Abstract
Feeding costs of farmed insects may be reduced by applying alternative nitrogen sources such as urea that can partly substitute true proteins. The aim of this study was to examine the effects of different nitrogen sources on body weight (BW) and survival rate (SR) of the Jamaican field cricket (JFC, Gryllus assimilis), the house cricket (HC, Acheta domesticus), yellow mealworm larvae (YM, Tenebrio molitor) and superworm larvae (SW, Zophobas morio). Crickets were either housed individually or in groups, and larvae were group-housed. Six isonitrogenous feeds composed of 3.52% nitrogen were designed for all four insect species using four independent replicates with micellar casein: urea proportions of 100–0%, 75–25%, 50–50%, 25–75%, 0–100% and 100% extracted soybean meal. All selected insect species were able to utilise urea. However, urea as the only nitrogen source resulted in low final BW. In the HC, the JFC, and the YM on nitrogen basis urea can replace 25% of micellar casein without having any negative effects on BW and SR in comparison to the 100% micellar casein group. In the SW, a 25% urea level did not have a significant effect on final BW, but SR decreased significantly.
Introduction
As the global population rises, the consumption of animal products is expected to increase by about 60–70% by the year 2050 (Wang et al., 2010; Makkar et al., 2014; Tilman and Clark, 2014). Insects are promising and sustainable alternative protein sources as food and feed (van Huis et al., 2013; Ribeiro et al., 2018). Insects also have the advantage of decreased impact on the environment compared to traditional livestock. Insect farming produces much less greenhouse gases and requires less land, water and feed (Oonincx and de Boer, 2012). Insects also have much lower energy requirements than mammals, while their feed conversion ratio (FCR) is like that of the monogastric farm animals (1.5–2.5 g g−1), unless their feed is very low in nutrients (Oonincx and de Boer, 2012; Melis et al., 2019; Rumbos et al., 2021). Commercial production of several insect species, including the house cricket (HC) (Acheta domesticus), the Jamaican field cricket (JFC) (Gryllus assimilis), the yellow mealworm (YM) (Tenebrio molitor) and the superworm (SW) (Zophobas morio) has already been developed (Cortes Ortiz et al., 2016; Ribeiro et al., 2018).
In the European Union, feeding legislation for farmed insects is like that of the farm animals (European Parliament and Council, 2009, 1069/2009). Insects, especially YM larvae can utilise waste and by-products low in nutrients (Cortes Ortiz et al., 2016; Morales-Ramos et al., 2020a). However, these diets tend to result in lower survival rate (SR), body weight (BW) and protein content (Oonincx et al., 2015; Bawa et al., 2020; Harsányi et al., 2020). Protein is a key macronutrient; the appropriate protein content of the feed leads to higher reproduction rate and better growth, while diets with lower protein content lead to delayed development time and decreased weight gain (Joern and Behmer, 1997; Patton, 1967). The protein content of mealworm feeds falls between 2 and 32%, but the optimal level is around 20–24% (Ramos-Elorduy et al., 2002; Morales-Ramos et al., 2020b; Kröncke et al., 2022). A protein-supplemented diet lowers the development time and improves the fecundity of YM (Morales-Ramos et al., 2013). Based on previous studies, crickets have much higher protein requirements than YM or SW larvae (Patton 1978; McCluney and Date, 2008; Lundy and Parrella, 2015; Oonincx et al., 2015; Dobermann et al., 2019). The optimum protein content in the diet of crickets should be 20–30% (Patton, 1967; Orinda et al., 2017). Feeding costs of farmed insects may be reduced by applying alternative nitrogen sources such as urea that can partly substitute true proteins. Diets of herbivorous insects (feeding on termites, cockroaches, leafhoppers, etc.) are often low in nitrogen. Symbiotic microorganisms such as Recilia dorsalis, Rhizopus oligosporus, Pseudomonas fluorescens, Serratia proteamaculans and Rahnella aquatilis can provide insect hosts with nitrogen through nitrogen fixation and nitrogenous waste recycling (Sabree et al., 2009; Ren et al., 2022; Huang et al., 2023). For example, in cockroaches, if dietary nitrogen is limited, the uric acid stored in their adipocytes is degraded into nitrogen which is then used to produce microbial protein (Ren et al., 2022).
There are 10 amino acids that are essential in arthropod diet: Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, Arg and His. Tyr is a main component of sclerotin and it is required in large amounts during moulting (Cortes Ortiz et al., 2016). The non-essential amino acids that are not necessary for the growth of the YM, include Ser, Tyr, Glu and Gly. Ala, Cys, Pro and Asp are semi-essential (Davis, 1975). Thr and Trp are also limiting amino acids for the YM (John et al., 1979; Ribeiro et al., 2018). Micellar casein consists of 20% whey protein and 80% casein and is considered to be a high-quality protein source, while soy is the most used plant-origin protein source in the feed industry. This study aimed to examine whether urea as a simple nitrogen source can be utilized by the four selected insect species. If yes, we wanted to examine how it can be used for replacing the valuable protein sources like micellar casein and extracted soybean meal. The parameters measured were BW, survival rate and FCR.
Materials and methods
The insects were reared and housed in the animal house of the Institute of Animal Breeding, Nutrition and Laboratory Animal Science, University of Veterinary Medicine, Budapest, Hungary. The temperature and the relative air humidity were measured with a digital thermometer/hygrometer (TFA 30.5027.01, TFA Dostmann). The temperature and the relative air humidity of the room were 27 ± 0.4 °C and 50–60% respectively. Ethical approval was not required.
