Aymar R Fogang
email: aymarfogang@yahoo.fr

Germain Kansci

Michèle Viau

Michèle Rodolphe Rougerie

Claude Genot

Aymar Rodrigue

Fogang Mba

A R Fogang Mba

Edible caterpillars of Imbrasia truncata and Imbrasia epimethea contain

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

With a booming world population which is anticipated to reach approximately 10 billion people in 2050, the global food demand will increase by as much as 70% compared with our current food requirements [START_REF] Zielińska | Selected species of edible insects as a source of nutrient composition[END_REF]. Food security stands on three pillars: food availability, food access and food use [START_REF] Dube | A survey on entomophagy prelence in Zimbabwe[END_REF]. Today, about 795 million people are undernourished worldwide with approximately 220 million in sub Saharan African countries (FAO -FIDA -WFP, 2015). Novel food sources should be investigated as alternative sources of proteins and energy, but also of other nutrients and micronutrients. Indeed food security requires a minimum daily energy intake but suboptimal diet is recognized as the leading risk factor for non-communicable diseases. An adequate diet should supply adequate amounts of nutrients and micronutrients. Proteins should carry adequate amounts of all essential amino acids (WHO/FAO/UNU, 2007). Adequate vitamins, minerals, essential fatty acids and dietary fibre are also required. Worldwide, intakes of polyunsaturated fats are far below optimal [START_REF] Micha | Global, regional and national consumption of major food groups in 1990 and 2010 : a systematic analysis including 266 country-speci fi c nutrition surveys worldwide[END_REF]. Extremely low or low levels for plant omega-3 (α-linolenic acid) and omega-6 (linoleic acid) and seafood omega-3 (long-chain polyunsaturated fatty acids such as EPA and DHA) were identified in several countries in Sub-Saharan, West and Central Africa [START_REF] Micha | Global, regional and national consumption of major food groups in 1990 and 2010 : a systematic analysis including 266 country-speci fi c nutrition surveys worldwide[END_REF]. Meta-analysis of randomized control feeding trials highlighted that polyunsaturated fatty acids (PUFA) have more favourable effects regarding improved glycaemia, insulin resistance, and insulin secretion capacity in comparison to carbohydrates, saturated fatty acids (SFA), or monounsaturated fatty acids (MUFA) [START_REF] Imamura | Effects of saturated fat, polyunsaturated fat, monounsaturated fat, and carbohydrate on glucose-insulin homeostasis : a systematic review and meta-analysis of randomised controlled feeding trials[END_REF]. Among PUFA, adequate equilibrium is also required between dietary omega-6 and omega-3 fatty acids to limit various diseases such as cardiovascular disease [START_REF] Calder | Functional roles of fatty acids and their effects on human health[END_REF][START_REF] Simopoulos | An increase in the Omega-6/Omega-3 fatty acid ratio increases the risk for obesity[END_REF]. The Food and Agriculture Organization recommends edible insects as an alternative source of food and feed [START_REF] Van Huis | Edible Insects[END_REF]. Entomophagy, i.e., eating insects, is popular among people of many regions of the world including Africa, Asia, Australia, Central Mexico, and South America (Defoliart, 1999). It is practiced by more than 2 billion people in the world [START_REF] Van Huis | Edible Insects[END_REF]. Insects are rich in proteins, lipids and several others micronutrients [START_REF] Bukkens | The nutritional value of edible insects[END_REF][START_REF] Ramos-Elorduy | Nutritional value of edible insects from the state of Oaxaca, Mexico[END_REF][START_REF] Rumpold | Nutritional composition and safety aspects of edible insects[END_REF]. The crude protein content of insects ranges from 40 to 75 g/100 g dry weight (DW), largely depending on species and stage in the life cycle. Most of these values could however be overestimated due to the use of inadequate nitrogen-to-protein conversion factor and of proteins tightly embedded in the chitin matrix, and not digested by humans [START_REF] Martinez | The high level of protein content reported in insects for food and feed is overestimated[END_REF]. The fat content ranges from 0.66 to 77 g/100 g DW, also depending on species, life stage and environment [START_REF] Rumpold | Nutritional composition and safety aspects of edible insects[END_REF]. Some edible insects contain dietary lipidswith essential PUFA) such as linoleic acid, α-linolenic acid; long-chain PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)having being detected in several species [START_REF] Raksakantong | Fatty acids and proximate composition of eight Thai edible terricolous insects[END_REF][START_REF] Rumpold | Nutritional composition and safety aspects of edible insects[END_REF][START_REF] Sihamala | Lipids and fatty Acid composition of dried edible red and black ants[END_REF][START_REF] Yang | Polyunsaturated fatty acid content of edible insects in Thailand[END_REF].

Caterpillars of Emperor Moths (vernacular name of African Saturniidae [START_REF] Oberprieler | Classification of the African Saturniidae (Lepidoptera) the quest for natural groups and relationships[END_REF]) are among the most widely eaten insects in several African countries (Democratic Republic of the Congo, Republic of the Congo, Cameroon, Ivory Coast, etc) where they are highly appreciated by populations [START_REF] Latham | Les chenilles comestibles et leurs plantes nourricières dans la province du Bas-Congo[END_REF][START_REF] Mabossy-Mobouna | Imbrasia truncata Aurivillius (Saturniidae): importance en Afrique Centrale, commercialisation et valorisation à Brazzaville[END_REF][START_REF] Okangola | Valeurs nutritionnelles des chenilles comestibles de la ville de Kisangani et ses environs (Province de la Tshopo, République Démocratique du Congo)[END_REF]. These insects are generally collected in the wild and sold in local markets. They contribute to the income of rural populations. They are also exported to Europe, particularly France and Belgium. In 2000, these two countries imported respectively from Democratic Republic of the Congo, 5 and 3 tons of dried Imbrasia sp. caterpillars, with an estimated value of 65,000 US $ and 41,500 US $.These insects are sold approximately 15.5 €/ kg in Belgium [START_REF] Tabuna | Evaluation des échanges des produits forestiers non ligneux entre l'Afrique Subsaharienne et l'Europe[END_REF]. In the European Union, insects have been recently covered by the new Novel Food Regulation, and may continue to be marketed throughout the EU (new Regulation (EU) 2015/2283).

Information about the nutritional composition of several Imbrasia species such as I. ertli, I. belina (mopane) and I. oyenensis are available in literature [START_REF] Akpossan | IJRB Nutritional characteristics of the caterpillars (Imbrasia oyemensis) from Côte d'Ivoire[END_REF][START_REF] Anvo | Nutritional qualities of edible caterpillars Cirina butyrospermi in southwestern of Burkina Faso[END_REF][START_REF] Foua | Biochemical and nutritional study of powder from caterpillars Imbrasia oyemensis eaten in west of Côte d'Ivoire[END_REF][START_REF] Glew | Gonimbrasia belina (Lepidoptera: Saturniidae): a nutritional food source rich in protein, fatty acids, and minerals[END_REF][START_REF] Kwiri | Mopane worm (Gonimbrasia belina) utilisation, a potential source of protein in fortified blended foods in Zimbabwe: a review[END_REF][START_REF] Oliveira | The nutritional value of four species of insects consumed in Angola[END_REF]. These studies evidenced that proteins are major components of these insects, with profile of essential amino acids comparable to soybean and fish meal. Lipids represent the second main components. I. truncata and I. epimethea carterpillars are two species of Emperor moths. I. truncata is a Central African species wheareas I. epimethea is a Central and Western African species. The two species have some similar host plants. I. truncata feeds on Petersianthus macrocarpus, Ricinodendron heudelotii and Uapaca guinensis; I. epimethea feeds on Petersianthus macrocarpus, Ricinodendron heudelotii, Holarrhena floribunda, Pycnanthu sangolensis, Funtumia africana and acacia auriculiformis [START_REF] Balinga | Contribution des insectes de la forêt à la sécurité alimentaire[END_REF][START_REF] Latham | Les chenilles comestibles et leurs plantes nourricières dans la province du Bas-Congo[END_REF][START_REF] Lisingo | Enquête sur les chenilles comestibles et les divers usages de leurs plantes hôtes dans les districts de Kisangani et de la Tshopo (R. D. Congo )[END_REF] [START_REF] Balinga | Contribution des insectes de la forêt à la sécurité alimentaire[END_REF][START_REF] Latham | Les chenilles comestibles et leurs plantes nourricières dans la province du Bas-Congo[END_REF][START_REF] Lisingo | Enquête sur les chenilles comestibles et les divers usages de leurs plantes hôtes dans les districts de Kisangani et de la Tshopo (R. D. Congo )[END_REF]. I. truncata and I. epimethea were found to have very close methods of preparations and nutritional compositions [START_REF] Kokondi | Intérêt nutritionnel de chenilles d'attacidés du Zaîre: composition et valeur nutritionnelle[END_REF][START_REF] Lautenschläger | Impact of different preparations on the nutritional value of the edible caterpillar Imbrasia epimethea from northern Angola[END_REF][START_REF] Mabossy-Mobouna | Apports nutritifs des chenilles d' Imbrasia truncata consommées au Congo-Brazzaville[END_REF][START_REF] Okangola | Valeurs nutritionnelles des chenilles comestibles de la ville de Kisangani et ses environs (Province de la Tshopo, République Démocratique du Congo)[END_REF]. To our knowledge, no data on their tocopherol (vitamin E) composition nor on the fatty acid composition of lipid fractions (neutral lipids, phospholipids and glycolipids) have been already published.

