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Summer Research Fellowship Programme of India's Science Academies

Haplotype diversity analysis of Achatina fulica from Indian sub-continent using 16S rDNA

Ansil P A

Department of Botany, University of Calicut, Thenhippalam, Malappuram, Kerala 673635

Dr. N A Aravind Madhyastha

Ashoka Trust for Research in Ecology and Environment post, Royal Enclave, Srirampura, Jakkur, Bengaluru, Karnataka 560064

Abstract

Achatina fulica is one among the worst invasive species that occur worldwide and is a threat to agricultural and horticultural crops as well as human health. This terrestrial snail is a native of east Africa and has made its inroad to different regions across the globe through human-mediated introductions. India has also witnessed an outburst of this snail population, especially in southern and Eastern part. The objective of this work is evaluating the haplotypic diversity of the giant African snail in India. We have sampled snails from different parts of India and also made use of secondary data available in GenBank. The haplotypic diversity has been analysed using the 16S rDNA sequences. This work also incorporated the sequence details of other global populations which are already sequenced. The sequences of haplotypes H and I are found similar in our analysis. Haplotype C, which is predominant in India, includes the Bangalore, Bihar, Andhra Pradesh and Odisha populations.

Keywords: invasive species, terrestrial snail, pest

Abbreviations

Abbreviations
COI Cytochrome c oxidase subunit 1 
rDNA Ribosomal DNA 

INTRODUCTION

An invasive species is one that is found in a location to which it is not native and can have adverse economic, environmental, and/or ecological effects on the invaded habitats and bioregions (Ehrenfeld, 2010). These non-indigenous plants or animals can dominate the region and cause harm to the native flora and fauna such as population reduction and extinction. These species can adversely affect the local environmental conditions as well. A common characteristic of successful invader is that they are highly adaptable and that allows them to outcompete native species for resources such as nutrients, light, physical space, water, or food (Ehrenfeld, 2010). This competition can be based on their feeding, micro-habitat preference, high reproduction and in some cases the species can interact more directly in case of parasites. The invasive species can act as vectors in bringing new diseases to native species (Crowl et al., 2008). They can also influence the ecosystem functioning. Invasive species can sometimes hybridize with the rare native species and the harmful effects of hybridization can led to a decline and even extinction of the rare native species (Simberloff, 2009). Even though many of the invasive species cause economic loss, some do offer potential economic benefits as well such as food for animals and humans.

The family Achatinidae, is native to Africa, is represented by about 200 species within 13 genera. Several members attained pest status within African range when the habitat was anthropogenically modified for settlement and agriculture (Raut and Barker, 2009). As part of the increased travel and trade, several Ach atinids were accidentally or purposefully transported to areas outside Africa (Gołdyn et al., 2016). Among them, the most notable is Achatina fulica Bowdich.

Achatina fulica has a narrow and long shell and contains 7 to 9 whorls when fully grown. The shell is generally reddish-brown in colour with weak yellowish vertical markings but the colouration may vary with environmental conditions as well as its diet. A light coffee colour is more common. Fully grown individuals of the species may exceed 20cm in shell length but generally average size is about 5 to 10cm. The average weight of the snail is approximately 32 grams.The life expectancy under captivity is 5-6 years and in wild it goes up to 10 years(Cooling, 2005).

ansil.png
    Global distribution of Achatina fulica

    Achatina fulica, the giant African snail, is considered as the most widely introduced and invasive terrestrial snail species around the world, and the most important land snail pest in agricultural ecosystems (Mead 1979; Karnatak et al. 1998; Raut and Barker, 2002). It is listed in top 100 worst invasive among the world’s worst invasive alien species, by the Global Invasive Species Database (http://www.issg.org/database/welcome/) (Lowe et al. 2000). This snail affects the farms, commercial, and domestic plantations of tropical and subtropical areas. A. fulica is known to eat more than 500 species of plants and also feed on decaying vegetation, dung, garbage, wet paper and cardboard, dead animals, and dead snails of its own kind (Srivastava, 1992). Achatina fulica acts as an intermediate host of the rat lung-worm Angiostrongylus cantonensis, the causative agent of abdominal angiostrongylosis (Chen, 1935) and as vector for a gram-negative bacterium, Aeromonas hydrophila, which causes a wide range of symptoms including severe headaches, nausea, vomiting, neck stiffness, seizures, and neurologic abnormalities (Mead, 1956, 1961; Wallace and Rosen, 1969; Dean et al. 1970; Mead and Palcy, 1992). The populations of A. fulica can reach huge densities in relatively short time and cause menace to humans as they can crawl up the house walls, spread over the sidewalks and there have also been reports of cars skidding on massed crushed snails on roads (Mead, 1961).

