Levv37n1 011

Reproductive Response Of Bulimulus Tenuissimus (Mollusca) As
An Intermediate Host Of Angiostrongylus Cantonensis

(Nematoda)
https://doi.org/10.56238/levv15n38-011
Florence Gonçalves Martins

Laboratório de Fisiologia das Relações Parasitárias, Departamento de Ciências Fisiológicas, Instituto
de Ciências biológicas e da Saúde, UFRRJ, Seropédica, RJ, BRAZIL.
Iza Patrício
Laboratório de Avaliação e Promoção da Saúde Ambiental, FIOCRUZ, RJ. BRAZIL.
Marcos Antônio José dos Santos

Departamento de Biologia Animal, Instituto de Ciências Biológicas e da Saúde. UFRRJ, Seropédica,
RJ. BRAZIL.
Jairo Pinheiro
Laboratório de Fisiologia das Relações Parasitárias, Departamento de Ciências Fisiológicas, Instituto
de Ciências Biológicas e da Saúde. UFRRJ, Seropédica, RJ. BRAZIL.
Juberlan Silva Garcia

Laboratório de Biologia e Parasitologia de Mamíferos e Reservatórios, FIOCRUZ. Rio de Janeiro,
RJ. BRAZIL.
ABSTRACT
The snail Bulimulus tenuissimus is a terrestrial gastropod widely distributed in the Americas, Asia,
Africa and some countries of other continents. It typically occurs in the same areas as Angiostrongylus
cantonensis, which needs a mollusk as intermediate host. In spite of this, the literature is scant about
the relationship of these organisms. Specimens of B. tenuissimus were experimentally infected with
L1 larvae of A. cantonensis. Weekly, until the third week post exposure, the reproductive parameters
were analyzed and snails were dissected for collection of hemolymph, along with the albumen gland
and tissues for histological analysis. The total number of eggs laid, total number of newly hatched
snails and number of newly hatched snails/eggs laid were higher in the infected snails, but the
differences observed were not significant. The galactogen content in the albumen gland was
significantly reduced from the second week post exposure onward. Also, in spite of presence of larvae
in the gonadal region of the snail with granuloma-like structures and some histological changes,
gametogenesis occurred regularly in the infected hosts. The results are worrisome, since they showed
that B. tenuissimus is an intermediate host to A. cantonensis which supports infection without changes
in its reproductive biology and activity. The present study sheds more light on a snail that is a newly
recorded experimental and natural intermediate host of A. cantonensis, which is widely distributed and
occurs in areas were neural angiostrongyliasis has been recorded. Therefore, this snail can act in the
nature as an important agent for dispersion of neural angiostrongyliasis, posing a serious problem to
human and animal public health.
Reproductive Response Of Bulimulus Tenuissimus (Mollusca) As An Intermediate Host Of Angiostrongylus Cantonensis (Nematoda)
LUMEN ET VIRTUS, São José dos pinhais, v. XV, n. XXXVIII, p.169-184, 2024 169
Keywords: comparative physiology, host-parasite relationship, reproductive biology,
Metastrongylidae, Bulimulidae.
1 INTRODUCTION
The molluscs are important elements in the life cycle of many parasites, thus attracting strong
interest as targets control programs. The snail Bulimulus tenuissimus (d’Orbigny, 1835) is a terrestrial
gastropod, from family Bulimulidae (Salgado and Coelho, 2003), widely distributed in North America
– specifically Mexico (Fahy 2003) and North Carolina (USA) (Robinson and Slapcinsky 2005) - and
South America (Agudo-Padrón 2014, Agudo-Padrón and Lenhard 2011, Breure and Avila 2016).
Despite its widespread distribution in the Americas, where it serves as an intermediate host for several
parasitic trematodes and nematodes, as well as attacking crops (mainly those for subsistence, such as
vegetable gardens), there are few studies of the physiology, biology and behavior of this snail species
(Silva et al. 2008, 2011).
The nematode Metastrogylidae Angiostrongylus cantonensis (Chen, 1935) is a parasite
naturally found in rodents, but occasionally parasitizes humans causing neural angiostrongyliasis or
eosinophilic meningoencephalitis (Hsu et al. 1990). The first case of neural angiostrongyliasis was
reported in 1945 (Australia) (Prociv and Carlisle 2001). Since then, the presence of human infections
by A. cantonensis has been recorded in Southeast Asia and the Pacific Islands (Kim et al. 2014) and in
different Brazilian regions (Thiengo et al. 1995, 2010, Caldeira et al. 2007, Morassuti et al. 2014). The
larval development of A. cantonensis occurs in a snail from L1 to L3, after which the larvae are shed
to infect a definitive host. In the literature several species of molluscs have been identified as A.
cantonensis intermediate host, but almost all studies have been conducted for epidemiological
purposes. Recently, B. tenuissimus was reported as a natural and experimental host of this nematode
(Ramos-de-Souza et al. 2018; Martins et al. 2018).
Brockelman performed the first studies of the host-parasite relationship focusing on
physiological changes in the terrestrial snil Achatina fulica induced by A. cantonensis infection in the
1970s, (Brockelman et al. 1976, Brockelman 1978, Brockelman and Sithitavorn 1980). Only in the
present decade, have new studies been conducted investigating different snail as potencial host and
their physiological response to parasitism by A. cantonensis (Tunholi-Alves et al. 2011, 2014, 2015).
Tunholi-Alves and co-workers were the first to report the reproductive changes caused in the
freshwater snail Biomphalaria glabrata infected with A. cantonesis, describing the occurrence of
partial parasitic castration of B. glabrata infected by A. cantonensis larvae, probably in response to the
depletion of energy reserves, since the authors did not observe injuries to the gonadal tissues (Tunholi-
Alves et al. 2011). Lima et al. (2017) also reported reproductive changes in Biomphalaria tenagophila
LUMEN ET VIRTUS, São José dos pinhais, v.37, n.16, p.169-184, 2024 170
and Biomphalaria straminea infected with A. cantonesis. But, there is still doubt about terrestrial
snails. Since the natural definitive hosts are rodents, why are there so few studies about host-parasite
relationship including A. cantonensis-terrestrial snails?
The present study aimed, for the first time, to find evidence of the changes in the reproductive
biology of B. tenuissimus, through biological, biochemical and histological analyses, as a consequence
of infection by A. cantonensis.
2 MATERIALS AND METHODS

