A Novel Strategy for the Production of Edible Insects: Effect of Dietary Perilla Seed Supplementation on Nutritional Composition, Growth Performance, Lipid Metabolism, and Δ6 Desaturase Gene Expression of Sago Palm Weevil (Rhynchophorus ferrugineus) Larvae

The nutritional value, growth performance, and lipid metabolism of sago palm weevil larvae (Rhynchophorus ferrugineus, SPWL) raised on plant-based diets (soybean, rice bran, and ground sago palm trunk (GSPT)), supplemented with various concentrations (0, 3, 7, 15, and 20%) of perilla seed (PS) were compared with traditional diets i.e., regular GSPT (control) and GSPT supplemented with pig feed. All supplemented diets rendered SPWL with higher lipid and protein contents (p < 0.05). Supplementing with 7–20% PS enhanced α-linoleic acid content in SPWL, resulting in a decrease in the n-6:n-3 ratio to a desirable level. Dietary PS supplementation increased Δ9 (18), total Δ9 and Δ5 + Δ6 desaturase indexes, fatty acid (FA) unsaturation, and the polyunsaturated FA:saturated FA ratio in SPWL, while lowering atherogenicity index, thrombogenicity index, and Δ6 desaturase (fads2) gene expression. Boosting with 7% PS improved the majority of growth parameters and enhanced essential amino acid and mineral contents (p < 0.05).


Introduction
Edible insects raised on farms can be regarded as an alternative food source that benefits the environment by emitting less greenhouse gases and ammonia, having greater feed conversion ratios, providing less disease and zoonotic risk, and using substantially less water than traditional livestock farming [1,2]. The use of insects as a food and feed source is thought to have major benefits for the economy, the environment, and food security. Although it is still a very small niche industry in the EU, insect farming for food and feed is growing in popularity [3]. Additionally, there is a challenge with the import of insects and insect-derived products into the EU for food and feed because the use of insects is more common outside the EU [3]. Considering the full supply chain, from farming to consumption, a risk profile and presentation of potential biological, chemical, allergenicity, and environmental concerns connected with farmed insects used as food and feed was developed [3]. This is the first study on the use of dietary supplementation from perilla seeds as a cutting-edge method for creating edible SPWL. The new findings could help increase the commercial value of this local insect for future food security and sustainability.

Diets and SPWL Rearing
To compare the effects of feed ingredients on nutrient composition, growth performance, lipid metabolism indices, and 6 desaturase (fads2) gene expression in SPWL, commercial pig feed (PF), mixed plant-based ingredients (PI, a 1:1 mixture of soybean and rice bran), and PI formulated with different levels of commercial PS (Bankongloi, Chiang Mai, Thailand) (0, 3, 7, 15, 20%, w/w) were supplemented to the basal GSPT (control). The supplemented diets were made by combining GSPT with each supplement in a 2:1 (w/w) ratio and used to rear SPWL in three separate lots. Table 1 lists the ingredients in the experimental diets. All the diets were analysed for their proximate composition (see Section 2.3) and FA composition (see Section 2.4) in comparison with the control diet. Experimental diet nomenclature: C: control, PF: diet supplemented with pig feed, P0-P20: diets contained 0-20% perilla seed, respectively.
According to Chinarak et al. [5], the SPWL were reared at room temperature (27-30 C) with a relative humidity of 70-80%. The Walailak University Animal Ethics Committee has evaluated and approved this rearing procedure (WU-AICUC-64029). In brief, 4 mature weevil pairs were grown in a spherical plastic container with 3 kg of individual feed meal and 1 L of water. The SPWL were formed after 20 days of rearing and fixed at 120 larvae/container with a total rearing period of 40 days. To avoid a feed shortage, new diets (1.5 kg each time) were added on days 20 and 30 of rearing. The SPWL were measured on days 20 and 40 to assess growth performance. The live larvae reared on day 40 were rinsed with tap water, blanched, and subjected to freeze-drying before being kept in a plastic box at −20 °C until further investigation. The nutritional composition, lipid metabolism indices, and gene expression of 6 desaturase (fads2) were also determined in freeze-dried SPWL.