The aim of the present study was to determine main nutritional features (proximate compositions, fatty acid and amino acid compositions, tocopherols) of two species of edible caterpillars in genus Imbrasia, (Emperor moths)and to provide new data about the characteristics of their lipid fraction (main lipid classes and their fatty acid compositions).

Materials and methods

Sampling, identification and preparation of insects

Three batches, each of approximately 500 g (71-84 animals) of two distinct species of edible caterpillars of Emperor moths were purchased alive at local markets of Yaoundé (Mvog Mbi, Mvog Atangana Mballa) and surroundings (Mfou). The living insects were washed with tap water, killed by freezing and stored at -20 °C for transportationto the Nantes laboratory where they were stored at -80 °C for about 1 month until further analysis. After morphological identification, including measurement of morphological parameters, their identity was confirmed by genetic identification using a DNA barcoding approach as described elsewhere [START_REF] Hebert | Biological identifications through DNA barcodes[END_REF]. Tissue samples (a small piece of thoracic leg) were processed through DNA extraction, PCR and sequencing of the standard barcode fragment of the COI mitochondrial gene following standard protocols at the Canadian Centre for DNA barcoding at the University of Guelph, Canada as described in [START_REF] Vaglia | Morphology and DNA barcoding reveal three cryptic species within the Xylophanes neoptolemus and loelia species-groups (Lepidoptera: sphingidae)[END_REF]. Vouchers for the two specimens sampled (Fig. 1) are preserved at the Muséum National d'Histoire Naturelle in Paris, with codes EL5922 and EL 5923 for I. epimethea and I. truncata respectively. Specimen data, photographs and sequence data are available in the Barcode of Life Datasystems (BOLD; www.boldsystems.org) within the publicly accessible dataset DS-IMBR01 (https://doi.org/10. 5883/ds-imbr01). Sequences are also available from GenBank with accession codes MH571150 for I. epimethea and MH571150 for I. truncata. Identifications using the two generated DNA barcodes were carried out using BOLD identification engine (http://www.boldsystems.org/index. php/IDS_OpenIdEngine), which compares query sequences to existing sequences within the database.

Prior to analysis, approximately 100 g of frozen caterpillars of all batches (n = 3) of each Imbrasiasp were thawn. With a scalpel, each insect underwent a longitudinal operation of the abdomen to empty the gut containing toxic substances from the plants they ate. The head and viscera (digestive tract) were separated from the body by hands, as performed traditionally by households. Before analysis portions of each batch of eviscerated caterpillars (2 × 3 batches) were mixed together to form a single batch. For lipid extraction and lipid analysis, around 100 g of eviscerated insects (around 15 caterpillars) were taken from the three different batches and mixed together. Lipid extraction was performed on these mixed samples of eviscerated insects (1 sample for I. truncata and 1 sample for I epimethea). For protein analysis, amino acids and ash analysis, more than 20 thawed and eviscerated insects were frozen again to be freeze-dried at -80 °C for 72 h. The freeze-dried insects were milled using a laboratory mill (20,000 rpm; M 20, IKA, Staufen, Germany) to prepare an homogenized insect flour sample, representative of the insects sold and consumed at Yaoundé. Protein, amino acid and ash analysis were performed on insect flour. The preparation and storage of insect flour followed the method described by Fogang [START_REF] Fogang Mba | Lipid and amino acid profiles support the potential of Rhynchophorus phoenicis larvae for human nutrition[END_REF].

Chemicals and solvents

d-Glucosamic acid solution (Ref 0259 > 98%), DL-norleucine (Ref N1398 > 99.9%), HCl 37%, barium hydroxide, ninhydrin reagent solution (Ref29670511) and BF 3 /CH 3 OH (14%) wereof analytical grade and supplied by Sigma-Aldrich (Saint Quentin Fallavier, France).Eluting solvents for amino acid analysis (methanol/trifluoroacetic acid/formic acid solvents A to F, pH 2. 94, 3.32, 4.48, 7.97and 10.5) and hydrochloric acid, lanthanum chloride, caesium chloride for mineral analysis were furnished by VWR (Strasbourg, France); chloroform (ethanol stabilized, HPLC grade), dichloromethane (amylenestabilized, HPLC grade), tert-butyl-methyl-ether (HPLC grade), n-hexane (HPLC grade), methanol absolute (HPLC Supra-Gradient), acetone (HPLC grade) were purchased from BiosolveChimie SARL (Dieuze, France). Cyclohexane (RS Plus for residual pesticide analysis) and ammonia (30% solution for analysis ACS) were purchased from Carlo Erba Reagents SAS (Val de Reuil, France).

Amino acid composition analysis

Amino acid composition of the caterpillars flours were determined with an amino acid analyser (Hitachi high-speed amino acid analyser L8900, Hitachi, Japan), paired with anion exchange chromatography column (125 mm L × 4 mm ID; polystyrene-divinylbenzene copolymer as stationary phase : Li-Form, PEEK), and with a mobile phase consisting in 6 successive lithium acetate buffers with increasing 2.94-10.51pHs. As previuously described (Fogang Mba et al., 2007;[START_REF] Latham | Les chenilles comestibles et leurs plantes nourricières dans la province du Bas-Congo[END_REF], detection was performed after post-column derivatization by ninhydrin, at 570 nm, except for proline at 440 nm.Two internal standards (norleucine and glucosamic acid) were added to the flour before protein hydrolysis to allow quantification of amino acids. To recover and quantify all amino acids, including the most sensitive ones, we combined the results obtained after different conditions of protein hydrolysis: acid hydrolysis(HCl 6 N, 110 °C) of 24 h to determine most amino acids, acid hydrolysis (HCl 6 N, 110 °C) of 48 h to determined branched amino acids (Val, Leu and Ileu), acid hydrolysis after oxidation by performic acid for the determination of sulfur amino acids (Met and Cys), and alkaline hydrolysis (Ba(OH) 2 , 4 N, 110 °C, 16 h) for tryptophan determination. According to apparatus provider (Hitachi), the method specifications are: Reproducibily of Peak Area = CV 1.0% (Gly, His) and Detection Limit = 3 pmol (S/N = 2, Asp). Individual amino acid residues and total amino acid residues concentrations were expressed in mg/g protein and in mg/100 g fresh weight. Indispensable amino acid scores were defined as the ratios of the contents in the insect proteins and in the reference protein for each essential amino acid (WHO/FAO/ UNU, 2007).

Total nitrogen content, nitrogen to protein conversion factor and non protein nitrogen determination

Total nitrogen content of the insect flours was determined using the Dumas method [START_REF] Dumas | Procédés de l'analyse Organique[END_REF] with an elemental analyser (Vario ISOTOPE Cube, Elementar, Frankfurt, Germany). To obtain nitrogen to protein conversion factors for Imbrasia species, their amount in total amino acid residueswere first calculated from amino acid composition (see parag. 2.3) and then divided by total nitrogen content (Dumas method) of the insect powders. Nitrogen content of total amino acid residues also calculated as the sum of nitrogen content of each amino acid residue. Non protein nitrogen was determined by subtracting mass of nitrogen of amino acid residues from total nitrogen content determined by Dumas method.

Determination of water, ash, protein and lipid contents and of energetic value

Water and ash contents of insect flours were assayed using the method of the Association of the Official Analytical Chemists [START_REF] Aoac | Official Methods of Analysis of AOAC International[END_REF] and calculated as described previously [START_REF] Fogang Mba | Lipid and amino acid profiles support the potential of Rhynchophorus phoenicis larvae for human nutrition[END_REF]. Protein content was determined by multiplying total nitrogen content with nitrogen to protein conversion factor, previously determined. Total lipidswere extracted from the raw thawed, eviscerated and ground insects according to [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF] with slight modifications as previously described [START_REF] Fogang Mba | Lipid and amino acid profiles support the potential of Rhynchophorus phoenicis larvae for human nutrition[END_REF]. Water, ash, nitrogen and lipid contents were analysed at least in triplicate. Carbohydrates were determined by difference, according to the following formula:

Carbohydrates content in g/100g = 100-[(Protein + lipid + ash + moisture) content in g/100g ]. Energy values were calculated using the Atwater coefficient: 16.2 kJ (4 kcal)/g for proteins and carbohydrates and 37.62 kJ (9 kcal)/g for lipids.

