1. Introduction
Insect protein meal is currently considered a viable alternative to meals made from fish (fishmeal) and other aquatic sources due to its nutrient profile, digestibility, and palatability. This view is confirmed and supported by several studies demonstrating positive growth and health performance in fish receiving insect meal [
1,
2,
3,
4].
The use of insect meal also implies relevant advantages in terms of environmental sustainability and, in perspective, economic viability [
5]. Farming insects might be highly convenient thanks to their favorable reproduction and growth rate, high feed conversion efficiency and because they can be reared on a wide array of bio-wastes. Insect production requires low energy input resulting in a low emission of greenhouse gases and ammonia [
2,
6,
7]. Nutrient composition of insect meal may significantly vary according to the species used, the nutritional composition of the rearing substrates and the production processes [
5]. Upon standardized and fully controlled production process, insect meal represents a very interesting protein source to be used for feeding many farmed fish species [
1,
3,
8,
9]. For the above-mentioned reasons, since 2017, its uses in aquafeeds have been approved in the EU (Commission Regulation 2017/893).
Among the five major groups of insects so far investigated (i.e., Black soldier fly, House fly, Mealworm beetles, Locusts-grasshoppers-crickets, and Silkworm), the Black soldier fly (
Hermetia illucens) has been defined as one of the most suitable species for insect meal production [
10]. Black soldier fly larvae meal is a high-value raw material rich in fat and protein [
2]. The crude protein (CP) can range from 30 to 60% [
11,
12], and it has been demonstrated to be a suitable replacement for soybean meal in pigs and poultry diets [
2,
13,
14]. Several studies have also shown that
Hermetia illucens meal can partially or fully replace fishmeal in diets for several fish species without a negative impact on fish growth performances and health [
3,
4,
12,
15,
16,
17,
18,
19,
20]. However, in some cases, a limited trend (not significant) to reduce growth performance was reported in Rainbow trout (
Oncorhynchus mykiss) and Atlantic salmon (
Salmo salar) [
15,
16]. In turbot (
Psetta maxima), a significant reduction of feed conversion and growth rates were observed when insect meal inclusion was higher than 33% [
17]. Some studies also reported that the dietary inclusion of Black soldier flies larvae meal did not alter the sensory quality of fillets [
16,
18].
In several terrestrial animals, such as poultry and pigs, diets significantly modified morphology, histology, and biochemical characteristics of the gastrointestinal tract [
21,
22]. The effect of insect meal inclusion on fish gastrointestinal tracts is still debated and not yet fully understood. In Jian carp (
Cyprinus carpio var. Jian), the dietary inclusion of
Hermetia illucens meal higher than 8% (75% fishmeal replacement rate) resulted in pathological alteration of intestinal tissue, a higher content of debris within intestinal microvilli, and an increase of HSP70 relative gene expression in hepatopancreas [
22]. Dumas et al. [
23] reported that shorter villi in Rainbow trout (
Oncorhynchus mykiss) fed a diet containing 26.4% of
Hermetia illucens meal, while no differences occurred using lower inclusion levels (6.6% and 13.2%). Conversely, Renna et al. [
24] demonstrated that a 40% inclusion rate of
Hermetia illucens meal in the Rainbow trout (
Oncorhynchus mykiss) diet did not affect villus length.
Consistently with recent studies, more effects have been reported on gene expression/modulation, including many gut-related genes [
25,
26,
27,
28].
The objective of this study was to investigate whether the dietary inclusion of
Hermetia illucens meal and fishmeal replacement rates higher than those previously investigated [
4] can determine the potential negative impact on fish growth performances as also suggested, for instance, by Kroeckel et al. [
17]. In detail, the effects of the dietary inclusion of 17%, 33% and 50% of insect meal (25%, 50% and 100% fishmeal replacement rate, respectively) on survival rate, growth performance, feed intake, feed conversion rate, intestine morphology, and gene expression, were investigated. To this purpose, adult Zebrafish (
Danio rerio) were used as fish model.
4. Discussion
The lack of feeding behavioral differences among treatments suggests that all feeds had acceptable palatability, independently of the inclusion level of the
Hermetia illucens meal in the feed. Moreover, in the present study, the inclusion level of the
Hermetia illucens meal was higher than those used in other studies [
4,
34], and the meal used was full fat rather than defatted [
34]; this aspect is quite relevant since fat might strongly impact the quantitative production of raw materials, the characteristics of the pellets, as well as the overall feed palatability.