2.1 MAINTENANCE OF THE SNAILS AND FORMATION OF GROUPS
Samples of the mollusc B. tenuissimus were manually collected, in the early morning, from
vegetable gardens located in Seropédica, RJ, Brazil (Latitude 22º 44' 38" S; Longitude 43º 42' 27" W;
Altitude 26m). The collected molluscs were maintained in plastic recipients under
laboratory conditions, with the bottom covered by vegetable soil (2cm), bedewed
on alternate days by using tap water.
The molluscs were fed with fresh lettuce leaves and slices of chayote, cucumber and carrot ad
libitum, with the food being replaced on alternate days.
The eggs were collected and transferred to new recipients prepared as described above. The
newly hatched snails that reached 90-100 days-old and had, at least, 10 mm in shell length, were used
to form six groups: three control (uninfected) groups (n=90), and three infected groups (n=90), each
group with 30 specimens. All the groups were formed using triplicates (ntotal=180). The experimental
observations lasted for three weeks, which corresponds to the pre-patent period of infection in the
intermediate host (Lima et al. 2017).
2.2 PARASITES
Molluscs A. fulica, naturally infected, were collected in the municipality of São Gonçalo, RJ,
Brazil in 2015, in the area surrounding the home of a patient diagnosed with eosinophilic
meningoencephalitis Third-stage larvae (L3) of A. cantonensis, obtained from naturally infected A.
fulica were inoculated in R. norvegicus in the Laboratory for Biology and Parasitology of Reservoir
Mammals (LBPMR) of Oswaldo Cruz Institute (Fiocruz), where the cycle is maintained under
laboratory conditions. All the first-stage larvae (L1) utilized in this study came from this
experimentally maintained life cycle of A. cantonensis.. This study was approved by the Oswaldo Cruz
Foundation Ethics Committee on Animal Use (CEUA/IOC-025/2018).
2.3 EXPERIMENTAL INFECTION OF THE SNAILS
Rodents R. norvegicus experimentally infected and maintained as previously described were
used to feces obtaining, from which the first-larvae were collected (Baermann technique - Willcox and
Coura 1989). Then, L1 larvae of A. cantonensis were spread on thin slices of fresh cucumber placed
in 24-well plates molluscs B. tenuissimus were individually placed on top of the cucumber slices, being
exposed to 1,200 L1 larvae. The plates were closed, allowing the contact among molluscs and larvae
for 24 h. The uninfected snails were submitted to the same procedure with distilled water instead of
the L1 larvae. The onset of infection was monitored under a stereomicroscope and some molluscs were
observed ingesting the larvae. After 24h the content of each well was observed under a
stereomicroscope to detect larvae (L1 stage) (Tunholi-Alves et al. 2011). After that, the molluscs
exposed/infected were transferred to the terrariums to form the experimental groups.
2.4 REPRODUCTIVE ACTIVITY ANALYSIS

