Physiological Ecology Environmental Entomology, XX(X), 2017, 1–9 doi: 10.1093/ee/nvx154 Research Assessment of Vegetable and Fruit Substrates as Potential Rearing Media for Hermetia illucens (Diptera: Stratiomyidae) Larvae Costanza Jucker,1 Daniela Erba, Maria Giovanna Leonardi, Daniela Lupi, and Sara Savoldelli Department of Food, Environmental and Nutritional Science (DeFENS), University of Milan, via G. Celoria 2–20133 Milano (MI), Italy 1 Corresponding author, e-mail: firstname.lastname@example.org All authors equally contributed to this work. Subject Editor: Melody Keena Received 10 March 2017; Editorial decision 31 August 2017 Abstract Hermetia illucens (L.) (Diptera: Stratiomyidae) is able to consume a wide range of organic matter and is of particular interest for waste management.The nutritional value of preimaginal stages, in particular the protein content, makes this species a valid candidate for use as feed for other organisms. Vegetables and fruits are promising rearing substrates for insects produced for this purpose according to the EU regulation. In order to examine the effects of diets on insect performance and chemical composition, larvae were reared on the following substrates: 1) fruit (apple, pear, and orange); 2) vegetable (lettuce, green beans, and cabbage); and 3) mixed fruits and vegetables. High percentages of survival were observed on all diets, but there were differences among weights of larvae, pupae, and adults, with weights of larvae reared on mixed fruits and vegetables lower than on other diets. Pupae reared on the mixed diet were heaviest, and also morphometric measurements of adults were highest. Larvae reared on fruit diets had the highest fat content, comprising mostly saturated fatty acids; the highest content of essential n-3 fatty acids was found in vegetable reared larvae and that of n-6 in mixed reared larvae. Larvae reared on the mixed diet had the highest protein content. Calcium contents were high and moderate amounts of iron and zinc were found. H. illucens showed the capability to develop on vegetable and fruits diets displaying different nutrient profiles and biological performances. The best-performing rearing strategy should vary in relation to the final use of H. illucens. Key words: nutritional composition, black soldier fly, preimaginal growth, biodegradation agent, protein content Hermetia illucens (L.) (Diptera: Stratiomyidae), also known as black soldier fly, is native to tropical, subtropical, and warm temperate areas of America, but is now widespread in tropical and temperate region of the world (Sheppard et al. 1994, Diener et al. 2011). Recent interest in this species is due to the fact that H. illucens is able to decompose large amounts of organic wastes and by-products contributing to the environmental problems connected with manure and other organic type of wastes (Zhou et al. 2013, Nguyen et al. 2015). Additionally, preimaginal stages of H. illucens have been shown to be a valuable alternative protein source for food and feed, in particular for farm animals (e.g., fish, poultry, and swine) (Belluco et al. 2013, Sánchez-Muros et al. 2014, van Huis 2013, Laureati et al. 2016). Chemical analysis showed that the dry matter (DM) of the preimaginal instars is high in protein (41–44%) and fat (15–49%) (Barroso et al. 2014, Makkar et al. 2014, Henry et al. 2015, Surendra et al. 2016). Saprophagous H. illucens larvae are able to develop on a wide range of organic decaying materials, ranging from animal waste and municipal garbage to fruits and vegetables, plant material, and market waste (St-Hilaire et al. 2007; Myers et al. 2008; Diener et al. 2009, 2011; Kalová and Borkovcová 2013; Cičková et al. 2014). They are able to consume twice their weight per day, reducing the volume of organic matter up to 42–75% (Sheppard et al. 1994, Newton et al. 2005, Diener et al. 2011). Larvae appear to be able to inactivate Escherichia coli and Salmonella spp. present in the substrates in which they live and feed in (Erickson et al. 2004, Liu et al. 2008). In addition, adults of H. illucens are not considered pests as they largely reside on vegetation, do not damage agricultural crops, do not cause illness or unproductivity in cattle, and do not vector human diseases or cause nuisance with baites (Cičková et al. 2014). Recently, H. illucens haemolymph has been considered a prospective component of artificial media for entomophagous insects rearing (Dindo et al. 2016). Diet quality can greatly affect development and adult performance in many arthropod species, including H. illucens (Kaspi et al. