Abstract
Farming edible crickets has environmental and nutritional benefits, as well as social benefits such as livelihood diversification. Commercial feeds for poultry and fish farming are often used to feed crickets, and in recent years, crop and food-processing by-products have also been used to improve sustainability. However, the design of feed for crickets has not been standardized. Here, we investigated growth and development of the Asian field cricket, Teleogryllus occipitalis (Audinet-Serville) (Orthoptera: Gryllidae), fed on different forms of the same diet. Body mass and the rate of development were significantly greater in crickets fed on millimetre-order granules than in crickets fed on micrometre-order powder. In addition, analysis of feeding behaviour revealed that crickets fed less frequently on the powdered diet than on the granulated diet. These results suggest that crickets have an avoidance behaviour towards fine particles, or that the granular form is easier for them to grasp and ingest than the powdery form, which may have contributed to growth performance. Simply feeding millimetre-order granules may contribute to the development of feed design for farming edible crickets.
1 Introduction
Edible-insect farming has attracted worldwide attention as an environmentally friendly source of animal protein and micronutrients (van Huis et al., 2013). Crickets are one of the most suitable insect groups for farming because of their rapid development, high fecundity, omnivorous nature, and ability to grow on dry feed as long as they are watered (Simmons, 2005; Gutiérrez et al., 2020). In addition, the feed conversion ratio of crickets is 5.9 times that of beef cattle (van Huis, 2013), and the amount of greenhouse gases emitted by cricket farming is about one-quarter (CO2 + CO2-eq.) of that emitted by beef cattle farming (Oonincx et al., 2010). The use of crickets as a food source is growing. In Thailand, approximately 20,000 farmers produce crickets, and annual production averaged about 7,500 t from 1996 to 2011 (Hanboonsong et al., 2013). The European house cricket, Acheta domesticus (L.) (Orthoptera: Gryllidae), is the third insect species to be authorized as a novel food by the European Commission (2022).
Commercial feed for poultry and fish farming has often been used as cricket feed (Hanboonsong et al., 2013; Miech et al., 2016). In poultry farming, it is well known that the aggregate size of feed affects growth performance. For example, the body mass of broilers fed a diet in crumb or pellet form tends to be higher than that of those fed mash, which is in powdered form (Reece et al., 1985; Svihus et al., 2004). Naser El Deen et al. (2022) reported that larvae of the mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), showed greater growth when fed on a diet with a particle size less than 2 mm. Patton (1967) described that A. domesticus nymphs tended to prefer smaller diet to larger one, but they presented no objective data such as size. No other studies have reported the effect of diet size on the performance of crickets.
Here, we show that diet size on its own affects developmental time and adult body mass of the Asian field cricket, Teleogryllus occipitalis (Audinet-Serville) (Orthoptera: Gryllidae), a traditional food species in East and South-East Asia.
2 Materials and methods
2.1 Insects
We used a population of Tel. occipitalis collected on Amami Ohshima (Kagoshima, Japan) and previously used for whole-genome sequencing (Kataoka et al., 2020). This population was maintained on chicken feed (Chougenki Edzukeyousuuyou; Nosan Corp., Yokohama, Japan) or goldfish feed (Kingyo Genki Probio Flake; GEX Corp., Osaka, Japan) and water in polypropylene containers at 25 °C with uncontrolled relative humidity (RH).
2.2 Diet processing and aggregate size distribution
2.3 Growth performance assay
A polypropylene container (239 mm × 176 mm × 91 mm) with a lid perforated with ∼100 holes (2.5 mm in diameter) for ventilation was used as a rearing container for each treatment. Non-woven fabric (Nougyouyoufushokufu; MonotaRO Co., Ltd., Osaka, Japan) was placed between the container and the lid to prevent escape of young nymphs. A polystyrene cup (V-9; As One Corp., Osaka, Japan) filled with water was prepared in the rearing container. A single hole (10 mm in diameter) was made in its lid, and a paper towel (Crecia EF Hand Towel Soft Type; Nippon Paper Crecia Co., Ltd., Tokyo, Japan) was placed through the hole and touching the water to provide a watering station. Two pieces (10 cm × 15 cm) of cardboard egg carton were put into each container as shelter to mitigate cannibalism. The diet was put into a polystyrene Petri dish (90 mm diameter) placed on the egg cartons and replenished as needed to prevent depletion. Kimtowels (Nippon Paper Crecia Co., Ltd.) were placed in each container to facilitate access for the crickets to the watering station and feeding area.