Tocopherol analysis

Tocopherol isomers (α, β, γ and δ tocopherols) were identified and quantified in the total lipid extracts following AOCS Ce-8-89method (AOCS, 1998) with minor modifications [START_REF] Fogang Mba | Growing conditions and morphotypes of African palm weevil (Rhynchophorus phoenicis) larvae influence their lipophilic nutrient but not their amino acid compositions[END_REF]. The compounds were identified by comparison of their retention time with that of authentic standards(tocopherol set 613424, purity ≥95% ; Calbiochem, Merk Chimie, Fontenay-Sous-Bois, France; γ tocotrienol, Ref. T0702, Sigma-Aldrich), also used for the external calibration. As previously stated, blank samples were run together with every sample series to check the column performance (Dionex Polar Advantage 2; 250 mm × 3 mm, 3µmwithhexane/di-ter-butyl-ethyl ester (95/5; V/ V) as mobile phase). The linear calibration curves for the different tocopherol isomers (α, β, γ and δ-tocopherol; 5-700 ng in n-hexane), obtained with a fluorescence detector (RF2000, Dionex; excitation; 295 nm; emission: 330 nm) wereperformed every day of analysis. The concentrations of the standard solutionswerepreviously checkedby the measurement of theirabsorbance at the maximum wavelength of each compound followed by concentration calculation by using their extinction coefficients. The calibration curves hadR 2 >0.99; and the method is characterized byLOD ≈ 5 ng; LOQ < 10 ng; RSD < 1.5%). The results for each batch were means of triplicate measurementsmade on 3 lipidextracts and expressed as mg tocopherols per 100 g fresh weight.

Fractionation of total lipids

Total lipids (TL)were fractionatedby adsorption chromatographywitha column (height: 36 cm; diameter: 2.5 cm; volume: 176 cm 3 ) packed with a mixture (1/0.5 ; w/w) of powdered silicic acid(MP Silica 63-100, 60A, MP Biomedicals) and Celite 545 (Acros organics from Fisher Scientific)according to [START_REF] Kates | Techniques for separation of lipid mixtures[END_REF]. The column was conditioned first with 50 ml of methanoland then by 5 ml of chloroform. Lipids (around 100 mg dissolved in 1mLchloroform) were then loaded onto the column.The neutral lipids (NL) were eluted with 150 ml of chloroform, glycolipid + sulfolipid fraction (GL + SL) with 100 ml of acetone and phospholipids (PL) with 100 ml of methanol. The collected fractions were dried under a stream of nitrogen and weighted to obtain the mass of each fraction. Each fraction was then re-dissolved in chloroform and stored at -20 °C until further analysis.

Determination of classes of neutral lipids and polar lipids

Approximately 5 μg of neutral lipid and polar lipid fractions in 10 μL chloroform were analysed by high-performance liquid chromatography (HPLC) with a modular UltiMate 3000 RS system (Dionex, Voisins Le Bretonneux, France) equipped with an UptisphereStrategy column (150 mm × 4.6 mm, 2.2 μm, 100 Å; Interchim, Montluçon, France) and coupled to an evaporative light scattering detector (ELSD) Sedex 85(Sedere S.A., Alfortville, France). Chromatographic conditions, identification and quantification of neutral lipids and phospholipids were described previously [START_REF] Fogang Mba | Lipid and amino acid profiles support the potential of Rhynchophorus phoenicis larvae for human nutrition[END_REF]. Lipid classes were identified bycomparison of their retention times to those of lipid standards and theirconcentrations calculated by the use of calibration curves (range concentrations)obtained witheach standard. Triolein, diolein, mono-olein, oleic acid, phosphatidyl-choline(PC), phosphatidyl-ethanolamine (PE), phosphatidyl-serine (PS),phosphatidy-linositol (PI), sphingomyelin (SPH) lyso-phosphatidyl-choline(LPC) were purchased from Sigma-Aldrich (Saint-QuentinFallavier, France).

Fatty acid composition of total lipids and main lipid fractions

Fatty acid compositions of total lipids were determined after transmethylation of 1 mg lipid with BF 3 /CH 3 OH14% in presence of an internal standard (C17:0),according to [START_REF] Morrison | Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron trifluoride methanol[END_REF]. Fatty acid methyl esters (FAMEs) analysis was performed on agas chromatograph (Clarus ® 680 GC, Perkin Elmer, Courtaboeuf, France) paired with a splitless injector and a flame ionization detector, and equipped with an HP 225 column (30 m × 0.25 mm, film thickness 0.25 µm, J&W Scientific, Chromoptic, Auxerre, France). Chromatographic conditions for fatty acid separation and their identification were performed as described previously [START_REF] Fogang Mba | Lipid and amino acid profiles support the potential of Rhynchophorus phoenicis larvae for human nutrition[END_REF], 2018). Individual fatty acids were identified by comparison of their retention times with those of standards (FAMEmix; Supelco, SigmaSaint Quentin Fallavier, France).Any loss of the separation capacity of the column was controlled on the chromatograms obtained with the fatty acid standards. Blank samples (n-hexane) were also injected every day to check the column performance. Results were expressed as percentage of each fatty acid (FA) to the total identified fatty acids (TFA) (g/100 g TFA) and in mg.g

-1 lipids and in g/100 g fresh weight. Limit of detection (amount of FA resulting in a peak with a height three times that of the baseline noise) was around 0.01 g/100 g total FA or 0.01 mg/100 g FW.

Data analysis

Except amino acid determination (n = 1), all analyses were performed in triplicates (n = 3). Results were expressed as the means ± standard deviations. Data were transformed with ARCSIN function in Excel 2007. The student t-test was used to compare significant difference between means at 0.05% probability level. Software GraphPad Prism 7.0 (GraphPad Software, San Diego, USA) was used for statistical analysis.

Results

Identification of the insects

Morphological identification of the larvae of the two Emperor moths butterfly was done from 10 individuals of each insect type whose morphologic criteria were compared to the descriptions made

U N C O R R E C T E D P R O O F
by [START_REF] Mabossy-Mobouna | Apports nutritifs des chenilles d' Imbrasia truncata consommées au Congo-Brazzaville[END_REF] and [START_REF] Mabossy-Mobouna | Clef de détermination des chenilles de dernier stade consommées au Congo-Brazzaville[END_REF]. The insects presented morphological characteristics typical of I. truncata and I. epimethea respectively (Fig. 1) [START_REF] Mabossy-Mobouna | Imbrasia truncata Aurivillius (Saturniidae): importance en Afrique Centrale, commercialisation et valorisation à Brazzaville[END_REF][START_REF] Rougerie | Morphology of the preimaginal stages of the African emperor moth Bunaeopsislicharbas (Maassen and Weyding): phylogenetically informative characterswithin the Saturniinae (Lepidoptera: saturniidae)[END_REF]. The insects identified as Imbrasia truncata (Fig. 1, B1 &B2) had a mean weight of 7.20 ± 0.96 g with a maximum weight of 9.51 g and a minimum weight of 5.98 g. The second species, identified as I. epimethea had a mean weight of 6.06 ± 1.25 g with a maximum weight of 7.81 g and a minimum weight of 4.21 g.

The comparison of DNA barcode sequences obtained fromtwo caterpillars belonging to the two morphologically recognized species confirmed unequivocally the identifications. The sequence of sample EL5922 matched with 100% similarity sequences for several records of I. epimethea on BOLD. There are actually 13 public records in BOLD for this species, all part of a cohesive genetic cluster registered as Barcode Index Number (BIN, see [START_REF] Ratnasingham | A DNA-based registry for all animal apecies : the barcode index number (BIN) system[END_REF]) BOLD:AAA8454. This BIN (http://v4.boldsystems.org/index.php/ Public_BarcodeCluster?clusteruri=BOLD:AAA8454) gathers 67 records on BOLD, with an average genetic distance of 0.39% (uncorrected p-distance as displayed on BOLD), a maximum distance of 2.45%, and a minimum distance to nearest neighbour of 7.3%with its relative I. ertli Rebel, 1904. Similarly, the sequence of EL5923 matched with 100% similarity the DNA barcodes of several records of I. truncata on BOLD. This species is represented by 9 public records on BOLD, all grouped in a single genetic cluster (BOLD:AAD8560; http://v4.boldsystems.org/index.php/Public_ BarcodeCluster?clusteruri=BOLD:AAD8560), with an average genetic distance of 0.65%, a maximum distance of1.38%, and a minimum distance to nearest neighbour of 4.3% with I. ertli.

Amino acid composition

Amino acid compositions of Imbrasia truncata and I. epimethea larvae are presented in Table 1. Eighteen amino acids were quantified in the two insects, including all of the indispensable amino acids (IAAs), even tryptophan. Glutamic acid was the major amino acid present in I. truncata (2772 mg/100 g FW) and I. epimethea (2789 mg/100 g FW). Indispensable amino acids represented 53.7 g/100 g of total amino acids (TAA) (10228 mg/100 g FW) in I. truncata and 51.3 g/100 g TAA (10310 mg/100 g FW) in I. epimethea.

