Wageningen Academic

Journal of Insects as Food and Feed, 2021; 8(11): 1173-1195 P u b l i s h e r s

SPECIAL ISSUE:Application of insect ingredients in sustainable aquaculture

Application of laboratory methods for understanding fish responses to black soldier fly
(Hermetia illucens) based diets
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M. Zarantoniello1, B. Randazzo1, G. Secci2, V. Notarstefano1, E. Giorgini1, E.J. Lock3,4, G. Parisi2 and I. Olivotto1*

1Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona,

Italy; 2Dipartimento di Scienze e Tecnologie Agrarie, Alimentari, Ambientali e Forestali (DAGRI), Università degli Studi di
Firenze, via delle Cascine 5, 50144 Firenze, Italy; 3Institute of Marine Research (IMR), 5817 Bergen, Norway; 4University
of Bergen, Department of Biological Sciences, P.O. Box 7803, 5020 Bergen, Norway; [email protected]

Received: 29 October 2020 / Accepted: 20 January 2021

© 2021 Wageningen Academic Publishers

OPEN ACCESS REVIEW ARTICLE

Abstract

A major challenge for development of sustainable aquafeeds is its dependence on fish meal and fish oil. Replacement with
more sustainable, nutritious and safe ingredients is now a priority. Over the last years, among several alternatives proposed,
insects have received great attention as possible candidates. In particular, the black soldier fly (Hermetia illucens; BSF)
represents a concrete example of how the circular economy concept can be applied to fish culture, providing a valuable
biomass rich in fat and protein valorising organic by-products. In the last decade, several studies have been published
about the use of different BSF dietary inclusion levels for various fish species including experimental models. Varying and
encouraging results have been obtained in this research field using a plethora of laboratory methodological approaches
that can be applied and coupled to obtain a comprehensive view of the BSF-based diets effects on fish physiology, health,
and quality. The present review aims to explore some of the most promising laboratory approaches like histology, infrared
spectroscopy, gut microbiome sequencing, molecular biology, fish fillets’ physico-chemical and sensory properties, essential
for a better understanding of fish welfare and fillet quality, when BSF is used as aquafeed ingredient. In particular, great
importance has been given to European finfish species and experimental models.

Keywords: insect meal, alternative proteins, fish welfare, circular economy, sustainable aquaculture

1. Introduction water, land and feed, the sustainability in aquaculture is

often restricted by a wide range of environmental concerns
Due to the global capture fisheries stagnation, aquaculture (Ahmed and Thompson, 2019; Naylor et al., 2005). Today,
is presently the fastest growing food production sector sustainable aquaculture technology and know-how have
worldwide (FAO, 2018). Over the last years, there has been significantly advanced and they can play a pivotal role
a significant increase in worldwide consumption of aquatic in achieving environmental sustainability goals. Long-
products (FAO, 2016) which is expected to further expand term growth of the aquaculture industry has to be based
over the next thirty years (Gutiérrez et al., 2020). In this on the concept of environmentally friendly practices and
context, the aquaculture industry will face an important sustainable resource management. Presently, after the
challenge: providing a world population estimated to reach launch of Horizon 2020 by the European Community,
9.7 billion by 2050 with a proper amount of nutritious aquaculture priorities can be summarised in three keywords:
and safe aquatic food (FAO, 2016; Godfray et al., 2010). In safe, sustainable, responsible. These words represent the
order to meet this challenge, aquaculture production has to basis for better animal’s welfare, less environmental impact,
grow further (FAO, 2018). Although there is a high market and the development of new approaches able to provide
demand for fish, a further development of aquaculture poses larger volumes of healthy and safe food.
serious environmental challenges. As a result of ecological
impacts of aquaculture inputs and resources, including

ISSN 2352-4588 online, DOI 10.3920/JIFF2020.01351173

 M. Zarantoniello et al.

Aquaculture feed accounts for 50-70% of production costs ABP derived from non-ruminant animals (Category 3)
(Van Huis et al., 2013) and represent an equal share of (Moutinho et al., 2017). European legislation states that
the CO2 footprint (Winther et al., 2020). Because of the Category 3 ABP can be processed for feeding aquatic
over-exploitation of pelagic fisheries, strategies to replace animals to contribute responsibly to both environment
fish meal (FM) and fish oil (FO) in aquafeeds became and public health (Gasco et al., 2020).
both a private and public priority (Tacon and Metian,
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2015). The study for low cost and sustainable feeding Recently, the circular economy concept has gained great
alternatives to totally or partially replace FM and FO attention within the European Community and the EC
have gained relevance (Henry et al., 2015; Voorhees et Directive No. 2008/98 (EC, 2008), which establishes the
al., 2019) and to help the aquaculture industry to expand order of importance in the choice of by-products treatment
and remain competitive. Alternative ingredients are, for (the first being their recycle and the last being their landfill
example vegetable proteins, including oilseeds (especially disposal) is playing a key role.
soybeans), and meat by-products (such as blood meal and
bone meal). Among others, plant meals (PM) are the most In order to meet the circular economy concept, aquafeed
widely used alternatives to FM since 2006 (Gatlin et al., production can take advantage of the great amount of
2007; Gerile and Pirhonen, 2017; Hardy, 2010). However, organic by-products which can be converted in a valuable
PM have unbalanced essential amino acids profile (due biomass rich in proteins and lipids, by using bio-converting
to the deficiency in methionine and lysine), low protein organisms (Lopes et al., 2020; Parodi et al., 2020; Truzzi
content, anti-nutritional factors and a significant amount et al., 2020; Zarantoniello et al., 2020b). Specifically,
of non-digestible carbohydrates, which often limit their bioconversion through insects grown on organic by-
use for carnivorous fish (Basto-Silva et al., 2019; Yasothai, products as feed source might represent a valuable solution
2016). In particular, non-digestible carbohydrates, including (Barroso et al., 2014; Belghit et al., 2019a; Henry et al., 2015).
non-starch polysaccharides, may bind to bile acids or It also represents a valid example of sustainable animal
obstruct the action of digestive enzymes as well as feed production in terms of land use, water consumption and
transit in the intestine reducing both nutrients digestibility CO2 production, because of the low energy requirements
and absorption (Francis et al., 2001). In addition, PM can during rearing procedures (Berggren et al., 2019; Smetana
cause inflammation of the digestive tract of fish (Blaufuss et et al., 2019). The nutritional values of the insects’ biomass
al., 2020; Henry et al., 2015) but also show low palatability are generally characterised by high fat and protein content
(Henry et al., 2015; Papatryphon and Soares, 2001). Over the (Barroso et al., 2014; Giannetto et al., 2020); however, the
years, several techniques have been developed to partially insects’ biomass nutritional composition depends on
overcome these issues including the use of enzymes, heat the quality and quantity of feed offered for their growth
processes and the inclusion of bioactive compounds in the (Gobbi et al., 2013; Pimentel et al., 2017; Truzzi et al., 2020;
formulated diets (Gatlin et al., 2007; Oliva-Teles et al., 2015). Zarantoniello et al., 2020b), with the fat content and quality
In recent years, PM registered a significant rise in prices varying the most (from 7 to 39% dry matter) (Barragan-
because of its increased use in human nutrition, causing Fonseca et al., 2017; Truzzi et al., 2020).
a competition between the animal feed and human food
sectors (Nogales-Mérida et al., 2018). As a consequence, Among the insects species used for aquafeed production,
PM industry has limited potential to expand its production Hermetia illucens (L.) (Diptera, Stratiomydae) (black soldier
without putting additional pressure on arable land use and fly; BSF) is one of the most promising species: it shows high
water consumption and shifting resource demand from efficiency as bio-converter of organic by-product during
oceans to land (Hua et al., 2019). the larval development (Salomone et al., 2017), a proper
protein content (up to 60% dry weight) and an essential
Another alternative ingredient to FM in aquaculture is amino acid pattern similar to that of FM (Müller et al., 2017;
animal by-product meal (ABP). These meals contain a Spranghers et al., 2017). Furthermore, BSF larvae represent
good balance of essential amino acids, show high protein a good source of lipids usually dominated by saturated fatty
content and good digestibility with features similar to those acids (SFA) (especially rich in lauric acid, 12:0), a medium
of FM (Barreto-Curiel et al., 2016; EL-Haroun et al., 2009; chain fatty acid which has anti-inflammatory and immune-
Hatlen et al., 2015). Although the ABP seems to be a good boosting properties (Gasco et al., 2018; Sealey et al., 2011).
and viable alternative, at technical and economic level, Their high content in SFA rather than in polyunsaturated
the consumer acceptance, a strict regulation and many fatty acid (PUFA) still represent a key-issue to be solved
restrictions for their use due to a lack of knowledge on the since this unbalanced fatty acid (FA) profile could impair
risk for developing human diseases are its main limitation fish growth, welfare and quality (Ewald et al., 2020).
(European Commission, 2013). In the European Union,
the use of ABP was prohibited from 1990 to 2000 due Recently, methods to modulate the final biomass’ nutritional
to the arising of the bovine spongiform encephalopathy profile by enriching the growing substrate have been
in ruminants and then, in 2013, was allowed only for developed (Liland et al., 2017; St-Hilaire et al., 2007). In

