Variability in nutrient composition of the edible long‐horned grasshopper (Ruspolia differens) in Uganda and its potential in alleviating food insecurity

Abstract Ruspolia differens Serville (Orthoptera: Tettigonidae) is a highly nutritious and luxurious insect delicacy that is consumed as a food source in many African countries. However, the nutrient profile of R. differens in different geographical regions have received limited research interest. Here, we provide comprehensive evidence of geographical impact on the nutrient profile of R. differens and its potential to meet the recommended dietary intake of the population. Our results demonstrated that proximate composition, fatty acids, amino acids, minerals, vitamins, and flavonoid contents of R. differens collected from five districts in Uganda varied considerably. The crude protein (28–45%), crude fat (41–54%), and energy (582–644 Kj/100 g) contents of R. differens exceed that reported from animal origins. The highest crude protein, crude fat, and carbohydrate contents of R. differens were recorded in Kabale, Masaka, and Kampala, respectively. A total of 37 fatty acids were identified with linoleic acid (omega‐6 fatty acid) being the most abundant polyunsaturated fatty acid in R. differens from Kabale, Masaka, and Mbarara. All essential amino acids were recorded in R. differens, particularly histidine with values exceeding the daily requirement for adults. Mineral and vitamin content differed significantly across the five districts. The highest quantity of flavonoids was recorded in R. differens from Hoima (484 mg/100 g). Our findings revealed that R. differens could be considered as functional food ingredients capable of supplying essential macro‐ and micronutrients that are critical in curbing the rising food insecurity and malnutrition in the regions.


| INTRODUC TI ON
Ruspolia differens Serville (Orthoptera: Tettigonidae) is an insect delicacy that is widely consumed as a main dish or snack in Eastern, Western, and Southern Africa (Agea et al., 2008;Mmari et al., 2017;van Huis, 2022). It has a rich profile of proteins (37%-54%) and essential amino acids that are not found in commonly consumed cereal proteins (Fombong et al., 2017;Ssepuuya et al., 2019;Zielińska et al., 2015). Its crude protein content exceeds values supplied by common animal protein sources (Orkusz, 2021). Thus, R. differens can serve as an alternative source of protein to supplement the increased demand for animal protein across the globe (Govorushko, 2019). R. differens contains fats (33%-49%) with high levels of polyunsaturated fatty acids (Fombong et al., 2017;Kinyuru et al., 2010;Nyangena et al., 2020). These polyunsaturated fatty acids are implicated in prophylaxis against autoimmune diseases, cancer, and osteoarthritis; at cellular level they influence the regulation of glucose levels, blood pressure, nervous system, blood clotting, and inflammatory reactions (Kapoor et al., 2021). The value of crude fats and energy in R. differens is higher than those obtained from pork, chicken, and beef (Orkusz, 2021). The high vitamins and mineral content of R. differens makes its consumption a critical option in addressing malnutrition and mineral deficiency especially in children and women of childbearing age in Sub-Saharan Africa (Kinyuru et al., 2010;Mwangi et al., 2018;Ssepuuya et al., 2019). The grasshoppers contain flavonoids and phenols which are antioxidant compounds that provide anticancer, antiviral, antibacterial, and antiinflammatory activities in human (Cheseto et al., 2020;Ssepuuya et al., 2019). The quantities of these antioxidant compounds in R. differens are comparable to values recorded for different fruits and vegetables (Ssepuuya et al., 2019).
The variability of nutritional profile of insects is influenced by stage of development, origin, and diet (Govorushko, 2019;Kouřimská & Adámková, 2016). Nutritional composition of insects is influenced by their diets which implies that phytophagous R. differens feeding on host plants in different agro-ecological zones would display considerable variation (Mwangi et al., 2018;Tang et al., 2019;van Huis, 2020). Nutritional composition of R. differens is known to be influenced by their diets (Lehtovaara et al., 2017;. This study, therefore, evaluated the variability of nutritional composition of R. differens collected from different districts in Uganda where commercial harvesting and trade of R. differens is predominant. The study assessed the proximate composition, fatty and amino acids profile, minerals, vitamins, and flavonoid content of R. differens from different locations. Where data were available, these parameters were compared against common animal protein sources and daily requirements for human. The finding of this study will generate more evidence on the influence of geographical location of collection on nutritional composition of R. differens that provide information on the nutritional potential of R. differens to supplement the existing animal protein sources to curb global increase in food insecurity and malnutrition.