Nitrogen to protein conversion factors and non-protein nitrogen

Nitrogen to protein conversions factors and non-protein nitrogen of Imbrasia truncata and I. epimethea larvae are presented in Table 2.Nitrogen to protein conversion factors equal to 6.01 ± 0.21 and 6.27 ± 0.15 were found for Imbrasia truncata and Imbrasia epimethea, respectively (Table 1). Non protein nitrogen represented 2.2 g/100 g total nitrogen in I. epimethea and 7.9 g/100 g total nitrogen in I. truncata.

Proximate composition

The proximate composition of Imbrasia truncata and I. epimethea larvae is presented in Table 3. The two insect species exhibited significantly similar water, protein and lipid contents (p > 0.05) but differed slightly but significantly in ash content (p < 0.05). Water content represented about 70.2 (69.7; 70.7) g/100 g fresh weight (FW) while proteins represented about 19.6 (19.1; 20.1) g/100 g FW; total lipid content represented around 6.8 (6.8; 6.7) g/100 g FW. Energy contents of I. truncata and I. epimethealarvae were also close: 633.0 kJ/100 g FW and 614.3 kJ/ 100 g FW respectively.

Vitamin E composition (α, β, γ and δ tocopherols)

Total tocopherol content was higher (p < 0.05) in Imbrasia truncata larvae: 119.7 ± 16.5 µg/g lipids (0.82 ± 0.11 mg/100 g FW) than in Imbrasia epimethea larvae: 224.4 ± 22.5 µg/g lipids (1.50 ± 0.15 mg/ 100 g FW) (Table 4).α-tocopherol was the major tocopherol isomer in I. truncata (76.6 ± 11.2 µg/g lipids)whileγ-tocopherol was major in I. epimethea (about 150 µg/g lipids), both accompanied by the other (α,β, γ, δ) isomers.α-Tocopherol supplied by 100 g of I. truncata is 0.52 ± 0.08 mg and 0.41 ± 0.06 mg by 100 g of I. epimethea larvae.

U N C O R R E C T E D P R O O F A.R. Fogang

Lipid fractions

The proportions of main lipid fractions in I. truncata and I. epimetheatotal lipids are presented in Table 5. For both insects, neutral lipid (NL) fraction was by far the major fraction:96.2 ± 0.2 g/100 g lipids for I. truncata; and 94.1 ± 1.1 g/100 g lipids for I. epimethea. In I. truncata, the fraction eluted by acetone (glycolipid (GL)+ sulfolipid fraction (SL)) accounted for 2.3 ± 0.4 g/100 g lipids and phospholipids (PL) 1.5 ± 0.2 g/100 g lipids. In I. epimethea, GL + SL fraction represented 3.3 ± 0.9 g/100 g lipids and PL 2.7 ± 0.3 g/100 g lipid.

Composition of the lipid fractions

The neutral lipids we recovered first from the total lipids extracts from I. truncata and I. epimethea larvae were almost exclusively com posed of free fatty acids as found on HPLC chromatograms of the fraction (Supplementary data, Figure A). This result suggested that the glycerolipids initially present in the living Imbrasia caterpillars were hydrolyzed during the steps that separated insects sampling to lipid extraction: freezing followed by storage at freezing temperature, thawing, evisceration and mincing. To verify this hypothesis, living caterpillars of the two Imbrasia species were frozen and then boiled during 15 min, to inactivate lipolytic enzymes. The insects were then eviscerated, grounded and finally kept frozen (-18 °C) until lipid extraction and fractionation. The neutral lipid fraction of these boiled I. epimethea larvae contained 43.5% of triglycerides and 56.5% of free fatty acids (Supplementary data, Fig. B). This result confirms that glycerolipids were present in the body of the living animals and that hydrolysis of lipids took place when the enzymes had not previously been denaturated. In contrast, the hydrolysis was partly inhibited by the thermal treatment. However, this treatment took place after a first step of freezing. Lipolytic enzymes, probably very active in the body of these caterpillars, are known to remain active at low temperature. Thus, they already had hydrolyzed a large fraction of the triacylglycerols during the first frozen storage step, explaining the free fatty acid level in the boiled I. epimethea larvae. Accordingly, the neutral lipid fraction of the boiled then frozen I. truncata still contained 99.46% of free fatty acids and only 0.54% of triglycerides. Phospholipid compositions of freeze, thawed and eviscerated I. truncata and I. epimethea are presented in Table 6. Interestingly, for both insects, the major phospholipid class was sphingomyelin with the highest level in I. epimethea(71.0 ± 9.3 g/100 g phospholipids). Phosphatidylcholine was the second major phospholipid, and the highest level was found in I. truncata (25.5 ± 1.5 g/100 g phospholipids). Apart from phosphatidylcholine whom contents were statistically different in both insects, all the other classes of phospholipids were present at similar percentages in I. truncata and I. epimethea. Little content of lysophospholipids (<9 g/100 g phospholipids) was found in the insects.

Fatty acid amounts of total lipids and lipid fractions

Fatty acid (FA) contents (mg/100 g FW) of I. truncata and I. epimethea larvae are shown in Table 7 and fatty acid composition of total lipids and lipid fractions (g FA/100 lipids) are presented in supplementary data (Table B).Thirteen fatty acids have been identified in I. truncata and fourteen in I. epimethea. These insects have fairly high levels of unsaturated fatty acids (UFA): 2633 ± 205 mg/100 g FW in I. truncata and 3243 ± 214 mg/100 g FWin I. epimethea. Three polyunsaturated fatty acids (PUFAs) were identified in total lipids of both insects ( 18:2n-6, 18:3n-3 and20:3n-3). The highest level of PUFAs was obtained in I. epimethea (2778 ± 194 mg/100 g FW) and the lowest in I. truncata(2183 ± 171 g/100 g FW). Alpha-linolenic acid (C18:3n-3) was by far the major fatty acid. It represented 1883 ± 146 mg/100 g FW in I. truncata and 2170 ± 131 mg/100 g FW in I. epimethea. Saturated fatty acids represented from 1846 ± 157 mg/100 g FW (I. truncata) to 1778 ± 189 mg/100 g FW (I.epimethea), mainly in the form of palmitic acid (C16: 798-1099 mg/100 g FW) followed by stearic acid (C18:710 -941 mg/100 g FW).Total fatty acid content was similar in I. truncata(4480 ± 361 mg/100 g FW) an in I. epimethea(5022 ± 26 mg/100 g FW). Only a small part of fatty acids were carried by polar lipids (Table 7).

Fatty acid composition of lipid fractions of Imbrasia truncata and I. epimethea are shown in Table B (Supplementary data). Fatty acid compositions of neutral lipid of I. truncata and I. epimethea caterpillars were similar to the compositions of total lipids. In contrast, the fatty acid compositions of PL of the two insects were strongly different from their neutral lipids and total lipid compositions. PL contained more saturated fatty acids than total and neutral lipids and within SFA, less C16, more C18 and C20 with the presence at minor levels of C22, 23 and C24 which were not detected in total and neutral lipids. PL also contained more 18:2 n-6 but less18:3 n-3 especially in I. truncataand globally less PUFA than total and neutral lipids.PL also contained small amounts of arachidonic acid (C20:4 n-6) while C20:3 n-3, present in total and neutral lipids was not detected in this fraction. Some differences were also observed in the FA compositions of the PL of the two insect species. The fatty acids C15 and C17:1 were absent in I. truncata and present in I. epimethea. Fatty acid compositions of the fractions eluted by acetone (GL + SL) differed also strongly from the fatty acid compositions of the NL and PL fractions. Major FA class in this fraction was PUFA with different contents in I. truncata (39.54 ± 2.66 g/100 g FA) and I. epimethea (43.47 ± 1.03 g/100 g FA) (Supplementary data, Table B).

