INTRODUCTION:

The Molluscas is estimated more than 35000 species, includes the family Subulinidae, which includes the Paropeas achatinaceum (L.P. Feiffer 1846) species and they lives in the most terrestrial lands (Asia, Europe, America and Africa) [1][2][3]. Terrestrial Mollusca were most abundantly in limestone areas, and they need the limestone to build their shells [4]. The Subulinidae family has a uniform colors, with cylindrical shapes and they diagnosed by shells sizes, and it is the most divers terrestrial gastropods, having widely variety of life style from climbing trees to hidden in soil, near human habitation (under trees, stones, grass root and in gardens) [5][6][7].

The Paropeas was a distinct genus in its class, its ear-breathing land snail, a terrestrial gastropod, eating plants and feed on roots [8]. The Paropeas can lives in artificial region such as parks, farm land gardens with natural pH (pH=7.4), and 26.7^ºC temperature [9,10]. Mitochondrial DNA markers have been widely used for identification of closely related species [11] and also for population genetic structure analyses [12]. Currently, the most widely used genes was cytochrome c oxidase subunit 1 (COI) for studying the phylogeny, systematics and the identification of species. The COI gene region was effective tool for delineating marine species of molluscs and for revealing overlooked species and this was confirmed by Layton [13]. The aim of this present work is to study the morphological description and the genetic identification at molecular level by using polymerase chain reaction (PCR) and mtDNA COI gene sequencing as DNA barcoding region in the Paropeas achatinaceum (L. P. Feiffer 1846).

MATERIALS AND METHODS:

Specimen:

Paropeas achatinaceum (L. P. Feiffer, 1846) (Gastropoda: Subulinidea) specimens were collected from four areas in Baghdad-Iraq from the period between June and July, 2017. DNA was extracted from fresh specimen and the other specimens were preserved in 70% ethanol for further studies.

Morphological, feeding and moving studies:

The collected Paropeas achatinaceum specimen was identified and classified according to Chat field and Pfleger [14] and Barker and Efford [15]. The snails were placed in ground areas with 1m² diminution. Each area surrounded by a closed metal net at 30 cm depth in soil and the same length high above the ground soil to protect them from external influence such as predators. The soil in each area was cleaned and removed from any plants residues. Temperature, humidity and pH of the soil were determined and maintained at optimal conditions for the snails breeding according to APHA American Public health association [16]. Fifty snails were used in this study and these snails were divided into two groups (each group contain 25 snails). Two environment conditions were used in this study. Natural environment considered as control. The experimental environment contains Citrus sinensis (L.) roots as snail’s source food. Roots of plant were added to the all areas. All of the roots were weighted every 12 hrs for three days. Each snail was weighted in gram unit before starting the experiments. Weights of each snail were recorded and compared with plant roots weights for observing the roots consuming by the snails. The main rate moving of snails was calculated by measuring the move rate per minute for 25 snails. The results for both feedings and moving were statistically analyzed.

DNA extraction protocol:

Total DNA from Paropeas achatinaceum specimen was extracted according to Doyle and Doyle [17]; Gittenberger et al. 2006 [18] and Huelsken et al. [19] and used for the molecular genetic analysis. The specimen was grind and dissolved by incubation at 60ºC for 2 hours in a mixture of 300 µl proteinase K (20 mg/ml) and 500 µl CTAB (Cetyltrimethyl ammonium bromide) buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl; for 10 ml of this buffer 0.4 g polyvinylpyrrolidone and 50 µl of β-mercaptoethanol were added before starting extraction). Five-hundred microliter of 24:1 (chloroform:isoamyl alcohol) was added and mixed well by shaking tubes and then centrifuged at 13000 g for 10 min . The aqueous phase was pipetted and placed into new eppendorf tube. Five-hundred microliter of cold isopropanol was added and left for standing in freezer for 15 min. The extraction then centrifuged at 13000 g for 3 min. Then the liquid was pipetted off and 700 µl of cold 70% ethanol was added. The extraction centrifuged at 13000 g for 1 min and the liquid was poured off. The tube was air dried for 20 min, and the pellet was re-suspended by adding 100 µl of 1X TE buffer (10 mM Tris-HCl, 1 mM EDTA). Four-microliter of RNase A was added to the extraction and incubated at 37 ºC for 1 hour. The DNA quality and quantity were tested by using Nanodrop (Act Gene, USA) and stored at-20 ºC for further use.