Diets and SPWL Rearing
To compare the effects of feed ingredients on nutrient composition, growth performance, lipid metabolism indices, and ∆6 desaturase (fads2) gene expression in SPWL, commercial pig feed (PF), mixed plant-based ingredients (PI, a 1:1 mixture of soybean and rice bran), and PI formulated with different levels of commercial PS (Bankongloi, Chiang Mai, Thailand) (0, 3, 7, 15, 20%, w/w) were supplemented to the basal GSPT (control). The supplemented diets were made by combining GSPT with each supplement in a 2:1 (w/w) ratio and used to rear SPWL in three separate lots. Table 1 lists the ingredients in the experimental diets. All the diets were analysed for their proximate composition (see Section 2.3) and FA composition (see Section 2.4) in comparison with the control diet. Experimental diet nomenclature: C: control, PF: diet supplemented with pig feed, P0-P20: diets contained 0-20% perilla seed, respectively.
According to Chinarak et al. [5], the SPWL were reared at room temperature (27-30 • C) with a relative humidity of 70-80%. The Walailak University Animal Ethics Committee has evaluated and approved this rearing procedure (WU-AICUC-64029). In brief, 4 mature weevil pairs were grown in a spherical plastic container with 3 kg of individual feed meal and 1 L of water. The SPWL were formed after 20 days of rearing and fixed at 120 larvae/container with a total rearing period of 40 days. To avoid a feed shortage, new diets (1.5 kg each time) were added on days 20 and 30 of rearing. The SPWL were measured on days 20 and 40 to assess growth performance. The live larvae reared on day 40 were rinsed with tap water, blanched, and subjected to freeze-drying before being kept in a plastic box at −20 • C until further investigation. The nutritional composition, lipid metabolism indices, and gene expression of ∆6 desaturase (fads2) were also determined in freeze-dried SPWL.

Growth Performance
Twenty larvae from each group were used to determine biometric characteristics. The larvae were individually measured for body weight (g) and total length (cm) at day 40 after rearing. SPWL biometric parameters were calculated using the following formulae: Dry matter content = (dry weight / live weight) × 100 (1) Condition factor = (body mass × 100) / body length 3 (2) Survival = (numbers of final larvae / numbers of initial larvae) × 100 Growth rate (g/day) = (ln final body weight − ln initial body weight) / day (4)

Proximate Composition and Mineral Profile
The proximate composition of the SPWL, including moisture, crude protein, crude fat, and ash, was investigated [17]. To avoid overestimation of protein content due to the presence of some non-protein nitrogen in SPWL, a conversion factor (CF) of 6.25 was used for the diets, but a CF of 5.6 was used for the SPWL [4,5]. Carbohydrate was calculated by subtracting 100 from the moisture, fat, protein, and ash contents. Inductive couple plasma (ICP) spectrometry was used to evaluate the elemental composition of SPWL, which contained potassium (K), phosphorus (P), sodium (Na), magnesium (Mg), calcium (Ca), zinc (Zn), manganese (Mn), iron (Fe), and copper (Cu) [17].

FA Composition
The FA composition of feed and SPWL was determined using a gas chromatography/quadrupole time of flight (GC/Q-TOF) mass spectrometer, as described by Chinarak et al. [5].
TRIZOL reagent (Invitrogen, USA) was used to extract total RNA from 40-day raised SPWL using the manufacturer's protocol. RNA concentration and purification were determined at 260 and 280 nm, respectively. RNA integrity was determined using electrophoresis on a 1% (w/v) agarose gel. Reverse transcriptase was used to reverse transcribe RNA into first-strand cDNA (iScript TM Select cDNA Synthesis Kit, Bio-Rad, Hercules, CA, USA).

Cholesterol Content
Cholesterol content of the whole SPWL sample was determined using a gas chromatography-triple quadrupole mass spectrometer (GC/QQQ, GC 7890B/MSD 7000D, Agilent Technologies, Santa Clara, CA, USA) connected to the PAL autosampler system (CTC Analytics AG, Zwingen, Switzerland). Data was acquired by the MassHunter software (Version 10.0, Agilent Technologies). The calibration curves were prepared using cholesterol at different concentrations ranging from 30 to 2000 µg/µL [6].

