As global aquaculture expands, sustainable alternative feed ingredients are needed to reduce reliance on fishmeal (FM) and conventional plant proteins. Herein, this study evaluated defatted black soldier fly (BSF; Hermetia illucens) larvae meal as a replacement mainly for soybean meal (SBM) and soy protein concentrate (SPC) in juvenile mirror carp diets. Fish (initial weight: 7.28 +/- 0.09 g) were reared in a recirculating aquaculture system and fed for 8 weeks on either a control diet or diets containing 20% (BSF20) or 40% (BSF40) BSF meal. After the trial, growth performance was significantly improved in BSF-fed groups, with higher final weight, weight gain, specific growth rate and a better feed conversion ratio compared with the control. Orthogonal polynomial contrasts indicated predominantly linear responses in growth and feed utilisation parameters. Histological analysis showed significantly enhanced intestinal morphology in BSF-fed groups, including increased mucosal fold length (MFL), microvilli length (MVL) and intraepithelial lymphocyte (IEL) density. At the molecular level, il10 and slc6a18 expression were significantly upregulated in the anterior intestine (AI) and posterior intestine (PI), respectively, of BSF-fed fish. The PI microbiota profiling via 16S rRNA gene metabarcoding showed that BSF inclusion promoted several beneficial microbial shifts. While the dominant phyla were Proteobacteria, Fusobacteria and Firmicutes, fish fed the BSF40 diet exhibited a higher relative abundance of Firmicutes and a reduced relative abundance of several potentially pathogenic phyla. The relative abundance of beneficial genera, including Bacillus and Enterococcus, increased significantly, whereas the relative abundance of potentially pathogenic genera such as Mycobacterium, Staphylococcus and Enterobacter were reduced by BSF meal. Overall, BSF meal represents a nutritionally viable and functional protein source that improves growth performance and intestinal health, and supports sustainable aquafeed development in juvenile mirror carp.

This study investigated the effects of dietary black soldier fly (BSF) larvae, Hermetia illucens, meal on growth performance, feed utilization, intestinal health, and immune response in Nile tilapia, Oreochromis niloticus, juveniles. The fish (initial weight: 7.28 ± 0.09 g) were reared in a recirculatory aquaculture system (RAS) and fed diets containing different levels of defatted BSF larvae meal: 0% (control diet), 20% (BSF20), and 40% (BSF40). At the end of the feeding trial, the final body weight, weight gain, specific growth rate (SGR), protein efficiency ratio (PER), and feed conversion ratio (FCR) were significantly ( p < 0.05) improved with increasing BSF meal inclusion levels in the diet. Histological analysis of the intestine revealed that mucosal fold length (MFL), muscularis thickness (MT), and goblet cell density (GCD) were significantly elevated by the BSF meal compared with the control. In addition, intraepithelial lymphocytes (IELs) and the enterocyte microvilli length were significantly elevated in BSF40‐fed fish compared with the control. Gene expression analysis showed that intestinal chitin digestion‐related ( endochitinase A ) and amino acid transporter ( Slc6a18 ) genes were not significantly affected by the BSF meal. However, the expression of peptide transporter Slc15a1a was upregulated in the fish fed BSF40 compared to the control. Moreover, posterior intestine and head kidney cytokine ( IL‐1β, TNF‐α , and IL‐10 ) gene expression levels were not significantly affected by the inclusion of the BSF meal. Therefore, these results suggest that the BSF meal up to a 40% inclusion level can be used as an alternative protein source in the diets of Nile tilapia juveniles to promote growth and intestinal health.

