Nutritional properties of giant water bug, Lethocerus indicus a traditional edible insect species of North-East India

ABSTRACT

Edible insects play an important role in human health and food security. Among those, the Giant water bug, Lethocerus indicus (Lep.& Ser.) is a widely used edible insect known for its aroma, flavor, and therapeutic purposes. In the present study, we investigated the nutritional profile, natural habitat, and feeding behavior of L. indicus in aquarium conditions. A comparative analysis of male and female insects’ aroma contents and fatty acid (FA) profiles was also conducted. A dry fried male insect yielded volatile oil of 0.96%/2 g body weight, whereas a dry fried female yielded 0.48%/5.36 g of body weight. In terms of lipids, fresh male insects had 0.15%/5.42 g of body weight and fresh female insects had 0.28%/9.48 g of body weight. There are 24 volatile compounds specific to males, 37 specific to females, and 13 commons to both were identified. 2-Hexen-1-ol, acetate, (Z)- which smells like banana, was prevalently found in males while 4-Octene, 2,6-dimethyl-, [S-(Z)] was prevalently found in female insects. Fatty acids profile analysis detected 32 FA with 12 unique FA from males whereas 22 FA and 3 unique FA were identified from female insects. The SFA percentage present in males and females was 77.44% and 85.21%. Males had 6.78% MUFA content while females have 4.75%. Males have 18% PUFA content enriched with DHA, and EPA, while females had 10.04%. This study revealed that with the presence of a banana-like smell of volatile compound and more MUFA and PUFA in males, the native people of North-East India preferred male over female insects for entomophagy.

GRAPHICAL ABSTRACT

Keywords:

• Lethocerus indicus
• entomophagy
• entomotherapeutic
• GCMS
• fatty acids
• SFA
• MUFA
• PUFA

1. Introduction

Giant water bugs are aquatic insects belonging to the Heteroptera suborder. Aquatic insects possess two types of scent glands: one that emerged from the thoracic region during the adult stage and one from the abdominal region during the larval stage [1]. These insects employ the chemical substances they emit to defend themselves from predators and to prevent microbial growth inside their bodies [2]. Lethocerus indicus, (Heteroptera: Belostomatidae) is one of the edible gigantic water bug insects and has long been a crucial flavoring agent in Manipur and Thailand. In contrast to female insects, male insects are well known for their flavor and odor [3,4]. Edible insects have become the source of future feed and food due to their beneficial nutritional characteristics and low environmental impact [5]. It is also safe and healthy for humans to eat insects as food [3]. It was predicted and hoped that malnutrition challenges may be resolved by providing 10% of the protein from insect sources rather than other meat sources. Insects are a good source of minerals, macronutrients, and other nutrients like sodium, calcium, and magnesium [6].

Because of metabolic mechanism variation based on diet, the nutritional component of aquatic insects varies from species to species and is dependent on the feeding habits available in their environmental niche [7]. Nutritional components of lipids present in different sexes of the species also vary due to sex-specific nutritional requirements of the body mechanisms [8]. A potent odorant compound (E)-2-hexenyl acetate which has been responsible for the aroma of the male L. indicus insects was reported in a study conducted in Thailand, but this study did not mention the aroma obtained from female insects [9]. The nine fatty acids identified from the L. indicus insects belonged to saturated and unsaturated fatty acids, but they did not mention the sex of the insect [10]. Lipids are the second largest constituent of insects after proteins [11]. Different fatty acids are utilized as ingredients in the food, pharmaceutical, and cosmetic, industries [12]. Cosmetic and personal care products derived from fatty acids obtained from the insects such as C12:0, C14:0, C16:0, and C18:1, are also available on the market [8]. For example, the insect oil extracted from Tenebrio molitor has positive effects on skin wound treatment [13] whereas fat oil extracted from black soldier fly larvae is used as a replacement for butter for making cookies, waffles, and cakes [14]. These potential properties obtained from the insects hypothesize that L. indicus must possess various properties which are economically important for human being.

Lethocerus indicus has a variety of roles in maintaining ecological balance, human health, and food security [7]. This study aimed to determine the fragrance and fatty acid differences between male and female Lethocerus indicus insects. To understand the adaptation of insects in their natural habitat, ecological conditions, and their feeding behavior a mimicking environment in an aquarium was also created and monitored. The nutritional value of this entomophagy insect has the potential to be very beneficial for bio economy strategies in the North eastern region of India.

2. Materials and methods

2.1. Sample collection and habitat observation

The giant water bugs were collected from the natural habitat of Doyapur Lake at Disagaphu (latitude: 25.7520540, longitude: 93.611874°) (Supplemental Fig. S1(a)) and reared in an aquarium dimension (40X60) cm.The aquatic plants associated with these insects were listed (Supplementary Table S1), and the water from the habitat was stored at 4°C for further analysis.

