Proximate and mineral compositions of Samia cynthia ricini and Dytiscus marginalis, commonly consumed by the Bodo tribe in Assam, India

Entomophagy, the consumption of insects, is a common practice among various tribal communities across the globe. Various factors such as flavour, nutrient content, availability and claimed medicinal values have contributed to the increased consumption of insects. Two commonly consumed insects among the Bodo tribe in Assam, namely, Samia cynthia ricini Boisduval, 1854 (Lepidoptera: Saturniidae) and Dytiscus marginalis Linnaeus, 1758 (Coleoptera: Dytiscidae), were procured from local markets in Kokrajhar and evaluated for their proximate and mineral contents using Association of Official Analytical Chemists (AOAC) guidelines and inductively coupled plasma-optical emission spectroscopy (ICP-OES). Both the insects are available throughout the year and are consumed on a large scale. S. cynthia ricini showed higher contents of moisture (7.89 ± 0.020%), ash (4.10 ± 0.077%), fat (22.23 ± 0.209%) and carbohydrate (7.78 ± 0.057%) whereas higher contents of crude fibre (14.28 ± 0.102%), protein (56.37 ± 0.366%) and nitrogen (9.02 ± 0.058%) were observed in D. marginalis. The calorific value of S. cynthia ricini was higher with 430.19 ± 2.241 kcal whereas D. marginalis showed a lower value of 382.58 ± 1.527 kcal. D. marginalis showed the presence of arsenic (As) (0.014 ppm), and sodium (Na) was not detected. S. cynthia ricini did not show the presence of As and magnesium (Mg). Lead (Pb) showed a value of 0.026 and 0.044 ppm, and cadmium (Cd) showed a value of 0.005 and 0.005 ppm in S. cynthia ricini and D. marginalis respectively. Both the studied insects showed high protein and fat contents and a high calorific value. Although the insects show the presence of several essential minerals in considerable amounts, they showed the presence of toxic heavy metals in trace amounts. Hence, their consumption must be done in a controlled manner owing to the presence of As, Pb and Cd which are capable of causing adverse reactions in its consumers.


Background
Insects are consumed in various countries across the world (Van Huis et al. 2013). About 2.5 billion people across the world supplement their food with insects (Van Huis 2016). With the rapid increase in population, there is a fear of food scarcity. Entomophagy may serve as an alternative solution to food scarcity (Kim et al. 2019). Insects are found to be rich sources of vitamins, proteins, carbohydrates, fats and minerals (DeFoliart 1989;Zielinska et al. 2018). Entomophagy has been reported from various parts of the world such as Sub-Saharan Africa, where about 250 insect species are consumed (Van Huis 2003) and in Thailand about 164 different species of insects are sold as food in open markets (Yhoungaree 2010). They can be used as emergency food in case of food scarcity, high cost of food grains, crop failure and seasonal disturbances (Gahukar 2018). The consumption of these insects depends on various factors such as their taste, availability, palatability, nutritional value and local and religious beliefs (Chakravorty et al. 2011).
In India, entomophagy is mainly practiced in the northeastern states predominantly by tribal population. In addition to these states, tribal communities in other states of India such as Kerala, Odisha, Jharkhand, Karnataka, Tamil Nadu, Chhatisgarh and Madhya Pradesh also practice entomophagy (Chakravorty 2014;Gahukar 2018). About 158 different species of insects are consumed in Arunachal Pradesh alone and about 40 are consumed by ethnic tribes in Assam (Ronghang and Ahmed 2010). The tribals in Mizoram consume about 60 species of insects (Meyer-Rochow 2005). About 42 species of insects are consumed in Nagaland (Meyer-Rochow and Changkija 1997) and about 41 insect species are consumed in Manipur (Shantibala et al. 2012).
Several workers have documented the use of insects as food by the Bodos; however, studies on their proximate and nutritional contents are lacking, or these studies portray the proximate contents only and not the mineral contents and vice versa (Narzari and Sarmah 2015a;Narzari and Sarmah 2015b;Ghosh et al. 2017a;Ghosh et al. 2017b;Hazarika 2018). Also, studies describing the presence of toxic heavy metals in edible insects are lacking. Toxic heavy metals such as lead (Pb), cadmium (Cd), arsenic (As) and mercury (Hg) are known to cause severe health complications in individuals exposed to them (Imathiu 2020). These heavy metals have been detected in some edible insects (Zhang et al. 2009). Consequently, this study was designed to evaluate the proximate, mineral and toxic heavy metal contents of S. cynthia ricini and D. marginalis commonly consumed by the Bodo tribe in Assam.