Discussion

Emperor moths (Imbrasia sp.) caterpillars could represent a good source of proteins and indispensable amino acids

Proteins contents of I. truncata and I. epimethea were in the range already reported for Imbrasia species [START_REF] Akpossan | IJRB Nutritional characteristics of the caterpillars (Imbrasia oyemensis) from Côte d'Ivoire[END_REF][START_REF] Kwiri | Mopane worm (Gonimbrasia belina) utilisation, a potential source of protein in fortified blended foods in Zimbabwe: a review[END_REF][START_REF] Bukkens | The nutritional value of edible insects[END_REF][START_REF] Mabossy-Mobouna | Apports nutritifs des chenilles d' Imbrasia truncata consommées au Congo-Brazzaville[END_REF]. As the lower limit for the food label 'high in protein' is 10 g/100 g edible portion (EP) [START_REF] Nowak | Review of food composition data for edible insects[END_REF], I. truncata and I. epimethea larvae, with approximately 20 g protein /100 g fresh weight, can be considered as high in protein. As the reference intake (PRI) of protein is estimated to be 0.83 g protein/kg body weight per day for an healthy adult(EFSA, 2012), that is62.25 g of protein daily for an adult of 75 kg, the consumption of a serving size (20 insects) of I. truncata (144 g) or I. epimethea caterpillars (121 g) will represent respectively 44.2% and 39.1% of protein daily reference intake. Other nutritional interest of insects is the amino acid composition of their proteins which in most [START_REF] Foua Bi | Étude de la qualité biochimique et nutritionnelle de la poudre séchée d'Embrasa oyemensis[END_REF]. Each individual indispensable amino acid was also at higher level in I. truncata and I. epimethea than in FAO reference protein (Supplementary data; Table A). Apart phenylalanine + tyrosine and methionine + cysteine, the other indispensable amino acids were lower in protein of I. truncata and I. epimethea than the amount reported for I. oyemensis [START_REF] Foua Bi | Étude de la qualité biochimique et nutritionnelle de la poudre séchée d'Embrasa oyemensis[END_REF]. These results showed that proteins of I. truncata and I. epimethea larvae contained good quality amino acids with high biological value. They have a real potential to be used as alternative source of food proteins. A complete evaluation of nutritional value of the protein of these insects would rather include a measurement of their digestibility. In insects, a part of the total protein can be non-digestible for humans and animals due to binding of some proteins to the chitin fibres (Jonas-levi and Martinez, 2017). In the studied Imbrasia caterpillars, even if the chitin fraction represented a limited fraction of the edible part of the insects (less than 5.3-11.1 g/100 g FW, Table 2), one cannot exclude such type of interaction decreasing protein digestibility. However, [START_REF] Kwiri | Mopane worm (Gonimbrasia belina) utilisation, a potential source of protein in fortified blended foods in Zimbabwe: a review[END_REF] reported low difference between crude protein content (55.4%) and digestible protein (53.3%) for Gonimbrasia belina caterpillars, which could indicate a low percentage of non-digestible proteins in these kinds of insects.

Imbrasia epimethea and Imbrasia truncata larvae contain non protein nitrogen but have fairly standard nitrogen to protein conversion factor

Nitrogen to protein conversion factors of Imbrasia truncata and Imbrasia epimethea were statistically similar (Table 1). That of I. truncata (6.01 ± 0.21) was lower than the value of 6.25 used for most meat products, while that of I. epimethea (6.27 ± 0.15) was close to this value. They were also higher than the value of 4.76 ± 0.09 measured for several edible insects [START_REF] Janssen | Nitrogen-to-protein conversion factors for three edible insects :Tenebrio molitor, Alphitobius, and Hermetia illucens[END_REF]. However, the percentages of protein nitrogen content, calculated from the composition in amino acid residues, that is for I. trunctata (92.1 g/100 g N) and I. epimethea (97.8 g/ 100 g N) confirmed that in our insects, a small percentage of nitrogen was no protein nitrogen but non protein nitrogen such as chitin, nucleic acids, and phospholipids.

Imbrasia caterpillars: a dietary source of n-3 fatty acids

Lipids represent after protein the second main component of I. truncata and I. epimethea caterpillars. Total lipid contents of I. truncata and I. epimethea (approximately 23 g/100 g DW) were higher than results obtained previously on I. epimethea: 12.4 g/100 g DW, Imbrasia truncata 15.2 g/100 g DW [START_REF] Bukkens | The nutritional value of edible insects[END_REF][START_REF] Mabossy-Mobouna | Apports nutritifs des chenilles d' Imbrasia truncata consommées au Congo-Brazzaville[END_REF] and Gonimbrasia belina: 16.37 g/100 g DW [START_REF] Kwiri | Mopane worm (Gonimbrasia belina) utilisation, a potential source of protein in fortified blended foods in Zimbabwe: a review[END_REF]. The contents were however similar to that reported for I. oyemensis: 23.10 ± 0.65 g/ 100 g DW [START_REF] Foua | Biochemical and nutritional study of powder from caterpillars Imbrasia oyemensis eaten in west of Côte d'Ivoire[END_REF].

Based on fresh weight, PUFA content of Imbrasia truncata(2.2 ± 0.2 g/100 g FW) and I. epimethea(2.8 ± 0.2 g/100 g FW) were higher than PUFA content of Rhynchophorus phoenicis larvae (0.76 ± 0.17 g/100 g FW) obtained by [START_REF] Fogang Mba | Lipid and amino acid profiles support the potential of Rhynchophorus phoenicis larvae for human nutrition[END_REF]. Based on total fatty acid content (Supplementary data, Table B), PUFA contents of total fatty acids of I. truncata and I. epimethea were close from resultsobtained by Mabossy-Mobouna (2017) onI. truncata: 51.63 ± 0.24 g/ 100 g TFA. They were far higher than the values we measured for R. phoenicis larvae: from 2.8 to 5.3 g/100 g TFA [START_REF] Fogang Mba | Lipid and amino acid profiles support the potential of Rhynchophorus phoenicis larvae for human nutrition[END_REF], 2018). Epidemiological studies and dietary interventions showed that fatty acid intake plays a key role in human health. PUFAs serve many important functions in the body, including reducing the risk of diabetes by reduction of glu cose intolerance and prevention of insulin resistance, regulating blood pressure and helping to synthesize and repair vital cell components/ parts [START_REF] Calder | Functional roles of fatty acids and their effects on human health[END_REF]FAO, 2010;[START_REF] Imamura | Effects of saturated fat, polyunsaturated fat, monounsaturated fat, and carbohydrate on glucose-insulin homeostasis : a systematic review and meta-analysis of randomised controlled feeding trials[END_REF][START_REF] Simopoulos | An increase in the Omega-6/Omega-3 fatty acid ratio increases the risk for obesity[END_REF]. I. truncata and I. epimethea are rich in PUFAs, particularly α-linolenic acid (C18:3n-3) (Table 7).Their contents were close from the value: 42.63 ± 0.33 g/100 g TFA, obtained by [START_REF] Mabossy-Mobouna | Apports nutritifs des chenilles d' Imbrasia truncata consommées au Congo-Brazzaville[END_REF] on I. truncata. They were higher than those obtained in I. belina larvae (19.60 ± 0.06 g/100 g FA) [START_REF] Ekpo | Pharmaceutical potentials of the oils of some popular insects consumed in southern Nigeria[END_REF]and I. oyemensis larvae (0.82 ± 0.02 g/100 g FA) [START_REF] Foua | Biochemical and nutritional study of powder from caterpillars Imbrasia oyemensis eaten in west of Côte d'Ivoire[END_REF].α-linolenic acid was also the major fatty acid in several lepidoptera larvae with contents up to 62 g/100 g FA [START_REF] Guil-Guerrero | Insects as food: fatty acid profiles, lipid classes,andsn-2 fatty acid distribution of lepidoptera larvae[END_REF].The presence of α-linolenic acid in the sn-2 position in TAG is of great importance in nutrition as it can affect their bioavailability [START_REF] Guil-Guerrero | Insects as food: fatty acid profiles, lipid classes,andsn-2 fatty acid distribution of lepidoptera larvae[END_REF]. Therefore, the regi specific distribution of α-linolenic acid in TAG of I. truncata nd I. epimethea larvae lipids need to be investigates. Others PUFAs (C18:2n-6 and C20:3n-3) are also present at low levels. Alpha-linolenic acid (ALA) and linoleic acid (LA) are respectively precursors of n-3 and n-6 long chain PUFAs families in humans. They are essential fatty acids that must be supplied by the diet of humans and other mammals because of the lack of endogenous enzymes for omega-3 desaturation [START_REF] Stanley-Samuelson | Fatty acids in insects: composition, metabolism, and biological significance[END_REF]. The concentration of C18:3n-3 in I. truncata (42.04 ± 0.20 g/100 g TFA) and I. epimethea larvae(43.21 ± 2.40 g/100 g TFA)agree with the value of 42.63 ± 0.33 g/100 g TFA found for I. truncata larvae [START_REF] Mabossy-Mobouna | Apports nutritifs des chenilles d' Imbrasia truncata consommées au Congo-Brazzaville[END_REF]. They are higher than the values of 36.8 g/100 g TFA for I. truncate and 35.1 g/100 g TFA for I. epimethea larvae [START_REF] Bukkens | The nutritional value of edible insects[END_REF]. Long-chain PUFA such as arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were however not detected in the larvae lipids contrary to Apkossan which reported having identified arachidonic acid in the lipids extracted from Imbrasia Oyemensis on the basis of GC retention times [START_REF] Akpossan | Fatty acids, mineral composition and physico-chemical parameters of Imbrasia oyemensis larvae oil with unusual arachidonic acid content[END_REF]. The abundance of n-3 PUFAs in the form of α-linolenic acid in Imbrasia larvae lipids are linked to their diet, which are mainly composed of leaves. The FA content of insect are linked to their diets [START_REF] Guil-Guerrero | Insects as food: fatty acid profiles, lipid classes,andsn-2 fatty acid distribution of lepidoptera larvae[END_REF]. Omega-3 fatty acids have strong anti-inflammatory effects, reduce blood pressure and insulin resistance [START_REF] Rustan | Fatty acids: structures and properties[END_REF]. The concentration of C18:2n-6 in I. truncata (6.33 ± 0.06 g/100 g FA) and I. epimethea larvae (11.70 ± 1.29 g/100 g FA) are close to the value of 6.58 ± 0.03 g/100 g FA measured in I. oyemensis larvae (Gonimbrasia (Nudaurelia) menalops) [START_REF] Foua | Biochemical and nutritional study of powder from caterpillars Imbrasia oyemensis eaten in west of Côte d'Ivoire[END_REF] and 6.02 g/100 g FA in I. belina larvae (Gonimbrasia belina) [START_REF] Ekpo | Pharmaceutical potentials of the oils of some popular insects consumed in southern Nigeria[END_REF]. Omega-6 fatty acids also have many beneficial effects with respect to cardiovascular diseases (hyperlipidaemia, atherogenesis, leucocyte reactivity, immunological functions and cardiac arrhythmias) [START_REF] Rustan | Fatty acids: structures and properties[END_REF]. Lack of essential fatty acids also promotes skin inflammation and delays wound healing [START_REF] Rustan | Fatty acids: structures and properties[END_REF]. The daily requirement intake for adults is 4.4 g/day for linoleic acid and 1.8 g/day for α-linolenic acid [START_REF] Anses | Actualisation Des Apports Nutritionnels Conseillés Pour Les Acides Gras. Rapport D'expertise Collective[END_REF]. A serving size (20 insects) of fresh I. truncata (144 g) and I. epimethea larvae (121 g)will provide respectively 2.72 g and 2.62 g α-linolenic acid. Thus they represent respectively 151% and 146% of daily requirement intake of this essential fatty acid. A serving size (20 insects) of fresh I. epimethea(144 g) and I. truncata(121 g) will provide respectively 0.41 and 0.71 g of linoleic acid. These will represent respectively 9.29% and 16.2% of daily requirement intake.