| Collection of R. differens
Raw R. differens were purchased from commercial harvesters from five different districts in Uganda: Kabale, Hoima, Mbarara, Kampala (Nakasero), and Masaka districts. These districts are known as the major areas where seasonal swamping of R. differens occurs within each year. The sample of the grasshoppers was purchased directly on-site from the commercial harvesters from each of the five districts. The samples were processed by plucking the wings, legs, and ovipositors. The samples had a mixture of the major morphotypes (brown, green, pink, or brown infused with green colored morphs).

| Sample preparation
Ruspolia differens samples were packed in polyethylene sterile Ziploc bags (SC Johnson brand, Size 15 × 13″) procured from a local supermarket in Kampala. The samples were labeled accordingly, packed with dry ice in cooler boxes, and sealed hermetically then transported by road to the laboratory in icipe Duduville campus in Nairobi A kilogram of frozen R. differens from each of the respective collection sites was allowed to thaw overnight under normal refrigeration at 5°C and rinsed with water to remove dirt. The samples were spread out evenly on aluminum foil and then oven dried (Model: SDO-225-CLAD-F-200 HYD; Wagtech Projects Ltd) at 60°C for 24 h (Fombong et al., 2017;Ochieng et al., 2022). The dried samples were ground using an electronic blender (Preethi TRIO, 500w, MG182/00), packed into Ziploc bags, and stored in a freezer for 24 h at −20°C prior to analysis at icipe's Behavioral and Chemical Ecology Unit (BCEU).

| Data collection
2.3.1 | Determination of proximate composition of R. differens Proximate composition was determined using the official methods of Association of Official Analytic Chemists (AOAC, 2012). Ash content was determined using a gravimetric method in a muffle furnace at 550°C for 3 h while moisture content was estimated as moisture loss after drying in an air oven at 105°C for 3 h. Kjeldahl method was used to determine the crude protein content of the samples following digestion in concentrated sulfuric acid. This was then computed using a 6.25 nitrogen-to-protein conversion factor. Soxhlet extraction method was used for the extraction of crude fat while crude fiber content was determined by acid digestion and evaluated by loss on ignition (Magara et al., 2019;Ochieng et al., 2022). The content of carbohydrates in each of the samples was estimated by subtracting the ash, moisture, fat, and protein content from 100%. Total energy (kJ/100 g) was computed using the formula: total energy = 4 × carbohydrate (%) + 4 × protein (%) + 9 × fat (%) (FAO, 2003).

