Traditional knowledge of invertebrates used for medicine and magical–religious purposes by traditional healers and indigenous populations in the Plateau Department, Republic of Benin

Background Since ancient times, invertebrates have played an important role in the traditional medicine in many parts of the world. In south-eastern Benin, more specifically in the Plateau Department, invertebrates are widely used in folk medicine. However, studies on their therapeutic use has been neglected and their magical–religious purposes are poorly understood. The present study aims to document traditional knowledge related to the use of invertebrates for medicinal and magical–religious purposes by traditional healers and indigenous people of Plateau Department. Methods An ethno-sociological survey was conducted with 145 informants (80 traditional healers, 12 merchants of medicinal animals and 53 households) belonging to six ethnic groups, in 20 villages located in Plateau of Benin. Data were collected through the participatory rural appraisal method involving individual interviews and direct observations with semi-structured questionnaires. The collected data regarding various medicinal and magical–religious uses of invertebrates were analysed through informant consensus factor (ICF), use value (UV) and, fidelity level (FL). Results A total of 20 families and 38 species of invertebrates, distributed among 6 taxonomic categories, were found to be used to treat 50 different ailments. Insects occupied 64.7% of the total invertebrates listed. The African earthworm Eudrilus eugeniae K. and African giant snail Achatina achatina L. had the highest use values. The highest ICF value (1.0) was cited for diseases of the blood or blood-forming organs. A principal component analysis (PCA) revealed the influence of ethnic groups in the diseases treated with invertebrates. The highest FL (100%) was recorded for 12 invertebrate species treating various ailments. Most of invertebrate-based remedies were associated with plant species. The mode of administration was mainly oral and topical. Most of the invertebrate drugs were traditionally collected in nature or imported, mainly from Nigeria. In addition, 7 magical–religious practices are documented. Conclusions Our results reveal that several invertebrate species play an important role in healing practices and magical–religious rituals in the Plateau Department. We suggest further studies to confirm the presence of any bioactive compounds on invertebrate species use in traditional medicine. In addition, this study highlights the need for ecological investigations of these species, in order to develop strategies for their conservation and sustainable use.

The exploitation of animals as zootherapeutic resources is one of the economic diversification strategies developed by local populations in Benin [7]. Indeed several animals, including invertebrates, are sold in local markets as medicine. However, several invertebrate groups are threatened with extinction although they are rarely considered in conservation policies [22]. Therefore, it is important to identify the invertebrates sold and the supply sources to devise strategies for their sustainable exploitation in Plateau and in other Departments in the Republic of Benin. This study aims to provide an overview of the use of invertebrates in traditional medicine and magico-religious purposes among traditional healers, merchants of medicinal animals and the people of the Plateau Department in the Republic of Benin.

Study area
The Plateau Department (7°10′ N and 2°34′ E) is located in south-eastern Benin, bordering Nigeria, and covers an area of 3264 km 2 (Fig. 1). This Department is subject to an equatorial Guinean coastal climate characterized by four seasons including two rainy alternating with two dry. The average annual rainfall in the area is 1300 mm. The average monthly temperatures are between 25 and 29°C while the relative humidity of the air oscillates between 68 and 85%. The vegetation is dominated by tree and shrub savannas, shrubby fallows, semideciduous forest patches, gallery forests and mangroves. The soil cover consists mainly of ferrallitic red soils formed on the Continental Terminal, vertisols and vertictic soils, hydromorphic soils and tropical ferruginous soils [23]. With a total population of 622,372 inhabitants, the religions practiced in this Department are Christianity, Islam and Animist [24]. Yorùbá-Nago and Holli are the main ethnic groups encountered in the study area. In each of the five municipalities (Kétou, Pobè, Adja-Ouèrè, Sakété and Ifangni) making up the Department, 20 villages were chosen based on two criteria: the ethnic groups they belonged to and accessibility (Fig. 1).

