The effect of different levels of Hermetia illucens oil inclusion on caecal microbiota of Japanese quails ( Coturnix japonica , Gould, 1837)

In this study, we investigated the effect of the dietary inclusion of Hermetia illucens larvae oil on the diversity and structure of the bacterial community of the caecal content of Japanese quails ( Coturnix japonica ). A total of 40 quails, equally selected for slaughter from 100 animals which were divided evenly into four treatment groups including control group (C) with a diet containing corn oil and 3 experimental groups with partial (25%, 50%) or total (100%) substitution of corn oil by H. illucens larvae oil, here referred to as Black soldier fly larvae oil (BSFO): BSFO 25, BSFO 50 and BSFO 100, respectively. After slaughtering (42 days of age), the microbiota of caecal samples was examined by high-throughput sequencing using the V4-V5 region of the 16S rRNA gene. In all the studied groups the dominant phylum was Firmicutes with prevailing families of Ruminococcaceae and Lachnospiraceae . Caecal microbiota was meaningly influenced on genus level. The linear discriminant analysis effect size (LefSe) analysis for the differential taxa abundance showed that Lactobacillus was significantly increased in BSFO 25 group, Fusicatenibacter was significantly enriched in all the experimental groups fed larvae oil (BSFO 25, 50 and 100) and Subdoligranulum was highly elevated in BSFO 100 group. The analysis revealed statistical dissimilarities between the control group (C) and the groups with 50% and 100% oil replacement (BSFO 50 and 100). The bacterial diversity was significantly suppressed in the samples of quails fed the diet with a total inclusion of H. illucens oil (BSFO 100). The results showed the considerable effect of Black soldier fly larvae oil on the caecal microbiota of Japanese quails.


Introduction
Poultry is nowadays the leading source of meat produced due to the remarkable feed conversion ratio, short lifecycle, and low greenhouse gas emission (FAO, 2020).Despite that chicken still dominate the poultry egg and meat production sectors, quails' production is a widely emerging branch in the poultry industry as it introduces diversity among both egg and meat yield and has been used extensively for both purposes (Minvielle, 2004;Nasr et al., 2017;Sabow, 2020).In Asia, Japanese quails are typically reared for their egg yield, while in the US and Europe their meat production is the core driver and in some Countries, such as Turkey for instance, Japanese quails are produced for both eggs and meat (Narinc et al., 2015).Reasons for interest in breeding these birds insist in their small size, rapid growth, early sexual maturity (7-8 weeks), short generation interval, and high laying rate (Du et al., 2020;Huss et al., 2008).In addition, quail are increasingly becoming an alternative to chicken, especially for health-conscious consumers, due to the higher levels of vitamins (A, C), minerals and amino-acids and lower content of fat and cholesterol in their meat (Fakolade, 2015;Glick and Fischer, 2013).Japanese quail (Coturnix japonica) moreover represents favoured animal model in the field of poultry research (Huss et al., 2008) and due to the well-researched and confirmed genetic similarities with chicken (Gallus gallus domesticus) it has been adopted as the best optimised model to study the poultry functional genomics (Minvielle, 2009;Shin, 2017).However, the number of intestinal microbiota studies, which has been an important topic in recent years, is surprisingly limited in these animals.It is now well accepted that the microbiota of digestive tract contributes to the overall health status of animals and their productivity.Even if the primary role of the gastrointestinal tract (GIT) microbes is digestion of food substrates, the microbiota is important in many other functions ranging from defense against pathogens, production of nutrients to maturation and regulation of the immune system (Aruwa et al., 2021;Carrasco et al., 2019;Clavijo and Flórez, 2018).
Up to now only few studies on the characterisation of quail intestinal microbiota have been done (Du et al., 2020;Ma et al., 2021;Su et al., 2014;Wilkinson et al., 2016).They differ substantially in intent and goals, either characterising the bacterial community profile along the whole GIT (Du et al., 2020;Su et al., 2014;Wilkinson et al., 2016), or focusing, from different reasons, on ileal microbiota (Borda-Molina et al., 2020;Vollmar et al., 2020), or describing composition of cultivable bacteria (Du et al., 2020;Su et al., 2014;Wilkinson et al., 2016), or studying caecal (Liu et al., 2015) or duodenal and ileal microbiota of atherosclerosis susceptible and resistant quail strains (Liu et al., 2018).An interesting article of Ma et al. (2021) provided the description of the gene catalog of the caecal bacteria of Japanese quail including the comparison of bacterial taxa and predictive metagenomic functions of male and female quail (Ma et al., 2021).
Research on altering the composition of the poultry intestinal environment with the use of insect feed additives as an alternative source of fats, proteins and/or antimicrobial peptides has made significant strides in recent years (Clavijo and Flórez, 2018;Shin, 2017).Hermetia illucens (also called black soldier fly) and Tenebrio molitor are the most studied insect species in poultry nutrition (Benzertiha et al., 2020;Bovera et al., 2018;Colombino et al., 2021;Kierończyk et al., 2018;Moniello et al., 2019;Secci et al., 2018Secci et al., , 2022) ) One of the major benefits of this insect is the ability to decompose organic waste to animal feed as a dietary source (Addeo et al., 2021;Surendra et al., 2016).
Corn production has previously showed negative environmental consequences, such as gas emissions, deforestation, and high-water costs (Holka and Bieńkowski, 2020), which is not the case for the environmentally friendly production of black soldier fly (BSF) feed materials, based on the use of plant-origin wastes, as a source of nutrients in vertical rearing systems, to limit greenhouse gas (GHG) emissions and to use frass as a fertiliser (Józefiak et al., 2016).Despite the higher cost of BSFO compared to corn oil, it can be employed as the basis of a highly promising technology to sustain a circular economy (Barragan-Fonseca et al., 2017).Up to date, only one study compared the usage of BSFO as an alternative source of corn oil in poultry, but the study focused on growth performance, serum parameters and carcass characteristics Kim et al., 2020).
In this study, we investigated the effect of H. illucens larvae oil on caecal microbiota of Coturnix japonica.
Commonly used corn oil (CO) was in quail diet partially (25%, 50%) or totally (100%) replaced by BSFL oil and the caecal content was analysed by highthroughput sequencing (HTS) of 16S rRNA fragments and evaluated for the bacterial diversity, community structures and taxonomic composition.