| Determination of fatty acid content of R. differens
Fat extraction from the samples was conducted by adding 1 g of sample into 15-mL falcon tube. The sample was mixed with 10 mL DCM: MeOH (2:1) in a hood. The mixture was vortexed for 10 s followed by sonication for 20 min and then allowed to stand for 1 h. The mixture was centrifuged for 10 min at 4800 g, then filtered into clean falcon tubes, and allowed to evaporate overnight in a hood until all solvents evaporated and crude oil extract remained.
Fatty acid methyl esters (FAMEs) were extracted as previously described by Cheseto et al. (2020). A quantity of 300 mg of oil extract was weighed into clean narrow neck vials. One and a half milliliters of sodium methoxide prepared by dissolving 2 g of sodium methoxide into 20 mL of dry methanol was added to the sample. The mixture was vortexed for 1 min, sonicated for 10 min, and then incubated in a water bath at 70°C for 1 h. Distilled deionized water (100 μL) was added to the mixture to quench the reaction and then vortexed for 1 min. GC-grade hexane (1000 μL; Sigma-Aldrich) was used to extract resultant FAMEs. A milliliter of hexane was added to the mixture, vortexed for 20 s, and then transferred to Eppendorf tubes prior to centrifugation at 16,000 g for 20 min. A quantity of 100 μL of supernatant was filtered into clean vials and dried through anhydrous sodium sulfate on insert fitted tips, followed by the addition of 900 μL hexane. The supernatant was analyzed using gas chromatography and mass spectrometry (GC-MS) on a 7890A gas chromatograph linked to a 5975C mass selective detector (Agilent Technologies Inc.).
The carrier gas was helium at a flow rate of 1.25 mL/min. Oven temperature was set to rise from 35°C to 285°C. The initial temperature was maintained for 5 min which rose at 10°C/min to 280°C at a hold time of 20.4 min. Quadrupole mass selective detector and ion source were maintained at temperatures of 230 and 180°C, respectively.
Mass spectra of electron impact (EI) were obtained at an acceleration energy of 70 eV while fragment ions were analyzed between 40 and 550 m/z mass range in full scan mode. Filament delay period was fixed at 3.3 min. Serial dilution of authentic standard methyl octadecenoate (0.25-125 ng/ μL) was analyzed in full scan mode by GC-MS to produce a linear calibration curve (peak area against concentration) using the equation: [y = 5E + 07x + 2E + 07] which yielded R 2 = .9997. The generated regression equation was used to quantify the different fatty acids externally.
ChemStation B.02.02 acquisition software installed in an HP (Hewlett-Packard: HP Z220 intel xeon) workstation was used to generate mass spectrum for each peak using the following integrators: initial threshold: 5, initial peak width: 0.1, initial area reject: 1, and shoulder detection: on. Identification of the compounds was done by comparison of mass spectral data and retention times with NIST 05, 08, and 11 authentic standards and reference spectra published by library-MS databases. Analysis for each sample was done in triplicates.

| Determination of amino acid composition of R. differens
The amino acid composition of R. differens collected from the diverse localities was analyzed using a modified protocol previously described by Musundire et al. (2016). A quantity of 100 mg of each sample was weighed into a 5-mL vial followed by 1.5 mL of 6 N HCL. The vial was capped following the introduction of nitrogen and then vortexed for 1 min. The samples were then placed in a GC oven at 110°C for 24 h to allow for complete hydrolysis.

| Mineral analysis of R. differens samples
Mineral composition was determined using ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometer) analysis (Campbell & Plank, 1991;Horwitz, 2000). Detector CCD, CCD; Purge gas, nitrogen; Shear gas, air and Plasma gas, argon. The minerals were measured at the following wavelength (nm): . ICP-OES mix standard CatNo. 43843 (Sigma Aldrich) was used for quantification. External standard calibration method was applied, and serial dilution of the standards was performed using 2% nitric acid to obtain calibration standards of 400, 800, 2000, and 4000 μg/L. Calibration was done using Perkin Elmer Winlab 32 software. This analysis was done in triplicate.