Survey
Surveys were carried out during the period from December 2018 to March 2019 through the 20 selected villages and public markets of the Department where animals and derivatives are sold for therapeutic purposes. Prior to every interview, we explained the aim of our research and informed consent was obtained from each informant for the use of their knowledge [25,26]. Data were collected using participatory tools and methods such as individual interview, open discussion and direct observation using a semi-structured questionnaire. Interviews were conducted with the help of local interpreters in the languages of the informants. A total of 145 individuals (80 traditional healers, 12 merchants of medicinal animals and 53 households) belonging to six ethnic groups (Yorùbá-Nago, Goun, Mahi, Tori, Holli, Ouémègbé) were interviewed. The surveyed traditional healers were identified with the help of village chiefs and by using the snowball sampling approach where community members were asked to locate neighbours fitting the criteria [26]. The interviewed traditional healers were only men, ranged in age from 25 to 80 years (average age 56) and the majority (47 people) were illiterate ( Table 1). The years of experience in traditional medicine practice of surveyed traditional healers ranged from 4 to 65 years (in average 24 years). The number of surveyed households per village varied from 1-4, with an average size of six individuals per household. Following methodology documented by Mahawar and Jaroli [27], surveyed households were selected based on their recognition as knowledgeable members concerning folk medicine. All surveyed persons in households were men, with age ranged from 26 to 72 years (43 years in average), of whom 26 people were illiterate. The number of years of experience in the use of animals as medicine by surveyed household ranged from 2 to 50, with 16 years on average. Survey data both for traditional healers and households included the sociodemographic characteristics of the interviewees, invertebrates used as remedy (local name, parts used, stage of development used, ailments treated, methods of preparation, administrated singly or in combination with other ingredients, use of live or dead, administered dose), invertebrate storage conditions, collection sites, how knowledge was acquired by the interviewees, any taboo associated with the traditional use of each invertebrates and the use of these animals for magico-religious purposes. In addition, traditional healers were asked to determine which of the invertebrate drugs in their own practices were most commonly prescribed, the most medically valuable and the most expensive [21]. We have translated the ethnopharmacological uses of each invertebrates into English medical terminologies with the help of educated (university and secondary school level) traditional healers. Visits were made to the main market of each of five municipalities of the department to interview merchants of medicinal animals. A total of 12 merchants of medicinal animals were interviewed, of whom 9 men and 3 women. The surveyed merchants were mostly illiterate with an average age of 49 years and an average of 20 years of experience in the sale of animals used in traditional medicine ( Table 1). The informants were asked to provide the vernacular name, origin, collection sites, conservation mode, commercial value, folk use, parts used and the modes of preparation as well as administration for each invertebrates traded.
Invertebrates revealed by the surveyed individuals were collected and stored in labelled boxes for later identification in the laboratory. With the aid of an insect taxonomist at the Biodiversity Resource Center of International Institute of Tropical Agriculture (IITA-Benin), some insects were identified at the specific level. The remaining zoological materials was identified with the aid of specialists through voucher specimens. Voucher specimens were deposited at the Faculty of Sciences and Technology of Dassa.

Data analysis
Similarly to Alves et al. [28], the ailments treated by invertebrate remedies were grouped in different categories according to the International Classification of Diseases (ICD-11) used by the World Health Organization (WHO). To determine the extent of utilization of each invertebrate species, we calculated the species use value (UV) following Alves et al. [28] using the formula: where U is the number of informants mentioning the use of the species and N is the number of informants that participated in the survey.
To estimate the level of agreement between interviewees over which invertebrates to use for each category, we calculated the informant consensus factor (ICF) [25]. This factor was calculated according to the formula used by Alves et al. [28]: where nur is the number of use reports in each category; nt is the number of species used; and ICF values range from 0 to 1. A high value (close to 1) indicates high consensus, whereby relatively few species are used by many people, and a value near zero indicates a high variation in the use of species for treating a particular illness [25,28]. ICF was also calculated for each category of ailments, in order to assess the informants agreement on treatments reported for that group of ailments.
To assess the importance of each invertebrate species for the reported ailments, the fidelity level (FL) was calculated following the formula: where Np is the number of informants that claim a use of a species to treat a particular disease or ailment and N is the total number of informants that use the invertebrates as a medicine to treat any given disease.
The recorded invertebrates were checked against the IUCN Red List Categories (Critically Endangered, Endangered, Vulnerable, Near Threatened, Least Concern, Data Deficient) for assessment of endangered status. Finally, the data obtained were subjected to principal component analysis (PCA) using Minitab 17 software, to describe the relationship between categories of diseases treated with invertebrates and the ethnic groups of the study area.