Housing and diet
In the present study, six experimentally composed isonitrogenous feeds containing 3.52% nitrogen (22% crude protein) were used (micellar casein, extracted soybean meal and corn starch adept for human consumption and bought in local shops; urea was purchased from a company distributing chemicals for laboratory purposes) to evaluate the growth performance of four different insect species. Table 1 shows the composition of the 6 isonitrogenous feeds. Nitrogen contents of feed ingredients were measured with standard methods (AOAC, 1990).
Ingredients of experimental isonitrogenous feeds on dry matter basis (g/100 g)
| Ingredients (g) | Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 |
| Micellar casein | 28.07 | 21.05 | 14.04 | 7.02 | – | – |
| Urea | – | 1.89 | 3.77 | 5.65 | 7.54 | – |
| Extracted soybean meal | – | – | – | – | – | 42.2 |
| Corn starch | 71.93 | 77.06 | 82.19 | 87.33 | 92.46 | 57.8 |
Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50–50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
The nymphs of each cricket species and YM and SW larvae were randomly chosen and assigned to one of six experimental feed groups with four replicates/group (Table 1). The feeding experiments were carried out using 1–1.5 mm cricket nymphs kept in plastic containers. Both cricket species were either group-housed (n = 15/group, ntotal = 60/group) or individually housed (n = 1/group). Paper towel was used as bedding material and paper egg holder as a surface maximiser. Wood flakes were placed in plastic containers of water to provide footholds for the crickets to avoid drowning while consuming water. For every diet treatment, 4 replicates were obtained. Water and feed were provided ad libitum. The group-housed JFCs were kept in 206 × 156 × 83 mm containers, while the group-housed HCs were kept in 160 × 115 × 55 mm containers. Round plastic containers (66 × 20 mm) were used to provide feed and water for crickets. Individually housed crickets were kept in 85 × 55 mm round plastic containers, feed was offered in the paper egg holder and water was provided in plastic bottle cups.
YM larvae (size: 10–10.3 mm, n = 20/group, ntotal = 80/group) and SW larvae (size: 10–10.5 mm, n = 15/group, ntotal = 60/group) were reared in 140 × 100 × 75 mm plastic containers containing 50 g of feed. The feed was offered ad libitum and no water was provided as they are able the use the moisture content of the air and can survive under dry conditions (Riberio et al., 2018; Adamaki-Sotiraki et al., 2022).
Data collection and statistical analysis
BW and feed consumption were measured weekly with a digital scale (Tecator 6110). The insects were counted weekly. Means and standard deviations (SD) were calculated for each dataset. Analyses were performed using R 4.0.3. software (R Core Team, 2020). P values lower than 0.5 were evaluated as significant.
The normality of the data was tested with Quantile-Quantile Plot. Variances of the data were tested with Levene's test. One-Way ANOVA tests were performed to compare normally distributed data. Tukey's post hoc analysis was performed, if the result of the AVOVA test was significant. Non-normally distributed data were analysed with the Kruskal-Wallis rank sum test. A post-hoc Dunn Test was performed, if the Kruskal-Wallis rank sum test result was significant.
The survival rate and final mean individual BW of crickets were tested on week 4. The development of crickets was followed for 13 weeks to see whether they reach sexual maturity (as indicated by fully developed wings or ovipositors). The survival rate and final mean individual BW of YMs were tested on week 14. Data of SWs were evaluated on week 5. FCR was calculated with the following formula: FCR = amount of ingested food (g)/weight gained (g).
Results
Crickets
The initial and final BWs of both cricket species were normally distributed and had equal variances (P > 0.05). The initial BWs of cricket species did not differ significantly. The survival rates of the HCs were normally distributed and had equal variances. The survival rates of the JFCs were non-normally distributed, with equal variances. Due to frequent technical problems (moisture absorption and defecation in the feed), the feed intake of crickets could not be evaluated.
Tables 2 and 3 show the data on the HC. There was a significant difference in the final BW (P = 0.014) of group-housed crickets on week 4, between Group 5-1 (P = 0.0447) and Group 5-6 (P = 0.0213). The mean individual BW was the lowest in Group 5 and the highest in Group 6. The survival rate of the group-housed crickets was poor. The highest survival was detected in Group 5 (28.3%) and the lowest one in Group 3 (18.3%), but the survival rates did not differ significantly (P = 0.854).