| Determination of vitamin content of R. differens
The composition of water-soluble vitamins was determined by The composition of fat-soluble vitamins (retinol and tocopherols) was determined by HPLC (High-Performance Liquid Chromatography) (Bhatnagar-Panwar et al., 2013;Hosotani & Kitagawa, 2003). A quantity of 0.5-g sample of ground grasshopper was weighed in a 25-mL tube in triplicate, mixed with 6 mL ethanol with 0.1% BHT (Butylated Hydroxytoluene), and homogenized for a minute. A quantity of 120 μL of potassium hydroxide 80% (w/v) was added to the solution and mixed by vortexing. The mixture was incubated for 5 min at 85°C, removed from the water bath, and immediately cooled in ice. Four milliliters of deionized water was added to each tube and then mixed in a vortex. Five milliliters of hexane was added to the tubes and then mixed in a vortex. The sample was centrifuged for 5 min at 3429 g. The upper phase (hexane) was transferred to a centrifuge tube using Pasteur pipette. This was extracted three more times with 4 × 3 × 3 mL hexane and the extract pooled into a 25-mL tube. Additional 5-mL deionized water was added to the extract, vortexed for a minute, and centrifuged at 3429 g for 5 min. The hexane layer was recovered into clean test tubes and then evaporated to complete dryness under nitrogen in the N-Evap. This was then reconstituted 1 mL of methanol: tetrahydrofuran (85:15 v/v), vortexed and sonicated for 30 seconds, and then transferred to 0.8 mL HPLC vials. HPLC system used was Shimadzu Nexera UPLC system linked to SPD-M2A detector with Reverse phase gradient HPLC method with the following specifications: Oven temperature, off; YMC C30, carotenoid column (3 μm, 150 × 3.0 mm, YMC); injection volume, 10 μL; Mobile phase A: Methanol/tert-butyl methyl ether/water (85:12:3, v/v/v, with 1.5% ammonium acetate in the water); Mobile phase B: methanol/tert-butyl methyl ether/water (8:90:2, v/v/v, with 1% ammonium acetate in the water); and a total flow rate of 0.4 mL/ min. Extraction, detection, identification, and quantification of each sample were done in triplicate.

| Determination of flavonoids
Flavonoid content of R. differens samples was determined using Aluminum Chloride calorimetry (Dewanto et al., 2002;Singleton & Rossi, 1965;Zhishen et al., 1999). Sample extraction was done by weighing 0.5 g into a clean propylene tube. This was followed by the addition of 10 mL of 80% methanol and then shaking in a mechanical shaker at 25°C for 24 h. The mixture was centrifuged for 10 min at 4571 g and the supernatant aliquot was taken out for the determination of total flavonoids. Approximately 20-μL aliquot of sample extract or standard solution of catechin (10, 20, 40, 60, 80, and 100 μg/ mL) was pipetted into microtiter well and then mixed with 80 μL of deionized distilled water. Ten microliters of 5% NaNO 2 was added to the mixture and mixed by priming. After 5 min, 10 μL of 10% ALCL 3 was added and mixed gently by priming. Five minutes later, 80 μL of 2 M NaOH was added to the mixture and mixed gently by priming.
The reaction was incubated at room temperature for 30 min. The absorbance of the samples and standards were read against the reagent blank using a Ultraviolet-visible spectrophotometer at a wavelength setting of 510 nm. Standard calibration (0.01-0.02-0.04-0.0 6-0.08-0.1 mg/mL) curve of catechin was plotted in 80% methanol.
Extraction, detection, identification, and quantification of each sample were done in triplicate.

| Data analysis
All analyses were performed using R version 4.1.2 (R Core Team, 2020), statistical software. Shapiro-Wilk's and Bartlett's tests were used to test for normality and homogeneity of data, respectively. All normal and homogenous data were subjected to one-way analysis of variance (one-way ANOVA) to test for differences in nutritional composition of R. differens collected from different sites. Nonnormal data were subjected to log transformation prior to ANOVA. Mean separation was done using Tukey's Honest Significant Difference (HSD), post hoc test where the differences were significant. Normal nonhomogeneous data were subjected to Welch ANOVA and differences in means separated using Games-Howell test where there were significant variations. All effects were considered significant at p < .05.

| Proximate composition of R. differens
Proximate composition of R. differens varied across the geographical locations ( Table 1). The highest crude protein from R. differens was obtained from Kabale, while higher values of crude fiber and carbohydrates were observed in Mbarara and Kampala, respectively. Ruspolia differens obtained from Masaka recorded the highest energy (645 ± 2.2 kj/100 g) and moisture (5.3 ± 0.0%) content while those from Hoima recorded the highest ash (2.8 ± 0%) content. The crude protein, crude fat, and energy content of R. differens collected from the various sites exceeded the values recorded for common animal protein sources (Orkusz, 2021;Williams et al., 2016).