2.2. Acclimatization of insects in the aquarium

Mass cultivation of L. indicus was performed in an aquarium by keeping the water at room temperature. The water level was 14 cm in the aquarium with the filter and aerators for the constant supply of oxygen. The selected aquatic plants available in the habitat such as Pontederia crassipes (water hyacinth) and Panicum hemitomon (Hub Napi), were added and reared in the aquarium (Figure 1(a,b)). The local fish species such as Trichogaster fasciata (Ngapemma), Puntius species (Phabounga), Lata Fish (Ngamu), Amblypharyngodon mola (Mukanga),Hydrophilus piceus (water beetle), Freshwater snails were also added as prey for L. indicus insects. In the aquarium,30% of the water was replaced daily, and dead fish and other unwanted materials were removed.

Figure 1. Acclimatization of Lethocerus indicus in the aquarium with the associated plants. The photo of a) L. indicus insects along with b) giant Zizania (Wild rice) grass and Pontederia crassipes (water hyacinth) aquatic plants inside the aquarium c) photo of L. indicus eggs laid inside the aquarium.

2.3. Water quality analysis

The collected water from the habitat and local pond stored at minus 4°C was brought at room temperature and the water quality parameters such as the potential of Hydrogen (pH), Dissolved Oxygen (DO), Total Dissolved Solids (TDS), Electrical Conductivity (EC), and salinity were tested using Aquaread AP-2000 [15]. The Chemical Oxygen Demand (COD) was analyzed following the protocol described in APHA [16].

2.4. Extraction of volatile oil from L. indicus

The male and female insects are identified based on the genital parts (Supplementary Fig. S1(b & c)).The selected insects were fried in a frying pan above the gas flame without oil as a traditional way roasting method for 10 minutes. The extraction of volatile oil was performed using n-hexane solvents from dry-fried males and females separately. Five fried males (total mass 10 g) and five females (total mass 26 g) were chopped into small pieces using a sterilized scissor and placed into a 500 ml glass bottle with 200 ml of Hexane and kept in the shaker oven at 30°C over night following the oil extraction method with slight modification [17]. The samples had allowed to cool down and filtered using a Whatman filter paper and transferred to the round flask bottles. The sample was concentrated using a rotary evaporator and stored at minus 4°C in an amber vial for further analysis. The total weight of the oil extracted was determined by subtracting the weight of the round flask bottle. The weight of oil obtained per 10 g male fried insect was 0.48 g and 26 g female insects were 0.63 g.

2.5. GC-MS analysis of the essential oil

The essential oil collected from the L. indicus male, and female were diluted in the ratio of 1:100 (essential oil: n-hexane). The 0.5 µl of the sample prepared was injected into the GC-MS. The GC-MS employed have Trace1300 as Gas chromatography and TSQ DUO as Mass Spectrophotometry. The GC have the silica capillary column (30 m X 0.25 mm; 0.25 µm film thickness), TG-5 MS interfaced with the MS.The carrier gas used in the instrument was helium with a flow rate of 1 ml/min for 60 minutes [18,19]. The ionization energy was set at 70 eV, and the mass transfer line temperature at 250°C and the ion source temperature at 280°C. The column temperature was programmed from 40°C for 1 min to 280°C at the rate of 5°C/min by a heating ramp for 20 min and held at 250°C.The inlet injector temperature was set at 240°C with a split mode of 1:20 maintained [18,19]. The mass spectra were filtered from the mass range from 35 to 450 Mw.The identification of the volatile compound was determined by comparing the spectra obtained from the sample with the reference to the mass spectrum given by the National Institute of Standards and Technology (NIST) GC/MS Libraries 2017 [19].

2.6. Total lipid extractions from L. indicus insects

The total lipid content in the fresh Lethocerus indicus of both male and female insects’ was determined using the method developed by Blight and Dyer [20]. The selected five fresh male insects with a total weight of 27.1 g and another five female insects with a total weight of 47.4 g were chopped into pieces and transferred to 200 ml of a mixture of chloroform and methanol solvents (1:2) contained in the 500 ml reagent bottle. The insect samples were kept in the shaking water bath for 4 hours at 30°C for homogenization. The homogenate samples were cooled down and filtered through Whatman No.1 Filter paper on a Coors No.3 Buchner funnel with a slight suction. The extracted lipid sample was transferred to a preweighed round bottle flask and concentrated using a rotary evaporator. The total lipid weight was determined after subtracting the weight of the round bottle flask.