Mean individual body weights and survival rates of group-housed house crickets on week 4
| House cricket | Number of live crickets | Mean individual body weight (g) | Survival rate | |||
| Week 1 | Week 4 | Week 1 | Week 4 | P-value | ||
| Group 1 | 60 | 13 | 0.0056 ± 0.0007 | 0.0247 ± 0.0048 | g1-g5 = 0.0447 g6-g5 = 0.0213 | 21.1% |
| Group 2 | 60 | 15 | 0.0058 ± 0.0007 | 0.0227 ± 0.0052 | 25.0% | |
| Group 3 | 60 | 11 | 0.0057 ± 0.0006 | 0.0167 ± 0.0051 | 18.3% | |
| Group 4 | 60 | 16 | 0.0056 ± 0.0008 | 0.0158 ± 0.0049 | 26.7% | |
| Group 5 | 60 | 17 | 0.0054 ± 0.0007 | 0.0112 ± 0.0046 | 28.3% | |
| Group 6 | 60 | 12 | 0.0058 ± 0.0008 | 0.0264 ± 0.0054 | 20.0% | |
Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50–50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
Mean individual body weights and survival rates of individually housed house crickets on week 4
| House crickets | Number of live crickets | Mean individual body weight (g) | Survival rate | |||
| Week 1 | Week 4 | Week 1 | Week 4 | P-value | ||
| Group 1 | 4 | 4 | 0.0049 ± 0.0049 | 0.0136 ± 0.0023 | g6-g1 = 0.03432 g6-g2 = 0.03933 g6-g3 = 0.01698 g6-g4 = 0.01354 g6-g5 = 0.00558 | 100% |
| Group 2 | 4 | 4 | 0.0050 ± 0.0050 | 0.0133 ± 0.0020 | 100% | |
| Group 3 | 4 | 4 | 0.0052 ± 0.0052 | 0.0126 ± 0.0021 | 100% | |
| Group 4 | 4 | 4 | 0.0052 ± 0.0052 | 0.0122 ± 0.0023 | 100% | |
| Group 5 | 4 | 4 | 0.0049 ± 0.0049 | 0.0092 ± 0.0024 | 100% | |
| Group 6 | 4 | 4 | 0.0050 ± 0.0050 | 0.0235 ± 0.0022 | 100% | |
Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50–50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
Table 3 shows the final BW of the individually housed HCs on week 4 that was significantly higher in Group 6 compared to the other groups. The mean individual BW was the lowest in Group 5 and the highest in Group 6. Significant differences with P-values are shown in Table 3. The survival rate of the individually housed crickets was 100% on week 4.
Tables 4 and 5 show the data on the JFC. There were significant differences in the final BW (P < 0.001) of the group-housed crickets on week 4. The mean individual BW was the lowest in Group 5 and the highest in Group 6. Significant differences with P-values are shown in Table 4. The survival rates of the group-housed JFCs were generally low, with the highest rates in Groups 2 and 4 (28.3%) and the lowest in Group 5 (16.7%), but the differences were not significant (P = 0.36).
Mean individual body weights and survival rates of group-housed Jamaican field crickets on week 4
| Jamaican field crickets | Number of live crickets | Mean individual body weight (g) | Survival rate | |||
| Week 1 | Week 4 | Week 1 | Week 4 | P-value | ||
| Group 1 | 60 | 12 | 0.0042 ± 0.0004 | 0.0238 ± 0.0034 | g5-g2 = 0.0320 | 20.0% |
| Group 2 | 60 | 17 | 0.0041 ± 0.0003 | 0.0256 ± 0.0038 | g2-g3 = 0.0254 | 28.3% |
| Group 3 | 60 | 15 | 0.0041 ± 0.0004 | 0.0186 ± 0.0037 | g2-g4 = 0.0435 | 25.0% |
| Group 4 | 60 | 17 | 0.0042 ± 0.0004 | 0.0192 ± 0.0035 | g2-g5 = 0.0064 | 28.3% |
| Group 5 | 60 | 10 | 0.0041 ± 0.0003 | 0.0160 ± 0.0037 | g1-g6 = 0.0036 | 16.7% |
| Group 6 | 60 | 12 | 0.0042 ± 0.0003 | 0.0333 ± 0.0035 | g2-g6 = 0.0165 | 20.0% |
| g3-g6 < 0.001 | ||||||
| g4-g6 < 0.001 | ||||||
| g5-g6 < 0.001 | ||||||
Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50–50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
Mean individual body weights and survival rates of individually housed Jamaican field crickets on week 4
| Jamaican field cricket | Number of live crickets | Mean individual body weight (g) | Survival rate | |||
| Week 1 | Week 4 | Week 1 | Week 4 | P-value | ||
| Group 1 | 4 | 4 | 0.0023 ± 0.0007 | 0.0150 ± 0.0028 | 100% | |
| Group 2 | 4 | 4 | 0.0024 ± 0.0005 | 0.0141 ± 0.0027 | g1-g5 = 0.0406 | 100% |
| Group 3 | 4 | 4 | 0.0024 ± 0.0006 | 0.0120 ± 0.0028 | g6-g3 = 0.0386 | 100% |
| Group 4 | 4 | 4 | 0.0025 ± 0.0007 | 0.0112 ± 0.0030 | g6-g4 = 0.0160 | 100% |
| Group 5 | 4 | 4 | 0.0025 ± 0.0005 | 0.0079 ± 0.0031 | g6-g5=<0.001 | 100% |
| Group 6 | 4 | 4 | 0.0023 ± 0.0006 | 0.0188 ± 0.0029 | 100% | |
Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50–50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
Table 5 shows the final BW of the individually housed JFCs with significant differences. The mean individual BW was the lowest in Group 5 and the highest in Group 6. Significant differences with P-values are shown in Table 6. The survival rate of individually housed crickets was 100% on week 4.