| Amino acid composition of R. differens
Nine essential and eight nonessential amino acids were detected in R. differens samples analyzed ( Table 3). The quantities of methionine (F 4,10 = 4.38, p = .026) and lysine (F 4,10 = 4.19, p = .030) varied by area of collection while the quantities of the other essential amino acids did not differ. Quantities of proline (F 4,10 = 3.52, p = .048) and glycine (F 4,10 = 3.66, p = .043) varied by area of collection while the quantities of other nonessential amino acids were not different. The leucine content of a gram of R. differens analyzed was higher than quantities obtained in common animal protein sources and exceeded the daily requirements for human adults (Orkusz, 2021;Rumpold & Schlüter, 2013a).

| Mineral composition of R. differens collected from different geographical locations
Several macro-and microminerals were obtained in varying quantities from R. differens collected from the five districts (Table 4).

| DISCUSS ION
The findings of this study demonstrate that R. differens is rich in macromolecules such as proteins, fats, and carbohydrates. The grasshopper is abundant in essential amino acids, unsaturated fatty acids, TA B L E 1 Proximate composition (% DM basis) of Ruspolia differens (mean ± SE) collected from different geographical locations versus common animal protein sources. Salama, 2020). The swarm of R. differens is composed of adult insects which possess wings and therefore able to fly. This implies that the observed variations in nutrient profile did not arise due to differences in the development stage of R. differens since swarms comprised adult insects (Matojo & Yarro, 2013). Ruspolia differens analyzed in the study were purchased from commercial harvesters from five different districts in Uganda. These districts are found in different agro-ecological zones which have diverse climatic conditions (Kabasiita et al., 2022). These districts were different from that reported by Ssepuuya et al. (2019), except for Masaka and Kampala.