    Achatina fulica natural range is between Natal and Mozambique in the south to Kenya and Italian Somaliland in the north (Pilsbry, 1904; Bequaert, 1950). A. fulica now occurs in the southern part of Ethiopia and Somalia, throughout Kenya and Tanzania and into northern Mozambique, Morocco, the Ivory Coast, and in Ghana of West Africa in African continent. The first introduction was from Madagascar to Mauritius as a food source and medicinal commodity happened around 150 years ago. From there it gradually spread east and reached the Seychelles and Re´union by nineteenth century, 1847 in India, Sri Lanka by 1900. It was present at Singapore by 1911 followed by other Malayan regions. In Borneo (Sarawak) it was spotted in 1928 and in Java and Sumatra by 1933. In 1931, it appeared in China and was found in Hong Kong in 1937. It appeared in south-east Asia, Japan, and islands of the western Pacific like Guam, the Marianas, and Palau in the 1920s and 1930s. It reached New Guinea by 1940s (Cowie, 2000). It came to Hawaii in 1936. It was introduced to the mainland United States (Florida) from Hawaii in 1966. There are numerous reasons for the worldwide introduction of Achatina fulica (Cowie and Robinson, 2003). It was introduced as food, pet, and for ornamental purposes and medicinal uses. The shipping of military equipment, agricultural, horticultural, and other commercial products may have also influenced the introduction (Mead, 1961).

    William Henry Benson, a pioneer in Indian Malacology, brought two live individuals of Achatina fulica from Mauritius. Later in April 1847, his friend released them in his home garden at Chowringhee in West Bengal. Although the initial spread was slow (Blanford, 1868), this innocent presented India with a significant agricultural pest that persists to this day. Oliver Collett introduced Achatina fulica in Sri Lanka around 1900 in his garden on a tea estate at Watawella. They could not flourish on the hills due to the cold climate. Eventhough Collett tried to destroy them, the survived ones initiated a colony at Beruwala, where the conditions were congenial for an optimum growth. Abundant populations of A. fulica in Nepal’s eastern urban areas were observed by Raut (1999). According to him the establishment might have happened 60-70 years ago. According to the study by Budha and Naggs (2008), A. fulica made its entry through the southern part of eastern Nepal and then spread to western limits. But they were unable to cross the Kathmandu valley because of the higher elevation and cold climate.

    The objective of the present study is to look for different haplotypes of Achatina fulica in the Indian subcontinent. The pioneering work studying the haplotype diversity was done by Fontanilla et al.,(2014). But the sampling was only from Maharashtra. Subsequent studies by Ayyagari and Sreerama (2017) included samples from few more states such as Bihar, Odisha, Karnataka and Andhra Pradesh. However, sampling from Kolkata, West Bengal, where the introduction happened in the first place was not done. For this study we have collected samples from localities of Tamil Nadu, Karnataka, Andhra Pradesh, Odisha, West Bengal, and Manipur. The molecular markers used are 16s rDNA and the cytochrome c oxidase subunit 1 (COI) gene from the mitochondrial genome. The haplotype diversity is identified and the haplotype networks have been developed by comparing the sequence of these markers with that of other global as well as Indian populations collected from the literature. Using the haplotype networks, the variation among the collected population is tested and the possibility of multiple introduction also is checked upon.