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: email@example.com. 1 Environmental Entomology, 2017, Vol. XX, No. X 2 2002, Tomberlin et al. 2002, Boggs and Freeman 2005, Hanks et al. 2005, Nguyen et al. 2013, Tschirner and Simon 2015, Favaro et al. 2017). Moreover, the final chemical composition of insects is not only species-dependent, but also variable with regards to life stages, rearing condition, and diet (Ujvari et al. 2009, Oonincx et al. 2011, Tschirner and Simon 2015). In particular, diet can largely influence the lipid content of insects (Stanley-Samuelson and Dadd 1983, St-Hilaire et al. 2007). In contrast, the crude protein (CP) content seems to be mainly a species-determined trait, although even within the same species slight differences have been found (Barroso et al. 2014). There is growing interest in vegetable substrates usable for H. illucens rearing (Hem et al. 2008, Parra Paz et al. 2015). According to the EFSA risk profile related to production and consumption of insects as food and feed (EFSA Scientific Committee 2015), vegetable wastes seem to have the greatest potential to be used as feed for insect production within the EU. In fact, as insects are considered ‘farmed animals’ according to Regulation (EC) No. 1069/20097 and the use or rearing substrates such as manure, catering waste, or former foodstuffs containing meat and fish is not allowed. It is also well known that fruit and vegetable commodities represent high proportion of waste and loss, especially in industrialized regions, mostly due to postharvest grading caused by quality standards set by retailers (FAO 2011). In addition, several byproducts coming from food industries processing food and vegetables (juices, ready-to-eat products, etc.) could be used as insect-rearing substrates. Despite the high interest, little research has been done on the life-traits, the development, the performance, or the chemical composition of H. illucens reared on organic matters of vegetal origin (Kalová and Borkovcová 2013, Nguyen et al. 2015, Parra Paz et al. 2015). Thus, the primary aim of this study was to evaluate the influence of different vegetable and fruit diets on life traits of H. illucens and on the variability of nutritional content of prepupae. The impact of the following three diets was assessed: 1) fruits; 2) vegetables; 3) mix of fruits and vegetables. The diets chosen for this study are representatives of common foods sold on the market year-round, and from which food waste is commonly generated. Larval development time, survival, and adult morphometric characteristics and performances were quantified. Finally, chemical composition of H. illucens prepupae was analyzed. Materials and Methods and orange (Citrus sinensis (L.)); 2) vegetable (V): lettuce (Lactuca sativa L.), string green beans (Phaseolus vulgaris L.), and cabbage (Brassica oleracea var. capitata L.); 3) mixed fruits and vegetables (FV): a mixture 1:1 of the diet F and V. Fruits and vegetables were purchased from a local market and used fresh. Each ingredient was finely chopped (5 × 5 × 5 mm) and was present in equal proportions in the specific diet (e.g., 1/3 apple, 1/3 pear, and 1/3 orange constituted the fruit diet). The chemical composition of each diet was extrapolated from a food composition database (http://nut.entecra. it) (Table 1). To synchronize egg hatching, cardboard strips for oviposition were inserted in stock-culture cages and removed after 1d. Six egg clutches for each diet were removed from the cardboard with a fine brush and positioned directly on the experimental diets. Finally, each container (500 ml) was placed into a climate chamber [T 25° ± 0.5°C, RH 60 ± 0.5%, photoperiod 12:12 (L:D)]. Once emerged, 9-d-old larvae were counted to obtain the specimens for experimentation. Three replicates of 200 larvae were used for each rearing diet. Larvae were placed in a plastic container (500 ml) with a perforated lid lined with mesh netting, for a total of nine containers. Food was provided ad libitum. All containers were stored in the climate chamber previously described. A sample of 10 larvae from each experimental container was collected daily and weighed on an analytical balance (SartoriusCP64, Germany). After weighing, larvae were returned to their respective containers. As in Nguyen et al. (2015), the mean time to reach prepupal stage (days ± SE) and the final mean larval weight (g ± SE) were estimated when 40% of the larvae reached the prepupal stage, indicated by the change in their color from creamy white to black. Prepupae were counted daily and transferred to labeled containers, covered with breathable gauze, and provided wood shavings to facilitate pupation and adult emergence. Twenty pupae from each container were weighed with the analytical balance and their length measured using an electronic caliper (Stainless hardened; 0–150/0.02 mm). Adults were counted, sexed, and then transferred into a rearing cage (60 × 44 × 35 cm) (one cage for each experimental diet) in order to determine fecundity assessed by oviposition success. The aforementioned