Component
Given that R. differens is known to be a highly oligophagous species  7.5%-28.2% recorded in other edible grasshopper species including Oxya fuscovittata (Orthoptera: Acrididae), Oxya hyla (Orthoptera: Acrididae), and Boopedon flaviventris (Orthoptera: Acrididae) (Anand et al., 2008;Blásquez et al., 2012;Ghosh et al., 2016). Carbohydrate content of R. differens obtained from Kampala was higher than those collected from the other locations. The high fat and carbohydrate content of the grasshopper supplies energy that is vital for the human body (Hlongwane et al., 2020). Carbohydrates derived from insects are also rich in polysaccharides that enhance immunity in humans (Kouřimská & Adámková, 2016). Limited data were obtained on carbohydrate content of common animal protein sources.
The crude fiber content of R. differens from the five districts was lower than quantities (6%-13%) recorded by several researchers (Bbosa et al., 2019;Ssepuuya et al., 2017); but within the same range as the values (4%-5%) observed by Kinyuru et al. (2010). Crude fiber content of R. differens from Mbarara was significantly higher than that of grasshoppers obtained from Masaka and Kampala but comparable to those collected from Hoima and Kabale.
Ruspolia differens contained more energy compared to beef, chicken, and pork (Orkusz, 2021). Limited data exist on energy content of R. differens; however, the values obtained in R. differens from the different sites were higher than findings by other researchers (Bbosa et al., 2019). However, the energy content observed in R. differens from Hoima, Kabale, Mbarara, and Kampala was lower than values recorded by some authors (Siulapwa et al., 2012). The highest energy content occurred in R. differens from Masaka which was probably due to the high crude fats and carbohydrate content of R. differens collected from the location.
The lowest ash content observed in R. differens collected from Masaka and Kampala was lower than previously recorded values for the species and other edible grasshopper species (Kinyuru et al., 2010;Rumpold & Schlüter, 2013a). The ash content of R. differens obtained from Hoima, Kabale, and Mbarara corroborated findings by Kinyuru et al. (2010) but were contrary to findings by other authors who recorded higher (Fombong et al., 2017;Ssepuuya et al., 2017) and lower ash content in R. differens (Siulapwa et al., 2012). The ash content of R. differens exceeded values recorded in beef, chicken, and pork (Orkusz, 2021;Williams et al., 2016).
The moisture content of R. differens collected from the different sites was lower than 50.4%-71.2% as previously reported (Kinyuru et al., 2010;Ssepuuya et al., 2017). The authors analyzed raw, wildcollected grasshoppers that may have had a higher moisture content compared to the oven-dried samples analyzed in this study.
However, the moisture content of R. differens collected from Masaka and Kampala was higher than the previous value obtained for ovendried samples (Fombong et al., 2017). in R. differens; however, this was only true for R. differens obtained from Masaka and Kampala (Malinga et al., 2020;. The high quantity of lauric acid, methyl 16 pentacosenoate, methyl heneicosanoate, and methyl nonadecanoate observed in R. differens obtained from some of the sites was contrary to the low proportions reported by other researchers (Fombong et al., 2017;Ssepuuya et al., 2020). Although oleic acid has been reported as the most abundant monounsaturated fatty acid in R. differens, our findings showed that it was only abundant in R. differens collected from Mbarara (Cheseto et al., 2020;Ssepuuya et al., 2019). Other abundant monounsaturated fatty acids observed in R. differens collected from the other areas included elaidolinolenic, methyl cis-13eicosenoate, and methyl cis-10-heptadecenoate. These compounds have previously been reported in R. differens oil and products made from their oils (Cheseto et al., 2020). Linoleic acid was the most abundant polyunsaturated fatty acid similar to findings by other authors (Cheseto et al., 2020;Malinga et al., 2020;Ssepuuya et al., 2019). Polyunsaturated fatty acids are implicated in the prevention of cancer, cardiovascular diseases, and diabetic neuropathy; however, these compounds cannot be manufactured by mammals and must, therefore, be supplied through diet (Govorushko, 2019;Yorek, 2018). Therefore, the inclusion of R. differens can be essential in supplying these polyunsaturated fatty acids.
Ruspolia differens samples contained almost all the essential amino acids most of which are not found in plant protein sources (Zielińska et al., 2015). This makes them a suitable alternative to plant proteins for fortification of food products such as flour and baked products in efforts to reduce malnutrition. It can also be utilized as a raw product in food processing to supply essential amino acids (Köhler et al., 2019;Ochieng et al., 2022). Other than leucine, the value of amino acids was lower than quantities obtained from pork, chicken, and beef; and was lower than the daily requirements for adults (Orkusz, 2021;Rumpold & Schlüter, 2013a;WHO, 2007).