Preventive health management is central to modern aquaculture practices, within which functional feed additives are a key tool to optimising fish robustness and disease resilience. Despite being well documented, only few studies have assessed the dose-effect of graded levels of S. cerevisiae yeast cell wall (YCW) on fish performance and mucosal barrier immunity. A 9-week trial was conducted to assess the impact of graded levels of YCW on the performance and mucosal barriers of the European seabass under period: (1) non-challenge (43 days) then (2) with an applied husbandry stressors (20 days) in the form of high stocking density, low water oxygen level, and repetitive handling. The four experimental diets were produced by incorporating graded levels of YCW (0.0, control; 1.5; 3.0 or 4.5 kg/MT) which were top dressed on the basal diet prior extrusion. At the end of the period 1, fish supplemented with YCW at 4.5 kg/MT revealed a 5% increase in body-weight compared to the control. Likewise, throughout the experimental period SGR and FCR were also improved by YCW supplementation (+ 5.8% and + 8.3% with YCW4.5, respectively). Supplemented diets increased intraepithelial lymphocytes density and the highest level of intake was associated with increased skin mucus secretion (+ 31% with YCW 4.5 kg/MT). Finally, we observed a linear relationship between fecal calprotectin-like levels and the YCW gradient: a positive correlation during period 1 and a negative correlation during period 2. In contrast, YCW supplementation levels were characterized by a positive linear relationship with fecal mucins across both periods. These results provide novel insights on the benefits and associated mechanisms of YCW extracts and reveal an optimal dose response with inclusion levels between 3.0 and 4.5 kg/MT. This study demonstrates that YCW dietary inclusion enhances growth performance, feed utilization, and mucosal immune barrier function in European seabass, with improved stress resilience evidenced by biomarkers fecal calprotectin-like and fecal mucins.

The host microbiome is a promising source of probiotics for aquaculture species including Nile tilapia. In this study, the probiotic potential of autochthonous bacterial isolates from Nile tilapia and carp mid-intestines were screened in vitro. Two isolates (C61 and T70), closely related to Bacillus subtilis, exhibited antagonistic activity against multiple pathogen species and demonstrated multiple digestive enzyme activities. Their antagonistic activity in Aeromonas hydrophila assays remained even under simulated intestinal juice (SIJ) exposure. Subsequently, C61 (PT1) and T70 (PT2) were added to experimental diets at log 7 CFU/g of diet, and fed to Nile tilapia (5.32 ± 0.12 g) for 40 days. There were no significant differences observed in the growth performance across treatments. Despite limited Bacillus intestinal recovery levels, 16S rRNA gene metabarcoding revealed subtle shifts in the intestinal microbial community composition of the probiotic-fed groups. In addition, the PT1 group showed significantly longer mucosal fold length, elevated intestinal and skin goblet cell levels, and higher skin goblet cell coverage compared to the control. These results indicate the potential benefits of the isolates as functional feed additives for enhancing the mucosal health of Nile tilapia, but their benefits were likely achieved through transient activity given the low level of Bacillus recovery in the intestine.

With the rising awareness of antimicrobial resistance, the development and use of functional feed additives (FFAs) as an alternative prophylactic approach to improve animal health and performance is increasing. Although the FFAs from yeasts are widely used in animal and human pharma applications already, the success of future candidates resides in linking their structural functional properties to their efficacy in vivo. Herein, this study aimed to characterise the biochemical and molecular properties of four proprietary yeast cell wall extracts from S. cerevisiae in relation to their potential effect on the intestinal immune responses when given orally. Dietary supplementation of the YCW fractions identified that the α-mannan content was a potent driver of mucus cell and intraepithelial lymphocyte hyperplasia within the intestinal mucosal tissue. Furthermore, the differences in α-mannan and β-1,3-glucans chain lengths of each YCW fraction affected their capacity to be recognised by different PRRs. As a result, this affected the downstream signalling and shaping of the innate cytokine milieu to elicit the preferential mobilisation of effector T-helper cell subsets namely Th17, Th1, Tr1 and FoxP3+-Tregs. Together these findings demonstrate the importance of characterising the molecular and biochemical properties of YCW fractions when assessing and concluding their immune potential. Additionally, this study offers novel perspectives in the development specific YCW fractions derived from S. cerievisae for use in precision animal feeds.

It is generally accepted that microbes play a critical role in maintaining gut barrier function, making them ideal to target in order to mitigate the effects of intestinal diseases such as inflammatory bowel disease with specialist supplementations such as probiotic or postbiotic preparations. In this study, specific strains of Lactobacillus helvictus both live and inactivated and Lactobacillus plantarum inactivated were fed to zebrafish at an inclusion level of 6 × 106 cells/g in order to assess the effects on gut barrier function and protection. Taken together, our results indicate that dietary administration of pro- or postbiotics strengthens the gut barrier function and innate immunity of healthy zebrafish in a strain-specific and process-dependent way. With some differences in the response intensity, the three treatments led to increased intestinal villi length and proportion of IELs, reinforcement of the GC population and up-regulated expression of biomarkers of AMP production and tight junction zona-occludin 2a (zo-2a). In addition, LPPost had an impact on the adaptive immune response, and we hypothesized that it conferred the potential to drive Th17/ILC3 immunity, as suggested by its effect on the gene expression of il22, of different AMPs, and the expression of zo2a. Moreover, LPPost showed the potential to drive Th1/ILC1-like immunity, with a higher percentage of CD8+ cells and higher ifnγ gene expression. In summary, the use of inactivated Lactobacilli species in this study represented a promising strategy for improving barrier function and regulating the immune fate of the intestinal mucosa in a strain-specific way.

