Ducetia japonica and Phyllozelus Sp.: Two Tettigoniid Species of Or- thopteran Insects Appreciated by Tribal People of Arunachal Pradesh (North-East India) May Serve as Future Alternative Food Source

Ducetia japonica and Phyllozelus sp., two tettigoniid orthopteran insect species appreciated as food by people of Arunachal Pradesh were analyzed to assess their nutritional potentials. Ducetia japonica and Phyllozelus sp. contained 56.28% and 61.57% protein, 14.99% and 7.93% fat, 11.84% and 8.30% fiber, 4.59 % and 3.01 % ash and 11.84% and 19.19% carbohydrates respectively. In both the species 18 amino acids were detected including essential amino acids and were found satisfying the recommended dosages (scores >100). Both the species contained 22.77% and 26.84% MUFAs and 18.93% and 41.73% PUFAs, respectively. The two orthopteran insects contained superior amount of micro minerals (Cu, Fe, Zn and Mn) and moderate amounts of macro minerals (Na, K, Ca and Mg). Thus, these two-orthopteran species could serve as an alternative food source and as a tasty and promising source of essential nutrients on par with or superior to conventional food of both plant and animal origin. They should be included in plans to rear and farm nutritionally valuable species of edible orthopterans.


Introduction
The population of especially India's northeast is expected to continue to increase, which means that food security for the inhabitants of the region becomes an increasingly important issue. Having noticed that members of various traditional societies made use of insects as food, [1] suggested that insects as an item in the human diet could ease the problem of global food shortages and Arunachal Pradesh is one of the regions of India where insects even today are still being valued as food [2]. Entomophagy, i.e. the consumption of insects by humans [3], has a long history and has been reported from many parts of the world [4]. It is estimated by [5] that even today worldwide there may still be 2 billion people that at least occasionally choose to eat some of the approximately 2,000 known species of edible insects [6]. Taking into consideration that many insect species are multivoltine and have a high fecundity, exhibit efficient feed conversion characteristics, possess low space requirements and may be reared on a variety of comestibles often considered inedible or unattractive by human, certain species of insects can indeed contribute to food security and represent an interesting alternative to conventional domestic animals.
With an area of 83,743 km 2 Arunachal Pradesh is the largest state in North-East India and home to numerous species of insects, a plethora of which, representing at least 12 orders, have been identified as edible and as highly appreciated by members of the local tribes [2,7]. In most cases, these insects are not consumed as a nutritional supplement or to ward off starvation but are esteemed for their taste and often regarded as a -frequently not even very cheap-delicacy. Analyses of the nutrient compositions of the locally highly regarded bug Aspongopus nepalensis [8], the weaver ant Oecophylla smaragdina and the termite Odontotermes sp. [9] have demonstrated that such insect species are of value as a source of essential amino and fatty acids, minerals and vitamins. Some of the most popular edible insects not only in Arunachal Pradesh, but worldwide, are members of the order Orthoptera and [10] in their thorough review, covering edible members of the families Acrididae, Pyrgomorphidae, Tettigoniidae and Romaleidae, provide a wealth of comparative data on the nutrient contents of these insects. The acridid grasshopper Chondracris rosea and the gryllid cricket Brachytrupes orientalis are two orthopterans appreciated by Arunachal Pradesh tribals belonging to the Adi, Apatani, Chakma, Deori, Galo, Nyishi, Singpho and Wancho and their nutrient contents have been published [11]. Two additional Orthopteran species, the tettigoniids Ducetia japonica and Phyllozelus sp, are equally appreciated, but have not yet had their chemical compositions analyzed, it is for this reason that the present study focuses on these two species, hoping that nutritious products based on these insects may be promoted in the future among the tribes of Arunachal Pradesh and elsewhere in India as food or feed or even a source of bioactive medicinal compounds [12].