Amino Acid Profile
The amino acid compositions of SPWL were determined using the Shimadzu-GCMS-TQ8050 NX (Kyoto, Japan). The amino acid content was given as g/100 g dry sample. Furthermore, the essential amino acid index (EAAI) and biological value (BV) were estimated using the formula given below by Chinarak et al. [4]: EAAI = 9 g of lysine in 100 g of analysis protein × 100 g of lysine in 100 g of reference protein × (etc. for other 8 EAA) (13)

Statistical Analysis
The Statistical Package for the Social Sciences (SPSS) 24 for Windows was used for statistical analysis. Each lot was tested three times. One-way ANOVA was used for statistical analysis. Duncan's multiple-range test was used to discover significant differences (p < 0.05) across samples when comparing means.

Basic Composition and FA Profile of Diets
The effect of PI formulated with various levels of PS (0-20%), then combined into GSPT in comparison to PF-formulated GSPT (2:1, w/w) and control GSPT alone on the proximate composition of the resulting diets is shown in Table 2. The amount of fat, protein, ash, and carbohydrate varied depending on the type of supplement used ( Table 2). Carbohydrate was the most prevalent composition in all diets (63.7-93.9%), due to the high content of starch in the GSPT [4]. The addition of PI, PI plus PS, and PF to GSPT resulted in a significant increase in protein (9.4-10.6 fold) and fat (6.3-22.4 fold) content over the common control diet (p < 0.05). The chemical compositions of mixed diets were primarily determined by the major component presented in individual supplements, in which the amounts of soybean, rice bran, PF, and PS formulated for each diet formula were varied (Table 1). Deng et al. [9] found that combining PS with other ingredients resulted in a difference in the composition of lamb diets, particularly protein and fat. FA composition of all diets is given in Table 3. The most abundant FA in the GSPT were C16:0 (palmitic acid), C18:0 (stearic acid), C18:1 (oleic acid), and C18:2 (linoleic acid), accounting for 62.45 g/100 g of lipid. It should be noted that the FA profiles were found to be similar in all test diets, though their contents differed based on the type of supplement used (Table 1). PS formulation led to a significant increase in C18:3 (all-cis-cis-9,12, 15-linolenic acid; ALA), which was 8-26 times greater than the control diet (p < 0.05). This led to an increase in n-3 FA enhancement following PS fortification into PI mixed GSPT diets, resulting in PS being an excellent source of ALA [19]. SFA, MUFA, and PUFA differed statistically between the test diets (p < 0.05), ranging from 16.87 to 47.26 g/100 g total lipid, 13.90 to 36.55 g/100 g lipid, and 8.96 to 66.94 g/100 g lipid, respectively ( Table 1). The n-3 PUFA content of the PI and PS added to the GSPT was primarily increased, leading to a low n-6/n-3 (Table 3). Table 4 shows the appearances and growth performance of SPWL fed on various test diets. When PI, PS, and PF were combined into GSTP, all growth factors of SPWL were significantly increased when compared to those fed a control diet (p < 0.05). The type of supplement and the concentration of PS had an impact on live weight, survival rate, and growth factors. All SPWL raised by 40 days had an 80% survival rate (Table 4). Increased PS levels in PI mixed diets had a negative impact on SPWL survival when compared to the control diet. This result was consistent with the findings of Chinarak et al. [5], who found that incorporating PS into GSPT reduced the survival rate of SPWL. The presence of antinutritive substances in PS, such as phytic acid, may have contributed to an imbalance in insect metabolism [5]. The antinutrient found in PS, specifically perilla ketone, a terpenoid composed of furan rings with six carbon chains and ketone functional groups, has been linked to the induction of pulmonary edema and symptoms of perilla mint toxicosis in cattle fed large amounts of PS [20]. Typically, anti-nutritional compounds have a significant negative impact on animal digestive performance [11]. According to Oonincx et al. [8], the addition of 0-4% flax seed oil to the diet of house crickets had a negative impact on survival rate, which decreased from 69% to 55%. SPWL fed PF, PI, and PI formulated with 3-15% PS-formulated GSPT had the highest live weight (p < 0.05). There were no significant differences in dry weight of SPWL between supplemented diet groups (p > 0.05). SPWL fed supplemental diets had increased dry weight, dry matter content, and condition factor compared to control SPWL (p < 0.05). Thus, the addition of supplements into regular GSPT could improve the growth performance of the SPWL. Each treatment had a slight variation in the condition factor. The incorporation of PI and PS into GSPT resulted in a slower growth rate than the PF-diet and control diet (Table 4). Table 3. Fatty acid composition of experimental diets.

