Effects of dietary replacement of fishmeal by mealworm meal on muscle quality of farmed shrimp Litopenaeus vannamei

Accepted Manuscript Effects of dietary replacement of fishmeal by mealworm meal on muscle quality of farmed shrimp Litopenaeus vannamei

Roseane L. Panini, Stephanie S. Pinto, Renata O. Nóbrega, Felipe N. Vieira, Débora M. Fracalossi, Richard I. Samuels, Elane S. Prudêncio, Carlos P. Silva, Renata D.M.C. Amboni PII: DOI: Reference:

S0963-9969(17)30584-7 doi: 10.1016/j.foodres.2017.09.017 FRIN 6966

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Food Research International

Received date: Revised date: Accepted date:

6 July 2017 6 September 2017 8 September 2017

Please cite this article as: Roseane L. Panini, Stephanie S. Pinto, Renata O. Nóbrega, Felipe N. Vieira, Débora M. Fracalossi, Richard I. Samuels, Elane S. Prudêncio, Carlos P. Silva, Renata D.M.C. Amboni , Effects of dietary replacement of fishmeal by mealworm meal on muscle quality of farmed shrimp Litopenaeus vannamei, Food Research International (2017), doi: 10.1016/j.foodres.2017.09.017

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Effects of dietary replacement of fishmeal by mealworm meal on muscle quality of farmed shrimp Litopenaeus vannamei

Roseane L. Panini a, Stephanie S. Pinto a, Renata O. Nóbrega b, Felipe N. Vieira b, Débora M. Fracalossi b, Richard I. Samuels c, Elane S. Prudêncio a, Carlos P. Silva d*, Renata D. M. C.

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Food Science and Technology Department, Federal University of Santa Catarina, Avenue Admar

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a

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Amboni a,*

Gonzaga, 1346, Florianópolis, SC, Brazil

Aquaculture Department, Federal University of Santa Catarina, Avenue Admar Gonzaga, 1346,

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b

Florianópolis, SC, Brazil

Departament of Entomology and Plant Pathology, State University of North Fluminense Darcy

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c

Ribeiro, Avenue Alberto Lamego, 2000, Campos dos Goytacazes, RJ, Brazil.

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Biochemistry Department, Federal University of Santa Catarina, Trindade, Florianópolis, SC, Brazil

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* Corresponding author:

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d

Silva).

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E-mail adress: [email protected] (Renata D.M.C. Amboni); [email protected] (Carlos P.

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ABSTRACT This study investigated the muscle quality of the shrimp Litopenaeus vannamei fed on a diet containing different proportions of mealworm meal (MW) (0, 25, 50, 75 and 100%) as a substitute for fishmeal, which is the normal diet used in shrimp commercial production. The proximate composition, fatty acid profile, colour and texture of the shrimps were

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evaluated. Moisture, protein, and ash content of shrimp muscle were not significantly altered

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when fishmeal was replaced by MW (p > 0.05). However, the replacement resulted in a linear

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increase in lipid content (p < 0.05). The fatty acid composition of the experimental diets directly mirrored the fatty acid composition of shrimp muscle. The absence of long-chain

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polyunsaturated fatty acids in MW composition resulted in a linear decrease in eicosapentaenoic and docosahexaenoic fatty acids in shrimp muscle with increasing levels of

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MW in the diet. The n-3/n-6 ratio ranged from 0.50 to 0.67. Colour and firmness were unchanged between the treatments. Although the use of MW as a fishmeal substitute in L.

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vannamei diets has affected the lipid and fatty acid composition of shrimp muscle, from a

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human nutritional perspective, the lipid content of the shrimps is considered low and the n3/n-6 ratio remained within the human dietary requirements. Therefore the use of a mealworm

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diets.

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diet for shrimp production is a viable alternative to increasingly expensive fishmeal based

Keywords: Pacific white shrimp, insect meal, proteins, fatty acids, colour, firmness.

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1. Introduction

There is increasing interest in edible insects as an alternative protein source for human consumption and animal feed (van Huis et al., 2013). Generally, depending on the species, stage of development and diet, insects can be a good source of proteins, lipids,

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minerals, vitamins, and energy (Makkar, Tran, Heuzé, & Ankers, 2014; Nowak, Persijn,

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Rittenschober, & Charrondiere, 2016). The insect species Tenebrio molitor, also known as the

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mealworm in the larval stage, is a coleopteran widely cultured for use as pet feed (Finke & Oonincx, 2014) and it has already been tested in diets to replace fishmeal in fish farming

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(Gasco et al., 2016; Piccolo et al., 2017; Roncarati, Gasco, Parisi, & Terova, 2015). Mealworms contain on a dry basis, high amounts of crude protein (47–60%) and lipid (31–

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43%), relatively low ash content (< 5%), and the fatty acid composition is rich in n-6 (Nowak et al., 2016; Siemianowska et al., 2013).

