Dietary zinc enrichment reduces the cadmium burden of mealworm beetle (Tenebrio molitor) larvae

The industrial production of Tenebrio molitor L. requires optimized rearing and processing conditions to generate insect biomass with high nutritional value in large quantities. One of the problems arising from processing is a tremendous loss in mineral accessibility, affecting, amongst others, the essential trace element Zn. As a feasible strategy this study investigates Zn-enrichment of mealworms during rearing to meet the nutritional requirements for humans and animals. Following feeding ZnSO4-spiked wheat bran substrates late instar mealworm larvae were evaluated for essential micronutrients and human/animal toxic elements. In addition, growth rate and viability were assessed to select optimal conditions for future mass-rearing. Zn-feeding dose-dependently raised the total Zn content, yet the Znlarvae/Znwheat bran ratio decreased inversely related to its concentration, indicating an active Zn homeostasis within the mealworms. The Cu status remained stable, suggesting that, in contrast to mammals, the intestinal Cu absorption in mealworm larvae is not affected by Zn. Zn biofortification led to a moderate Fe and Mn reduction in mealworms, a problem that certainly can be overcome by Fe/Mn co-supplementation during rearing. Most importantly, Zn feeding massively reduced the levels of the human/animal toxicant Cd within the mealworm larvae, a technological novelty of outstanding importance to be implemented in the future production process to ensure the consumer safety of this edible insect species.

Mealworm drying even aggravates this problem 20 . A promising strategy for improving the mineral quality of T. molitor could be the use of Zn-enriched feed during breeding 20 . In this regard, the mealworm Zn tolerance needs to be determined, in order to minimize feed-aversion induced growth delays and mortality during rearing 21 . Moreover, excess Zn and minerals ingested with the feed may compete for intestinal transporters and intracellular chaperones in order to be efficiently delivered across the gut epithelium into the hemolymph. For mammals, intestinal competition between Zn and several other essential micronutrients (Cu, Fe, Ca) or toxic heavy metals (Pb, Cd) has been described 22,23 . Hence, the aim of this study was to investigate the potential dimensions of Zn enrichment in T. molitor during late instar larval development, when providing a ZnSO 4 -spiked wheat brain diet. Furthermore, it was to be investigated to what extent this fortification impacts the larval composition in essential and toxic elements, respectively. These results will be of utmost importance when aiming to improve T. molitor processing technologies, a basic prerequisite for utilizing mealworms as novel food or animal feed in the future.

Results
Larval growth parameters. Diet composition is one of the main variables determining the efficiency of feed conversion into biomass for a given insect species in industrial mass rearing. T. molitor larvae fed over the whole period with Zn basal wheat bran (Fig. 1) did effectively increase their starting L 3-4 weight by a factor of 40,  Table 1. Larval growth and feed utilization parameters. T. molitor larvae (start: 60 larvae; developmental stage L 3-4 ) were fed with Zn-spiked wheat bran feed for 8 weeks (end: developmental stage L 9-10 ); # see Table 2 for total Zn content of the wheat bran material. LWG total / total live weight gain larvae; average larval weight; FC/ total feed consumption; ECI/ efficiency of ingested feed conversion; FCR/ feed conversion ratio; total feces release during rearing; FA/ feed assimilation; ECD/ efficiency of digested feed conversion; Data are shown as means ± SEM of 5 replicates. Statistically significant differences from control are indicated (*p < 0.05, **p < 0.01, ***p < 0.001) one-way ANOVA with Dunnett's post hoc test. www.nature.com/scientificreports/ with very low mortality rate (Table 1). Overall 15.0 ± 0.5 g (around 75%) of the total offered Zn basal wheat bran material was consumed (FC) with a total assimilation (FA) of 6.7 ± 0.3 g. The increase in larval body weight is reflected in a favorable feed conversion ratio (FCR 5.1 ± 0.1) and feed conversion efficiency (ECI 19.5 ± 0.3%) ( Table 1). Mealworms grown in any of the Zn-enriched wheat bran materials also remained highly viable until late instar, but were significantly lower in fresh weight at stage L 9-10 (52.3 ± 1.6 mg Zn basal vs. 42.4 ± 1.8 mg Zn 40 ; ANOVA with Dunnett's post hoc test Zn basal vs Zn 40 p < 0.01), corresponding to a loss in biomass of around 20% (Table 1). Total feed consumption over the eight weeks feeding period was almost the same for the different groups, suggesting that the animals had no aversions against the spiked wheat bran, even though the added ZnSO 4 was in the range of gustatory impact (0.35% w/w in the Zn 40 wheat bran 21 ). Likewise, total feces production was comparable, indicating that the overall amount of feed available for assimilation and growth (FA) was similar between the different feeding groups. Nevertheless, the slightly diminished feed conversion efficiency (ECI and ECD) of the Zn-enriched wheat bran (Table 1) suggests certain post-absorptive nutrient imbalances of the larvae, when starting from Zn-enriched bran, which seems to be restrictive for mealworm growth and development.