Mean individual body weights and survival rates of yellow mealworm larvae (YM) on week 14
| Yellow mealworm | Number of live mealworms | Mean individual body weight (g) | Survival rate | |||
| Week 1 | Week 14 | Week 1 | Week 14 | P value | ||
| Group 1 | 80 | 47 | 0.0104 ± 0.0003 | 0.0405 ± 0.0044 | g1-g2 = 0.0012 | 58.8% |
| Group 2 | 80 | 55 | 0.0105 ± 0.0004 | 0.0250 ± 0.0044 | g1-g3,4,5 | 68.8% |
| Group 3 | 80 | 62 | 0.0105 ± 0.0004 | 0.0188 ± 0.0047 | <0.001 | 77.5% |
| Group 4 | 80 | 50 | 0.0104 ± 0.0003 | 0.0183 ± 0.0044 | g2-g5 = 0.0172 | 62.5% |
| Group 5 | 80 | 55 | 0.0105 ± 0.0003 | 0.0134 ± 0.0044 | g6-g3 = 0.0094 | 68.8% |
| Group 6 | 80 | 15 | 0.0105 ± 0.0003 | 0.0313 ± 0.0049 | g6-g4 = 0.0067 | 18.8% |
| g6-g5< 0.001 | ||||||
Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50–50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
The growth tendencies and survival rates of the HCs and the JFCs were similar. The final BW was the highest in Group 6 and the lowest in Group 5. The survival rates were equally poor in both cricket species. However, the survival rate in Group 5 was the highest among the HCs but the lowest among the JFCs. In Group 6, both sexes of HCs and JFCs reached maturity on week 11 and 12, respectively.
Yellow mealworm
The initial and final BW of the YMs were normally distributed with equal variances (P > 0.05). The initial BWs were not significantly different. Table 6 shows the data on the YM larvae. There were significant differences in the final BW (P < 0.001) of the YM larvae on week 14. The mean individual BW was the lowest in Group 5 and the highest in Group 1. Significant differences with P-values are shown in Table 7. Only one individual reached pupation, in Group 1, on week 12.
Substrate reduction of yellow mealworms
| Yellow mealworm | Substrate weight (g) | ||
| Week 1 | Week 14 | Feed consumption | |
| Group 1 | 50.17 ± 0.2034 | 45.85 ± 0.72 | 4.32 |
| Group 2 | 50.18 ± 0.2131 | 45.56 ± 0.71 | 4.73 |
| Group 3 | 50.20 ± 0.2054 | 44.71 ± 0.66 | 5.43 |
| Group 4 | 50.18 ± 0.2046 | 44.30 ± 0.65 | 5.88 |
| Group 5 | 50.20 ± 0.2116 | 43.41 ± 0.66 | 6.79 |
| Group 6 | 50.17 ± 0.2027 | 46.52 ± 0.71 | 3.65 |
Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50–50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
Table 6 shows the survival rates of the YMs, all above 50%, except for Group 6 that had a very poor survival rate (18.8%). The survival rates were normally distributed with equal variances. Groups 6-1, 2, 3, 4, 5 differed significantly (<0.001).
Table 7 shows the substrate reduction (feed consumption) of the YM groups. The highest substrate reduction was noted in Group 5 and the lowest in Group 6. The substrate reduction differed significantly between Groups 4 and 6 (P = 0.0149) and between Groups 5 and 6 (P = 0.0035).
Table 8 shows the FCR of the YMs. The FCR seemed to gradually worsen with the increase of the urea level. The FCR was the best in Group 6 followed by Group 1 and the worst in Group 5.
Feed conversion ratio of yellow mealworms
| Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | |
| FCR | 4.16 ± 0.66 | 4.43 ± 0.70 | 4.88 ± 0.39 | 5.73 ± 0.13 | 6.65 ± 0.53 | 3.49 ± 0.77 |
FCR: Feed Conversion Ratio, Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50-50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
Superworms
The initial BW of the SWs were normally distributed with equal variances (P > 0.05). Table 9 shows the data on the SWs. The initial BWs were not significantly different. The final BWs were non-normally distributed with equal variances. The mean individual BW was the lowest in Group 5 and the highest in Group 6.
Mean individual body weights and survival rates of superworm larvae on Week 5
| Super-worms | Number of live superworms | Mean individual body weight (g) | Survival rate | |||
| Week 1 | Week 5 | Week 1 | Week 5 | P-value | ||
| Group 1 | 60 | 45 | 0.0281 ± 0.0014 | 0.0449 ± 0.0039 | g1-g4 = 0.0488 g1-g5 = 0.0202 g2-g5 = 0.0233 g4-g6 = 0.0101 g5-g6 = 0.0029 | 75.0% |
| Group 2 | 60 | 29 | 0.0289 ± 0.0014 | 0.0453 ± 0.0033 | 48.3% | |
| Group 3 | 60 | 34 | 0.0285 ± 0.0012 | 0.0404 ± 0.0035 | 56.7% | |
| Group 4 | 60 | 22 | 0.0283 ± 0.0011 | 0.0352 ± 0.0034 | 36.7% | |
| Group 5 | 60 | 22 | 0.0283 ± 0.0014 | 0.0317 ± 0.0033 | 36.7% | |
| Group 6 | 60 | 20 | 0.0281 ± 0.0014 | 0.0506 ± 0.0038 | 33.3% | |
Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50–50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
The survival rates were normally distributed with equal variances. The survival rate decreased with the gradual increase of the urea level. The highest was in Group 1 and the lowest was in Group 6. The difference was significant between Group 1-2 (P = 0.0106), 4, 5, 6 (P < 0.001) and Group 3-6 (P = 0.0185).