Wet cotton on which cricket eggs were laid was collected from the stock population and placed in the polystyrene cup. The cup was covered with a non-perforated lid and maintained in an incubator (MIR-554-PJ; PHC Holdings Corp., Tokyo, Japan) at 30 °C with uncontrolled RH (but the inside of the cup would be at saturated water vapor pressure) until hatching. First-instar nymphs hatched within 24 h were collected and allocated among the containers (50 in each) on day 0, and reared under a 16-h light and 8-h dark cycle (LD 16:8) at 30 °C with uncontrolled RH. The number of individuals in each container was recorded on days 6, 13, 20, 27, 34 and 41. Fresh body mass of 10 nymphs randomly collected from each container was individually measured on an electronic balance (FX-500i; A&D Co., Ltd., Tokyo, Japan) on days 20, 27, 34 and 41. When adults emerged, we calculated the developmental day and measured the fresh body mass on the electronic balance and the head width, pronotum length, pronotum width, forewing length and hind-leg femur length with a digital calliper (CD67-S15PS; Mitsutoyo Corp., Kawasaki, Japan). Adults were removed from each container after measurement and the bioassay was continued until all surviving crickets had reached the adult stage. The bioassay consisted of three independent experimental runs.
2.4 Calculation of population growth rate
2.5 Morphological analyses of mouthparts
First-instar nymphs and adults were anaesthetised, decapitated, mounted on holders using double-sided carbon tape, and the mouthparts of non-treated samples were observed through a scanning electron microscope (VHX-DF510; Keyence Corp., Osaka, Japan) at 1.2 kV acceleration voltage. From the images, widths of the labrum were calculated from the images as an approximation of the mandibular range.
2.6 Feeding behaviour assay
The feeding behaviour of eight cricket nymphs weighing 0.18 ± 0.01 g (mean ± SE) was recorded to compare preference between the granular (n = 4) and powdered (n = 4) diets. The recording system was adapted from Hayakawa et al. (2024). The nymphs were individually placed in a transparent acrylic box (137 mm × 65 mm × 37 mm) containing a polystyrene Petri dish (35 mm diameter) filled with 1.5 g of the granular or powdered diet, and a wet hemp string as a watering device, one end of which penetrated the wall of the each box and the other end of which was connected to a water tank (V-3; As One Corp.) located outside of the each box. To make the individuals invisible to each other, cardboard walls (50 mm high) were placed between each box. After a 24-h acclimation period for the nymphs in the box, time-lapse photography was performed using an infrared camera module 3 (Raspberry Pi NoIR Wide; 4,608 × 2,592 pixels) connected to a Raspberry Pi 3 model B (Raspberry Pi Foundation, Cambridge, UK), which captured RGB images of the crickets at 1-min intervals for 24 h. During this period, diets and water were not replenished or replaced. The camera was positioned 350 mm above the bottom of the acrylic boxes. The experimental system was installed in an incubator (MIR-253; Sanyo Electric Co., Ltd., Osaka, Japan) at 30 °C with uncontrolled RH. Illumination was provided by white LEDs (Timely, Tokyo, Japan) and infrared LEDs (Broadwatch, Tokyo, Japan). The white LEDs were connected to a time switch to ensure the LD 16:8 cycle. From the time-lapse images, the frequency of the nymphal head located on the diet dish was counted to estimate the feeding frequency.
2.7 Statistical analyses
All data analyses and visualizations were performed in R v. 4.4.1 software. Statistical differences between the means of growth performance and feeding behaviour data were analysed with Student’s t-test, Welch’s t-test, the Wilcoxon–Mann–Whitney U-test, or the Brunner–Munzel test, according to the data distribution and variance. Statistical differences of the Kaplan–Meier survival curves were analysed by log-rank test. A statistical difference between two categorical variables, such as adult emergence, was analysed with Fisher’s exact test. Pearson’s or Spearman’s rank correlation coefficient tests were performed for all correlation analyses according to the data distribution.
