Population characterization and parasitological assessment of the giant African snail ( Achatina fulica) in urban areas of Cartagena, Colombia

Background: The giant African snail, Achatina fulica, is an invasive species recognized for being a serious agricultural pest and an intermediary vector for diverse parasites that cause diseases in humans. The knowledge of the state of African snail populations in urban areas is of great ecological and public health importance. Therefore, our objective was to characterize the status of giant African snail populations present in the city of Cartagena, Colombia, including the assessment of nematode parasites in the specimens. Methods. Sites were visited following information from citizens affected by the presence of the African snail. The specimens were collected and transported to the laboratory; subsequently, they were weighed, measured, and classified by size. Dissections of lung tissue and soft organs were performed to search for and identify nematode parasites. Size measurement between the sampled sites was statistically compared and density and biomass indicators were established. Results. In total, 204 snails were collected distributed among four sites within Cartagena city. Of these, 50% were juvenile specimens (10-40 mm). The size of the specimens showed significant differences between the sampling areas. The calculated density was between 0.0019-0.68 ind/m 2 and the biomass between 3.92-48.75 kg/ha. No presence of nematode parasites was observed in these specimens. Conclusions. Densities and biomasses of A. fulica in Cartagena do not reach levels considered highly harmful. On the other hand, although no parasites were found in these snails, it is relevant to continue with studies on the human health risks that represent the presence of this invasive species in urban areas of Cartagena.


Introduction
One of the greatest threats to global biodiversity, livelihoods, human and animal health, agriculture, and the local economy are invasive alien species 1 . The giant African snail, Achatina fulica, (Bowdich 1822) is a gastropod native to Africa and considered one of the 100 worst invasive alien species in the world 2,3 . Currently, this species is widespread in tropical and subtropical areas on all continents due to the growth of international trade and increased transport that facilitate the introduction of this species, accidentally through agricultural or horticultural products or intentionally as a source of food or as a pet 2,4,5 .
The giant African snail is recognized as an agricultural and garden pest 5 . Its great reproductive capacity allows it to reach high densities and biomass in short times 3,5 , and its broad spectrum herbivorous diet allows it to consume more than 50 species of native and agricultural plants, all of which makes this species a serious pest with the ability to modify habitats and displace native species 5 . The giant African snail is an intermediate host for various parasites, among which the nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis stand out, which cause eosinophilic meningoencephalitis and abdominal angiostrongyliasis in humans, respectively 6,7 . Recently, the presence of A. cantonensis in African snails in Colombian territory was confirmed, putting the population on alert due to the exposure of humans to the parasitic load of this snail, leading to the consideration of eosinophilic meningitis as a possible emerging disease in Colombia 8 . Risk factors for infection in humans and pets by this and other nematodes include eating infected snails raw or undercooked, or food contaminated by snail slime or feces 9,10 .
In Colombia, the presence of A. fulica was registered for the first time in 2009 11 , since then the mollusk has been distributed in more than 20 departments, registering in all regions of the country 12 . Particularly in the Colombian Caribbean Region, the growth of A. fulica populations has been documented in the department of Sucre [12][13][14] . In Cartagena, Bolívar, journalistic media reported the presence of the giant African snail south of the city in 2016. However, since its appearance, there has been no scientifically documented record of the presence and distribution of this species in urban areas of the city, as well as its impact on human health. Therefore, the objective of this study was to generate the baseline of reference information on the African snail populations present in the Cartagena city, including the assessment of nematode parasites in the specimens.

Study area
Cartagena (10 ° 23'59 '' N 75 ° 30'52 '' W), capital of the department of Bolívar located within the Colombian Caribbean region, is a city of low ecotope or coastal, presenting altitudes between 1 and 9 meters above sea level, a temperature average of 27.5°C, a relative humidity of 80% and an annual rainfall of 870 mm.

