Effects of full replacement of dietary fishmeal with insect meal from Tenebrio molitor on rainbow trout gut and skin microbiota

Background:Aquaculture must continue to reduce dependence on fishmeal(FM)and fishoil in feeds to ensure sustainable sector growth.Therefore,the use of novel aquaculture feed ingredients is growing.In this regard,insects can represent a new world of sustainable and protein-rich ingredients for farmed fish feeds.Accordingly,we investigated the effects of full replacement of FM with Tenebrio molitor(TM)larvae meal in the diet of rainbow trout(Oncorhynchus mykiss)on fish gut and skin microbiota.Methods:A feeding trial was conducted with 126 trout of about 80 g mean initial weight that were fed for 22 weeks with two isonitrogenous,isolipidic,and isoenergetic extruded experimental diets.Partially defatted TM meal was included in one of the diets to replace 100%(TM 100)of FM,whereas the other diet(TM 0)was without TM.To analyse the microbial communities,the Illumina MiSeq platform for sequencing of 16S rRNA gene and Qiime pipeline were used to identify bacteria in the gut and skin mucosa,and in the diets.Results:The data showed no major effects of full FM substitution with TM meal on bacterial species richness and diversity in both,gut mucosa-and skin mucus-associated microbiome.Skin microbiome was dominated by phylum Proteobacteria and especially by Gammaproteobacteria class that constituted approximately half of the bacterial taxa found.The two dietary fish groups did not display distinctive features,except for a decrease in the relative abundance of Deefgea genus(family Neisseriaceae)in trout fed with insect meal.The metagenomic analysis of the gut mucosa indicated that Tenericutes was the most abundant phylum,regardless of the diet.Specifically,within this phylum,the Mollicutes,mainly represented by Mycoplasmataceae family,were the dominant class.However,we observed only a weak dietary modulation of intestinal bacterial communities.The only changes due to full FM replacement with TM meal were a decreased number of Proteobacteria and a reduced number of taxa assigned to Ruminococcaceae and Neisseriaceae families.Conclusions:The data demonstrated that TM larvae meal is a valid alternative animal protein to replace FM in the aquafeeds.Only slight gut and skin microbiota changes occurred in rainbow trout after total FM replacement with insect meal.The mapping of the trout skin microbiota represents a novel contribution of the present study.Indeed,in contrast to the increasing knowledge on gut microbiota,the skin microbiota of major farmed fish species remains largely unmapped but it deserves thorough consideration.

Comprehensive evaluation of the metabolic effects of insect meal from Tenebrio molitor L.in growing pigs by transcriptomics, metabolomics and lipidomics

Background:The hypothesis was tested that insect meal(IM)as protein source influences intermediary metabolism of growing pigs.To test this,30 male,5-week-old crossbred pigs were randomly assigned to 3 groups of 10 pigs each with similar body weights(BW)and fed isonitrogenous diets either without(CON)or with 5%IM(IM5)or 10%IM(IM10)from Tenebrio molitor L.for 4 weeks and key metabolic tissues(liver,muscle,plasma)were analyzed using omics-techniques.Results:Most performance parameters did not differ across the groups,whereas ileal digestibilities of most amino acids were 6.7 to 15.6%-units lower in IM10 than in CON(P<0.05).Transcriptomics of liver and skeletal muscle revealed a total of 166 and 198,respectively,transcripts differentially expressed between IM10 and CON(P<0.05).Plasma metabolomics revealed higher concentrations of alanine,citrulline,glutamate,proline,serine,tyrosine and valine and a lower concentration of asparagine in IM10 than in CON(P<0.05).Only one out of fourteen quantifiable amino acid metabolites,namely methionine sulfoxide(MetS),in plasma was elevated by 45%and 71%in IM5 and IM10,respectively,compared to CON(P<0.05).Plasma concentrations of both,major carnitine/acylcarnitine species and bile acids were not different across groups.Lipidomics of liver and plasma demonstrated no differences in the concentrations of triacylglycerols,cholesterol and the main phospholipids,lysophospholipids and sphingolipids between groups.The percentages of all individual phosphatidylcholine(PC)and phosphatidylethanolamine(PE)species in the liver showed no differences between groups,except those with 6 double bonds(PC 38:6,PC 40:6,PE 38:6,PE 40:6),which were markedly lower in IM10 than in CON(P<0.05).In line with this,the percentage of C22:6n-3 in hepatic total lipids was lower in IM10 than in the other groups(P<0.05).Conclusions:Comprehensive analyzes of the transcriptome,lipidome and metabolome of key metabolic tissues indicate that partial or complete replacement of a conventional protein source by IM in the diet has only a weak impact on the intermediary metabolism of growing pigs.Thus,it is concluded that IM from Tenebrio molitor L.can be used as a dietary source of protein in pigs without causing adverse effects on metabolism.

