The use of fish and herptiles in traditional folk therapies in three districts of Chenab riverine area in Punjab, Pakistan

Background Like botanical taxa, various species of animals are also used in traditional and modern health care systems. Present study was intended with the aim to document the traditional uses of herptile and fish species among the local communities in the vicinity of the River Chenab, Punjab Pakistan. Method Data collected by semi-structured interviews and questionnaires were subsequently analyzed using relative frequency of citation (FC), fidelity level (FL), relative popularity level (RPL), similarity index (SI), and rank order priority (ROP) indices. Results Out of total 81 reported species, ethnomedicinal uses of eight herptiles viz. Aspideretes gangeticus, A. hurum, Eublepharis macularius, Varanus bengalensis, Python molurus, Eryx johnii, Ptyas mucosus mucosus, Daboia russelii russelii and five fish species including Hypophthalmichthys molitrix, Cirrhinus reba, Labeo dero, Mastacembelus armatus, and Pethia ticto were reported for the first time from this region. Fat, flesh, brain, and skin were among the commonly utilized body parts to treat allergy, cardiovascular, nervous and respiratory disorders, sexual impotency, skin infections, and as antidote and anti-diabetic agents. Hoplobatrachus tigerinus, Duttaphrynus stomaticus, and Ptyas mucosus mucosus (herptiles), as well as Labeo rohita, Wallago attu, and Cirrhinus reba (fish) were top ranked with maximum informant reports, frequency of citations, and rank order priority. Uromastyx hardwickii, Ctenopharyngodon idella, H. molitrix, Cirrhinus mrigala, C. reba, L. rohita, L. calbasu, L. dero, and Pethia ticto were the species with 100% fidelity level. Furthermore, medicinal uses of Aspideretes gangeticus, Aspideretes hurum, Calotes versicolor, Daboia russelii russelii, Hypophthalmichthys molitrix, Cirrhinus reba, Labeo dero, Mastacembelus armatus, Pethia ticto, and Gagata cenia were reported for the first time. Conclusion About half of the reported species depicted zero similarity index with previously reported literature, which indicates strong associations of local inhabitants with animal species, particularly for therapeutic purpose. Inclusive studies on composition and bioactivities of the species with maximum use reports may contribute significantly in animal-based novel drugs discovery.


Introduction
The multipurpose usage of animal species, e.g., as food, medicine, entertainment, magic, music and religion, tools for art, and in trade, is well known [1][2][3][4][5][6][7][8][9]. It has been reported that loving, watching, and working with animal species is beneficial to lower heartbeat and control stroke [10]. Animal-based products are used as traditional medicines, and an estimated 8.7% of the vital chemicals used in modern healthcare systems are extracted from different animal species [11]. However, compared to plant species, animal-based products are widely neglected [12]. Inhabitants of rural areas are more depended on animal-based products as food and medicines, and possess significant traditional knowledge of zootherapies [9,11,13]. Many species of animals, either wild or domesticated, are important to humans [13]. Wild animal species are often under threat due to anthropogenic activities like illegal hunting and trade for food, medicines, and ornamental purposes, deforestation, agriculture intensification, urbanization, and industrialization [3,[14][15][16][17][18][19].
Herptiles and fish are recognized as extremely fascinating and important animal species [20][21][22]. In many societies, different species of herptiles and fish are used in ethnomedicine and folklore to treat health disorders [16,22,23]. An estimated 10,450 species of reptiles and 7850 amphibian species have been reported [24] globally. In Pakistan, 195 species of reptiles [25] and 24 species of amphibians [26] have been documented so far. The Asian region has also a high diversity of marine and freshwater fish species (22907 and 10036 species, respectively) [24,27]. An estimated 186 species of freshwater fish and 719 species of marine water fish have been reported so far in Pakistan [28]. However, traditional uses of animal species, particularly to treat diseases in humans and other animals, have rarely been documented in Pakistan [6,7,[29][30][31][32]. To best of our knowledge, traditional uses of herptiles and fish species have never been reported before in Pakistan. Therefore, the present study was planned to document herptiles and fish species used to treat various diseases by the local communities residing along the Chenab riverine areas, i.e., Gujranwala, Gujrat, and Sialkot districts in the Punjab province of Pakistan. Qualitative indices were used to elucidate commonly utilized species with high fidelity level and frequency of citation. We hope the data provided will be of significant value for pharmaceutical industries to discover animal-based novel drugs to meet the recent challenges to human health.