Zn-enrichment in T. molitor larvae. Based on FC, total Zn intake for the Zn basal group during the feeding period was estimated to be 1.4 ± 0.1 mg ( ∼ 24.3 ± 0.9 µg Zn/ animal; Fig. 2B). Total Zn content in the final biomass of the Zn basal L 9-10 larvae was 116.4 ± 4.3 mg kg −1 dry weight ( Fig. 2A). Yet the larvae are higher in total Zn than the feeding material, indicating an accumulation of the essential mineral within the animals (Fig. 2C). Wheat bran contains significant amounts of phytate, an anti-nutrient inhibiting intestinal Zn absorption in both invertebrates and vertebrates 24,25 . Assuming a typical phytate content of ∼ 6000 mg/100 g wheat bran 26 , the estimated phytate/Zn molar ratio for the Zn basal feed corresponds to 66, which might be disadvantageous for larval midgut Zn accessibility. In the Zn-spiked feed this ratio is shifted in favor of Zn, decreasing the molar phytate/Zn-ratio to 3.8 for the Zn 5 -wheat bran and down to 1.7 for Zn 40 -wheat bran. We observed a rise in larval Zn content with elevated level of Zn in the wheat bran, yielding up to a maximum of 309.0 ± 0.5 mg Zn kg −1 larval dry mass for the Zn 40 group ( Fig. 2A). The Zn larvae /Zn wheat bran ratio, however, decreased with increasing Zn concentration in the feed from 1.

Discussion
The need to find innovative sources for food and feed production has led to an increased recognition of insects 2,27,28 . Currently, about 92% of the ~ 2000 edible insect species worldwide is wild harvested 29 . However, this is no longer an option with regard to economics of food and feed production, as there is a compelling need to farm the insects in mass quantities. Consequently, present-day insect industry is looking for reliable, consistent ways to scale their production in order to compete with other sources of livestock feed while guaranteeing safety and high nutritive quality of insects 2 .
T. molitor L. is a promising candidate and already grown by mass rearing 10,11 . There are several studies examining rearing conditions and the effect of different substrates on mealworm larval development 16,17,30,31 . The current study confirmed the larval fitness and growth response when grown on pure wheat bran 20 . The estimated FCR for the Zn basal group (value 5.1) is almost identical to the one published by Melis et al. 2019 for wheat bran-fed mealworm larvae 32 , confirming the potency of this species to convert the bran feed into body weight of appropriate proximate composition 20,32 . However, dietary efficiency seems to be related to the micronutrient composition of the diet, as biomass output decreased with elevated Zn feeding. Feed avoidance due to excessive heavy metal concentrations (> 0.1% Zn 2+ ) is common in terrestrial insects 21 , but was not observed in the present study. Recent observations depict the role of the Zn 2+ -gated midgut sensor Hodor in controlling Drosophila melanogasters food intake behavior, to ensure insect growth even under macronutrient-scarce conditions 33 . This mechanism, however, seems to be of minor importance in the present study, as mealworms were fed with a rather high-calorie wheat bran diet.
