Elsevier

Acta Tropica

Volume 222, October 2021, 106067
Acta Tropica

Sublethal concentrations of usnic acid potassium salt impairs physiological parameters of Biomphalaria glabrata (Say, 1818) (Pulmonata: Planorbidae) infected and not infected with Schistosoma mansoni

https://doi.org/10.1016/j.actatropica.2021.106067Get rights and content

Abstract

Schistosomiasis is a public health problem in many developing countries. The mollusc Biomphalaria glabrata is the most important vector of Schistosoma mansoni in South America. The population control of this vector to prevent the spread of schistosomiasis is currently done with the application of highly toxic molluscicide to the environment. The screening of substances in sublethal concentrations that have deleterious effects on physiological parameters is very relevant for the control of schistosomiasis, since the effectiveness of disease prevention increases if it acts on population control of the vector and on reproduction and elimination in S. mansoni cercariae. The objective of this study was to evaluate the reproductive parameters (fecundity and fertility), intra-mollusk effect (sporocysts I (72 h) and II (14 days after)) on the development of cercariae of S. mansoni and the immune cell profile of B. glabrata exposed to sublethal concentrations (LC25 - 0.5 µg/mL and LC50 - 0.92 µg/mL) of the usnic acid potassium salt (potassium usnate). LC 25 and LC 50 significantly reduced (p < 0.05) the fecundity of B. glabrata when treated infected and/or not exposed to infection, while unviable embryos were not observed in sporocyst stage I, being only significant (p < 0.05) for mollusks infected and treated with LC50 on sporocyst II. LC25 and LC50 of the potassium usnate caused significant reductions (p < 0.05) in the production and cercarial shedding when evaluated on sporocysts I and II. In addition, the mortality of infected and treated B. glabrata in the sporocyst II phase was quite marked after the 9th week of infection. Regarding the immunological cell profile of uninfected B. glabrata, both concentrations led to immunomodulatory responses, with significant morphological changes predominant of hemocytes that entered programmed cell death (apoptosis). It was concluded that the application of LC25 and LC50 from the potassium usnate could be useful in the population control of B. glabrata, since it interferes both in their biology and physiology and in the reproduction of the infectious agent of schistosomiasis mansoni.

Introduction

Schistosomiasis is one of the main diseases of socioeconomic importance and public health in the world, especially for developing countries (Utzinger et al., 2011; Savioli et al., 2017). Freshwater snails of the genus Biomphalaria naturally infected are responsible for the transmission of the disease in South America, mainly in Brazil (Caldeira et al., 2016). Within the genus, mollusks of the species B. glabrata (Say, 1818) are considered the main intermediate hosts of this parasitosis, due to their greater susceptibility to infection and effectiveness in the transmission of schistosomiasis. It has also been widely used as a study model due to its wide geographic distribution, long service life, short embryo cycle and easy maintenance in the laboratory (Scholte et al., 2014; Cousteau et al., 2015; Famakinde et al., 2017).

The population control of the intermediate hosts of Schistosoma spp. is carried out through the application of molluscicides. It is estimated that more than 7000 chemicals have been tested to control these mollusks, such as copper sulphate, gramoxone, calcium hydroxide, N-tritylmorpholin (Frescon) and niclosamide, the latter still in use (Oliveira-Filho et al., 2010; WHO, 2017). Although this method is considered one of the most effective (King et al., 2015; Sokolow et al., 2016; 2018), the application of niclosamide causes high fish mortality, among other non-target organisms present in the aquatic ecosystem (Oliveira- Filho and Paumgartten, 2000; WHO, 2017; Zhu et al., 2020). Due to the impact on the local fauna and flora, for example, government technical guidelines in Brazil recommend the use of niclosamide only for areas with a high prevalence of the disease (Brasil, 2006).