Polymerase chain reaction (PCR):

The mtDNA cytochrome c oxidase subunit I (COI) gene was used as DNA barcode and amplified using the polymerase chain reaction (PCR) approach. Primers for COI gene was used depending on Folmer et al. [20], and synthesized in Alpha DNA Company (Canada). Two Primer sequences of the COI gene with approximately 710 bp products size were used. The forward primer (LCO1490) was 5´-GGTCAACAAATCATAAAGATATTGG-3´ and the reverse primer (HC02198) was 5´-TAAACTTCAGGGTGACCAAAAAATCA-3.

AccuPower^® PCR PreMix (Bionner, Korea) was used for amplifiying COI gene by PCR. One-microliter of each primer with 1 µM concentration and 5µl of DNA template with 100 ng/µl concentration were added to PCR PreMix tubes. The final volume for PCR reaction was made up to 20 µl with nuclease-free water. The reaction mixers were placed in thermal cycler (Esco, Singapore). The optimized PCR program was described in Table (1).

The PCR products were resolved on 1.5% agarose gels prepared in 1x TBE buffer (Bioner, Korea). The 100 bp DNA ladder (Bioner, Korea) was loaded on the agarose gel. One-microliter of Bromophenol blue dye was also loaded with the reaction mixer sample. The gel electrophoresis was performed by using 75V for 2 hrs. Ethidium bromide dye (Promega, USA) was used for staining the agarose gel for 30 min. The agarose gel was documented with gel documentation system (Biocom, USA).

Table 1: PCR conditions for detecting of COI gene

Steps	Temperature (ºC) and Cycles		Time
Denature template	95		3 min
Initial denaturation	95	35 Cycles	30 sec
Annealing	45		1 min
Extension	72		45 sec
Final Extension	72		7 min
Incubation	4		5 min

DNA sequencing:

PCR products for amplified COI gene were subject to DNA sequencing. PCR products were purified by using Accu Prep®PCR Purification kit (Bioneer Corp., Korea) before sequencing. DNA sequencing was done by using genetic analyzer 3500 instrument (Applied Biosystems, USA). The used protocol was BDxStdSeq50_POP7_1, BigDye terminator V3.1, which provided by Applied Biosystems.

Statistical analysis:

The data was analyzed by using Statistical Package for Social Sciences ver. 22 (SPSS). LSD test was done for comparing the means of the treatment experiments in comparison with the control. DNA sequencing was analyzed by using two specific programs; Chromas Pro. Version 1.6, 2012, which made by Technelysium Pty Ltd and BioEdit Sequence Alignment Editor [21].

RESULTS AND DISCUSSION:

Morphological Study:

The collected snail has a pale yellow color with small shell (11mm) having eight right convex whorls. The top of the shell was not sharp and the aperture has small oval shape, rounded in the base and pointed shape in the top. The head holds a pair of tentacle and the eyes located at the tip of long stalk (Figure 1).

Figure 1: External feature of the Paropeas achatinaceum

Feeding and Moving of snails:

The natural and experimental soil pH and temperature ranging between 7.4–7.6, and 30–33 ^oC, respectively. In these conditions the animal produced eggs and multiplied, that is the animals is considered under appropriate condition [22], and was found in the number of 28–32 individual in the vicinity of one square meter of its natural soil. Wu et al. [23] found that the optimum thermal level for this animal is ranging between 25.3 and 26.7 ^oC in other parts of the world and this means that the degree of the thermal tolerance of the animal is 25.3–33 ^oC. The mean (X^-±S.E.) of snail weight was 0.034±0.0042g ranging between 0.027 to 0.045g in the natural environment (control), while the mean snail weight was (0.028±0.003) g in the experimental environment with range (0.020–0.033) g. In the other hand, the mean weight average of the Citrus sinensis (L.) roots as snail’s source food supplemented every 12 hours per animal was 0.057±0.008 g with range (0.041 – 0.078) g. The comparison result between snail weights in the nature and experimental environment was not significant (0.497, 95% confidence interval [CI] 0.01209–0.02309) when LSD test was done. Also, the comparison between snail weights in the nature environment and the food mean weight was significant (0.014, 95% confidence interval [CI] 0.00591-0.04109), while the comparison between snail weights in the experimental environment and the food mean weight was significant (0.005, 95% confidence interval [CI] 0.01141-0.04659). Since it feed on the root of plants so it causes a damage on it [24, 25] especially on Citrus, We may know the damage caused by this animal in the long term on agricultural places because we found the animal near the roots of Citrus sinensis, Especially that it was found a year before now in the same place and recorded for the first time by Al-Doori et al. [26]. The results of snail's mean speed rates was 6.225±0.085 cm/min.

MtDNA COI gene amplification and sequencing:

MtDNA COI gene was detecting by using PCR technique with two universal primers (LCO1490 and HC02198). The primer pair successfully amplified a part of this gene from the Paropeas achatinaceum specimen. One band was observed with 710 bp (Figure 2).

Figure 2: Gel electrophoresis for mtDNA COI gene in the Paropeas achatinaceum specimen (Gel electrophoresis was performed by using 1.5% agarose gel concentration at 75 Volts for 2 hours).

The similarity of COI gene sequences of this specimen was observed by alignment with the same gene sequences database which published in NCBI. The sequencing results revealed a high similarity (98%) with the Paropeas achatinaceum (accession number MF415354.1; specimen collected from Sri Lanka), which published in NCBI (Figure 3). The calculated base composition average of these sequences was to be 137 (26.20%) adenine, 203 (38.81%) thiamine, 100 (19.12%) guanine and 83 (15.87%) cytosine. The G+C content was (34.99%), while the A+T content were 65.01%. The sequence data was submitted in the NCBI and the accession number of the COI gene sequences was MG747646.1.

The new classification of organisms are now depends on molecular levels and the genes barcodes are useful for such studies. In Iraq, there are no such studies specially to re-classify the snail at molecular level. Paropeas achatinaceum was founded in different parts in the world, but it’s the first time was recorded in Iraq. For confirming the classification of this genus; studies on morphological and genetic were done. New possibilities of dealing with taxonomic problems were offered by using the application of molecular biology methods [27]. Mitochondrial DNA (mtDNA) offer a powerful tool in molecular phylogenetic of marine organisms [28] little information was available on the amplification success of the COI gene fragment in gastropod tissues, also sequencing of nuclear nucleotides and MtDNA analyses were provides us a basis for taxonomic and phylogenetic considerations [29]. Currently, one of the most widely used genes for systematics, phylogeny and the identification of species is COI [19]. Sequences of cytochrome c oxidase subunit I (COI) and barcode gene, may serve as additional valuable character set for phylogenetic analyses and subsequent identification [30]. The current study revealed some differences in nucleotide sequences in comparison with the same nucleotide sequences for Paropeas achatinaceum which published in NCBI data. This study revealed a good similarity with Paropeas achatinaceum specimen published in NCBI data and these results were confirmed that the collected genus is Paropeas achatinaceum.

CONCLUSIONS:

In conclusion, the Paropeas achatinaceum specimen was first recorded in Iraqi environment, and the comparisons between snail weights in the nature and experimental environments and the food mean weight were significant. The nucleotide sequences of mtDNA COI gene was showed a high similarity with the same genus, which published in NCBI data with accession number MF415354.1. The sequences results confirm that the collected specimen is Paropeas achatinaceum. The sequence data of mtDNA COI gene was submitted in NCBI and the accession number of these sequences is MG747646.1.

DISCLOSURE STATEMENT:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Received on 12.08.2018 Modified on 31.08.2018

Research J. Pharm. and Tech 2018; 11(10): 4267-4271.

DOI: 10.5958/0974-360X.2018.00782.5