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The shrimp Litopenaeus vannamei, also known as the Pacific white shrimp, is one of

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the most highly consumed farmed shrimp in the world (FAO, 2017). They are an extreme good source of protein, minerals and vitamins, particularly essential amino acids, calcium,

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iron, zinc, and vitamin B12 (Puga-Lopéz et al., 2013). Additionally, shrimps contain high levels of n-6 and especially n-3 polyunsaturated fatty acids (PUFA), for example

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eicosapentaenoic (C20:5n-3, EPA) and docosahexaenoic (C22:6n-3, DHA) acids (Harlıoğlu et al., 2015), considered essential for the human health (Orsavova, Misurcova, Ambrozova, Vicha, & Mlcek, 2015). Moreover, the red/orange colour and firm texture have been associated with freshness and good product quality (Niamnuy et al., 2008; Parisenti et al., 2011a). Farmed shrimps are fed manufactured diets, which typically contain approximately 25% fishmeal (Samocha, Davis, Saoud, & DeBault, 2004). However, in the last decade, the

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increasing demand for fishmeal, combined with over exploitation of fish stocks, directly affected the formulation costs of diets for farmed species in aquaculture, which makes the search for cheaper and sustainable protein ingredients necessary (FAO, 2012). Moreover, the chemical composition of the ingested feed could modify the quality of shrimp muscle, such as texture characteristics (Brauer et al., 2003; Rivas-Vega et al., 2001) or the chemical

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composition of shrimp muscle (Martínez-Córdova et al., 2013). Hence, the present study was

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conducted to evaluate the effects on proximate and fatty acid composition, colour and texture

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of the shrimps muscle following a gradual substitution of fishmeal with mealworm meal.

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2. Materials and Methods

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2.1 Samples

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A total of 450 shrimps were used to stock fifteen blue dark polyethylene tanks (400 L;

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30 shrimps per tank) corresponding to the treatments evaluated and their repetitions (n = 3) as previously described by Panini et al. (2017). The shrimp were fed with five experimental diets

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containing fishmeal which was gradually substituted by MW (0, 25, 50, 75, and 100%) (Table 1). Following six weeks, the shrimps (average weight 9.23 ± 0.36 g) were harvested, washed,

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and transported on ice to the laboratory within 4 h of capture. To understand the effect of gradual substitution of fishmeal with mealworm, shrimps were divided into two groups: one group with 10 shrimps from each tank which were beheaded and stored at -20 ºC for determination of muscle composition (total moisture, protein, lipid, ash and fatty acid composition). For fatty acid composition, shrimps were freeze-dried. Another group (6 shrimps from each tank) was used immediately for colour and texture analyses.

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2.2 Chemical analyses

Proximate composition of experimental diets, MW and shrimp muscle were analysed according to AOAC procedures (AOAC, 2005). Moisture, crude protein, lipid, ash and fibre

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were determined according to methods 950.01, 945.01, 920.39C, 942.05 and 962.09,

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respectively. Gross energy content of diets and MW were determined with an adiabatic bomb

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calorimeter (IKA, Heitersheim Gribheimer, Germany) at CBO Laboratory (Campinas, São Paulo, Brazil).

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The fatty acid composition was analysed from the total lipid extracts (Folch, Lees, & Sloane, 1957) of MW, experimental diets samples and freeze-dried muscle samples obtained

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from shrimps fed on different diets. Subsequently, fatty acids were esterified into methyl esters (O’Fallon, Busboom, Nelson, & Gaskins, 2007) and analysed using gas

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chromatography (CG–2014, Shimadzu, Kyoto, Japan) with a capillary column (RTX® 2330,

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90% biscyanopropyl, 10% phenylcyanopropyl polysiloxane, 105 m x 0.25 mm ID, 0.20 μm film thickness; Restek®, Bellefont, USA). The column flow rate was 1 mL min-1, carrier gas

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was synthetic air and the makeup gas was nitrogen. The operation parameters were: injector temperature 250°C; volume injected 1.0 µL, split ratio 1:40; flame ionization detector (FID)

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260°C; column heating ramp: initially at 130°C for 5 min, followed by a gradient of 130 to 180°C at 5°C min-1, 180°C maintained for 10 min, 180 to 240°C at 3°C min-1 and 240°C maintained for 13 min. For fatty acid identification and quantification, retention times and peak areas of methyl esters were compared to external standards (37 component fame mix and PUFA No. 3 from menhaden oil, Supelco, Bellefonte, U.S.A.) and to the internal standard 23:0 (tricosanoic acid, Sigma-Aldrich, St. Louis, U.S.A.). Peak areas were corrected by theoretical relative FID response factors. The analyses were performed in duplicate.