1174 Journal of Insects as Food and Feed 8(11)

  Laboratory approaches for testing insect based aquafeeds

particular, Truzzi et al. (2020) demonstrated that growing Presently, the fast development of science and the
BSF prepupae on a rearing substrate enriched with 10% development of several new laboratory techniques allow
Schizochytrium sp. can improve insect’s final biomass, scientist to have a deep and comprehensive approach when
especially in terms of some FA like PUFA. studying the effects of new practical diets in aquaculture.
Numerous laboratory techniques like histology, infrared
The inclusion of new ingredients in aquafeeds must spectroscopy, gut microbiome sequencing, molecular
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be approached cautiously, since it is well-known that biology as well as fish fillets’ physico-chemical and sensory
modulatory effects of different feed ingredients on fish properties are now available for a better understanding
physiological responses, quality and safety exist (Li et al., of the above-mentioned aspects related to fish farming,
2019; Rimoldi et al., 2019). The scientific community should representing an up-to date approach for a better under­
take into consideration productivity, fish welfare and end- standing of the effects of insect-based diets on fish welfare
product’s quality in order to guarantee both animals and and production.
humans wellbeing.
The present review aims to provide an overview on the main
While productivity is not affected by proper insect meal analytical methods presently available for understanding
inclusion levels in aquafeeds (Belforti et al., 2015), the main welfare and quality of fish when fed on diets including
problem found in the use of insect meal in fish production BSF meal. Emphasis is given to a number of fish species
is changes in the FA profile of the fish product (Ferrer of interest for the European aquaculture as well as to
Llagostera et al., 2019). When fed on insect meal based experimental models.
diets, fish fillets generally showed a reduced n-3/n-6 ratio,
as well as low PUFA content, reducing the nutritional 2. Fish gut and liver
quality of fish for human consumption (Devic et al., 2018;
Ruxton et al., 2004). In addition, since a large variety of Histology
organic by-products can be used as feed for insects,
potentially toxic elements and pathogen microorganisms Novel feed formulations for fish need to evaluate not only
can enter the food production chain and be potentially economical cost/benefit but also the effect on fish welfare.
harmful to animals and humans (Bosch et al., 2019; The gastrointestinal system is the primary target of dietary
Swinscoe et al., 2019). Therefore, to meet a safe production changes and challenges (Giorgini et al., 2018a).
of insects, a strict chemical and microbiological monitoring
is necessary. Histological analysis of the digestive apparatus is considered
one the main approaches to evaluate fish welfare and
During the last decade, the issue of farmed fish welfare nutritional status (Raskovic et al., 2011). In particular,
has raised increasing public and scientific concern intestine and liver are the most important organs involved
(Panagiotaki and Malandrakis, 2019). Animal welfare has in digestive and immune functions and, consequently, are
been defined as ‘the aptitude of an animal to familiarise to of particular interest when new ingredients are applied
its environment and maintain good health, while living a to aquafeed formulation (Ray and Ringø, 2014; Robaina
natural life and show its natural behaviour’ (Ashley, 2007; et al., 1995; Zhang et al., 2020).Intestinal morphology is
Brijs et al., 2018). Much research on the welfare of farmed considered one of the main indicators of fish health since
fish is thus required to provide recommendations for best its morphological structure rapidly and often reversibly
practices and future legislation (Ashley, 2007; EFSA, 2009). changes in response to dietary inputs. Alteration of
Farmed fish are usually exposed to a variety of stressors gut integrity may modify nutrient absorption and thus
including handling, stocking density and nutrition which fish welfare and growth, possibly affecting productivity
long-term exposure could have negative effects on health (Krogdahl et al., 2015, 2010; Penn et al., 2011; Santigosa
and growth performances (Barton, 2002; Olivotto et al., et al., 2011; Voorhees et al., 2019; Zhang et al., 2013).
2002; Piccinetti et al., 2015; Tocher, 2010). When it comes Morphometric assessment of intestine architecture is
to new feed ingredients the gastrointestinal tract plays a widely used for studying intestine response to dietary
key role. It constitutes an important barrier to the external challenges and involves the measurement of a number of
environment (Groschwitz and Hogan, 2009), providing histopathological parameters, in fish species (Baeverfjord
defence against pathogens and tolerance to dietary antigens and Krogdahl, 1996; Daprà et al., 2011; Gu and Li, 2004;
(Peterson and Artis, 2014) while playing, at the same time, Krogdahl et al., 2003; Swatson et al., 2002).
a fundamental role in the absorption of nutrients (Uran
et al., 2008) and on the innate and adaptive immunity of Histological analysis relies on the use of tissue staining
fish (Donaldson et al., 2015; Kinnebrew and Pamer, 2012). techniques to visualise intestinal morphology and specific
Therefore, intestine integrity is considered to be essential cell markers. Among the most widely used stainings,
to sustain a proper fish growth and welfare. haematoxylin and eosin staining (H&E) is commonly
applied to provide general morphological information and

Journal of Insects as Food and Feed 8(11) 1175

 M. Zarantoniello et al.

to detect and discriminate inflammatory cells (lymphocytes, showed that a partial (40%) up to a total substitution of
granulocytes, melanomacrophages, etc.) (Mokhtar, FM with full-fat (Lock et al., 2016) or partially defatted (Li
2017) based on their acidophilic (eosin) or basophilic et al., 2020b) BSF prepupae meal did not cause negative
(haematoxylin) features. effects on post-smolt Atlantic salmon intestine morphology,
indicating a high tolerance of salmonids to high dietary BSF
Other stainings allow to obtain more accurate information meal inclusion levels.
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about the chemical composition of some cell categories,

by virtue of specific chemical reactions (Dama and Pathan, The inclusion of a full-fat BSF prepupae meal in pre-smolt
2019). As for example, periodic-acid Schiff (PAS) staining Atlantic salmon diet showed beneficial effects on fish
and Alcian blue pH2 (AB) staining are elective for neutral intestine by reducing the enterocyte hyper-vacuolisation
and acid (PAS and AB, respectively) mucins in mucous which usually characterises intestine of fish fed diets
cells (Cardoso et al., 2015; Purushothaman et al., 2016). including high levels of soybean meal (Heikkinen et al.,
2006; Li et al., 2019) and decreased steatosis in proximal
The analysis of mucous cells in the intestinal epithelial intestine (Li et al., 2020b).
layer is also of particular interest (Cardinaletti et al., 2019;
Zarantoniello et al., 2021). These cells are able to produce and Moreover, studies performed on rainbow trout showed
release defensive substances including mucins, lectins, toxins, that up to a 50% dietary substitution of FM with partially
immunoglobulins and antimicrobial peptides in response to defatted (Dumas et al., 2018; Renna et al., 2017) BSF
specific dietary stimuli or mechanical injury (Bosi et al., 2017; prepupae meal did not affect anterior or distal intestinal
Hasnain et al., 2013; Lazado and Caipang, 2014). tracts morphology. However, Józefiak et al. (2019a) showed
a slight decrease of villi height in trout proximal intestine
Aside providing information on the cell type and tissue fed a 50% dietary full-fat BSF inclusion level with respect
morphology, histological analysis can be useful to obtain to FM. In another recent study performed on rainbow
a number of histopathological parameters able to provide trout fed diets containing 25 or 50% full-fat BSF meal with
(semi-) quantitative information on possible intestinal respect to FM (Cardinaletti et al., 2019), no intestine severe
alterations through a multi-grade scoring system (Silva inflammatory events were highlighted but a significant
et al., 2015). mucosal folds height reduction was observed in both
groups, respected to control. In addition, Randazzo et
The most traditional histopathological indexes include: al. (2020b, 2021b) showed a dose dependent increase of
mucosal folds morphology (length, width, fusion), distal intestine mucous cells in rainbow trout fed diets
enterocytes supranuclear vacuolisation, lamina propria in which 30 or 60% of vegetable proteins were replaced
and submucosa width, leucocyte infiltration (also named with defatted BSF prepupae meal. This result suggests a
‘cellularity’), and mucous cells abundance (Baeverfjord possible involvement of undigested chitin in inducing a
and Krogdahl, 1996; Knudsen et al., 2008; Laporte and higher lubrication of the intestine final tract. Similarly,
Trushenski, 2012; Morris et al., 2005; Penn et al., 2011; it was demonstrated that dietary administration of full-
Uran et al., 2008). Since insect meal is known to include fat BSF prepupae meal (25 or 50% with respect of FM)
different molecules like chitin and short-medium FA, which stimulated a higher secretion of neutral mucins rather than
may have an important role in gut welfare regulation (Bruni the acidic ones, along the entire digestive tract of rainbow
et al., 2018; Gasco et al., 2018; Rimoldi et al., 2019), the trout (Cardinaletti et al., 2019). These results suggest a
analysis of all these parameters has recently been applied to possible involvement of undigested chitin in inducing a
several studies in order to provide information on possible higher lubrication of the intestine final tract. Differently,
inflammation and/or alterations in the nutrient transport Elia et al.(2018) did not evidence significant differences
in fish (Li et al., 2020b). in mucous cells (both rich in neutral and acidic mucins)
in rainbow trout, independently of partially defatted BSF
Most of the histological studies have been performed on larvae meal dietary inclusion (25 or 50%). However, the
Atlantic salmon (Salmo salar) (Li et al., 2019; Lock et al., role of chitin in inducing mucous cells proliferation has
2016), rainbow trout (Oncorhynchus mykiss) (Cardinaletti still not been directly demonstrated and further studies
et al., 2019; Renna et al., 2017) and zebrafish (Danio rerio) are necessary.
(Zarantoniello et al., 2019, 2020b), while a few studies are
available on Siberian sturgeon (Acipenser baerii) (Caimi To date, no studies are available on the effects of BSF meal
et al., 2020a,b; Józefiak et al., 2019b; Zarantoniello et al., dietary inclusion on European sea bass (Dicentrarchus
2021) and Japanese sea bass (Labrax japonicas) (Wang et labrax) intestinal histology, while one has recently been
al., 2019). published on gilthead seabream (Sparus aurata) by Randazzo
et al. (2021a). In addition, a recent study on Japanese sea bass
As regards Atlantic salmon, studies which adopted histo­ showed that a replacement up to 64% of FM by defatted BSF
logical analyses in support to other laboratory techniques, meal did not affect intestine integrity (Wang et al., 2019).