Proximate Composition, Cholesterol Content, and Mineral Profiles of SPWL
The SPWL fed with the PF-formulated diet had the highest cholesterol content, while the SPWL fed with the GSPT control diet showed the lowest cholesterol content (p < 0.05). SPWL fed PI-enriched or PI with PS diets (3-20%) had significantly lower cholesterol levels than SPWL fed a PF-fortified diet (p < 0.05) ( Table 5). This finding was in line with the observations of Batkowska et al. [25], who found that the addition of linseed and soybean oils to hen diets led to a significant reduction in cholesterol content in the yolks. The cholesterol content of SPWL fed with PS-formulated diets differed slightly (Table 5). Cholesterol is a sterol found in animal-based foods that is naturally produced by animal cells [26]. It serves as a substrate for the manufacture of important molecules such as steroid hormones, vitamin D, and bile acids, as well as maintaining cell membrane integrity [27]. A high intake of cholesterol has been connected to an increased risk of hypercholesterolemia, Foods 2022, 11, 2036 9 of 18 cardiovascular disease, and coronary artery disease [28,29]. The adult population has been advised to ingest less than 300 mg of cholesterol per day [30].
Minerals are required for the maintenance of some life-sustaining physicochemical processes, despite the fact that they do not produce energy, and they play important roles in many bodily activities [31]. Natural minerals found in edible insects include Ca, Cu, Zn, Fe, Mn, Mg, Na, and P [24]. Table 5 shows the macrominerals found in SPWL, which include K, P, Mg, Ca, and Na. K was the most plentiful element in the SPWL (6421-9265 mg/kg) ( Table 5). It was greater than the comparative values of conventional meats such as pork (5043 mg/kg), beef (6247 mg/kg), and chicken (5557 mg/kg) [23]. The P content of SPWL ranged from 2926-4273 mg/kg (Table 5), with 3% PS diet-reared SPWL having the greatest P content (p < 0.05). SPWL fed PI and PI with PS-supplemented diets had significantly higher P levels than those fed PF-added diet (p < 0.05). As a result, SPWL fed PI or PI with PS-containing diets had comparable or even higher P content than those fed the control diet. However, the P content of SPWL fed all diets was enough for a daily requirement of 700-4000 mg/day for good health as informed by the World Health Organization [32]. Ghosh et al. [23] stated that, in contrast to plant-based P, P in insects is readily bioavailable, indicating a potential source for humans. The SPWL fed with the 3% PS diet had the highest Mg content; however, SPWL fed PI or higher PS than 3% added diets had greater Mg content than those fed with the control diet (p < 0.05). All SPWL raised on PI or PI with PS-formulated diets had higher Mg levels than those fed the CF diet (p < 0.05). Mg content in the SPWL was significantly greater than that reported by Araújo et al. [24] for Gryllus assimilis (271 mg/kg) and Zophobas morio (391 mg/kg). The Ca content of SPWL ranged from 441 to 699 mg/kg (Table 5), which was greater than that of conventional animal foods such as pork (379 mg/kg), beef (187 mg/kg), and chicken (323 mg/kg), with the exception of chicken eggs (2348 mg/kg) [23]. The Na content of SPWL ranged from 986 to 1351 mg/kg (Table 5), with less Na present in SPWL after all supplemental diet feeding than after control diet feeding (p < 0.05). The Na content of SPWL fed only PI or PI-formulated with different PS concentrations differed slightly, but all were higher than that of SPWL fed the PF diet (p < 0.05). Na is required for cellular homeostasis and physiological functions; however, excessive Na consumption has been linked to hypertension and non-communicable disease. SPWL raised on a 3% PS added diet had the highest Ca concentration, which was comparable to those fed with a control diet (p > 0.05). The feeding of only PI or PI with PS higher than 3% based diets resulted in lower Ca content in the resulting SPWL, but their contents were still higher than that fed on the PF diet (p < 0.05). Ca is primarily an essential element, with its phosphate salts playing critical roles in neuromuscular function as well as many enzyme-mediated activities such as blood clotting, bone formation, and tooth formation [33]. The most important micro-minerals in humans are Zn, Mn, Fe, and Cu, which are depicted in Table 5 for current SPWL. The insects are thought to aid in the supply of minerals, particularly Fe and Zn [24], with current SPWL compost containing 81.8-104.6 mg/kg of Zn and 14.2-19.0 mg/kg of Fe, respectively ( Table 5). The increased PS level in the diets resulted in lower Zn and Fe contents in the resulting SPWL (p < 0.05), except in the SPWL fed a 3% PS diet. Surprisingly, the highest Zn and Fe concentrations were found in SPWL fed on a 3% PS-formulated diet (p < 0.05), which could be attributed to the proper absorption of both microelements from the feed ingredients. However, higher PS intake in SPWL fed on high PS-formulated diets may alter mineral absorption from the gut by chelating with phytate, resulting in lower element uptake. The Zn content of these SPWL was higher than that of Gryllus assimilis (52.2 mg/kg) and Zophobas morio (24.7 mg/kg), with both insects having higher Fe content (27.8 and 22.7 mg/kg, respectively) than the SPWL [19]. Infants, children, teenagers, and women of childbearing age, particularly pregnant women, are the most vulnerable to iron deficiency [33]. Thus, SPWL can be used as an iron alternate. The Mn concentration of SPWL ranged from 10.4 to 49.9 mg/kg (Table 5), with SPWL fed all supplemental-added diets having significantly lower Mn content than those raised on the control diet (p < 0.05). This finding may be related to the reduction of Mn availability caused by dietary factors, specifically the lower total concentration of Mn in the experimental diet affected by the dilution of GSPT content in the supplemental-added diets. The Mn in this current SPWL was higher than Tribolium castaneum (4.9 mg/kg) [21], but lower than Allomyrina dichotoma (86.4 mg/kg), and Protaetia brevitarsis (58.9 mg/kg) [23]. The Cu content of SPWL ranged from 10.2-26.1 mg/kg ( Table 5). The greatest Cu content was found in the SPWL reared on the PF-formulated diet (p < 0.05). SPWL reared on PI and PI withPS-added diets had a significantly lower Cu content than those fed on PF-added diet (p < 0.05), though the Cu concentrations were slightly greater or comparable to that reared on the control diet. This could be explained by the PF's high Cu content, which was consistent with our previous study on the same insect [4]. Despite the fact that SPWL has a lower Cu content, it can be used as an alternative Cu source. As a result, incorporating 3% PS into PI and GSPT could significantly improve the overall element content in SPWL, which could play a significant role in food security. On the other hand, this current study addressed the availability of minerals as influenced by the experimental diet composition, which has not previously been addressed in this insect.

