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Evaluation of Genetic Diversity of Golden Apple Snail, Pomacea Canaliculata

ABSTRACT Genetics is a trend these days especially that, DNA barcoding has been developed.DNA barcoding is an important tool in categorizing the taxa of different species and it tells so much about the species’ traits, including genetic diversity.The Pomacea canaliculata was introduced in different parts of Asia and had been an invasive species and a pest in different ecosystems ever since the introduction.

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In understanding this species of snails, samples were collected, DNA’s were extracted, undergone PCR and electrophoresis, and was sequenced and analyzed.

The analysis was qualitative In the Philippines while quantitative in China. In the Philippines, the cytochrome oxidase subunit 1 (COI) genes was used and compared among the species collected and when it was sequenced, it showed differences due to localized gap, mismatch and non-correspondence of bases. And in China, the diversity was analyzed by means of Nei’s gene diversity, Shannon’s information index, percentage of polymorphic bands (PPB) an AMOVA anlysis. Both the quantitative and qualitative showed that there was diversification within and among the populations of these snails.

INTRODUCTION Genetics is the study of the genes, and the heredity and variation of individuals. Understanding the genome, which is the complete set of chromosomes or the entire genotype of an individual, is important because it helps in the taxonomy of species, especially nowadays wherein advancements in science needs more specific information, and that basing on morphological features is not enough. Life is specified by genomes which contain all the biological information which is encoded in its deoxyribonucleic acid (DNA) and divided into units or the genes.

The genes are the blueprint for life because it is the particulate determiner of hereditary traits. Hence, DNA barcoding became a trend for scientists and researchers for the understanding of the different variations in the traits of different organisms. The golden apple snails (Pomacea canaliculata) originated from the South America, Central America, the West Indies and the Southern USA (Pain 1972) and was spread in the past decades to the different parts of Southeast Asia, namely Philippines, China, Thailand, Cambodia, Hong Kong, Indonesia and Japan.

The introduction of the P. canaliculata without prior studies caused damages to the different plants and it became an invasive species which resulted to becoming pests to humans and competitors to other local snails, example of which are those from the genus Pili. The P. canaliculata was observed to have different growth and reproduction in different parts of Asia, together with their external characteristics due to the different habitats and environmental conditions (Keawjam, 1986 and 1987), therefore there is the possibility to misidentify two sympatric species as one.

On the other hand allopatric populations inhabiting different habitats may show ecomorphological variations and questionable species status and it was also suggested that the golden apple snails had high adaptability hence it was easier for them to form new populations (Dong et al. 2011). The understanding of the genomes of different species is a trend for scientists these days but the information about the different mollusks is still limited. The basic information on the number of species and/or population is of help for conservation programs (Carvalho and Hauser, 1994) and for building appropriate management schemes.

In contribution, the studies aims are to evaluate the genetic diversity of the golden apple snail population in Asia, namely, Philippines and China via molecularly characterizing the P. canaliculata and to find different ways of analyzing the gathered data from the sequenced DNA of the said species. REVIEW OF RELATED LITERATURE Genetic diversity (Reed 2005) The significance of genetic diversity arose from two necessities: genetic diversity is required for populations to evolve in response to environmental changes and heterozygosity levels are linked directly to reduce population fitness via inbreeding depression.

The amount of genetic variation a population contains is predicted to correlate with current fitness and, in the case of heritabilities (which can remain high or even increase despite severe reductions in population size) with evolutionary potential. This correlation between fitness and levels of genetic variation, however, may be weak or nonexistent due to the neutrality of molecular markers used in estimating heterozygosity, nonadditive genetic variation and the purging of deleterious alleles because of increased selection against homozygotes.

There is a body of literature that suggests that allozyme heterozygosity is a good measure of population fitness and adaptive potential. Others caution though that such molecular genetic data generally reflect only a small portion of genome and thus may not be indicator of adaptive genetic differences. But molecular markers may be useful for assessing the extent of genetic drift. Moreover, deleterious alleles, in mutation-selection balance, are responsible for at least half of the genetic variation in fitness.

Selection has the tendency to purge the population of the deleterious recessive alleles which in theory creates inbred populations with a higher fitness than their outbreed progenitor. In other words, inbred populations with less genetic diversity would have higher fitness if the population is not kept small enough for a long enough to allow the fixation of deleterious alleles to occur. Fitness and future adaptability are reduced in smaller populations of plants and animals due to drift and inbreeding depression.