1176 Journal of Insects as Food and Feed 8(11)

  Laboratory approaches for testing insect based aquafeeds

Only recently, a few studies focused on the effects of BSF medium-chain SFA and MUFA rather than in long-chain
prepupae meal dietary inclusion on Siberian sturgeon gut PUFA. Liver lipid and glycogen deposition can provide
morphology: Józefiak et al. (2019b) showed that a diet in important information on the effects and suitability of
which 15% of FM was replaced by full-fat BSF meal caused BSF-based diets (Bruni et al., 2020b; Cardinaletti et al.,
a reduction in intestinal muscular and mucosal thickness 2019; Li et al., 2016). While traditional staining (H&E)
but did not affect mucosal folds length, while Caimi et al. allows to detect hepatocytes lipid storage, histochemical
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(2020a) did not report histological alterations of the distal PAS staining helps in detecting liver glycogen accumulation
intestine in fish fed highly defatted BSF meal (25 or 50% (Aziza et al., 2013; Bui-Nguyen et al., 2015).
replacement with respect to FM).
In post-smolt Atlantic salmon fed full-fat BSF prepupae
Conversely, Zarantoniello et al. (2021), in juvenile sturgeons, meal diets (from 33 up to 100% with respect to FM), a
demonstrated that a 50% dietary inclusion level of full-fat dose-dependent increase in liver lipid accumulation was
BSF meal with respect to FM induced mucosal folds atrophy observed (Belghit et al., 2019a), while only minor effects
and a dramatic decrease of enterocyte vacuolisation. on liver lipid deposition were observed in Japanese sea bass
fed diets including defatted BSF prepupae meal (Wang et
Recently, the effects of the administration of BSF-based al., 2019). Similarly, an increase in liver lipid deposition was
diets in the experimental model zebrafish were deeply evidenced in rainbow trout fed diets containing 25 or 50%
investigated. Particularly, a general increase in mucous cells full-fat BSF meal with respect to FM (Bruni et al., 2020b;
number was observed in zebrafish larvae fed exclusively Cardinaletti et al., 2019).
on full-fat BSF prepupae meal (Vargas et al., 2018). On
the contrary studies in which zebrafish were fed on Studies performed on zebrafish suggested that the
diets including increasing inclusion levels of full-fat BSF n-6/n-3 ratio is a key factor in determining hepatic lipid
prepupae meal (with respect to FM) did not show intestine accumulation. The higher the ratio value, the higher
morphology changes (Zarantoniello et al., 2019, 2020a,b). was the hepatic lipid accumulation (Vargas et al., 2018;
Zarantoniello et al., 2018, 2019, 2020b). Finally, Li et al.
In addition to intestine, liver is often considered a second (2017) evidenced a reduction in liver lipid deposition in
target organ when testing new aquafeed ingredients. This is the herbivorous species Jian carp fed on defatted BSF-
particularly important when testing insect meal since its FA based diets.
composition, rich in SFA, may alter the lipid accumulation
in this organ (Vargas-Abúndez et al., 2019). Liver welfare With respect to glycogen deposition, to date, only a few
is a key aspect for fish production since this organ plays studies applying a histological approach are available. PAS
a central role in many of the fish metabolic pathways was used to discriminate the contribution of glycogen in
and its morphological structure and macromolecular hepatocytes composition of juvenile clownfish (Amphiprion
composition are deeply influenced by the diet (Bruni et ocellaris) fed partially defatted BSF larvae meal (25, 50 or
al., 2020b; Cardinaletti et al., 2019; Novriadi et al., 2018; 75% replacement with respect to FM) and rainbow trout fed
Vargas-Abúndez et al., 2019). full-fat BSF prepupae meal (25 or 50% with respect of FM).
However, no significant differences among the experimental
Several histological analyses, using the above-mentioned groups were detected in terms of glycogen accumulation
histological stainings, may be applied to the liver, providing (Cardinaletti et al., 2019; Vargas-Abúndez et al., 2019).
information about lipid accumulation, inflammation,
necrosis, and glycogen deposition in this organ. From this overview it is clear that fish responses are not
only species-specific but also dependent on fish life stage
In 1997, McFadzen et al. (1997) proposed a criteria and on the characteristics of BSF meal (full-fat or defatted).
scale for the analysis of fish liver in order to determine A traditional and well-established method like histology
the nutritional conditions and this approach has been plays a pivotal role in the analysis of fish gut and is still
successfully applied over the years for studying the effects used as gold standard serving scientific research because
of dietary challenges on farmed fish species (Sabbagh et of its validity and accuracy. These types of analysis could
al., 2019). Liver histological parameters are responsive provide the community with essential insights on the effects
to food quality and availability (O’Connell, 1976) and of BSF meal on fish.
are particularly addressed to provide information on
hepatocyte morphology, with emphasis on the relative Fourier transform infrared spectroscopy: hyperspectral
amount of intracellular vacuolisation associated with lipid imaging analysis and biomolecular composition
and glycogen storage.
Fourier Transform Infrared (FTIR) spectroscopy is a
The fatty acid profile of insects does not always match the well assessed analytical tool for the analysis of biological
nutritional requirements of fish since insects are rich in samples, such as tissues, cells and biological fluids