The molluscs placed in each terrarium were observed daily for three weeks. The number of live
snails was recorded and the earth of each terrarium was gently examined to look for the eggs. Next,
the eggs found were counted and transferred to new terrariums (nursery) and daily observed until their
hatching.
The reproductive parameters observed were the following:: total number of eggs laid, total
number of newly hatched snails, and number of newly hatched snails/eggs laid.
2.5 DETERMINATION OF THE GALACTOGEN CONCENTRATION

To galactogen determination, albumen gland was obtained from dissection of 25 molluscs (each
group, ntotal=25) under stereomicroscope, without anesthesia, with tissues maintained in ice bath during
dissection and stored at -20 ºC. Galactogen was extracted according to Pinheiro and Gomes (1994),
and its content was determined by the 3,5 DNS technique (Sumner 1924) and expressed as mg
galactose/g tissue, wet weight.
2.6 HISTOLOGICAL AND HISTOCHEMICAL ANALYSES

Five molluscs foram each group (ntotal=15) had their shells of removed and the tissues were
fixed in Duboscq-Brasil fixative (Fernandes, 1941), for 24 hours at 4 ºC, and were processed according
routine histological techniques (Humason 1979). Sections (5 µm) were obtained. The slices were
stained using hematoxylin and eosin or Gomori’s trichrome techniques to observe the tissue
morphology. The galactogen deposits were evidenced by Periodic Acid Schiff (PAS) reaction
(Humason, 1979).
2.7 STATISTICAL ANALYSES
The total number of eggs laid, total number of newly hatched snails, and number of newly
hatched snails/eggs laid results were expressed as mean ± standard error of mean (X ± SEM). The
relation between the parameters analyzed and the time of infection was determined by polynomial
regression test (Graph Pad Prism, Prism Inc.). The means observed in the different periods were
analyzed by Tukey-Kramer test and ANOVA (InStat, v. 4.00, Prism Inc.).
3 RESULTS
The total number of eggs laid by B. tenuissimus did not vary significantly between uninfected
and A. cantonensis-infected snails or during the different infection periods (Tab. 1). The mean number
of eggs laid was higher in infected snails throughout the three weeks after exposure, where the highest
value was observed in the first week post exposure (236.43 ± 45.94), 7.0% higher than that observed
among uninfected snails. One-way ANOVA indicated no significant difference among the values
observed in the different weeks (P<0.05). Polynomial regression revealed a weak positive relation
between the period of infection and the total number of eggs laid by infected B. tenuissimus (Fig. 1A),
showing a tendency to reach the normal oviposition value at the end of pre patent period.
The viability of the eggs laid by B. tenuissimus infected with A. cantonensis was evaluated by
analyzing the total newly hatched snails from eggs laid by infected snails in comparison with
uninfected ones. In spite of the absence of significant difference among the values obtained (P<0.05),
the same variation pattern observed for the number of eggs laid was observed here. The greatest
hatchability was observed in the third week post exposure (241.75 ± 34.69), 23.30% higher than that
observed in the control group (Fig. 1B).
The ratio increased continuously with the progress of infection reaching the maximum value in
the third week after exposure (1.07 ± 0.15), an increase of 20.49% in relation to the control group
(Table 1). Also, this was the only period of infection in which the ratio between the newly hatched
molluscs and the laid eggs was significantly higher than that observed in the control group (P<0.05).
In addition, there was a weakly significant relationship between the ratio (number of newly hatched
snails/eggs laid) and the period of infection (Fig. 1C).
The galactogen content showed a weak positive relation with infection period. However,
despite the reduction (Fig. 1D) in the third week after exposure, one-way ANOVA (P<0.05) did not
revealed significant differences between the values observed for the control group and the three periods
of infection analyzed.
Histological analyzes of uninfected snails showed ovotestis with normal characteristics,