FA Composition and Health Promoting Indices of SPWL
The content and composition of lipids in insects is determined by feed or de novo synthesis, and they are stored as body fat before being degraded, digested, and delivered to their final target cell [34]. Table 6 shows the FA composition of the lipids presented in SPWL as affected by dietary ingredients. The FA composition of the experimental diets, as expected, significantly altered the FA composition of the resulting insect. Increased PS-formulated concentrations in PI-mixed regular GSPT had a significant effect on FA composition, specifically SFA, MUFA, and PUFA of SPWL (Table 6). In SPWL fed with increasing PS levels, there was a progressive decrease in total SFA with increasing PUFA compared to those raised on a control diet (p < 0.05). The highest C16:1 content was found in SPWL reared on a 7% PS-added diet (p < 0.05), whereas there was no significant difference in C18:1 content between SPWL fed PF-, PI-, and PI with 3-15% PS-formulated diets (p > 0.05). Certain ingredients, particularly soybean meal and rice bran, may contribute to the presence of C18:1 in the resulting SPWL, as these are excellent sources of linoleic acid [35]. It should be noted that the SPWL fed with PF-, PI-, and PI with 7% PS-added diets had the highest MUFA content, with no difference among them (p > 0.05). The highest C18:2 content was observed in SPWL raised on a PF-diet (p < 0.05), whereas feeding SPWL on PI-and PI with 3-20% PS diets resulted in an FA which was 1.4-3 fold lower ( Table 6). The SPWL reared on all supplemental diets had a higher C18:2 concentration than those reared on the control diet (p < 0.05). This finding indicated that PF and all plantbased ingredients contribute to the C18:2 content of the SPWL [5]. The addition of 3 to 20% PS to the experimental diets increased C18:3 content in the SPWL by 12.40-105 fold and 5.80-49.40 fold, respectively, compared to those fed control and PF-added diets (p > 0.05). Notably, adding PF to the GSPT diet did not improve C18:3 content in SPWL compared to feeding GSPT alone (p > 0.05), while 3% PS formulation did not significantly increase C18:3 content in SPWL compared to rearing on the PI-added diet (p > 0.05). The SPWL fed PF, 7% PS, and control diets had the highest C20:4 content (arachidonic acid, ARA), while those raised on 7% PS-added diets had the highest C20:5 content (EPA), with no significant difference between the other groups (p > 0.05). SPWL fed a PI-formulated diet had the highest C22:6 (DHA) content, while SPWL reared on other diets had no significant difference in DHA concentration (p < 0.05). When PS inclusion levels in the experimental diets were increased, there was a reduction in total SFA with an increment in PUFA in SPWL, resulting in an increase in the ratio of PUFA to SFA (p < 0.05).