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2.3 Colour analysis

The colour analysis of whole shrimp body (in natura and cooked for one minute in 100 mL of boiling water) was determined using a Minolta Chroma meter CR-400 (Minolta

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Co., Osaka, Japan) calibrated against a standard white plate. Two measurements from close to

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the head of the shrimp (one from each side) were performed on three shrimps from each tank.

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The colour is reported in terms of L*, a* and b* values. The total colour difference (ΔE) was calculated according to the following equation Cruz-Romero et al. (2007):

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√

Where: ΔL*, Δa* and Δb* are the differences between the 0% fishmeal replacement and each

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one of the other fishmeal replacement diets in L*, a* and b*, respectively. The perception of the total colour difference (ΔE) varied according to the sensitivity and the observed colour by

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the human eye, which only distinguishes colour difference if ΔE is higher than 3 (Martínez-

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2.4 Textural analysis

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Cervera, Salvador, Muguerza, Moulay, & Fiszman, 2011).

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Firmness analysis of shrimp muscle (in natura and cooked for one minute in 100 mL of boiling water) was determined using a TA-XT2i texturometer (Stable Micro Systems Ltd., Surrey, UK) equipped with a 25 kg load cell according to the methodology described by Niamnuy et al. (2008). Firmness was reported in terms of kPa values, which resulted from the maximum compressive force measured during compression divided by the contact area between the shrimp and the probe. The measurements were performed on three shrimps from each tank.

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2.5 Statistical analysis

The data analysis was carried out with STATISTICA 12.0 software (StatSoft Inc., Tulsa, USA). The results were subjected to normality and homogeneity of variance testing

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submitted to regression analysis at a significance level of 5%.

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(Levene’s test). The chemical composition, colour and texture of shrimps postharvest were

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3.1 Proximate composition of L. vannamei

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3. Results and Discussion

The proximate composition of shrimp muscle is shown in Table 2. Moisture, protein

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and ash contents in shrimp muscle were not significantly influenced by replacing fishmeal

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with MW (p > 0.05), while lipid content increased linearly with reduced levels of fishmeal replaced by MW (p < 0.05). The moisture and protein content of shrimp muscle seen in this

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study are in agreement with the literature, which reports values of between 70 and 79% for moisture and around 20% for protein (Silva and Chamul, 2000; Puga-Lopéz et al., 2013).

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Martínez-Córdova et al. (2013) evaluated the postharvest quality of L. vannamei fed on live insects (Trichocorixa sp.) as a replacement for formulated feed and their results were similar to the present study, but with a lower protein content (145.6 to 158.8 g kg-1) and a higher lipid content (13.6 a 15.3 g kg-1). These authors observed that shrimps fed with higher percentages of live insect replacement diets when compared to the standard formulated feed, resulted in a higher lipid deposition in the muscle and they attributed this effect to the high lipid content of the insects. González-Félix et al. (2002a) also noted that increasing the dietary lipid level has

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an effect on the total lipid composition of shrimp, by increasing lipid deposition in the hepatopancreas and muscle tissue. As mealworm is not only a source of protein but also contains a high amount of lipids (Table 1), consequently, the lipid content in the diets increased with increased substitution of fishmeal by MW, which probably had a direct influence on the increase in the lipid deposition in shrimp muscle. Other studies that used

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MW as fishmeal replacement in African catfish and tilapia farming, observed the same pattern

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in the body lipid composition of these two species (Ng, Liew, Ang, & Wong, 2001; SánchezMuros et al., 2015). However, the shrimp muscle lipid content found in this study was very

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low (4.1 – 5.4 g kg-1). Bragagnolo and Rodriguez-Amaya (2001) reported that the total lipid

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3.2 Fatty acid composition of total lipids

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content in shrimps captured from nature, ranged from 9 to 11 g kg-1.

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Polyunsaturated fatty acids (PUFAs) play vital role in maintaining health and

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wellbeing in humans by minimizing the risk of neurodegenerative and cardiovascular disease, certain types of cancer, arthritis and diabetes (Orsavova et al., 2015). Since the human body is

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unable to synthesise PUFAs in sufficient quantities, they are essential fatty acids which need to be obtained through the diet (Timilsena, Wang, Adhikari, & Adhikari, 2017).