This study was conducted to evaluate the mucosal immune responses of rainbow trout when supplementing an experimental formulated feed with multi-strain yeast fraction product ( Saccharomyces cerevisiae and Cyberlindnera jardinii ). In total, 360 fish (initial BW 23.1 ± 0.2 g) were randomly allotted into three dietary treatments in an 8-week feeding trial. The dietary treatments included basal diet (control) and control + 1.5 g/kg multi-strain yeast fraction product (MsYF) fed continuously and pulsed every two weeks between control and MsYF diet. No negative effects on growth performance of feeding the MsYF supplemented diet were observed. SGR and FCR averaged 2.30 ± 0.03%/day and 1.03 ± 0.03, respectively, across experimental groups. Muscularis thickness in the anterior intestine after 8 weeks of feeding was significantly elevated by 44.3% in fish fed the MsYF continuously, and by 14.4% in fish fed the MsYF pulsed ( P < 0.02). Significant elevations in goblet cell density in the anterior and posterior (>50% increase) intestine were observed after 8 weeks of feeding the MsYF supplemented diet ( P < 0.03). In contrast, lamina propria width was significantly lower in fish fed the experimental diets (>10% reduction). The gene expression analysis of the intestine revealed significant elevations in expression of tlr2 , il1r1 , irak4 , and tollip2 after 4 weeks of feeding the MsYF. Significant elevations in effector cytokines tnfα , il10 and tgfβ were observed after 4 weeks of feeding the MsYF regime. After 8 weeks significant elevations in the gene expression levels of il1β , ifnγ , and il12 were observed in fish fed the MsYF. Likewise, the expression of the transcription factor gata3 was significantly elevated ( P <0.01). Supplementation of the multi-strain yeast fraction product positively modulates the intestinal mucosal response of rainbow trout through interaction with toll-like receptor two signalling pathway and potential for increased capacity of delivery of antigens to the underlying mucosal associated lymphoid tissue.

Background: Increasing reliance on non-medicinal interventions to control sea lice in the Atlantic salmon (Salmo salar) farming industry imposes a high level of skin mucosal disturbance and indirect health issues. Dietary supplementation with yeast-based MOS products is widely used to support intestinal homeostasis across farmed species. Evidence of their effect on skin mucosa is increasing in aquatic species but it remains inconsistent and someway short of a clear contribution to sea lice management. A tank-based trial was performed to test the effect of a yeast-based MOS functional compound (sMOS) on the skin mucosal layer and its protective effects against sea lice (Lepeophtheirus salmonis). Results: The test compound significantly increased skin mucus (+46%) and goblet cell density (+25%) after 6 weeks of dietary supplementation when positive effects on intestinal villi-length (+10.9%) and goblet cell density (+80.0%) were also documented. Following dietary supplementation, a 16.6% reduction in susceptibility to an acute standard copepodid challenge was measured alongside an earlier increase in skin lysozyme activity widely used as an index of innate immunity. Conclusion: The study provides functional evidence that the benefits of dietary sMOS reach beyond the intestine to the skin mucosa. Bolstering of the Atlantic salmon skin barrier and immune functions and the resulting lower susceptibility to sea lice has the potential to reduce the need for delousing interventions and the impact of non-medicinal interventions on the animal's health and welfare.

