egg mass. In addition to this, these R. auricularia forms had variations in their distributions. Form E had the highest spread percentage (33%) followed by 17, 15, 14, 11 and 10% for forms B, D, C, A and F, respectively. These forms had also variations in their morphological characteristics. The largest form was the form D whereas the smallest form was the form B but other forms A, C, E and F were similar and had moderate sizes.Thus, the current study concludes the existence of R. auricularaia snails in Basrah as a representative city for the southern part of Iraq and also confirmed the presence of the polymorphism phenomenon in the members of R. auricularaia obtained from the AL-Sewaib river.


INTRODUCTION
Lymnaeidae has many freshwater snails that involve in the different helminth life cycles (9,13). These snails gain the large parasitological significance as they are mainly worked as first intermediate hosts for several trematodes. The transmission of trematode parasites by a specific lymnaeid species typically depends on the parasite-host specificity as well as depend on the geographical region considered and local ecological characteristics (12 (15,21). Globally, F. hepatica is usually found in temperate regions while F. gigantica is mainly found in tropical regions. In Iraq, the F. hepatica parasite and its intermediate host G. truncatula are found in the northern and north-eastern parts while the F. gigantica parasite and its intermediate host R. auricularia are found in the middle and southern parts (6). In the last two decades, the majority of studies have been reported the presence of F. gigantica and its intermediate host in the southern part of Iraq (1-3, 7, 8). In contrast to this, only two studies reported that there is no L. auricularia (R. auricularia) in the southern part of Iraq, in particular in Basrah province (11,19). This needs to be investigated further. Thus, the present study aimed to use the morphometric and bioinformatics analyses in the determination of Radix sp. serving as an intermediate host of some trematode parasites in Basrah province.

MATERIALS AND METHODS Bioinformatics
A search was done of the Barcode of Life Data System (BOLD) (22) to find all data regarding Radix spp. collected from Basrah province, Iraq. In the BOLD identification system, the essential barcode sequence for individuals from the animal kingdom is the mitochondrial cytochrome c oxidase subunit I (COI) (5,18). In the search results, there were seven different COI sequences under the name Radix sp. that obtained from Basrah province. In order to determine the species of Radix in Basrah province, the seven different COI sequences from Basrah in addition to other COI sequences for Radix species from different countries were obtained from the "Nucleotide Database" on the National Center for Biotechnology Information (NCBI) website. The sequences obtained in this way were aligned using Clustal X version 2.1 (17). Then the Neighbor-joining method (23), accessed through Molecular Evolutionary Genetic Analysis (MEGA) version X (26), was utilised to produce a phylogenetic tree. The identity matrix of nucleotide sequences was calculated using Clustal Omega (25).

Sample collection and breeding
Four hundred R. auricularia snails were collected from the AL-Sewaib river, Basrah province during May 2018 to March 2019. Collected snails were distributed depending on the color and form of shells onto six plastic tanks (20 x 50 x 10 cm). These tanks were supplied with dechlorinated water and aerated for 24 h. The snails were fed with dried celery and incubated at room temperature for three weeks (3). Egg masses were collected from each tank after 1-2 weeks. Morphometric analysis of the R. auricularia shells Shell distances for each R. auricularia form measured using Vernier Caliper as previously described (3). These shell distances included shell length, shell width, spire length and body whorl length. Twenty replicates were used to each R. auricularia form. Statistical analyses SPSS version 25 was employed in statistical analyses and data were analyzed utilizing oneway analysis of variance (ANOVA). Significant differences were counted when the P value was < 0.05. Figure 1 shows a phylogenetic analysis of relationships between the COI sequences of

R. auricularia forms and their egg masses:
The current study showed that there were six different forms of R. auricularia snails collected from the AL-Sewaib river, Basrah and these forms were separate based on the colour and form of shells (Fig. 3). The colours of these forms were dark red (A), dark moderate red (B), very dark desaturated red (C), brown (D), dark moderate orange (E) and black (F). Figure 3 also revealed the rate of eggs in the egg mass of R. auricularia forms. The R. auricularia form A had the highest rate of eggs in egg masses which reach about 56 eggs per an egg mass compared with other R. auricularia forms. This rate of eggs per an egg mass in the Form A was somewhat higher than that for the form B (48 eggs per an egg mass) but it was not statistically significant. However, it was statistically significantly higher than that for either forms C, D, E or F. In contrast to this, the form F had the lowest rate of eggs in egg masses which reached about 30 eggs per an egg mass and it was similar to the form E in the number of eggs per an egg mass (34). Moreover, there were no significant differences between forms E and F.  Figure 4. These percentages were varied between forms. The highest distribution percentage was observed in the form E (dark moderate orange) which reached up to 33% compared with 11, 17, 14, 15 and 10% for the form A, B, C, D and form F, respectively. In addition to this, the statistical analysis revealed the distribution percentage of form E was statistically significantly higher than that for other forms. In contrast to this, the lowest distribution percentage was noted in the form F (black) which reached up to 10%. The distribution percentage of form F was statistically similar to either forms A, C or D but was statistically significantly lower than the distribution percentages of either forms B or C. These results indicated that the R. auricularia form E possessed the highest spread percentage in the AL-Sewaib river compared with other forms.  Table 2 shows morphological characteristics for the R. auricularia forms (A, B, C, D, E and F). Results of the present study showed that the six R. auricularia forms had some variances in the shell length, shell width, spire length and body whorl length. The R. auricularia form D had the highest values in the shell length (14.52 mm), shell width (9.45 mm), spire length (3.80 mm) and body whorl length (10.72 mm) compared with other R. auricularia forms. Moreover, these values were statistically significantly higher than those for other R. auricularia forms. In contrast to this, the R. auricularia form B had the lowest values in the shell length (8.31 mm), shell width (5.12 mm), spire length (2.79 mm) and body whorl length (5.52 mm) compared with other R. auricularia forms and these values were statistically significantly lower than those for other R. auricularia forms. Unlike R. auricularia forms B and D, the forms A, C, E and F had almost similar shell measurements (moderate) with some statistical differences. These results indicated that the largest form was the form D whereas the smallest form was the form B but other forms A, C, E and F were similar and had moderate sizes.