Ethics statement
The animals were treated in accordance with the EC Directive 63/2010/EEC on the protection of the animals used for experimental and other scientific purposes.The experimental procedures were approved by the Ethical Animal Care and Use Committee of the Department of Veterinary Medicine and Animal Production of the University of Napoli Federico II, Italy (prot.N. 2017/0017676).The trial was carried out on a private quail's farm in Sardinia (Italy).

Animals and experimental design
A total of one hundred 7 days old Japanese quails (Coturnix japonica) were equally divided in 4 groups (5 replicates of 5 birds/replicate) in galvaniased metal cages (100 cm length × 50 cm depth × 25 cm height), where they were reared until 42 days of age.At the beginning of the trial the quails were housed at 32-35 °C using the infrared heat lamps (150 W) situated at 25 cm from the floor of the cages.Then, the temperature was gradually decreased until the end of fourth week to reach final temperature of 23-24 °C.The lighting programme was 16:8 h light: dark.

Diet
The birds were fed 4 isoproteic and isoenergetic diets, which differed only in the source of fat (oil).The control group (C) was fed a basal diet containing corn oil (CO) and the three experimental groups (BSFO 25, BSFO 50 and BSFO 100) were fed the same diet, in which, CO was partially (25%, 50%) or totally (100%) substituted by BSF oil (BSFO) extracted from BSF larvae by cold press extraction technique, as commercially available from PROTIX, Dongen, the Netherlands to keep the amount of 50 g of added oil per kg of diet.PRO-TIX, recognised as a leading insect farming company in EU, assure the purity of their industrial products as they must conform with the relevant EU legislations by ensuring high standards of animal welfare in insect production.Feed and fresh water were administered ad libitum.The analysis of the diets' contents was performed according the AOAC (2005) methods: for crude protein (method 978.04), ether extract (method 920.39), crude fiber (method 978.10), dry matter (2001.12)and ash (method 930.05).Based on the composition of the diet's constituents, the content of amino acids was estimated.
From the diets' chemical composition, the metabolisable energy (ME) was calculated based on the NRC, (1994) equations.Detailed information about the experimental diets and their nutritional characteristics are shown in the Table 1.
To perform a fatty acids (FAs) transmethylation analysis, a base-catalysed procedure was used, as reported by Christie et al. (1982) and modified by Chouinard et al. (1999).A gas chromatograph (Agilent technologies, model 5890, Santa Clara, CA, USA), equipped with a fused SP-2560 silica capillary column L × I.D. 100 m × 0.25 mm, df 0.20 μm (Supelco, Inc., Bellefonte, PA, USA) was used for FAs methyl esters quantification.Helium was used as carrier gas, with 280 kPa of constant pressure, 50 mL/min splitting flow, and 1 μL of injection volume.Concerning the column parameters, for 15 min the column temperature was sustained at 170 °C, then increased up to 240 °C by adjustment of 5 °C/min.The overall execution duration was 64 minutes.The FAs peaks were categorised through comparision of the retention times of a commercial standard comprising 37 methyl esters of FAs (Sigma-Aldrich, St. Louis, MO, USA).Controlling of the CLA isomers retention time was done by the elution of the commercial standard (Larodan AB, Solna, Sweden) of these fatty acids.By applying a percentage calculation to the overall area of the eluted peaks, the area of each specific FA found in the sample was determined.The fatty acid profile analysis of H. illucens oil, corn oil and the diets used in this experiment are shown in Table 2.