A study was conducted to evaluate the probiotic effect of Pediococcus acidilactici MA18/5M on rainbow trout, Oncorhynchus mykiss. Fish (310 +/- 9 g) were fed a control diet or a P. acidilactici-supplemented diet (at 2.4 x 10(6) CFU/g) for 4 weeks. The probiotic was observed to populate the intestine with levels ranging from log 3.7 to 5.4 CFU/g. Furthermore, these populations were able to persist for at least 24 hr after the cessation of probiotic feeding. High-throughput sequencing analysis of bacterial 16S rRNA libraries demonstrated that P. acidilactici was able to modulate the gut microbiome of rainbow trout and that the probiotic was detected as a common taxon on the mucosa and in the digesta of the probiotic fish (p < .05). Real-time polymerase chain reaction demonstrated that feeding the probiotic upregulated pro-inflammatory cytokines, interleukin-1 beta, and interleukin-8 and downregulated anti-inflammatory interleukin-10 compared to the control-fed fish. Furthermore, the mRNA levels for the mucosal antibody immunoglobulin T was also elevated in probiotic-fed fish. These findings help to explain some of the mechanisms behind the previously reported observed benefits of using this probiotic in the intestinal morphology and immunity of rainbow trout.

Fish were fed a single‐strain yeast fraction (SsYF; 2 g/kg) or a multistrain yeast fraction (MsYF; 0.8 g/kg) for 10 weeks. The results demonstrated significant (p ≤ 0.03) elevations in weight gain, specific growth rate, protein efficiency ratio, and feed conversion ratio in fish fed the yeast fraction‐supplemented diets. In the distal intestine, a significant elevation in microvilli density was observed after 5 and 10 weeks of dietary supplementation with MsYF and SsYF, respectively, compared to control fed fish (p < 0.001). A significant elevation (p = 0.02) in the perimeter ratio was observed in fish fed diets supplemented with the yeast fractions. After 10 weeks of feeding on the experimental diets, Rt‐qPCR demonstrated a significant downregulation (p < 0.05) in the stress response genes, heat‐shock protein 70 (hsp70) and proliferating cell nuclear antigen (pcna), in fish fed diets supplemented with the yeast fractions. Significant (p < 0.05) elevations in interleukin 1‐beta (il1β) and interleukin‐10 (il10) gene expression were observed in fish fed diets supplemented with the MsYF compared to the other dietary groups. These findings suggest that feeding an MsYF specifically at a lower incorporation rate < 1 g/kg, compared to a commercial SsYF at 2 g/kg, is effective in improving the intestinal health status and growth performance of European seabass.

The aim of this study was to assess the effect of the transfer from freshwater to seawater on the distal intestinal bacterial communities of Atlantic salmon (Salmo salar L.) and to evaluate the effect of dietary inclusion of Pediococcus acidilactici MA18/5M (at 1.19 × 106 CFU/g). In this context, fish health and antiviral response were also investigated. A 12-week feeding trial was conducted in a flow-through rearing system involving 6 weeks in freshwater and 6 weeks in seawater. Fish received a control and probiotic diet. The composition of the salmon gut bacterial communities was determined by high-throughput sequencing of digesta and mucosa samples from both the freshwater and seawater stage. The main phyla detected during both freshwater and seawater stages were Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria. Significant differences were observed between the intestinal microbiota in the digesta and the mucosa. Both probiotic supplementation and the seawater transfer (SWT) had a substantial impact on the microbial communities, with most pronounced changes detected in the mucosal communities after SWT. This last finding together with a significantly higher antiviral response (mx-1 and tlr3 gene expression) in the distal intestine of fish fed the probiotic diet suggest a causal link between the microbiota modulation and activation of antiviral response. Feeding probiotics during the freshwater stage did not significantly increase survival after infectious pancreatic necrosis virus (IPNV) challenge after SWT, although higher survival was observed in one out of two replicate challenge tanks. In conclusion, this study demonstrated that both dietary probiotic supplementation and transfer from freshwater to seawater have an important role in modulating the bacterial communities in the distal intestine of Atlantic salmon. Furthermore, supplementation of the diet with P. acidilactici MA18/5M can modulate antiviral response.