cardboard oviposition substrates were provided to adults. Cardboard strips were checked every 2 d for egg masses. Ten egg clutches for each replicate were weighed and the eggs counted. To compare adult weights, 60 adults from each experiment were weighed following desiccation at 105°C for 48 h. H. illucens Stock Culture A laboratory stock culture of H. illucens was founded from field-collected larvae in Lombardy (northern Italy) (45°19ʹ54ʺN; 9°05ʹ58ʺE), retrieved from inside a compost container. Larvae were reared in a climate chamber [T 25° ± 0.5°C, RH 60 ± 0.5%, photoperiod 12:12 (L:D)] at the University of Milan on hen feed mixed with water (500 g/800 ml water) until pupation. Pupae were then transferred into a container with wood shaving to prevent desiccation (Holmes et al. 2013). Once emerged, adults were placed in a cage (100 × 80 × 60 cm) at room temperature of 25 ± 0.5°C and 25 ± 0.5% RH where they could mate. Oviposition sites consisted of strips of corrugated cardboard (2 mm in width; three flute openings per cm) (Booth and Sheppard 1984) and were positioned on a plastic container (20 × 10 × 5 cm) filled with hen diet mixed with water as attractant for females. Chemical Analyses For chemical analyses of prepupae, six synchronized egg clutches from the H. illucens stock culture were transferred to each experimental diets (F, V, FV) in a rearing containers (2000 ml). Larvae were fed ad libitum as previously described and maintained in the climate room, at the aforementioned conditions. Table 1. Chemical composition of rearing substrates (F, V, and FV)a (g/100 g FW) F V FV Moisture Protein Lipid Carbohydrate Fiber 86.6 0.4 0.1 8.9 2.7 92.3 2 0.2 2.4 2.3 89.5 1.2 0.2 5.6 2.5 Experimental Trials Three organic diets were tested: 1) fruit (F): apple (Malus domestica Borkh. var. golden delicious), pear (Pyrus communis L. var. Kaiser), a Data were calculated on the basis of food composition database (http:// nut.entecra.it), and expressed on FW. Environmental Entomology, 2017, Vol. XX, No. X When larvae reached the prepupal stage, they were collected and placed in a ceramic mortar, flash-frozen with liquid nitrogen, and ground to powder with a pestle. Aliquots of the homogenized samples were used for moisture and ash determination (AOAC 950.46 and 920.153). The remaining samples were lyophilized via freezedrier laboratory equipment (Edwards Freeze Drier Pirani 1001, IVT, Milano, Italy), and used for CP and lipid analyses in accordance with AOAC standard methods (method 981.10 and 960.39); results were then reported on fresh weight (FW). Nitrogen-free extract contents were calculated by difference (Barroso et al. 2014). For mineral analyses, lyophilized samples were weighed and dry-ashed (550°C, one night; AACC method 40–70.01, 1999) in a muffle furnace (Cavallo, Italy). Gray ashes were treated with high purity hydrogen peroxide (H2O2 30%, Suprapur Merck, Germany), obtaining white ashes that were dissolved with acid solution (2 ml HCl 30%, Suprapur Merck, Germany) and diluted with distilled water in volumetric flasks. Mineral concentrations (Zn, Fe, Cu, and Ca) were determined by atomic absorption spectrometry (AAnalyst 800 Perkin Elmer, United States) in relation to external standard curves, following the instrumental condition recommended for each mineral (Ca 422.7 nm, Cu 324.8 nm, Zn 213.9 nm, and Fe 248.3 nm). Measurement of mineral content was checked using certified values of reference material (NCS ZC 85006). All analyses were performed in triplicate and the results are expressed as means ± sd on a DM basis (mg/100 g DM). Finally, the fatty acid composition of the lipid fraction of the lyophilized samples was analyzed by chloroform/methanol extraction using a 2:1 (v/v) ratio mixture according to the method of Folch et al. (1957). Methyl esters were prepared from the total lipids following the method of Ackman (1986). Fatty acid methyl esters were analyzed by a Varian 3400 CX gas liquid chromatograph equipped with a OMEGAWAX AX 320 column (Supelco, Bellefonte, PA) and a flame ionization detector. Injector and detector temperatures were 250 and 260°C, respectively. The initial oven temperature was 140°C and was increased by 2°C/min to 200°C and held at this temperature for 25 min; hydrogen at a flow rate of 2.0 ml/min was used as carrier gas. A standard fatty acid methyl ester mixture (Omegawax Column test Mix 4-8476 Supelco) was run and retention times were used in identifying the sample peaks; nitrogen at a flow rate of 2.0 ml/min was used as carrier gas. A response factor was calculated to correct the GC response of each fatty acid ester to bring them to a common baseline. The methyl ester of pentadecanoic acid (C15:0) was used as an internal standard. Fatty acid levels were estimated on the basis of peak areas of the standards. Fig. 1. Larval growth of H. illucens over time on F, V, and FV diets. 