2.7. Thin layer chromatography (TLC) analysis

TLC was performed to identify lipid profile components of lipids extracted from the L. indicus male and female insects. The TLC aluminum sheets coated with 0.2 mm thickness silica 60F₂₅₄ from E Merck, India was used by spotting a 5 µl sample. The lipid contained in the sample was separated by using a mixture of solvent systems containing hexane, diethyl ether, and acetic acid in the ratio of 80:20:1 [21]. The separation of the lipid was conducted inside a glass chamber, and spots of the samples were visualized with the help of iodine vapors.

2.8. Fatty acids profiling of insect lipid by gas chromatography

Transesterification of extracted lipid from male and female L. indicus insects was conducted using methanol and H₂SO₄ reagents at 100°C for 1 hr. The fatty acid methyl esters (FAMEs) were dissolved in hexane and allowed to dry at room temperature [21]. The dried FAME samples obtained from male and female insects were analyzed in a GC/MS (6850N, Agilent Technologies) attached to an FID detector along with Agilent DB-225 capillary column. The oven temperature program was set from 160°C for 2 minutes to 230°C at the ramping rate of 5°C/min and held till 230°C for 20 min. The temperature of the injector and detector was maintained at 230°C to 270°C. The sample injection volume was 1 µl and the carrier gas was nitrogen with a flow rate of 1 ml/min. The area percentage for each chromatogram was recorded with a standard HP-chemstation data system. The FAME analysis data were identified by comparing their fragmentation pattern with the internal standard obtained from Sigma Aldrich, India.

2.9. Statistical analysis

Experiments were carried out in triplicate and data were represented as mean ± standard error (SEM) for habitat water parameter analysis.

3. Results

3.1. Observation of the habitat and feeding behavior in the aquarium

The habitat observation for L. indicus aquatic insects was conducted to mimic the natural habitat under aquarium conditions for the large scale production and also for the consistent nutritional profile maintenance. The habitat of Lethocerus indicus Doyapur Lake at Disagaphu has a swampy natural environment, the plentiful aquatic flora in habitat. The water analysis of Doyapur Lake is represented in Supplementary TableS 2. The data showed that water is acidic, with a pH of 5.4, a dissolved oxygen value of 7.5 mg/L, TDS of 0.0083 ppm, EC of 0.129 s/cm, a salinity of 0.04 ppt, and COD of 879 mg/L respectively. The water analysis of habitat help in understanding the adapted environment of L. indicus and mimicking the mass cultivation of insect in the aquarium condition. Under aquarium conditions, L. indicus primarily attacked the caudal peduncle, caudal fin region, head, operculum, lateral line, and spiny region of the local fish preys as in Figure 2(a–f). The disturbance that occurs when fish were attacked in the area mentioned slows down their swimming speed inhibits them from sensing the surrounding water pressure, throws off their swimming balance, and weakens them by interfering with their respiratory systems. All these attacked preferences will help in immobilizing their prey and make it easier to trap them as their food source. These insects were hunting in groups for the larger prey. The freshwater snail was attacked at the region of the digestive gland, stomach, anterior kidney, and testis while the water beetle was assaulted between the head and shoulder and between labrum and labium. Their prey’s tissue was dissolved and sucked the pre-digested food into their proboscis. These insects adapted to the aquarium environment and were able to endure it for six months. Insects began to lay eggs in the adapted aquarium conditions (Figure 1(c)).The giant predatory aquatic water bug Lethocerus indicus habitat observational study revealed that these insects required a habitat that is swampy and less polluted with specific water parameters. The habitat of this insect’s surrounding area is a thinly populated rural area with less human disturbance and encircled by paddy fields. Such conditions are ideal for their survival in their natural habitat.

Figure 2. Feeding behavior observation in the aquarium. a) attacked at the caudal peduncle and caudal fin region of Trichogaster fasciata. b) attacked at the head region, operculum as well as a lateral line of Puntius species. c) attacked in between the shoulder and head part of Hydrophilus piceus. d) attacked at the caudal fin and lateral portion close to the intestine of Amblypharyngodon mola; e) attacked at the caudal fin, head, and spiny region of Lata fish. f) feeding on freshwater snails by piercing at digestive gland, stomach, anterior kidney, and testis portion.