The few mortality events observed in the present study were mainly recorded in the early stages of the experimental period when fish were still in a fast-growing phase and not yet in the adult stage. After that, mortality events were mainly associated with the procedures related to the periodic measurements (handling, anesthesia, etc.) and thus to the handling stress (i.e., capture, anesthesia, photographic detection, and weighing) rather than dietary treatments. Overall, the results suggested that the dietary inclusion of
Hermetia illucens meal up to 50% (100% replacement of fishmeal) did not negatively affect fish survival rate and feed intake. On the contrary, FCR was significantly more favorable when
Hermetia illucens meal was included even at the higher level, and totally replacing fishmeal. Similar results were observed in a previous study when lower inclusion rates (but similar fishmeal replacement rates) were used [
4]. The only difference of interest in this work is related to the FCR values. When the dietary inclusion rate of
Hermetia illucens meal was high, as in the present study (from 17% to 50% and from 33% to 100% fishmeal replacement rate), they decreased from 2 to 1.8. However, the FCR values observed were consistent with those reported in Zebrafish by Yossa et al. [
35]. Also, Stejskal et al. [
34] reported similar FCR values (1,81) in pikeperch (
Sander lucioperca) when 36% of
Hermetia illucens meal (defatted) was included in the diet; on the contrary, the diets characterized by a lower level of
Hermetia illucens meal showed much more favorable FCR values (1,28 and 1,26 with 9 and 18%
Hermetia illucens meal, respectively). This means that while Stejskal et al. [
34] in pikeperch observed a negative correlation between
Hermetia illucens meal (defatted) inclusion level, the results of the present study (carried out on zebrafish) suggest a positive effect of the inclusion of the
Hermetia illucens meal (full fat) on the FCR.
In the lack of specific studies, this result can be hypothetically explained based on the dietary differences and notably with the slightly higher crude lipid content and lower fiber, starch and ash content in the diet containing HI meal. Similarly, another hypothesis might be related to the dietary amino acids profile, notably for Lysine and Methionine and Cysteine, among others (
Table 1).
Partial or total fishmeal replacement with insect meal has demonstrated no negative effects on feed utilization and mortality in several other species of interest for the aquaculture sector [
36,
37]. Kroeckel et al. [
17] reported that the dietary inclusion of
Hermetia illucens meal from 17 to 33% for replacing fishmeal showed no negative effect on survival, growth, and feed utilization in turbot. Other studies [
11,
19] reported similar results on other species, such as Atlantic salmon [
18], Gilthead seabream (
Sparus aurata), European seabass (
Dicentrarchus labrax) and Japanese seabass (
Lateolabrax japonicus). Furthermore, several studies reported that fishmeal replacement with
Hermetia illucens meal up to 50% resulted in similar growth performances and feed utilization in Rainbow trout (
Oncorhynchus mykiss) [
16,
24,
36,
38,
39], Jian carp (
Cyprinus carpio var. Jian) [
22] and Nile tilapia (
Oreochromis niloticus) [
40]. Moreover, comparable yield data with no significant alterations (i.e., off-flavors) were reported by several authors in trout and salmon meat [
16,
18].
Similar results [
41,
42] were also obtained by replacing fishmeal with
Tenebrio molitor meal in some fish species, such as Rainbow trout (
Oncorhynchus mykiss) and Blackspot seabream (
Pagellus bogaraveo). In Blackspot seabream [
42], no significant decrease was recorded for FI, FCR, BWg and fillet characteristics (i.e., texture properties and water holding capacity). In Rainbow trout (
Oncorhynchus mykiss), the feeding rate was lower in the group fed a diet including 50%
Tenebrio molitor meal and 25% fishmeal compared to the group fed a fishmeal-based diet (control; 75% fishmeal). This lower FI did not negatively affect the BWg but rather resulted in better FCR and SGR, as well as better PER (protein efficiency ratio). Also, the survival rate followed the same trend, being higher for the groups fed Tenebrio molitor meal [
41].
Notably, insect meal protein inclusion in fish diets might also either reduce or improve fish growth performance and feed utilization, essentially depending on its inclusion level and the fish species. In turbot, according to Kroeckel et al. [
17],
Hermetia illucens meal inclusion level higher than 33% (from 49 to 76%) demonstrated a decrease of BWg, FI and FCR. Similarly, Xiao et al. [
12] observed a BWg reduction in Yellow catfish when the fishmeal replacement rate was higher than 65%. Moreover, low FCR and growth performance [
15] were also observed in other fish species fed a high level of
Hermetia illucens meal, such as Channel catfish (
Ictalurus punctatus) and Blue tilapia (
Oreochromis aureus), but some doubts may arise in relation to the characteristics of the meal production process.
Concerning the aspects related to Zebrafish growth performances, the results observed in the present study differed from those reported in a previous one [
4]. The results also differed from those described by Kroeckel et al. [
17] on turbot. Cumulative BWg was significantly higher in treatments IM17 and IM50, in comparison to control (IM0), and FCR resulted higher in the control treatment than in the experimental treatment. In addition, fish final BW did not result in significant differences between the control and experimental treatments. However, the highest final BW (not significantly) was recorded in treatments IM17 and IM50. Therefore, these results suggest that in Zebrafish level of
Hermetia illucens, meal inclusion higher than 33% lead to better growth performance.