3 Statistical Analyses Recorded data were analyzed using SPSS® Statistic (Version 23 for Windows, SPSS Inc. Chicago, IL). All percentage values were transformed to the arcsin of the square root of each value before statistical analysis but are presented as untransformed means. To test significant differences, prior to analyses, all data were examined with Levene’s test for homogeneity of variance and with Shapiro Wilk test for normal distribution. Log transformation was applied to make data conform to normality, when necessary. A one-way analysis of variance was used to compare data recorded on time of larval development, final larval and pupal weight, adult weight and length, survival of different stages, and sex ratio. Where significant differences occurred, Tukey–Kramer’s Honestly Significant Difference multiple comparisons test was applied for mean separation (P < 0.05) between tested diets. The same method was applied to analyze differences in egg clutches, including weight and number of eggs, and chemical composition of H. illucens prepupae. Pearson’s Correlation analysis and regression was applied to determine the relationship between clutch egg weight and number of eggs for each treatment. The two-tailed Student’s t-test was used to determine significant differences between male and female development time, weight, and length (P < 0.05). Results Preimaginal Stages The duration of the larval period (measured from egg eclosion to the 40% of the prepupae presence) was significantly impacted by the diets (F = 200.33; df = 2, 6; P < 0.001) (Fig. 1; Table 2). Larvae took more days to reach the prepupal stage when fed F and V diets, while on the FV diet larvae took 36.67 ± 0.33 d to reach the wandering phase. As represented in Fig. 1, larvae gained weight most rapidly on the F diet during the first 24 d, followed by FV and V. Although individuals on the FV diet initially exhibited lower rates of growth than those on F, their rates of growth increased over time and eventually exceeded the rates for those on the F diet. On V diet, the gap was 5 d, and the delay was not compensated by a rapidly increasing larval weight. The final larval weight differed among diets (Table 2) (F = 5.53; df = 2, 6; P < 0.05). Larvae reared on V diet were the heaviest, significantly different from the ones fed with the mix (FV diet); larvae reared on the F diet were intermediate in weight. Also, the mean pupal weights and lengths differed significantly among Environmental Entomology, 2017, Vol. XX, No. X 4 Table 2. Effect of diet on mean larval duration, mean larval and pupal weights, and mean pupal length (mean ± SE) Diet Larval development (d)a n = 3 Final larval weight (g) n = 3 Pupal weight (g)a n = 60 Pupal length (mm)a n = 60 F V FV 52.00 ± 1.00a 48.33 ± 0.33b 36.67 ± 0.33c 0.174 ± 0.009ab 0.184 ± 0.005b 0.154 ± 0.003a 0.081 ± 0.003a 0.084 ± 0.004ab 0.094 ± 0.002b 15.79 ± 0.16b 16.00 ± 0.18b 14.80 ± 0.25a Significant differences among means in a column are indicated by different lower case letters (Tukey’s test, P < 0.05). Values in column not followed by letters are not significantly different across treatments. a Data were log-transformed to meet normality assumptions. Table 3. Effect of diet on percentage of larval survival, adult emergence, and total survival of H. illucens (mean ± SE) Diet Larval survival to prepupae Adult emergence from prepupae Total survival (young larvae-adults) F V FV 96.44 ± 2.62 88.67 ± 6.12 93.67 ± 4.10 90.12 ± 0.61c 52.11 ± 2.14a 63.55 ± 3.76b 86.89 ± 1.94b 46.00 ± 2.00a 59.83 ± 6.07a Significant differences among means in a column are indicated by different lower case letters (Tukey’s test, P < 0.05). Values in column not followed by letters are not significantly different across treatments. Percentages data were transformed to the arcsin of the square root before analysis but are presented as untransformed means. the three treatments (weight: F = 3.43; df = 2, 6; P < 0.05; length: F = 12.17; df = 2, 6; P < 0.001) (Table 2). In particular, pupae reared on the F diet were significantly lighter than the ones reared on the FV diet; pupae reared on the V diet were in the middle. Pupae from the FV diet were the smallest, significantly different from the others. Percentage of larval survival from newly emerged larvae to pupae of H. illucens on the different diets did not differ significantly among diet treatments (F = 0.39; df = 2, 6; P = 0.73), with percentages from 88.67 to 96.44%: most larvae pupated (Table 3). Higher mortality occurred during the pupal stage: differences in the adult emergence were observed among diets (F = 75.90; df = 2, 6; P < 0.001), with greater survivorship for insects fed F diet than for either FV or V diets. With regard to the overall survival from young larva (handled larva) to adult, a significantly higher