3 Results
3.1 Aggregate size distribution
Aggregate sizes (median [first quartile, third quartile]) were 2.287 mm [2.593 mm, 2.800 mm] in the granular diet (n = 170) and 25.74 μm [16.20 μm, 39.83 μm] in the powdered diet (n = 327) (Figure 1). The size distributions did not overlap.
3.2 Growth performance of crickets
Fresh body mass of nymphs reared on the granular diet was significantly higher than those of nymphs reared on the powdered diet at 20, 27, 34 and 41 days after hatching (
3.3 Distance between mandibles of nymphal and adult crickets
The labrum width (mean ± SE) as an approximation of the distance between the mandibles (Figure 5A-D) was 0.223 ± 0.002 mm (n = 9) in first-instar nymphs and 1.853 ± 0.023 mm (n = 9) in adults. As the mandibles of crickets are often covered by the labrum when closed, it is difficult to measure their range. However, as the mandibles open during CO2 anaesthesia (personal observation), to assess the validity of labrum width as an approximation of the mandibular range, labrum width and the range of forced-open mandibles were measured in nymphs and adults (n = 9), which were different individuals from those mentioned above. The range of forced-open mandibles by CO2 anaesthesia showed a trend towards a positive correlation with the labrum width (
3.4 Preference of dietary form
Nymphal feeding frequencies (mean ± SE) on the granular and powdered diets, measured at a resolution of 1 min for 24 h, were 268.3 ± 33.4 (n = 4) and 24.3 ± 7.7 (n = 4), respectively, and there was a significant difference between them (
4 Discussion
As far as we know, ours is the first study to demonstrate that diet size affects the growth performance of crickets. Most of the growth parameters of Tel. occipitalis fed a millimetre-order granular diet were superior to those fed a micrometre-order powdered diet, even though the nutrient composition of the diets was identical.
Although the survival of crickets from hatching to first adult emergence (41 days after hatching) did not differ between the two diets, the body mass of nymphs and adults fed on the granular diet was higher than those of crickets fed on the powdered diet (Figure 2). However, because the body mass of insects often varies greatly depending on the timing of feeding and the water intake, the head width, pronotum length, pronotum width, forewing length and hind-leg femur length are often used as more accurate growth parameters of orthopteran insects (Arai and Watanabe, 2019). The lengths of all five of these body parts were significantly greater in adults that emerged from nymphs fed on the granular diet (Figure 3), and they had significant positive correlations with the adult body mass (Figure 4A-E); thus, body mass is also an accurate growth parameter, at least in Tel. occipitalis adults within a day after emergence. In contrast, adult body mass had a significant negative correlation with developmental time (Figure 4F). This result is not consistent with Masaki (1978) that head width and developmental time were positively correlated in the lawn ground cricket, Polionemobius taprobanensis (Walker) (Orthoptera: Trigonidiidae). In addition, there was no correlation between adult body mass and developmental time in Tel. occipitalis fed on chicken feed (unpublished data). The correlation between developmental time and adult body mass of crickets may vary with nutritional conditions during the nymphal period. Further studies are needed to investigate this hypothesis.
The feeding frequency, estimated from the position of the nymphal head that could access the diet, was significantly higher for the granular diet than for the powdered diet (Figure 6). The low frequency of nymphal access to the powdered diet (Figure 6B) suggests their avoidance of fine particles rather than the attractiveness of granular forms to them. The German cockroach, Blattella germanica (L.) (Blattodea: Blattellidae), is known to groom itself to remove dust from its body surface (El-Awami and Dent, 1995). In the field cricket, Tel. oceanicus (Le Guillou) (Orthoptera: Gryllidae), grooming is thought to be important for maintaining the working condition of the sensory apparatus (Lefebvre, 1981). Thus, at least for insects that exhibit grooming behaviour, fine particles adhering to the body are an object to be avoided, and this may be the reason why the frequency of feeding on powdered diet was lower than that on granulated diet.