Table 10 shows the substrate reduction (feed consumption) of the SWs. The feed intake increased with increasing urea inclusion level. The substrate reduction did not differ significantly (P = 0.2076).
Substrate reduction of superworm larvae
| Superworm | Substrate weight (g) | ||
| Week 1 | Week 5 | Feed difference | |
| Group 1 | 50.24 ± 0.20 | 49.58 ± 0.3884 | 0.6567 |
| Group 2 | 50.19 ± 0.22 | 49.54 ± 0.4220 | 0.6545 |
| Group 3 | 50.23 ± 0.20 | 49.56 ± 0.4294 | 0.6697 |
| Group 4 | 50.21 ± 0.20 | 49.2971 ± 0.41 | 0.9161 |
| Group 5 | 50.23 ± 0.21 | 49.0427 ± 0.38 | 1.1907 |
| Group 6 | 50.24 ± 0.22 | 49.6283 ± 0.38 | 0.6169 |
Proportions of nitrogen sources in the different groups: Group 1: 100% micellar casein, Group 2: 75% micellar casein, 25% urea; Group 3: 50–50% micellar casein and urea, Group 4: 25% micellar casein, 75% urea, Group 5: 100% urea, Group 6: 100% extracted soybean meal.
The FCR of the SWs seemed to gradually worsen with the increase of the urea level. The FCR was the best in Group 6 followed by Group 1 and the worst in Group 5. Overall, the FCRs were very poor over 15 kg kg−1.
Discussion
Crickets
Several studies showed that the amount, source and quality of protein influence the growth rate of insects. It seems that, in general, poultry diets containing soy are optimal for insects (Sorjonen et al., 2019; Harsányi et al., 2020; Zim et al., 2021). This was also supported by this study as Group 6 (100% extracted soybean meal as a protein source) reached the highest final BW. As previously mentioned, crickets require protein-rich diets (Patton 1978; McCluney and Date, 2008; Lundy and Parrella, 2015; Oonincx et al., 2015; Dobermann et al., 2019; Sorjonen et al., 2019; Morales-Ramos et al., 2020a). Only a few studies tested milk powder or skimmed milk (Patton, 1978; Morales-Ramos et al., 2020a), thus it is difficult to compare the results of this study with those of others. Crickets consuming diets containing skimmed milk developed faster than crickets reared on defatted dry or whole dry milk (Patton, 1978; Morales-Ramos et al., 2020a). The latter study showed a higher consumption of milk powder and of whole milk than of extracted soybean meal. The mean individual BW of group-housed crickets of Group 1 (100% micellar casein) and Group 6 (100% extracted soybean meal) had similar mean individual BWs with crickets kept on chicken feed, while the performance of the other groups was lower (Harsányi et al., 2020). Different harvesting time of insects in other studies makes the comparison of final BWs difficult (Bawa et al., 2020). Higher BW of individually housed crickets in comparison to group-housed individuals was also described by McFarlane (1965). Urea proportions above 25% negatively impacted the final BWs of both group- and individually housed crickets.
The survival rates of group-housed crickets were low. As the proportion of urea did not significantly influence the survival rate, some other reasons must have led to this phenomenon. One reason could be cannibalism, as the survival rates were much better in the individually housed crickets. High mortality was also noted in the study of Oonincx et al. (2015), with survival rates of 6–55% depending on the quality of the feed. The high protein (22.9%) and low fat (1%) diet resulted in the lowest survival rate, while this parameter was the best in the control group (17.2 CP and 4% fat). This might be explained by the presence of cinnamon in the cookie remains which comprised 50% of that diet. On the other hand, Sorjonen et al. (2019) described 80% overall, 94% and 91% survival rates on medium- (22.5%) and high-protein (30.5%) diets in HCs, respectively. In the same study, the overall survival rate of the JFC was 44%, showing better results compared to the ones fed with high-protein (30%) turnip rape and chicken feed (15.2% protein). The proportion of urea did not have a negative impact on the survival rate of crickets. Interestingly, it was the highest (28%) in the 100% urea groups of group-housed crickets. The low survival rate might also be the consequence of a densovirus (AdDNV), which is abundant in European and North American house cricket facilities, interfering with the absorption of nutrients, decreasing growth rates and increasing mortality (Liu et al., 2011; Szelei et al., 2011; Pham et al., 2013; Oonincx et al., 2015).
Crickets reared on chicken feed reached maturity much faster (HC: 34 days and two-spotted cricket [Gryllus bimaculatus]: 24 days) than crickets fed on low-protein barley (45 and 28 days respectively, Sorjonen et al. (2019)). In the study of Oonincx et al. (2015), the development period was between 48 and 167 days depending on the diet. The low protein (12.9%) and high fat (14.6%) diet showed the slowest development time, this paremeter being the highest in the control group (17.2 CP and 4% fat). In the current study, it was difficult to evaluate the development time as only few crickets reached maturity in Group 6 (100% extracted soybean meal). Therefore, the period of 77–84 days was much longer than the results of Sorjonen et al. (2019), but closer to the results of Oonincx et al. (2015).