percentage survived on F diet than on the other treatments (F = 32.13; df = 2, 6; P < 0.001). Adults Total development time from egg hatch to 50% adult emergence was different among sexes and diets (Table 4). Males needed fewer days to complete their preimaginal development than females on all diets (F: t = 6.21; df = 390; P < 0.001; V: t = 3.93; df = 274; P < 0.001; FV: t = 5.60; df = 356; P < 0.001). Among diets, males took a significantly longer time to emerge when reared on the F and V diets than when reared on FV (F = 14.71; df = 2, 6; P < 0.05). Females developed more slowly on F diet, while no differences were observed between V and FV diets (F = 27.46; df = 2, 6; P < 0.01). Mean adult emergence period, from the first to the last day of emergence, was affected by the diet (F = 47.20; df = 2, 6; P < 0.001), and was shorter in V and FV, while on F diet was twofold longer (Fig. 2; Table 4). Adults started to emerge from the F diet, followed by FV and V. Fifty percent of adult emergence in the diets was reached after 33 d in F, 20 in V, and 16 in FV, counted from the presence of the first adult. Mean sex ratio (males/females) ranged from 0.56 to 0.99, with significant differences among diets (F = 10.46; df = 2, 6; P < 0.05) (Table 4). In particular, V diet statistically differed from the others with a higher number of females emerged. Adult weight significantly differed among diets, both for males (F = 15.93; df = 2, 177; P < 0.001) and females (F = 16.69; df = 2, 177; P < 0.001) (Table 4). Adults that emerged from the FV diet weighed significantly more than the others, in both sexes. In all the diets tested, females weighed significantly more than males (F: t = 14.71; df = 118; P < 0.001; V: t = 14.45; df = 118; P < 0.001; FV: t = 13.85; df = 118; P < 0.001). Length of males and females was affected by the treatments (males: F = 19.61; df = 2, 177; P < 0.001; females: F = 16.50; df = 2, 177; P < 0.001): in particular, the adults of both sexes reared on the FV diet as juveniles were statistically bigger than the ones reared on the other diets. Females reared on the three diets were always significantly bigger than males (F: t = 6.48; df = 118; P < 0.001; V: t = 2.94; df = 118; P < 0.05; FV: t = 4.68; df = 118; P < 0.001). Egg Masses No statistical differences were observed for mean egg clutch weight (F = 0.54; df = 2, 27; P = 0.589), number of eggs per flute (F = 0.41; df = 2, 27; P = 0.665) or egg weight (F = 3.18; df = 2, 27; P = 0.057) among the tested diets (Table 5). Number of eggs for each clutch ranged from a minimum of 125 in V to a maximum of 744 in V. The mean number of eggs per flute was 300.90 ± 56.92 for adults reared on V, 318.20 ± 37.58 on F, and 365.20 ± 57.97 on FV. The mean egg weight was similar for all tested diets. Pearson’s Correlation between clutch weight and egg number and the regression analysis showed a positive trend between the two parameters. Composition Analyses The chemical composition of prepupae was affected by the experimental diet: the main differences were found for moisture, fat, and CP (Fig. 3). The lowest level of moisture was found in F specimens, followed by FV and V specimens (F = 8129.3; df = 2, 6; P < 0001). In contrast, fat content displayed the opposite trend with F significantly higher than FV and V samples (F = 7811.7; df = 2, 6; P < 0.001). CP content was highest in the FV group (17.6 g/100 g FW), while the other two groups showed lower levels of about 30%, although significantly different from each other (13.2 and 11.7% for V and F, respectively; F = 2530.3; df = 2, 6; P < 0.001). H. illucens ash contents were similar regardless of diet, and comprised between 3.0 and 3.4% on FW. With regard to minerals (Table 6), the highest level of Fe was measured in FV specimens, while the specimens containing Development time was calculated from egg hatching to 50% of the adult emergence. Means within males and females within the same treatment in the same row followed by different capital letter differ significantly (t-test, P < 0.05). Means within column followed by different lower case letters differ significantly (Tukey’s test, P < 0.05). Values not followed by letters are not significantly different across treatments. 