INTRODUCTION

The giant African snail Achatina fulica (Bowdich 1822) is a gastropod mollusk from the Achatinidae family, characterized by having a conical light- to dark-brown shell, with a distinctive banding pattern. Adults reach up to 30 cm in size (1). They are considered protandric hermaphrodites (they develop the masculine sex first). Giant African snails reach sexual maturity at five months, and under optimal conditions of temperature, humidity, and food availability, can oviposit hundreds of eggs (1,2,3,4,5).

Attributes such as having a high fertility rate and few habitat requirements, being a superior competitor easily transported by humans, and being economically attractive, placed the African snail as one of the 100 most dangerous invasive species worldwide (6,7,8,9). Since its departure from its natural distribution range in Africa 200 years ago, this species has caused a wide variety of environmental damage and public health issues because it is an intermediary host in the life cycle of several parasitic species, such as nematodes of the Angiostrongylus genus, which cause death in humans from eosinophilic meningitis.Furthermore, this species can lead to significant economic losses by affecting agricultural crops and requiring high investments to implement management, control and eradication measures (1,9,10). These direct and indirect impacts of A fulica on human health and the economy of the regions, have provided the impetus for research into the ecology, physiology and genetics of this mollusk, to obtain the necessary tools to strengthen management plans and propose integrated exploitation measures for this species (9-10).

The first record of A. fulica in Latin America occurred in Brazil during the decade of the 1980s where it was introduced for heliculture. Until the present time this species has been recorded in Cuba, Venezuela, Argentina, Paraguay, Ecuador, Peru, and Colombia (1,3-5). According to Resolution 0848 of 2008 by the Ministry of Environment, Housing, and Land Development, A. fulica is considered an exotic invasive species in Colombia. This resolution establishes the responsibilities and general guidelines of response of the environmental institutions faced with this threat, and defines a “national inter-institutional plan of the environmental, agriculture and livestock, health and defense sectors for the management, prevention, and control of the giant African snail (A. fulica)” (3,11,12).

The current hypothesis is that there was a sole point of entry into Colombia from Brazil, as according to De la Ossa-Lacayo et al (3), the first report of this species in Colombia was given by Corpoamazonia in 2010. In the next five years, the giant African snail was reported in over half of the country. The first official record of the giant African snail in Valle del Cauca Department occurred in 2011. Several control efforts have been carried out since its appearance, and some biological and ecological studies have been undertaken since 2013 (3,11).

Likemost other invasive species (13,14,15), the giant African snail shows low genetic diversity compared with the populations in its natural distribution range in Africa, due to a founder effect that occurs at each introduction event, and to the bottlenecks that occur after establishment and acclimation to the conditions of the new habitat (1,4). Although from the ecological point of view A. fulica populations can be considered successful at their initial invasion stages, the population dynamics tend to stabilize or even decay naturally several decades after the invasion (1, 15). Several hypotheses have been postulated to explain this phenomenon, including genetic causes, phenotypic plasticity, behavioral causes, or multiple introductions that compensate the genetic drift suffered by the founding individuals (14, 15).

Several studies of giant African snail population genetics have been carried out over large spatial scales (> 500 km) (1, 4). However, there is a lack of information on genetic variability of these populations at regional scales, and on the genetics of giant African snail populations in locations where invasion is recent. Taking into account that the giant African snail has been present in the Valle del Cauca Department for less than five years and that the geographical size of this region is in the order of 200 km, in this study we used microsatellite molecular markers (11, 16) to establish the intrapopulation genetic diversity of A. fulica at a regional scale.

MATERIALS AND METHODS.