Growth performance
The live weights (LW) of birds were recorded individually at the beginning (day 7) and the end of the experiment (day 42).The amounts of administered feed and leftovers were measured daily to calculate the birds' feed intake.Average daily feed intake (ADFI), average daily gain (ADG) of live weight, and feed conversion ratio (FCR) were calculated for the entire experimental period (35 days).

Samples collection
Ten quails (42 days old) were picked from each group (2 birds from each replicate) and slaughtered by cervical dislocation.The intestine was immediately removed from the carcasses and the luminal contents from the caeca were collected, packed and sealed in sterilised micro-centrifuge tubes (2.0 ml, Eppendorf®), immediately refrigerated (for about 1 hour) and transported into laboratory.The samples were frozen at −80 °C and lyophilised using Heto powerdry LL3000 freeze dryer (Thermo Fisher Scientific, Wilmington, DE, USA).The dried samples were transported to the Institute of Animal Physiology and Genetics of the Czech Academy of Sciences (Prague, Czech Republic) for further analysis.
The list of samples is summerised in Supplementary Table S1.

DNA extraction
The DNA was extracted from the dry caecal samples using PowerSoil DNA Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.The concentration and quality of the nucleic acids were measured using a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and the DNA was stored at −20 °C until further use.

Amplification and purification of 16S rRNA fragments
The DNA isolates were diluted 10-fold in nuclease-free water and 2 μl (~20 ng/μl) were used as templates for the PCR reaction.The V4-V5 region of the 16S rRNA gene was amplified using the specific primer pair, BactB-F (GGA TTA GAT ACC CTG GTA GT) and BactB-R (CAC GAC ACG AGC TGACG) (Fliegerova et al., 2014), using the EliZyme™ HS FAST MIX Red Master Mix (Elisabeth Pharmacon, Brno, Czech Republic).Thermal cycling conditions included a denaturation step for 5 min at 95 °C, followed by 25 cycles of 30 s at 95 °C, 30 s at 57 °C and 30 s at 72 °C, a final elongation step at 72 °C for 5 min.The length and quality of PCR amplicons were checked by 1.5% agarose gel electrophoresis.
The amplicons were purified using the Monarch® PCR and DNA Cleanup Kit (New England BioLabs, Ipswich, MA, USA) and their concentration was checked using a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

Library preparation and next generation sequencing
The libraries were prepared from purified amplicons using the NEBNext Fast DNA Library Prep Set kit (New England BioLabs, Ipswich, MA, USA) and the Ion Xpress Barcode Adapters 1-96 Kit (Thermo Fisher Scientific, Waltham, MA, USA).The quality of the libraries was checked by the Agilent 2100 Bioanalyser instrument using the Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA).The quantification of the libraries was done using the KAPA Library Quantification Kit (KAPA Biosystems, Roche, Pleasanton, CA, USA).A volumetric pooling of each library was done after its normalisation by dilution to achieve a 30 pM concentration and used for the template amplification and enrichment by the Ion OneTouch™ 2 instrument using the Ion PGM™ HiQ™ View OT2 Kit-400   (Thermo Fisher Scientific, Waltham, MA, USA).The enriched template was sequenced with the Personal Genome Machine (PGM™) System (Thermo Fisher Scientific, Waltham, MA, USA) using the Ion PGM™ Hi-Q™ View Sequencing solutions kit and the Ion 316™ Chip v2 BC according to the manufacturer's protocols.