Table 2. Morphological characteristics of R. auricularia shells collected from the AL-Sewaib river
In the South of Iraq, there are too many infections with F. gigantica transmitted to water buffalos, cattle and sheep by an intermediate host from R. auricularia known as L. auricularia (2,4,7,8). Surprisingly, Al-Waaly (10) and Naser et al. (19) reported that R. auricularia snails are not present in the Southern regions of Iraq including Basrah province. If this situation is true, infections with fascioliasis in these regions of Iraq cannot happen due to the absence of the most important intermediate host. Thus, the current study was performed to answer this and to identify the morphometric characteristics of Radix species in Basrah as representative species for the Southern part of Iraq. Based on the phylogenetic analysis, the Radix COI sequences from Basrah were more closely related to R. auicularia COI nucleotide sequences than that for other Radix species. In the present study, the Radix COI nucleotide sequences from Basrah were highly identity to R. auicularia COI nucleotide sequences from Iran than those R. auicularia COI nucleotide sequences from European countries and Russia. Al-Waaly (10) reported the Radix COI nucleotide sequences from Basrah share 87% identify with R. auicularia (Croatia (EU818826.1) and France (JN614403.1)), R. nataleinsis and R. relicta. According to this, Al-Waaly (10) recorded Radix samples from Basrah as a new species called Radix sp. This was not correspondent with the results obtained in the present study which confirms the presence of R. auricularia in Basrah province. In addition to this, an alignment of COI amino acid sequences indicated that Radix COI sequences from Basrah were highly similar to the R. auricularia sequences from Iran. In total, the results of present study completely disagree with Al-Waaly (10) study and suggest that the Radix sp. in Basrah belongs to R. auricularia. Radix spp. snails have been used in many studies including parasitology, biodiversity and ecotoxicology (24). Nevertheless, a few studies have been focused on the polymorphism phenomenon in these snails (10,16,19). The current study revealed the presence of six different forms of R. auricularia in the AL-Sewaib river that had variations in the colour and form of shells as well as variations in the number of eggs per an egg mass in each form. This was consistent with previous studies that showed the presences of the polymorphism phenomenon in Radix sp. (R. auricularia) snails (10,14,19). In Iraqi, Al-Waaly (10)  the present study from the number of R. auricularia forms [3] obtained in the Al-Waaly (10) study could be due the geographic area that collected snails from. The polymorphisms phenomenon in R. auricularia snails reflected on the distribution of these snails and the number of eggs per an egg mass in each R. auricularia form. For instant, unlike other R. auricularia forms recorded in the present study, the R. auricularia form E had the largest distribution percentage (33%) compared with other forms (10 -17%) collected from the AL-Sewaib river but it shared the similar rate of eggs per an egg mass with the R. auricularia form F. Thus, the distribution percentage of the R. auricularia form E was consistent with the distribution percentage (27.4 -37.1%) of the R. auricularia forms collected from Basrah in another study (10). The variation in R. auricularia forms in the AL-Sewaib river also extended to include the variation in the length, width, spire length and body whorl length of shells. The R. auricularia form D, had the highest rate in the all morphological characteristics compared with the rest of R. auricularia forms obtained in the present study. Al-Waaly (10) reported that there were variations in morphological characteristics of Radix sp. (R. auricularia) obtained from three cities (Basrah, Kerbala and Diwaniya) in Iraq. For the R. auricularia obtained from Basrah, these morphological characteristics were 10.24 mm for shell length, 6.07 mm for shell width and 3.07 mm for spire length (10). These morphological characteristics were partially similar to R. auricularia forms A, C and E obtained in the present study. The current study shows based on the bioinformatics analysis the presences of the main vector of fascioliasis, R. auricularia, in Basrah province. This study also revealed the occurrence of the polymorphism phenomenon in R. auricularia snails collected from the AL-Sewaib river. As a result, there were six different forms of R. auricularia in the AL-Sewaib river that had variations in the colour and form of shells as well as variations in the number of eggs per an egg mass in each form. In addition to this, these R. auricularia forms had variations in their distributions and morphological characteristics.