Mealworms and superworms
Casein supplementation (3 and 20%) increases the BW of YM larvae from 4.08 to 6.16 g (Davis, 1970). In this study, the casein group (Group 1, 100% micellar casein) reached the highest final BW. Likewise, the amino acid composition of milk-derived products seems to be adequate for the YM (Davis and Leclercq, 1969). The proportion of the urea above 25% negatively impacted the final BWs of YMs and SWs. The mean individual BW and weight gain of YMs was lower than that of the larvae kept either on chicken feed or wheat bran (Morales-Ramos and Rojas, 2015; Harsányi et al., 2020). In the study of Oonincx et al. (2015), the determining factor for development was the dietary protein content. Development was faster and survival was better on high-protein diets (22–23%) than on low-protein diets. In the current study, only one individual reached pupation in Group 1 (100% micellar casein) on week 12 which is close to the results of the van Broekhoven study (2015), where it varied between 79 and 168 days. On the other hand, the rest of the mealworms did not reach pupation which requires further investigation. In case of the SW, the isolation prolonged larval development time in comparison to group-housed larvae (Quennedey et al., 1995).
A high protein diet (>21%) improves the survival rate (67%, Oonincx et al., 2015; 80%, van Broekhoven et al., 2015) of the YM, especially if the diet is also rich in carbohydrates. The mortality rate was of >70% during the first 2 months in a study where larvae were reared on either bread or oat flour (Dreassi et al., 2017). The best feed to reach a 50% pupation rate (96.2 ± 3.834 days) was the mixture of beer yeast (5%), wheat flour (47.5%) and oat flour (47.5%). Cannibalism occurs often in mealworm species and has a negative impact on the survival rate (Ichikawa and Kurauchi, 2009). It is difficult to compare the results of this study with those of others, as survival rates were recorded when pupation reached a certain percentage. In the current study, the survival rates were over 50%, but the pupation rate was almost zero. The proportion of urea did not have a negative impact on the survival rate of YMs. Interestingly, it was the second highest (68.8%) in the 100% urea groups. In contrast, the survival rate of SWs was negatively influenced by the increasing urea level which highlights the difference between the two species.
The YM FCRs of the current study were similar to that of Oonincx et al. (2015). It was clear that the FCR of Group 1 (100% micellar casein) and Group 6 (100% extracted soybean meal) were the best, as these feeds were the highest in true protein. FCRs tended to worsen with increasing urea levels, resulting in the worst FCR in Group 5 (100% urea). The SW is not as well studied as the YM, thus the evaluation of FCR results is difficult. Both BW gain and FCR were poor. These findings are in agreement with other studies where feed intake and, consequently, FCR were disadvantageous when larvae were fed with low-quality feeds (Oonincx et al., 2015).
Conclusions
In the HC, the JFC, and the YM on nitrogen basis, urea can replace 25% of micellar casein without having a negative effect on the BW and survival rate, in comparison to the 100% micellar casein group. In the SW, urea supplementation is suggested to be kept below 25%, as higher proportions decreased the survival rate. Further research is needed to find the reasons of high mortality rate of group-housed HCs and SWs, as well as the lack of pupation in YWs.
References
Adamaki-Sotiraki, C., Rumbos, C. I. and Athanassiou, C. G. (2022): Developmental plasticity among strains of the yellow mealworm, Tenebrio molitor L., under dry conditions. J. Insects Food Feed 8, 281–288. https://doi.org/10.3920/JIFF2021.0087.
AOAC (1990): Official Methods of Analysis of the Association of Official Analytical Chemists, Fifteen. ed, Assosiaciation of Official Analytical Chemists. Arlington, VA, USA.
Bawa, M., Songsermpong, S., Kaewtapee, C. and Chanput, W. (2020): Effect of diet on the growth performance, feed conversion, and nutrient content of the House cricket. J. Insect Sci. 20, 1–10. https://doi.org/10.1093/jisesa/ieaa014.
Cortes Ortiz, J. A., Ruiz, A. T., Morales-Ramos, J. A., Thomas, M., Rojas, M. G., Tomberlin, J. K., Yi, L., Han, R., Giroud, L. and Jullien, R. L. (2016): Insect mass production technologies. In: Dossey, A.T., Morales-Ramos, J.A., Rojas, M.G. (Eds.), Insects as Sustainable Food Ingredients. Elsevier Academic Press, Amsterdam, the Netherlands, pp. 153–201.
Davis, G. R. F. (1970): Protein nutrition of Tenebrio molitor L. XII. Effects of dietary casein concentration and of dietary cellulose on larvae of race F. Arch. Physiol. Biochem. 78, 37–41.
Davis, G. R. F. (1975): Essential dietary amino acids for growth of larvae of the yellow mealworm, Tenebrio molitor L. J. Nutr. 105, 1071–1075.
Davis, G. R. F. and Leclercq, J. (1969): Protein nutrition of “Tenebrio molitor” L. IX. Replacement caseins for the reference diet and a comparison of the nutritional values of various lactalbumins and lactalbumin hydrolysates. Arch. Physiol. Biochem. 77, 687–693. https://doi.org/10.3109/13813456909059786.
Dobermann, D., Michaelson, L. and Field, L. M. (2019): The effect of an initial high-quality feeding regime on the survival of Gryllus bimaculatus (black cricket) on bio-waste. J. Insects Food Feed 5, 117–123. https://doi.org/10.3920/JIFF2018.0024.
Dreassi, E., Cito, A., Zanfini, A., Materozzi, L., Botta, M. and Francardi, V. (2017): Dietary fatty acids influence the growth and fatty acid composition of the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae). Lipids 52, 285–294. https://doi.org/10.1007/s11745-016-4220-3.