Journal of Insects as Food and Feed 8(11) 1177

 M. Zarantoniello et al.

(Giorgini et al., 2018b; Notarstefano et al., 2019, 2020). MUFA and PUFA were found in control and Hi25 groups,
The interaction between the electromagnetic radiation Hi50 showed higher SFA and lower PUFA contents. This
in the mid-infrared spectral range (4,000-800 cm-1) and study suggested that the substitution of FM with BSF
the samples causes vibrational transitions at the level of meal up to 25% in zebrafish female diet did not affect
chemical bonds, allowing to identify the presence of specific reproductive performance, while a higher inclusion (50%
chemical groups (Talari et al., 2017). This potentiality with respect to FM) resulted in reproductive impairments,
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coupled with the optical microscopy allows to perform specifically in terms of number of spawned eggs.
the hyperspectral imaging analysis of selected areas inside
the analysed samples. In addition, this tool lets combine Since high dietary SFA and n-6 PUFA intake have often
the topographical distribution of the most relevant been related to behavioural and cognitive impairments
biomolecules (in terms of lipids, proteins, carbohydrates in humans and rodents, FTIR was also applied to better
and nucleic acids) with meaningful information on the understand the FA composition of zebrafish brain in
biochemical composition and the occurrence of specific response to dietary BSF meal inclusion. Although specific
biological mechanisms and pathways (Giorgini et al., 2018a; behavioural tests such as open-field and photic entrainment
Randazzo et al., 2020a; Zarantoniello et al., 2020a). tests did not evidence differences among the experimental
groups, the FTIR analysis highlighted that increasing dietary
FTIR spectroscopy has recently been applied to evaluate the full-fat BSF meal inclusion levels caused a drastic decrease
physiological responses of fish species to BSF-based diets, of unsaturated FA and carbohydrates in zebrafish brain
including rainbow trout, Siberian sturgeon, and zebrafish. samples. These results suggest possible compensatory
Most of the studies applying this specific technique have pathways developed by the fish (Zarantoniello et al., 2020a).
been performed on zebrafish, where administration of diets
with increasing full-fat BSF meal levels (0, 25, 50, 75 or 100% For commercially relevant species, only a few studies that
with respect to FM; Hi0, Hi25, Hi50, Hi75 and Hi100 fish applied FTIR technique are available. Recently, Giorgini
groups, respectively) was tested during the larval, juvenile et al. (2018a) analysed by FTIR spectroscopy cryosection
and adult phases. of medium and hind intestinal tracts of rainbow trout,
with emphasis on the mucosa layer. The spectral outcomes
As regards the zebrafish larval phase, due to the small were compared to those obtained through the classical
sample size, FTIR analysis was performed on lyophilised histological analysis, based on three different staining
samples. Statistically significant higher amounts of overall methods. The hyperspectral imaging analysis confirmed that
lipids and SFA were found in Hi50, Hi75 and Hi100 groups the distribution of the most represented macromolecules
with respect to control (Hi0) and Hi25, together with a followed the well-known arrangement of intestine tissues.
decrease of the unsaturated FA; conversely, no statistically In addition, the hyperspectral imaging provided a proper
significant changes were detected in terms of protein outline on the macromolecular composition and the
composition (Zarantoniello et al., 2020b). building blocks of rainbow trout intestinal mucosa, through
a semi-quantitative information obtained by univariate
In juvenile and adult zebrafish, cryosections (10 µm thick) analysis of the spectral data.
of specific target organs (such as liver and ovaries) were
analysed. In liver, the hyperspectral imaging analysis Moreover, the effects of practical diets with increasing
provided details on the biochemical composition of full-fat BSF meal levels (0, 25 and 50% with respect to FM)
this organ which were subsequently coupled to the on the macromolecular composition of rainbow trout
histological data to provide a more comprehensive overview juvenile’s liver have been investigated (Cardinaletti et al.,
(Zarantoniello et al., 2019, 2020a). As an example, liver 2019). Results showed that liver samples from fish fed diet
samples of juvenile zebrafish fed diets including increasing with 50% BSF meal inclusion level (with respect to FM)
BSF meal levels (0, 25, 50, 75 or 100% with respect to FM; contained higher amounts of lipids and glycogen compared
Hi0, Hi25, Hi50, Hi75 and Hi100 groups, respectively) to a control group.
showed an increment of total FA together with a decrease
of both carbohydrates and phosphates. Specifically, in Finally, FTIR was used to assess the liver macromolecular
Hi75 and Hi100 liver samples, higher amounts of total composition and small intestine nutrient absorption in
lipids and lower ones of proteins were detected, while the Siberian surgeon fed diets in which 50% of dietary FM was
lowest glycogen levels were found in Hi50 and Hi75 groups replaced by full-fat BSF prepupae meal (Zarantoniello et
(Zarantoniello et al., 2020a). al.,2021). As regards liver, a significant decrease of total
lipids, FA and glycogen was observed compared to the
Recently FTIR was used to characterise the macromolecular control group fed a FM control diet. Conversely, no change
composition of class IV oocytes of zebrafish adult females was observed in the macromolecular composition of small
fed over a 12 months period on BSF-based diets (Randazzo intestine samples.
et al., 2020a). Specifically, while similar amounts of SFA,

1178 Journal of Insects as Food and Feed 8(11)

  Laboratory approaches for testing insect based aquafeeds

Based on these results, FTIR spectroscopy represents a next-generation sequencing tool allows to inexpensively
reliable tool for the analysis of different fish organs and produce large volumes of sequence data and to have a
tissues, providing at the same a correlation between higher power of resolution in detecting microbial species
chemical and morphological features and, as further extent, compared to conventional methods (McAdam et al., 2014;
improving histological outcomes. Metzker, 2010). Illumina sequencing was also utilised in
studies performed on Atlantic salmon (Li et al., 2020a)
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Microbiome and Siberian sturgeon (Zarantoniello et al., 2021) that,

in agreement with the previous studies, highlighted an
Dietary composition is one of the key factor in shaping fish increase in microbial community diversity in fish fed diets
gut microbial communities (Egerton et al., 2018; Wang et including 15% of partially defatted BSF larvae meal or 50%
al., 2018) which in turn can modulate fish metabolism, of full-fat BSF prepupae meal, respectively.
intestinal mucosa development and maturation, immunity
and disease resistance (Llewellyn et al., 2014; Maslowski A higher microbiome diversity is generally considered
and MacKay, 2011). as an indicator of improved gut health, while a reduced
diversity has frequently been associated to gastrointestinal
The use of BSF prepupae meal as aquafeed ingredient has tract colonisation by pathogens (Apper et al., 2016; Sekirov
been shown to positively affect fish intestinal microbiota et al., 2010).
biodiversity, regardless of the insects’ life-cycle stage and/
or defatting process, the insects’ dietary percentage of The overall increased diversity and the positive modulation
inclusion or the fish species analysed (Huyben et al., 2019; of intestinal microbial communities in response to different
Józefiak et al., 2019a,b; Li et al., 2020a; Rimoldi et al., 2019). levels of dietary BSF meal inclusions have been mainly
Presently, most of the studies on the effects of BSF-based attributed to chitin. Chitin is generally considered not easily
diets on the microbiome have been performed on rainbow digestible by fish but represents one of the main growth
trout (Bruni et al., 2018; Huyben et al., 2019; Józefiak et al., substrates of lactic acid bacteria (LAB) (Askarian et al.,
2019a; Rimoldi et al., 2019; Terova et al., 2019). 2012; Ringø et al., 2012). LAB are related to Firmicutes
and Actinobacteria phyla which usually represent the
Bruni et al. (2018) explored the effects of 25 or 50% of ‘core gut microbiota’ in different marine and freshwater
FM replacement with partially defatted BSF prepupae species (Ghanbari et al., 2015; Givens et al., 2015; Li et al.,
meal in rainbow trout using the denaturing gradient 2014; Ringø et al., 2016; Wong et al., 2013). These bacteria,
gel electrophoresis (DGGE), highlighting an increase in using chitin as prebiotic, are crucial in making available
intestinal bacterial diversity in fish fed BSF-based diets. indigestible carbohydrates leading to a better nutrient
DGGE is a well-established, reproducible, rapid and less accessibility and utilisation for fish (Beier and Bertilsson,
expensive molecular tool based on genetic fingerprinting. 2013). In addition, LAB contribute to the synthesis of
DNA or RNA extraction is followed by the amplification vitamins and short-chain FA, considered as the primary
of genes encoding the 16s rRNA and then by the analyses enterocyte’s energy source (Ghanbari et al., 2015), or of
of amplification products by a denaturing gradient gel important anti-inflammatory molecules, like butyrate
electrophoresis. In addition, the identification of community (Rimoldi et al., 2016; Terova et al., 2016).
members is made possible by the sequencing of excised
bands or by hybridisation analyses by specific probes Firmicutes and Actinobacteria phyla have been shown
(Muyzer, 1999; Osimani et al., 2019). to increase in intestinal mucosa and digesta of rainbow
trout fed with defatted BSF prepupae meal (up to 50% of
An increased biodiversity was reported also by Józefiak et inclusion; Bruni et al., 2018; Rimoldi et al., 2019; Terova
al. (2019a), through the fluorescent in situ hybridisation, et al., 2019) or full-fat BSF prepupae and larvae meal (30%
including 20% of full-fat BSF prepupae in rainbow trout of inclusion; Huyben et al., 2019). In particular, Terova et
diet. This hybridisation technique based on nucleic acids al. (2019) reported that the dietary defatted BSF inclusion
allows the identification of microbial species using group or increased the relative abundance of Lactobacillales, mainly
species-specific fluorescent labelled oligoprobes avoiding represented by Lactobacillaceae and Leuconostocaceae,
the use of DNA extraction and polymerase chain reaction involved in starch and fibres digestion as well as of bacteria
(PCR) (Kumar et al., 2018). from the order Clostridiales that includes many butyrate
producers like Clostridium butyricum.
These results were in line with further studies performed
on rainbow trout (Huyben et al., 2019; Rimoldi et al., Similarly, an enhanced colonisation of Lactobacillus sp.
2019; Terova et al., 2019) that evidenced an increased was observed in rainbow trout fed a diet including 20%
microbiome biodiversity with dietary administration of full-fat BSF prepupae meal (Józefiak et al., 2019a) and
of up to 30% of defatted BSF prepupae meal, using high in Siberian sturgeon fed 15% of full-fat BSF larvae meal
throughput sequencing method (Illumina MiSeq). This (Józefiak et al., 2019b).