Invertebrates used for medical purposes
In total, 38 invertebrate species were reported by interviewees as used for 50 medicinal purposes. The reported species were distributed among 20 zoological families. Among them, insects occupied 64.7% of the total invertebrates reported followed by arachnids (8.8%), gastropods (8.8%), clitellata (7.4%), diplopods (7.4%) and crustaceans (2.9%). Insects were the taxonomic group with the largest numbers of animal species and medicinal uses (Fig. 2). The traditional healers prescribed 35 of the 38 invertebrates recorded as used for medicinal purposes, while surveyed households used only 18 of them (Table 2). Camponotus maculatus Fabricius, Phaneroptera nana sparsa Stål and Trithemis arteriosa Burmeister mentioned as home medicinal drug were not used by traditional healers. The great majority of registered invertebrate species (32) have between 1 and 5 different medicinal uses. Only 6 invertebrate species showed more than 5 medicinal uses. The use value of invertebrate species ranged from 0.007 to 0.293 ( Table 2). The species which attained the highest use value were Eudrilus eugeniae K. (0.293) and Achatina achatina L. (0.255). A total of 13 invertebrate species were considered by traditional healers as most prescribed by them (Table 3). Among them, A. achatina was mentioned by 29 of the surveyed traditional healers as the most prescribed invertebrate. This gastropod was considered by 7 surveyed traditional healers as having the highest medicinal value.

Ailments treated with invertebrates
Based on the information obtained from the traditional healers and surveyed households in the study area, all the 50 reported ailments were categorized into 16 categories of diseases ( Table 4). The category 'undefined illnesses' which includes all diseases with unspecific description of symptoms included the largest number of ailments (15) treated by invertebrates in the study area. Moreover, the majority of recorded invertebrate species (17) is used to treat undefined illnesses (Table 5). Ten (10) invertebrate species are used as remedies for diseases of the skin, nine (09) against diseases of the circulatory system and eight (08) respectively against diseases of the musculoskeletal system, certain infectious and parasitic diseases. Only one invertebrate species is used to treat diseases of the blood or blood-forming organs. Invertebrate-based remedies used for pregnancy, childbirth or the puerperium showed the most important number (34) of use citation after those of undefined illnesses ( Table 5). The highest Informant Consensus Factor (ICF) values were for diseases of the blood or blood-forming organs (ICF = 1), pregnancy, childbirth or the puerperium (ICF = 0.88), and for injury, poisoning or certain other consequences of external causes (ICF = 0.87). Figure 3 shows the results of principal component analysis (PCA) made to determine the relationship between categories of diseases treated with invertebrates and ethnic groups. The results show that the first component explains 56.4% of the information and that the first two components account for 76.5% of the information sought (Fig. 3a). The correlation graph revealed that Mahi, Holli, Yorùbá-Nago and Goun ethnic groups were positively correlated with the first axis (Fig. 3a). The Tori ethnic group was positively correlated with the second axis, and members of the Ouémégbé ethnic group were negatively correlated with the same axis. Our study revealed a difference between ethnic groups of Plateau Department in the use of invertebrates for medicinal purposes (Fig. 3). The projection of the categories of diseases treated with invertebrates in the first two axes shows that Mahi, Holli, Yorùbá-Nago and Goun ethnic groups use invertebrates more to treat undefined illnesses (Fig. 3b), while the Tori ethnic group uses it more for treating diseases of the skin and nervous system. The Ouémègbé ethnic group uses invertebrates more to treat diseases of the circulatory system and for pregnancy, childbirth or the puerperium (Fig. 3b).
Most invertebrate medicines (67.2%) have no dosage and are taken at will (Table 6). When considering the fidelity level of each invertebrate species for the reported diseases, the most quoted species (FL = 100) were C. maculatus (alcoholism treatment), Macrotermes bellicosus Smeathman (arthritis treatment), P. nana sparsa Stål (athlete's foot treatment),