63.33 ± 3.18b 32.67 ± 2.40a 36.67 ± 1.33a 88.67 ± 1.67bB 80.67 ± 0.33aB 77.00 ± 1.00aB 80.67 ± 2.03bA 79.00 ± 1.00bA 71.00 ± 0.58aA F V FV a 14.62 ± 0.13aB 14.34 ± 0.13aB 15.38 ± 0.13bB 13.47 ± 0.12aA 13.83 ± 0.11aA 14.51 ± 0.13bA 0.016 ± 0.001aA 0.017 ± 0.001bA 0.020 ± 0.001cA 0.99 ± 0.05b 0.56 ± 0.08a 0.86 ± 0.08b (53–119) (61–90) (57–95) 0.021 ± 0.001aB 0.023 ± 0.001bB 0.026 ± 0.001cB Females Males Males First-last day Interval Females Males Sex-ratio Adult emergence period (d) Development time from egg to adult (d)a Diet Table 4. Total development time of H. illucens reared on the three diets and parameters of adults emerged (mean ± SE) Adult weight (g) n = 60 Females Adult length (mm) n = 60 Environmental Entomology, 2017, Vol. XX, No. X 5 Fig. 2. Trend of male and female emergence from the different diets (percentage of emergence). Days are calculated from egg hatching. (a) F diet, (b) V diet, (c) FV diet. the most Ca were V; both those samples displayed similar levels of Zn. Levels of Cu in H. illucens were not affected by the dietary treatments. The experimental diets also influenced fatty acid composition (Table 7): in general, H. illucens prepupae showed consistent percentages of saturated fatty acids (SFA), especially those of medium and long chain (lauric, miristic, and palmitic acids), and moderate contents of oleic acid (18:1 n-9, monounsaturated fatty acids – MUFA), linoleic (18:2 n-6, polyunsaturated fatty acids – PUFA), and α-linolenic acids (18:3 n-3, PUFA). Moreover, fruit diet (F) resulted in higher levels of SFA and lower PUFA than the other two samples, while vegetable diet (V) was associated with high levels of MUFA. Taking into consideration the PUFA/SFA percentage ratio, the best results were for larvae reared on FV diet: a higher ratio is generally considered more favorable for human health (Skeaff et al. 2009) and a ratio of 1 is present in commercial fish meal (Barroso et al. 2014). Notwithstanding, the high content of SFA is worthy of particular attention. Discussion This study has shown that H. illucens successfully completed development when reared on the three tested diets, indicating that all diets were suitable. However, some life traits of the species Environmental Entomology, 2017, Vol. XX, No. X 6 Table 5. Effect of diet on egg production for H. illucens emerged from the three diets: clutch weight (mg), egg number (n), and egg weight (mg) (mean ± SE) Diet Clutch weight (mg) Egg number Egg weight (mg) Correlation Clutch weight/egg number (P < 0.05) Regression F = 109.45; df = 1, 8; P < 0.001; R2 = 0.92 24.29x + 69.78 F = 94.50; df = 1, 8; P < 0.001; R2 = 0.92 27.45x + 86.52 F = 49.22; df = 1, 8 P < 0.001; R2 = 0.84 34.56x + 40.71 (n min–max) F 10.27 ± 1.50 318.20 ± 37.58 (207–550) 0.031 ± 0.002 r = 0.97; df = 10; P < 0.001 V 7.81 ± 1.99 300.90 ± 56.92 (125–744) 0.025 ± 0.002 r = 0.96; df = 10; P < 0.001 FV 9.39 ± 1.56 365.20 ± 57.97 (135–605) 0.026 ± 0.002 r = 0.93; df = 10; P < 0.001 Results of correlation between clutch weight and egg number and regression analysis for egg number per clutch (P < 0.05). Fig. 3. Chemical composition of H. illucens prepupae reared on F, V, and FV substrates (data are expressed as g/100 g FW and as means ± sd). NFE, nitrogen-free extract (Barroso et al. 2014). Within the same analysis, data not sharing common letters are significantly different (P < 0.05). were strongly affected by the diets. Larval development time was longer on all diets tested than on diets described by other authors (Nguyen et al. 2013, Zhou et al. 2013). Slower larval development seemed to be related to poor food quality, in particular the low protein content in the diets (Gobbi et al. 2013, Oonincx et al. 2015). Significant differences were observed among dietary treatment: larvae reared on fruit diet, with lowest protein content, had the longest development period. Moreover, as the three tested diets have low lipid content, the energetic demand should be supplied by carbohydrates, mainly provided by fruits. Even if in insects, the amount and the quality of the protein component in the diet seems to be the main factor that influences development time (Friend 1958, House 1961, Oonincx et al. 2015), the faster larval development observed on FV diet could result from a better balance of energy from protein and carbohydrates (Table 1). The diets significantly impacted the final larval weight, but the weights of larvae reared on different diets were similar to weights observed by Tomberlin et al. (2002) and Zhou et al. (2013), and were higher than those obtained by Nguyen et al. (2013) of larvae reared on a mix of fruits and vegetables. This result suggests that H. illucens larvae are able to exploit poor nutritional diets to reach a body mass comparable to that obtained with richer diet, but with a penalty of a prolonged feeding period to reach the prepupal stage. Pupal and adult weights were significantly different among diets, increasing from F to FV to V. The weights were correlated with the weights of the last-instar larvae from which they developed, as holometabolous insects must reach a critical weight to trigger the hormonal cascade that leads to cessation of feeding and metamorphosis (Davidowitz et al. 2003, Nijhout 2003, Stern 2003). Moreover, the nutritional supply strongly affected endocrine events influencing the final body size: therefore, diet quality, in addition to the quantity, may impact weight during different developmental stages (Nijhout 