Study area
The River Chenab is the combination of two main streams, i.e., Chandra and Bhaga originating from the Himalayan region of Himachal Pradesh in India. After passing through the Siwalik Range in the south, and the Lesser Himalayas in the north of Indian Jammu and Kashmir, it continues into Pakistan [33]. The present study was conducted in three districts of the Chenab riverine area, i.e., Gujranwala, Gujrat, and Sialkot ( Fig. 1) from March 2016 to April 2017. The study area covers 9830 km 2 , with temperature ranges from around 0°C in December to 50°C in June [34][35][36]. This region has a high diversity of wild fauna, comprising 150 species of birds, 47 herptiles, 34 fish, and 15 mammalian species [15,[37][38][39]. Demographically almost 52% of the population are male, and 48% are female. The major population is rural, and encompasses Arain, Gujjar, Jutt, Sheikh, Rana Butt, Malik, and Mughal casts. Punjabi is the common language spoken, although some people speak Siraiki and Urdu, while educated people can also speak English to some extent [34][35][36].

Data collection and analysis
Following the Nagoya Protocol, prior informed consent was taken from local informants for data collection and publication. In addition, the International Society of Ethnobiology Code of Ethics (http://www.ethnobiology.net/) was also followed. Ethnomedicinal uses of herptiles and fish species along with cultural importance were collected from local informants (n = 100) using semistructured interviews and group discussions. Informants including farmers, fishermen, hunters, teachers, and health practitioners were selected based on their traditional knowledge of animal species, i.e., herptiles and fish. Animals were identified using "The Amphibian and Reptiles of Pakistan" [25], and "Freshwater Fish of Pakistan" were also consulted for correct classification and identification of fish of the study area [40]. Different indices, i.e., relative frequency of citation (RFC), fidelity level (FL), relative popularity level (RPL), rank order priority (ROP) and similarity index (SI), were used to analyze that data.
Relative frequency of mention (RFC) was calculated using formula as reported previously [41].
Where FC is the frequency of citation for an ethnomedicinal or cultural use of a specific species and N is the total number of informants.
Fidelity level (FL) was obtained using the method explained earlier [42] based on formula where N p indicates number of informants reporting major ailment for a specific species of herptiles or fish and FC is the frequency of citation for ethno-medicinal or cultural use of that species. Relative popularity level (RPL) of the reported species was elucidated as reported by [43,44]. Herptiles and fish species were classified into two groups (i) "popular" and (ii) "unpopular." Popular herptiles and fish species were those having more than half of the maximum frequency of citation (FC), whereas the left-over herptiles and fish were documented as unpopular. For popular herptiles and fish species, a horizontal line was imaginary, namely the average number of uses per species is independent of the frequency of citation (FC), who recognizes the herptiles and fish; therefore, the average numeral of uses of a popular herptile and fish species does not enhance with the add to frequency of citations who cite a herptile and fish for any medical use. For the popular herptiles and fish, the RPL was chosen to one (1). For herptiles and fish in the unpopular group, the relative popularity level value is less than 1.0.
Rank order priority (ROP) is used to grade plants and animal species and was calculated as explained earlier [43,44] and was analyzed by the following formula ð Þ M s = Alike number of medicinal uses in the previous and present research records for a specific herptiles and fish species. M t = Total number of medicinal uses in the present research reports for a specific herptiles and fish species.
Principal component analysis (PCA). Data were statistically analyzed with the help of principal component analysis by using Past software Version 3 [45].

Results and discussion
Demography Data were collected from 100 informants of an age between 18 and 75 years (Fig. 2). About 70% informants were literate, and participants had finished having primary, matric, intermediate, bachelors, and master levels (23, 24, 21, 8, and 3, respectively). The majority of the informants (76%) were from rural areas with agricultural background.