Invertebrates used in magical-religious practices
In the study area, at least one mystical-religious use of invertebrates was noted for all the surveyed ethnic groups, except the Goun ethnic group. A total of 12 magical-religious practices were recorded in the study area (Table 7). Twenty-two invertebrate species are considered by 74 traditional healers and 10 surveyed households as having magical or supernatural properties. Most of the invertebrate species (15) were used by the Yorùbá-Nago ethnic group for protection against evil spirits (Table 7). However, most of the invertebrate species were used for multiple magical purposes. For example, Apis mellifera L. was used for protection against evil spirits (FL = 4.3), bewitchment (FL = 11.1), for good luck (FL = 28.6) and use by hunters to reach animals (FL = 100). Similarly, E. eugeniae was used for protection against evil spirits (FL = 13.1), bewitchment (FL = 11.1), to have good luck (FL =7.1), and love potions (FL = 50.0). The highest FL value (FL = 100) calculated was for A. achatina (used for purification), Pandinus imperator Koch (protection against accidents), S. nitens (protection against thieves) and A. mellifera (use by hunters to reach animals).

Commercialization of invertebrates for zootherapy and ritual practices
The majority of mentioned invertebrate species (31)

Constraints of used of invertebrates in zootherapy and ritual practices
Six constraints related to the use of invertebrates in zootherapy and ritual practices have been recorded in the study area (     nitens, T. arteriosa, C. glabrum, P. nana sparsa and M. religiosa) were of least concern (LC) according to the IUCN Red List of threatened species, and 1 species, the bee A. mellifera was listed as Data Deficient.

