Hermetia illucens in diets for zebrafish (Danio rerio): A study of bacterial diversity by using PCR-DGGE and metagenomic sequencing

Autoři: Andrea Osimani aff001;  Vesna Milanović aff001;  Andrea Roncolini aff001;  Paola Riolo aff001;  Sara Ruschioni aff001;  Nunzio Isidoro aff001;  Nino Loreto aff001;  Elena Franciosi aff002;  Kieran Tuohy aff002;  Ike Olivotto aff003;  Matteo Zarantoniello aff003;  Federica Cardinali aff001;  Cristiana Garofalo aff001;  Lucia Aquilanti aff001;  Francesca Clementi aff001
Působiště autorů: Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, Università Politecnica delle Marche, Ancona, Italy aff001;  Food Quality and Nutrition Department (DQAN), Research and Innovation Center, Fondazione Edmund Mach (FEM), San Michele all’Adige, Italy aff002;  Dipartimento di Scienze della Vita e dell'Ambiente, Università Politecnica delle Marche, Ancona, Italy aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0225956


In the present research, bacterial diversity was studied during a 6-month feeding trial utilizing zebrafish (Danio rerio) fed Hermetia illucens reared on different substrates with an emphasis on fish gut bacterial diversity. A polyphasic approach based on viable counting, PCR-DGGE and metagenomic 16S rRNA gene amplicon target sequencing was applied. Two different H. illucens groups were reared on coffee by-products (C) or a mixture of vegetables (S). Viable counts showed a wide variability based on substrate. PCR-DGGE and Illumina sequencing allowed the major and minor bacterial taxa to be detected. Both samples of larvae and their frass reared on the S substrate showed the highest richness and evenness of bacterial communities, whereas zebrafish (ZHC) fed H. illucens reared on substrate C and zebrafish (ZHS) fed H. illucens reared on substrate S had the lowest bacterial richness and evenness. A stimulating effect of bioactive compounds from coffee by-products on the occurrence of Lactobacillaceae and Leuconostoccaceae in H. illucens reared on substrate C has been hypothesized. Zebrafish gut samples originating from the two feeding trials showed complex microbial patterns in which Actinobacteria and Alteromonadales were always detected, irrespective of the diet used. Enterobacteriaceae in fish guts were more abundant in ZHS than in ZHC, thus suggesting an influence of the bioactive compounds (chlorogenic and caffeic acids) in the substrate on Enterobacteriaceae in fish guts. ZHC showed a higher abundance of Clostridia than did ZHS, which was likely explained by stimulating activity on the bacteria in this class by the bioactive compounds contained in H. illucens reared on substrate C. An influence of the microbiota of H. illucens or insect-derived bioactive compounds on the gut microbiota of zebrafish has been suggested. The presence of bacteria consistently associated with zebrafish guts has been found irrespective of the diet, thus attesting to the likely stability of the core fish microbiota.

Klíčová slova:

Diet – Enterobacteriaceae – Insects – Larvae – Microbiome – Sequence databases – Vegetables – Zebrafish


1. Oonincx DG, de Boer IJ. Environmental impact of the production of mealworms as a protein source for humans—a life cycle assessment. PLoS One. 2012; 7: e51145. doi: 10.1371/journal.pone.0051145 23284661

2. Oonincx DG, van Itterbeeck J, Heetkamp MJ, van den Brand H, van Loon JJ, van Huis A. An exploration on greenhouse gas and ammonia production by insect species suitable for animal or human consumption. PLoS One. 2010; 5: e14445. doi: 10.1371/journal.pone.0014445 21206900

3. Rumpold BA, Schlüter OK. Nutritional composition and safety aspects of edible insects. Molecul Nutr Food Res. 2013; 57: 802–823.