Myths about herptiles and fish
Some common myths on snake and fish species were also noted during the field survey. These myths were comparable to previous reports [15].
It is a common assumption that if Eryx johnii (common sand boa) bites somebody, it will bite on the arrival of the new moon in each month. If the male or female ofa snake pair is killed by someone, then the other will certainly take revenge from the assassin. Naja naja changes into a human after 100 year of age and can transfer poison to a person if it sniffs someone. Some people of the study area believe that Python molurus (rock python) has seven mouths.
Most people believe that a special bone called Mankana is present in the snake head, and that this bone can absorb poison from snake-bitten people. Seeing a snake in a dream means that an enemy may attack a person. Every species of fish has a special kind head like a human and body like a fish, which locally known as Jal Pari. Turtles have a blade which can cut anything.

Ethnomedicinal uses herptiles and fish
Inhabitants of the study area possessed significant knowledge on the medicinal as well as cultural uses of animal species, particularly that of herptiles and fish (Fig. 3). As mentioned in Table 1, 38 species of herptiles and fish were used to treat various health disorders such as allergy, cardiovascular, nervous and respiratory disorders, sexual impotency, skin infections, and as antidote and anti-diabetic agents in human and livestock ( Table 2).

Body part(s) used
In both herptiles and fish, as shown in Fig. 4, fat was the most commonly utilized body part and was used in the preparation of 19 recipes to treat a number of diseases, followed by flesh, brain, and skin, used in 14, 11, and 3 recipes, respectively. In the present study, the application of body fat of herptiles and fish served to treat rheumatic problems, eye diseases, sexual impotency, cold, fever, skin infections, nervous and cardiovascular diseases, diabetes, respiratory tract infections, urine problem, liver infections, heal wounds, and as antidote. This might be related to the presence of health beneficial metabolites, i.e., omega-3 fatty and omega-6 fatty acids, etc. It has been reported that these acids contribute substantially in the treatment of neurological disorders, thrombotic, atherosclerosis, and act as anti-aging agents [73]. Beneficial effects of omega-3 fatty acids on atherosclerosis are mainly through their actions on plasma lipids. Likewise, their role in the reduction of blood pressure and plasminogen activator inhibitor, improvement of metabolic syndrome, and maintain endothelial function may be due to other potential antiatherogenic factors. As atherosclerosis is inflammatory diseases, omega-3 fatty acids offer protection through their anti-inflammatory effects [74][75][76]. The major benefit of omega-3 fatty acids in patients with type 2 diabetes mellitus is the enhancement in their highly atherogenic lipid profile. It has been reported less development of pancreatic islet-cell autoimmunity and reduction in insulin resistance was observed in mice treated with omega-3 fatty acids [75,77].
Inhabitants of the study area use carapace of the turtle to treat internal injuries, allergy, cough, and as a sexual stimulant. These pharmacological properties of turtle carapace are mainly due to the presence of β-pleated sheet keratin [83], keratinocytes, melanocytes, lipids [84], and mineralized collagen fibrils [85]. Likewise, fish brain is used to treat joint pain, Alzheimer, heart diseases, for sexual potency, to improve eye-sight, as an anti-depressant and anti-diabetic. The health beneficial properties of fish brain mainly attributed to rich compounds of docosahexaenoic acid, omega-3 fatty acids, and proteins present in it [76,86].