Specimen Acquisition and Sample Preparation
Both Ducetia japonica and Phyllozelus sp. were collected from a variety of places around campus, e.g. rice and sugarcane fields, forests, fruit orchards and hills, trees, shrubs, herbs and grasses. Sampling occurred throughout the year for two consecutive years, 2013 to 2015. Only adult insects were collected. The sexes were not separated, because firstly, no taboo had been reported regarding the sex of the selected insects, secondly male and female specimens differed little in size and coloration and thirdly, both were equally appreciated as food.

Sample Preparation
In the laboratory, the insects were washed thoroughly, blotted and then oven-dried (50-60°C). Ground to a powder, they were then stored in deep-freeze (-20°C) under vacuum for proximate, amino acid, mineral, and fatty acid analyses. The analyses were carried out in triplicate and completed within ten days following the collection of the insects. All of the solvents and chemicals used in this study were of analytical grade and care was taken that the glassware was meticulously clean.

Analyses Macronutrients
Proximate analyses of the insect samples to determine moisture content, crude protein, crude fat, crude fibre, ash and Nitrogen Free Extract (NFE), were based on standard methods [13]. Moisture percentage was calculated by drying the sample in an oven at 100°C for 2 h. Crude protein was determined by the Kjeldahl method and total protein content was subsequently calculated by multiplying the nitrogen content by a factor of 6.25 [14]. Fat percentage was calculated by drying fats after extraction in a Soxhlet apparatus, using petroleum benzene. Carbohydrate content was estimated by subtracting the sum of the weights of crude protein, lipid, ash and fibre from the total dry matter and reported as NFE by difference or as nitrogen free extracts. Crude fibre was determined through double digestions, first with sulphuric acid and then with sodium hydroxide. The ash content was determined by combusting the insect sample in silica crucibles in a muffle furnace at 620°C for 3 hours. The calorific value (kcal/100 g) was calculated by multiplying the factors for carbohydrate and protein by 4 each and that of fat by 9 and then taking the sum of the products. All of the analyses were performed in triplicate and expressed as mean ± standard deviation.

Fatty Acids
The methyl ester of the fat of the insect sample was prepared according to [15]. The fatty acid composition of the methyl ester was determined by Gas-Liquid Chromatography (GLC) according to [16] and based on three samples of 30g dry weight each.

Amino acid
Amino acid composition was determined by HPLC (Agilent 1100) according to the standard method recommended by the [13]. The ground samples were hydrolyzed in 6 N HCl for 18h at 120°C under nitrogen atmosphere and then concentrated. The concentrated samples were reconstituted with 20 mM HCl and derivatized with borate buffer. The hydrolyzed samples were analyzed for amino acid composition. All the analyses were performed in triplicate and expressed as a percentage of individual amino acid in the protein (i.e. total amino acids) fraction. Amino acid scores were calculated based on the recommendations by [17].

Minerals
Minerals were determined by Atomic Absorption Spectrophotometry (AAS) after dry-ashing the samples followed by acid dilution [13]. The ash was digested with HCl made up to 100 ml and filtered before the mineral elements were determined by AAS. All the analyses were performed in triplicate and expressed as mean ± standard deviation.