Black soldier fly defatted meal as a dietary protein source for broiler chickens:Effects on growth performance,blood traits,gut morphology and histological features

Background: The present study has evaluated the effects of different inclusion levels of a partially defatted black soldier fly(Hermetia illucens L.; HI) larva meal on the growth performance, blood parameters and gut morphology of broiler chickens. A total of 256 male broiler chickens(Ross 308) were reared from d 1 to d 35 and assigned to4 dietary treatments(8 replicates/treatment and 8 birds/replicate). HI larva meal was included at increasing levels(0, 5%, 10% and 15%; HI0, HI5, HI10 and HI15, respectively) in isonitrogenous and isoenergetic diets formulated for 3 feeding phases: starter(1–10 d), growing(10–24 d) and finisher(24–35 d). Two birds per pen were slaughtered at d 35 and morphometric investigations and histopathological alterations were performed.Results: The live weight(LW) showed linear and quadratic responses to increasing HI larva meal(maximum for HI10 group). Average daily gain(ADG) showed a linear and quadratic responses to HI meal(maximum for HI10 group) during starter and growing periods. A linear decrease was observed for ADG during the finisher period. The daily feed intake(DFI) showed a linear and quadratic effect during the starter period(maximum for HI10 group).Linear and quadratic responses were observed for the feed conversion ratio(FCR) in the growing period and for the whole period of the experiment. The FCR showed a linear response in the finisher period(maximum for HI15).No significant effects were observed for the blood and serum parameters, except for the phosphorus concentration,which showed linear and quadratic responses as well as glutathione peroxidase(GPx) activity, the latter of which showed a linear response. The HI15 birds showed a lower villus height, a higher crypt depth and a lower villus height-to-crypt depth ratio than the other groups.Conclusions: Increasing levels of dietary HI meal inclusion in male broiler chickens may improve the LW and DFI during the starter period, but may also negatively affect the FCR and gut morphology, thus suggesting that low levels may be more suitable. However, no significant effects on the haematochemical parameters or histological findings were observed in relation to HI meal utilization.

Black soldier fly and gut health in broiler chickens:insights into the relationship between cecal microbiota and intestinal mucin composition

Background:The relationship between diet and intestinal microbiota and mucin composition appears to be fundamental for poultry gut health.The effects of insect meal(whose role as alternative feed ingredient is now well recognized)on gut microbiota and mucin composition have recently been reported in Tenebrio molitor-fed free-range and broiler chickens,but no data are currently available for Hermetia illucens(HI)-fed broilers.The present study evaluated the effects of dietary HI meal inclusion on cecal microbiota and intestinal mucin composition of broiler chickens.Results:A total of 256 male broiler chickens were allotted to 4 dietary treatments(control diet[C]and 5%,10%and 15%HI meal inclusion,with 8 replicate pens/treatment and 8 birds/pen)and slaughtered at 35 d of age(2 animals/pen,16 birds/diet).The cecal microbiota assessment by 16S rRNA amplicon based sequencing showed lower alpha diversity in HI15 chickens(Shannon,P<0.05)and higher beta diversity(Adonis and ANOSIM,P<0.001)in birds fed HI diets than C.Furthermore,HI15 birds displayed significant increase of the relative abundance of Proteobacteria phylum(False Discovery Rate[FDR]<0.05)when compared to HI10.L-Ruminococcus(Ruminococcus from Lachnospiraceae family),Faecalibacterium,Blautia and Clostridium genera were found to be characteristic of HI5 cecal microbiota(FDR<0.05),while broiler chickens fed HI10 and HI15 diets were characterized(FDR<0.05)by Lactobacillus and Ruminococcus(HI10)and Bacteroides,Roseburia and Helicobacter genera(HI15).Periodic-acid Schiff,Alcian Blue pH 2.5 and high iron diamine staining on small and large intestine also demonstrated lower mucin staining intensity in the intestinal villi of HI10 and HI15 birds than C(P<0.05).Conclusions:Dietary HI meal utilization at low inclusion levels(i.e.,5%)positively influenced either the cecal microbiota or the gut mucin dynamics in terms of selection of potentially beneficial bacteria and increase in villi mucins.However,high inclusion levels(in particular the 15%)may have a negative influence in terms of partial reduction of microbial complexity,reduction of potentially beneficial bacteria,selection of bacteria with mucolytic activity and decrease in villi mucins.

Effects of dietary Hermetia illucens meal inclusion on cecal microbiota and small intestinal mucin dynamics and infiltration with immune cells of weaned piglets