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The fatty acid composition of MW (Table 1) showed elevated levels of saturated (C16:0), monounsaturated (C18:1n-9) and polyunsaturated n-6 (C18:2n-6) fatty acids. However, MW had a low level of C18:3n-3 polyunsaturated fatty acid and no long-chain PUFAs (i.e., EPA and DHA). These results are similar to those reported by Siemianowska et al. (2013) except for EPA, which was detected in their study. Yang et al. (2006) testing six species of insects, found EPA content in the range of 57 to 264 mg 100 g -1, but only in aquatic species (giant water bugs, water beetles and true water beetles). Terrestrial insects

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have lower levels of EPA and DHA than aquatic insects, possible due to the delta-5 and delta6 desaturase enzymes that are either lacking or of low activity in the larvae (Mba et al., 2017). Moreover, according to Barroso et al. (2014), the variations in the fatty acid composition of MW could be related to the type of feed consumed during the development of the insects and processing methods. In a recent study, Barroso et al. (2017) related that the fatty acid profiles

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of Hermetia meal, a Diptera fly, could be easily modified by dietary manipulation of larvae,

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to improve the percentages of the healthy EPA and DHA. Furthermore, van Broekhoven et al.

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(2015) observed that increasing levels of dietary polyunsaturated fatty acids generally lead to a higher proportion of polyunsaturated fatty acids in the insect tissue and a lower proportion

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of monounsaturated fatty acids.

Table 3 shows the fatty acid composition of L. vannamei shrimp muscle. The major

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fatty acids in shrimp muscle were, in decreasing order, linoleic (C18:2n-6), palmitic (C16:0), oleic (C18:1n-9), stearic (18:0), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids.

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However, with increasing the percentage of MW, replacing fishmeal this order changed, with

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oleic acid became the major fatty acid. The fatty acid composition of the experimental diets directly mirrored the fatty acid composition of shrimp muscle. These results are in line with

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other studies, which reported that the fatty acid pattern of shrimp muscle reflected that of dietary fatty acids (González-Félix, Lawrence, Gatlin, & Perez-Velazquez, 2002; Ouraji et al.,

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2009; Sánchez-Muros et al., 2015). The amount of monounsaturated fatty acids (MUFA) increased linearly with increasing levels of fishmeal substituted by MW, particularly due to oleic acid (C18:1n-9). However, the amount of polyunsaturated n-6 and n-3 (PUFAs) did not follow the same trend, decreasing linearly with the increase in MW in the diets, as shown in Figure 1. A similar pattern was observed by Sánchez-Muros et al. (2015) for tilapia fed with mealworm diets, which also showed a significantly decrease of the PUFAs n-3 in fish muscle.

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The concentrations of the fatty acids arachidonic (ARA), EPA and DHA in the shrimp muscle were higher than the dietary concentrations, which suggest that the shrimps may have performed a selective retention of these essential fatty acids during the culture period. Moreover, the shrimps probably elongate and desaturate LA and LnA to synthesise HUFA (as ARA, EPA and DHA) as described by González-Félix et al. (2002b). A quadratic effect was

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observed for the ratio of n-3/n-6 of the shrimps fed with the experimental diets, decreasing

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from 0.69 to 0.52 when higher amounts of MW were used. The levels of n-3/n-6 ratio of 0.5

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to 1.5 were seen by Ouraji et al. (2009) in farmed Indian white shrimp, and these levels occurred due to the type of diet, which in their experiment was rich in PUFA n-6. In tilapia

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muscle, Sánchez-Muros et al. (2015) found n-3/n-6 ratios of 0.74 when fish were fed with mealworm diet. Nevertheless, from a human nutritional perspective, food with a high n-3/n-6

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ratio has been considered as an indication of high nutritional value, and the optimal

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concentration for n-3/n-6 human intake varies from 0.25 to 1(Simopoulos, 2002).