Discussion
The predominance of men among the surveyed healers and households could be explained by the fact that in Yoruba ethnic groups, traditional healers are men and traditional medicine practice is dominated by males due to secrecy in transmitting the knowledge from generation to generation [29][30][31]. Indeed, to become qualified to practise Yoruba traditional medicine, it would be necessary to go to apprenticeship (ranging from 2 to 30 years) followed by initiation into the Ifa cult [32]. The presence of women among the surveyed merchants of medicinal animals is not surprising because in the Yoruba socio-cultural area, women are more involved in the sale of traditional medicine products [31,33]. Zootherapy is well established in the Plateau Department where people use invertebrates to treat both common and rare diseases. Our study revealed that 38 medicinal invertebrates were being used in the study area, indicating very rich ethnomedical knowledge of indigenous people of the Plateau Department. Insects were the most important medicinal invertebrates with the most species and uses. In fact, their immunological, antiviral, analgesic, antibacterial, anti-cancer, diuretic,  anaesthetic, antioxidant, anti-inflammatory, anti-rheumatic and immunomodulatory properties are well recognised [16,34,35]. However, the African earthworm E. eugeniae and giant land snail A. achatina had presented the highest use value. In fact, like in our study, many peoples throughout the world had use earthworms to treat diseases such as haemorrhoids, arthritis, postpartum weakness, digestive ulcer, earache and epilepsy [36][37][38][39]. Earthworms possess antipyretic, antispasmodic, diuretic, detoxifying, antiasthmatic, spermatocidal, antihypertensive and antiallergenic effects [13,16,36]. Similarly, A. achatina which is known to have hemagglutination potential [40] is also used by Nigerian people to treat haemorrhage, suppression of convulsion and eye problems [41]. Sodjinou et al. [42] also reported a similar use of A. achatina by inhabitants of southern Benin for wound healing, to treat epilepsy and difficult childbirth. The widespread use of invertebrates throughout the world suggest that traditional knowledge on zootherapy is to be studied more seriously, in order to lead to the discovery of new sources of drugs [43].
Our study suggests a wide knowledge of the use of invertebrates in medicine in the surveyed households, compared with the number of common medicinal invertebrates used by traditional healers (15 of the 38 invertebrates). Indeed, for most of the surveyed households, knowledge on invertebrate-based remedies comes mainly from forefathers through informal training or verbal discussion. It is known that elderly persons and traditional healers are the custodians of indigenous knowledge systems [44]. In the current context of erosion of the traditional knowledge system, it is important to preserve this medicinal indigenous knowledge for the benefit of future generations. Of note, three of the identified invertebrate species (C. maculatus, P. nana sparsa and T. arteriosa), only recorded in surveyed households, are not known in the literature as medicinal animals. Knowing that the endogenous knowledges of the populations is built on their observations that have stood the test of time [45], it is important to conduct further studies to confirm the presence of any bioactive compounds in traditional remedies based on these three insects.
Our results showed that some invertebrates had high fidelity level (100%), which indicates that all of the use reports mentioned the same method for using the animal for treatment for the same diseases [46]. However,  most medicinal invertebrate species (80%) are used by traditional healers and households to treat more than one ailment. This trend is a common practice observed in folk medicine in different parts of the world [46][47][48][49], and biological reasons to explain this and the fact that often different invertebrate species are used to treat seemingly identical ailments are given by Meyer-Rochow in [16]. On the other hand, different invertebrate species were used to treat the same ailment. For instance, in our study, ten invertebrate species were used to treat headache and haemorrhoids respectively. The use of different invertebrate-based remedies for the same ailment allows for adaptation to the availability of the possible animals and suggests that these animals can share similar medicinal properties [50,51]. For instance, skin diseases have been treated with more invertebrates compared with other disease categories. Similarly, invertebrates were more used to treat skin diseases in contemporary Spanish ethnoveterinary medicine [52].
In the study area, ailments included in the undefined illness category were the most treated with invertebratebased remedies. These results are in accordance with those of Chakravorty et al. [53] who observed that common ailments encountered in day-to-day life were most treated with animal-derived treatments. Diseases of the blood or blood-forming organs have presented the most important ICF. In general, high ICF value allows to identify interesting species for the search of bioactive compounds [54]. However, in this case, the high ICF value reflects the fact that only one species was listed for the treatment of one disease (sickle cell disease), with two use citations, rather than a high cultural importance. The same trend was observed for the category 'ophthalmological diseases' where only one disease was treated by one animal with two use citations [47].
Our results also reveal that the categories of diseases treated with invertebrates were influenced by ethnic groups. The main ethnic groups (Yorùbá-Nago, Mahi, Holli and Goun) in the study area mostly used invertebrate-based remedies to treat various undefined illnesses. Similar trends were observed in Brazil, where the community of Queimada city uses more animal-based remedies to treat diseases with unspecific symptoms [20]. The reason why the Tori ethnic group uses invertebrates more for treating diseases of the skin and nervous system and Ouémègbé ethnic group for treating diseases of the circulatory system and for pregnancy, childbirth or the puerperium could be related to the high prevalence of these disorders in the areas these groups occupy.