Study area and the people
The Bodo tribe is of Tibeto-Burman origin and belongs to the Tibeto-Chinese linguistic stock. They are the largest group among the Plains Tribal population in Assam today. They racially belong to the Mongoloid stock (Census of India 2011). According to the 2011 census, they number about 1,416,125 persons in the state of Assam (Census of India 2011). The Bodo-dominated areas fall under the Bodoland Territorial Autonomous Districts (BTAD), namely, Kokrajhar, Chirang, Baksa and Udalguri.

Experimental procedure
Fresh insects were procured from local markets in Kokrajhar, Assam, from August to November 2019. They were degutted, weighed and sun-dried. They were identified using literature and taxonomic keys. The samples were then made into powder using a kitchen blender. The powdered samples were subjected to proximate, mineral and heavy metal content analyses.

Studied insects
A preliminary survey across the markets and villages was performed. The data collected is depicted in Table 1. It was found that these insects are usually eaten due to their taste which is usually from their fat content. They are very commonly available, and Samia cynthia ricini is reared at home by several families.

Samia cynthia ricini Boisduval, 1854
Samia cynthia ricini (Family: Saturniidae) is commonly called as the Indian eri silkworm. It belongs to the order Lepidoptera and is a commercial silk-producing insect (Vijayan et al. 2006). It is believed to have originated in the Brahmaputra valley (Jolly et al. 1979). Although it feeds on several other plant species, its preferred host plant is the castor plant (Naika et al. 2003). It is found in tropical and temperate eastern Asia (Naumann and Peigler 2001;Peigler and Naumann 2003). It is abundant in northeastern India, particularly in the states of Assam and Meghalaya (Renuka and Shamitha 2014). The collected worms (prepupae) were bulky and measured about 3.5 to 4 cm in length with a bright yellow texture ( Fig. 1).

Dytiscus marginalis Linnaeus, 1758
Dytiscus marginalis (Family: Dytiscidae) is commonly called as the great diving beetle. They belong to the adephagous order Coleoptera. They are predatory aquatic insects inhabiting stagnant freshwater bodies (Lundkvist et al. 2003). They are commonly found in Asia and Europe (Nilsson 2003). They can be serious pests at commercial fisheries (Frelik 2014). Characteristics include threadlike antennae with 11 segments; long, flattened hind legs with five-segmented, tapering tarsi bearing swimming hairs; and hind coxae with rounded, pointed or truncate processes (Foster and Friday 2011). The collected beetles showed a shiny black texture and measured about 3 cm in length (Fig. 2).
i. Moisture content: After drying the samples in an incubator for 4 h at 105°C, the samples were cooled in desiccators and then weighed. The moisture content was obtained from the difference between wet weight and dry weight using the following formula: Moisture% ¼ weight of fresh sample − weight of dried sample weight of fresh sample Â 100 ii. Ash content: After moisture content analysis, the samples were placed on porcelain dishes and then placed in a furnace at 550°C for 4 h. The ash content was calculated using the following formula: Ash% ¼ weight of ash weight of dried sample Â 100 iii. Crude fibre content: The dried samples were digested with 1.25% sulphuric acid, filtered and washed and then digested with 1.25% sodium hydroxide, filtered, washed and dried. This dried sample was then ignited. The crude fibre content was calculated using the following formula: Crude fibre% ¼ weight of sample after ignition weight of sample Â 100 iv. Fat content: The samples were kept in diethyl ether for 2 h at 90°C. After extraction, the samples were then kept at 70°C in an incubator for 30 min, cooled in desiccators and weights were taken. The fat content was calculated using the following formula: Crude fat% ¼ original weight of the sample − weight of the sample after extraction original weight of the sample Â100 v. Protein content: The nitrogen content was evaluated by Kjeldahl method. The crude protein content was calculated by multiplying the crude nitrogen content by a factor of 6.25.

Mineral analysis
The samples were processed using acid digestion method, cooled, filtered with distilled water, final volume made to 50 ml and the filtrate was used for the analysis of mineral contents. Mineral contents were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) (Thermo Fisher iCAP 7600).

Statistical analysis
Each experiment was performed thrice, and data was analysed using OriginPro v8. All data are represented as mean ± standard error of mean (SEM).

Proximate compositions
D. marginalis showed higher crude fibre, protein and nitrogen contents, whereas S. cynthia ricini showed higher moisture, ash, fat and carbohydrate contents. The calorific value of S. cynthia ricini was higher with 430.19 ± 2.241 kcal whereas D. marginalis showed a lower value of 382.58 ± 1.527 kcal (Table 2).

Mineral contents
D. marginalis showed higher contents of potassium (K), calcium (Ca), copper (Cu), iron (Fe) and zinc (Zn) than S. cynthia ricini. High level of Mg of 54.323 ppm was observed in D. marginalis. S. cynthia ricini showed higher levels of manganese (Mn) and boron (B). It, however, did not show the presence of Mg. A high level of 80.400 ppm Na was observed in S. cynthia ricini. Pd, Cd, and As were detected in D. marginalis whereas, S. cynthia ricini showed the presence of Pb and Cd only, and As was found to be absent (Table 3).