Commonly used surrogates for fitness such as heritabilities, heterozygosity, and population size, significantly correlate with fitness and explain 15-20 % of the variation in fitness. Correlations suggest that many populations have reduced fitness as a result of inbreeding depression and genetic drift. There is much fuss and concern thus, over genetic variation because of the fact that endangered species typically have lower levels of heterozygosity and the loss of adaptive genetic variation and inbreeding depression puts wildlife populations at an increased risk of extinction.

Finally, this increase occurs as a result of the reduction of productive fitness because of inbreeding depression or due to the failure of tracking the change in abiotic and biotic environment of the population as a result of the loss of genetic variation through drift.

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DNA barcoding (Moritz & Cicero 2004) At the very core, the purpose of DNA barcoding is for large scale screening of one or a few reference genes in order to assign unknown individuals to species and enhance discovery of new species.

In the hope of developing a comprehensive database of sequences that will serve as a comparison tool to sequences from sampled individuals, proponents used DNA barcoding. There is, however, nothing new with DNA barcoding as it is an offshoot of the use molecular markers for the very same purpose except, in DNA barcoding, there is an increased scale and proposed standardization. The selection of one or more reference genes characterizes standardization, with regards to microbial community and in stimulating large scale phylogenetic analyses if of proven value, though whether or not one gene fits all remain to be a question.

Presently, most methods of DNA barcoding are tree-based and can fall into two broadly defined classes. One class is the distance-based, wherein it is based on the degree of DNA sequence variation within and between species. This kind of approach converts DNA sequences into genetic distances and then uses these distances to establish identification schemes. It further defines a similarity threshold below which a DNA barcode is assigned to a known or a new species. There is also the mention by several authors of a “barcoding gap”, a distance-gap between intra- and interspecific sequences, for species identification.

However, the distance-based approach seems to be ill suited as a general means for species identification and the discovery of new species. One reason is that substitution rates of mitochondrion DNA vary between and within species and between different groups of species. The varied substitution rates can result in broad overlaps of intra- and interspecific distances, and hinder the accurate assignment of query sequences. Another class which is the monophyly-based requires the recovery of species as discrete clades (monophyly) on a phylogenetic tree and is used to assign unknown taxa to a known or new species.

Similarly, some issues complicate the use of monophyly in a barcoding framework. For example, the long-recognized problem of incomplete lineage sorting will yield gene genealogies that may differ in topology from locus to locus. The recently divergent taxa may not be reciprocally monophyletic due to lack of time needed to coalesce. In addition, the gene trees are not necessarily congruent with species trees, and the monophyly, while a discrete criterion is arbitrary with respect to taxonomic level.

Moreover, there is a recently applied new technique that has been proposed as an alternative to tree-based approaches for DNA barcoding, the so called character-based DNA barcode method, which is based on the fundamental concept that members of a given taxonomic group share attributes that are absent from comparable groups. It is the kind of method that characterizes species through a unique combination of diagnostic characters rather than genetic distances. The four standard nucleotides (A,T,C,G) if found in fixed states in one species can be used as diagnostics for identifying that species.

This way, species boundaries can be defined by a diagnostic set of characters which can be increased to any level of resolution by applying multiple genes. Presently, character-based DNA barcode method has been proved useful for species identification and discovery of several taxa. In the view that single-gene sequence should be the primary identifier of species, a contention arises that if that is the case then there’ll be a real need to connect different life history stages and increase the precision and efficiency of field studies involving diverse and difficult-to-identify taxa.

Although the DNA barcoding community has put emphasis on the importance of large-scale sequence database within the existing framework and practice of systematics, it should be bore in mind that DNA barcoding is not the primary answer in resolving the tree of life. Furthermore, as much as the term “DNA barcoding” appealing, it implies, however, that each species has a fixed and invariant characteristic. But this kind of implication renders unrest to the minds of evolutionary biologists.

In evaluating thus, the promise and pitfall of DNA barcoding, two areas of application should be distinguished: the molecular diagnostics of individuals relative to described taxa and DNA-led discovery of new species. And although there is little doubt that large-scale and standardized sequencing, when integrated with existing taxonomic practice, can contribute significantly to the challenges of identifying individuals and increasing the rate of discovering biological diversity as presented by this study, the general utility of DNA barcoding still requires further scrutiny.

PCR (Moore 2005) In rapidly copying a selected template sequence from a DNA mixture in vitro, PCR offers a wide range of applications such as sequence detection and isolation for research, forensics and species identification through the PCR itself and in combination with other techniques. PCR’s new technique uses flourescent probes to monitor the amounted product at end of every cycle and PCR machines look for the cycle at which the can readily detect flourescence.