Bioinformatic and statistical analysis
The growth performance data were subjected to a oneway ANOVA with diet as a fixed effect, following the GLM procedure (General Linear Model) of the IBM SPSS Statistics package (IBM Corp. Released 2011.IBM SPSS Statistics for Windows, Version 20.0.Armonk, NY, USA).The raw sequences retrieved in FASTQ format from the Ion Torrent software were analysed using Qiime2 version 2020.2 software (Bolyen et al., 2019).The sequences were quality filtered, and denoised using DADA2 and chimeras were removed (Callahan et al., 2016).The rarefaction was conducted to ensure a uniform sampling depth, the dataset was subsampled to a minimum of 5000 reads per sample.The rarefaction curves reached a plateau, showing that the depth of sequencing was adequate and all the species in the samples were sufficiently covered (Supplementary Figure S1).The sequences were clustered into Amplicon Sequence Variants (ASVs) by VSEARCH, and the taxonomic assignment was achieved with a BLAST search against the SILVA database (version 132) with a 97% threshold (Rognes et al., 2016).The bacterial diversity was assessed using alpha diversity indices (Faith's Phylogenetic Diversity, Pielou Evenness, and Shannon Entropy), the comparison between the groups was done with the Kruskal-Wallis H test and visualised using the qiime2R, tidyverse and ggplot2 packages in R-Studio (version 4.2.1)(Bisanz, 2018;Wickham, 2016;Yan Hui, 2021).Beta diversity was calculated using the Jaccard distance matrix.The principal coordinate analysis (PCoA) was used for the visualisation, and the resultswere plotted using EMPeror (Vázquez-Baeza et al., 2013).The permutational multivariate analysis of variance (PERMANOVA) with 999 permutations was performed to determine the statistical differences between the groups, PERMDISP test was also performed to check the homogeneity of dispersions among the ani-mal groups.Linear discriminant analysis (LDA) with an effect size (LEfSe) algorithm (Segata et al., 2011) was done on the Galaxy web module (http://huttenhower .sph.harvard.edu/galaxy/(accessed on 15 August 2022) to identify the significantly differentially abundant taxa, with the following parameters: alpha = 0.05 and a minimum LDA score = 2.0.
The sequence information was deposited in the Sequence Read Archive under accession number: PRJNA871111.

Fatty acid composition of the dietary fat sources
Oil sources (50 g/kg) were added into a soybean meal and corn base diet.The analysis of the experimental diets showed a similar content of crude protein, ether extract and ash, but the fatty acid composition of the oil sources clearly differed as shown in Table 2. BSF larvae oil was rich in saturated fatty acids (SFA) as the result of high concentrations of lauric (C12:0) and myristic (C14:0) acids, while corn oil had higher content of monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) concentrations represented by oleic (C18:1 cis-9) and linoleic acid (C18:2cω6), respectively.

Growth performance
No statistically significant differences (P > 0.05) were recorded for the birds' live weights (LW), average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR).Results are summarised in Table 3 showing no influence of the different dietary treatments on the animal growth parameters.

Diversity and similarity of bacterial communities
The bacterial population in the caecal samples of quails of the 4 groups was qualitatively and quantitatively analysed for species richness, evenness and phylogenetic diversity.The analysis showed a lower diversity richness and evenness mainly in the samples of quails fed diet with a total replacement of corn oil by H. illucens larvae oil, as shown in the Supplementary Table S2.
Mainly, the Shannon entropy revealed a significant difference in richness between the control group and the BSFO 100 group (P = 0.03), also between the BSFO 25 group and the BSFO 100 group (P = 0.03), as shown in Figure 1A.Similarly, Pielou evenness index showed a significant difference between the control group and the BSFO 100 group (P = 0.02), also between the BSFO 50 group and BSFO 100 group (P = 0.04), as shown in Figure 1B.However, the Faith's phylogenetic distance showed no significant differences among the groups.
The results of the alpha diversity assessment are shown in Supplementary Table S3.The Beta diversity, which evaluates the similarity/differences in bacterial composition among the groups, was assessed using Jaccard's non-phylogenetic distance matrix.A Principal Coordinate Analysis (PCoA) plot (Figure 2) shows the spatial separation of the samples and statistical analysis revealed significant differences among all the studied group (PERMANOVA P < 0.05) except the comparison of control group with BSFO 25 group (P > 0.05).PERMDISP showed no statistical differences among the groups (P < 0.05), indicating a low intergroup variability (Table 4).