Some of the invertebrate species used in folk medicine in the study area are also used in very similar ways by people throughout the world [16]. For example, similarly to inhabitants of the Yoruba tribe of southwestern Nigeria, A. mellifera is used to treat madness and Archachatina marginata Swainson to treat haemorrhoids [19]. Likewise, M. religiosa is used by traditional healers and indigenous people in India to treat ear wounds [49]. Moreover, Lawal and Banjo [19] and Costa-Neto [12] report a use of the housefly Musca domestica to treat eye problems. The fact that invertebrate species are being used for the same purpose by several communities might indicate their pharmacological effectiveness. However, the treatment of some diseases by invertebrate-based remedies followed folk logic. For example, the centipede T. niger with their numerous legs, feet and articulated body segments are used for foot problems such as paralysis and swollen foot. Similar trends are observed in Korean traditional medicine, where centipedes (Scolopendra spp.) are used for leg, foot and joint problems [4,16]. Likewise, the firefly Luciola discicollis Castelnau, which possesses a bioluminescent abdomen, is used in the study area to treat myopia. Another reasoning of folk logic in traditional medicine is based on the negative interactions that the invertebrates have with people. For instance, scorpions (P. imperator) whose sting causes pain has its venom used to treat pain and snake bites as also observed in Korean traditional medicine [4,16]. Similarly, in our study, the ant species Pachycondyla tarsata Fabricius, which is also called 'cadaver ant' because of it's strong, putrid and smell, is used to treat alcoholism.
The majority of invertebrate-based remedies preparations were used in combination with plant species or No sales market for invertebrates -- 11 11 Difficult to find some invertebrates in the markets 15 -3 18 High cost 7 -5 12 Difficult conservation 6 2 2 10 Very low demand -9 -9 *N number of surveyed people plant derivatives for the treatment of single ailment. Similar combinations were also reported by Chakravorty et al. [53], which observed that in members of the Nyishi and Galo tribes (India), the use of treatments solely based on animals or animal products is rare and that treatments involving animal material frequently contain a plant component as well. Some medicinal preparations where both plants and animals are utilized in combination are also reported from Brazil [50] and India [49].
In the study area, the Guinea pepper (Xylopia aethiopica (Dunal) A. Rich.) was the most cited plant used in combination with the invertebrate-based remedies. This plant was found to possess anti-microbial [55], antifungal [56], anti-helmintic [57], anti-cancer [58], antianaphylactic and anti-inflammatory [59], cardiovascular and diuretic [60] activities. The analgesic effect of the fruits of Guinea pepper could explain their mixing with invertebrate-based remedies in the study area to treat pain disorders including arthritis, stomachaches, earaches, headaches, menstrual cramps, haemorrhoids and neuralgia [61]. Oral and topical applications were the most commonly used routes of invertebrate-based remedies application. This finding is in agreement with the result of various zootherapy studies conducted in Brazil [48], India [49] and in fact throughout the world [16]. As in Yoruba medicine of Nigeria, in the study area, health and religion are tightly interrelated [29]. The use of invertebrates for magical-religious practices has been also observed in several other countries throughout the world such as Brazil [2,20,24], Nigeria [19], India [62] and Mexico [63]. In our study, invertebrates were in the majority used by the Yorùbá-Nago ethnic group for spiritual protection against evil spirits. Indeed, Yorùbá people believe that illness can be caused by entities such as witchcraft, sorcery, a god or ancestors [64]. Unlike of the Tribal Adi of North-East India, which believed that some insects are representatives of ghosts and evil spirits [29], the surveyed ethnic groups use insects and others invertebrates for their protection. Similarly to this study, the same invertebrate can have several magical uses and one of these species, M. domestica, is used by Yoruba tribe of southwestern Nigeria for spiritual protection [19]. The highest FL (100%) presented by four invertebrate species used for magical-religious purposes might give some useful leads for further esoteric research.
The low demand of medicinal invertebrates, which is the main constraint on their sale according to merchants, is not surprising because, like almost everywhere in the world, most of medicinal invertebrates are collected in nature [47]. Except for M. religiosa, which is listed as least concern by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species, the other invertebrates sold at the markets of the study area were not listed. Indeed, invertebrates are rarely considered in conservation policies although several species are in the process of extinction [21]. As in the case of medicinal mammals [7], Nigeria is the main importing country of medicinal invertebrates used in the study area. Further studies must be done to evaluate the abundance and distribution of the sold invertebrate species in Benin and Nigeria for a development of conservation strategies. Moreover, in view of the scarcity of some of these invertebrates during the dry season, it is important to develop techniques for rearing the main invertebrates used as medicinal products in the study area.

Conclusions
This study is the first one in Benin that documents the use of invertebrates in traditional medicine. The results showed that inhabitants from the Plateau Department use several invertebrate species for healing practices and magical-religious rituals. Since most of the invertebrate species used in the study area are not listed in the IUCN Red List of Threatened Species, we suggest that future studies be conducted for their conservation and sustainable use. Further studies must also be done to confirm the presence of any bioactive compounds in invertebrate species used in traditional medicine in the study area.