Collection of specimens
The search for, and collection of, the snails was carried out between July and October 2019, a period that included the rainy season in the region and which the proliferation of the giant African snail is favored. The sampling consisted of visiting the zones within the neighborhoods of the city Cartagena previously informed by citizens affected by the presence of the snail in their homes and surroundings. At each place, a 50-minute sampling effort was made by two people in a single visit. During the inspections, searches were carried out in gardens, under undergrowth and rubble, or between crevices where they could take refuge. The specimens were collected manually using gloves and face masks and transported in easy-sealing bags to the laboratory of the UNIMOL research group in the University of Cartagena. As this was a monitoring study, all the snails available during collections were included for analyzes, no exclusion criteria were applied for the collection of the snails.

Morphometric analysis
The collection included live individuals of all sizes, which were measured using a digital caliper (0.01 mm) and weighed in a weighing machine (1 gr). The length of the shell was used as a descriptor of the total size of each individual, so size classes were defined as the following: class 1, to which newly hatched individuals and up to 10 mm were assigned; class 2, juveniles (10-40 mm); class 3, young adults (40-70 mm) and class 4, adults (> 70 mm) 15 .

Parasitological analysis
Specimens larger than 50 mm have a greater probability of carrying nematode larvae 16 , even so, we decided to evaluate specimens greater than 30 mm to increase the probability of findings. The snails were washed externally and were put to sleep by thermal shock by introducing them in ice water, once they were numb, the shell was broken by using dissection scissors to extract the lung tissue and the rest of the soft organs. The search and collection methods for nematode parasites described by Córdoba et al. 17 were used. Briefly, the lung tissue was cut in 1 × 1 cm pieces and placed on microscope slides for observation under a stereoscope. Likewise, the other organs were cut and examined by compression between two glass pieces.

Data analysis
As data were not normally distributed, the snail size measurements between the sites were compared using the Kruskal-Wallis and Bonferroni tests post hoc, using the statistical software SPSS v. 19.0 (IBM Corp, Armonk, NY) with a statistic significance defined as p<0.05. Also, the population density of each zone was calculated according to the number of individuals collected per effective area sampled (Individuals/m 2 ) and the biomass

Amendments from Version 1
In this version we have corrected the year of the first report of the giant African snail in Colombia, we have also provided more details on the collection methods, data analysis, and the characteristics of the sites where the snails were found. Finally, in Table 1, it was indicated that "total" refers to the total of individuals collected.
Any further responses from the reviewers can be found at the end of the article REVISED per unit area (kg/hectares) was established. The google earth Pro 7.3.3.7786 polygon was used to determine the sampled area of each zone.

Ethical aspects
Giant African snail has been declared in Colombia as an invasive species, for which legal research permits are not required. This study was adjusted to the management regulations for A. fulica issued by the Ministry of Environment, 2011 18 . Also, during the execution of the study, efforts were made to reduce animal suffering during sacrifices, for this, the snails were put to sleep in ice water until their death before the dissections.

Results
During the period studied 204 snails were collected, distributed in four zones of four Cartagena neighborhoods, for which the presence of A. fulica was indicated by the community (Figure 1). The inspected sites where the snails were collected corresponded principally to extensive gardens and other spaces inside of private properties and residential condominiums (Manga, Las Gavias, and Serena del Mar). At these sites the snails were found in different substrates, such as plants, tree roots, grasses, under litter, flowerpots, and ornamental plants; In Zaragocilla, snails were found aggregated in rubble inside an educational institute.
Mean size and weight per site is shown in Table 2. The Kruskal-Wallis test showed a significant difference between the sizes of the most abundant African snail population groups  (Zaragocilla, Manga, and Las Gavias sites) (p<0.01). Likewise, Bonferroni-adjusted pairwise comparisons indicated that differences existed between each site (p<0.01 between each pairing). The population density established in each point, as well as the estimated biomass according to the calculated total weight, is presented in Table 3.
Regarding the parasitological analyzes, all the observed lungs were found healthy (free of cysts), likewise, no free larvae were observed in the slime or the rest of the soft organs, showing that these specimens were not infected with nematode parasites according to the methodology used.