Nutritionally, the n-6/n-3 ratio is also important. The n-6/n-3 ratios in total lipids of I. truncata and I. epimethea larvae were lower than 0.3. [START_REF] Guil-Guerrero | Insects as food: fatty acid profiles, lipid classes,andsn-2 fatty acid distribution of lepidoptera larvae[END_REF] obtained ratios lower than the value of one for 12 lepidoptera larvae. They are lower than the maximum target value of 5 [START_REF] Anses | En Acides Gras De La Population Vivant En France Et Comparaison Aux Apports Nutritionnels Conseillés Définis En[END_REF] and thus may participate to the re-equilibration of the current lipid dietary intake, characterized in most countries by too high n-6/n-3 levels. Many studies suggested that an unbalanced consumption of n-6/n-3 ratio is associated with human chronic diseases. An equilibrated n-6/n-3 ratio has positive effects on

U N C O R R E C T E D P R O O F
the prevention of coronary heart disease (CHD), hypertension, reducing total mortality; it also would decreases the risk of breast cancer and many other diseases (diabetes, arthritis, autoimmune and neurodegenerative diseases [START_REF] Simopoulos | An increase in the Omega-6/Omega-3 fatty acid ratio increases the risk for obesity[END_REF][START_REF] Simopoulos | The omega-6/omega-3 fatty acid ratio: health implications[END_REF] even if the possible mechanisms and effectiveness remain highly debated [START_REF] Albracht-Schulte | Omega-3 fatty acids in obesity and metabolic syndrome: a mechanistic update[END_REF][START_REF] Wei | The association and doseresponse relationship between dietary intake of α -linolenic acid and risk of CHD : a systematic review and meta-analysis of cohort studies[END_REF].

Imbrasia caterpillars as complementary source of energy

Energy content of I. truncata and I. epimethea were respectively 633.0 kJ (151.3 kcal)/100 g FW and 613.3 kJ(146.6 kcal)/100 g FW. As the dietary energy intake of a healthy young men (between 18 and 30 years old, 65 and 70 kg) in active or moderately active lifestyle is 3 100 kcal/day (FAO, 2001), a serving size (20 insects) of fresh I. truncata (144 g) and I. epimethea larvae (121 g) will provides respectively 217 and 180 kcal of energy. Energy content of I. truncata and I. epimethea larvae were lower than that of palm weevil (Rhynchophorus phoenicis) larvae (940.0 kJ (224.7 kcal)/100 gFW) [START_REF] Fogang Mba | Lipid and amino acid profiles support the potential of Rhynchophorus phoenicis larvae for human nutrition[END_REF]. These two types of insects, with different nutritional characteristics, are consumed by same population. Contrary to palm weevillarvae which can be considered as an energetic food due to their high lipid contents, the caterpillars of I. truncata and I. epimethea can be used to re-equilibrate high lipid content diets.

Imbrasia truncata and Imbrasia epimethea are source of Vitamin E

Tocopherol contents of lipids of Imbrasia truncata(119.7 ± 16.5 µg/ g lipids)and Imbrasia epimethea(224.4 ± 22.5 µg/g lipids)were higher than the contents of mophane (I. belina) oil: 73 µg/g lipid [START_REF] Yeboah | Further lipid profiling of the oil from the mophane caterpillar, Imbrasia belina[END_REF]. Our results showed different repartition of tocopherol isomers in the two Imbrasia species. The major tocopherol isomer was α-tocopherol in I. truncata (77 ± 11 µg/g lipids), while it was γ-tocopherol in I. epimethea(150 ± 12 µg/g lipids) (Table 4). α-tocopherol content of total lipids of I. truncata and I. epimethea was different to α-tocopherol content of mophane oil: 72 µg/g lipids [START_REF] Yeboah | Further lipid profiling of the oil from the mophane caterpillar, Imbrasia belina[END_REF]. α-tocopherol supplied by 100 g I. truncata or I. epimethea larvae (0.41 to 0.52 mg/100 g FW) is also lower than the value reported for Tenebrio molitor larvae (1.9 mg/100 g FW) [START_REF] Nowak | Review of food composition data for edible insects[END_REF].Total tocopherol content of I. truncata(1.5 ± 0.2 mg/100 g FW) and I. epimethea (0.8 ± 0.1 mg/100 g FW) larvae were in the same order range than that of cricket (2.3 ± 1.8 mg/100 g FW), honey bee (0.6 ± 0.4 mg/100 g FW) and black soldier fly (0.62 mg/100 g FW) [START_REF] Payne | A systematic review of nutrient composition data available for twelve commercially available edible insects, and comparison with reference values[END_REF]. They were lower to the values found for palm weevil (Rhynchophorus Phoenicis) larvae: 2.3 to 4.8 mg/100 g FW, depending on larvae morphotype [START_REF] Fogang Mba | Lipid and amino acid profiles support the potential of Rhynchophorus phoenicis larvae for human nutrition[END_REF], 2018). As the recommend nutrient intake of α-tocopherol is 10 mg/day (FAO, 2001), the consumption of 100 g of these insects will contribute up to 15% of Vitamin E recommend nutrient intake. These insects can be considered as source of vitamin E. The presence of tocopherols in these insects may also be useful in protection of the lipids of these insects from oxidation during processing of raw insects into derived products (drying, cooking, etc). These tocopherol contents of Imbrasia truncata and Imbrasia epimethea should be carefullyconsidered. Indeed, it could vary with the season, as it is known that the tocopherol content of plants, on which insect feed, varies with the season and vegetation. This is the case, for example, with flax [START_REF] Teneva | Changes of composition in triacylglycerols, sterols and tocopherols of flax during the vegetation[END_REF].

Lipolytic enzymes are very active in Imbrasia truncata and Imbrasia epimethea

Total lipids of I. truncata and I. epimethea were majorly composed of neutral lipids.The neutral lipid fractions extracted from the larvae killed by freezing and further stored at frozen state were majorly composed of free fatty acids. In animal as in vegetal worlds, the molecular form for storage lipids is triacylglycerols. These storage molecules are hydrolysed in vivoto liberate free fatty acids available for body physiological needs. Hydrolysis also takes place when tissues and cellular injuries provoke contact between the lipolytic enzymes and their substrates. We assume that the presence of free fatty acids was due to the hydrolysis of the triacylglycerols taking place between the harvest of the larvae and lipid extraction and that the viscera and other organs of Imbrasia larvae contained very active lipases which are able to efficiently hydrolyse lipids. Boiling of insects immediately after their killing by freezing had accordingly permitted to reduce the level of free fatty acids in the neutral lipids of I. truncata larvae. Free fatty acids are generally considered as lowering food quality because their favour enzymatic and non-enzymatic oxidation of PUFAs. Thus, the possibility to inactivate efficiently the lipolytic activities in the Imbrasia larvae, just after their harvest, should be investigated.

Polar lipids of Imbrasia truncata and Imbrasia epimethea larvae present untypical composition

Polar lipids (phospholipids and the fraction soluble in acetone) were found in low proportions in total lipids of Imbrasia truncata and Imbrasia epimethea, less than 6 g/100 g total lipids (Table 5). [START_REF] Ekpo | Pharmaceutical potentials of the oils of some popular insects consumed in southern Nigeria[END_REF] found higher proportions of polar lipids in I. belina total lipids: 4.16 ± 0.21 g/100 g for glycolipids and 9.87 ± 0.35 g/100 g for phospholipids.