Developmental retardation has already been observed in Zn feeding trials with other terrestrial insects 34,35 . This could be the outcome of systemic Zn intoxication. Besides, Zn might restrict the systemic availability of other essential minerals in the larvaes' midgut 22,36 . Like any other organism, T. molitor requires trace elements for survival. A 6 ppm zinc supplementation was established by Fraenkel 37 for optimal larval growth in synthetic casein/glucose-based minimal media. Hence, feeding wheat bran material containing a total of 89.4 mg Zn kg −1 (89.4 ppm) is seemingly an excess of Zn. However, the abundance of the anti-nutritive mineral antagonist phytate in wheat bran (estimated phytate/Zn molar ratio of 66) provides a rather unfavorable matrix for intestinal Zn  38 . Still, the measured total Zn content for Zn basal T. molitor larvae of around 120 mg kg −1 dry weight surpasses the wheat bran (Zn larvae /Zn wheat bran ratio > 1). Accordingly, the mealworm larvae seem, at least partially, to abolish the Zn-antagonist throughout the digestion process, either by activating phytogenic phytase or phytases of gut-associated bacteria 17,39 . Feeding Zn-enriched wheat bran led to the intended Zn fortification of the insects, in the same order of magnitude as in a previous study 40 . Considering the estimated Zn-bioaccessibility upon human intestinal digestion of 40% 20 , an intake of ~ 30 g Zn 40 -freeze dried  Table 1). (A) Larval Zn content determined by ICP-MS normalized to the weight of the freeze dried animals. Statistically significant differences from control are indicated (*p < 0.05, **p < 0.01, ***p < 0.001; oneway ANOVA with Dunnett's post hoc test) (B) Total Zn intake over the feeding period was calculated from feed consumption data (see Table 2). (C) Zn larvae /Zn wheat bran ratio. Data are shown as means + SEM of five replicates. www.nature.com/scientificreports/ mealworms would be more than sufficient for an adult to replenish the daily zinc losses 41 . Due to their reduced total Fe content, consumption of Zn 40 -T. molitor larvae would result in lower Fe intake compared to food prepared from conventionally reared animals. Yet, the Fe content is still in excess of most other food sources, making even Zn 40 -larvae potentially useful for preventing Fe deficiency and contributing significantly to meeting the nutritional Fe requirements 42,43 . In any way, introducing Zn-biofortified mealworm larvae for use in food and feed will necessitate a copious assessment of the potential implications not just of their Fe content, but of their total macronutrient and vitamin composition as well as an examination of biological and chemical contaminants. This is crucial for avoiding long-term risks and adverse health effects for consumers [5][6][7] . The Zn larvae /Zn wheat bran ratio decreased inversely related to its concentration in the diet. Thus T.molitor larvae, similar to the mass rearing insect Hermetia illucens 44 , appear to be metal-deconcentrators 45 , able to adjust their internal Zn levels through homeostatic adaption of Zn absorption and excretion. Movement of Zn ions across the insect midgut epithelium, nowadays best described for the holometabolous insect D. melanogaster, requires the concerted interplay of solute carrier (SLC)39/Zrt, Irt-like protein (ZIP) Zn importers, SLC30/Zn transporter (ZnT) Zn exporters and cysteine-rich metal-binding metallothioneins (MT). Amongst the 10 ZIP and 7 ZnT proteins encoded in the Drosophila genome, the two importers dZIP42C.1 and dZIP42C.2, along with the exporter dZnT63C, are predominantly involved in dietary Zn absorption. Moreover, their expression is sensitive to both excessive and insufficient Zn supply 46 . Currently, 1219 insect genome-sequencing projects have been registered within the National Center for Biotechnology Information, among them 174 for Coleoptera, but the nuclear genome of T. molitor has not yet been unraveled 47 . According to our BLAST searches homologs of D. melanogasters dZIPs and dZnTs as well as the metal-responsive transcription factor-1 (dMTF-1) are encoded in Tenebrionidae genomes (Suppl Tables 1 and 2; Suppl Fig. 1-3). Notably, insects within this superfamily seem to lack the classical Cys-MTs, as the metal-buffering activity within midgut cells of T. molitor was shown to be mediated by other low molecular weight proteins rich in Asp/Glu 48 . Elucidating the expression, activity and tissue distribution of all these proteins during the lifecycle of T. molitor would not only contribute to the overall knowledge on Zn homeostasis in insects, but particularly provide decisive advances when aiming to raise the mealworm Zn enrichment quota by implementing Zn transporter-targeted strategies into rearing 49 .