3.2. Characterization and comparative analysis of the volatile compound from L. indicus insects

L. indicus male insects are preferable entomophagy insects for their aroma and flavor by the local people of northeast India, Manipur. We aim to compare the volatile compound present in both male and female insects. The male and female insects acclimatized in the aquarium conditions were used for the volatile oil extraction. The amount of volatile oil extracted from fried insects was 0.96%/2 g male body weight and 0.47%/5.36 g female body weight. 50 volatile compounds from female insects (Table 1) and 37 from male insects (Table 2) were identified using GC-MS analysis. Thirteen of these chemicals are common to both male and female insects, whereas 37 of these compounds are specific to female insects, and 24 of these compounds are specific to male insects (Supplementary Fig. S2). We also identified 13 functionally uncharacterized volatile compounds from male and 19 functionally uncharacterized volatile compounds from female insects. The functionally characterized volatile compounds of male L. indicus were 64.9% and their respective functions are represented in the (Supplementary Fig. S3) whereas 62% of the female volatile compounds are functionally characterized as represented in the (Supplementary Fig. S4). The functionally characterized volatile compounds obtained from male and female insects are used as an ingredient in food as well as cosmetic industry. Some of them are used in the pharmaceutical industry and some other compounds are used as Surfactant, Refrigerant, Heat and cooking etc.

Table 1. Volatile compounds detected from the female L. indicus insects.

Sl.no	RT	Compound name	Odor Description	Common/Unique	RSI	Area %	Mol. Wt.
1	28.11	4-Octene, 2,6-dimethyl-, [S-(Z)]-		Unique	901	42.28	140
2	23.11	1-Undecanol	Citrus, apple & banana	Unique	941	21.6	172
3	28.27	1-Hexadecanol	Waxy & rose	Unique	904	10.2	242
4	40.63	à, à-dimethyl-Palmitoleamide		Unique	881	6.96	253
5	59.91	Andrographolide	Aromatic	Unique	842	1.73	350
6	49.14	Cyclopropane,(R,R)-1-((Z)-hex-1’−enyl)-2-ethenyl- enyl-		Unique	908	1.27	150
7	46.95	10-Pentadecen-5-yn-1-ol, (E)-	Waxy, fatty & rosy	Common	943	1.21	222
8	10.58	2-Hexen-1-ol, acetate	Fruity, apple, waxy & oily	Common	905	0.94	142
9	53.56	2-Methyl-1-undecanol	Herbaceous & orange	Unique	914	0.89	186
10	43.71	2-Hexyl-1-octanol	Waxy	Unique	911	0.88	214
11	45.53	Nonacos-1-ene	Lotus	Unique	931	0.84	406
12	37.3	1-Hexyl-2-nitrocyclohexane		Unique	967	0.47	213
13	37.63	Nonanamide	Pungent	Unique	837	0.42	157
14	37.44	3-Ethyl-2,6,10-trimethylundecane		Common	878	0.4	226
15	8.92	Hydroperoxide,1-methylpentyl		Unique	846	0.37	118
16	35.81	10-Heneicosene (c, t)		Common	823	0.35	294
17	39.53	2-Heptadecenal		Unique	896	0.3	252
18	8.64	Ethanone, 1-(3-ethyloxiranyl)-		Common	851	0.27	114
19 20	46 47.58	Heptane, 3,3-dimethyl- Acetic acid, trifluoro-,3,7-dimethyloctyl ester	Petroleum	Unique Unique	886 863	0.27 0.25	128 254
21	32.89	7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione		Unique	896	0.21	276
22	38.93	Z-2-Dodecenol	Pleasant fatty	Unique	857	0.19	184
23	42.6	Pentane, 2,2,3,3-tetramethyl-		Unique	869	0.19	128
24	39.06	1,6:3,4-Dianhydro-2-deoxy-á-d-lyxo-hexopyranose		Common	827	0.18	128
25	29.35	Tridecanol, 2-ethyl-2-methyl-		Unique	857	0.17	242
26	34.7	Ethyne, fluoro-		Unique	968	0.16	44
27	38	2,7-Octadiene-1,6-diol,2,6-dimethyl-, (E)-		Unique	916	0.16	170
28	41.03	3,8,11-Trioxatetracyclo [4.4.1.0(2,4).0(7,9)] undecane,(1à,2á,4á,6à,7á,9á)-		Unique	867	0.16	154
29	13.15	Propane, 2-methyl-1-nitro-		Common	864	0.14	103
30	40.94	Sulfurous acid, nonyl pentylester		Unique	851	0.14	278
31	56.09	3-Hexyne, 2-methyl-	Fruity green	Unique	868	0.14	96
32	32.77	Nonadecane, 2-methyl-		Unique	862	0.12	282
33	36.72	Octadecane, 2-methyl-	Floral	Common	837	0.12	268
34	13.43	Propanoic acid, 2-oxo-, ethyl ester	Ethereal fruity sweet	Unique	902	0.11	116
35	35.64	2-Heptanone,6-methyl-5-methylene-	Citrus fruity	Common	909	0.11	140
36	40.29	2-Butenoic acid, 2-methoxy-, methyl ester, (Z)-		Unique	859	0.11	130
37	48.26	1,1,1,5,7,7,7-Heptamethyl-3,3-bis(trimethylsiloxy)tetra siloxane		Unique	805	0.11	444
38	50.68	Cyclopentadienylrhodiumbis (trimethyl phosphite)	Pungent	Unique	819	0.1	416
39	61.28	s-Triazolo[4,3-a] pyridine,5-methyl-3-phenyl-		Unique	848	0.10	209
40	33.89	2-Methyl-1-hexanol		Unique	829	0.09	116
41	37.81	2,7-Octadiene-1,6-diol,2,6-dimethyl-, (Z)-		Common	917	0.09	170
42	29.8	N-(2,6-Dimethylphenyl)-N-[(2E)-3-methyl-1,3-thiazinan-2-ylidene] amine		Unique	893	0.08	234
43	35.95	1-Allyl-cyclohexane-1,2-diol		Unique	897	0.08	156
44	46.35	1,2-Pentadiene,4-methoxy-4-methyl-		Common	938	0.08	112
45	48.69	3-Methyl-2-(2-oxopropyl) furan	Caramellic	Unique	800	0.08	138
46	12.47	p-Cresol	Strong floral	Common	864	0.07	108
47	24.63	Dodecane, 2,6,11-trimethyl-		Unique	853	0.07	212
48	51.61	1,1,1,3,5,5,5-Heptamethyltrisiloxane		Unique	960	0.07	222
49	23.78	Propane		Common	876	0.06	44
50	49.41	Silabenzene,1-methyl-		Unique	852	0.06	108