Study area. Considering the strong association of A. fulica with urban centers, the specimens used in the present study were obtained from the cities of Buenaventura (3º53’ N, 77º 05’ W), Bugalagrande (4º12’ N, 76º18’ W), Cali (3º26’ N, 76º31’ W), Cartago (4º44’ N, 75º54’ W), Jamundí (3º15’ N, 76º32’ W), Palmira (3º31’ N, 76º81’ W), Tuluá (4º05’ N, 76º12’ W), and Buga (3º54’07’ N, 76º18’4’ W)(Figure 1) during a study of African Snail´s population and reproductive ecology in the Valle del Cauca (11). The city of Buenaventura is located within the Chocó biogeographic region, at a mean 7 m above sea level. The vegetation is typical of very humid tropical forest. The mean annual precipitation is 7650 mm per year, the mean relative humidity is 89%, and the annual mean temperature is between 25ºC and 28ºC. The remaining cities are located in the geographic valley of the Cauca River. The dominant vegetation is dry tropical forest; it has a flat topography, mean elevation of 1000m, annual mean precipitation of 900 mm, and average temperature of 23.6ºC (17).

Field work. The sampling protocol carried out in the population and reproductive study of A. fulica consisted in delimiting the search area of African snails in three sectors, randomly selected, of each city. To identify the sampling sectors, each urban perimeter was divided into 2 km2 cells, and then randomly select three cells. Within each cell, three sampling plots of 16 m2 were located and all the African snails detected were harvested. These sampling plots corresponded to urban green areas, road separators or abandoned lots, where plant material, building debris or garbage was accumulated.

Individuals were collected manually, following the sanitary recommendations provided by the environmental authority (12), stored in individual zip-lock bags, and transported to zoology laboratory at Universidad del Valle in Cali. After measuring and weighing each captured snail, 20 individuals from each city, were randomly selected to remove 30 mg of foot tissue using a sterilized surgical blades. Tissue samples were deposited at 96% ethanol in 1.5 mL eppendorf tubes and stored at -20ºC. Biological waste was disposed following guidelines stated in Resolution 654 of 2011 of the Ministry of Environment, Housing and Land Development of Colombia (12).

Laboratory Methods. DNA extraction was performed using the protocol of Fontanilla (1) and the recommendations of the protocols of the commercial houses ThermoScientific® and Qiagen®. 30 μL of proteinase K and RNAase were added to 20 mg of chopped tissue for at least 5 hours of lysis in water bath at 55 ° C, for a final extraction reaction volume of 200 μL. The 10 Microsatellite loci (GENBANK KM104170-KM104179) were amplified under the concentrations and reaction conditions proposed by Morrison et al (16). Equal concentrations (0.5 µM)of primer forward and primer reverse were used, and DNA was diluted to a concentration of 20 ng/µL. The amplification was verified usingelectrophoresis in 10% polyacrylamide gels (8.75 mL of polyacrylamide solution 2X; 26.25 mL of TBE 1X; 300 µL of ammonium persulfate, and 30 µL of TEMED), with a 10 min pre-run at 50V, and a three hour run at 180V.

To continue with the molecular analysis, the samples from each city were assigned to four geographical zones: zone 1 or Pacific zone contained the samples of Buenaventura, zone 2 or Center zone contained the samples of Buga, Tulua and Bugalagrande, zone 3 or North zone contained the samples of Cartago, and zone 4 or South zone contained the samples of Cali, Palmira, Jamundí. Eight samples of randomly pre-amplified DNA were selected for each zone and displayed in gel using silver nitrate staining and two weight markers, one of 100 base pairs long and other one of 25 base pairs long Generuler ThermoScientific®. Gels were photographed and edited in Photoshop®, and genotyped using the ImageJ® program.

Ethical aspects. As the giant African snail has been declared an exotic invasive species, no permits were required from environmental authorities. The safety guidelines dictated by the Ministry of Environment were followed (12).