In accordance with other studies carried out on Zebrafish by Zarantoniello et al. [
43,
44], Vargas et al. [
45], and Fronte et al. [
4], intestinal morphometry did not show any histological alteration and significant differences in villus length of zebrafish. This result might confirm the hypothesis that this ingredient is devoid of allergens and other substances that are detrimental to enterocytes, as suggested by Li et al. [
22]. The absence of intestinal histological alteration was also previously reported in several fish species fed a diet partially or totally composed of
Hermetia illucens meal, such as Atlantic salmon, Rainbow trout (
Oncorhynchus mykiss) and Japanese seabass [
18,
23,
24,
46]. In Jian carp, pathological intestine damage (i.e., tissue disruption) was observed when the fishmeal diet was replaced with insect meal for more than 75% [
22]. Black soldier fly meal inclusion higher than 50% (75 and 100%) also showed hepatic steatosis, microbiota modification, higher lipid content, fatty acid modification and higher expression of immune response markers in zebrafish [
44]. Therefore, as previously suggested by Li et al. [
22], the results obtained in this study seem to confirm that a 50% inclusion level of Black soldier fly meal in the diet for zebrafish represented the best compromise between ingredient sustainability and proper fish growth and welfare.
Data available in the scientific literature on insect meal inclusion effect on intestine histology are still yet variable and not exhaustive. In Rainbow trout (
Oncorhynchus mykiss), a significant reduction of villus length in the anterior intestine was observed when fed a diet containing partially defatted Black soldier fly larvae meal at 26.4% inclusion level (100% fishmeal replacement). Morphological changes in the anterior intestine were also detected in Jian carp-fed defatted Black soldier fly larvae meal at 8% inclusion [
22].
Noteworthy is that, as already reported in a previous study [
4], villus length in the upper intestinal lumen surface was significantly higher than those in the lower one. However, no differences were recorded in villus length in the upper and lower intestinal lumen surface among the considered groups, control included (no Black soldier fly meal). This suggests that the differences in villus length between the upper and the lower intestinal lumen surface of zebrafish are not diet related. Further investigation to assess whether these differences are due to an anatomical characteristic of zebrafish or to other factors (i.e., age or rearing system) is needed.
Concerning goblet cells, no differences were recorded among treatments for density and area of Alcian blue-positive goblet cells as well as for density of PAS-positive goblet cells. Conversely, the area of PAS-positive goblet cells resulted significantly higher in the IM33 diet group than in the control group and IM50. The intestinal barrier efficiency depends on host-microbial balance as well as mucin production [
47,
48,
49]. Other studies showed that diet composition could strongly affect mucins production by goblet cells in European seabass (
Dicentrarchus labrax), Common carp (
Cyprinus carpio) and Gilthead seabream (
Sparus aurata) [
21,
50,
51,
52]. However, although it seems that diet plays a key role in goblet cell size and density, based on the available knowledge, it is not possible to formulate a specific hypothesis for explaining the mechanism that underlies this finding. Hence, further investigations on the biological response of the digestive tract to different diets are necessary.
Because of its relevance in animal nutrition,
Slc15a1/
pept1 is one of the best-studied solute carriers amongst those characterized in teleost fish [
53,
54,
55]. It is thus adopted as a marker of functional and regulatory expression of the correct setup of the absorptive potential of the intestinal epithelium in all vertebrates, teleost included. Due to its importance at the intersection of the alimentary functions of the gut, its response under physiological and dietary solicitations, and its possible involvement in examples of total body plasticity, such as growth and compensatory growth,
pept1 represents a highly specific sensor [
56]. On these bases, the results of the present study suggest that the considered diets were comparable in relation to dietary effectiveness and, particularly, to digestion and absorption of the protein components.
Besides
pept1, data on
gata4 transcriptional expression are coherent according to the equivalence of standard and insect meal treatments.
Gata4 is a key regulator of epithelial cell differentiation and epithelial-mesenchymal interactions in the gut epithelium of zebrafish [
57].
Gata4 is not significantly regulated by fishmeal replacement with insect meal in the Zebrafish gut, thus indicating no specific impact on the epithelium. Nevertheless, the results hint at an up-regulating trend of
gata4 expression consequent to an increase of insect meal inclusion; interestingly [
58],
gata4 is a crucial “housekeeping” of the barrier function of the gut epithelium (i.e., it controls mucosal integrity) in vertebrates. In this view, the insect meal dietary assumption might be furtherly investigated to assess the ability to improve the barrier performance of intestinal epithelium after long-term feeding, which in turn might improve the healthy metabolism of fish.
In this regard, an added value comes from the assessment of the invariant expression of
nfkb1b mRNA, whose protein product is a component of the
NF-kB complex. In fact,
NF-kB is a key node of innate immunity and inflammatory pathways in the fish gut, zebrafish included [
59,
60]. Overall, the results of the gene expression suggest a safe use of Black soldier fly meal and possible ameliorating effects at the intestine epithelium level and consequently on the whole fish’s health and growth performances.