PCR is also being used to monitor RNA through the addition of reverse transcriptase enzyme at the beginning to generate DNA template. In addition, there are now new applications of PCR like single nucleotide polymorphism detection and screening. Cytochrome Oxydase subunit 1 (COI) (Buhay 2009) COI plays a significant role in documenting biodiversity and remains to be the choice for phylogenetic and phylogeographic studies. COI is a mitochondrial protein-coding gene which is a widely accepted marker for molecular identification across diverse taxa.

Mitochondrial DNA (mtDNA) have a relatively fast mutation rate, thus they result in significant differences between species. With respect to this, the mitochondrial cytochrome c oxidase subunit I (COI) gene with ~700bp was proposed to be a potential barcode or marker for molecular identification across various taxa. Furthermore, COI is a protein coding gene that has an open reading frame and in thecase of barcoding, COI can be highly divergent from the actual COI sequences which may cause major problems because species identification is based on sequence similarity.

Pomacea canaliculata (Cowie 2002) The Pomacea canaliculata belongs to the family Ampullariidae. Its structure appears to have a slight dimorphism in shape of aperture and operculum. Females have broader mouth and concave operculum while convex in male. In terms of reproduction, oviposition often takes place at night or at early morning or evening about 24 hrs after copulation up to two weeks after mating (occurs three times per week) which occurs anytime of the day or night although there may be some diurnal rhythm.

On each oviposition occasion a single clutch is laid of highly variable egg number. Moreover, the interval among successive ovipositions for p. canaliculata has been reported to be about five days and hatching generally takes place about two weeks after oviposition. The P. canaliculata breeds only during summer and grows into maturity in less than two months. P. canaliculata is said to be prolific and hence has rapid succession of generations which leads to rapid population expansion.

They relatively inhabit still water and in water temperatures above 32 degree Celsius, it has been observed that the mortality of p. canaliculata is high. Whereas in low temperature p. canaliculata can survive 15-20 days at 0 degree Celsius, 2 days at -3 degree Celsius but only 6 hrs at -6 degree Celsius. And it is sufficiently tolerant of sea water to survive long enough to be carried by currents from one stream mouth to another, thereby expanding its distribution. P. canaliculata shows preferences among food plants.

Its rate of growth has a direct correlation with its feeding on the preferred plant. Moreover, it is able to detect its food plants from some distance using chemical cues in the water. P. canaliculata, however, appears to be relatively generalist and indiscriminate that it is viewed to be particularly voracious compared to other Ampullariids. METHODOLOGY Sampling Snail samples were identified and collected from 2 countries in Asia, specifically in the Philippines and in China, where the P. canaliculata was introduced. In the Philippines; Los Banos (Dong et al. 011, p. 1778), 2 barangays in Tarlac (Brgy. Cabayaoasan, Paniqui and Brgy. Pance, Ramos) and Iloilo (Chichoco & Patdu 2012, p13), 44 snail samples were collected. And in China, specifically from Yuyao and Taizhou in Zhejiang province, Fuzhou in Fujian province, Guangzhou in Guangdong province, Nanning in Guangxi province, Kunming in Yunnan province, wherein a total of 120 samples were identified with the conserved sequence by Matsukura et al. (2008) and Pan et al. (2009) and then was collected (Dong et al. 2011, p. 1778).

The snails were then stored, either by wrapping in paper, freezing or preserving it in ethanol, and brought into the respective labs in each country for the next steps; DNA extraction, PCR, electrophoresis and sequencing. DNA extraction The two studies used the phenol-chloroform method (Bergallo et al. 2006) with an alternative of the Qiagen’s Dneasy extraction kit for China. The DNA concentration was determined spectrophotometrically and adjusted by a mini-gel method (Maniatis et al. , 1982) when the extracted DNA was enough, it was stored at 4oC to -20oC until needed. PCR and Electrophoresis

The PCR method was basically done by choosing the right primers that will yield clearly reproduced bands and they tested the proper amounts and amplification effects of the components of PCR, which were the Mg2+, dNTP’s, DNA templates and polymerase, and the primers. After the mixture of the components and the DNA extracted, it was carried out in the thermocycler programmed for pre-denaturing at 94°C for 3 min, followed by 26 cycles of 94°C for 10-30s, 36-52°C for 30-45s, extension of 65-72°C for 60-90s, and the final extension for 5-7mins at 72°C for final extension with 38-48 cycles.