The inclusion of R. differens in diets can, therefore, supplement the essential amino acids supplied by the animal protein sources. The quantities of essential amino acids such as leucine, arginine, threonine, valine, histidine, isoleucine, and phenylalanine did not vary significantly among the five districts. Quantities of leucine obtained were comparable to values reported in Kenya and Uganda but higher than values recorded in R. differens collected from Zambia (Fombong et al., 2017;Siulapwa et al., 2012;Ssepuuya et al., 2019). However, the quantities of arginine, threonine, valine, histidine, isoleucine and phenylalanine, methionine, and lysine were lower than previously recorded values (Fombong et al., 2017;Siulapwa et al., 2012;Ssepuuya et al., 2019). Similarly, the quantities of nonessential amino acids such as glutamic acid, aspartic acid, alanine, cysteine, arginine, glycine, proline, serine, and tyrosine detected in R. differens collected from the different sites were lower than the values reported by other researchers (Fombong et al., 2017;Siulapwa et al., 2012;Ssepuuya et al., 2019). The low amino acid content recorded in the study could be attributed to the heat processing (oven drying) method that was applied prior to analysis of the samples. Processing is associated with alteration of nutritional profile of R. differens (Fombong et al., 2017;Nyangena et al., 2020).
Ruspolia differens showed a rich profile of macro-and microminerals that are vital for human health and development (Kinyuru et al., 2010;Mwangi et al., 2018;Silva et al., 2019). Minerals function as cofactor for diverse enzymes that are essential for different physiological processes in the human body (Gupta & Gupta, 2014).
Minerals are required in diverse quantities and obtained through diet (Silva et al., 2019). Consumption of R. differens can, therefore, mitigate mineral deficiencies in humans. Phosphorous content of R. differens collected from Hoima, Kabale, Masaka, and Mbarara was lower than the least previously recorded values (121 mg/100) (Kinyuru et al., 2010). The significantly higher value of phosphorus recorded in R. differens collected from Kampala was higher than the values observed by Kinyuru et al. (2010) but lower than the findings by Fombong et al. (2017) and Siulapwa et al. (2012). The quantity of phosphorus obtained in R. differens collected from Masaka was almost 2.5 times lower than the least recorded quantity in the other sites.
Significantly higher potassium content was obtained in R. differens collected from Kabale which exceeded the highest value previously observed in the grasshopper (Fombong et al., 2017). The least potassium content occurred in R. differens collected from Masaka which was lower than previously recorded value (Kinyuru et al., 2010). The potassium content of R. differens obtained from Kampala, Hoima, and Mbarara was comparable to findings documented by other researchers (Fombong et al., 2017;Ssepuuya et al., 2019). A higher content of calcium occurred in grasshoppers from Mbarara followed by Hoima, Kabale, and Kampala. This was comparable to quantities observed by Ssepuuya et al. (2019), higher than the values obtained by Kinyuru et al. (2010), but lower than the findings of other authors (Fombong et al., 2017;Ssepuuya et al., 2017).  (Fombong et al., 2017;Kinyuru et al., 2010;Ssepuuya et al., 2019). Acrididae), and Trimerotropis pallidipennis (Orthoptera: Acrididae) (Finke, 2015;Ladeji et al., 2003;Oibiokpa et al., 2017). The iron content of R. differens analyzed in the study was lower than quantities observed by Fombong et al. (2017). Grasshoppers obtained from Hoima contained the highest iron content followed by those collected from Kabale. These grasshoppers recorded a higher iron content compared to values recorded by Kinyuru et al. (2010) and Acrididae), M. foedus, and Z. variegatus (Alamu et al., 2013;Ladeji et al., 2003;Oibiokpa et al., 2017). R. differens was more abundant in zinc and iron compared to other animal proteins; therefore, the consumption of R. differens can curb zinc and iron deficiencies that are prevalent among children and women of childbearing age (Mwangi et al., 2018;Orkusz, 2021).  (Ghosh et al., 2016;Kinyuru et al., 2009;Oibiokpa et al., 2017). Gamma tocopherol was observed in grasshoppers collected from Hoima, Kabale, and Kampala but was not detected in grasshoppers obtained from Masaka and Mbarara.
Flavonoid content observed in the study was within the same range previously recorded value in processed R. differens (Ochieng et al., 2022). Flavonoids are rich sources of antioxidants that possess antimicrobial, anti-inflammatory, antiallergenic, and anticancer properties, critical for human health (Cheseto et al., 2020;Pal & Dubey, 2013). The flavonoid content of the grasshopper exceeds the quantities of flavonoids obtained in several vegetables and fruits in some countries (Ssepuuya et al., 2020).

| CON CLUS ION
The

ACK N OWLED G M ENTS
The authors would like to thank Xavier Cheseto and Jactone Kongere for their technical support in sample preparation and Brian Ochieng for support in data organization. We acknowledge the support of Eric Ibrahim in data analysis.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All relevant data are within the paper and are available upon request from the authors.