presenting oocytes and spermatozoa in different developmental stages (Fig. 2A), and the albumen
gland with regular tubular structure in the uninfected snails (Fig. 2B). The presence of oocytes and
spermatozoa in the ovotestis of B. tenuissimus experimentally infected with A. cantonensis was
confirmed throughout the prepatent period of infection (Figs. 2C-D), indicating the normal functioning
of the gonads. Only in the first week post exposure, were larvae observed in the ovotestis tissues. Fig.
2C shows a granuloma-like structure with two larval profiles, where the tissues as a whole are
structurally changed in comparison with the uninfected snails. Nevertheless, the observation of
histological sections of other infected snails in the same period after exposure (Fig. 2D) indicated a
gonad with normal appearance, evidencing differences in the individual response to parasitism in this
host-parasite system. Similar results were observed in the snails in the third week after exposure, where
some had morphological alterations (fig. 2E) and others had no alterations in the structure of the
ovotestis (Fig. 2F). Despite the changes observed, all the sections analyzed contained oocytes and
spermatozoa with morphology preserved.
4 DISCUSSION
Changes in reproductive biology have been recorded in many relationships between different
organisms for more than 100 years (McCrady 1874, Malm 1881). However, Giard, in a sequence of
studies published from 1886 to 1913 (Giard 1886, 1887, 1889, 1911-1913, Giard and Bonnier 1887)
was the first to propose the use of the term “parasitic castration” to refers to reproductive changes
observed in host-parasite relationships. Research in this field has been focused on the effects of
trematode larval development and the consequences on the reproduction of the snails’ first intermediate
host during their life cycle (McClelland and Bourns 1969, Becker 1980, Tunholi et al. 2011).
In many experimental models, total or partial inhibition of the reproductive activity in the
relationships between helminths and molluscs has been recorded (Baudoin 1975). In spite of the great
emphasis on the issue in the international scientific literature, the effects of the parasitic infection by
larval nematodes on the reproduction of intermediate snail hosts have largely been neglected. This lack
of information is particularly glaring regarding the relationship of A. cantonensis with its intermediate
snail host. Although the species was first described in 1935, the first studies about physiological
changes in A. fulica infected with A. cantonensis were only published in the 1970s (Brockelman 1978,
Brockelman et al. 1976).
This study presents the first report of the effects of experimental infection with A. cantonensis
L1 larvae on the reproductive biology of B. tenuissimus and our data indicate the infection did not
significantly affect the reproductive parameters analyzed. Lima et al. (2017) compared the effects of
the infection by this species of nematode on the reproduction of B. straminea and B. tenagophila and
observed marked parasitic castration in both parasitized molluscs. Tunholi-Alves et al. (2011) observed
similar results for B. glabrata infected by A. cantonensis larvae. Interestingly, even though this parasite
uses a terrestrial rodent as definitive host, most studies focus on interactions with the intermediate
hosts freshwater snails.
The total number of eggs laid by infected B. tenuissimus varied more in the snails in the first
week after exposure compared to the uninfected ones, but from this point onward only small
differences were observed with the values of the control group. Based on the number of newly hatched
snails, the infection seemed not affect the viability, since mean values were always higher than those
observed among uninfected snails, resulting in a rising mean ratio between these parameters
throughout the development of the infection. These results differ from those recorded by the authors
in both studies mentioned above. The increasing ratio of newly hatched snails/eggs laid showed that
the infection leads to higher energy demand and burden on reproductive biology of B. tenuissimus in
the first week after exposure. However, with the progress of the infection, a reduction of the
