An Acad Bras Cienc (2020) 92(1): e20190298 DOI 10.1590/0001-3765202020190298 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal BIOLOGICAL SCIENCES Behavioral response of Biomphalaria glabrata Running title:Behavioral response exposed to a sublethal concentration of B. glabrata exposed to E. milii of Euphorbia milii var. hislopii latex Academy Section: BIOLOGICAL SCIENCES ANNA CARLA ALBERTO-SILVA, RODOLFO A. CUNHA, VALDIR A. DA COSTA, EVERTON GUSTAVO N. DOS SANTOS, MAURÍCIO C. DE VASCONCELLOS, CLÉLIA CHRISTINA MELLO-SILVA & CLÁUDIA P. SANTOS e20190298 Abstract: The Euphobia milii var. hislopii latex has been tested in the control of schistosomiasis but its action in the locomotor activity of Biomphalaria glabrata 92 is unknown. The objective of this work was to study the locomotor and reproductive (1) behaviors of B. glabrata exposed to E. milii var. hislopii latex. For this, 96 snails were 92(1) individually exposed to the latex (LC50 - 0.53 mg / L) for 24 hours. The specimens were submitted to biomonitoring for image analysis to record the locomotor parameters at different times: before exposure (control), one day post exposure (group 1 d-p-e) and 30 days post exposure (group 30 d-p-e). The reproductive parameters were recorded weekly for 10 weeks. All locomotor activities of group 1 d-p-e decreased significantly. The egg/egg mass ratio decreased the week after exposure while there was an increase in the hatching rate. After 30 days, these reproductive parameters were similar to those observed in these same snails before exposure. However, the number of hatched snails declined after exposure until the end of the experiment. The influence of the latex in the parameters of B. glabrata added further evidence that this natural water-soluble product can be an important tool for the control of schistosomiasis. Key words: behavior, biomonitoring, Biomphalaria glabrata, control of schistosomiasis, Euphorbia milii var. hislopii, freshwater environments. INTRODUCTION because they occupy intermediate trophic levels in the ecosystem and act as fundamental links Alterations of freshwater environments are in the food chain (Abílio et al. 2007). Aquatic caused by contamination by organic and snails have been the subject to toxicological inorganic wastes, environmental catastrophes studies because despite being tolerant to and climatic changes, interfering in the trophic organic pollution, they are vulnerable to other relations and the flow of energy and biomass pollutants even at low concentrations (Oliveira- (Perkins et al. 2010, Ledger & Milner 2015, Filho & Paumgartten 1997, Abd Allah et al. 2003). Woodward et al. 2016). Freshwater organisms Schistosomiasis is the main helminth- are influenced by these environmental factors, borne disease in the world and Biomphalaria so they are often used as bioindicators of glabrata (Say, 1818), the main intermediate contamination (Hook et al. 2014, Habib et al. host of Schistosoma mansoni (Sambon 1907) in 2016). Among the aquatic organisms used in Brazil, has been used as a study model due to biomonitoring programs, mollusks are relevant its wide geographical distribution, long life cycle An Acad Bras Cienc (2020) 92(1) ANNA CARLA ALBERTO-SILVA et al. Behavioral response of B. glabrata exposed to E. milii and easy maintenance in the laboratory (Hotez were also observed the infected snails, which et al. 2009, Scholte et al. 2014). The chemical were eliminating cercariae were less motile compound most commonly used in campaigns and the number of cercariae shed was directly to control these vectors is niclosamide, which associated with the reduction/interruption causes high mortality of fish, among other non- in egg-laying with an increase in random target organisms (Vega et al. 1988). Due to the movement (Alberto-Silva et al. 2015). However, impact of this product on the environment, the these experiments did not include the use of Ministry of the Environment recommends its the natural moluscicide E. milli var. hislopii latex. use in Brazil only for areas with high prevalence The E. milii var. hislopii latex has been (Brasil 2006). As regards natural molluscicides, tested in the control of schistosomiasis but its Euphorbia milii var. hislopii satisfies the action in the locomotor activity of Biomphalaria recommendations of the WHO for efficiency in glabrata is unknown. The objective of this work the control of mollusks at lethal concentrations was to study the locomotor and reproductive (LC90) between 1.5 mg/L and 5.0 mg/L. The latex behaviors of B. glabrata exposed to E. milii var. photo degrades over a period of 30 days (Schallet hislopii latex. al. 1992, Oliveira-Filho & Paumgartten 1997) and has no carcinogenic, mutagenic or embryotoxic effects on mammals at lethal concentrations MATERIALS AND METHODS to snails (Vasconcellos & Amorim 2003). The latex significantly reduces the elimination of Ethics S. mansoni cercariae by snails and has been This study