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3.3 Colour analysis

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One important aspect of the shrimps is their pigmentation, as the consumer is attracted by the bright and appropriate coloration, which is associated with freshness and quality of the

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product (Parisenti et al., 2011a). Shrimps colouration is dependent upon the presence of carotenoid pigments (predominantly astaxanthin, 3,3′-dihydroxy-β,β-carotene-4,4′-dione) present in the external tissues, particularly in the exoskeleton and in the epidermal layer between the abdominal muscle and the exoskeleton (Boonyaratpalin, Thongrod, Supamattaya, Britton, & Schlipalius, 2001; Tume, Sikes, Tabrett, & Smith, 2009). For shrimps in natura, astaxanthin is linked with the caroteno-protein complex, which has a green, blue or purple colour and a wavelength of 580 nm. The dissociation of this complex occurs after cooking

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and free astaxanthin is seen (yellow, orange, and red colouration) at a wavelength of 470-472 nm. This change in wavelength is called the bathochromic effect and it is biologically important since changes in shell colour are responsible for the camouflage mechanism essential as a defence strategy against predators (Cianci et al., 2002). The colour parameters for the shrimp samples are shown in Table 4. For all treatments,

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the colour of shrimps in natura fed fishmeal replaced by MW showed low values for the L*

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parameter and negative values for a* and b* parameters, indicating low luminosity and a

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tendency to the colour green and blue, respectively. This blue-green colour occurred probably because to the blue dark colour of the tanks in which the shrimp were housed. Parisenti et al.

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(2011b) also observed that colour of in natura shrimps cultivated in the dark was more green (a* -2.42) and more blue (b* -2.85) than when cultivated in white tanks (a* -0.77, b* 3.88).

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In contrast, cooked shrimps showed high luminosity, and positive values for the a* and b* parameters, indicating a tendency to the colour red and yellow. The cooking process

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affects the colour of shrimps through chemical changes in the nature of the pigment

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(Boonyaratpalin et al., 2001; Tume et al., 2009) and physical state, due to protein aggregation and increasing opacity. Consequently, this results in an increase in the luminosity value (L*)

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(Larsen, Quek, & Eyres, 2011). The parameters a* and b* observed in cooked shrimps showed similar values as related by Parisenti et al. (2011b) in shrimps cultivated in dark

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tanks (a* 21.68, *b 33.22). In another study, Parisenti et al. (2011a) supplemented the L. vannamei diet with carotenoids sources and they noted a similar value to the parameter a* observed in the present study, however, with a lower value for the parameter b*. This difference could occur due to the carotenoid content in the shrimp body (Tume et al., 2009) or high dietary lipid content consumed by the shrimp (Larsen et al., 2011). The desired redness in salmon is achieved in fish with a higher oil content because there is a greater refractive index between muscle and air than between muscle and oil, which result in less light

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scattering and more redness (Larsen et al., 2011). Regarding the total colour difference (ΔE), for both in natura and cooked shrimps, this was lower than 3, i.e, the difference cannot be distinguishable by the human eye (Martínez-Cervera et al., 2011) .

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3.4 Texture analysis

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The protein in shrimp muscle can be divided into three main groups: myofibrillar

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(main protein in muscle fibre), sarcoplasmic (located in the interstitial region between individual muscle fibres) and stroma (connective tissue) protein (Niamnuy et al., 2008). The

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texture of the muscle is associated mainly with the integrity of myofibrillar proteins and connective tissue, which can be affected by ante-mortem and post-mortem factors and also by

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the type of processing (Dunajsky, 1979). Moreover, changes in texture were observed by Rivas-Vega et al. (2001) when the protein concentration in shrimp feed was modified.

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Martínez-Córdova et al. (2013) observed that shrimps from treatments using high

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concentrations of insects had high lipid contents and consequently low firmness. Dunajski (1979) stated that water and lipid reduced the structural factors of muscle tissue, lowering its

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mechanical strength, i.e., fish with higher lipid or moisture content was softer in texture. In the present study, the firmness of both in natura and cooked shrimp muscles was not affected

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by replacing fishmeal with MW (Table 5). However, an increase in muscle firmness was observed after cooking. According to Niamnuy et al. (2008), this increase of firmness could occur due to the changes in meat structure after cooking, such as destruction of cell membranes, shrinkage of muscle fibre, aggregation of sarcoplasmic protein and shrinkage and solubilisation of stroma protein.

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4. Conclusion

The quality of the shrimp L. vannamei muscle fed on fishmeal substituted by mealworm was not affected, even though, shrimps fed on diets above 25% fishmeal substitution showed an increase in lipid content and a decrease in the poly-unsaturated fatty

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acids. However, for a human nutritional perspective, the lipid content of the shrimps is

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considered low and the n-3/n-6 ratio has been considered within the optimal concentration for human intake. It is also important to optimise the fatty acid profile of MW, since it reflects in

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the shrimp’s fatty acid profile, possibly by addiction of polyunsaturated fatty acid sources to

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the insect diet.