RT-(retention time).

RSI-(reverse search index on TG-5 MS capillary column).

RA%-(Peak area relative to the total peak area).

Table 2. Volatile compounds detected from the male L. indicus insects.

Sl.no	RT	Compound name	Odor description	Common/Unique	RSI	Area %	Mol. Wt.
1	10.59	2-Hexen-1-ol, acetate, (Z)-	Banana & waxy	Common	926	54.86	142
2	59.95	Cholesterol		Unique	890	7.83	386
3	46.99	10-Pentadecen-5-yn-1-ol, (E)-		Common	937	6.39	222
4	43.54	1-Hexyl-2-nitrocyclohexane		Common	983	3.19	213
5	45.54	Heptacos-1-ene		Unique	938	2.55	378
6	34.86	2-Heptanone,6-methyl-5-methylene- 6-methyl-5-methylene-	Banana fruity	Common	925	1.6	140
7	46.2	5,10-Pentadecadiyne, 1-chloro-		Unique	916	1.43	238
8	34.97	Propanoic acid, 2-(aminooxy)-	Acidic	Unique	860	1.18	105
9	8.93	Acetoacetic acid, 1-thio-, S-allyl ester		Unique	860	1.16	158
10	12.46	p-Cresol	Sweet tarry	Common	916	0.61	108
11	38.01	5,10-Pentadecadien-1-ol, (Z, Z)-		Unique	961	0.55	224
12	9	Nitroxide, bis (1,1-dimethyl ethyl)		Unique	835	0.39	144
13	42.6	Octadecane, 2-methyl-		Common	901	0.31	268
14	56.07	5,10-Pentadecadienal, (E, Z)-		Unique	874	0.31	222
15	18.69	Indole	Mothball, camphor green and fecal.	Unique	910	0.3	117
16	49.43	5,10-Pentadecadiyn-1-ol		Unique	932	0.29	220
17	40.84	2,7-Octadiene-1,6-diol, 2,6-dimethyl-, (Z)-		Common	871	0.25	170
18	6.21	Propane, 2-methyl-1-nitro-		Common	902	0.24	103
19	42.23	10-Heneicosene (c,t)		Common	922	0.18	294
20	8.65	Ethanone, 1-(3-ethyloxiranyl)-		Common	833	0.17	114
21	9.75	Propane		Common	859	0.14	44
22 23	42.35 47.63	Pentacos-1-enetert-Butyl methyl carbonate tert-Butyl methyl carbonate		Unique Unique	868 839	0.13 0.13	350 132
24	37.3	9,9-Dimethoxybicyclo [3.3.1] nona-2,4-dione		Unique	857	0.12	212
25	48.9	3,3-Diphenyl-2-(2-bromocyclopropyl)-propane nitrile		Unique	820	0.12	325
26	44.16	Decane, 3-Bromo-		Unique	844	0.11	220
27	47.68	6-Tetradecanesulfonic acid, butyl ester		Unique	800	0.11	334
28	48.29	5,6,8,4’-Tetrahydroxy-7,3’−dimethoxy flavone		Unique	894	0.1	346
29	45.11	1,6-Methanonaphthalene, decahydro-		Unique	839	0.08	150
30	57.37	8,13,17-Trioxo-5,6,7,8,13,14,15,16-octahydro-6,15-methanobenzo(A)naphto(2,3-F) cyclodecene		Unique	843	0.08	342
31	13.5	1,6:2,3-Dianhydro-4-O-acetylá-d-talopyranose		Unique	851	0.07	186
32	43.94	1,6:3,4-Dianhydro-2-deoxy-á-d-lyxo-hexopyranose		Common	860	0.07	128
33	44.24	2-Methyl-3-(2,2-dimethylpropyl)-butadiene		Unique	872	0.07	138
34	51.4	1,2-Pentadiene,4-methoxy-4-methyl-		Common	921	0.07	112
35	59.29	Propane, 1,1,1-trifluoro-		Unique	919	0.07	98
36	23.57	Hydroxylamine, O-decyl-		Unique	824	0.06	173
37	40.43	5,10-Pentadecadienoic acid, (Z, Z)-		Unique	892	0.06	238