Taxonomical composition
In total, the caecal bacterial community consisted of 5 phyla including 110 bacterial phylotypes.3 and listed in the Supplementary Table S4.

Determination of taxonomic biomarkers
Linear discriminant analysis effect size (LEfSe) was performed to determine the bacterial taxa with significantly different levels of abundance between the control group and the other groups of quails in which the H. illucens larvae oil was included in diet.

Discussion
In this study, we examined the influence of black soldier fly larvae oil (BSFO) on bacterial community of the caecal content of Japanese quails.BSFO has attracted attention in the recent years as an alternative oil source in poultry nutrition, because this insect oil, which is rich in medium-chain fatty acids (MCFA), especially lauric acid (Ewald et al., 2020), could have a positive effect on growth performance, gut health and meat quality in broiler chickens (Cullere et al., 2019;Kim et al., 2020;Schiavone et al., 2018;van Immerseel et al., 2006).BSFL  Taxa with a relative abundance lower than 1% are grouped as 'Others' .
and its oil moreover exhibit antioxidant, antimicrobial and immune-modulating properties (Lee et al., 2018;Mlcek et al., 2014).The research on the effect of the insect oil on poultry is still scarce and insufficient and completely lacking on quails.Therefore, we focused on the study of the influence of BSFO on the caecal microbiota of Japanese quails.Even if the whole poultry digestive tract is important and each part plays a specific role in the digestion process and absorption of nutrients, the caecum represents the primary site of fermentation in the avian GIT responsible for transformation of indigestible carbohydrates (cellulose, starch) into shortchain fatty acids (SCFA) (Józefiak et al., 2004), whose processes are closely related to productivity (Díaz Carrasco et al., 2018;Waite and Taylor, 2014).With up to 1011 cells per gram, caeca have the greatest bacterial number and biodiversity along the poultry GIT (Grant et al., 2018) and it is dominated by the phyla Firmicutes, Bacteroidetes and Proteobacteria (Oakley et al., 2014).
The dietary treatments used in this study did not significantly affect the animals growth performance parameters (ADFI, ADG and FCR).Results in respective poultry literature regarding an influence of insect oil on feed intake and body weight gain are not consistent describing positive (Benzertiha et al., 2019a), or, more often, no significant effects on growth of broiler chickens (Benzertiha, et al., 2019b;Chen et al., 2022;Kawasaki et al., 2019;Kim et al., 2020;Schäfer et al., 2023), which is in agreement with our study.The discrepancies can be attributed to different experimental conditions, variations in animal age, breed, trial settings and management, feed composition, and the wide range in FA contents of insect oils.However, a broader view of the influence of BSF oil as part of the poultry diet indicates the beneficial effect on the animal health and production (Gasco et al., 2018).
Regarding the influence of the diet on the diversity of the caecal microbiota, the richness and equity in bacterial species abundance was significantly lower in the BSFO 100 group compared to the Control group.The Principal Coordinate Analysis (PCoA) based on the Jaccard distance matrix, showed the spatial separation of the samples, with statistical differences between the studied groups.This is in agreement with Biasato et al. (2020) who showed a lower alpha diversity in broiler chicken fed 15% H. illucens meal and a significant beta diversity (using the weighted UniFrac distance matrix) between birds fed H. illucens diets and control diet fed a maize, corn gluten and soybean meals (Biasato et al., 2020).In contrast, Dabbou et al. (2021) study didn't find differences in alpha diversity but the Principal component analysis (PCA) based on OTUs relative abundance showed a clear separation of the control samples fed with soybean oil from sample of the BSF oil group diet.The significant beta diversity differences were also described in several other insect-fed poultry studies (Biasato et al., 2018(Biasato et al., , 2019;;Borrelli et al., 2017;Dabbou et al., 2021).Our results are partially in agreement with Chen et al. (2022) who also found a lower richness in alpha diversity in the chickens fed different levels of BSF oil, however the PCoA based on OTUs revealed no significant difference in beta diversity between the studied groups.A decreased bacterial diversity is in general a negative phenomenon because the species diversity plays an important role in maintaining the stability of the intestinal ecosystem and its normal ecological function.A loss of diversity could initiate immune-mediated disorders, which can result in the development of inflammatory disease.This is well documented mainly in human research (Al Bander et al., 2020) but also in poultry science (Wickramasuriya et al., 2022;Yang et al., 2022).In poultry research the GIT bacterial species diversity is considered a key factor of pathogen exclusion (Pedroso et al., 2021).