Diseases treated
As mentioned in Fig. 5, joint pain, eye diseases, sexual impotency, common cold, and fever were among the top ranked diseases treated with maximum number of animal-based recipes. Lack of hygiene, nutritional deficiency, and "community evils" were among the major factors involved in the high prevalence of diseases in the study area. Comparative analysis of the present findings with previous reports on medicinal uses of herptiles and fish species indicate that different methods of treatments and body parts were used in study area ( Table 1). The inhabitants of the study area use skin of Indus valley toad (Duttaphrynus stomaticus) to treat skin infections, while the same species have been reported to treat allergy, thelitis, bolianerengia, bronchial pneumonia, dermatitis, abscess, and to heal wounds [46][47][48]. Body fat of the Indian bullfrog (Hoplobatrachus tigerinus) was used to treat backbone pain, sexual impotency, and joint pain; but in previous studies [49][50][51][52], different body parts of this species have been reported against acidity, burn, cold, cough, diarrhea, dysentery, and to heal wounds. In the study area, carapace ash, fat, and oil of the Indian flap-shelled turtle (Lissemys punctata andersoni) were used to enhance sexual potency and in the treatment of internal injuries, allergy, and cough. However, this species has also been reported to treat acne, piles, arthritis, asthma, bronchitis, burns, cough, dermatitis, epilepsy, backbone pain, diabetes, urinary obstruction, diarrhea, indigestion, lung diseases, malaria fever, menorrhagia, rashes, wound healing, and tuberculosis [48,[53][54][55]. Fat, oil, and bile of Agror agama (Laudakia agrorensis) were used for joint pain; sexual potency; snake, spider, wasp, and scorpion sting; as well as body pain. Same species are used to treat jaundice, joint pain, malaria, arthritis, burn, cough, fever, and skin disease [47,48,50,52,[56][57][58]. The Indus spiny-tail lizard (Uromastyx hardwickii) is used in the treatment of body pain, joint pain, sciatica pain, and for sexual potency,   whereas [48,59] it was reported that the same species is useful against ear pain, backache, joint pain, and headache. Local people used body fat, skin, and oil of the Indian cobra (Naja naja naja) to treat sciatica, snakebite, to improve eye sight, and as sex stimulant. This species has been reported to cure arthritis, cancer, leprosy, muscular pain, as aphrodisiac, and as antidote [48,51,52,59]. Fat and oil of Russell's chain viper (Daboia russelii russelii) were used as a remedy for urine problem and hemorrhoids. However, in previous studies [50,59,87], different parts of this species have been reported as used against weak eye sight, urination, stool, flatus, and as anti-venom. Likewise, body fat and oil of the Sind valley saw snake viper (Echis carinatus sochureki) were used to treat joint pain, snakebite, weak eye sight, and to enhance sexual desire [48,59]. Inhabitants of the study area preferred Ctenopharyngodon idella for the treatment of eyesight, night blindness, fever, cold, and joint pain, while the same species was reported to treat erectile disinfection, cold, to enhance memory, and sexual power and showed 0.20 similarity index with previous reports [31,48]. Our findings revealed that Cyprinus carpio and Cirrhinus mrigala were effective against weak eyesight, night blindness, fever, cold, and joint pain. In previous studies [48,60], C. carpio has been reported as used for CNS, erysipelas, lumbago, to enhance memory, enhance energy, sexual power, to reduce overweight, and against cold and has depicted similarity index = 0.20. Likewise, C. mrigala was reported to reduce weight, to treat joint pain, to enhance memory and sexual power, to provide energy, and to treat against cold [48,59]. Labeo rohita and L. calbasu were used for the treatment of joint pain, body pain, sexual potency, eyesight, depression, diabetes, Alzheimer, and heart diseases.