With regard to molluscicidal candidates, the search for molluscicides of natural origin/derived has gained prominence all over the world, with the objective of obtaining efficient products for the control of mollusc populations of medical epidemiological importance (Bakry et al., 2009; 2011; Oliveira-Filho et al., 2010; Rizk et al., 2012; Albuqurerque et al., 2014; Martins et al., 2014; Rocha-Filho et al., 2015; Araújo et al., a,2018b,2018c;2018d). Lichens are a symbiotic association of interspecific mutualism between fungi (mycobiont) and algae or cyanobacteria (photobiont). Lichens produce about 1000 secondary metabolites, of which more than 80% are exclusive to the mononuclear, aromatic, depsidone, diphenyl ether and dibenzofuran (usnic acid) classes (González-Burgos et al., 2019; Ranković and Kosanić, 2021). Usnic acid is uniquely found in lichens, and is especially abundant in genera such as Alectoria, Cladonia, Usnea, Lecanora, Ramalina and Evernia widely distributed in all continents and territories of the globe (Ahti et al., 1993; 2000; Ingólfsdóttir, 2002; Calcott et al., 2018; Ranković and Kosanić, 2021). Usnic acid is found naturally in two enantiomeric forms, such as (-) levogyrous and (+) dextrogyrous, due to the angular projection of the methyl group located in position 9b (Ingólfsdóttir, 2002; Cazarin et al., 2021). Although usnic acid is a molecule with an initial report available in the literature in the 1950s, whose objective was the search for new antibiotics (Bustinza, 1952), even today several biological activities and toxicity attributed to usnic acid are reported (Galanty et al., 2019; González-Burgos et al., 2019; Macedo et al., 2020), among them we highlight antimicrobial, (Goel et al., 2021), antifungal (Kumar et al., 2019), antiviral (Sokolov et al., 2012), antitumor properties (Zakharenko et al., 2019), antiparasitic (Si et al., 2016), insecticide (Martins et al., 2018) and recently molluscicide (Araújo et al., 2018a;2018b). However, usnic acid has low water solubility, a limiting factor for its use, especially against target organisms in aquatic environments (Jin et al., 2013). Furthermore, usnic acid presents significant toxicity to environmental bioindicators in molluscicide concentrations (Araújo et al., 2018a;2018b). In an attempt to increase solubility and reduce environmental toxicity, usnic acid can be used in an acid-base reaction, giving rise to its derivative the usnic acid potassium salt (potassium usnate), a molecule that is completely soluble in water.

The potassium usnate falls within the technical recommendations of the World Health Organization, proving to be an efficient molluscicide in the population control of adult molluscs of B. glabrata, with a lethal concentration (LC50) of 0.92 µg/mL and lethality for all molluscs at 1 µg/mL without toxicity to Artemia salina nauplii, environmental bioindicator (World Health Organization, 1965; Martins et al., 2014). Recently, Araújo et al. (2018c,2018d) confirmed the efficacy of the potassium usnate as a promising molluscicide by observing a toxic and teratogenic effect in the different embryonic stages of B. glabrata and mortality of S. mansoni cercariae (infectious agent). In addition, the potassium usnate showed a schistosomicidal effect on evolutionary stages of S. mansoni, absence of cytotoxicity on human cells and absence toxicity to mammals (Araújo et al., 2019a;H.D.A. 2019b; 2020a;2020b) in lethal concentrations for mollusks.

The candidates for molluscicides when applied to water resources in sublethal concentrations represent a favorable way for the population control of molluscs, causing no significant changes in the environment, taking into account that molluscs are also affected by the infection of the miracidium of Schistosoma spp. (Cousteau et al., 2015; Famakinde et al., 2017). The penetration of the miracidium occurs in any point of the exposed parts of the snail, however, the cephalopodal mass, mainly the base of the antennae and the foot are the points of preference. The larva establishes itself in the subcutaneous tissue and subsequently migrates to the digestive or hepatopancreas glands where, during this path, anatomophysiological changes occur, causing the larva to be called primary sporocyst after 72 h (I). On the 14th day after the penetration of the miracidium, the germ cells are in intense multiplication (polyembryony), with the formation of the secondary sporocyst (II). Migration starts around the 18th day for the digestive gland and, less frequently, for the mollusk's ovotestis (reproductive gland) (Coelho et al., 2008). The formation of cercariae, until their release into the aquatic environment, can occur within a period of 27 to 30 days and a single miracidium can generate about 300 thousand cercariae (Brasil, 2014).