European Parliament and Council (2009): Regulation (EC) No 1069/2009: laying down health rules as regards animal by-products and derived products not intended for human consumption and repealing Regulation (EC) No 1774/2002 (Animal by-products Regulation). Off. J. Eur. Union 300, 1–33.
Harsányi, E., Juhász, C., Kovács, E., Huzsvai, L., Pintér, R., Fekete, G., Varga, Z. I., Aleksza, L. and Gyuricza, C. (2020): Evaluation of organic wastes as substrates for rearing Zophobas morio, Tenebrio molitor and Acheta domesticus larvae as alternative feed. Insects 11, 604. https://doi.org/10.3390/insects11090604.
Huang, Q., Feng, Y., Shan, H. W., Chen, J. P. and Wu, W. (2023): A novel nitrogen-fixing Bacterium Raoultella electrica isolated from the midgut of the leafhopper Recilia dorsalis. Insects 14:431. https://doi.org/10.3390/insects14050431.
Ichikawa, T. and Kurauchi, T. (2009): Larval cannibalism and pupal defense against cannibalism in two species of tenebrionid beetles. Zoolog. Sci. 26, 525–529. https://doi.org/10.2108/zsj.26.525.
Joern, A. and Behmer, S. T. (1997): Importance of dietary nitrogen and carbohydrates to survival, growth, and reproduction in adults of the grasshopper Ageneotettix deorum (Orthoptera: Acrididae). Oecologia 112, 201–208. https://doi.org/10.1007/s004420050301.
John, A. M., Davis, G. R. F. and Sosulski, F. W. (1979): Protein nutrition of Tenebrio molitor L. XX. Growth response of larvae to graded levels of amino acids. Arch. Physiol. Biochem. 87, 997–1004.
Kröncke, N. and Benning, R. (2022): Self-Selection of feeding substrates by Tenebrio molitor larvae of different ages to determine optimal macronutrient intake and the influence on larval growth and protein content. Insects 13, 657. https://doi.org/10.3390/insects13070657.
Liu, K., Li, Y., Jousset, F-X., Zadori, Z., Szelei, J., Yu, Q., Pham, H. T., Lepine, F., Bergoin, M. and Tijssen, P. (2011): The Acheta domesticus densovirus, isolated from the European house cricket, has evolved an expression strategy unique among parvoviruses. J. Virol. 85, 10069–10078. https://doi.org/10.1128/JVI.00625-11.
Lundy, M. E. and Parrella, M. P. (2015): Crickets are not a free lunch: protein capture from scalable organic side-streams via high-density populations of Acheta domesticus. PLoS One 10, 1–12. https://doi.org/10.1371/journal.pone.0118785.
Makkar, H. P. S., Tran, G., Heuzé, V. and Ankers, P. (2014): State-of-the-art on use of insects as animal feed. Anim. Feed Sci. Technol. 197, 1–33. https://doi.org/10.1016/j.anifeedsci.2014.07.008.
McCluney, K. E. and Date, R. C. (2008): The effects of hydration on growth of the house cricket, Acheta domesticus. J. Insect Sci. 8, 1–9. https://doi.org/10.1673/031.008.3201.
McFarlane, J. E. (1965): Studies on group effects in crickets - I. Effect of methyl linolenate, methyl linoleate, and vitamin E. J. Insect Physiol. 12, 179–188.
Melis, R., Braca, A., Sanna, R., Spada, S., Mulas, G., Fadda, M. L., Sassu, M. M., Serra, G. and Aneddaet, R. (2019): Metabolic response of yellow mealworm larvae to two alternative rearing substrates. Metabolomics 15, 113. https://doi.org/10.1007/s11306-019-1578-2.
Morales-Ramos, J. A., Rojas, M. G., Shapiro-Llan, D. I., Tedders, W. L. (2013): Use of nutrient self-selection as a diet refining tool in Tenebrio molitor (Coleoptera: Tenebrionidae). J. Entomol. Sci. 48, 206–221. https://doi.org/10.18474/0749-8004-48.3.206.
Morales-Ramos, J. A. and Rojas, M. G. (2015): Effect of larval density on food utilization efficiency of Tenebrio molitor (Coleoptera: Tenebrionidae). J. Econ. Entomol. 108, 2259–2267. https://doi.org/10.1093/jee/tov208.
Morales-Ramos, J. A., Rojas, M. G., Dossey, A. T. and Berhow, M. (2020a): Self-selection of food ingredients and agricultural by-products by the house cricket, Acheta domesticus (Orthoptera: Gryllidae): a holistic approach to develop optimized diets. PLoS One 15, 1–30. https://doi.org/10.1371/journal.pone.0227400.
Morales-Ramos, J. A., Rojas, M. G., Kelstrup, H. C. and Emery, V. (2020b): Self-selection of agricultural by-products and food ingredients by Tenebrio molitor (Coleoptera: Tenebrionidae) and impact on food utilization and nutrient intake. Insects 11, 1–15. https://doi.org/10.3390/insects11120827.
Oonincx, D. G. A. B. and de Boer, I. J. M. (2012): Environmental impact of the production of mealworms as a protein source for humans – a life cycle assessment. PLoS One 7, 1–5. https://doi.org/10.1371/journal.pone.0051145.