Cultural values of herptiles and fish
Cultural values of the reported species of herptiles and fish are given in Table 2. Local people of the study area used the skin of the black cobra in magic. Likewise, different species of snakes like the Indian cobra (Naja naja naja), Indian rat snake (Ptyas mucosus mucosus), and buff striped keelback snake (Amphiesma stolatum) were used for pleasure of the public such as the mongoose competition with a snake. According to local informants, the presence of the yellow belly common house gecko (Hemidactylus flaviviridis) in a home is considered as bad omen for residents. The Bengal monitor (Varanus bengalensis) is tight knot with rope and with help of that rope a person can climb walls. Fish species were not only used in the treatment of various diseases but also as nutritious food. Local inhabitants used fish flesh as bait for varieties of fish species from rivers, as reported earlier by [88].
Inhabitants of the study area used different species of fish for commercial purposes. Likewise, Indian soft shell, Peacock soft shell, Indian Flap-shelled Turtle, and Indian Bullfrog were captured and sold for lab practice. Only four species of herptiles such as Indian soft shell (Aspideretes gangeticus), Peacock soft shell (Aspideretes hurum), Indian Flap-shelled Turtle (Lissemys punctata andersoni), and common leopard gecko (Eublepharis macularius) were exported from the area, and are used as food and for medicines. Two species of fish, i.e., Bronze featherback (Notopterus Notopterus) and Indian glassy fish (Parambassis ranga), are ornamental fish for aquaria.
The animal species reported by the maximum number of respondents were frequently used to treat various diseases, and exhibited high FC (Frequency of Citation) ranging from 5 to 90 (Fig. 6), i.e., Labeo rohita (rohu) had a maximum FC (90), followed by Wallago attu (wallago catfish) and Cirrhinus reba (Reba carp) (68 and 67, respectively).
The fidelity level is utilized to recognize species that are commonly favored by people to treat different diseases [89,90]. The FL of herptile and fish species in this study varied from 2.9 to 100% ( Table  2). Nine species, including U. hardwickii (Indus spiny-tail lizard), C. idella (grass carp), H. molitrix (silver carp), L. dero (dero), C. mrigala (mrigal carp), C. reba (reba carp), L. rohita (rohu), L. calbasu (orange fin labeo), and P. ticto (ticto barb) which were used for sexual potency, fever, cold, and to treat eyesight, body pain, and joint disorders, depicted 100% FL (Fig. 5). These findings indicate the prevalence of particular diseases in the area that were cured with species having high FL. The animal species with maximum FL were highly used in the area, as compared to species having low FL. The FL of herptile and fish species was documented for the first time, and species with highest FL might be subjected to in-depth compositional analysis and bioactivities in pharmaceutical industries, as possible sources to manufacture novel drugs.
The relative popularity level (RPL) of the reported species is given in Table 2. Both herptile and fish species were classified as popular and unpopular categories based on RPL (Fig. 7), which were comparable to [43,44]. During the study, we noted that C. idella, C. carpio, H. molitrix, C. reba, C. mrigala, L. rohita, L. calbasu, L. dero, G. catla, C. punctata, C. marulius, O. niloticus, R. rita, B. bagarius, M. armatus, and W. attu were most popular with RPL = 1.0 while all other species were ranked as unpopular. The high popularity of these species might be attributed to their high efficiency utilized as medicine.
The healing potential of herptile and fish species was observed using FL values, while rank order priority (ROP) was utilized to assign a suitable grade to all species with various FL values. The measured levels of ROP of each herptile and fish species are cited in Table 3. The ROP of seven species used to treat different diseases was above 100. These species were C. idella (eyesight and cold); C. carpio (fever); H. molitrix and C. reba (fever and cold); and L. rohita, L. calbasu, and L. dero used to treat joint pain, fever, and cold and for sexual potency. A decrease in ROP may be due to the decline in the popularity of ethnomedicinal and ethnocultural uses of fauna among local peoples. Moreover, the respondents of the rural area have more information and interaction with cultural and medicinal uses of animals compared to urban areas. The findings of the present survey were analogue to previous results for medicinal species of animal species in Palestine [44].
This study, for the first time, reported the use of flesh ash, fat, and oil of Aspideretes gangeticus (for skin diseases and sexual potency), Aspideretes hurum (backbone/joint pain), flesh ash of Calotes versicolor (for foot and toe injuries), fat oil of Daboia russelii russelii (to treat urinary problems and hemorrhoids), brain oil of Hypophthalmichthys molitrix (to improve eyesight, night blindness, and to treat fever, cold, joint pain). In addition, the brain oil of Cirrhinus reba and Labeo dero was used to treat eyesight, night blindness, fever, cold, and joint pain; the flesh of Mastacembelus armatus was used to improve sexual potency and body weakness; the brain of Pethia ticto was used to treat night blindness, eyesight, and to develop central nervous system; and the brain of Gagata cenia was used to treat urinary problems (Table 1).
Zoonoses with a wildlife reservoir are a major public health issue, affecting the whole world. Various pathogens and different modes of transmission are present, and many variables impact the epidemiology of different zoonoses. The recognition and importance of wildlife as a reservoir of zoonoses are increasing [91,92]. The prevalence of transmission of disease-producing driving forces from fish to humans is however very low. In general, humans contract fish-borne disease through ingestion of tissues, or by contamination of the skin [93]. Human sensitivity to amphibian proteins in a laboratory setting is rare. It remains possible, however, to become sensitized to amphibian proteins through inhalation or skin contact [94].