Phospholipids of I. truncata and I. epimethea larvae contained 62-71 g/100 g sphingomyelin (Table 6). This is highly higher than in muscles of most animal and current foods [START_REF] Weihrauch | Phospholipid content of foods[END_REF]. As compared to neutral lipids, phospholipids contained lower levels of unsaturated fatty acids. This lower level of unsaturated fatty acids can be explained by the abundance of sphingomyelin in polar lipids. More study must be done to clearly define if the high level of sphingomyelin in phospholipid composition is related to the insect species or to their stage of development.

The knowledge of lipid classes and their fatty acid compositions are important as they can affect nutritional impact of dietary lipids [START_REF] Meynier | Molecular and structural organization of lipids in foods: their fate during digestion and impact in nutrition[END_REF]. It can also be important for a further valorisation and transformation of the insects for marketing and industrial applications. Indeed, dietary lipids are not only sources of energy and nutrients, but are actively involved in properties such as texture, sensory properties and participate to the overall acceptability of foods [START_REF] Meynier | Molecular and structural organization of lipids in foods: their fate during digestion and impact in nutrition[END_REF].

Conclusion

The results presented in this article indicate that I. truncata and I. epimethea larvae have a great potential to be used as source of dietary proteins and dietary lipids. These insects also contain indispensable amino acids at levels higher than the protein reference of FAO. They could thus serve as an alternative dietary supplement of proteins, able to narrow the amino acid deficits that are prevalent in many developing countries. These insects also contain important amounts of α-linolenic acid, the consumption of 100 g of fresh insect covering the daily reference intake. Their n-6/n-3 ratios were lower than 0.3 which is appreciable in prevention of several diseases. These insects also contain tocopherols. Indeed, if they have an appreciable nutrient composition, many others factors can contribute to limit their consumption, among them the efficient lipolysis of their neutral lipids. Researches are needed to optimize their lipid extraction without lipolysis. Besides, detailed analysis of these Imbrasia species for other nutrients, anti-nutrients and secondary metabolites with medicinal potential should be considered. The results from the present analysis might strengthen the use of this insect as a supplement of protein, amino acids and α-linolenic acid in foods.

Fig. 1 .

 1 Fig. 1. Imbrasia epimethea (A1: alive; A2: dried) and Imbrasia truncata caterpillars ((B1: alive; B2: dried). EL5922 and EL5923 are Sample ID codes referring to their respective records in the Barcode of Life Datasystems (BOLD: www.boldsystems.org) dataset: DS-IMBR01 (https://doi.org/10.5883/ds-imbr01).

  . Fogang Mba et al. Journal of Food Composition and Analysis xxx (xxxx) xxx-xxx

Table 1

 1 Amino acid content of Imbrasia truncata and Imbrasia epimethea caterpillars and comparison with other protein sources and FAO/WHO/UNU nutritional recommendations.

	Imbrasia truncata	Imbrasia epimethea	Fish (flesh)(1)	Beef and veal (1)	Chicken (flesh)(1)	WHI RV (2)
	ALA ARG ASP GLU GLY PRO SER Total NIAA HIS ILE LEU LYS THR VAL PHE TRP TYR AAA CYS MET SAA Total IAA Total AA residues	(mg/100 g fresh weight) 716 930 1126 971 1100 1066 1668 2029 1947 2772 2789 2655 840 858 906 1018 1098 692 834 970 816 8819 9773 666 685 665 809 865 900 1148 1250 1445 1449 1479 1713 1036 1207 861 864 1049 1150 799 1114 737 245 266 2647 1821 689 3692 3201 1426 237 205 220 326 370 539 564 575 759 10228 10310 8919 19047 20082 18338	1033 1118 1590 2703 860 628 713 603 852 1435 1573 812 886 778 637 1415 226 478 704 8280 17163	682 1114 1834 3002 1059 829 781 525 1069 1472 1590 794 1018 800 205 669 1469 262 502 764 8906 18206	690 1380 1794 2070 1058 1794 276 1748 1012 9062

AA: amino acids; IAA: indispensable amino acids; NIAA: non indispensable amino acids; AAA: aromatic amino acid (Phe + Tyr + Trp);*SAA: sulfur amino acid (Met + Cys); FAO: Food and Agriculture Organisation, WHO: World Health Organization; UNU: United Nation University. (1) : (FAO, 1970); (2) : WHO RV: World Health Organization Reference Values (WHO, 2007) for the daily amino acid requirements of a 55 kg woman consuming 46 g of protein per day.

Table 2

 2 Nitrogen(Dumas method) to protein conversion factors for Imbrasia truncata and Imbrasia epimethea caterpillars.

		TAAR (g/ 100 g FW)	N (g/100 g FW)	CF	Nitrogen in TAAR (g/100 g FW)	% PN (g/ 100 g N)	% NPN (g/ 100 g N)
	Imbrasia truncata Imbrasia epimethea	19.05 20.08	3.17 ± 0.11 3.20 ± 0.08	6.01 ± 0.21 6.27 ± 0,15	2.92 3.13	92.11 97.78	7.89 2.19
	Value presented in the table is mean ± s.d.

CF: conversion factor; FW: fresh weight; N: total nitrogen (Dumas method); NPN: non-protein nitrogen; PN: protein nitrogen; TAAR: total amino acid residues.

Table 3 :

 3 Mba et al. Journal of Food Composition and Analysis xxx (xxxx) xxx-xxx Proximate composition of Imbrasia truncata and Imbrasia epimethea caterpillars (n = 3).

		Water	Total lipids	Proteins	Carbohydrates *	Ash	Energy (kJ/kcal)
	Fresh weight (g/100 g FW) Imbrasia truncata Imbrasia epimethea Dry weight (g/100 g DW) Imbrasia truncata Imbrasia epimethea	69.7 ± 1.5 70.7 ± 0.9 / /	6.8 ± 0.6 6.7 ± 0.2 22.6 ± 2.1 22.8 ± 0.8	19.1 ± 0.7 20.1 ± 0.5 62.9 ± 2.1 B 68.6 ± 1.6 A	3.4 0.9 11.1 5.3	1.03 ± 0,01 A 0.97 ± 0,01 B 3.41 ± 0,03 A 3.32 ± 0,04 B	633.0 (151.3) 613.3 (146.6) 2088.7 (499.1) 2094.6(500.6)
	Value presented in the table is mean ± s.d. * : by difference (Cabohydrates g/100g FW = 100 -[Proteins + lipids + ash + water] g/100g FW ); For a given proximate compound, means with different capital letter within a colunm are significantly different (p < 0.05). FW: fresh weight; DW: Dry weight.

Table 4

 4 Tocopherol composition in Imbrasia truncata and Imbrasia epimethea larvae (n = 3).

		Imbrasia truncata	Imbrasia epimethea	DRI*(mg/ Day)
	α-tocopherol (µg/g lipids) β-tocopherol (µg/g lipids) γ-tocopherol (µg/g lipids) δ-tocopherol (µg/g lipids) Total tocopherol (µg/g lipids) α-tocopherol (mg/100 g FW) β-tocopherol ((mg/100 g FW) γ-tocopherol (mg/100 g FW) δ-tocopherol (mg/100 g FW) Total tocopherol (mg/ 100 g FW)	76.6 ± 11.2 10.9 ± 2.3 A 21.9 ± 3.3 A 10.4 ± 1.1 119.7 ± 16.5 A 0.52 ± 0.08 0.07 ± 0.02 A 0.15 ± 0.02 A 0.07 ± 0.01 0.82 ± 0.11 A	62.1 ± 9.0 B 3.3 ± 1.0 149.7 ± 12.1 B 9. 2 ± 0.8 B 224.4 ± 22.5 0.41 ± 0.06 B 0.02 ± 0.01 B 1.00 ± 0.08 0.06 ± 0.01 1.50 ± 0.15	12-15

B

Value presented in the table is mean ± s.d. FW: Fresh weight; DRI*: Dietary Reference Intake for an adult,

(FAO, 2001)

. For a given tocopherol isomer, means with different capital letter within a row are significantly different (p < 0.05).

Table 5

 5 Lipid fractions in total lipids of Imbrasia truncata and Imbrasia epimethea caterpillars (n = 3).

	Insect type	Neutral lipids	Acetone-eluted fraction	Phospholipids
	Imbrasia truncata Imbrasia epimethea	g/100 g total lipids 96.2 ± 0.2 A 2.3 ± 0.4 94.1 ± 1.1 B 3.3 ± 0.9	A 1.5 ± 0.2 2.7 ± 0.3

B

Value presented in the table is mean ± s.d. For a given lipid fraction, means with different capital letter within column are significantly different for the two Imbrasia species (p < 0.05).