Background: The constant interaction between diet and intestinal barrier has a crucial role in determining gut health in pigs. Hermetia illucens(HI) meal(that represents a promising, alternative feed ingredient for production animals) has recently been demonstrated to influence colonic microbiota, bacterial metabolite profile and mucosal immune status of pigs, but no data about modulation of gut mucin dynamics are currently available. The present study evaluated the effects of dietary HI meal inclusion on the small intestinal mucin composition of piglets, as well as providing insights into the cecal microbiota and the mucosal infiltration with immune cells.Results: A total of 48 weaned piglets were randomly allotted to 3 dietary treatments(control diet [C] and 5% or10% HI meal inclusion [HI5 and HI10], with 4 replicate boxes/treatment and 4 animals/box) and slaughtered after61 days of trial(3 animals/box, 12 piglets/diet). The cecal microbiota assessment by 16 S r RNA amplicon based sequencing showed higher beta diversity in the piglets fed the HI-based diets than the C(P < 0.001). Furthermore,the HI-fed animals showed increased abundance of Blautia, Chlamydia, Coprococcus, Eubacterium, Prevotella,Roseburia, unclassified members of Ruminococcaceae, Ruminococcus and Staphylococcus when compared to the C group(FDR < 0.05). The gut of the piglets fed the HI-based diets showed greater neutral mucin percentage than the C(P < 0.05), with the intestinal neutral mucins of the HI-fed animals being also higher than the sialomucins and the sulfomucins found in the gut of the C group(P < 0.05). Furthermore, the piglets fed the HI-based diets displayed lower histological scores in the jejunum than the other gut segments(ileum [HI5] or ileum and duodenum [HI10], P < 0.05).Conclusions: Dietary HI meal utilization positively influenced the cecal microbiota and the small intestinal mucin dynamics of the piglets in terms of selection of potentially beneficial bacteria and preservation of mature mucin secretory architecture, without determining the development of gut inflammation. These findings further confirm the suitability of including insect meal in swine diets.

Evaluation of growth performance,feed efficiency and nutrient digestibility of red hybrid tilapia fed dietary inclusion of black soldier fly larvae(Hermetia illucens)

Insects,particularly black soldier fly larvae(BSFM),Hermetia illucens,have been regarded as promising protein and lipid sources in animal feed,including fish.Digestibility is the key factor that affects nutrient utilization,feeding physiology and efficiency.In the present work,the effect of BSFM on the digestibility of red hybrid tilapia(RHT)compared to fish meal(FM)was ultimately unveiled.A 56-day growth trial was performed with 60 RHT(~30 g)randomly distributed into six tanks and fed two experimental diets containing FM as a reference diet and BSFM as a test diet.In addition,faeces were collected to determine their chromium concentration and proximate composition.The apparent digestibility coefficient(ADC)of dry matter,crude protein and energy in the BSFM diet were observed to be significantly higher than those in the FM diet.Besides,the growth performance of fish fed with the BSFM diet exhibited significantly higher(P<0.05)weight gain,specific growth rate,protein efficiency ratio and lower feed conversion ratio than those in the FM group.This study shows that the BSFM feed was well digested by tilapia and improved its growth performance.

Defatted black soldier fly (Hermetia illucens) in pikeperch (Sander lucioperca) diets: Effects on growth performance, nutrient digestibility, fillet quality, economic and environmental sustainability

The use of insect meal in aquafeed formulations has recently gained attention.Detailed knowledge about the inclusion levels for pikeperch(Sander lucioperca),a promising candidate for intensive aquaculture in Europe remains,however,fragmented.In the present study,4 isoproteic(45%dry matter)and isoenergetic(21 MJ/kg)diets were formulated,including a control diet(H0)containing 30%fishmeal(FM)on an as-fed basis and the other 3 diets in which FM protein was replaced by defatted black soldier fly(Hemetia illucens)meal(HIM)at 25%,50%,and 100%(diet abbreviation H9,H18 and H36,corresponding to an inclusion level of 9%,18%and 36%,respectively).The feeding trial was performed in triplicate groups of 50 juvenile pikeperch(mean weight,68.7 g)fed with experimental diets for 84 d during which the growth performance,nutrient digestibility,fillet quality and economic and environmental sustainability of rearing pikeperch were evaluated.Our findings indicated that pikeperch in H0,H9,and H18 groups displayed better results regarding growth performance indices,except for survival rate where no significant difference among groups was recorded(P?0.642).A significantly lower organ-somatic index,including hepatosomatic,viscerosomatic and perivisceral fat index,was found in fish in H18 groups than other groups(P<0.05).Inclusion of HIM affected the digestibility of the nutrients and resulted in an almost linear reduction in the apparent digestibility coefficient of dry matter and protein.Concerning the fillet quality,dietary HIM negatively affected the protein and ash contents of the fish fillets,while the crude fat remained unchanged.Dietary HIM did not significantly modify total saturated,monounsaturated and polyunsaturated fatty acids in the fillets of fed pikeperch(P>0.05)but did reduce total n-3 fatty acids(P?0.001)and increased total n-6(P<0.001).Increasing inclusion levels of HIM reduced the environmental impacts associated with fish in-to-fish out ratio but entailed heavy burdens on energy use and eutrophication.Low and moderate inclusion levels of HIM did not negatively affect land use and water use compared to an HIM-free diet(P>0.05).The addition of HIM at a level as low as 9%elicited a similar carbon footprint to that of the control diet.The economic conversion ratio and economic profit index were negatively affected at increased insect meal inclusion levels.This study has shown that the incorporation of HIM in feed formulations for pikeperch is feasible at inclusion levels of 18%without adverse effects on growth performance parameters.The feasibility also highlighted the environmental benefits associated with land use and marine resources required to produce farmed fish.