4. van Huis A, Van Itterbeeck J, Klunder H, Mertens E, Halloran A, Vantomme P. Edible insects: Future prospects for food and feed security. Rome: Food and Agriculture Organization of the United Nations. FAO Forestry Paper, FAO. 2013; 187 pp. Eds. http://www.fao.org/docrep/018/i3253e/i3253e14.pdf

5. EFSA Scientific Committee, 2015. Scientific opinion on a risk profile related to production and consumption of insects as food and feed. EFSA J. 13(10) (4257), 60. http://dx.doi.org/10.2903/j.efsa.2015.4257

6. ANSES (2015) Opinion of the French Agency for Food. In: Environmental and Occupational Health & Safety on “the Use of Insects as Food and Feed and the Review of Scientific Knowledge on the Health Risks Related to the Consumption of Insects” Maisons-Alfort, France.

7. Garofalo C, Milanović V, Cardinali F, Aquilanti L, Clementi F, Osimani A. Current knowledge on the microbiota of edible insects intended for human consumption: A state-of-the-art review. Food Res Int. 2019; 125: 108527 doi: 10.1016/j.foodres.2019.108527 31554102

8. Commission Regulation (EU) 2017/893 of 24 May 2017 amending Annexes I and IV to Regulation (EC) No 999/2001 of the European Parliament and of the Council and Annexes X, XIV and XV to Commission Regulation (EU) No 142/2011 as regards the provisions on processed animal protein. Official Journal of the European Union, L 138/92-116. Available at: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32017R0893&from=IT

9. Cardinaletti G, Randazzo B, Messina M, Zarantoniello M, Giorgini E, Zimbelli A, et al. Effects of Graded Dietary Inclusion Level of Full-Fat Hermetia illucens Prepupae Meal in Practical Diets for Rainbow Trout (Oncorhynchus mykiss). Animals. 2019; 9, pii:E25. doi: 10.3390/ani9010025

10. Cutrignelli MI, Messina M, Tulli F, Randazzo B, Olivotto I, Gasco L, et al. Evaluation of an insect meal of the Black Soldier Fly (Hermetia illucens) as soybean substitute: Intestinal morphometry, enzymatic and microbial activity in laying hens. Res Vet Sci. 2018; 117: 209–215. doi: 10.1016/j.rvsc.2017.12.020 29304440

11. Diener S, Zurbrügg C, Tockner K. Conversion of organic material by black soldier fly larvae: Establishing optimal feeding rates. Waste Manage Res. 2009; 27: 603–610.

12. Vargas A, Randazzo B, Riolo P, Truzzi C, Gioacchini G, Giorgini E, et al. Rearing Zebrafish on Black Soldier Fly (Hermetia illucens): Biometric, Histological, Spectroscopic, Biochemical, and Molecular Implications. Zebrafish. 2018; 15: 404–419. doi: 10.1089/zeb.2017.1559 29589997

13. Zarantoniello M, Randazzo B, Truzzi C, Giorgini E, Marcellucci C, Vargas-Abúndez JA, et al. A six-months study on Black Soldier Fly (Hermetia illucens) based diets in zebrafish. Sci Rep. 2019; 9: 8598. doi: 10.1038/s41598-019-45172-5 31197206

14. Vogel H, Müller A, Heckel DG, Gutzeit H, Vilcinskas A. Nutritional immunology: Diversification and diet-dependent expression of antimicrobial peptides in the black soldier fly Hermetia illucens. Dev Comp Immunol. 2018; 78: 141–148. doi: 10.1016/j.dci.2017.09.008 28966127

15. Bruno D, Bonelli M, De Filippis F, Di Lelio I, Tettamanti G, Casartelli M, et al. The Intestinal Microbiota of Hermetia illucens Larvae Is Affected by Diet and Shows a Diverse Composition in the Different Midgut Regions. Appl Environ Microbiol. 2019; 85, pii: e01864–18. doi: 10.1128/AEM.01864-18 30504212