Table 6 :

 6 Phospholipid classes of Imbrasia epimethea and Imbrasia truncata caterpillars (g/100 g phospholipids; n = 3).

	g/100 g PL	Imbrasia truncata	Imbrasia epimethea
	PE LPE + PI PC SPH LPC	7.5 ± 1.7 2.9 ± 0.2 25.5 ± 1.5 A 61.8 ± 2.2 2.3 ± 1.1	6.7 ± 1.1 3.3 ± 0.9 13.4 ± 4.6 B 71.0 ± 9.3 5.6 ± 6.2
	Value presented in the table is mean ± s.d. PE: phosphatidyl-ethanolamine; LPE: phosphatidyl-serine; PI: phosphatidyl-inositol; PC: phosphatidyl-choline; SPH: lyso-phosphatidyl-ethanolamine; PS: sphingomyelin; LPC: lyso-phosphatidyl-choline. For a given phospholipid class, means with different capital letter within a line are significantly different for the two Imbrasia species (p < 0.05).

Table 7

 7 Fatty acid amounts (mg/100 g FW) contained in main lipid fractions of Imbrasia truncata and Imbrasia epimethea larvae (n = 3). .R. FogangMba et al. Journal of Food Composition and Analysis xxx (xxxx) xxx-xxx case contain appreciable levels of indispensable amino acids. Total IAA of I. epimethea and I. truncate (513.3-536.6 mg/g protein; Supplementary data; TableA) were by far higher to the FAO/WHO amino acid reference pattern (269 mg/g protein) established for Humans (WHO/ FAO/UNU, 2007) but lower than that of Imbrasia oyemensis (588.4 mg/ g protein)

		Total lipids		Neutral lipids		Acetone-soluble fraction	Phospholipids
		I. truncata	I. epimethea	I. truncata	I. epimethea	I. truncata	I. epimethea	I. truncata	I. epimethea
	C14 C15 C16 C18 C20 C22 C23 C24 C16:1n-9 C16:1n-7 c17:1 C18:1n-9 C18:1n-7 C20:1n-9 C22:1n-9 C18:2n-6 C20:4n-6 C18:3n-3 C20:3n-3 SFA MUFA PUFA UFA TFA N3 N6 N6/N3	18.1 ± 1.9 A 9.6 ± 0.9 A 1099 ± 95 A 710 ± 60 B 9.6 ± 1.7 B nd nd nd 2.6 ± 4.5 A 31 ± 3 A 7.0 ± 0.4 A 385 ± 28 A 25 ± 2 A nd nd 284 ± 24 B nd 1883 ± 146 A 17 ± 2 A 1846 ± 157 A 450 ± 34 A 2183 ± 171 B 2633 ± 205 B 4480 ± 361 A 1900 ± 147 A 284 ± 24 B 0.15 ± 0.00 B	11.0 ± 3.5 B 8.7 ± 0.3 A 798 ± 63 B 941 ± 121 A 20.1 ± 3.1 A nd nd nd 6.3 ± 0.4 A 16 ± 1 B 7.0 ± 0.1 A 407 ± 22 A 18 ± 1 A 11.5 ± 5.2 nd 588 ± 68 A nd 2170 ± 131 A 20 ± 3 A 1778 ± 189 A 465 ± 20 A 2778 ± 194 A 3243 ± 214 A 5022 ± 26 A 2190 ± 129 A 588 ± 68 A 0.27 ± 0.02 A	20.0 ± 0.2 A 10.6 ± 0.2 A 1248 ± 31 A 823 ± 28 A 11.9 ± 1.1 B nd nd nd 7.7 ± 0.1 B 28 ± 6 A 8.6 ± 0.1 446 ± 16 A 29 ± 1 A nd nd 32 ± 610 B nd 2181 ± 70 A 22 ± 2 A 2113 ± 60 A 519 ± 21 A 2529 ± 81 A 3048 ± 102 A 5161 ± 162 A 2203 ± 71 A 326 ± 10 B 0.15 ± 0.00 B	12.1 ± 1.3 B 7.9 ± 0.5 B 678 ± 48 B 828 ± 54 A 16.6 ± 0.3 A nd nd nd 12.5 ± 1.1 A 0.0 ± 0.0 B tr 323 ± 17 B 14 ± 1 B 8.7 ± 0.7 nd 464 ± 38 A nd 1712 ± 104 B 16 ± 1 B 1542 ± 102 B 365 ± 28 B 2192 ± 136 B 2557 ± 163 B 4099 ± 256 B 1728 ± 105 B 464 ± 38 A 0.27 ± 0.02 A	0.11 ± 0.01 A 0.05 ± 0.00 A 4.7 ± 0.3 A 2.7 ± 0.1 B 0.11 ± 0.00 A 0,08 ± 0,00 A nd nd 0.03 ± 0.02 A 0.19 ± 0.03 A 0.0 ± 0.0 A 1.12 ± 0.05 B 0.08 ± 0.00 A 0.06 ± 0.02 A 0.12 ± 0.03 A 0.79 ± 0.04 B 0.03 ± 0.01 A 5.17 ± 0.36 B nd 7.71 ± 0.41 B 1.60 ± 0.07 B 5.99 ± 0.40 B 7.60 ± 0.41 B 15.30 ± 0.00 B 5.17 ± 0.36 B 0.82 ± 0.04 B 0.16 ± 0.00 B	0.14 ± 0.03 A 0.08 ± 0.02 A 5.03 ± 0.13 A 5.06 ± 0.21 A 0.17 ± 0.05 A 0,09 ± 0,03 A nd nd 0.05 ± 0.01 A 0.14 ± 0.01 A 0.01 ± 0.02 A 1.56 ± 0.03 A 0.06 ± 0.01 A 0.07 ± 0.03 A 0.15 ± 0.11 A 2.07 ± 0.09 A 0.04 ± 0.00 A 7.47 ± 0.30 A nd 10.57 ± 0.06 A 2.04 ± 0.16 A 9.58 ± 0.22 A 11.6 ± 0.06 A 22.2 ± 0.00 A 7.47 ± 0.30 A 2.11 ± 0.08 A 0.28 ± 0.02 A	0.04 ± 0.00 A nd 0.90 ± 0.07 A 1.73 ± 0.03 B 1.12 ± 0.03 B 1,52 ± 0,05 B 0,12 ± 0,00 A 0,13 ± 0,02 B 0.03 ± 0.01 A 0.03 ± 0.00 B nd 0.55 ± 0.06 B 0.04 ± 0.01 A nd 0.17 ± 0.08 A 1.09 ± 0.16 B 0.06 ± 0.00 B 2.77 ± 0.18 B nd 5.56 ± 0.07 B 0.83 ± 0.05 B 3.93 ± 0.10 B 4.75 ± 0.07 B 10.32 ± 0.00 B 2.77 ± 0.18 B 1.16 ± 0.16 B 0.42 ± 0.09 A	B 0.07 ± 0.01 0.08 ± 0.01 A 1.07 ± 0.09 A 2.96 ± 0.20 A 2.62 ± 0.10 A 2,89 ± 0,01 A 0,13 ± 0,01 A 0,24 ± 0,02 A 0.06 ± 0.02 A 0.06 ± 0.00 0.16 ± 0.03 A 0.85 ± 0.04 A 0.04 ± 0.01 nd A 0.29 ± 0.15 A 1.61 ± 0.12 A 0.18 ± 0.05 A 4.86 ± 0.06 nd 10.05 ± 0.34 A A 1.46 ± 0.23 A 6.65 ± 0.10 A 8.11 ± 0.34 A 18.2 ± 0.00 A 4.86 ± 0.06 A 1.79 ± 0.17 0.37 ± 0.04

A

Value presented in the table is mean ± s.d. FW: fresh weight; FA: fatty acids; SFA: saturated fatty acids; UFA: Unsaturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids; nd: not detected (Limit Of Detection (LOD) ≈ 0.01 mg/100 g FW). For a given lipid fraction and a given fatty acid, means with different capital letter are significantly different for Imbrasia truncata and Imbrasia epimeathea (p < 0.05).

A

Acknowledgements

The authors wish to thank Christian Blassel, Véronique Solé-Jamault, Lucie Ribourg and Nordine Hafnaoui for helping us respectively in insect lyophilization and water analysis, protein analysis, tocopherol analysis, and amino acid analysis.

P R O O F

A.R. Fogang Mba et al. Journal of Food Composition and Analysis xxx (xxxx) xxx-xxx Funding This work was supported in part by the SCAC (Service de Coopération et d'Action Culturelle) of the French Embassy of Yaoundé -Cameroon [Code projet Prisme/Numéro Prisme: 0185 CMR B16 0011]. https://cm.ambafrance.org/Bourses-du-gouvernement-francais

The DNA barcoding process was funded through the international Barcode of Life project (iBOL) supported by Genome Canada through the Ontario Genomics Institute, Canada Foundation for Innovation, and by the Ontario Ministry for Research and Innovation.

Conflict of interest

The authors declare no conflict of interest Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jfca.2019.03.002.