Oonincx, D. G. A. B., van Broekhoven, S., van Huis, A. and van Loon, J. J. A. (2015): Feed conversion, survival and development, and composition of four insect species on diets composed of food by-products. PLoS One 10, 1–20. https://doi.org/10.1371/journal.pone.0144601.
Orinda, M. A., Mosi, R. O., Ayieko, M. A. and Amimo, F. A. (2017): Growth performance of common house cricket (Acheta domesticus) and field cricket (Gryllus bimaculatus) crickets fed on agro-byproducts. J. Entomol. Zool. Stud. 5, 1664–1668.
Patton, R. L. (1967): Oligidic diets for Acheta domesticus (Orthoptera: Gryllidae). Ann. Entomol. Soc. Am. 60, 1238–1242.
Patton, R. L. (1978): Growth and development parameters for Acheta domesticus. Ann. Entomol. Soc. Am. 71, 40–42.
Pham, H. T., Yu, Q., Bergoin, M. and Tijssen, P. (2013): A novel ambisense densovirus, Acheta domesticus mini ambidensovirus, from crickets. Genome Announc. 1, 2012–2013. https://doi.org/10.1371/10.1128/genomeA.00914-13.
Quennedey, A., Aribi, N., Everaerts, C. and Delbecque, J. P. (1995): Postembryonic development of Zophobas atratus Fab. (Coleoptera: Tenebrionidae) under crowded or isolated conditions and effects of juvenile hormone analogue applications. J. Insect Physiol. 41, 143–152. https://doi.org/10.1016/0022-1910(94)00091-T.
R Core Team (2020): R: A Language and Environment for Statistical Computing. R Found. Stat. Comput. Vienna, Austria. https://www.r-project.org/.
Ramos-Elorduy, J., González, E. A., Hernández, A. R. and Pino, J. M. (2002): Use of Tenebrio molitor (Coleoptera: Tenebrionidae) to Recycle Organic Wastes and as Feed for Broiler Chickens, J. Econ. Entomol. 95, 214–220. https://doi.org/10.1603/0022-0493-95.1.214.
Ren, X., Guo, R., Akami, M. and Niu, C. (2022): Nitrogen acquisition strategies mediated by insect symbionts: a review of their mechanisms, methodologies, and case studies. Insects 13, 84. https://doi.org/10.3390/insects13010084.
Ribeiro, N., Abelho, M. and Costa, R. (2018): A review of the scientific literature for optimal conditions for mass rearing Tenebrio molitor (Coleoptera: Tenebrionidae). J. Entomol. Sci. 53, 434–454. https://doi.org/10.18474/JES17-67.1.
Rumbos, C. I., Bliamplias, D., Gourgouta, M., Michail, V. and Athanassiou, C. G. (2021): Rearing Tenebrio molitor and Alphitobius diaperinus larvae on seed cleaning process byproducts. Insects 12, 293. https://doi.org/10.3390/insects12040293.
Sabree, Z. L., Kambhampati, S. and Moran, N. A. (2009): Nitrogen recycling and nutritional provisioning by Blattabacterium, the cockroach endosymbiont. Proc. Natl. Acad. Sci. U S A. 106, 19521–19526. https://doi.org/10.1073/pnas.09075041.
Sorjonen, J. M., Valtonen, A., Hirvisalo, E., Karhapää, M., Lehtovaara, V. J., Lindgren, J., Marnila, P., Mooney, P., Mäki, M., Siljander-Rasi, H., Tapio, M., Tuiskula-Haavisto, M. and Roininen, H. (2019): The plant-based by-product diets for the mass-rearing of Acheta domesticus and Gryllus bimaculatus. PLoS One 14, e0218830. https://doi.org/10.1371/journal.pone.0218830.
Szelei, J., Woodring, J., Goettel, M. S., Duke, G., Jousset, F. X., Liu, K. Y., Zadori, Z., Li, Y., Styer, E., Boucias, D. G., Kleespies, R. G., Bergoin, M. and Tijssen, P. (2011): Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics. J. Invertebr. Pathol. 106, 394–399. https://doi.org/10.1016/j.jip.2010.12.009.
Tilman, D. and Clark, M. (2014): Global diets link environmental sustainability and human health. Nature 515, 518–522. https://doi.org/10.1038/nature13959.
van Broekhoven, S., Oonincx, D. G. A. B., van Huis, A. and van Loon, J. J. A. (2015): Growth performance and feed conversion efficiency of three edible mealworm species (Coleoptera: Tenebrionidae) on diets composed of organic by-products. J. Insect Physiol. 73, 1–10. https://doi.org/10.1016/j.jinsphys.2014.12.005.
van Huis, A., Van Itterbeeck, J., Klunder, E., Halloran, A., Muir, G. and Vantomme, P. (2013): Edible Insects: Future Prospects for Food and Feed Security, Forestry P. ed, Food and Agriculture Organization of the United Nations. Rome. https://edepot.wur.nl/258042.
Wang, Q., Liu, H. L. and Cheung, Y. M. (2010): Food security: the challenge of feeding 9 billion people. Science 327, 194–197. https://doi.org/10.1126/science.1185383.
Zim, J., Lhomme, P., Takhim, A., Sarehane, M. and Bouharroud, R. (2021): Growth performance, conversion and survival rates of Tenebrio molitor (Coleoptera: Tenebrionidae) reared on various livestock diets. Mor. J. Agri. Sci. 2, 161–164. https://doi.org/10.1007/s42690-021-00707-0.