FA Metabolic Indices and ∆6 Desaturase (fads2) Gene Expression
rearing SPWL with PS-formulated diets (Table 6), which was well related to the low fads2 gene expression (Figure 2b). Despite the fact that SPWL can enzymatically synthesize n-3 longer chain PUFA from ALA, as evidenced by the presence of estimated 5 + 6-desaturase activity and fads2 gene expression (Figures 1b and 2a), the extent of all n-3 longer chain PUFA, particularly EPA, was low in this study (Table 6). Due to a lack of desaturase/elongase activity, the SPWL may only synthesize EPA de novo from ALA in small amounts through specific tissues. This result was consistent with the findings of Mattioli et al. [41], who found that Tenebrio molitor larvae were nearly unable to produce long-chain PUFA when fed high levels of ALA-containing diets. The larvae simply bioaccumulated ALA and converted roughly two-thirds of it to SFA, most likely lauric acid or myristic acid [36]. The current study first addressed the relationship between n-3 long-chain PUFA extent, desaturation/elongation enzyme systems, and fads2 gene expression as altered by high ALA-containing diet feeding in SPWL, which still has some gaps to fill in order to fully understand their lipid metabolism. Values are given as mean ± standard deviation from triplicate determinations. Different letters in the same attribute indicate significant differences (p < 0.05). Experimental diet nomenclature: C: control, PF: diet supplemented with pig feed, P0-P20: diets contained 0-20% perilla seed, respectively. DI = desaturase index, EI = elongase index, TI = thioesterase index. Table 7 shows the amino acid profile of the SPWL as influenced by feeding composition. The highest EAA in the SPWL was lysine, which was increased after adding PI and PS into the diets (p < 0.05). There was a non-significant difference in lysine content between SPWL reared on PI and different PS contents in diets, which was greater than that raised on the control and PF added diets by 1.09-1.34 fold and 1.71-2.11 fold, respectively. The second most prevalent EAA in the SPWL was leucine, which was followed by valine, isoleucine, histidine, threonine, phenylalanine, methionine, and tryptophan ( Table 7). Fortification of PI and PI with PS into the experimental diets increased all EAA in the resulting SPWL except threonine, indicating that mixed plant-based ingredients and PS were a rich source of EAA. It should be noted that the high concentration of PS inclusion in the diets, ranging from 15-20%, resulted in a reduction in the individual EAA counterpart in SPWL. This could be due to an imbalance in insect metabolism as a result of feeding high lipid-containing PSadded diets. SPWL contained tryptophan in concentrations ranging from 0.1 to 0.2 mg/g, a typical limited amino acid in insects [42,43]. Supplemental diets increased tryptophan levels in SPWL by approximately twofold when compared to control diets (p < 0.05). The total EAA of the SPWL ranged from 77.8 to 121.7 mg/g ( Table 7). The highest total EAA content was found in SPWL raised on 3-7% PS added diets (p < 0.05). The total EAA content of SPWL was similar to soybean, which was superior to other vegetable proteins but inferior to livestock proteins [44]. However, the EAA content of the SPWL (except when raised on PF diet) was greater than that of Rhynchophorus bilineatus (40 mg/g) and Cladomorphus phyllinum (83.2 mg/g), as reported by Köhler et al. [45] and Botton et al. [42]. It should be noted that SPWL raised on PI and PI with PS had higher total EAA content than the control and PF-added diets (p < 0.05), suggesting that PI and PS are rich sources of EAA [5]. most prevalent non-EAA in this insect ( Table 7). As a result, this diet formula containing 3-7% PS yielded the highest total AA content of SPWL, ranging from 241.1-254.6 mg/g, and could potentially be used to improve AA content in this insect. All EAA presented in SPWL met the FAO recommended level [47], with the exception of methionine, tryptophan, and phenylalanine, which are the limiting EAA with the lowest EAA score in SPWL (Table 8). In comparison to the reference protein, this result indicated that SPWL was a promising source of high-quality protein. The SPWL fed a 7% PS-diet had relatively high valine, leucine, isoleucine, histidine, and methionine + cysteine scores than that raised on a commercial PF-added diet (p < 0.05). However, there was no difference in threonine, lysine, phenylalanine, or total EEA score among SPWL raised on all experimental diets (p > 0.05). Interestingly, all SPWL had a very high lysine score of 4.15-5.28 fold when compared to the reference protein, which was independent of the diet formulations (p < 0.05). Our findings indicated that adding PI and PS to a regular diet could improve the protein quality of SPWL, which was 1.56-1.81 fold higher than the FAO reference protein [47]. As indicated by protein quality, rearing SPWL with a proper plant-based ingredient formulation could completely replace commercial animal feed. This strategy has the potential to boost commercial competitiveness in the Muslim market while also ensuring long-term sustainability. Values are given as mean ± standard deviation from triplicate determinations. Different letters in the same row indicate significant differences (p < 0.05). Experimental diet nomenclature: C: control, PF: diet supplemented with pig feed, P0-P20: diets contained 0-20% perilla seed, respectively. * Numbers in the parentheses represent the score ratio for each EAA in the SPWL fed with different diets and in the reference protein (EAA score). # FAO [47].