Mantids – predatory polyneopteran insects – grasp their prey in their forelegs. The preferred prey size of the mantid, Hierodula crassa Giglio-Tos (Mantodea: Mantidae), is predictive of the lengths of the femur and tibia of the foreleg and the angle between them (Holling, 1964). Crickets, which are also polyneopteran insects, grasp their food mainly in their mandibles (Winkler et al., 2024). The width of labrum for estimating the mandibular range in Tel. occipitalis was 0.223 mm in first-instar nymphs and 1.853 mm in adults (Figure 5). Therefore, crickets that ingested granular diet (2.56 ± 0.03 mm; Figure 1) larger than their mandibular range first had to gnaw it down to a suitable size. In contrast, the powdered diet, with a smaller size (25.74 μm; Figure 1), was less suitable for grasping in the crickets’ mandibles. Initially, we hypothesized that the powdered diet with smaller aggregate sizes was difficult for crickets to grasp with their mandibles, resulting in reduced feeding compared to the granular diet and thus inhibiting their growth and development. However, if this is true, it would be reasonable to expect that crickets would increase their feeding frequency in order to obtain nutrients from the powder diet, which is potentially hard to grasp with their mandibles. In fact, however, the frequency of feeding on the powdered diet was clearly lower than that on the granular diet (Figure 6), suggesting that in the present study, avoidance behavior of crickets toward fine particles, rather than ease of grasping with their mandible, affected their growth and development.
5 Conclusion
The growth and development of Tel. occipitalis reared on a granular diet were significantly superior to those of Tel. occipitalis reared on a powdered but otherwise identical diet. This difference may be primarily due to the reduced frequency of feeding on the powdered diet, indicating crickets may exhibit avoidance behavior toward fine particles. In addition, a size that crickets’ mandibles can grasp could affect their ingestion. We plan to investigate these potential effects in order to optimize the form of diet in the production of edible crickets.
Corresponding author; e-mail: tszk@cc.tuat.ac.jp
Supplementary material
Supplementary material is available online at: https://doi.org/10.6084/m9.figshare.26983534
Acknowledgements
We thank Dr Masanobu Yamamoto, Mr Takuma Takahashi, and Dr Wuled Lenggoro of the Tokyo University of Agriculture and Technology (TUAT) for useful discussions and for sharing knowledge of particle suspensions in water. We also thank Dr Susumu Inasawa of TUAT for his guidance in the use of the scanning electron microscope. This work was supported partly by the Cabinet Office, Government of Japan Cross-ministerial Moonshot Agriculture, Forestry and Fisheries Research and Development Program, “Technologies for Smart Bio-industry and Agriculture” (funded by the Bio-oriented Technology Research Advancement Institution) (JPJ009237).
Conflict of interest
The authors have no conflict of interest to declare.
References
Arai, T. and Watanabe, U., 2019. Life history of Phonarellus ritsemai (Orthoptera: Gryllidae) (1) Egg development and egg period. Archives of Yamaguchi Prefectural University 12: 9-15. (In Japanese with English abstract).
El-Awami, I.O. and Dent, D.R., 1955. The interaction of surface and dust particle size on the pick-up and grooming behaviour of the German cockroach Blattella germanica. Entomologia Experimentalis et Applicata 77: 81-87. https://doi.org/10.1111/j.1570-7458.1995.tb01988.x
European Commission, 2022. Commission Implementing Regulation (EU) 2022/188 of 10 February 2022 authorising the placing on the market of frozen, dried and powder forms of Acheta domesticus as a novel food under Regulation (EU) 2015/2283 of the European Parliament and of the council, and amending Commission Implementing Regulation (EU) 2017/2470. Official Journal of the European Union 30: 108-114. Available at: http://data.europa.eu/eli/reg_impl/2022/188/oj