After which, the amplified products together with negative controls were run in electrophoresis to be separated and tested for contaminations, respectively. The products were then purified later on with the respective kits present in each lab. In the Philippines, the reaction was done with 2? L MgCl2, 5? L PCR buffer, 1? L dNTP, 2. 5 ? L of the primers, which were the LCO1490 and HCO2198, distilled H2O with 22. 75 ? L, 0. 25 Taq, and 10 ? L Q-buffer. The electrophoresis was done after the ethidium bromide staining (Maniatis et al. , 1982), analyzed through 1. % agarose gels and visualized under a transilluminator. In China, they made use of the ISSR-PCR analysis where they got four primers, which produced clearly reproduced bands, out of the 90 that was screened from the University of British Columbia’s primer set and the reactions were done with a volume of 20 ? l, containing 0. 2 mM of each dNTP, 1. 5 mM MgCl2, 0. 5 ? M primers, 1 U Taq polymerase and 10 ng DNA template, and also with the determination of the optimal reaction system of ISSR for P. canaliculata (Dong et al. 2011, p. 1779).

The products’ sizes after the amplification was estimated using DNA marker DL2000 and then was run in electrophoresis, which was done on 6% polyacrylamide gels, visualized by silver staining and then photographed (Li et al. , 2009). Sequencing/ Data analysis Chichioco and Patdu (2012) sent the DNA samples to the First Base Laboratory in Singapore for sequencing and the results were sent back to the DNA barcoding Laboratory after a week. The COI sequences were aligned in the BLAST, specifically the STADEN package version 1. 5. 3 and Bioedit Sequence Alignment Editor version 7. 0. 9. 0.

Aside from the sequences sampled, other sequences and their haplotypes from the GenBank were also compared and matched. In Dong’s (2011) research, he made use of the RAPD fragments by labeling them into binary matrices, used them to get the similarity index, Sxy = 2nxy / nx+ ny, where nx and ny represent the number of RAPD bands in individuals x and y, and nxy represents the number of shared bands between individuals, as stated by Nei and Li (1979), then averaging it across all the possible comparisons between individuals within a geographic sample to get the within samples similarity (Si).

Between sample similarity corrected by within sample similarity Si and Sj of geographic samples i and j, respectively) is also calculated between pairs of individuals across samples i and j using the equation; S’ij = 1 + Sij – (Si and Sj)/2. Genetic distance between paired samples was then calculated as D’ij = 1- S’ij (Lynch, 1990). RESULTS AND DISCUSSION In the Philippines (Chichioco & Patdu 2012, p. 18-31) The collected samples from Brgy. Cabayaoasan were found in the elevated parts of a rice paddy, specifically, it was a muddy substrate with decaying leaves from the rice plants and surrounding trees while those that was found in Brgy.

Pance was in the muddy bottom of the shallow fish pond in the roots of water lilies and grasses. The samples from the two barangays in Tarlac and Iloilo had relatively different colors and sizes. Those that were collected from Brgy. Cabayaoasan had the largest size and they are colored black while those in Brgy. Pance had brown in color and still, those in Iloilo had very small sized specimens and some of the specimens can be mistaken as Pila conica snails if not examined properly. Primers affect the amplification success greatly, since according to Hajibabaei (2005) a 95% success is necessary for barcoding.

The primers LCO1490 (SENSE) and HCO2198 are generally used for the amplification of forward and reverse fragments from COI genes. The DNA samples were subjected to the PCR and agarose gel electrophoresis (AGE), and they produced single discrete bands that suggest that the fragments were homogenous and start and end at the same point (Reece 2004). The bands that were brighter and distinct are more appropriate for sequencing because it means that the DNA fragments were well amplified. The best DNA’s were chosen and forwarded to the First Base Laboratory in Singapore for sequencing.

At the return of the results, other sequenced DNA barcodes were also collected and was aligned and compared with the Basic Local Alignment Search Tool (BLAST) database. Fig 1. Alignment of the COI gene sequences of the Pomacea canaliculata (CPT1-5 from Brgy. Cabayaoasan, PRT 7,9,10 from Brgy. Pance, IICK & IIPC1,3 from Iloilo) from the Philippines using Bioedit Sequence Alignment and ClustalW multiple Alignment (Chichioco & Patdu 2012, p. 26) By aligning the sequenced data, it can be seen that there are both similarities and differences among the genetic make-up of the samples.