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Acknowledgments

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The authors are thankful to the CNPq (National Counsel of Technological and Scientific

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Development) for their financial support, the CAPES (Coordination of Improvement of Higher Education Personnel) for the scholarship. Débora M. Fracalossi, Felipe do Nascimento

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Vieira, Richard Ian Samuels, Elane Schwinden Prudêncio, Carlos P. Silva and Renata D.M.C. Amboni are CNPq research fellows.

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The authors declare no conflict of interest.

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coacervation: A review. Food Hydrocolloids, 69, 369–381. Tume, R. K., Sikes, A. L., Tabrett, S., & Smith, D. M. (2009). Effect of background colour on the distribution of astaxanthin in black tiger prawn (Penaeus monodon): Effective method for improvement of cooked colour. Aquaculture, 296, 129–135. van Broekhoven, S., Oonincx, D. G. A. B., van Huis, A., & van Loon, J. J. A. (2015). Growth

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Figure 1 Polyunsaturated fatty acids measured in the muscle tissue of Litopenaeus vannamei raised on diets replacing fishmeal by mealworm after a six weeks culture period (LA, Linoleic acid; ARA, Arachidonic acid; LnA, Linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid).

ACCEPTED MANUSCRIPT 21 Table 1 Composition of the experimental diets and mealworm.

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94.34 194.05 227.57 23.33 1.40 4.92 7.25

51.5 228.8 250.0 213.0 2.5 0.00 20.3 1.4 50.0 113.0 15.0 30.0 15.0 1.0 6.5 2.0

0.00 305.0 250.0 213.0 9.8 0.00 20.3 1.4 50.0 80.5 15.0 30.0 15.0 1.0 6.5 2.5

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Mealworm (MW)

901.8 377.0 300 98.5 203.9 320.6 4084 3011

906.5 381.9 300 111.4 187.5 319.2 4230 3086

908.2 385.2 300 143.3 158.4 313.1 4525 3301

962.8 558.2 423.0 346.4 30.3 18.6 7744 5150

112.08 248.88 183.80 15.43 1.20 3.71 5.83

125.24 290.53 148.37 9.47 0.62 2.19 2.94

118.49 272.31 118.69 6.38 0.74 3.27 3.18

162.10 430.07 144.00 2.77 nd nd nd

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897.2 371.6 300 99.7 211.4 317.3 4028 3028

103.0 152.5 250.0 213.0 2.5 9.7 20.3 1.4 50.0 130.6 15.0 30.0 15.0 1.0 4.5 1.5

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154.5 76.3 250.0 213.0 2.5 30.3 20.3 1.4 50.0 135.2 15.0 30.0 15.0 1.0 4.5 1.0

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Constituents (g kg-1 dry weight) Fishmeal 206.0 Mealworm 0.00 Soybean concentrate protein 250.0 Wheat bran 213.0 Cod liver oil 2.5 Soybean oil 50.9 Soybean lecithin 20.3 Cholesterol 1.4 Carboxymethyl cellulose 50.0 Kaolin 139.9 Vitamin premix 15.0 Mineral premix 30.0 Sodium chloride 15.0 Vitamin C 1.0 Bicalcium phosphate 4.5 DL-Methionine 0.5 -1 Nutrients (g kg dry basis) Dry matter a 891.7 Crude protein 367.2 Digestible protein b 300 Lipid 101.9 Ash 220.7 c Nitrogen-free extract 310.2 Energy (kcal kg-1) 3972 a -1 Digestible energy (kcal kg ) 3044 -1 d Fatty acids (mg g dry lipid basis) C16:0 85.29 C18:1n-9 154.67 C18:2n-6 273.27 C18:3n-3 29.29 C20:4n-6 1.57 C20:5n-3 5.03 C22:6n-3 7.87

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Composition

g kg-1 wet basis Digestibility used in the diet formulation of this experiment was previously assessed by Panini et al. (2017). c Calculated value: 100-(moisture+crude protein+crude lipid+ash). d Only major acids are present; nd, not detected (<0.05%); each sample was measured in two analytical replicates. b

ACCEPTED MANUSCRIPT 22 Table 2 Proximate composition of Litopenaeus vannamei muscle when shrimps were fed on diets with fishmeal replaced by mealworm after six weeks culture period. Nutrients -1

(g kg wet basis) Moisture Protein Lipid Ash

75 755.9 ± 6.9 194.4 ± 7.2 5.2 ± 0.2 14.2 ± 0.2

100 761.6 ± 4.0 203.0 ± 7.7 5.4 ± 0.1 13.7 ± 0.4

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When regression was significant this equation was used: Lipid, y = 0.0012x + 0.4257 (R2 = 0.71)