Statistical analyses. In order to stablish the possible clusters in the population and their possible coincidence with the geographic delimitations established for this research, we used the Bayesian algorithm from the Geneland package of the R program (18), based on the genotype databases and the geographic coordinates of the sampled plots. This algorithm infers and locates genetic discontinuities between populations, obtaining geospatial plots of the posteriori distribution probability using kriging interpolation techniques (18). A null alleles model was run with one million iterations, of which 1% were retained and 20% were discarded as burn-in. Based on the information obtained with geospatial distribution plots, the allelic frequencies and Hardy-Weinberg equilibrium were calculated for the groups and for the population, as well as genetic diversity and polymorphism estimators such as effective number of alleles (Ea), mean number of alleles (Na), percentage of polymorphic loci, effective number of migrants (Nm), observed heterozygosity (Ho), expected heterozygosity (He), fixation index (F), genetic identity, and Nei´s genetic distance(D). A Mantel test was performed using the obtained matrix of distances of the plot geographic coordinates, and the matrix of genetic distances. These tests were performed using the GeneAlEx v.6.502® program (19). The FST and FIS statistics and null allele frequencies were calculated using the Geneland program; significance was established through a molecular variance analysis (AMOVA).

RESULTS

Eighty percent of loci were polymorphic. The Aful012 locus was not present in the sampled individuals, and the Aful030 locus was monomorphic. The Bayesian analysis defined two aggregations for the evaluated samples with a probability density higher than 50%, indicating that the giant African snail in the Valle del Cauca Department comprises two groups. The assignation probability was 96% (Table 1).

The first (Southern) group comprisedcitiesin the west and south of the department: Buenaventura, Cali, Palmira, and Jamundí (Figure 2). The second (Northern) group comprised cities in the north and center of the department: Cartago, Buga, Tuluá, and Bugalagrande (Figure 3). Of the eight polymorphic loci, the Aful032 locus had less allelic bands than the base pairs recorded in a previous study, and it was monomorphic in the Southern population. The locus with the highest number of alleles was Aful069 with five alleles, followed by Aful061 with four alleles, and Aful086 with three alleles. The northern group had a higher frequency of unique alleles than the southern group (Table 2). The approximate effective number of alleles was conserved in the groups as well as in the population. The mean values of observed heterozygosity were lower than the expected values, except for theloci Aful082 and Aful086 loci, which had negative fixation index values, contrary to the Aful080 locus that had the highest statistical value, indicating a heterozygote deficit in most loci (Table 3).

The frequency of null alleles in A. fulica specimens in Valle del Cauca was 0.3, with four loci with an effective number of migrants above four, which would indicate high genetic flow for those loci, especially for Aful082 and Aful086. On the other hand, the χ² test showed that Aful080, Aful069, Aful086, and Aful061 were not in Hardy-Weinberg equilibrium (p<0.05) (Table 4). This was probably due to extreme Nm values and a heterozygote deficitrevealed by the significant (p<0.05) FIS values, which suggests that there was non-random pairing within the groups that comprise the population. A significant moderate genetic differentiation was detected between the northern and southern groups (FST0.163; p<0.05) due to isolation by distance revealed by the Mantel test (p 0.01; R2 0.06), and there was a slight difference inferred from the genetic identity value (0.841) (Table 5).

DISCUSSION

Microsatellites have been widely used in population genetics as molecular markers, as they allow the estimation oflevels of genetic variability within populations, and of genetic diversity, among other advantages. These molecular markers have been used to elucidate the genetic state of populations and their demographic history, as they are in most cases “neutral” markers and have a high degree of polymorphism (20).

In the case of the giant African snail, the degree of polymorphism reported by Morrison et al (2014) was higher for all loci compared with this study, due to the monomorphism and lack of one locus in this study (16). This is a reflection of the scale used in the two studies. Although the sample size was similar, the samples used by Morrison et al(2014) (16) came from countries in different continents, whereas in the present study the scale was regional, and the described loci had not yet been recorded in individuals from Latin America. However, taking into account that A. fulica is an invasive species, we would expect that polymorphism would decrease with decreasing geographic scale due to the high endogamy levels that are usually found in this species populations (1, 20, 21).