Gutiérrez, Y., Fresch, M., Ott, D., Brockmeyer, J. and Scherber, C., 2020. Diet composition and social environment determine food consumption, phenotype and fecundity in an omnivorous insect. Royal Society Open Science 7. https://doi.org/10.1098/rsos.200100
Hanboonsong, Y., Jamjanya, T. and Durst, P.B., 2013. Six-legged livestock: edible insect farming, collecting and marketing in Thailand. FAO, Bangkok, Thailand. Available at: http://www.fao.org/docrep/017/i3246e/i3246e00.pdf
Hayakawa, S., Kataoka, K., Yamamoto, M., Asahi, T. and Suzuki, T., 2024. DeepLabCut-based daily behavioural and posture analysis in a cricket. Biology Open 13: bio060237. https://doi.org/10.1242/bio.060237
Holling, C.S., 1964. The analysis of complex population processes. The Canadian Entomologist 96: 335-347. https://doi.org/10.4039/Ent96335-1
Kataoka, K., Minei, R., Ide, K., Ogura, A., Takeyama, H., Takeda, M., Suzuki, T., Yura, K. and Asahi, T., 2020. The draft genome dataset of the Asian cricket Teleogryllus occipitalis for molecular research toward entomophagy. Frontiers in Genetics 11: 470. https://doi.org/10.3389/fgene.2020.00470
Lefebvre, L., 1981. Grooming in crickets: Timing and hierarchical organization. Animal Behaviour 29: 973-984. https://doi.org/10.1016/S0003-3472(81)80050-4
Masaki, S., 1978. Climatic adaptation and species status in the lawn ground cricket: II. Body size. Oecologia 35: 343-356. https://doi.org/10.1007/BF00345141
Miech, P., Berggren, Å., Lindberg, J.E., Chhay, T., Khieu, B. and Jansson, A., 2016. Growth and survival of reared Cambodian field crickets (Teleogryllus testaceus) fed weeds, agricultural and food industry by-products. Journal of Insects as Food and Feed 2: 285-292. https://doi.org/10.3920/JIFF2016.0028
Naser El Deen, S., Spranghers, T., Baldacchino, F. and Deruytter, D., 2022. The effects of the particle size of four different feeds on the larval growth of Tenebrio molitor (Coleoptera: Tenebrionidae). European Journal of Entomology 119: 242-249. https://doi.org/10.14411/eje.2022.026
Oonincx, D.G.A.B., van Itterbeeck, J., Heetkamp, M.J.W., van den Brand, H., van Loon, J.J.A. and van Huis, A., 2010. An exploration on greenhouse gas and ammonia production by insect species suitable for animal or human consumption. PLOS ONE 5: 1-7. https://doi.org/10.1371/journal.pone.0014445
Patton, R.L., 1967. Oligidic diets for Acheta domesticus (Orthoptera: Gryllidae). Annals of the Entomological Society of America 60: 1238-1242. https://doi.org/10.1093/aesa/60.6.1238
Reece, F.N., Lott, B.D. and Deanton, J.W., 1985. The effects of feed form, grinding method, energy level, and gender on broiler performance in a moderate (21 C) environment. Poultry Science 64: 1834-1839. https://doi.org/10.3382/ps.0641834
Schneider, C.A., Rasband, W.S. and Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9: 671-675. https://doi.org/10.1038/nmeth.2089
Simmons, L.W., 2005. The evolution of polyandry: sperm competition, sperm selection, and offspring viability. Annual Review of Ecology, Evolution, and Systematics 36: 125-146. https://doi.org/10.1146/annurev.ecolsys.36.102403.112501
Svihus, B., Kløvstad, K.H., Perez, V., Zimonja, O., Sahlström, S., Schüller, R.B., Jeksrud, W.K. and Prestløkken, E., 2004. Physical and nutritional effects of pelleting of broiler chicken diets made from wheat ground to different coarsenesses by the use of roller mill and hammer mill. Animal Feed Science and Technology 117: 281-293. https://doi.org/10.1016/j.anifeedsci.2004.08.009
van Huis, A., 2013. Potential of insects as food and feed in assuring food security. Annual Review of Entomology 58: 563-583. https://doi.org/10.1146/annurev-ento-120811-153704
van Huis, A., van Itterbeeck, J., Klunder, H., Mertens, E., Halloran, A., Muir, G. and Vantomme, P., 2013. Edible insects: future prospects for food and feed security (No. 171). FAO, Rome, Italy. Available at: https://www.fao.org/4/i3253e/i3253e.pdf
Winkler, D.E., Seike, H., Nagata, S. and Kubo, M.O., 2024. Mandible microwear texture analysis of crickets raised on diets of different abrasiveness reveals universality of diet-induced wear. Interface Focus 14: 20230065. https://doi.org/10.1098/rsfs.2023.0065