In this work, the bacterial community composition in the quail caecal samples was also dominated by Firmicutes, regardless of the type of diet, which is in agreement with the previous studies on quails (Du et al., 2020;Su et al., 2014;Wilkinson et al., 2016) and other poultry (Andreani et al., 2020;Biasato et al., 2020;Cardenas et al., 2021;Costa et al., 2017;Danzeisen et al., 2011;Józefiak et al., 2020;Moula et al., 2018;Wei et al., 2013;Yeoman et al., 2012;Zou et al., 2018).At the family level, the dominance of the families Ruminococcaceae and Lachnospiraceae (>80% in all treatment groups) in the caecal microbiota is in agreement with many previous studies, (Andreani et al., 2020;Biasato et al., 2020;Danzeisen et al., 2011;Józefiak et al., 2020;Moula et al., 2018;Wei et al., 2013;Wilkinson et al., 2016).These two families from the order Clostridiales are highly specialised for the degradation of complex plant material and have the considerable capacity to break down a full range of plant-derived substrates including cellulose, hemicellulose and starch (Biddle et al., 2013;Broom, 2018;Stanley et al., 2016;Yang et al., 2017).Their production of SCFA (mainly acetate, butyrate and propionate) improves feed efficiency and butyrate supports gut health.The production of butyrate is an essential energy source for colonocytes (Jung et al., 2015), it ameliorates the integrity of tight-junctions (Peng et al., 2009) and has an antiinflammatory effect or role by reducing the inflammatory response (Furusawa et al., 2013).The two dominant families, Ruminococcaceae and Lachnospiraceae, however, responded differently to the inclusion of different levels of black soldier fly larvae oil in the quail diet.In BSFO 25 group the Ruminococcaceae decreased (30.9%) and Lachnospiraceae increased (50.5%), while in BSFO 100 the shift was the opposite with Ruminococcaceae forming (55.3%).In addition, an increase in Lactobacillaceae and a decrease in Streptococcaceae was observed in the BSFO 100 group.Lactobacillaceae are known to be beneficial in improving the intestinal health and growth performance of poultry (Chateau et al., 1993;Yan et al., 2017).While Streptococcaceae is a source of streptococcosis in poultry and its reduction thus can be considered as the beneficial effect of HI oil inclusion.On the other hand, regardless of the diet, the abundance of Clostridiaceae and Bacteroidaceae families was found to be low, which is in disagreement with previous studies on quails (Du et al., 2020) and chickens (Biasato et al., 2020;Cardenas et al., 2021).
At the genus level, the core bacterial genera were Subdoligranulum, Unclassified genus within family Lachnospiraceae, Blautia, and Clostridiales bacterium CHKCI001, which represented together 56.3% of the sequences in the control group, 60.7% in the BSFO 25 group, 63% in the BSFO 50 group and 71% in the BSFO 100.This is in good agreement with previous studies describing a predominance of these taxa in healthy broiler chickens (Biasato et al., 2020;Cardenas et al., 2021;Clavijo and Flórez, 2018;Emami et al., 2021;Hou et al., 2016;Ijaz et al., 2018;Kong et al., 2021;Polansky et al., 2016).The gradual effect of increasing doses of black soldier fly larvae oil is well seen at the genus level (Figure 3B), which is evident in particular by the increasing amount of Subdoligranulum sp.This genus belongs to the Ruminococcaceae family and is a butyrate-producing organism closely related to the Faecalibacterium genus.Subdoligranulum has been linked to multiple beneficial health effects on host energy metabolism and several interesting findings supported using this organism as promising probiotic (van Hul et al., 2020).This genus promotes the development of intestinal epithelial cells, which can minimise Salmonella invasion and colonisation (Eeckhaut et al., 2008).Danzeisen et al. (2011) stated that the use of antibiotics in poultry feed increased the prevalence of Subdoligranulum, leading to an increased approval of the hypothesis that the inclusion of BSF oil can be a substitute for conventional antibiotic usage, probably due to the high lauric acid content in its fatty acid composition (Boyen et al., 2008).With respect to other highly abundant