The application of molluscicidal agents or candidates can cause adverse effects on molluscs, causing changes in their homeostasis that need to be evaluated (Rizk et al., 2012; Wang et al., 2017). The adverse effects and homeostatic changes in B. glabrata exposed to sublethal concentrations (LC25 and LC50) of the potassium usnate have never been investigated. Therefore, the present study aimed to evaluate the reproductive parameters (fecundity and fertility) of B. glabrata, survival and reproduction after infection by S. mansoni in the development stages of sporocysts I and II and the answer of exposure to sublethal concentrations LC25 and LC50 of potassium usnate in the reproduction and elimination of cercariae. Finally, to evaluate the immunological aspects, through the evaluation of the hemocyte cell profile and possible morphological changes after treatment with both concentrations of potassium usnate.

Section snippets

Lichen collected, purification of usnic acid and synthesis of potassium usnate

Samples of Cladonia substellata (Vainio, 1887) were collected in February 2019 in the city of Mamanguape, Paraíba (Northeastern Brazil), on the margins of the Federal Highway BR-101, at coordinates 6º42′17.7′'S 35º7′3.4′'W . The specimen was deposited in the Geraldo Mariz herbarium of the Department of Botany at the Federal University of Pernambuco, Recife/PE, Brazil voucher nº 85.216.

Organic extracts were obtained by an exhaust system at room temperature (28 ± 2 °C) from dry lichen thallus

Chemical analyzes to confirm the molecular structure of the usnic acid and potassium usnate

Purified usnic acid was obtained from ethereal, chloroform and acetone extracts from C. substellata. Data on purified substance were analyzed by TLC and HPLC with Rf value of 0.81 and RT 20.12 min, respectively (99.59% purity), consistent with standard usnic acid (Rf 0.81, RT 20.49 - (99.83% purity, Merck®, Darmstadt, Germany). The optical rotation of usnic acid was α25D 478.2200 (c 1.0 acetone). In this way, the usnic acid used in our study is dextrorotary. The molecular structure of usnic

Conclusions

Exposure of B. glabrata to sublethal doses LC25 and LC50 of potassium usnate reduced the fecundity pattern of molluscs. This pattern was intensified when they were infected with S. mansoni and treated. LC25 and LC50 showed suppression in the transmission of schistosomiasis, reducing infectivity in molluscs, with less production and cercarial shedding, demonstrating an immunomodulation of hemocytes and the presence of apoptotic cells caused by exposure to potassium usnate. LC25 and LC50 of the

Author contributions

H.D.A. Araújo and A.L. Aires designed the study protocol; H.D.A. Araújo, H.M.F. Silva, W.N. Siqueira, M.V. Lima, V.H.B. Santos, J.G. Silva Júnior, N.H. Silva, M.C.P.A. Albuquerque, A.M.M.A. Melo, and A.L. Aires carried out the assays and were involved in analysis and interpretation of all the data; H.D.A. Araújo, A.L. Aires and L.C.B.B. Coelho contributed to drafting the manuscript and/or critically revising the paper and intellectual content. All authors read and approved the final manuscript.

Author statement

The authors confirms sole responsibility for the following: study conception and design, data collection, analysis and interpretation of results, and manuscript preparation.

Declaration of Competing Interest

The authors have no conflict of interest.

Acknowledgments

The authors express their gratitude to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and L.C.B.B. Coelho thanks CNPq by Research Professor, PhD Fellowship (309923/2019-0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE). H.D.A. Araújo thanks FACEPE for the Researcher Fixation Scholarship (BFP-0080-2.08/20). The authors would like to thank Dr. Maria de Lourdes Buril for

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