16. De Smet J, Wynants E, Cos P, Van Campenhout L. Microbial Community Dynamics during Rearing of Black Soldier Fly Larvae (Hermetia illucens) and Impact on Exploitation Potential. Appl Environ Microbiol. 2018; 84, pii: e02722–17. doi: 10.1128/AEM.02722-17 29475866

17. Wynants E, Frooninckx L, Crauwels S, Verreth C, De Smet J, Sandrock C, et al. Assessing the Microbiota of Black Soldier Fly Larvae (Hermetia illucens) Reared on Organic Waste Streams on Four Different Locations at Laboratory and Large Scale. Microb Ecol. 2019; 77: 913–930. doi: 10.1007/s00248-018-1286-x 30430196

18. Varotto Boccazzi I, Ottoboni M, Martin E, Comandatore F, Vallone L, Spranghers T, et al. A survey of the mycobiota associated with larvae of the black soldier fly (Hermetia illucens) reared for feed production. PLoS One. 2017; 12: e0182533. doi: 10.1371/journal.pone.0182533 28771577

19. Zarantoniello M, Bruni L, Randazzo B, Vargas A, Gioacchini G, Truzzi C, et al. Partial Dietary Inclusion of Hermetia illucens (Black Soldier Fly) Full-Fat Prepupae in Zebrafish Feed: Biometric, Histological, Biochemical, and Molecular Implications. Zebrafish. 2018; 15: 519–532. doi: 10.1089/zeb.2018.1596 29912648

20. Osimani A, Cardinali F, Aquilanti L, Garofalo C, Roncolini A, Milanović V, et al. Occurrence of transferable antibiotic resistances in commercialized ready-to-eat mealworms (Tenebrio molitor L.). Int J Food Microbiol. 2017; 263: 38–46. doi: 10.1016/j.ijfoodmicro.2017.10.009 29028569

21. Garofalo C, Silvestri G, Aquilanti L, Clementi F. PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone. J Appl Microbiol. 2008; 105: 243–254. doi: 10.1111/j.1365-2672.2008.03768.x 18312562

22. Milanović V, Osimani A, Garofalo C, De Filippis F, Ercolini D, Cardinali F, et al. Profiling white wine seed vinegar bacterial diversity through viable counting, metagenomic sequencing and PCR-DGGE. Int J Food Microbiol. 2018; 286: 66–74. doi: 10.1016/j.ijfoodmicro.2018.07.022 30048915

23. Ampe F, Ben Omar N, Moizan C, Wacher C, Guyot JP. Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations. Appl Environ Microbiol. 1999; 65: 5464–5473. 10584005

24. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215: 403–410. doi: 10.1016/S0022-2836(05)80360-2 2231712

25. Baker GC, Smith JJ, Cowan DA. Review and re-analysis of domain-specific 16S primers. J Microbiol Method. 2003; 55: 541–555.

26. Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, et al. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res. 2010; 38: e200–e200. doi: 10.1093/nar/gkq873 20880993

27. Osimani A, Milanović V, Cardinali F, Garofalo C, Clementi F, Pasquini M, et al. The bacterial biota of laboratory-reared edible mealworms (Tenebrio molitor L.): from feed to frass. Int J Food Microbiol. 2018; 272: 49–60. doi: 10.1016/j.ijfoodmicro.2018.03.001 29525619

28. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushma FD. Costello E.K., et al. QIIME allows analysis of highthroughput community sequencing data. Nat Methods. 2010; 7: 335–336. doi: 10.1038/nmeth.f.303 20383131

29. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holme S.P. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016; 13: 581. doi: 10.1038/nmeth.3869 27214047

30. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30: 772–780. doi: 10.1093/molbev/mst010 23329690

31. Price MN, Dehal PS, Arkin AP. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol. 2009; 26: 1641–165030. doi: 10.1093/molbev/msp077 19377059

32. Vazquez-Baeza Y, Pirrung M, Gonzalez A, Knight R. EMPeror: a tool for visualizing high-throughput microbial community data. GigaScience. 2013; 2: 16. doi: 10.1186/2047-217X-2-16 24280061