Cu, Mn, and Fe contents of T. molitor larvae grown on non-spiked wheat bran were close to values reported for mealworm larvae in other feeding trials 19,50 , underlining the micronutrient quality of the applied bran material. Yet, the metals were differently impacted by Zn-biofortification. The Cu status of the mealworms remained stable, irrespective of Zn dosing. This is contrary to the situation in mammals, where prolonged high Zn feeding causes systemic copper-deficiency, possibly due to overexpression of intestinal apo-MT retaining the copper ions inside the enterocytes 51 . Insect midgut function seems to be strictly regionalized 52,53 . Cu uptake occurs primarily within the so-called "copper cell region", where, at least in D. melanogaster, Zn was shown to slightly colocalize 33,54 . Nevertheless, the bivalent Zn 2+ does not compete with cellular Cu uptake via the high-affinity Cu + transporter Ctr1 55 . Cellular Zn-sensing by the metal regulatory transcription factor MTF-1 is critical with www.nature.com/scientificreports/ regard to triggering the expression of Zn-sensitive genes (e.g., metallothionein genes) in any species-including insects 56 . In addition to its conserved zinc fingers within the DNA-binding domain D. melanogaster MTF-1 contains a unique C-terminal cysteine-enriched copper-cluster that allows specific intracellular copper monitoring; consequently dMTF-1 keeps the cellular metallothionein gene transcription low under limited Cu supply and enhanced upon copper overload 57 . A homologous Cu-cluster sequence is present in the predicted protein of the beetle Tribolium castaneum, wheras Asbolus verrucosus MTF-1 is C-terminally truncated, thus missing this domain (Suppl Fig. 3). The outcome of the 1KITE (1 K Insect Transcriptome Evolution) and the "i5k" (Sequencing Five Thousand Arthropod Genomes) 58 initiative will soon provide insight into the regulation of Zn and Cu homeostasis in several other insects designated for food/feed production. The reduction in total Fe and Mn content in the Zn 40 larvae is probably due to a competition with Zn for intestinal transporters at the site of absorption. Malvolio (Mvl), an insect homolog of the divalent metal ion transporter 1 (DMT1), is the likeliest candidate for the luminal uptake of Fe 2+ and Mn 2+ 59 in the mealworm midgut (see Suppl Table 2). Although Zn 2+ is a rather weak Mvl substrate 60 , there might be a competition with the aforementioned metal ions, especially under massive nutritional Zn excess. Accordingly, a Zn/Fe/Mn-mixed co-supplementation strategy during mealworm rearing would be a worthwhile option when aiming to counterbalance the other trace element delivery quotas while simultaneously enhancing larval growth/biomass output.
As T. molitor larvae are aimed to be rated as novel food or feed, further quality and safety concerns should be addressed and strictly monitored in addition to their macro-and micronutrient content. In fact, insects might accumulate hazardous chemicals during growth, amongst them heavy metals that may pose a risk to humans or animals 7,61 . As a natural resource, grain material varies considerably in its elemental composition, which is also reflected in slightly different toxic metal contents in T. molitor within various studies 61 . The EU maximum level for Cd in complete feed for farm animals has been set at 0.5 mg kg −1 (relative to a moisture content of 12%) 62 . From a safety perspective, the mealworm larvae grown on Zn basal wheat bran in the present study would be acceptable for feed application, consistent with results of other studies using the same feeding substrate 63,64 . Insects intended for human consumption in Europe are covered by the Novel Food Regulation EU 2015/2283 3 , and must therefore be authorized by EU institutions 61 . The EU "ALARA" (as low as reasonably achievable policy) precautionary principle becomes relevant when setting maximum limits for contaminants, including heavy metals, in foodstuff for public health protection purposes (Regulation 315/93/EEC 65 ). The introduction of Zn-enriched wheat bran material into mealworm breeding is certainly an effective strategy to reduce the amount of the human/animal toxicant Cd within the mealworm larvae. Zn-competitive transport routes for cellular Cd uptake in insects were already discussed 66 , yet their molecular identity and mechanisms need to be clarified when intending to further target the Cd burden of insects during rearing. Aside from interfering with the uptake of this toxic element, Zn triggers synthesis of the aforementioned MT-like proteins within the midgut epithelium, providing a stable pool of midgut Cd-trapping molecules. In fact, these proteins were described to be released into the feces during cell gut renewal 44,48,63 , explaining the decreased mealworm larval Cd bioaccumulation factor observed in the present study. As a substantial part of Cd enters the cells via Ca 2+ channels 67 , it might be useful to include this macromineral in future mealworm enrichment strategies. Here, Zn/Fe/Cu/Mn/Ca-supra-supplemented wheat bran should provide an ideal trace element composition when aiming to produce mealworm larvae enriched in multiple essential micronutrients and low in toxic metals, which is worthwhile to be tested and introduced into T. molitor rearing on an industrial scale.