    LITERATURE REVIEW

    Distribution

    The haplotypic analysis of Achatina fulica has only been done in the recent times and hence understanding on the subject is limited. However, the distributional status and feeding behaviours of the species were mostly studied. According to Albuquerque et al., (2008) The abundance and distribution of A. fulica in Brazil was most representative in urban area, mainly near to the coastline. A. fulica started their activity at the end of the evening and stopped in mid-morning. Their preferred food included plants such as Hibiscus syriacus, Ricinus communis, Carica papaya, Galinsonga coccinea, Lippia alba, Ixora coccinea, Musa parasidisiaca, Mentha spicata and Cymbopogon citrate. Hayes and Meyer (2009) observed Achatina fulica preying on Veronicellid slugs in Hawaii and estimate that A. fulica may pose a greater threat to terrestrial mollusc conservation than previously imagined. Thiengo et al., (2007) also states that Achatina fulica occurs in dense populations in urban areas where it is a pest in garden plants, vegetable gardens, and small-scale agriculture. It can also act as an intermediate host of Angiostrongylus cantonensis, a nematode that can cause meningoencephalitis in people, and it may be a potential host of A. costaricensis, which causes abdominal angiostrongylosis. The most threatened and infested areas were the coastal and Amazonian regions of the Ecuador (Goldynet al.2016). According to the reports from government agencies, A. fulica most often affected cocoa and banana plantations, but was also known to forage on host of other cultivated plant species. Buddha and Naggs (2008) reported the spread of African giant snail in Nepal and the consequences as well as the eradication measures taken. The distribution of Achatina fulica in the Indian subcontinent was first reported by the studies of Godwin-Austen (1908). He depicts the tale of arrival of the snail species to India through William Benson. According to him several individuals of the species were collected along a railway line at Rajmahal which is around 170 km away from Kolkata.

    Sridhar, Vinesh and Jayashankar (2014) mapped the potential distribution of A. fulica the Majorly prone areas fall in parts of Rajasthan, Gujarat, Karnataka, Andhra Pradesh, Kerala and Tamil Nadu and in minor levels at Himachal Pradesh, Uttarakhand, Jammu and Kashmir, parts of North-Eastern India. Sarma, Munsi and Aravind (2015) studied the Effect of Climate Change on Invasion Risk of Giant African Snail using ecological niche modelling approach. The results indicated high risk of invasion in Eastern India, peninsular India and the Andaman and Nicobar Islands and currently invaded areas including parts of Bihar, Southern Karnataka, parts of Gujarat and Assam would be more prone to future invasion. The work also states that the understanding of the invasion pattern could help in better management of this invasive species. The occurrence of the snail in various coffee growing areas of Karnataka, India was recorded by Kumar et al., (2018).

    Genetic Analysis

    The genetic analysis of Achatina fulica in Cameroon is done by Woogeng et al., (2017) and he arrives at a conclusion that the diversity in the introduced species was much lower, which is likely due to introduction of limited numbers. The extent of genetic diversity of the snail in global population was done by Fontanilla et al., (2014). They used mitochondrial 16S ribosomal RNA gene and a total of 560 individuals from 39 populations were taken into consideration. Results reveal that out of 18 distinct haplotypes; 14 are found in East Africa and the Indian Ocean islands, but only two haplotypes from the Indian Ocean islands emerged from this region, the C haplotype, now distributed across the tropics, and the D haplotype in Ecuador and Bolivia. Haplotype E from the Philippines, F from New Caledonia and Barbados, O from India and Q from Ecuador are variants of the emergent C haplotype. For the non-native populations, the lack of genetic variation points to founder effects due to the lack of multiple introductions from the native range. The study suggests Indian Ocean islands and East African regions as the earliest known common source of the species.

    In India, the evaluation of haplotype diversity of the species was done by Ayyagari and Sreerama (2017) using 16S ribosome RNA gene and the phylogenetic relationship of Indian haplotypes with other global populations were also studied. According to them, haplotype ‘C’ was the predominant and a new haplotype ‘S’ from the state of Odisha was observed. The other observations by them include the association of haplotype S with the haplotype H of Mauritius which indicate the possibility of multiple introductions.

    MATERIALS AND METHODS

    For this work, different populations of Achatina fulica from Kolkata, Assam, Manipur, Odisha, Andhra Pradesh and Karnataka were used.

    lissachatina_fulica_madagascar_light_juvenil_01big.jpg
      Achatina fulica adult​ Source : (https://landsnails.org/en/Sale/Snails/Achatinidae/Lissachatina/Lissachatina_fulica/Lissachatina_fulica_fulica/Lissachatina_fulica_Madagascar_light)​

      Systematic Position of the Species

      Kingdom : Animalia

      Phylum : Mollusca

      Class : Gastropoda

      Order : Styllomatophora

      Family : Achatinida

      Genus : Achatina

      Species: Achatina fulica Bowdich

      Equipment and Chemicals

      The materials and equipment include Petri plates, Forceps, Absolute Ethanol, Micro pipettes, Distilled water, Tissue papers, Surgical blade, 1.5mL and 2mL centrifuge tubes, CD –markers, Refrigerator, thermoshaker, Water bath, Metallic beads, Bead beater, Centrifuge, Vortex mixer, Tube rotator, Laminar air flow cabinet, Nanodrop spectrophotometer, C-TAB solution, β-mercaptoethanol, Tris EDTA buffer, Chloroform-isoamyl alcohol, Sodium acetate

      Tissue Preservation

      Small amount of the tissue was taken from each sample and kept in absolute ethanol under refrigeration.