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Fishmeal replacement (%) 0 25 50 758.2 ± 5.8 762.5 ± 2.6 767.8 ± 6.4 202.6 ± 1.4 203.3 ± 0.4 201.0 ± 4.2 4.1 ± 0.2 4.7 ± 0.4 4.8 ± 0.4 14.8 ± 1.2 11.8 ± 1.7 12.2 ± 0.2

p value1 0.50 0.62 < 0.05 0.91

ACCEPTED MANUSCRIPT 23 Table 3 Fatty acid composition (mg g-1 dry lipid basis) of Litopenaeus vannamei muscle fed on diets with fishmeal replaced by different percentages of mealworm after a six weeks culture period. p value1 100 < 0.05 < 0.05 < 0.05 < 0.05

1.23 ±0.22 10.42 ±0.91 167.28 ±8.42 5.00 ±0.17

0.69 < 0.05 < 0.05 < 0.05

117.36 ±5.57 8.39 ±0.16 11.80 ±1.52 4.74 ±0.25 1.42 ±0.05 36.15 ±2.04 2.93 ±0.24 36.14 ±1.60 43.89 ±3.90 646.84 ±6.22 211.02±10.76 193.16 ±8.50 242.66 ±7.00 140.50 ±4.79 82.16 ±3.66 0.58

< 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05

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38.03 ±9.52 1.52 ±0.20 108.65 ±2.99 51.88 ±2.82

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Fatty acid Fishmeal replacement (%) (FA) 0 25 50 75 Saturated fatty acids(SFA) C10:0 50.89 ±6.75 46.40 ±3.99 32.52 ±2.46 35.98 ±1.66 C14:0 0.61±0.16 1.05 ±0.16 1.24 ±0.08 1.33 ±0.22 C16:0 136.78 ±6.29 121.08 ±3.52 117.03 ±5.36 104.19 ±1.58 C18:0 78.04 ±4.27 67.40 ±2.32 72.81±3.32 51.24 ±3.16 Monounsaturated fatty acids (MUFA) C16:1n-7 1.17 ±0.11 1.33 ±0.03 1.16 ±0.46 1.02 ±0.29 C18:1n-7 15.63 ±1.25 12.54 ±0.54 10.57 ±2.32 9.20 ±0.32 C18:1n-9 108.29 ±7.66 115.61±3.32 164.04 ±1.26 148.74 ±4.61 C20:1n-11 3.86 ±0.22 3.72 ±0.30 3.78 ±0.54 3.86 ±0.11 Polyunsaturated fatty acids (PUFA) C18:2n-6 175.76 ±10.62 143.78 ±6.71 162.78 ±3.31 119.27 ±7.74 C20:2n-6 17.79 ±0.51 13.46 ±1.10 14.09 ±0.93 8.93 ±0.98 C20:4n-6 19.98 ±0.83 18.34 ±1.55 14.93 ±0.72 11.94 ±0.80 C18:3n-3 10.56 ±0.61 8.79 ±0.27 8.52 ±0.97 5.80 ±0.41 C20:3n-3 2.58 ±0.29 2.14 ±0.47 1.88 ±0.29 1.09 ±0.28 C20:5n-3 62.10 ±1.96 52.08 ±3.01 38.97 ±0.89 31.45 ±1.06 C22:5n-3 6.48 ±0.29 5.41±0.30 4.88 ±0.42 3.11 ±0.22 C22:6n-3 61.45 ±1.71 51.57 ±4.14 41.93 ±0.83 32.01 ±0.48 Others2 80.12 ±0.69 64.50 ±0.75 55.60 ±0.74 50.54 ±0.67 Total FA 832.09 ±27.65 729.20 ±20.93 746.73 ±4.00 619.70±13.52 ∑SFA3 274.73 ±4.37 243.06 ±7.49 229.23 ±3.60 199.07 ± 4.75 ∑MUFA4 146.97 ±8.73 148.06 ±3.91 191.13 ±2.26 169.31 ±3.82 ∑PUFA5 410.38 ±14.76 338.08 ±13.45 326.36 ±2.65 251.33 ±9.28 ∑PUFA n-6 217.70 ±12.20 179.16 ±6.56 193.17 ± 3.04 142.89 ±8.00 ∑PUFA n-3 143.43 ±4.82 119.98 ±6.36 96.19 ±1.20 74.06 ±1.91 n-3/n-6 0.66 0.67 0.50 0.52 Mean values ± SD (n=3).