Journal of Insects as Food and Feed 8(11) 1179

 M. Zarantoniello et al.

As reported by Huyben et al. (2019), the abundance of biodiversity showed by the group fed 50% substitution of
Bacillaceae (phylum Firmicutes) was higher in rainbow FM with full-fat BSF prepupae meal as well as by the Vibrio
trout fed BSF-based diets and this increase was attributed abundance which was negatively influenced by the dietary
to the high level of dietary chitin since these bacteria are increasing inclusion of full-fat prepupae meal (Zarantoniello
able to use chitin through the endogenous production of et al., 2020b). Conversely, the same BSF dietary inclusion
chitinase. Furthermore, the same authors demonstrated that levels caused in zebrafish juveniles, a reduction of microbial
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the presence of Corynebacterium (phylum Actinobacteria) diversity (Zarantoniello et al., 2020a). However, the overall
was higher in rainbow trout fed the full-fat BSF larvae or fish gut health was preserved since BSF prepupae meal is
prepupae meal with respect to the defatted larvae meal. This particularly rich in lauric acid which is known to possess
underlies the ability of Corynebacterium to use dietary lipid anti-inflammatory and antimicrobial properties against
through the activation of endogenous lipases (Brennan et Gram-positive bacteria (Skřivanová et al., 2005, 2007;
al., 2002). An increased abundance of Corynebacterium was Spranghers et al., 2018).
also observed in Atlantic salmon fed on partially defatted
BSF-based diet (15% of inclusion) with respect to the control It should be pointed out that to fully unveil the response
diet (Li et al., 2020a). of intestinal microbiota to BSF dietary inclusion levels,
differences between mucosa-adhered and gut digesta
Furthermore, chitin and its deacetylate derivate chitosan communities must be considered since some allochthonous
have antimicrobial properties and bacteriostatic effects, species could poorly colonise host intestine with a lower
particularly on some Gram-negative pathogen bacteria biodiversity with respect to the intestinal content (Kim
(Nawaz et al., 2018; Qin et al., 2014; Udayangani et al., et al., 2007). Different studies on BSF prepupae meal
2017). This defence mechanism could be enhanced by administration in salmonids reported that microbial
LAB which are able to produce bactericidal compounds diversity was higher in gut digesta than in intestinal
like lactic acid, hydrogen peroxide and biosurfactants mucosa (Bruni et al., 2018; Li et al., 2020a). For that reason,
preventing the adhesion to intestinal mucosa of pathogens analysing only gut digesta- or intestinal mucosa- associated
like Staphylococcus aureus, Streptococcus agalactiae and microbiota or a mixture of both of them could partially
Pseudomonas aeruginosa (Gajardo et al., 2017; Gudiña et underestimate microbial community response to dietary
al., 2015). changes, while it is recommended to profile mucosal and
digesta communities separately when feasible.
Several studies on rainbow trout fed defatted BSF prepupae
meal diets (up to 50% of inclusion; Bruni et al., 2018; Molecular biology (real-time PCR): growth factors,
Rimoldi et al., 2019; Terova et al., 2019) or full-fat BSF immunity, chitinases, lipid metabolism, appetite stimulus
prepupae and larvae meal diets (30% of inclusion; Huyben
et al., 2019) revealed a reduction in both gut digesta One of the most robust and widely used methods for gene
and mucosal-adhered Proteobacteria, a Gram-negative expression quantification is the real-time PCR which is
phylum containing pathogens. In particular, Rimoldi et al. able to distinguish and amplify a specific nucleic acid
(2019), reported that 20 or 30% of defatted BSF prepupae sequence in a sample, monitoring the amplification process
meal dietary inclusion increased the number of bacteria through the correlation between product concentration
belonging to Mycoplasma genus which is associated to a and fluorescent intensity (Olivotto et al., 2011; Valasek and
beneficial effect on host health due to the production of Repa, 2005; Wong and Medrano, 2005).
lactic and acetic acids as main metabolites (Razin, 2006).
Similarly, rainbow trout fed 25 or 50% of defatted BSF This technique has been used in some of the most important
prepupae meal inclusion in diets were particularly rich in finfish species like gilthead sea bream (Psofakis et al.,
Pseudomonas stutzeri (Bruni et al., 2018) which possess 2020), European sea bass (Kokou et al., 2019), Atlantic
antiviral activity and was listed as a probiotic bacterium salmon (Belghit et al., 2019b), Siberian sturgeon (Luo et al.,
(Balcázar et al., 2006; Nayak, 2010). In contrast, in rainbow 2019), sole (Piccinetti et al., 2015), ballan wrasse (Piccinetti
trout, a 20% full-fat BSF prepupae meal inclusion caused an et al., 2017) and rainbow trout (Lindholm-Lehto et al.,
increase in the Clostridium coccoides abundance (Józefiak 2019). However, because of the lack of complete and easy
et al., 2019a) which is known to play an important role in genomic information availability about these last species
fish immune response and pathological processes (Liu et its application is still limited (Ribas and Piferrer, 2014).
al., 2016).
With the use of BSF meal in aquafeed formulation, studies
Finally, recent studies performed on zebrafish fed diets about the application of real-time PCR over the last few
with increasing inclusion levels of full-fat BSF prepupae years are quite limited and mainly focused on growth
(0, 25, 50, 75 or 100%) showed contradictory results. In factors, fish immune and stress response, appetite stimulus,
zebrafish larvae, the positive effects of dietary BSF inclusion lipid metabolism and chitinolytic activity.
was highlighted by the highest microbial community

1180 Journal of Insects as Food and Feed 8(11)

  Laboratory approaches for testing insect based aquafeeds

Considering growth factors [insulin-like growth factors et al., 2020b; Zarantoniello et al., 2019, 2021); however,
(igf1, igf2a) and myostatin (mstn)], BSF meal dietary no direct correlation between chitin feed content and fish
inclusions usually resulted in good growth performance growth reduction has yet been demonstrated. Chitin can
in most of the studies. In both zebrafish larvae and in also have a beneficial activity on the fish immune system,
rainbow trout juveniles, an increase in growth parameters lower stress response and improved gut health (Józefiak et
was well supported by the hepatic gene expression of the al., 2019a; Nogales-Mérida et al., 2018; Ringø et al., 2012).
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growth factors (Cardinaletti et al., 2019; Zarantoniello et