The differences are due to localized gap, mismatch and non-correspondence of bases along the COI fragments as pointed out in fig. 2 Fig 2. Comparisons of the COI sequences of the P. canaliculata samples from 35bp- 120bp (Chichioco & Patdu 2012, p. 28) As emphasized in fig. 2, on the 55bp-58bp, a sequence from CPT1 was observed having (5’-AATT-3’) while all the others have (5’-GGTA-3’). Even though this is a noticeable difference and could have been caused by mutation or variation, the difference is still low enough and less that 1% difference to be considered significant.

But on the other hand, the PCa1 sample had 36 different base pairs compared with the others, which was 5. 5% difference and is high enough and can be considered significant since it is ;4% divergent(Meyer and Paulay 2005). Then with a ~98% confidence, it could be said that PCa1 is from an independent evolutionary lineage and might indicate a divergence within or outside its population or might have occurred due to relationships and interactions among the other species.

The introduction of the P. canaliculata to different places may have an effect on its intra- and interpopulation and might be why it has various genetic sequences although it goes against the theory that introduced species becomes a founding population in a new habitat thus they have a limited gene pool and as a consequence genetic drift, which removes variability since it affects all genes, and bottleneck might occur, which reduces the new species to have a reduced genetic diversity.

To observe the genetic variability, the sequences collected were compared with those from GenBank with the use of the BLAST software. As a result from 81 COI barcodes and 55 haplotypes, the samples collected showed 99% and 100% similarities with the different haplotypes thus it showed that the species has a high diversity within the populations. The phylogeography within and among the species does not apply on the P. analiculata since intra- and interpopulation diversity was observed which was shown by the multiple introduction throughout the Philippines, hence the different times of the introduction contributed more to its diversity and it coincides with the migrant pool model that says that the introduced population acquires more genetic variability because of the multiple sources of genetically divergent populations as compared to that of the local species (Slatkin 1997, Sakal et al 2001). In China (Dong et al 2010) The chosen primers an average of 124. bands, since they generated a total of 498 bands, which ranged from 150-2500bp and qualifies them for barcoding, as seen in table 1. Among the 140 individuals, 435 bands were polymorphic which was different for each primer. In table 2, Nei’s gene diversity (H) varied between 0. 2612 and 0. 3340, with an average of 0. 3044, and arranged in a descending order the populations, LB ; KM ; NN ; FZ ; TZ ; GZ ; YY while the Shannon’s information index (I) ranged from 0. 3910 to 0. 4856, with an average of 0. 4499.

At the species level, the values of Nei’s and Shannon’s showed the same trend as that of PPB. AMOVA analysis showed that there are highly significant (P ; 0. 001) genetic differences among the seven populations of P. canaliculata. The genetic diversity was mostly due to the differences within the population (92. 76%) while the rest was due to among populations. The analysis tells the same as that of the Nei’s and Shannon’s information, which says that there was a relatively high level of genetic differentiation among populations. CONCLUSION

Genetics of different species are studied by means of DNA barcoding, mostly of the COI gene in the mitochondria, to know the taxon of a species and to understand their trends and characteristics not only morphologically but also genetically. The diversity of a species can also be tested by means of DNA barcoding as seen in the study of the Pomacea canaliculata. The P. canaliculata was introduced in Asia for agricultural purposes and was seen for its benefits but not its drawbacks, which later on resulted to it being invasive and a pest for both humans and other species.

To understand the P. canaliculata further, its diversity was studied by means of DNA barcoding and was analyzed both qualitatively and quantitatively in the Philippines and China, respectively. Both the analysis showed the same outcome, wherein the results showed high levels of genetic diversity among populations. Because invasive species tends to give a negative feedback to those species in the local area, it is important to understand these alien species and to know how diverse they are so that proper management of these species could be done. REFERENCES Barker, G.

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Main Library, University of the Philippines Baguio, Baguio City. Cowie, R. H. 2002. Apple snails (Ampullariidae) as agricultural pests: their biology, impacts and management. In: Molluscs as Crop Pests (ed. G. M. Barker), p. 145-192. CABI Publishing, Wallingford. Dodson, Edward O. 1956. Genetics: The Modern Science of Heredity. Philadelphia: W. B. Saunders Company. Dong, S. , Shentu, X. , Pan, Y. , Yu, X. , Wang, H. 2011. Evaluation of genetic diversity in the golden apple snail, Pomacea canaliculata (Lamarck), from different geographical populations in China by inter simple sequence repeat (ISSR).

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