According to official records the giant African snail was introduced six years ago for commercial purposes into the Valle del Cauca Department (3, 11).Taking into account that under the assumption of microsatellite neutrality genetic diversity patterns are determined basically by the interaction of genetic flow and genetic drift (20), the moderate differentiation (FST 0.16) of the population in two groups could have occurred due to the species being introduced at more than one location, in this case in the north and south-west of the department. This hypothesis is supported by the position of the Valle del Cauca, as a region of commercial encounter of the southwestern Colombian. To Buenaventura arrived goods and perishables from Asia, and this stuff are transported by truck or train to the interior of the Valle del Cauca (22, 23). This route could be functioning as a dispersal path for the African snail into the Valle del Cauca. By the south, the African snail could be entering into Valle del Cauca associated with the agricultural products that are transported from the departments of Cauca and Nariño to the interior of the Country (11). On the other hand, the African snail also could be entering by the North of the Valle del Cauca associated to the agricultural transport coming from the department of Tolima and the coffee field of Risaralda, Caldas or Quindío (22, 23).Both situations could be promoting small scale intrapopulation genetic structure of A. fulica described in this study for the Valle del Cauca Department.

Multiple spatial and temporal origins are part of a demographic hypothesis to explain the paradox of invasive species, as a way to offset the deleterious effects of genetic drift from bottleneck and founder effects that are produced at the beginning of biological invasions (14, 15, 16, 20). This deleterious effects tend to produce slight differences in the populations ofinvasive species, and particularly seems to be true for A. fulica, since genetic studies carried out in populations of this species, where the introduction occurred more than 50 years ago, are obtained similar results to our work (1, 4).Therefore, to understand the dynamics of invasion it is important to know the demographic history with as much detail as possible.

The demographic history of A. fulica in Colombia is still controversial, since there is a chronological lag in the records of the occurrence of this species in the different geographical areas of the country. However, this situation could be indicating that A. fulica arrive to Colombia in different moments and by different points of entry. In less than two years, since the first formal technical report of presence of A. fulica in Colombia was carried out by Corpoamazonia, the African snail was recorded in more than half of the national territory. Moreover, this species was recorded in Santander Department the same year in which it was reported in the Valle del Cauca Department, even though these two departments are separated by over 700 km and by the Andes montains (3, 11, 24). This situation makes even more probable intrapopulation genetic differentiation at the regional scale, such as was described in this study.

The endogamy (FIS 0.4) and high genetic identity (0.84) recorded in this study could be the result of a recent invasion process. Theoretically animals that arrived as juveniles could still be alive at the same time as the first generation born in the study zone. The founder effect should therefore still be acting on the population. However, if multiple temporal introductions had not occurred and the generation time was short, the polymorphism of the marker would have been lost oversubsequent generations. The population controls that are carried out continuously in the Valle del Cauca Department have eliminated a high proportion of adult individuals (11), which probably led the population to face a bottleneck, increasing endogamy and genetic identity (15, 20).

In conclusion, it is probable that the giant African snail population of the Valle del Cauca Department had more than one point of introduction according to the two groups observed. As it is an invasive species that has been a short time in the study area, the population and detected groups exhibit high endogamy, modulated by the founder effect and possible multiple introductions. However, the control actions carried out by environmental authorities have probably exposed the A. fulica population in the Valle del Cauca Department to abottleneck effect, which could have led to moderate differentiation between groups. The intrapopulation genetic characteristics of A. fulica in the Valle del Cauca Department coincide with what has been described for invasive species, where high endogamy, high genetic identity, violation of the Hardy-Weinberg equilibrium, and extreme values of the fixation index have been reported (1, 20, 21). Alleles tend to be fixed in some loci, and in others there could possibly be an excess of heterozygotes as evidence of the multiple introductions (1, 20).

The results of our research, open a new research line which seeks to apply molecular techniques to improve our knowledge about invasive species. Probably, the use of other molecular markers or techniques of analysis such as capillary electrophoresis or sequencing, should be explored in the future.

Acknowledgments

To Wilmar Bolívar, Ángela María González, Rodrigo Lozano, Natalia Rivera, Fernando Díaz and Mario F. Garcés for their contribution in different stages of the investigation. To the laboratory of Molecular Biology and Human Molecular Genetics of the Universidad del Valle. This study was co-financed by CVC and Univalle (agreement 054/2014).vv

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