      Softening of Tissues

      The tissue samples are blotted to remove the absolute ethanol and kept in 400μL of Tris EDTA buffer. The tubes are then kept in thermoshaker for one hour under 25°C at 400rpm.

      Tissue Digestion

      A 0.2% solution of β-mercaptoethanol in C-TAB is made and it is kept in waterbath at 60°C. The tissue is then blotted to remove the Tris EDTA buffer and is then transferred to a 2mL tube filled with 400μL of 0.2% β-mercaptoethanol in CTAB solution. 20μL of Proteinase enzyme is added to the tubes followed by the metallic beads and are subjected to bead-beating with a frequency of 15/min for 1 minute. The tubes are then subjected to a short centrifugation and are kept for waterbath at 60°C overnight.

      DNA Extraction

      To the sample tubes, 400μL of chloroform-isoamyl alcohol is added and is mixed in vortex followed by centrifugation for 5 minutes under 4°C at 12,000rpm. The supernatant is then collected to another 2mL tube and again 400μL of chloroform-isoamyl alcohol is added, mixed by vortex and is centrifuged for 5 minutes. This step is repeated once more for increasing purity. The supernatant is collected in 1.5mL tubes followed by the addition of 40μL sodium acetate and 800μL of chilled absolute ethanol. The contents are kept in tube rotator for 10 minutes under 25rpm to ensure proper mixing. The tubes are then centrifuged for 10 minutes under 4°C at 12,000rpm. DNA pellets can be observed at the bottom of the tube.

      For the purification of the DNA pellets thus formed, after decanting the liquid phase except the pellet, chilled 70% ethanol is added to the tubes followed by centrifugation under 4°C at 12,000rpm for 10 minutes. The ethanol is then removed from the tube and the tubes are kept open under laminar airflow for drying. After the tubes are dry, 50μL of Tris EDTA buffer is added and the tubes are kept under -20°C after vortexing so that the pellet gets dissolved in the buffer.

      DNA Amplification

      DNA sequences are amplified exponentially using PCR and is doneto generate a large number of copies of a particular DNA segment within a short span of time. The main reagents which are employed for the PCR are forward and reverse primers, DNA polymerase enzyme, MgCl2 and the dNTPs (deoxyribonucleotide triphosphates).

      During the process of denaturation, the DNA double helix is physically separated to individual strands and this process occurs at a high temperature. In the second step called annealing, the temperature is decreased and the primers attach to the complementary sequences of DNA. The DNA polymerase enzyme then assembles a complimentary new DNA strand from the free dNTPs. During further increase of temperature, the newly formed DNA fragment undergoes denaturation and act as template for the synthesis of newer complimentary strands. This process then repeats to produce multiple copies of the desired DNA fragment at an exponential rate. After ‘n’ cycles, PCR generates a 2n-fold increase in the target DNA.

      The PCR mastermix is a solution containing combinations of premixed PCR chemicals. It normally contains all the components needed for the smooth running of the process. It includes, the buffer, dNTPs, a thermostable DNA polymerase enzyme, primers (forward and reverse), DNA template and enough double distilled water to make upto the required volume. The buffer in the mixture creates optimal pH for the proper activity of the polymerase. It also contains Mg2+ ions which act as the cofactor for the polymerase. The PCR primers provide a “free” 3’-OH group to which the DNA polymerase sequentially add the dNTPs. The MgCl2 concentration influences the stringency of the primer- template interaction. Low MgCl2 concentrations can help to eliminate non-specific priming when looking for high fidelity DNA. However very less concentration of Mg2+ ions result in a low PCR yield. Even if high concentrations of MgCl2 help to stabilize the primer-template interaction, the very high Mgcl2 can also result in non-specific binding of primers and lead to unwanted PCR output. Here the enzyme used is the Taq polymerase which is a thermostable high fidelity DNA polymerase. Higher Taq concentrations may cause non-specific amplification. In the presence of inhibitors in the DNA samples or the reaction mix, higher amounts of Taq may be needed for a better amplification. QIAGEN® PCR Buffer comes along with the QIAGEN® amplification kit minimizes the requirement for optimization of the PCR mix.