genera, Blautia was increased in BSFO 25 and unclassified genus within family Lachnospiraceae was increased in BSFO 50.Clostridiales bacterium CHKCI001 and Streptococcus were suppressed in BSFO 50 and 100.With respect to less abundant genera, the Ruminococcus torques group was increased in the BSFO 50 group and Lactobacillus was elevated in the BSFO 100 group.Both these two bacteria are supposed to have positive effect.The Ruminococcus torques has been shown to be more abundant in broiler chickens fed a diet with a blend of medium-chain fatty acids (MCFA) (Kers et al., 2019) it and has been associated with improved performance in the broiler caeca (Torok et al., 2011).Lactobacillus was identified as characteristic OTU of the caecal microbiota of broiler chickens fed 10% of H. illucens meal inclusion (Biasato et al., 2020) and in general it is well known for its positive effect on gut health and the immune cells homeostasis (Ren et al., 2016;van Tassell and Miller, 2011).
The LEfSE analysis identified taxa with significantly different abundances in each animal group.The increased relative abundances of Anaerotruncus (family Ruminococcaceae) in the control group and Fusicatenibacter (family Lachnospiraceae) in all three groups including H. illucens oil were the common features resulting from all three analyses.Anaerotruncus may enhance the absorption of volatile fatty acids to increase the energy utilisation of recipients, leading to an increased muscle fiber diameter and decreased drip loss (Lei et al., 2022).Fusicatenibacter is SCFA-producing taxon which produce formate and acetate and it can protect the equilibrium of the intestinal microbial community (Qiu et al., 2020;Takada et al., 2013), suppresses the intestinal inflammation (Takeshita et al., 2016), reduces diarrheal symptoms (Yang et al., 2021) and regulates colonic motility (Zhang et al., 2021).As well, Subdoligranulum, which was the dominant genus among the four groups and was significantly enriched in the BSFO 100 group compared to the control group (LDA > 4.8), can have positive outcomes as discussed above.The other taxa significantly increased in BSFO 25 group, compared to Control, also can be considered beneficial.The Eubacterium halii group (family Lachnospiraceae) includes only uncultured bacteria, but Eubacterium hallii itself is a butyrate producing organism important for intestinal metabolic balance (Duncan et al., 2004;Engels et al., 2016).Lactobacillus genus also have positive effects on the animal health and performance by producing antimicrobial substances (Oakley et al., 2014), short chain fatty acids, exopolysaccharides and additional sources of energy (Pajarillo et al., 2015).The effect however highly depends on species (Brisbin et al., 2015).The comparison of bacterial shift in BSFO 50 and Control group however seems to be more beneficial to the control group.The increased abundance of Bacilli phylotypes (Bacillales, Bacillaceae and Bacillus) and butyrate-producing Coprococcus3 genus in could have a positive impact on nutrient absorption (Aliakbarpour et al., 2012).Similar conclusions could be partially assessed for the comparioson of BSFO 100 group with its respective Control group.Taxa Eubacterium brachy group, Lachnoclostridium and Anaerostipes are butyrate producers (Pryde et al., 2002;Ríos-Covián et al., 2016) and Coriobacterial phylotypes (Coriobacteriia, Coriobacteriales and Eggerthellaceae) are involved in the conversion of bile salts and steroids as well as the activation of dietary polyphenols (Clavel et al., 2014).On the other hand, the increase of Erysipelotrichales phy-lotypes in control group seems to be negative, because they were shown to be associated with lipid metabolism and inflammation (Kaakoush, 2015).From this point of view the replacing corn oil with 25% of H. illucens oil would be the best dietary recommendation.
Quails are an economically advantageous type of poultry with growing popularity among consumers and an increasing interest in their research can be expected.Our work on the effect of insect oil on the caecal microbiota proves that the diet is rightly considered to be the most influential factor on the composition of the gut microbiota.The GI tract has the most extensive exposed surface in the body and a wide variety of factors associated with diet can positively or negatively affect the delicate balance among the components of the poultry gut.Therefore the research in this area is of great importance.The understanding of the efficient conversion of feed into its basic components for optimal nutrient absorption is vital for both broiler production and welfare and for broiler breeder.

