Distribution of cysts of Strongyluris sp . ( Nematoda ) in the pallial system of Achatina fulica Bowdich , 1822 from Vila Dois Rios and Vila do Abraão

This work aimed to assessing Strongyluris sp. cysts distribution pattern in the several inner organs from pallial system of Achatina fulica Bowdich, 1822. Also we verified if there is a relationship between the mollusk size and the number of specimens from parasites collected from two touristic villages in Ilha Grande (Angra dos Reis, Rio de Janeiro state): Vila Dois Rios (VDR) and Vila do Abraão (ABR). The samples were obtained through a field work conducted bimonthly during 2007, 2008, 2010, and 2011, at both locations. Height and width were measured from shells collected, and the all specimens were classified in different classes: class 1 <4.0 cm, class 2 4.1-9.0 cm and class 3 < 9.0 cm. After the specimens were dissected in order to find and count the number cysts in the pallial system. In specimens from both locations, the pulmonary and secondary veins showed a high number of cysts. No significance difference was found both in the abundance of cysts among the specimens in ABR (p=0.138) and VDR (p=0.181). Achatina fulica showed different intensities of cyst infection based on the size classes: the class-3 specimens, at both locations, showed the greatest cyst average (ABR Anova F= 3.8; p=0.02); (VDR T of Student T= -2.04; p=0.04). The results suggested that the highest number of cysts in the vascularized area in pallial system of A. fulica was a consequence of a greater hemolymph circulation in that area, delivering more nutrients for larvae development. We think that bigger individuals host a higher number of cysts, as they usually present a larger biomass and a larger area of the pallial system, allowing an efficient parasite colonization. Other possible explanation could be the long exposure of the molluscs of class 3 to the parasites, which allowed a longer time to the larvae to allocate themselves.


Introduction
The gastropod Achatina fulica Bowdich, 1822, native from Eastern Africa (Mead, 1961), is considered one of the hundred worst invasive species in the world (IUCN, 2013).One of the hypothesis on the invasion of this mollusk in Brazil establish that A. fulica was introduced in the 1980's, in a farming fair promoted by the Brazilian government, as a commercial purpose of replacing the escargot (usually Cornu aspersum Müller, 1774).Then, the country became one of the main producers for the European market (Colley and Fischer, 2009).Currently, the mollusk is present in 24 of the 26 Brazilian states, including islands and environments of protected areas (Thiengo et al., 2008;Faraco, 2011).
Achatina fulica is considered a harmful species to the environment, as it can possibly change the structure of communities and the composition of native populations (CBD, 2011), caused by the direct competition for resources against native mollusk species (Simberloff, 2002), such as Megalobulimus spp.and Thaumastus spp.(Carvalho et al., 2003;Fischer and Colley, 2004;Thiengo et al., 2008;Zenni and Ziller, 2010;Faraco, 2011).
In 2008, Strongyluris larvae were found encysted inside the pallial cavity of specimens of A. fulica from Ilha Grande (Thiengo et al., 2008).According to Seehabutr (2005) and Franco-Acuña et al. ( 2009) several nematodes are parasites of A. fulica, but the understanding of their relationship has not yet been clarified.Moreover, as it is a possible paratenic host, facilitated by its soft body, habitat, and generalist habits (Fischer et al., 2010), A. fulica can harbor countless nematodes, which could parasitized the vertebrate fauna when ingested.When spread to new areas, the mollusk also would introduce new parasite species, contributing to the parasitism of the local fauna (Simberloff, 2002;Thiengo et al., 2008).
In order to better understand and clarifie the role of A. fulica as an intermediate host for Strongyluris larvae, this work have as goal aimed to assessing the Strongyluris spp.cysts distribution pattern inside the organs of the pallial system and evaluating if exist a relationship between the mollusk size and the number of parasited specimens in Ilha Grande.