      For the amplification of the 16S rDNA, the primers, 16Sar-L 5’-CGCCTGTTTATCAAAAACAT- 3’and 16Sbr-H 5’- CCGGTCTGAACTCAGATCACGT- 3’and the QIAGEN PCR chemicals were used for a final PCR volume of 12.5μL. The master mix consisted of 1.25 μL of 10X PCR buffer, 0.5 μL of 10mM dNTPs, 2.5 μL of Q solution, 0.125 μL of 10 μM forward and reverse primers, 1 μL of 25mM Mgcl2, 0.25 μL of Taq pol and 5.75 μL of molecular biology water. Negative control for the reaction was also used. PCR was carried out in a thermal cycler (Eppendorf, Germany) with the following cycling conditions: 95 ° C for 3 min, followed by 40 cycles of 94 ° C for 45 s, 50 ° C for 45 s and 72 ° C for 2 min and a final extension at 72 ° C for 10 min followed by an infinite hold at 4°C.

      Amplicon Visualization and Gel Documentation

      The amplicons were visualized using agarose gel electrophoresis. Here DNA based on their size and negative charge will travel through the gel at different speeds, towards the positive electrode allowing them to be separated from one another. The success of the amplification is assessed by comparing the amplicon bands to a standard "ladder DNA" made up of DNA fragments of known sizes. The 2% gel is casted using 1X Tris-Acetate EDTA buffer with 1μL of 10,000X Gel red per 10 mL of the agarose solution. The Tris-Acetate EDTA buffer is also used as the electrolyte. The process of electrophoresis is carried out for 40 minutes under 70V potential. While the amplicons move through the gel, The GelRed®nucleic acid stain intercalates with the DNA and will fluoresce when exposed to UV light. The bands obtained were visualized using gel documentation system (VILBER, Germany).

      07.05.2019 Achatina 16S.JPG
        Gel visualisation showing formation of primer dimmers

        Sequence Analysis and Haplotype Networking

        Since the conditions for 16S rDNA amplification could not be standardized owing to the limited time of the project, we have collected the 16S rDNA sequences of A. fulica from NCBI GenBankTM for the construction of haplotypic network and study the relationships between different haplotypes described from India and different countries of the world so far. Multiple sequence alignment was performed in MEGA v 10 (S.Kumar et al., 1994 ) using the inbuilt MUSCLE (Edgar, 2004) alignment programme using the default programme parameters. The aligned sequences were exported to FASTA file format using the same software, Selection of conserved blocks from alignments was done using GBlocks (Castresena Lab) and linear ALTER (Daniel Glez et al., 2010). The file is then subjected to Median joining network calculation using the Network 4.6 (Röhl, Bandelt and Forster, 1999) software in default parameters and saved as ‘.out’ file format which is then used to draw the haplotype network. The haplotype network is also drawn using the same Network software and is exported to ‘.bmp’ format.

        RESULTS

        The standardisation of amplification protocol of the 16S rDNA was unsuccessful for most of the samples, and the sequencing and the haplotyping of the snails from different part of the country could not be completed given the very limited amount of time. The current PCR conditions and master mix compositions resulted in smears rather than discrete bands. Haplotype analysis was carried out using secondary data downloaded from GenBank. The studies conducted in the haplotype diversity of Achatina fulicain India are very less and sampling and sequencing from all parts of India is needed to derive a conclusion regarding the path of introduction and distribution of the snail in the country.

        Network Analysis

        16S haplotypes edited.jpg
          Haplotype network of Achatina fulica populations

          The median joining network calculations are done and the haplotype network is generated using the NETWORK v4.6. The sequences of haplotypes H from Mauritius and I from Tanzania are found similar in our analysis and are hence represented as haplotype H in the network. From the generated network it is evident that haplotype C which is predominant in India include the Bangalore, Bihar, Andhra Pradesh and Odisha populations and the haplotypes P, Q, N, O, F and D differ by a single nucleotide change. Similarly, haplotypes A, B, G, O are different from the haplotype D by a single nucleotide change and the haplotypes K, R, J, N, M Tanzania by two such changes. Haplotype L from Tanzania is the most distant from haplotype D of Ecuador with 7 base changes.

          DISCUSSION

          According to the studies done by Fontanilla et al. (2014) India harbours haplotype C and P of the land snail Achatina fulica. Haplotype S was further added to the list according to investigation by Ayyagari and Sreerama (2017).