reproductive efforts by the infected snails clearly occurred, increasing the number of newly hatched
snails/eggs laid, which can be interpreted as rising reproductive success of the parasitized hosts. At the
end of the prepatent period, the infected snails had a reproductive success rate 20.49% higher than the
uninfected ones, showing the occurrence of a fecundity compensation process in response to the
parasitism by A. cantonensis larvae. Minchella called fecundity compensation the sharp increase of
snail’s egg laying following parasite exposure (Minchella and LoVerde 1981; Minchella 1985)
describing it as an adaptive physiological response to trematode parasitism. Davis et al. (2022)
described this phenomenon as “life-history response, occurring when hosts increase their current
reproductive output to make up for expected losses in future reproduction due to parasitic infection”.
The term is currently used more broadly to indicate patterns of increased reproductive effort in host-
parasite relation, as a way for compensate future or past losses. In addition, Tunholi et al.(2011)
showed that the infection of the freshwater snail B. glabrata triggered the same kind of response to
Echinostoma paraensei larval infection. Duffield et al. (2017) stated that reproductive investment
made in fecundity compensation “can be allocated towards an increase in the quantity and/or quality
of offspring”, and our results showed that B. tenuissimus infected with A. cantonensis, not only increase
the eggs production (number of eggs/snails), but also produces embryos with high viability and
hatchability.
Galactogen is a galactose polymer restricted to the albumen gland. It is produced as an
important source of energy for the embryo during its development in the egg (Goudsmit and Ashwell
1965, Nieland and Goudsmit 1969). Therefore, this polysaccharide is an essential component to assure
the normal development of the embryo and newly hatched snails. Therefore, the progressive reduction
observed in our study seems to be more related to the use of this substance by the snail to the increase
egg production, since galactogen reduction is inversely related to the reproductive success, expressed
as number of newly hatched snails/number of eggs laid by A. cantonensis-infected B. tenuissimus. A
similar pattern was observed in B. straminea infection (Lima et al. 2017). Also, Tunholi-Alves et al.
(2011) observed that in B. glabrata infected with A. cantonensis, all the reproductive parameters
analyzed were significantly reduced. In turn, Pinheiro and Amato (1995) observed parasitic castration
in Bradybaena similaris infected with larvae of the digenetic trematode Eurytrema coelomaticum, but
fecundity compensation did not happen in this host-parasite relationship. This diversity of reproductive
responses observed in the different host-parasite associations, indicates that each relationship involves
a particular response, reflecting the coevolution of the organisms and their adaptations to parasitic
coexistence. For this reason, further studies about these new interrelationships are needed, to obtain
information that in the future, can be used in programs to control parasitic diseases caused by these
helminths.
The histological analysis showed that changes in the gonad tissues may occur, but it was not a
general response of all the infected snails, reflecting an intraspecific variation pattern in the tissue
response. Even when histological changes were observed, it was possible to observe gametes in
different developmental stages, indicating that gametogenesis was occurring normally. These
morphological findings reinforce the oviposition activity and egg viability found in our study.
Thompson and Kavaliers (1994) stated that in many host-parasite systems, parasitism does not result
in physical damage or destruction of the host's reproductive organs, but even so, parasitic castration
occurs. In B. tenuissimus-A. cantonensis system it seems that the opposite occurred: in spite of the
presence of larvae and the disorganization of the structural features of the ovotestis and albumen gland
tissues, the reproductive activity proceeded normally.
In conclusion, the results presented in our study are very worrisome and denotes are important