2003). Indeed, lower weights were observed in insects fed on the most unbalanced diet tested. While in all treatments larval survivorship was similar to that reported in other studies of different diets, not of vegetable origin (Tomberlin et al. 2002, Myers et al. 2008, Oonincx et al. 2015), survival through all stages of metamorphosis was strongly affected by the three diets in this study. In particular, low adult emergence from larvae reared on the F and FV diets seemed to be correlated with a diet poor in carbohydrate, the primary energetic source of the diet. Nutrients, supplied by diet, are utilized both for growth and storage, mainly used in post-feeding development (Nijhout 2003). The importance of energetic supply during metamorphic processes was supported by the high percentage of emergence observed in H. illucens fed only on fruits, comparable with data reported by Tomberlin et al. (2002) for wild populations. Successful emergence of adults was correlated with fat content in prepupae. Percentages of emerged adults from FV and V were higher than the ones observed in sub-optimal rearing conditions by Tomberlin et al. (2002). Additionally, adults emerged from FV and V presented shorter and more synchronized emergence periods compared with adults emerged from F, as expected in the presence of a higher protein content (Kaspi et al., 2002). As previously observed (Tomberlin et al. 2002), H. illucens females were always significantly bigger than males. Moreover, vegetable diet significantly affected sex ratio. Several factors including moisture (Fatchurochim et al. 1989) and mineral contents determined the food quality and influenced insect growth and development. A previous study indicated that the moisture level of the larval substrate principally affected the pupal stage (Jucker et al. 2016). Also, Fatchurochim et al. (1989) determined that H. illucens optimally developed in manure with 40–60% moisture, while survivorship was significantly reduced at higher moisture levels. Environmental Entomology, 2017, Vol. XX, No. X 7 Table 6. Mineral contents of H. illucens reared on F, V, and FV substrates (data are expressed as mg/100 g DM, mean ± sd) (mg/100 g DM) Ca Fe Zn Cu F V FV 1421.3 ± 45.2a 4.2 ± 0.2a 2.1 ± 0.4a 0.8 ± 0.1a 2703.7 ± 25.5c 9.1 ± 0.5c 11.1 ± 0.1b 0.9 ± 0.1a 1897.3 ± 82.4b 21.9 ± 0.1b 10.3 ± 1.1b 0.8 ± 0.1a Data in the same row not sharing common letters are significantly different (P < 0.05). Table 7. Fatty acid content (as a percentage of total fatty acids) of H. illucens reared on F, V, and FV substrates (%) 10:0 12:0 14:0 16:0 16:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-3 Total n-3 Total n-6 n-6 / n-3 SFA MUFA PUFA P/S* F V FV 1.1 ± 0.1 68.0 ± 0.1 7.6 ± 0.1 8.3 ± 0.1 3.8 ± 0.1 0.9 ± 0.1 7.1 ± 0.1 2.3 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 2.3 ± 0.1 4.6 86.0 ± 0.1 11.2 ± 0.1 2.8 ± 0.1 0.03 1.5 ± 0.1 25.0 ± 0.1 5.4 ± 0.1 15.4 ± 0.1 10.0 ± 0.1 4.9 ± 0.1 11.8 ± 0.1 7.5 ± 0.1 5.8 ± 0.1 6.8 ± 0.1 8.3 ± 0.1 1.2 56.5 ± 0.1 27.2 ± 0.1 16.2 ± 0.1 0.29 1.0 ± 0.1 41.5 ± 0.6 7.4 ± 0.1 12.8 ± 0.3 1.4 ± 0.1 1.7 ± 0.1 8.7 ± 0.2 21.2 ± 0.4 2.6 ± 0.1 2.8 ± 0.2 20.4 ± 0.6 7.3 65.0 ± 0.6 11.0 ± 0.1 24.1 ± 0.3 0.37 *indicates PUFA/SFA. Although in insects nutritional reserves affect eggs production (Chippindale et al. 1993, Kaspi et al. 2002, Tomberlin et al. 2002), no differences were recorded among dietary treatments in egg number and weight of clutches. However, Tomberlin et al. (2002) highlighted that eggs collected from one flute may not represent a clutch from a single female, as flute size influences the number of deposited eggs, and multiple individuals could deposit eggs in a single flute. In any case, the weight of a single egg was in line with previously published data (Booth and Sheppard 1984, Tomberlin et al. 2002). In contrast, egg number per flute in this study was lower than that observed by Booth and Sheppard (1984) in flutes with the same size, and by Tomberlin et al. (2002) in clutches of individuals reared on more balanced diets and deposited in smaller flutes. This observation suggested that all tested diets did not provide enough nutritional reserves to support optimal egg production. Nutritional analysis revealed that the three dietary treatments clearly affected the chemical composition of H. illucens prepupae. Particularly, the fruit-based diet increased not only the total fat content but also affected the fatty acid composition of prepupae, resulting in a higher percentage of SFA and a lower PUFA/SFA ratio with respect to the other diets. Overall, lipid levels of H. illucens reared on F diet was higher than values observed in the literature (Sheppard et al. 2002, FAO/INFOODS 2013), while the high content of SFA, especially of 12:0, was already found in H. illucens at larval stage, but not at the pupal stage (Barroso et