Results and Discussions
Moisture: (Table1) The moisture content of both species (58.48% in Ducetia japonica and 57.78% in Phyllozelus sp.) was found to be rather similar to that of beef (USDA database), but somewhat higher than that known from the orthopteran Chondacris rosea [11] and lower than that of the meadow grasshopper Chorthippus parallelus [18]. Moisture content of a food item can be an indicator of its stability and susceptibility to microbial spoilage [19] and therefore a lower value can be expected to improve the shelf life and the retention of nutrients of the food item in question. Crude fibre: (Table 1) Crude fibre is the nonstructural carbohydrate or the indigestible part of an insect. The present study shows that the species analysed are rich in fibre content reaching a value of 11.84% in Ducecia japonica and 8.30% in Phyllozelus sp. In terms of fibre content this places the species within the range of 3 -12.2% for 25 orthopteran species as reported by [20] and members of the family Tettigoniidae [18]. It was also comparable to the dietary fibre content (g/100 g) of rice (4.1), wheat (12.5), whole Bengal gram (28.3), lentil (15.8), cabbage (2.8), green colocasia (6.6) and yam (4.2) [21]. Fibre enriched food is considered advantageous for our health as it reduces constipation by stimulating bowel movement, is useful in the context of weight control and fat reduction since it gives a feel of satiety even when only a small amount of such food is eaten. Nitrogen Free Extract: (Table 1) Besides crude fibre, considerable amount of nitrogen free extracts indicative of carbohydrates is found in Ducecia japonica and Phyllozelus sp. and amounted to 11.84% and 19.19% respectively. These values are comparable to those (0.19 to 22.64%) reported by [20,22] and for other Orthopteran species, but considerably higher than carbohydrate values given for 3 tettigoniid species listed in [10]. Differences like these may be species-specific and depend on the season, nutritional state and age of the insect, as shown for lipophilic contents of Rhynchophorus phoenicis larvae by [23] but are also likely due to differences in the methods used to determine carbohydrate content.
Protein and Amino Acids: (Table 1 and 2; Figure 1,3 and 4) Respective crude protein values of 56.28% and 61.57% of Ducetia japonica and Phylozellus sp. were higher than those for other Orthopterans reported by [20,25] but in line with acridid grasshoppers [10,11]. When compared with the protein content of some conventional sources of meat like beef, pork and chicken, that of insects generally was often higher, indicating the potential of edible insects in mitigating protein malnutrition. The amino acid composition of proteins determines the quality of proteins. Humans and insects have the same amino acid requirements [28]. In Ducetia japonica and Phyllozelus sp. proteins are composed of 18 amino acids, including all the amino acids regarded as essential for humans and satisfying (scores > 100) the recommended dosages suggested by [17]. As with other insect species (including orthopterans: [10,29] all of the essential amino acids were present [20,30]. Of particular interest were the high levels of valine, leucine, lysine, isoleucine and threonine, which together with lysine, phenylalanine, and histidine surpass the values recommended by [17] for the "chemical score" of amino acid as an index of protein quality. Lysine and threonine are the limiting amino acids in conventional diets of rice, wheat, cassava and maize prevalent in the developing world [31]. A comparison of the amino acid composition of Ducetia japonica and Phyllozelus sp. with conventional animal foods (USDA database) indicates the supply of some of these essential amino acids from the two-insect species would either be similar or even superior to those found in conventional food items.
An appropriate balance of EAA and NEAA and other nitrogenous compounds is necessary for an effective utilization of dietary proteins. In both of our species, the NEAA were also present in substantial amounts (2.7 to 4.8 g/100 g), which were higher than or comparable to those of other edible insects [10,20,30] Figures 4 (a and b): Comparison of amino acid composition (% of total amino acid) of Ducetia japonica, Phyllozelus sp. and some conventional foods of both plant and animal origin (source USDA database www.ndb.nal.usda.gov).  Fat Content: (Table 3; Figure 2,3 and 5) With respective fat contents in Ducetia japonica and especially Phyllozelus sp. of 14.99% and 7.93%, these insects were leaner than many other insect species appreciated as food or conventional food sources [8,9,20,23,[32][33][34], something not exactly undesirable. When compared with conventional meats (USDA database) both of the species are lower in fat content.