Conclusion
To best of our knowledge, this is the first study evaluating the effect of H. illucens larvae oil (BSFL oil) inclusion in quails' diet on the caecal bacterial diversity and composition.The results suggest that insect larvae oil can produce changes in the caecal microbiota by enhancing bacterial genera known for their positive effect on gut health.However, the total replacement of corn oil in quails' diet needs to be further studied, due to the adverse effects on bacterial richness and evenness.Based on our results the substitution of corn oil by 25% of BSF oil could be suggested and adopted in quails' diet due to an increase in beneficial bacteria without any significant alteration in richness or evenness of caecal bacterial microbiota and growth performance.However more studies about other parameters, especially regarding meat quality, are necessary to perform to support this hypothesis.The use of BSFL oil for the manipulation of intestinal microbiota, thus should be applied with caution and more research is required to better understand the potential effects of insect oil on intestinal bacteria.
BSFO = black soldier fly oil; IBW = initial body weight; FBW = final body weight; ADFI = average daily feed intake; ADG = average daily gain; FCR = feed conversion ratio; SEM = standard error of the mean.

Figure 1 Figure 2
Figure 1 Comparison of diversity indices for caecal bacterial communities of four groups of quails fed different diets.(A) The bacterial diversity estimated by the Shannon entropy.(B) The bacterial evenness estimated by Pielou evenness index.The Kruskal-Wallis pairwise test was used for sample comparison.
ing the control group C to BFSO 100 resulted in the highest number of taxa with significantly different relative abundances.A total of 16 differentially abundant bacteria (LDA score > 2.0) were found.Fourteen taxa had significantly higher relative abundance in control group C (green bars) and only 2 were significantly more abundant in the BSFO 100 group (red bars).In the control group C, the increased phylum Actinobacteria (LDA score > 3.0) included several coriobacterial phylotypes (Coriobacteriia, Coriobacteriales and Eggerthel-laceae).Increased class Erysipelotrichia included several Erysipelotrichales phylotypes.The genera Eubacterium brachy group (Family XIII), Anaerotruncus and Negativibacillus (belonging to Ruminococcaceae family), Coprococcus 3, Anaerostipes and Lachnoclostridium (belonging to Lachnospiraceae family) and finally Bacillus (family Bacillaceae) were also significantly higher in the control group.On the other hand, in the BSFO 100 group, the genera Fusicatenibacter (family Lachnospiraceae) and Subdoligranulum (family Ruminococcaceae) were significantly enriched (LDA score > 3.6) (Figure4C,F).

Figure 3
Figure 3 Relative abundance of caecal bacteria in four groups of quails fed different diets illustrated at family (A) and genus level(B).Taxa with a relative abundance lower than 1% are grouped as 'Others' .

Figure 4
Figure 4 Linear discriminant analysis (LDA) scores on different taxonomical levels of four groups of quails fed on different diets: phylum(p), class(c), order(o), family(f), genus(g).(A), (B) and (C) represent the histogram plots of LDA scores for differentially abundant taxa among the groups.The length of the bar represents the log10 transformed LDA score, indicated by vertical dotted lines.Positive LDA scores (green bars) and negative LDA scores (red bars) represent bacterial taxa over-abundant in the corresponding group.(D), (E) and (F) represent the cladograms showing the phylogenetic relationship among different groups of organisms with significantly different levels of abundance.

Table 2
Fatty acid profile of the dietary fats and experimental diets (% total FA)

Table 3
Effect of the dietary inclusion of Hermetia illucens larvae oil on the growth performance of broiler Japanese quails