The growth performance, immunological status, intestinal morphology and microbiology of tilapia, Oreochromis niloticus, were investigated after dietary administration of the commercial probiotic AquaStar (R) Growout. Tilapia (29.02 +/- 0.33 g) were split into five treatments; control (CON), 1.5 g kg(-1) probiotic (PRO-1.5), 3 g kg(-1) probiotic (PRO-3), pulsed probiotic feeding (PRO-PULSE) or an initial pro biotic feed followed by control feeding (PRO-INI). After six weeks of experimental feeding, fish fed PRO-3 displayed significantly higher final weight, weight gain and SGR compared to the CON or PRO-INI treatments. Supplementation of the probiotic at this dose induced an up-regulation of intestinal caspase-3, PCNA and HSP70 mRNA levels compared to the CON fed fish. Immuno-modulatory pathways were also affected; significantly higher expression of TLR2, pro-inflammatory genes TNF alpha and IL-1 beta, and anti-inflammatory genes IL-10 and TGF beta suggest that the probiotic may potentiate a higher state of mucosal tolerance and immuno-readiness. Histological appraisal revealed significantly higher numbers of intraepithelial leucocytes in the intestine of PRO-3 fed fish compared with treatments CON, PRO PULSE and PRO-INI but not PRO-1.5. Additionally, fish receiving PRO-3 had a significantly higher abundance of goblet cells in their mid-intestine when compared with fish from all other treatments. Together, these data suggest that continuous provision of AquaStar (R) Growout at 3 g kg(-1) can improve tilapia growth and elevate the intestinal immunological status of the host. (C) 2015 Elsevier Ltd. All rights reserved.

An investigation was conducted to evaluate the effect of feeding selected exotic ingredients on immune responses and expression of immune related genes in mirror carp (Cyprinus carpio). Fish were fed diets for a total of 56days. Fishmeal served as the main protein source in the control diet and two experimental diets consisted of fishmeal fixed at 34% provision of protein and the remaining 66% protein was provided either by earthworm meal (EW diet) or a combination of whey protein concentrate (8%) and casein (58%) (WPC diet). At the start of the trial fish were injected intraperitoneally with Aeromonas hydrophila bacterin and samples of carp blood were taken for gene expression, haematological and serological analysis. Compared to fish fed fishmeal, a significant increase in mRNA expression of the pro-inflammatory cytokines IL-1β (24h post injection) and TNFα (at 12h and 48h post injection) was observed in fish fed EW. Moreover a similar trend was observed for complement 3 (C3) gene, where fish fed EW showed significant elevations in mRNA expression values at both 12 and 48h post injection compared to control fed fish. In contrast, fish fed WPC showed a significant decrease in C3 and TNF-α mRNA expression compared to fish fed fishmeal (48 h post injection). Fish fed EW and WPC showed a significant increase in peripheral leukocyte levels compared to fish fed fishmeal 14d post injection. Fish fed fishmeal presented significantly higher circulatory IgM levels at 7d post injection compared to fish fed EW and WPC. In contrast, fish fed EW and WPC showed a significant increase in IgM levels at 28d post injection. The present study shows an up-regulation of all immune related genes in fish fed EW, compared to fish fed fish meal, which is indicative of an acute inflammatory response. In contrast it appears feeding WPC has an immunosuppressive effect towards TNFα and C3 expression in carp blood. This may be offset by the fact that feeding WPC to carp appears to aid in the increased production of circulatory IgM and leukocyte levels compared to fishmeal fed fish.

The application of probiotics in aquaculture has received concerted research efforts but the localised intestinal immunological response of fish to probiotic bacteria is poorly understood. Therefore, a study was conducted to evaluate the probiotic effect of Pediococcus acidilactici on Nile tilapia (Oreochromis niloticus) with specific emphasis on intestinal health and probiotic levels as well as system level responses such as growth performance, feed utilization and haemato-immunological parameters under non-challenged conditions. Fish (9.19 ± 0.04 g) were fed either a control diet or a P. acidilactici supplemented diet (at 2.81 × 106 CFU g−1) for six weeks. At the end of the study the probiotic was observed to populate the intestine, accounting for ca. 3% (1.59 × 105 CFU g−1) of the cultivable intestinal bacterial load. Real-time PCR indicated that the probiotic treatment may potentiate the immune-responsiveness of the intestine as up-regulation of the gene expression of the pro-inflammatory cytokine TNFα was observed in the probiotic fed fish (P < 0.05). Light microscopy observations revealed elevated intraepithelial leucocyte (IEL) levels in the intestine of P. acidilactici fed tilapia after six weeks (P < 0.05) of feeding and a trend towards elevated goblet cells was also observed after six weeks feeding (P = 0.08). Concomitantly at week six, along with elevated IELs and elevated TNFα mRNA levels in the intestine, an increased abundance of circulating neutrophils and monocytes were observed in fish fed the probiotic supplemented diet (P < 0.05). This haemopoietic expansion of innate immune cells could be reflective of an elevated state of immuno-readiness. Together these results suggest that the probiotic has a protective action on the intestinal mucosal cells, stimulating the innate immune response after feeding for a period of six weeks. These immunological modulations did not impair growth performance or the remaining haematological and zootechnical parameters compared to the control group (P > 0.05).