          It is obvious that the African subcontinent consist of much diverse haplotypes than any other part of the world. Being a native of the Eastern Africa, Achatina fulica reached different parts of the world due the process of introduction mediated by humans, which can be either accidental or intentional (food, animal feed and pet trade). Since the introductions happened in a relatively small course of time in comparison with the evolutionary process (Raut and Baker, 2002), the different haplotypes present in a region should be due to introduction rather than changes due to evolution. The higher number of haplotypes in the Madagascar and Mayotte regions (Fontanilla et al., 2014) is probably due to multiple introductions (Ayyagari and Sreerama, 2017). Since no fossil evidences have been being obtained from Madagascar, Mauritius and Mayotte, It suggests Eastern Africa to be the place of origin of Achatina fulica (Raut and Baker, 2002). From Africa, A. fulica was introduced to Madagascar before 1800 (Raut and Baker, 2002). The comparatively low haplotypic diversity in other parts of the world including India points to the less frequency of introductions to these regions. However, more populations from Indian region especially from the sea port regions where the invasion of giant African snails are high could give better picture regarding the introduction events in India. The emergence of newer haplotypes at different regions outside Africa can be due to the course of evolution.

          More sampling from Africa is required to trace back the point of origin of haplotype C which is now predominant in India (Ayyagari and Sreerama, 2017). As the story goes, two individuals were introduced to Kolkata from Mauritius, and the haplotypes of those remain unknown because of the lack of sampling from Kolkata. There are possibilities that those individuals could be haplotype C or any other haplotype from which the present day C haplotype emerged (Ayyagari and Sreerama, 2017). Since the haplotype F present in Barbados and D of Ecuador possess only one mutational variation from that of the haplotype C in India, there can be a possibility of introduction from India to these places or vice versa. The haplotypes A, B, G of Mayotte and O of Uganda is different from the haplotype D of Ecuador by only one base change and the haplotypes K, R, J, N, M and L of Tanzania (East Africa) by only two base changes suggesting the introduction of the snail to the Tanzanian region from Ecuador. In this analysis, the Tanzanian haplotypes M and I present in the literature were found similar. The haplotypes P and S of India could be emerged from haplotype C. However extensive sampling is needed from inside and outside India to derive a clear picture regarding the possibility of evolutionary emergence of newer haplotypes and multiple introductions.While the snail is terrestrial, the absence of strict quarantine can be the reason for the transfer of these snails across countries and the porous borders. The spread of Achatina fulica within the country can be controlled by creating awareness among the general public regarding the harmful effects these snails can have on agriculture as well as human health. The changes in climatic patterns within the country increases the chances for the infection of new areas by the species (Sarma, et al. 2015). This work is intended to provide information regarding the haplotypic diversity of A. fulica population in the country.

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          ACKNOWLEDGEMENTS

          My sincerest gratitude to the following personalities who made my work smooth.

          Dr. N A Aravind Madhyastha, my guide, for accepting my proposal, giving me the great opportunity to work in his lab, for all the guidance regarding my work and for making effort arrange my stay near the institution. Sudeshna Chakraborty, my supervisor, for teaching me a lot of things, from DNA extraction, amplification to network building and for all fruitful discussions together. Also for helping me with my writing part and for being patient with me.

          Nipu Kumar Das and Biswa Bhusana Mahapatra, for their cooperation and advice on various aspects of my work as well as my time in Bangalore.

          The Ashoka Trust for Research in Ecology and Environment (ATREE) for their facilities and cooperation during my work.

          All the collectors for generously providing me with Achatina specimens to work on.

          Bipin Charles, Poorna Bhat, Madhushree Mudke, Anushree Jadhav, Dr Maitreya Sil, Richa, Abishikta, Pallavi and Shubhanki for all the good times together;

          Aswaj, Dr. Priyadarshanan, Dr. Bijoy, Dr. Seena, Ramesh for helping me find an accomodation nearby.

          I also express my sincere gratitude to the ATREE-team for helping to complete my work successfully.

          I take this opportunity to sincerely acknowledge the Indian academy of sciences (IAS) for selecting me as summer research fellow and giving this golden opportunity do this wonderful summer project and also for providing necessary infrastructure, resources and fellowship.

          And above all, My family, for their effort to send me there, financial support and understanding.

          Source

          • Fig 1: OSGeo4W
          • Fig 2: (https://landsnails.org/en/Sale/Snails/Achatinidae/Lissachatina/Lissachatina_fulica/Lissachatina_fulica_fulica/Lissachatina_fulica_Madagascar_light)​
          • Fig 4: Network 4.6
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