al. 2014). Low percentages of α-linolenic acid, a precursor of the n-3 series, have been found (V>FV>F), while LC-PUFA (long-chain PUFA), like DHA (C22:6 n-6) and EPA (C20:5 n-3), were not present, as it is common in terrestrial insects (Barroso et al. 2014). In accordance with the studies of Beenakkers et al. (1971), the results of this study suggest that the fatty acid profile of insects most likely is affected by diet composition. In particular, fruit diet appears to be the most lipogenic diet, causing high deposition of SFA in H. illucens. The percentage of CP of H. illucens prepupae was similarly influenced by dietary treatment: on average, our results (about 45 g CP/100 g on DM) are consistent with data in the literature (Newton et al. 1977, Sheppard et al. 2002, Arango et al. 2004). H. illucens reared on FV diet contained significantly higher CP than the other two groups on FW bases. However, expressing CP as percentage of DM, the best protein yield was found for the vegetables group (60.1 g/100 g DM). Given that the potential use of insects as feed or food ingredients should be on dry basis, this parameter is noteworthy. H. illucens are characterized by high ash content with respect to other insects: Barroso et al. (2014) reported 9.3% ash in larvae and 19.7% in pupae on DM, and similar percentages were also found by Newton et al. (1977). Our data on H. illucens prepupae ranged from 5.6% in F to 14.3% in V samples on DM; this evidence suggests that ash contents are influenced by the insect developmental stage (larvae < prepupae < pupae) and, in relation to the present results, are also influenced by diet. The ash levels of our samples are indicative of contents of inorganic matter. Calcium levels are usually low in insects: in the review of Rumpold and Schluter (2013) which covered 85 insect species, the median value of Ca content was 73 mg Ca/100 g DM (0.04– 2010.00 mg Ca/100 g DM), and few species surpass 1,000 mg/100 g DM. Few studies in the literature have evaluated the mineral profile of H. illucens (Newton et al. 1977, Arango et al. 2004); the recent review of Makkar et al. (2014) reports that Ca content in H. illucens larvae ranged between 5.0 and 8.6 g/100 g DM. In this study, Ca levels in H. illucens prepupae, comprised between 1.4 and 2.7 g/100 g DM (F and V samples, respectively), were lower: this result could be due to different insect developmental stages, as suggested earlier, or to the different rearing diets. For instance, the Ca contents reported by Makkar et al. (2014) were from insects reared on farm animal manure, thus the present results confirm that the nutrient composition of rearing diets plays a role in determining the chemical composition of H. illucens, as already proposed (Ramos-Elorduy et al. 2002, Tschirner and Simon 2015), making comparison among studies difficult. Finally, the high accumulation of one mineral in H. illucens does not necessarily imply an overall better mineral profile: in our study, V sample displayed the highest Ca content, but FV had the highest Fe content, while the contents of Zn were similar in both groups. This study is among the few reporting information about H. illucens reared on a plant matter diet, comparing fruits and vegetables. Our research provides additional information on the life history and on the chemical composition of H. illucens reared on the three diets considered. Nutritional values of preimaginal stages confirmed H. illucens as a species of interest for use as feed for other organisms. Overall, the results suggest, once again, that diet can influence the development and the final nutrient composition of this species. The selection of a specific diet in order to obtain specimens with a particular nutrient profile seems feasible, but the best-performing strategy may vary in relation to the desired objectives. Up to now, few studies have been conducted on the potential risk associated with the use of insects as a source of protein for animal feed, in particular the presence and accumulation of pesticides residues, heavy metals, mycotoxins, and various pathogens (Belluco et al. 2013, van der Spiegel et al. 2013, Charlton et al. 2015). Further studies are necessary to assess the safety of H. illucens prepupae for 8 feed use, reared on organic diets, and in particular on vegetable and fruit wastes. Additionally, the efficacy of biodegradation of organic matter by H. illucens should be considered as vegetables represent a high volume of low-quality waste that can be converted into substrates rich in proteins, lipids, and minerals. Acknowledgments We thank Dr Marco Palamara Mesiano for his help in the laboratory and Dr Alessio Scarafoni for first larval supply. We are grateful to Ruby Elisabeth Harrison (University of Georgia) for her language revision. 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