Conservation status of the reported species
Knowing the background of interaction and exploitation between humans and natural resources is vital for the implementation and development of animal and landscape conservation strategies [95]. Ethnozoological studies provide necessary information and contribute significantly to animal conservation because in addition to incorporating biological factors, and providing traditional knowledge on medicinal and cultural values of animal species in any area, such studies also cover Fig. 7 The relationship between informant numbers and the percentage of informants who argued similar use for that herpetofauna and fish; circled numbers show herpetofauna and fish names as given in Table 1 cultural, social, economic and traditional roles of fauna in human civilization [96].
Based on the cultural uses of herptiles and fish species (Table 2), it was observed that 47% of the reported species are listed as least concern (LC), 44% are not evaluated (NE), 0.04% species (i.e., Indian soft shell, Peacock soft shell, Rock pathon, and Common carp) are vulnerable (V), and 0.03% species (i.e., Silver carp, Foji Khaga, and wallago catfish) are listed as near threatened (NT) globally by the International Union of Conservation of Nature (IUCN). Interestingly, most of the herptiles and fish species (74/91%) showed threats, and only 9% of the species were listed as threatened by IUCN as mentioned above. Use of animal species in traditional therapies and for cultural purpose by humans is not the only threat to animal biodiversity in any region. Factors also include changes in climate and various types of interactions in an ecosystem, i.e., food chain, food webs also contribute significantly in threatening animal population and diversity [96,97]. Given the great need to find solutions to deal with the current crisis of biodiversity loss [98], more specifically that of animal species, it is obligatory to adopt strategies that address the problem in all its complexity. And for this, ethnozoology presents itself as an interdisciplinary tool, approaching the issue in an additional comprehensive method [99].

Principal component analysis
The ethnomedicinal data were analyzed through principal component analysis (PCA), which allowed for the ordination of plots in terms of three variables viz.
Informant of major ailment (IMA), fidelity level (FL), and rank order priority (ROP). The result of the PCA showed the sum of all the eigenvalues with total inertia of 3105.67. The first eigenvalue was high (2881.04) which showed high gradient strength in distribution of indigenous knowledge along the first axis (PC1). The total variation explained along this axis is (92.77%). The first two axes of the principal component analysis showed 99.99% variation in samples (component 1: 92.77%; component 2: 7.23%); therefore, only two axes were considered in Fig. 8. The variables IMA (r = 0.33506), FL (r = 0.57662), and ROP (r = 0.74514) positively correlated with first axis (PC 1) while IMA (r = − 0.23734) and ROP (r = − 0.52551) was negatively correlated with component 2 and FL (r = 0.81701) was positively correlated with component 2 (PC 2), which were comparable with previous studies [100].

Conclusion
Traditional uses of various herptiles and fish species were recorded, and to the best of our knowledge, the ethno-pharmacological applications of 11 herptiles and seven fish species were reported for the first time from this region. Our findings revealed that the indigenous communities of the study possess significant traditional knowledge because of their strong relation with the nearby fauna. These results could be valuable for sustainable utilization and conservation of animal species. Additionally, detailed investigations on pharmacologically active substances and in vitro and/or in vivo of biological activities of compounds from herptiles and fish species with highest FL and FC could be interesting for  Table 1). The positions of the arrows relative to components 1 and 2 show how strongly independent variables (IMA, ROP and RI) are correlated the development of novel animal-based drugs to treat various health disorders.