The fatty acid composition determines the quality of the fat. The fatty acid compositions of both Ducetia japonica and Phyllozelus sp. revealed the presence of 14 and 19 Fatty acids, respectively. Ducetia japonica and Phyllozelus sp. contained 57.96% and 30.74% saturated (SFA), 22.77% and 26.84% Mono-Unsaturated (MUFA) and 18.93% and 41.73% Poly-Unsaturated Fatty Acids (PUFA), respectively. Major components of the SFA fraction commonly found in both the species were 14:0 myristic, 16:0 palmitic, and 18:0 stearic acids. The amounts of oleic acid in both Ducetia japonica and Phyllozelus sp. were 18.93% and 24.48% respectively. In Ducetia japonica the UFA fraction was 41.70%, which is lower than the SFAs present. In Phyllozelus sp. the proportion of unsaturated fatty acids (UFAs 68.57%) was higher than that of Saturated Fatty Acids (SFAs) and a similar situation was reported for many other edible insects [10,33,[35][36][37][38][39][40]. In Ducetia japonica alpha-linolenic acid was only recorded to 1.50%, but another PUFA, i.e., ω-6 linoleic acid reached 8.51% in Ducetia japonica and 14.60% in Phyllozelus sp. and was therefore more prevalent in these insects than in most other conventional foods of animal origin like lamb, veal, beef and cow milk. In Phyllozelus sp. alpha-linolenic acid (a PUFA) reaches 26.15%, which is significant for human nutrition as it is thought to considerably help reducing the occurrence and effects of cardiovascular, hypertensive, inflammatory, and auto immune disorders as well as depression and certain neurological functions [41,42]. Thus, Ducetia japonica and Phyllozelus sp. could be an excellent source of quality fat, due to the presence of PUFAs and stearic acid as part of the SFA content. With an SFA: UFA ratio of 1.390 and 0.448 in Ducetia japonica and Phyllozelus sp., (a little higher than the reported range of 0.262 to 0.96 for other insects: [9,30,33,[37][38][39]. The presence of both SFA and UFA could be seen as an advantage as they may complement each other's physiological functions. However, not all the saturated fatty acids have the same effect on cholesterol synthesis in the liver. Only saturated fats with chain lengths of (12, 14, and 16: lauric, myristic and palmitic acids) have been shown to elevate blood cholesterol level and stearic acid (C18, saturated) is actually credited with lowering body cholesterol by 21% [43].   Ash and Mineral: (Table 1 and 4; Figure 6) After the removal of water and organic substances by intense heating the remaining ash contents of 4.59 % in Ducetia japonica and 3.01 % in Phyllozelus sp. are comparable to those of other orthopterans [10] and insect species, generally [20,30,32,44]. Ash content of a species is an indicator of its mineral content.
The mineral composition (mg/100g) of Ducetia japonica and Phyllozelus sp. showed the presence of at-least eight important minerals. Macro-minerals (mg/100g) Ca, Mg, Na and K as well as micro-minerals (mg/100g) i.e. Cu, Fe, Zn and Mn were present in appreciable amounts. However, the rank of micro-nutrients was superior to macronutrients as per requirement proposed by [45]. Both insects contained moderate amounts of macro minerals (Na, K, Ca and Mg and although their levels in Ducetia japonica and Phyllozelus sp. were low as per the [45] recommendation, they lay within the range known from other insect species [32,46,47]. With the exception of Zn and Fe, which were somewhat lower in our two species than what had been reported for other orthopterans [10]; the micronutrient assemblage was generally comparable to or even higher than that known from conventional foods of animal origin [48]. Availability and uptake of iron represent core public health problems, especially in developing countries, where often the food that children, elderly and nursing women get is deficient in this and some of the other micronutrient minerals [49]. About 100 g of Phyllozelus sp. can provide a sizeable amount of the recommended daily doses of zinc and iron and although 100 g of both Ducetia japonica and Phyllozelus sp. may not be able to supply all the minerals or their amounts required by humans, the two insects can make a considerable contribution to the provision of much needed trace elements in the nutrition. Figures 6 (a and b): Comparison of mineral content (mg/100 g DM) of Ducetia japonica, Phyllozelus sp. and some conventional foods of both plant and animal origin (source USDA database www.ndb.nal.usda.gov).

Conclusion
Based on our chemical analyses, Ducetia japonica and Phyllozelus sp. can be recommended as a nutritional supplement to combat malnutrition among Arunachal Pradesh's inhabitants and by inference, all those elsewhere in the world, which have access to the species under debate. Insects like the Ducetia japonica and Phyllozelus sp. studied in this paper must be seen as a tasty and promising source of essential nutrients on par with or superior to conventional meats of vertebrate origin. They should be included in plans to rear and farm nutritionally valuable species of edible orthopterans [18,50].