Material and Methods
The specimens were obtained from Vila Dois Rios (23°10'55.73"S;44°11'30.81"W)and Vila do Abraão (23°08'20.30"S;44°10'07.19"W),located at Ilha Grande, a township of Angra dos Reis, in the south of the Rio de Janeiro state (Figure 1).The samples were collected bimonthly in 2007, 2008, 2010, and 2011, at both communities.According to the sugestion made by Fischer and Colley (2004) and Albuquerque et al. (2008), the sampling localities were selected based on the mollusk preference in terms of rest and foraging places (hedges, vegetable farming, gardens, garbage, rubble, empty lots, and woods edges).
The specimens collected in 2007 and 2008 were sacrificed by drowning, fixed, and preserved with 70 o GL alcohol, following the technique described in Thomé (1975).The animals collected in 2010 and 2011 were immediately dissected, after sacrifice (Salgado, 2010).
The height and width of the shells were measured with a 0.05 mm-accuracy Mitutoyo caliper rule, according to Salgado (2010); the specimens were classified as <4.0 cm -class 1, 4.1-9.0cm -class 2, and <9.0 cm -class 3 (Tomiyama and Nakane, 1993;Fischer et al., 2008).In order to retrieve the animal, the shells were cracked with a hammer, wrapped in a newspaper to reduce the impact caused by the hits and to avoid damaging the pallial system.The dissection was conducted by inserting fine point scissors, from the mantle edge, at the pneumostome level, initially cutting near the anus and then along the rectum, exposing the pallial cavity roof, folded to the left.This animal was throughtly examined with naked eye and a Leica MZ6 stereomicroscope in order to find and count the cysts in different organs.
Nematode identification was based on morphology characters according described in Bain ( 1970 Non-parametric Kruskal-Wallis test (H test) was used to analyze the cyst distribution pattern in the soft parts of A. fulica.The comparison between the size classes and the number of cysts was evaluated by parametric Student t test and ANOVA.After that, we conducted the a posteriori Tukey test to compare the average pairs after the variance analysis.
In order to compare the size classes, 95 infected specimens from ABR were analyzed (one of them presented a broken shell apex and was removed from the analysis, because it was not possible to measure its height), and found that 10% (10) belonged to class 1, 62% (59) to class 2, and 28% (26) to class 3.In VDR, 44 specimens were assessed, and 59% of them (26) belonged to class 2, and 41% (18) to class 3.There was not observed parasitized specimens in class 1. Achatina fulica presented different intensities of infection based on the size classes.The results showed that the specimens of class 3, in both locations, presented a higher average of cysts.The variance analysis performed for the three size classes in ABR revealed that difference: class 1: 2.1 ± 1.5; class 2: 8.7 ± 24.8; class 3: 28 ± 49.5; Anova F= 3.8; p=0.02.Tukey post-test revealed a remarkable difference (p= 0.03) only between class 1 and the others.Student's t-test for the VDR data also indicated an significant difference in the average of cysts between the size classes (class 2: 8.15 ± 16.1 and class 3: 22.3 ± 29.7); (Student's t-test= -2.04; p=0.04).
Then, we can consider that the infection of Achatina fulica by Strongyluris spp.larvae could be explained by the role of the snail as an intermediate host, as observed by Valente et al. (2016).This hypothesis can be accepted, as Fischer et al. (2010) mentions that the habitat and habits of A. fulica facilitate the parasitism in this species.
Its is suggested that the greatest number of cysts in the vascularized area of A. fulica was a consequence of a great hemolymph circulation and, consequently, nutrients availability in that area, which delivered more requirements for larval development, as proposed by Bain (1970), based on experiments.The author infected cockroaches Blatella germanica species (Linnaeus, 1775) and mosquitoes Culex sp. with larvae of S. brevicaudata Mueller, 1849.As a result he noted that third-stage larvae were found in the general body cavity, region rich in hemolymph in the two species.In the mosquitoes, besides the presence of larvae in the body cavity, some were located in the chest within thin-walled cysts filled with fat globules.The search for nutrients for larva development by parasites in the intra-mollusk phase can be observed in an essay with Bradybaena similaris (Férrussac, 1821) infected by a larval trematode, conducted by Pinheiro and Amato (1994).The authors noticed an outstanding decrease in the hemolymph glucose content of the parasitized mollusk after a few days of infection.