Conclusions
The 7% PS supplementation in the mixed PI/GSPT diet resulted in positive growth performance and nutritive quality of SPWL. The enrichment diet with 7-20% PS effectively increased ALA content, resulting in a desirable n-6/n-3 ratio in the subsequent SPWL. Dietary PS formulation raised the ∆9-DI (18), total ∆9-DI, and ∆5 + ∆6 DI activities in SPWL, which was linked to an increase in FA unsaturation and PUFAs/SFAs ratio while reducing the n-6/n-3 ratio, AI, and TI of SPWL lipids. The higher dietary PS content formulated in the diets reduced the fads2 gene expression of the resulting SPWL. When compared to the reference protein reported by FAO, the SPWL raised on 7% PS-added diet had superior EAA with high BV. Overall, SPWL fed 7% PS-formulated into a mixed PI/GSPT diet demonstrated superior nutritional quality, particularly lipids and proteins, as well as proper growth performance. This diet formulation rendered a well-balanced nutritional composition in SPWL which is satisfactory for human requirements. As a result, the proper dietary PS formulation resulted in SPWL with an excellent source of nutrients for food security and sustainability, as well as a less expensive food source that is easily accessible and affordable to locals. However, investigations should pay attention to other concerns including pesticide residues, polycyclic aromatic hydrocarbon (PAH) contamination, biogenic amine production, and microbiological quality in order to comply with safety requirements.