Conclusion
Global population growth will increasingly challenge the food industry in the coming years. The yellow mealworm (T. molitor) is a sustainable alternative source to animal-derived protein and lipids, suitable for mass rearing and large-scale industrial production. Yet, the industrial technology needs to be optimized and standardized to process the insects in the best possible way, from an economic, food/feed safety and nutritive point of view. Summarizing this study's results, a Zn-spiked wheat bran feeding strategy is an easy and inexpensive approach to produce T. molitor valorized in its content of the essential micronutrient Zn. Zn-biofortification led to a moderate Fe and Mn reduction in mealworms, reflected in reduced feed conversion efficiency. Nevertheless this can easily be counteracted in the future by Fe/Mn co-supplementation. Importantly, Zn-enriched rearing reduced the levels of the human/animal toxicant Cd within the mealworm larvae, a technological novelty of outstanding importance to be implemented in the future production process to ensure the consumer safety of this edible insect species. Overall, these results provide relevant insight for the development of optimized strategies to process T. molitor in the future, both in terms of nutrient quality and quantity. Experimental design. T. molitor beetles of University of Applied Sciences Bremerhaven own breeding were placed on wheat flour for oviposition (see Fig. 1). After 7 days, the eggs were removed from the laying substrate with a fine-mesh sieve (1 mm mesh size) and placed in the breeding container. Freshly hatched larvae were allowed to grow in the wheat bran substrate. 6 weeks post hatching T. molitor larvae (stage L 3-4 , average starting weight 1.23 ± 0.01 mg) were seeded into 400 ml glass beakers on Zn-spiked wheat bran feed (Zn basal to Zn 40fold basal ; further details for wheat bran preparation are provided in the "feed spiking" section). Each Zn treat-Scientific Reports | (2020) 10:20033 | https://doi.org/10.1038/s41598-020-77079-x www.nature.com/scientificreports/ ment was performed in 5 biological replicates, where each replicate comprised 60 mealworm larvae in a density of 142 larvae/dm 2 growth surface. All beakers were incubated at 27 °C with a humidity of 75% with no day/night rhythm for light, temperature and humidity for an 8 week period 68 . Each feeding group was fed as soon as the feed in the beaker had been consumed, with a total of 20 g feed over the whole feeding period. The exact amount of feed supply and feces production was recorded separately for every larval replicate. After harvesting at day 98 post hatching, the number and larval weight of living L 9-10 animals was registered to assess the growth performance and feed utilization. Prior to metal analysis, larvae were starved for 24 h to diminish gastrointestinal feed residues. Following freeze-drying in a Christ Beta  Table 2).

Materials and methods
Larval growth performance and feed utilization parameters. Larval growth performance was evaluated based on the larval fresh weight and the survival rate (SR, Eq. 1) of L 9-10 animals.
Larval weight data from the end (stage L 9-10 ) and the beginning (stage L 3-4 ) of the feeding period along with total feed consumption (FC, Eq. 2) were further used to calculate the efficiency of ingested feed conversion (ECI, Eq. 3) and the feed conversion ratio (FCR; feed input per unit of fresh product; Eq. 4) on fresh matter basis 16,31,69 .