When regression was significant this equation was used: C10:0, y = 0.0035x2 - 0.4923x + 52.342 (R2 = 0.58); C14:0, y = 0.0084x + 0.732 (R2 = 0.77); C16:0, y = - 0.2926x + 132.18 (R2 = 0.76); C18:0, y = - 0.2739x + 77.968 (R2 = 0.74); C18:1n7, y = - 0.055x + 14.422 (R2 = 0.62); C18:1n-9, y = 0.6044x + 110.57 (R2 = 0.74); C20:1n-11, y = 0.0003x2 - 0.02x + 3.9345 (R2 = 0.72); C18:2n-6, y = - 0.5652x + 172.05 (R2 = 0.70); C20:2n-6, y= - 0.934x + 17.201 (R2 = 0.86); C20:4n-6, y = 0.0911x + 19.955 (R2 = 0.88); C18:3n-3, y = - 0.0585x + 10.61 (R2 = 0.91); C20:3n-3, y = -0.0135x + 2.4951 (R2 = 0.67); C20:5n-3, y = - 0.2901x + 58.652 (R2 = 0.81); C22:5n-3, y = - 0.0376x + 6.4418 (R2 = 0.92); C22:6n-3, y = - 0.2807x + 58.656 (R2 = 0.84); Total FA, y= - 1.9202x + 810.93 (R2 = 0.77); ∑SFA, y = - 0.6858x + 265.71 (R2 = 0.81); ∑MUFA, y = 0.4545x + 147 (R2 = 0.61); ∑PUFA, y = - 1.6888x + 398.2 (R2 = 0.92); ∑PUFA n-6, y = - 0.7626x + 212.81 (R2 = 0.79); ∑PUFA n3, y = - 0.6739x + 136.86 (R2 = 0.86); n-3/n-6, y = 0.00003x2 - 0.0047x + 0.6893 (R2 = 0.61). 2 Fatty acids detected and summed but not expressed: C15:0, C17:0, C22:0, C24:0, C14:1n5, C15:1n5, C17:1n7, C24:1n9, C16:2n4, C22:2n6, C18:3n6, C20:3n6, C22:3n3, C22:4n6.

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ACCEPTED MANUSCRIPT 24 Table 4 Colour parameters and firmness of muscle tissue from Litopenaeus vannamei fed on diets with fishmeal replaced by mealworm analysed immediately after a six weeks culture period. 100

38.84 ± 1.35 -3.75 ± 0.36 -2.36 ± 1.30 0.69 71.17 ± 1.00 19.76 ± 1.48 33.71 ± 1.90 0.74

39.33 ± 1.74 -3.97 ± 0.60 - 3.31 ± 1.57 0.50 71.22 ± 1.56 19.20 ± 1.40 35.48 ± 2.61 0.78

38.62 ± 1.41 -3.98 ± 0.40 -1.06 ± 1.37 1.32 70.80 ± 1.74 19.66 ± 2.22 34.93 ± 3.06 0.31

241.6 ± 25.1 536.0 ± 75.6

221.3 ± 28.6 505.9 ± 40.7

225.5 ± 30.4 528.0 ± 60.5

38.56 ± 1.57 -4.39 ± 0.55 -1.37 ± 2.18 0.49 71.16 ± 1.79 19.47 ± 1.90 36.07 ± 1.69 2.53

0.70 <0.05 <0.05

239.7 ± 46.6 503.0 ± 30.4

0.60 0.10

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0 Colour parameters In natura L* 38.68 ± 1.68 a* -4.05 ± 0.57 b* -2.42 ± 2.90 ΔE 0 Cooked L* 70.15 ± 1.71 a* 20.31 ± 2.36 b* 34.61 ± 2.75 ΔE 0 Firmness (kPa) In natura 222.4 ± 46.0 Cooked 555.8 ± 53.4

p value1

Fishmeal replacement (%) 25 50 75

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Shrimp

When regression was significant the equations were: raw shrimp a*, y = -0.0001x2 + 0.0105x – 4.0265 (R² = 0.13) and b*, y = 0.0003x2 – 0.0144x – 2.437 (R² = 0.07); cooked shrimp b*, y = 0.0002x2 – 0.003x + 34.392 (R² = 0.05).

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0.17 0.21 <0.05

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Tenebrio molitor

meal

Extruded feed

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mealworm

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Litopenaeus vannamei

Proximate composition

Firmness

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Graphical abstract

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Colour

Fatty acid profile

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Highlights:

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Protein content in the shrimp muscle was not altered by fishmeal replacement. Mealworm diets resulted in increases in the lipid content of the shrimp muscle. EPA and DHA decreased linearly with increased levels of mealworm in the diet. Colour and firmness were unchanged when fishmeal was replaced by MW.

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