RT-(retention time).

RT-(retention time).

RSI-(reverse search index on TG-5 MS capillary column).

RA%-(Peak area relative to the total peak area).

3.3. Comparative analysis of volatile compounds obtained from L. indicus male and female insects

The most prevalent volatile substances found in the female insects were 4-Octene, 2,6-dimethyl-, [S-(Z)]-with 42.28%, 1-Undecanol (21.6%), 1-Hexadecanol (10.2%) à, à-dimethyl-Palmitoleamide (6.96%). Other volatile substances from female insects were found in the range of 0.06 to 1.73% (Table 1). The most prevalent volatile substance discovered in male insects was 2-Hexen-1-ol, acetate, (Z)- with an area percentage of 54.86, followed by cholesterol with 7.83%, 10-Pentadecen-5-yn-1-ol, (E)- with 6.39%, 1-Hexyl-2-nitrocyclohexane with 3.19%, and heptacos-1-ene with 2.55%. Other volatile substances from male insects were found in the range of 0.06 to 1.6% (Table 2). The comparative analysis of abundant volatile compounds illustrates that male has higher composition of 2-Hexen-1-ol, acetate, (Z) volatile substance which has been used as ingredient in food and cosmetic industry due to its fruity, apple & waxy oily aroma. The most abundant substance present in female insect 4-Octene, 2,6-dimethyl-, [S-(Z)] function is not reported in the industry whereas its second abundant volatile compounds (1-Undecanol) are used in flavor and cosmetic industry with Citrus, apple & banana aroma.

3.4. Characterization of fatty acid from L. indicus insects

The male L. indicus is more palatable than the female insects. We aimed to compare the Fatty acids present in both male and female insects. The total amount of lipid extracted from the acclimatized fresh L. indicus insects were 0.28%/5.42 g from males and 0.15%/9.48 g from female insects. The extracted lipids profile contained TAG (triacyl glycerides), FFA (free fatty acids), DAG (diacyl glycerides), and MAG (monoacyl glycerides) were observed in the TLC plate (Supplemental Fig. S5). Male and female L. indicus insect’s fatty acid methyl ester (FAME) analysis showed 31 fatty acids from the male and 15 fatty acids from the female. The fatty acids profiles of both male and female insects are represented in (Table 3). Insect fatty acids are enriched with saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) containing omega 3 and 6. Both male and female insects detected all four types of saturated fatty acids although only short-chain MUFAs were present whereas PUFA detected from these insects fall into two categories: short-chain and long-chain fatty acids (PUFAs). The important omega −3 fatty acids found among PUFAs in insects are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), which are essential fatty acids required for humans and animals.

Table 3. Fatty acid composition obtained from L. indicus.