Given that the mantle region is one of the main storage sites of glycogen in terrestrial gastropods, the hemolymph that supplies this area is rich in glucose (Pinheiro, 2007).The mantle region is a strategic site to warrant larval development by providing the types and amount of nutrients required.
Hemolymph in A. fulica also features high cholesterol levels, a result of the metabolism of lipids in these animals (Lustrino et al., 2010).This factor can also explain the preference of larvae allocation in the mantle organs, as for filling the cysts and folding the larvae, are necessary amounts of fat that possibly were easily acquired from hemolymph flowing in this region.
We believe that larger individuals house a higher number of cysts, as they usually show a larger area in the pallial cavity roof which in turn allows an efficient parasite colonization, or because the longer exposure of the molluscs from class 3 to the parasites, which allowed a longer time to the larvae to allocate themselves (Franco-Acuña et al., 2009).The non-registration of cysts on individuals of class 1, may have been due to not verifying the tissues through histological sections, to the search for microscopic sizes cysts.
Another hypothesis can also be explained by the probable relation between the food preference and the ontogenetic development of the species (Fischer et al., 2008).Fischer et al. (2008) observed in a study of the African snail diet that individuals who reach larger sizes (adults) consumed specially plant litter.With this behavior in mind, we can perceive a possible infection route of that species by the Strongyluris spp., as the nematode eggs are certainly being offered on the soil and the plant litter by the definitive host, in order to be freely ingested by A. fulica adult specimens.Other possible infection routes could take place through geophagia, coprophagia, and necrophagy, considering that these sources are contaminated.These eating habits are also common in larger specimens (Mead, 1961;Albuquerque et al., 2008).The immunological stress caused by the senescence can also increase animals vulnerability to parasites (Mead, 1961).The gradual reduction in the organism physiological functions of old mollusks (class 3) probably facilitated the access and the establishment of parasites in the studied specimens.
This work stresses the need for more studies about the relation of the helminth fauna with the invasive mollusks and the potential role of the African snail as a intermediate host for parasites that can reach the wild fauna in Brazil (Thiengo and Fernandez, 2010).These data contribute to the increase of information on the role of A. fulica in life cycle of parasites of medical and veterinary significance (Faraco, 2011).Besides increase of knowledge the present study show and reinforce others as Franco-Acuña et al. (2009) andMaldonado Júnior et al. (2010), that, in spite of the great importance of A. fulica acts as intermediate host to A. cantonensis, as proven by other authors, the risk that this snails bring to public health and environment is greater than usually we can imagine.There are some kind of parasites that are found in A. fulica that are not known and we do not even know what kind of damages they can cause to human or domestic and wild animals.By this reason, the present study, supported by previous ones, focused the importance to analyze the parasitic fauna in A. fulica, as well as the possible relationship between the parasite and the snail and vertebrate host.

Figure 1 .
Figure 1.Location map of Ilha Grande in Rio de Janeiro State showing the collecting sites, Vila do Abraão and Vila Dois Rios, pointed out by the red squares.Scale in kilometers.Map: L. E. M. Lacerda.

Figure 3 .
Figure 3. Achatina fulica Bowdich, 1822 from Vila Dois Rios, Ilha Grande, Angra dos Reis, RJ, showing cysts of Strongyluris sp. in the roof of pallial cavity.(A) cyst within the pulmonary vein indicated by the black square; the arrow indicates the large pulmonary vein; (B) cysts inside the secondary veins indicated by the white square.Scale bar = 1mm.Photos: S. B. Santos.

Table 1 .
Organs of Achatina fulica Bowdich, 1822 from Vila do Abraão and Vila Dois Rios, Ilha Grande, Angra dos Reis, Rio de Janeiro, parasitized by Strongyluris sp. by locality, total number of cysts and average of cysts per organ.