AimTo assess the effects of dietary Saccharomyces cerevisiae -(1,3)(1,6)-d-glucan supplementation (MacroGard((R))) on mirror carp (Cyprinus carpio L.) intestinal microbiota and ultrastructure of the enterocyte apical brush border.Methods and ResultsCarp were fed either a control diet or diets supplemented with 01, 1 or 2% w/w MacroGard((R)). Culture-dependent microbiology revealed that aerobic heterotrophic bacterial levels were unaffected by dietary MacroGard((R)) after 2 and 4weeks. No effects were observed on the allochthonous lactic acid bacteria (LAB) populations at either time point; however, reduced autochthonous LAB populations were observed at week 4. PCR-DGGE confirmed these findings through a reduction in the abundance of autochthonous Lactococcus sp. and Vagococcus sp. in MacroGard((R))-fed fish compared with the control-fed fish. Overall, sequence analysis detected microbiota belonging to the phyla Proteobacteria, Firmicutes, Fusobacteria and unidentified uncultured bacteria. DGGE analyses also revealed that dietary MacroGard((R)) reduced the number of observed taxonomical units (OTUs) and the species richness of the allochthonous microbiota after 2weeks, but not after 4weeks. In contrast, dietary MacroGard((R)) reduced the number of OTUs, the species richness and diversity of the autochthonous microbiota after 2weeks, and those parameters remained reduced after 4weeks. Transmission electron microscopy revealed that intestinal microvilli length and density were significantly increased after 4weeks in fish fed diets supplemented with 1% MacroGard((R)).ConclusionsThis study indicates that dietary MacroGard((R)) supplementation modulates intestinal microbial communities of mirror carp and influences the morphology of the apical brush border.Significance and Impact of the StudyTo the authors' knowledge, this is the first study to investigate the effects of -(1,3)(1,6)-d-glucans on fish gut microbial communities, using culture-independent methods, and the ultrastructure of the apical brush border of the enterocytes in fish. This prebiotic-type effect may help to explain the mechanisms in which -glucans provide benefits when fed to fish.

In recent years, aquaculture research has focused on probiotics, prebiotics, and β-glucans, in order to improve health status and growth performance. Information regarding the effects of β-glucan on growth performance and intestinal immunity of mirror carp (Cyprinus carpio L.) is scarce. An experiment was therefore conducted to investigate the effects of a yeast β-glucan preparation (MacroGard(®) ) on growth performance, intestinal morphology and haemato-immunological indices of mirror carp. Carp (initial weight 11.1 ± 0.0 g) were fed highly purified diets supplemented with 0% (control), 0.1%, 1% or 2% MacroGard(®) for 8 weeks. Fish fed diets containing 1% and 2% MacroGard(®) showed significant improvements in weight gain, specific growth rate and feed conversion ratio compared to fish fed both the control and the 0.1% MacroGard(®) containing diet. Histological appraisal of the intestine showed a significantly higher infiltration of leucocytes into the epithelial layer of fish fed diets supplemented with 1% and 2% MacroGard(®) in the anterior intestine compared to fish fed the control and 0.1% MacroGard(®) diet. This effect was not observed in the posterior intestine. There were no significant differences in the intestinal absorptive surface area and number of goblet cells in either intestinal region. At the end of the experiment, the haematological status of the fish was examined. Compared to control fed fish, the haematocrit value was significantly elevated in fish fed the 2% MacroGard(®) diet. Furthermore, the blood monocyte fraction was significantly higher in fish fed the 1% and 2% MacroGard(®) diets. No significant changes were observed in the other blood parameters assessed. The present study shows that high dietary β-glucan inclusion increases growth performance without detrimental effects on the health indicators assessed. Increased intraepithelial leucocytes in the anterior intestine may indicate a localized immune response; no detrimental effects on intestinal morphology were observed.