Common name of fatty acids	Fatty acids	Types of Fatty acids	Area % for L. indicus(F)	Area % for L. indicus(M)
Butyric acid	C4:0	SFAs	0.73	0.63
Valeric acid	C5:0	SFAs	ND	0.05
Caproic acid	C6:0	SFAs	0.33	0.37
Pelargonic acid	C9:0	SFAs	0.95	1.34
Lauric acid	C12:0	SFAs	0.41	0.43
Tridecylic acid	C13:0	SFAs	2.12	2.05
Myristic acid	C14:0	SFAs	ND	0.12
Pentadecylic acid	C15:0	SFAs	2.03	1.17
Palmitic acid	C16:0	SFAs	27.12	27.37
Tricosylic acid	C23:0	SFAs	3.63	4.62
Lignoceric acid	C24:0	SFAs	1.18	1.11
Arachidic acid	C20:0	SFAs	2.88	3.18
Behenic acid	C22:0	SFAs	2.08	ND
Heneicosylic acid	C21:0	SFAs	21.81	ND
Nonadecylic acid	C19:0	SFAs	0.96	14.8
Margaric acid	C17:0	SFAs	5.57	2.02
Stearic acid	C18:0	SFAs	12.06	11.64
Palmitoleic acid	C16:1	MUFA	0.91	1.69
cis-10-Heptadecenoic acid	C17:1	MUFA	ND	0.28
oleic acid	C18:1	MUFA	3.76	1.62
Nonadecenoic acid	C19:1	MUFA	ND	0.1
Linoleic acid	C18:2	Omega-6	ND	3.12
α-Linolenic acid	C18:3	Omega-3	4.31	0.7
linoleic acid	C19:2	omega 6	ND	0.11
Dihomo-γ-linolenic acid	C20:3	Omega-6	ND	2.26
Arachidonic acid	C20:4	Omega-6	ND	0.2
Eicosapentaenoicacid	C20:5	Omega-3	ND	2.3
Docosatetraenoicacid	C22:4	Omega-6	0.84	0.78
Docosapentaenoic acid	C22:5	Omega-3	1.76	1.46
Hexadecadienoic acid	C16:2	PUFA	ND	0.13
Hexadecatrienoic acid	C16:3	PUFA	ND	1.24
Heneicosatrienoic acid	C21:3	PUFA	ND	3.01
Eicosadienoic acid	C20:2	PUFA	1.4	ND
Cervonic acid	C22:6	DHA	1.57	1.66

3.5. Comparative analysis of fatty acids obtained from L. indicus male and female insects

FAME-GCMS analysis of female insects reveals a higher concentration of saturated fatty acids than unsaturated fatty acids (Figure 3). The male and female insects contain 12 common fatty acids with 19 unique fatty acids in male insects and 3 unique fatty acids in female insects (Figure 4). The abundance of SFA fatty acids (Figure 5) in male and female insects have palmitic acid (C16) whereas elaidic acid(C18:1) MUFA and the PUFA, omega-6 (C22:5) docosapentaenoic acid (DHA) are present in female insects. The MUFA (C16:1) palmitoleic acid and PUFA omega-6 (18:2), linoleic acid, and omega −3 (C21:3).Docosapentaenoic acids are the abundant fatty acids present in male insects. The abundant fatty acids MUFA and PUFA fatty acids detected in male insects are not present in female insects.

Figure 3. SFAs, MUFA, and PUFA detected from male and female L. indicus insects.

Figure 4. Common and unique fatty acids characterized from males and females L. indicus insects.

Figure 5. Abundant fatty acids; SFA, MUFA, and PUFA present in male and female insects.

4. Discussion

The volatile compounds found in L. indicus are employed as a component in the manufacture of lubricants, emulsifiers, solvents, rubber surfactants, paints, coatings, household detergents, and products for the food, cosmetics, and pharmaceutical industries [22,23]. The chemical 1-Hexyl-2-nitrocyclohexane, which has neuroactive, anti-inflammatory, and analgesic properties, is also present in both male and female insects [22,23]. Nonacos-1-ene, a female-specific chemical, is one of the pheromone elements utilized for insect communication [24], and 2-Heptadecenal, a substance with lipophilic characteristics aids in the production of propolis bee glue is also detected [25]. Among the unique volatile compounds of male insects, indole has been used as a promising agent for the treatment of antimicrobial activity, malaria, diabetes, cancer, migraines, convulsions, and hypertension [26–28]. 2-Methyl-3-(2,2-dimethyl propyl)-butadiene, present in the insect is reported as having antioxidant and antibacterial properties [29].

The most pungent volatile chemicals detected from Thailand male L. indicus insects were E-2- (E)-2-hexenyl acetate and (E)-2-hexenyl butanoate [9]. Among these two volatile compounds (E)-2-hexenyl acetate has been used as a flavoring agent commercially in Thailand [30]. The major volatile compound detected from the Indian male insect sample was 2-Hexen-1-ol, acetate whereas the female has 4-Octene, 2,6-dimethyl-, [S-(Z)]. The number of volatile compounds detected in the present study was 50 for females and 37 for male L. indicus insects, whereas the Thailand study reported the identification of only 23 volatile compounds.

Previous studies on L. indicus insects from Thailand showed the presence of four SFA, two MUFA fatty acids and three PUFA fatty acids [10] (Supplemental Table S3) whereas the FAME analysis performed on L. indicus collected from North-east India, obtained 17 saturated fatty acids, 4 MUFA fatty acids, and 13 PUFA fatty acids from both male and female insects. Fatty acids obtained from L. indicus could be bioaccumulated from their diet available in their aquatic ecosystem [31]. C18:2 Fatty acids might have synthesize in their body as insect posses Δ12 desaturase which convert (C18:1) fatty acid to (C18:2) fatty acids, which is essential for the entire animal [31,32].