to veterinary medicine and public health. Pien and Pien (1999) stated that A. cantonensis has spread
from Southeast Asia to the South Pacific, Africa, India, the Caribbean, Australia and North America,
but this distribution certainly became wider in the ensuing decade (Foronda et al. 2010). In the present
study, B. tenuissimus was able to withstand exposure to 1,200 L1 larvae and the prepatent
intramolluscan larval development did not significantly affect the reproductive activity, according to
the parameters analyzed, or the internal morphology.
Three factors should be considered: 1 - the presence of snails of the B. genus in many countries
of North and South America, in many of which the occurrence of A. cantonensis has also been
recorded; 2 – the snail B. tenuissimus is frequently found in gardens and other planted areas, especially
subsistence crops; and, 3 - the habits of smallholders of not using personal protective equipment. These
characteristics cause a high risk of exposure to infection by A. cantonensis larvae.
Neural angiostrongyliasis is a neglected disease that is now disseminated in many countries.
Our results bring new information and call attention to a largely overlooked snail host, but one whose
infection can became a serious problem to public and wildlife health.
FUNDING
This study was supported by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do
Rio de Janeiro (FAPERJ, process number: E-26/203.004/2016) and Conselho Nacional para o
Desenvolvimento Científico e Tecnológico (CNPq, process numbers: 305587/2015-3; 447211/2014-
5; 303248/2018-1). This study was also financed in part by the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES, finance code 001).
DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
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FIGURES
Figure 1. Reproductive changes in Bulimulus tenuissimus experimentally infected with 1,200 L1 of Angiostrongylus
cantonensis for three weeks. Week 0 represents the average of the control groups during the three weeks of analysis, since
there was no significant difference during the three weeks.
Figure 2. Histological sections of the ovotestis region of Bulimulus tenuissimus experimentally infected with 1,200 L1
larvae of Angiostrongylus cantonensis, for three weeks. A. Uninfected snail showing normal aspect of the gonad, containing
sperm (s) at different stages of development and oocytes (ov). B. Albumen gland (a) of uninfected snail. C-F. Ovotestis
region of infected snail, with larvae developing in the tissues of the organ (white arrow) along with hemocyte infiltrates
(black arrow), albumen gland (a), but still with the presence of sperm (s) and oocytes (ov) in different stages of
development. Granuloma (g) formed around the developing larva.A -B, stained by hematoxylin and eosin; C- F, stained by
Gomori's trichromic.
Table 1 Changes in the total number of eggs laid and number of newly hatched snails/eggs laid by Bulimulus tenuissimus
during three weeks after exposure to Angiostrongylus cantonensis L1 larvae. Week 0 represents the average of the control
groups during the three weeks of analysis, since there was no significant difference among the values observed in the three
weeks analyzed.
Total number of Number of hatched

eggs laid snails/eggs laid
Period of
infection
(Weeks) X±SD (%)
0 237.00 ± 33.55 0.89
1 236.43 ± 45.94 0.92
2 209.00 ± 25.47 0.93
3 226.00 ± 30.09 1.07

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Reproductive Response Of Bulimulus Tenuissimus (Mollusca) As

An Intermediate Host Of Angiostrongylus Cantonensis

Florence Gonçalves Martins

Marcos Antônio José dos Santos

Juberlan Silva Garcia

2 MATERIALS AND METHODS

2.4 REPRODUCTIVE ACTIVITY ANALYSIS

2.5 DETERMINATION OF THE GALACTOGEN CONCENTRATION

2.6 HISTOLOGICAL AND HISTOCHEMICAL ANALYSES

DECLARATION OF COMPETING INTEREST

Agudo-Padrón, A. I., 2014. Inventario sistemático de los moluscos continentales ocurrentes en el

Giard, A., (1911-1913). “Oeuvres Diverses.” Laboratoire d’Evolution, Etres Organ,

McClelland, G., Bourns, T. K. R. 1969. Effects of Trichobilharzia ocellata on growth, reproduction,

Total number of Number of hatched

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