al., 2020b). These results support the hypothesis that BSF The use of molecular markers represents a valid and up to
meal possesses a proper amount of proteins (up to 60% dry date tool since several biomarkers are available, for different
weight) and a well-balanced amino acid (AA) profile (similar fish species, to assess stress and immune response. Studies
to that of FM) which are key dietary features to guarantee demonstrated the correlation between increased gene
a proper aquaculture production (Barroso et al., 2014). expression levels of glucocorticoid receptor and heat shock
proteins and exposure to stressors (including malnutrition;
Fish growth is strictly related to feed intake and the brain Piccinetti et al., 2015). Pro-inflammatory cytokines such as
(particularly the hypothalamus) is a key-actor in regulation interleukin 1-beta (il1b), tumour necrosis factor a (tnfa),
of energy metabolism, nutrient absorption, and the control anti-inflammatory cytokines like interleukin-10 (il10), and
of feeding activity. However, it should be considered that the inflammation mediators such as nuclear factor kappa-light-
gastrointestinal tract is connected to the brain in metabolic chain-enhancer of activated B cells (nfkb) and myeloid
and appetite control. The gut-brain crosstalk occurs through differentiation primary response 88 (myd88) represent
the release of a number of gut peptides that exert responses useful markers of inflammation, able to provide early
within the brain, as well as through neuroendocrine and information when testing new ingredients in aquafeed
sensory inputs from the gut (Sadoul and Vijayan, 2016). formulation (Fehrmann-Cartes et al., 2019; Marjara et
Other signals are also involved such as nutrient levels, al., 2012; Seierstad et al., 2009). However, the presently
through central nutrient sensing systems, and the presence/ available results are still controversial and fish responses
absence of food in the gastrointestinal tract, through vagal seem to be species and stage specific as well as related to
afferents projecting to the brain (Bertucci et al., 2019). BSF meal dietary inclusion levels.
To date, studying the gene expression of both orexigenic
[ghrelin (ghrl), neuropeptide y (npy) and cannabinoid Gut gene expression of immune response markers in adult
receptor 1 (cnr1 or cb1)] and anorexigenic [leptin (lepa) and zebrafish (il1b, il6 and tnfa; Zarantoniello et al., 2019), pre-
melanocortin 4 receptor (mc4r)] signals represents a valid smolt (il4, tgfβ1, il10, ifnγ, il8 and myd88; Li et al., 2019)
tool to better understand fish responses to new formulated and seawater-phase (il1b, il17a, myd88, il8, il4, mhcl, il10,
diets. No significant variation in ghrl gene expression in ifnγ, tgfβ1, cd8β, cd3γδ and foxp3; Li et al., 2020b) Atlantic
medium and distal intestine of rainbow trout juveniles salmon was not negatively affected by the BSF-meal dietary
fed with practical diets containing 30 or 60% substitution inclusion level up to 50, 60 or 100%, respectively.
levels of vegetable proteins with BSF prepupae meal was
recently evidenced (Randazzo et al., 2020b). However, in the Conversely, a dietary full-fat BSF inclusion of 50% or higher
same experimental group, but considering brain, appetite resulted in the immune response activation in both larval
signals analysed (cb1, npy and mc4r) showed a significant and juvenile zebrafish intestine (il1b, il10, il6 and tnfa;
downregulation (Randazzo et al., 2020b). Differently, in Zarantoniello et al., 2018, 2020ab) and in rainbow trout
zebrafish larvae and juveniles, gene expression of orexigenic juveniles medium intestine (il10, tnfα and tlr-5; Cardinaletti
signals (ghrl, npy and cb1 analysed in whole fish samples et al., 2019) as well as in the stimulation of regulatory T
for zebrafish larvae; ghrl and cb1 in intestine samples for cell activity in the proximal and distal intestine of Atlantic
zebrafish juveniles) increased with the increasing dietary salmon (cd3γδ and foxp3; Li et al., 2019).
BSF prepupae meal inclusion levels (Zarantoniello et al.,
2020a,b) fully supporting growth. Inflammatory events detected by real-time PCR can be
coupled with a higher stress response: an increase in the
While BSF meal has been proven to be an adequate protein hepatic hsp70.1 gene expression was detected in rainbow
source, its inclusion in aquafeeds is still possibly limited trout juveniles (Cardinaletti et al., 2019) and pre-smolt
by the chitin content (Olsen et al., 2006; Spranghers et Atlantic salmon (Li et al., 2019). Accordingly, in zebrafish
al., 2017) and its (often) unbalanced FA profile (Ewald et larvae and juveniles, an increased dietary full-fat BSF
al., 2020). prepupae meal inclusion resulted in a higher hepatic
hsp70.1 and glucocorticoid receptor (nr3c1) gene expression
Over the last years a controversial role has been attributed (Zarantoniello et al., 2020a,b). However, in Atlantic salmon
to chitin in aquafeed formulation (Henry et al., 2015). fed diets with higher full-fat BSF meal inclusion (85% of the
High dietary inclusion levels of BFS meal (and possibly diet protein content) the hepatic expression of hsp70.1 was
of chitin) often induced a fish growth reduction (Caimi not affected by the diet (Belghit et al., 2019b).

Journal of Insects as Food and Feed 8(11) 1181

 M. Zarantoniello et al.

Finally, due to the unbalanced fatty acid profile of BSF To date, researchers have useful tools to objectify many of
meal, with emphasis on their lack of PUFA, a number of the quality parameters, such as physical properties, chemical
biomarkers related to long-chain PUFA biosynthesis and composition, sensory properties, and consumers’ liking
FA metabolism is now available and represents a great which have been applied to answer if BSF meal inclusion in
opportunity for better understanding the biosynthetic fish diets may affect fish quality and consumers’ acceptance.
pathways in different fish species in response to dietary
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treatments. Most of the studies so far published focused Physical attributes, such as fillet colour, water holding
their attention on elongase and desaturase gene expression. capacity (WHC) and texture are commonly evaluated by
Specifically, Bruni et al. (2020b) demonstrated that the instrumental methods. For instance, colorimeters have
elovl2 and fads2 gene expression in the pyloric caeca been utilised by different authors while performing a colour
of rainbow trout juveniles tended to increase with evaluation of fillets from Atlantic salmon (Bruni et al.,
the increasing amount of the dietary full-fat BSF meal 2020a) and rainbow trout (Bruni et al., 2020b; Renna et al.,
inclusion (25 and 50%). Accordingly, in both zebrafish 2017; Secci et al., 2019) fed diets with different inclusion
larvae (Zarantoniello et al., 2020b) and adults (Randazzo levels of partially or not defatted BSF larvae or prepupae
et al., 2020a; Zarantoniello et al., 2019), an upregulation of meals. This instrument is designed to assess the colour
elovl2, elovl5 and fads hepatic gene expression was observed of a sample in a user-friendly way (easy to use and fast
in experimental groups fed the highest full-fat dietary BSF response); however, the punctual reading of the colorimeter
prepupae meal inclusion (100 and 50%, respectively). The requires at least two- or three-points determinations in
dietary lack of PUFA in freshwater fish fed BSF-based diets the same sample. Mancini et al. (2018) instead, captured
could enhance the conversion of shorter-chain FA in highly the whole fillet image through a digital camera, then the
unsaturated ones through the activation of elongation and colorimetric measurement was carried out by ImageJ
desaturation pathways (Tocher, 2010). Furthermore, an software. Irrespective to the instrument utilised, results
increasing expression of markers involved in FA (cd36, are generally presented using the CIELab colour space (CIE,
fabp2) and cholesterol (npc1l1) uptake has been observed in 2018), thus splitting the colour into three axes explaining
the proximal intestine of Atlantic salmon fed diets including the lightness (L*), redness (a*), and yellowness (b*) values of
60% of full-fat BSF meal (Li et al., 2019). the sample. In this way, colour perception and its variations
are difficult to interpret. Indeed, data showed as L*, a*, and
While real time PCR may represent a valid tool for the b* values do not allow an inexpert reader to understand the
early detection of many physiological changes in fish, if overall sample colour perception. As a consequence, the
applied to nutritional studies its employment should be differences observed through these methods among the
coupled with other laboratory techniques, like histology and samples did not answer the question: ‘were the differences
infrared spectroscopy, to obtain a clearer overview. Further among colour of the samples perceived by an observer?’.
research is necessary to overcome the lack of complete A useful tool to distinguish different colours is the Delta
and easily available genomic sequences for farmed fish E (ΔE) which is defined as a difference in sensation, as
species and to make real-time PCR a ‘routine technique’ revealed by the name itself: Delta is a Greek letter that
in nutritional studies. stands for the incremental change of a variable, while
the ‘E’ is the abbreviation for ‘Empfindung’, the German
3. Fish quality word for sensation. As a whole, ΔE shows the distance
between two colours and it is calculated as proposed by
The consumers’ perception process of food quality has been Sharma and Bala (2002). Furthermore, Mokrzycki and Tatol
summarised by Fernqvist and Ekelund (2014). The authors (2011) reported different ΔE ranges based on the observer
showed how it is influenced by intrinsic quality attributes perception of the colour difference, as follows: 0<∆E<1 –
(i.e. sensory properties) and intrinsic and extrinsic quality observer does not notice the difference; 1<∆E<2 – only
cues which compose quality expectations. Among the firsts, experienced observer can notice the difference; 2<∆E<3.5
attributes perceived before the consumption like colour, – inexperienced observer also notices the difference;
size, and damages are listed. The latter aspects refer to 3.5<∆E<5 – clear difference in colour is noticed; ∆E>5 –
label, packaging, and other external factors. In addition to observer notices two different colours. While looking at the
this, consumers are increasingly focusing on health, eco- results showed in the literature, it seems that the dietary
friendliness, sustainability of food (Fernqvist and Ekelund, treatments with BSF did not affect colour values of the
2014) suggesting that people are looking for deeper sensory rainbow trout fillets (Renna et al., 2017), with the exception
pleasures from foods, moving from the sensory properties of yellowness which was, in some cases lowered (Bruni et
to health and ethical motivations (Lee and Hwang, 2016). al., 2020b; Mancini et al., 2018). In the case of Mancini et
On this regard, the aspects related to animal farming, such al. (2018), where ΔE was proposed, control (C) and fillet
as animal right, welfare, and feed assurance, are embedded from trout fed BSF at 25% of substitution (HI25) showed
into the ‘new’ concept of meat quality (Bernués et al., 2003). the same perceived colour (ΔE value=0.39), whereas the ΔE
values calculated between fillets from C and trout fed BSF