Discussion
Since these insects are consumed as food, knowledge on their mineral contents is essential. Earlier studies on various insects do not possess information on their mineral contents (Narzari and Sarmah 2015b;Narzari et al. 2017). Proximate compositions of 20 insects consumed by the Bodo tribe were analysed by Narzari and Sarmah (2015b). The study revealed moisture content of the insects ranging from 1.72 to 8.59%, nitrogen content  Similarly, the proximate contents of both the studied insects ranged within the values described in the above study and showed high contents of fat and protein and a high calorific value of 430.19 kcal (S. cynthia ricini) and 382.58 kcal (D. marginalis) indicating that these insects could act as alternatives to other foods which are usually costlier, particularly in rural areas (Patel et al. 2019). Consumable food items should be examined for the presence of essential elements. Rumpold and Schlüter (2013) analyzed 236 different edible insects and found that most edible insects contain considerable amounts of energy, protein, unsaturated fatty acid and nutrients such as Cu, Fe, Mg, Mn, biotin and pantothenic acid in trace amounts. Another similar study by Kim et al. (2017) on 5 edible insects consumed in Korea revealed that Ca concentration ranged from 349.2 to 2282, phosphorus ranged from 5105 to 8875.1, Mg ranged from 502.2 to 2522.9, Zn ranged from 75.6 to 189.1, Fe ranged from 49.5 to 99.7, Cu ranged from 9.4 to 27.3, Mn ranged from 7 to 48.2, B ranged from 14.6 to 22.3 and molybdenum (Mo) ranged from 0.2 to 1.5 mg/kg dry weight. The results of this study revealed that the studied insects possess considerable amounts of Na, K, Ca, Mg, Zn, Fe, Cu, Mn and B. Toxic elements could also be present in insects, and their ingestion can cause mental retardation and damage to the central nervous system. They also alter blood parameters and cause damage of vital organs such as the liver, kidneys and lungs (Hajeb et al. 2014). Several studies on mineral content of insects have not evaluated the content of toxic elements (Shantibala et al. 2014;Ghosh et al. 2017a). A study by Kweon et al. (2012) on elemental analysis of Bombyx mori analysed the contents of both essential and toxic metals. The study detected the presence of elements such as Zn and Cu and although toxic heavy metals such as Pd, Cd and As were absent, mercury (Hg) was found in trace amounts. This undertaken study revealed the presence of toxic heavy metals Pb and Cd in both the studied insects in trace amounts. Earlier, Narzari and Sarmah (2015b) had estimated the proximate contents of D. marginalis, but mineral contents including toxic heavy metal were lacking. D. marginalis showed the presence of As, whereas it was not detected in S. cynthia ricini. The highest risk of As exposure takes place through ingestion (Cooper et al. 2020) leading to bioaccumulation in the consumers which ultimately results in adverse health conditions.

Conclusions
Edible insects can be sources of nutrients such as proteins, fats, carbohydrates and minerals. Although insects have high nutrition value, the current status of edible insect is still insufficient to replace other traditional foods worldwide (Kim et al. 2019); hence, more studies to evaluate their nutrient contents needs to be carried out. Heavy metal accumulation in edible insects depends on many factors such as insect species, growth phase and feed substrate (EFSA 2015). Hence, every edible insect must undergo proper studies to evaluate their mineral contents involving toxic elements as well. Such studies will help in avoiding future health complexities due to their consumption or can act as food supplements during food shortage.
Abbreviations AOAC: Association of Official Analytical Chemists; ICP-OES: Inductively coupled plasma-optical emission spectroscopy; SEM: Standard error of mean KC supervised the work, collected the insects and edited the manuscript. DS carried out investigation of the samples and performed the calculations. PJS carried out investigation of the samples and performed the calculations. ADS conceptualised the study, carried out the statistical analysis and wrote the original draft. The final draft was verified, read and approved by all the authors.
Authors' information KC is currently the Head of the PG Department of Zoology at Science College, Kokrajhar. He specialises in teaching Entomology and has been actively teaching pre university, undergraduate and post graduate students. DS is currently an Assistant Professor in the PG Department of Zoology at Science College, Kokrajhar. She specialises in teaching Entomology and has been teaching undergraduate and postgraduate students. PJS is also currently Assistant Professor in the PG Department of Zoology at Science College, Kokrajhar. She has been actively teaching undergraduate and postgraduate students. ADS earlier served as an Assistant Professor in the Department of Zoology at St. John College, Dimapur and Union Christian College, Shillong, teaching undergraduate students. He is currently an Assistant Professor in the PG and Research Department of Zoology at B.