Discussion
Understanding the populations of giant African snail present in different areas of Colombia is of great ecological, agricultural, and public health importance. The current study constitutes the first scientifically documented record of the presence of the species A. fulica in urban areas of the city of Cartagena.
It is known that the African snail can reach sizes of up to 300 mm in the length of the shell 19 15,29 . It has been mentioned that the areas highly affected by the giant African snail present densities of 10 ind/m 2 or more 12,30 ; this similarly occurs with regards to biomass, where devastating values of up to 780 kg/ha are estimated for areas which are highly affected 12,31 .
If that is the case, the densities and biomasses obtained in the snail populations of Cartagena are not very harmful.
Nematodes such as A. cantonensis and A. costaricensis cause eosinophilic meningoencephalitis and abdominal angiostrongyliasis, respectively, in humans. The life cycle of these nematodes involves rodents as the definitive host and mollusks of various species, including A. fulica, as intermediate hosts 7 . The snails examined in this work did not show evidence of infection by nematode parasites, one of the reasons could be the environment in which these snails were found. It has been noted that the African snail prefers sites such as garbage dumps, landfills, and empty lots, places where there is a greater probability of contact with rats, necessary for the nematodes to complete their life cycle 17 . In our case, more than 80% of the analyzed snails were collected in gardens of remarkably well-kept properties given the exclusivity of the sites, and according to interviews with various neighbors, rodents do not frequent these spaces.
In conclusion, although the densities of A. fulica estimated do not reach values considered highly harmful, the presence of this invasive species in properties and residential areas of Cartagena is worrying. Most of the snails were found associated with gardens, which suggests that the introduction and dispersal of this species within the city are related to the transport of organic fertilizers or other garden products, which could convert this species into a serious pest. Similarly, although no parasites were found in these snails, it is necessary to continue carrying out studies on the effect of this species on human health, as well as training campaigns on the management and control of the giant African snail in the city.

Open Peer Review
provide details of how the variables were analyzed in the model?
It would perhaps be helpful if the authors provided information (in form of the column data) of the calculated population density and biomass per unit area in the raw data file.

2.
Can the authors clarify why they decided not to normalize the data? The weight data can be ln-transformed, and the shell length can normalize using sqrt function. 3.
The column "number of snails collected per site" is missing or if it is in the data file, the column title is missing.

4.
Can the authors please elaborate on the column shell length? What do the two values indicate and some rows do not have two values.

5.
Graphical representation of the statistical analysis will help in understanding the paper more as it will indicate the significant differences between the population density across the different collection sites.

6.
The authors mention the Giant African snail affect the citizens of Cartagena; it would be helpful if they can shed light on what those effects are? For example, they are in general a nuisance or their presence or the sight of the snails.

7.
Under the section "Morphometric analysis" it can be useful if the authors provide information as standard errors as "+/-" for digital callipers and weighing machine. 8.
I would like to see some more discussion about the different phases of the A.fulica establishment and its potential impact (if any) in the future in Cartagena.

9.
I would like to see some more discussion on the population density of the snails in Cartegena and what it can possibly imply for the future. 10.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? materials and methods, nor in the information analysis. However, no mention is made of the community's reaction to the presence of the snail. There is mention of a potential impact on public health, but no mention of consumption habits or home gardens with the data of interviews. Please clarify.
6.The conclusion emphasizes the significance of continuing to conduct studies and management and control campaigns; I propose that it be stated whether they exist in Colombia or should be designed, and whether they should be articulated with the ICA or the National Ministry of Health.
expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The present study focuses on an invasive alien species with potential effects on human health and agriculture. The population and parasitological analysis in urban areas of Cartagena, Colombia is highlighted, in such a way as to evaluate the role of these snails as potential transmitters of parasites. As the authors postulate, these studies must be constant, since in Colombia itself parasites have already been registered in this snail. With regard to data collection, the estimate of density is not clear, nor is the number of samplings. Did they go to the same site more than once? Is a site the same as a house? These points should be better clarified in the methodology and in the results tables.

Minor Comments
Keywords: Achatina fulica must go in italic.  Table 1 you must indicate if it is the total number of individuals collected.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?