1182 Journal of Insects as Food and Feed 8(11)

  Laboratory approaches for testing insect based aquafeeds

at 50% of substitution (HI50) or between HI25 and HI50 substitution levels (commonly 25, 50, 75% of FM replaced)
fillets were 2.96 and 2.60, respectively, thus being perceived and the investigated species (rainbow trout: Borgogno
as different. The inconsistent results found until now can et al., 2017; Secci et al., 2019; Atlantic salmon: Bruni et
be due to the complex interactions composing the overall al., 2020a). Analogous outputs were obtained while using
sample colour. For instance, the flesh colour may be directly Tenebrio molitor larvae meal as protein source at 25 or
affected by the pigments contained in the administered diets 50% of inclusion for feeding blackspot sea bream, Pagellus
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(i.e. carotenoids), especially carried by vegetable ingredients bogaraveo (Iaconisi et al., 2017) or rainbow trout (Iaconisi et
(soybean, corn) and BSF meal, in a minor extent (Secci al., 2018). In conclusion, from the instrumentally evaluation
et al., 2019). The addition of astaxanthin in the diet for of the physical properties of fish fillets emerged that BSF did
salmonids, inferable from the high a* values reported for not impair these items even if colour modification needs
Atlantic salmon (Bruni et al., 2020a) and rainbow trout to be considered when formulating the experimental diets.
(Mancini et al., 2018) fillets, could drown out the effect of
dietary ingredients on the different indexes. In addition, Fish are an important source of energy, essential and non-
lipid oxidation might produce yellow pigments, hence essential AA, minerals (iodine, selenium, calcium) and
high b* values can be found in fillets with high malondial­ vitamins (A, D) and a functional food, being one of the
dehyde equivalents content (MDA-eq.), as in the case of main sources of the long-chain PUFA, as eicosapentaenoic
rainbow trout fillets fed on BSF meal-free diet (b*: 5.57; and docosahexaenoic acids (EPA and DHA, respectively).
MDA-eq.: 0.55 mg/kg fillet) or containing 25% of full-fat A weekly consumption of around 1.75 g (for adults) and 3.5 g
BSF prepupae meal (b*: 4.21; MDA-eq.: 0.30 mg/kg fillet) (during pregnancy and lactation) of EPA+DHA is highly
(Bruni et al., 2020b). recommend while following a healthy diet (EFSA, 2015),
since they play positive roles in the prevention of body
Considering WHC and texture, these two physical overweight and obesity (Buckley and Howe, 2009) and in the
properties are strictly connected, since the WHC (defined protection against cardiovascular diseases (Bernstein et al.,
as the ability of a food to retain its own water after the 2012). Hence, fish quality needs to be evaluated considering
application of a pressure, centrifugation, or heating) has flesh chemical composition, by means at least of the AA and
proved to play a key role in the formation of food texture FA profiles. The methods utilised for these analyses were
(Picard et al., 2017). The most common method adopted well established and no significant implementations have
for WHC assessment is the one proposed by Hultmann and occurred in the recent published articles (Belghit et al., 2018;
Rustad (2002) and modified by Iaconisi et al. (2017) based Borgogno et al., 2017; Bruni et al., 2020b,a; Stadtlander et
on a centrifugation at 510×g for 5 min. of a 2 g sample with al., 2017). Specifically, total AA composition is commonly
known water content. Applying this method to fillets from evaluated using HCl to hydrolyse fish samples prior to
BSF meal fed fish, Bruni et al. (2020a,b) did not find any assess a liquid chromatography, as ultra-performance or
significant difference nor among rainbow trout or Atlantic high pressure ones. Results are commonly expressed as mg
salmon samples. Similarly, Secci et al. (2019) did not show AA/g muscle. Concerning FA, once total lipids are extracted
any significant WHC modifications of rainbow trout fillets from the flesh (methanol:chloroform 2:1 v/v being the most
as affected by BSF meal inclusion in fish diet, either after utilised solution) and quantified (gravimetrically), they
frozen storage (120 days at -10 °C) and cooking (boiling at are subdued to a saponification, then they are methylated
95-98 °C for 5 min.). An indirect measurement of WHC to obtain fatty acid methyl esters (FAME). An internal
is obtained by calculating the cooking loss as the weight standard as C19:0 (Belghit et al., 2018, 2019a) or C23:0
difference of the fillet before and after the cooking process, (Borgogno et al., 2017; Bruni et al., 2020a,b; Secci et al.,
according to the formula: 100 × [(raw fillet weight − cooked 2019) is added to the samples. Finally, FA composition is
fillet weight) / raw fillet weight]. Consistently with the determined using a gas-chromatograph (GC) coupled with
WHC assessment, both Borgogno et al. (2017) and Secci a flame ionisation detector. The comparison between the
et al. (2019) did not underline significant dietary effects recorded chromatograms with the one of a pure standard
on this parameter. Texturometer is generally utilised for FAME mix allows the researchers to qualitatively determine
texture measurements. The instrument can be equipped the FA profile, thus the results are expressed as % of total
with a variety of load cells (as 1 kN or less) and probe FAME. A quantitative analysis is also possible when using
(cylindrical, straight blade, Warner-Bratzler) based on a calibration curve obtained with the FAME standard
the type of test the users want (shear test, texture profile mix and it is highly suggested. Indeed, the FA content
analysis, etc.). Other parameters, such as cross-head speed (g/100 g muscle) is necessary to retrieve the fundamental
and the percentage of total deformation have to be set. information on the EPA+DHA level in the sample, hence,
For this reason, results obtained with different methods to know if the samples make a valuable contribution to a
and by different researchers are not comparable. Despite balanced and healthy diet. Nevertheless, the FA content
this, the textural properties of the fillets from fish fed diets of fillet from fish fed BSF meal is still scarcely depicted
containing BSF meal as alternative protein source seemed (Belghit et al., 2019a; Bruni et al., 2020b; Renna et al., 2017;
not to vary with respect to a control diet, irrespective to the Secci et al., 2019). However, as showed by several studies,

Journal of Insects as Food and Feed 8(11) 1183

 M. Zarantoniello et al.

fillet FA profile represents the main weakness of BSF meal For this purpose, the extracted samples were inserted in a
inclusion in fish diet, while the total AA both of Atlantic vial and heated (60 °C for 30 min.) to collect VOCs in the
salmon muscle (Belghit et al., 2018, 2019a) and rainbow headspace of the vial. The VOCs were absorbed on a fibre,
trout plasma (Sealey et al., 2011) were scarcely affected. as Carboxen™/Polydimethylsiloxane (75 μm) (Mancini et
Since the early work of St-Hilaire et al. (2007), it was evident al., 2018) prior to be GC-analysed. The identification of
that the partial substitution of fishmeal (or of the overall components is generally conducted by consulting available
https://www.wageningenacademic.com/doi/pdf/10.3920/JIFF2020.0135 - Monday, October 21, 2024 7:21:19 PM - IP Address:2800:e2:3580:1a4:e0dc:7a69:6fd0:3f84