was approved by the Animal Ethics tested as a natural product to control the Committee of Oswaldo Cruz Foundation (CEUA/ transmission of schistosomiasis (Augusto et IOC 016/2015), in accordance with the guidelines al. 2015). Recently, Augusto et al. (2016, 2017) of the Brazilian Society of Laboratory Animal analyzed the chemical composition of the latex Science (COBEA). This study is registered in the and confirmed the efficacy of this latex as a National System of Management of Genetic natural molluscicide for schistosomiasis vector Heritage and Associated Traditional Knowledge control using comparative transcriptomics and - SisGen (no. A9666E5). proteomics. It was proved that the latex changes the morphological structure of adult parasites obtained from cercariae exposed to the latex, Euphorbia milii var. hislopii latex thus impeding the normal development of The E. milii var. hislopii latex was collected in adults. the Ilha do Governador district (22°48´09´´S/ Behavioral, reproductive and physiological 43°12´35´´W), Rio de Janeiro, Brazil, on May 4, changes which may occur in B. glabrata have 2015 by the first author. been reported in different situations, such as The latex in natura was lyophilized on 6th parasitic infection, starvation, aestivation and May of the same year and used in the trial in exposure to molluscicides (White et al. 2007, the months of July and August 2015, according to Faro et al. 2013, Mello-Silva et al. 2011). In our the method described by Augusto et al. (2016). previous paper we video-tracked the locomotor The lyophilized pellet obtained was diluted in activity of B. glabrata before and after infection distilled water and homogenized by sonication by S. mansoni. Their reproductive parameters (UltraSonic Clean S-1600/HM 230V, frequency of An Acad Bras Cienc (2020) 92(1) e20190298 2 | 10 ANNA CARLA ALBERTO-SILVA et al. Behavioral response of B. glabrata exposed to E. milii 40 kHz) for 20 min. After this, the stock solution concentration (LC50 - 0.53 mg / L) of the latex at a concentration of 100 mg/L was prepared and for 24 hours. After this period, they were placed from this solutions at different concentrations back in the dechlorinated water. were prepared for use in the bioassays (0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 5.0 and 10.0 mg/L). This Locomotor activity of Biomphalaria glabrata procedure followed the method described and The analyses of the snails’ locomotor activity employed by Vasconcellos & Amorim (2003). The were performed before and after exposure to the sublethal concentration (LC50) in this experiment latex, using the image analysis biomonitoring was 0.53mg/L. system (IABS) (Columbus Instruments, Ohio, USA) with the Travelled Distance of Multiple Objects Raising Biomphalaria glabrata and exposure software, according to Alberto-Silva et al. (2015). to E. milii var. hislopii latex The biomonitoring was realized with the same In this experiment, we used 96 specimens of B. snails at three times: before exposure (control glabrata (BH lineage) aged six weeks, raised in group), one day post-exposure (1 d-p-e) and 30 the Schistosomiasis Laboratory of Fiocruz, which days post-exposure (30 d-p-e.). Each analysis has a quality policy with traceability system of was performed for 1 hour 20 minutes, with the all parameters of their lyfe-cycle. Therefore, first 20 minutes being for acclimation and 1 ween sure that the snails used in this study had hour of video analysis recorded at 60 intervals the same age, since they came from egg-laying of 1 min each. All values for each interval of masses of the same week. For this experiment, five parameters of locomotor activity were we monitored this group of mollusks until they used for statistical analysis: distance traveled, reached sexual maturity around five weeks ambulatory time, stereotypic time, resting time, of age (young). The mollusks of this group and average speed (Alberto-Silva et al. 2015). measuring 8 to 12 mm were selected to be tested. In this 60-day period (observation time of this Reproductive parameters of Biomphalaria experiment), the mollusks are still considered glabrata young. Under optimal laboratory conditions, The reproductive parameters were also analyzed the mollusks have a longer life spanup to 24 weekly, five weeks before exposure (control months (Brasil 2008). The mean weight of the group) and five weeks afterward (1 d-p-e and specimens was 0.34g (SD: 0.09, range 0.10 -0.27g) 30 d-p-e groups). We analyzed the following and the shell diameters ranged from 8 to 12 mm. reproductive parameters: fecundity- total number The snails were individually kept in beakers of egg masses, total number of eggs, number of (100 mL) with dechlorinated water, which was eggs/egg mass; and fertility- number of hatched changed weekly. Pieces of styrofoam were put eggs and hatching rate. All parameters were on the water for egg laying and also replaced analyzed using a stereomicroscope.