33. Lalander C, Diener S, Magri ME, Zurbrügg C, Lindström A, Vinnerås B. Faecal sludge management with the larvae of the black soldier fly (Hermetia illucens)—From a hygiene aspect. Sci Total Environ. 2013; 458–460: 312–318. doi: 10.1016/j.scitotenv.2013.04.033 23669577

34. Costa ASG, Alves RC, Vinha AF, Costa E, Costa S.G, Nunes MA, et al. Nutritional, chemical and antioxidant/pro-oxidant profiles of silverskin, a coffee roasting by-product. Food Chem. 2018; 267: 28–35. doi: 10.1016/j.foodchem.2017.03.106 29934169

35. Filannino P, Bai YP, Di Cagno R, Gobbetti M, Gänzle MG. Metabolism of phenolic compounds by Lactobacillus spp. during fermentation of cherry juice and broccoli puree. Food Microbiol. 2015; 46: 272–279. doi: 10.1016/j.fm.2014.08.018 25475296

36. Fritsch C, Heinrich V, Vogel RF, Toelstede S. Phenolic acid degradation potential and growth behavior of lactic acid bacteria in sunflower substrates. Food Microbiol. 2016; 57: 178–86. doi: 10.1016/j.fm.2016.03.003 27052717

37. Parkar SG, Stevenson DE, Skinner MA. The potential influence of fruit polyphenols on colonic microflora and human gut health. Int J Food Microbiol. 2008; 124: 295–298. doi: 10.1016/j.ijfoodmicro.2008.03.017 18456359

38. Osimani A, Milanović V, Garofalo C, Cardinali F, Roncolini A, Sabbatini R, et al. Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR. Int J Food Microbiol. 2018; 276: 54–62. doi: 10.1016/j.ijfoodmicro.2018.04.013 29665523

39. Berini F, Katz C, Gruzdev N, Casartelli M, Tettamanti G, Marinelli F. Microbial and viral chitinases: Attractive biopesticides for integrated pest management. Biotechnol Adv. 2018; 36: 818–838. doi: 10.1016/j.biotechadv.2018.01.002 29305895

40. Kroeckel S, Harjes AGE, Roth I, Katz H, Wuertz S, Susenbeth A, et al. When a turbot catches a fly: Evaluation of a pre-pupae meal of the Black Soldier Fly (Hermetia illucens) as fish meal substitute—Growth performance and chitin degradation in juvenile turbot (Psetta maxima). Aquaculture. 2012; 364–365: 345–352.

41. Jiang CL, Ji W.Z, Tao XH, Zhang Q, Zhu J, Feng SY, et al. Black soldier fly larvae (Hermetia illucens) strengthen the metabolic function of food waste biodegradation by gut microbiome. Microb Biotechnol. 2019; 12: 528–543. doi: 10.1111/1751-7915.13393 30884189

42. Kämpfer P, Martin K, Schäfer J, Schumann P. Kytococcus aerolatus sp. nov., isolated from indoor air in a room colonized with moulds. Syst Appl Microbiol. 2009; 32: 301–305. doi: 10.1016/j.syapm.2009.05.004 19541443

43. Zhang Y, Wang Y, Chen D, Yu B, Zheng P, Mao X, et al. Dietary chlorogenic acid supplementation affects gut morphology, antioxidant capacity and intestinal selected bacterial populations in weaned piglets. Food Funct. 2018; 9: 4968–4978. doi: 10.1039/c8fo01126e 30183786

44. Mitchell RF, Hanks LM. Insect frass as a pathway for transmission of bacterial wilt of cucurbits. Environ Entomol. 2009; 38: 395–403. doi: 10.1603/022.038.0212 19389288

45. Anbutsu H, Togashi K. Oviposition deterrence associated with larval frass of the Japanese pine sawyer, Monochamus alternatus (Coleoptera: Cerambycidae). J Insect Physiol. 2002; 48: 459–465. doi: 10.1016/s0022-1910(02)00067-7 12770095

46. Yang CC, Kim MS, Millner P, Chao K, Cho BK, Mo C, et al. Development of multispectral imaging algorithm for detection of frass on mature red tomatoes. Postharvest Biol Technol. 2014; 93: 1–8.