protein content) with BSF meal dramatically increased libraries and comparing with mass spectra and retention
rainbow trout fillet SFA while decreasing the PUFA fraction. times of commercial standards. At the best of authors’
Numerous subsequent studies have confirmed this finding knowledge, the articles showing the VOCs content are
irrespective the fish species investigated (Belghit et al., limited in literature. Among these, the paper by Mancini
2018, 2019a; Borgogno et al., 2017; Bruni et al., 2020a,b; et al. (2018) offers interesting results while assessing VOCs
Mancini et al., 2018; Renna et al., 2017; Sealey et al., 2011; profile of BSF larvae meal, rainbow trout feeds (control, 25
Secci et al., 2019; Stadtlander et al., 2017; Zarantoniello or 50% of FM substitution with partially defatted BSF larvae
et al., 2019). Since BSF meal is rich in lauric acid (12:0), meal), and fish fillets. Although the dietary VOCs profile
fillet increase in SFA was expected in fish fed BSF diet. In was effectively different, Mancini et al. (2018) showed that
addition, while adding BSF meal to the diet, a reduction the VOCs profile of fillets was scarcely affected by the
of FO, which is the main source of n-3 PUFA, could be presence of BSF in the diet for rainbow trout, irrespective
necessary to obtain isolipidic and isoenergetic diets. The the substitution level. Such result could be attributable
sum of these elements leads PUFA depletion in fish flesh both to a molecule degradation, occurring during the
which, in turn, lowers the overall nutritional quality of the digestive process of the fish, and/or to an absent muscle
fillet. For this reason, mitigation actions were investigated deposition. An innovative approach was adopted by Bruni et
such as the use of partially defatted BSF meal (Renna et al. (2020a) while evaluating the volatile composition of raw
al., 2017), the use of BSF grown on substrates rich in n-3 Atlantic salmon fed diets containing 25 to 75%. BSF meal
PUFA (St-Hilaire et al., 2007; Truzzi et al., 2020), or the larvae. The proton transfer reaction-time of flight-mass
avoidance of FO reduction in feeds (Bruni et al., 2020a). spectrometer (PTR-ToF-MS) technique offers technical
However, interesting information comes from the recent advantages as a rapid data collection (100 s for each sample)
research of Bruni et al. (2020b) who observed attenuated and the possibility to evaluate VOCs of the whole sample
FA profile differences in rainbow trout fed full-fat BSF at room temperature, hence avoiding sample manipulation
prepupae meal (inclusion levels set at 0, 25 or 50%) relative (mincing, solid phase extraction), and heating. Despite its
to the dietary differences. Surprisingly, the n-3 PUFA as pros, PTR-ToF-MS technique requires numerous and time-
well as EPA and DHA amounts were not impaired by the consuming offline activities, corresponding to the spectra-
dietary intervention, thus resulting in a well-balanced FA calibration, raw data acquisition and correction (elimination
profile of the fillets. Standing on our knowledge, Bruni et of peaks imputable to water chemistry, interfering ions,
al. (2020b) linked for the first-time fillet quality and fish and also elimination of all peaks for which the average
lipid metabolism, finding that the formulated practical diets concentrations are lower than an established threshold), and
containing full fat BSF meal were effective in increasing tentative peak identification based on literature or libraries.
pyloric caeca elovl2 and fads2 gene expression which in This last step appears to be critical, since 18 out of 29 VOCs
turn positively modified the fillet dietary FA profile. This were quantified without being identified, as depicted in
topic deserves further investigations. Bruni et al. (2020a) results. Concerning the effect of BSF
meal on Atlantic salmon, data agree with Mancini et al.
The modifications occurring both on lipid and protein (2018) since no different VOCs profiles emerged among
fractions while handling fish fillets, i.e. storage and cooking, the samples.
promote the development of the fish volatile profile
(referred as volatile organic components; VOCs). Recently, In conclusion, the inclusion of BSF meal up to 25% did not
Nieva-Echevarría et al. (2018) found significant different jeopardise fillet physical and nutritional characteristics,
volatiles comparing wild and farmed European sea bass while upper inclusion levels mainly affect colour and
which the authors associated to contaminants and diets. In FA composition. Despite this, understanding if and how
addition, an interaction between fish growing conditions the diet affects fillet sensory properties and consumers’
and cooking methods was highlighted by the same authors, liking is necessary to positively judge BSF meal as protein
because of a different lipid content and FA profile. Hence, a source for aquafeeds. The early study by Sealey et al. (2011)
possible effect of BSF meal inclusion in the diet on farmed approached the topic through a sensory evaluation of
fish can be also hypothesised. The most common method rainbow trout fillets conducted by 30 untrained panellists
to evaluate VOCs composition is based on a solid-phase (14 males and 16 females, from 18 to 65 years old). The
extraction, followed by the GC-mass spectrometry (MS) method adopted was a triangle difference test, which
analysis of the sample headspace, as described in Iglesias consists to indicate the odd sample in a set of three blinded
and Medina (2008) and modified by Fratini et al. (2012). samples. Panellists were also free to express the reason of

1184 Journal of Insects as Food and Feed 8(11)

  Laboratory approaches for testing insect based aquafeeds

their choice and what were the attributes making different source in aquafeed. The authors served Atlantic salmon
the sample perception. Sealey et al. (2011) showed that blinded samples at 80 consumers, asking them to express
people were unable to discriminate between fillets from their like or dislike (using a 9-points scale) for a series
rainbow trout fed a control diet (containing anchovy meal) of sensory attributes. Interestingly, Ferrer Llagostera et
and the experimental diets containing normal and enriched al. (2019) highlighted that people had a good opinion of
(with n-3 PUFA) BSF prepupae meal. Recently, the intensity gilthead sea bream farmed using insects as feed ingredients
https://www.wageningenacademic.com/doi/pdf/10.3920/JIFF2020.0135 - Monday, October 21, 2024 7:21:19 PM - IP Address:2800:e2:3580:1a4:e0dc:7a69:6fd0:3f84

of the perceived sensory attributes of fish fed BSF meal was but they expected these fish to taste ‘less’. On the contrary,
evaluated by using trained (Belghit et al., 2018; Borgogno the hedonic evaluation proposed by Bruni et al. (2020a),
et al., 2017) or untrained assessors (Stadtlander et al., conducted under no informed condition (consumers did
2017). For instance, Stadtlander et al. (2017) proposed an not know what was the dietary treatment), gave important
organoleptic test on steam-cooked fillets from rainbow trout information. Firstly, people perceived as too pale the colour
fed a diet where 46% of FM was substituted by BSF meal. of fillets from groups fed diets with 66 and 100% BSF
Fifteen untrained panellists were asked to rate different meal inclusion levels and the flesh resulted slightly firmer
sensory characteristics (odour, colour, texture and taste) than the control group (0% BSF meal), in line with the
on a scale between 0 (does not apply) to 9 (applies fully). instrumental analyses (Belghit et al., 2018). Moreover, Bruni
Similarly, Belghit et al. (2019a) tested raw and baked fillets et al. (2020a) point out that increasing substitution level of
from Atlantic salmon fed diets at increasing substitution BSF meal (0, 33, 66 and 100%) in Atlantic salmon diet did
level of FM with BSF meal. Ten trained assessors were not counteract consumers’ overall liking and intention of
asked to evaluate the intensity of several sensory attributes re-consumption (>70% of positive answers). The results
(odour, colour, texture) by using a 15-cm non-structured from Spanish and Italian research groups give reason to
continuous scale. Results from the previous mentioned hope for a complete consumers’ acceptance of insect meal
studies mostly agreed. Indeed, the sole significant variation as protein source in the aquaculture sector.
detected was a difference in fillet colour even if Stadtlander
et al. (2017) found darker flesh in fillets from trout fed 4. Conclusions and future perspectives
insect meal, while the salmon fed 66% of BSF meal had
fillets significantly less coloured than those of the group fed Several analytical methods and laboratory techniques are
the control diet (without BSF) in Belghit’s et al. (2018). An presently available to deepen our knowledge about fish
innovative approach distinguishes the work of Borgogno welfare and quality in response to diets including BSF
et al. (2017), who found that BSF larvae meal inclusion meal. While these laboratory techniques have been used
at 20 and 40% in rainbow trout diets affected the fillet in fish and other animal’s responses to all kind of alternative
sensory profile when assessed through a descriptive analysis ingredients, a number of studies have recently showed their
and a temporal dominance of sensation (TDS) methods suitability for a better understanding of fish responses to
by trained panellists. Several outcomes can be retrieved BSF based diets. Results evidenced that certain dietary
from the proposed methodology, both in terms of sensorial inclusion levels of BSF meal are able to promote fish health,
attributes related to aroma, texture, appearance, and tactile welfare and quality of the product. However, there is a lack
sensations and of the sensory perception process during all of information about long-term use of these diets. For this
the chewing. The main criticism of these methods is about reason, scientists should perform further studies over longer
the training sessions. Indeed, both descriptive analysis and periods of time and possibly over the whole fish life cycle.
TDS require three training sessions of about 60 min. each, On this regard, emphasis should be given to nutritional
that means overall 6 sessions dedicated for training more programming experiments as well as the possible effect
the time necessary for sensory evaluation. Consumers’ of these new diets on fish reproduction. The laboratory
expectation and willingness to pay were investigated by methodological approaches included in this review article
Ferrer Llagostera et al. (2019) while Bruni et al. (2020a) may serve as a starting point for this further research.
focused on consumers’ liking. On one hand, discrete choice
experiment (DCE) was chosen by Ferrer Llagostera et al. Considering these last aspects, the authors suggest a
(2019) to understand the preferences of Spanish people constructive crosstalk between research and industry to
towards farmed gilthead sea bream fed diets containing sustain the development of a high quality and sustainable
insect meal. DCE is basically an on-line questionnaire which aquaculture.
proposes different products (in this case gilthead sea bream
fed insect meal) at different price levels in several purchase Acknowledgements
situations. In the case of Ferrer Llagostera et al. (2019),
the purpose was to identify the consumers’ trade-offs in This study was partially funded by Ricerca Scientifica
their choice decision. On the other hand, consumers’ test 2017 Cariverona, NUTRIFISH project N. 2017.0571 to
was selected by Bruni et al. (2020a) to investigate Italian’s Ike Olivotto.
liking and intention of re-consumption of Atlantic salmon
obtained by administering BSF meal to fish as protein

Journal of Insects as Food and Feed 8(11) 1185

 M. Zarantoniello et al.

Conflict of interest Barton, B.A., 2002. Stress in fishes: a diversity of responses with
particular reference to changes in circulating corticosteroids.
The authors declare no conflict of interest. Integrative and Comparative Biology 42(3): 517-525. https://doi.
org/10.1093/icb/42.3.517
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