The fatty acids derived from insects have been used in cosmetic and personal care products. Lauric acid (C12:0) has been used in 92 products; myristic acid has been used in 45 personal care products; palmitic acids (C16) have 255 products and oleic acid (C18:1) has been used in 367 cosmetic and personal care products [8]. In the present scenario, the insect as a source of fatty acids has become the most promising solution for use in cosmetic and personal care applications [33]. Due to ethical issues, animal-source fatty acids were replaced by plant-source fatty acids, but there were some drawbacks, such as the use of valuable land for non-food use, transport cost, and the release of pollutants throughout the process. So, insects can be an alternative resource for industries [33].

The saturated and unsaturated fatty acids used in the cosmetic industry were detected in male insect samples collected from India whereas myristic acid is not found in female insects and samples from Thailand present only C16 and C18:1 fatty acids [10]. We have demonstrated that male from various locations possesses different fatty acid and volatile chemical compositions. Another finding is that females synthesize unique volatile fatty acids compared to male insects.

L. indicus insects also posses fatty acids such as EPA and DHA which have anticancer characteristics [34]. ALA supports the heart, immunological system, and nervous systems. Additionally, EPA has anti-inflammatory, anti-atherosclerotic, and anti-depressive effects [35]. DHA makes up 8% of the brain weight and supports brain function and helps in brain development. This fatty acid also supports the prevention of type 2X-linked adrenoleukodystrophy [35,36]. Both male and female insects contain omega-6 fatty acids, which also have anti-inflammatory, anti-cancer, and immune-regulatory effects and support brain development [35,36]. All these important fatty acids found in L. indicus suggest that these insects are medically important entomophagy insects.

The consumption of aquatic species from the polluted habitat causes harm to human health due to the intake of heavy metals (Pb, and Cd) that are accumulated by the insects [37]. The long-term accumulation of Cd causes cancer and is harmful to the skeletal and respiratory systems of the human body whereas Pb causes harm to the gastrointestinal, nervous, hematologic, cardiovascular, and renal systems. Arsenic (As) is also hazardous to the neurological and pulmonary systems and has been linked to skin, lung, liver, and bladder cancer. However, it is important to prevent water pollution to protect aquatic species [38]. The population of L. indicus is also disappearing from the habitats of Manipur Lake has an impact on the ecosystem and food culture of the people which is inherited from the old-age tradition of consuming giant water bugs as their food. Aquaculture has been contributing to the supply of fish and shellfish in the market with a consistent nutritional profile than the wild fishes [39]. Mass cultivation under controlled conditions will also be safer for human health as compared to insects collected from polluted areas that may accumulate heavy metals which are hazardous to human health. The observational study data for L. indicus under aquarium condition showed that this insect can be cultivated in aquariums safely without any contamination from harsh pollutants.

Another significance of this insect is its economic value to local ladies who harvest them and sell at the market at Rs.30 per insect. It became a livelihood during the season from January to April. Once the rainy season arises in June, this insect are not able to be caught due to the increase in water level in its habitat. Furthermore, the Lethocerus genus used to fly from August to November. It is a highly demanded insect on the market because of its aroma, flavor, and therapeutic purposes. However, mass production of these insects in pollutant-free water is important to fulfill the demand for these insects in the market.

5. Conclusion

The present study highlighted that the volatile oil extracted from male insects is rich in volatile flavoring compounds 2-Hexen-1-ol, acetate with 54.86% a banana-like odor, Monounsaturated fatty acids, and Polyunsaturated fatty acids unique to male insects. The most abundant volatile compound found in female insects was 4-octene, 6, dimethyl with 42.28% whereas 2-Hexen-1-ol, acetate with 0.94% was detected. The pharmaceutical, cosmetic, and personal care industries, as well as the detergent and surfactant industries, use saturated and unsaturated fatty acids. The functional characterization of 32 volatile compounds is another area for future research, to aid the scientific community and better comprehend the vital roles these compounds play in nature. The mass cultivation of these entomophagy insects has the potential to be a sustainable bioresource that is very valuable for the bioeconomy approaches of NER regions and the conservation of this species.

Credit authorship contribution statement

RM: Writing – original draft and experimental design of the research work, SBU: Review, editing, and supervision of experimental work, AB: review& editing, supervision&guiding the feeding behavior assay in the aquarium. VR: Supervision. YR: Review & editing, supervision& guiding on volatile compound work.

Acknowledgments

The authors would like to thank the Director, Institute of Bioresources and Sustainable Development for providing support and Dr. Muthu Arumugam, Microbial Process Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum for extensive support for GC-MS analysis of the fatty acid profile of insect oil and IBSD manuscript No: IBSD/MS/2020/01/112.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21655979.2023.2252669

Additional information

Funding

This work was supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India, grant No. DBT-NER/Agri/24/2013.