47. Grabowski NT, Klein G. Bacteria encountered in raw insect, spider, scorpion, and centipede taxa including edible species, and their significance from the food hygiene point of view. Trends Food Sci Technol. 2017; 63: 80–90.

48. Milanović V, Cardinali F, Aquilanti L, Garofalo C, Roncolini A, Sabbatini R, et al. A Glimpse into the Microbiota of Marketed Ready-to-Eat Crickets (Acheta domesticus). Indian J Microbiol. 2019; https://doi.org/10.1007/s12088-019-00817-x

49. Nziza J, Tumushime JC, Cranfield M, Ntwari AE, Modrý D, Mudakikwa A, et al. Fleas from domestic dogs and rodents in Rwanda carry Rickettsia asembonensis and Bartonella tribocorum. Med Vet Entomol. 2019; 33: 177–184. doi: 10.1111/mve.12340 30390316

50. Breitschwerdt EB, Maggi RG, Chomel BB, Lappin MR. Bartonellosis: an emerging infectious disease of zoonotic importance to animals and human beings. J Vet Emerg Crit Care (San Antonio). 2010; 20: 8–30.

51. Fernandez-Gomez B, Lezama A, Amigo-Benavent M, Ullate M, Herrer M., Martín MA, et al. Insights on the health benefits of the bioactive compounds of coffee silverskin extract. J Funct Foods. 2016; 25: 197–207.

52. Qiao R, Deng Y, Zhang S, Wolosker MB, Zhu Q, Ren H, et al. Accumulation of different shapes of microplastics initiates intestinal injury and gut microbiota dysbiosis in the gut of zebrafish. Chemosphere. 2019; 236: 124334. doi: 10.1016/j.chemosphere.2019.07.065 31310986

53. Xue S, Xu W, Wei J, Sun J. Impact of environmental bacterial communities on fish health in marine recirculating aquaculture systems. Vet Microbiol. 2017; 203: 34–39. doi: 10.1016/j.vetmic.2017.01.034 28619164

54. Rimoldi S, Gini E, Iannini F, Gasco L, Terova G. The Effects of Dietary Insect Meal from Hermetia illucens Prepupae on Autochthonous Gut Microbiota of Rainbow Trout (Oncorhynchus mykiss). Animals (Basel). 2019; 9, pii: E143.

55. Nurul ANA, Muhammad DD, Okomoda VT, Nur AAB. 16S rRNA-Based metagenomic analysis of microbial communities associated with wild Labroides dimidiatus from Karah Island, Terengganu, Malaysia. Biotechnol Rep (Amst.). 2019; 21: e00303.

56. Mills CE, Tzounis X, Oruna-Concha MJ, Mottram DS, Gibson GR, Spencer JP. In vitro colonic metabolism of coffee and chlorogenic acid results in selective changes in human faecal microbiota growth. Br J Nutr. 2015; 113: 1220–1227. doi: 10.1017/S0007114514003948 25809126

57. Bruni L, Pastorelli R, Viti C, Gasco L, Parisi G. Characterisation of the intestinal microbial communities of rainbow trout (Oncorhynchus mykiss) fed with Hermetia illucens (black soldier fly) partially defatted larva meal as partial dietary protein source. Aquaculture. 2018; 487: 56–63.

58. Huyben D, Vidaković A, Werner Hallgren S, Langeland M. High-throughput sequencing of gut microbiota in rainbow trout (Oncorhynchus mykiss) fed larval and pre-pupae stages of black soldier fly (Hermetia illucens), Aquaculture. 2019; 500: 485–491.

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden