Tetlow, Ellen
(2019)
Manipulation of the nutritional composition of Tenebrio monitor larvae (yellow mealworms) for use as a feed ingredient in aquaculture.
PhD thesis, University of Nottingham.
Abstract
The larvae of Tenebrio molitor, a species of coleopteran, have received increasing attention in recent years as a potential sustainable protein source to replace fishmeal (FM) in the feeding of fish. FM is produced using small pelagic fish and is a high price commodity that is considered to be a finite resource. The high price of FM, as a high-quality resource, limits the expansion possible in aquaculture pro- duction systems that are reliant on it as the major protein provision in the feed of farmed fish. Provision of a protein source that is sustainable, both environmentally and economically, will enable aquacultural systems to expand and meet the rising demand for healthy animal proteins. Fish have been associated with health benefits to consumers and have a high abundance of long-chain poly-unsaturated fatty acids (LC-PUFA) of the n-3 series, linked to anti-inflammatory effects. Fish require a dietary source of LC-PUFA, which they gain from FM and FM by-product fish oil (FO) in aquacultural systems. Consumption of feeds containing FM and FO helps to meet their nutritional requirements and prevent pathologies in the fish. In addition, FM and FO consumption results in an accumulation of LC-PUFA in the nutritional composition of the fish, from which humans can gain health benefits from consuming them. In order for aquacultural system expansion to help meet an increased meat demand per capita and a growing population com- plete replacement protein sources for FM will be required that provide a source of LC-PUFA in addition to suitable protein contents. If T. molitor have the endogenous capacity for LC-PUFA synthesis from dietary precursors, they could be a suitable FM and FO replacement.
The aims of this work were to successfully produce T. molitor larvae on a non-competing protein feed source and to determine the capacity to manipulate their nutritional content with feeding inputs. Of particular interest was the manipulation of specific fatty acids (FA) through various oil feeding inputs, as well as determining whether T. molitor have the ability to synthesis LC-PUFA endogenously. Further to this, an assessment of gene expression was undertaken in T. molitor larvae using RNA-Sequencing and de novo transcriptome assembly to establish potential target gene transcripts involved in endogenous lipid metabolism. Finally, techniques for production of T. molitor larvae were further assessed and optimised and their suitability as a FM replacement was assessed in Danio rario over a 10-week trial period.
The results indicated that T. molitor larvae can be as successfully produced on a non-competitor protein source, wheatbran (WB), over sow feed (SF), a conventional production animal feed. Larvae attained the same final weights and had significantly increased crude protein (P<0.001) and reduced crude lipid (P<0.001) contents, compared to SF treated larvae. Further to this FA composition in T. molitor larvae was successfully manipulated by oil inputs in feeds (P<0.05). T. molitor larvae mimicked feeding inputs of specific oils and achieved a >5-fold ac- cumulation in n-3 alpha-linolenic acid (ALA) through feeding with linseed oil in wheatbran (P<0.001).
Feeding with linseed oil at 5% (w/w) in a basal feed of WB resulted in 2-fold and significant differential expression in 41 gene transcripts (P<0.05). The up-regulation of genes associated with transcription factor, immunity and lipolysis function were of particular interest and suggested potential for n-3 PUFA modulated effects in larvae gene expression. Furthermore, the potential identification of a delta-12 desaturase enzyme means T. molitor have additional FA synthesis capacities above that observed in mammals, birds and fish which is a novel finding and of particular interest. The presence of this enzyme suggests T. molitor have the capacity to elongate to n-6 linoleic acid (LA) from n-9 oleic acid, without the requirement of dietary inputs. Mammals, birds and fish require a dietary source to obtain n-6 LA as they are unable to sythesise LA endogenously.
In comparison to larvae grown in a 13h:11h, light:dark environment, larvae grown in complete darkness had significantly increased growth rates, (P<0.001), whilst consuming the same amount of feed and had unaltered gross energy, crude fat, crude protein and FA compositions. These findings will enable production cost savings on both electricity and space, in addition to an increased efficiency and biomass accumulation in T. molitor.
Finally, T. molitor were found to successfully replace FM in the feeds of D. rario at 20% replacement of FM and inclusion of T. molitor meal (TM) prepared by feeding WB with an addition of rapeseed oil, which had an overall FA composition that was below the n-3 ALA requirement of the fish. Fish feed containing T. molitor larvae supplemented with 5% linseed oil, in a WB basal feed, resulted in significantly reduced deaths in fish compared with TM feeds with additions of rapeseed and/or linseed oil during feed formulations, indicating increased palatability. Replacement of FM in fish with various TM meals had no impact on the FA profiles for LC-PUFAs ARA, EPA and DHA in the fish, which could support the presence of LC-PUFA in T. molitor larvae that meet fish requirements or suggests 30% FM that remained in the treatment feeds is enough to meet the LC-PUFA requirements of the fish.
This thesis increased the body of knowledge on the suitability of T. molitor larvae as an alternative for FM in the feeding of fish. The novel findings, that T. molitor have higher crude protein content when fed on a non-production animal feed indicates that there is a further benefit of producing them on WB, which is a non-competing protein source. In addition, optimisation of rearing methods, such as the increased efficiency of larvae produced in complete darkness, will enable considerable cost reductions. Furthermore, the ability to manipulate FA content of T. molitor with feeding oils, similar to other monogastric animals, have great implications for their use as an n-3 PUFA-enriched protein source in the future. In turn, T. molitor successfully replaced 20% FM in a freshwater fish species.
The discovery of a non-mammalian desaturase activity in T. molitor is of great interest, as mammals cannot insert unsaturation above delta-9 position. A delta-12 desaturase means that T. molitor are not reliant on plant sources for provision of LA which is a n-6 LC-PUFA precursor. Further evaluation of this DN 53206 T. molitor transcript is necessary as well as annotation of the 33 T. molitor gene transcripts that were not classified. The large variation exhibited within treatment groups suggests biological variation, which could be exploited through selective breeding programs.
There is a requirement for further work to discover the actual capacity of T. molitor for endogenous LC-PUFA synthesis, which was inconclusive in this work and further fish trial work replacing FM in fish species consumed by humans, particularly carnivorous species, with a greater requirement for LC-PUFA.
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