Dietary Iron Fortification Did Not Affect the Intestinal Microbiome for Channel Catfish (<scp><i>Ictalurus punctatus</i></scp>) Juveniles, but Decreased Their Resistance Against <scp><i>Edwardsiella ictaluri</i></scp>

Idiopathic catfish anaemia (ICA) has been an ongoing issue affecting the United States catfish industry for decades (Klar, Hanson, and Brown 1986); (Plumb, Horowitz, and Rogers 1986). This disease typically manifests in early spring or late fall; however, due to its sporadic nature and occurrence, its aetiology has remained elusive (Camus et al. 2014). While catfish anaemia has been linked to nutrition, parasite infestation, and waterborne toxins (Yamamoto et al. 2023), recent farm cases appear to rule out feed-related anaemia, as outbreaks tend to occur in isolated ponds, while other ponds receiving the same feed show no signs of disease. To mitigate this issue, catfish feed mills have supplemented their diets with iron sulphate to improve erythropoiesis and, in turn, prevent the onset of anaemia (Buyinza et al. 2023). Despite no empirical evidence was presented to support this iron supplementation, a linear increase in blood haematocrit was later observed for hybrid catfish (Ictalurus punctatus × I. furcatus) fed graded levels of iron sulphate (Yamamoto et al. 2023). Nevertheless, a numerical reduction in survival was also observed for fish fed iron supplemented diets when challenged with the causative bacteria of enteric septicaemia of catfish (Edwardsiella ictaluri) (Yamamoto et al. 2023). Iron is an essential micronutrient for teleost fish, and supplementation of this element in the mineral premix is a standard practice in aquafeeds. The mineral requirements for farmed aquatic species typically ranges from ~60–170 mg/kg (Prabhu, Schrama, and Kaushik 2016). However, the catfish industry has consistently included 700–1000 mg/kg, surpassing the normal range by at least 5.5-fold. This practice raises concern, as vertebrates use physiological mechanisms to sequester iron from circulation during bacterial infections, thereby depriving invading microorganisms of this essential nutrient (Santander et al. 2012). It is thought that excess dietary iron may compromise this host defence strategy, particularly against enteric pathogens. The present study evaluated dietary supplementation of iron sulphate at either 0 or 1000 mg/kg to better understand the implications of iron supplementation on production performance, the intestinal microbiome in channel catfish (Ictalurus punctatus) and subsequent susceptibility to E. ictaluri infection. A 9-week feeding trial was conducted to evaluate the supplementation of iron sulphate (1000 mg/kg) using naïve channel catfish juveniles reared indoors at the Thad Cochran National Warmwater Aquaculture Center in Stoneville, MS. Four hundred and eighty fish (~13.2 g) were equally distributed in 16 aquaria (110-L; 30 fish/tank) operating as a flow-through system supplied with ground water (2 L/min; ~27°C) and supplemental aeration supplied by a regenerative blower (Dissolved oxygen: > 95%). Each tank was assigned a dietary treatment in a completely randomised design (n = 8). The control diet was formulated with plant-based ingredients to meet the dietary requirements of catfish with 32% crude protein and 5% lipid (Table 1) (Li and Robinson 2021). The iron-fortified diet included a ferrous sulphate premix in its formulation and was prepared as previously described (Yamamoto et al. 2023) to provide a final concentration of 1000 mg of Fe/kg of diet. Fish were fed to apparent satiation twice daily for 63 days, and water quality parameters were steady throughout the trial. At the end of the feeding trial, fish were fasted for 24 h, and group weighed to calculate the production performance as described previously (Yamamoto et al. 2021). A subset of three fish per aquaria were anaesthetised with MS-222 (100 mg/L) and blood samples collected using heparinised syringes to measure haematocrit, then centrifuged to collect plasma. Plasma protein was measured by a handheld refractometer. The same fish were then euthanised with an overdose of MS-222 (~300 mg/L) and individually weighed to compute condition indices (Yamamoto et al. 2021). The remainders of these fish made a composite sample, which was frozen at −20°C to analyse the whole-body proximate composition (AOAC 2005) and protein conversion efficiency (NRC 2011). The remaining fish in each tank were fed for an additional week to collect intestinal digesta, and sampling followed the methods described by Older, Griffin et al. (2023). The intestinal microbiota characterisation via amplification of the 16S rRNA (primers 515F and 806R) and sequencing using an Illumina MiSeq (Older, Yamamoto et al. 2023). Samples were subjected to diversity analyses after being rarefied to 27,964 reads per sample, resulting in the exclusion of four samples (two to each treatment). One day after the digesta sampling, catfish were pooled by treatment and redistributed in nine aquaria per treatment [40 L; 18 fish/tank; 162 fish (nine tanks) per treatment], where they were exposed to 6.6 × 106 CFU/mL of E. ictaluri (S97-773) for 1 h, by immersion. Culturing conditions of the bacterium were the same as previously described (Yamamoto et al. 2023). While the tanks were offered their assigned experimental diets once a day, fish mortality was recorded for 21 days, and classic ESC signs were observed on deceased fish. Production performance, condition indices, whole-body proximate composition, blood haematocrit and plasma protein data were analysed for normal distribution using the Shapiro–Wilk test and subjected to a one-way ANOVA (JMP Software v.14.0; SAS Institute, California, USA). If significant differences were observed (p < 0.05), then homogeneity of variances was validated using Brown–Forsythe test, and comparison of means was performed using the Student's t-test. Sequencing data was processed and analysed as described by Older, Griffin et al. (2023). Survival data after bacterial challenge was performed using Kaplan–Meier survival test (SAS v. 9.0; SAS Institute). No differences were observed for production performance, condition indices, or whole-body proximate composition (Table 1). These findings are in agreement with previous studies evaluating graded levels of iron supplementation for hybrid catfish (Yamamoto et al. 2023) and for channel catfish (Barros, Lim, and Klesius 2002; Buyinza et al. 2023). Interestingly, at the end of the feeding trial, survival was numerically higher for the control group but presenting a non-significant P-value (p = 0.08). Another trend was observed for plasma total protein (p = 0.10), where fish-fed diets supplemented with iron had a numerically higher protein concentration compared to plasma from fish offered the control diet. In addition, blood haematocrit ratio was significantly greater for catfish juveniles fed iron supplemented diets, consistent with results from previous studies (Yamamoto et al. 2023). In this study, the intestinal microbiome presented a predominance of Fusobacteria (65.5%), followed by Proteobacteria (17.7%), Firmicutes (15.1%) and Bacteroidota (0.5%) (Figure 1). The communities were mainly comprised of the genus Cetobacterium spp. (~65%), followed by Unclassified Enterobacterales spp. (17%), and Turicibacter spp. (~10%). These intestinal community profiles are consistent with channel catfish reported literature (Bledsoe et al. 2018; Older, Griffin et al. 2023). No differences were observed in terms of relative abundance, alpha- or beta-diversity of the channel catfish digesta microbiota after being offered the experimental diets for 70 days (Figure 1; Supplementary Table 1). These results contradict with observations in largemouth bass (Micropterus salmoides) where doses ranging from 40 to 200 mg/kg significantly impacted the diversity and composition of bacterial communities (Mao et al. 2024). Nevertheless, it was surprising to not observe differences in the intestinal microbiota in the present study, when iron supplementation of this magnitude could have induced oxidative stress in the intestine (Loveikyte et al. 2023). Studies of iron deficiency or fortification in humans and murine models were associated with reduction in alpha-diversity, reduction in short chain fatty acid producing bacteria, and possibly proliferation of pathogenic enteric bacteria (Loveiktye et al., 2023). Iron-deficient diets have been shown to reduce the numbers of short-chain fatty acid producing bacteria in mice, while subsequent iron supplementation could reverse some of these effects (Dostal et al. 2012). A reduced survival rate followed by the bacterial challenge was observed for the fish-fed iron fortified diets (Figure 2; p-value = 0.0002). This finding corroborates with our previous hypothesis that iron supplementation compromises the host ability to resist against bacterial infections (Yamamoto et al. 2023). It is unclear whether injuries related to iron-induced oxidative stress (Buyinza et al. 2023) could have jeopardised the host immunological responses to combat the invading pathogens; when iron supplemented diets did not induce histopathological modifications for several tissues in hybrid catfish (Yamamoto et al. 2023). The observed results strengthen our hypothesis that the physiological mechanisms limiting the circulating iron may be overwhelmed by the concentration provided in the feed, which could allow an excess of iron for pathogenic bacteria to thrive on. As stated in the previous study, idiopathic catfish anaemia has higher incidence in harvestable size fish (> 550 g) (Yamamoto et al. 2023), but due to the more controlled environment provided by aquaria, it is more opportune to conduct feeding trials using juvenile fish. Therefore, it is important that future studies assess if larger catfish fed iron supplemented diets, would also be more susceptible to bacterial disease. In conclusion, iron should be supplemented judiciously in catfish diets, and preferably only during periods when ICA is most likely to occur. Further studies are needed to evaluate if iron-supplemented diets would also pose risk of bacterial infection to market-sized catfish. Until comprehensive studies are completed, caution is advised when supplementing grow-out diets with iron sulphate. Based on these findings, it is not recommended to offer fortified diets throughout the growing season, particularly during periods of increased risk for bacterial disease. Fernando Y. Yamamoto: investigation, writing – original draft, funding acquisition, visualization, methodology, software, formal analysis, supervision, resources, data curation, project administration, conceptualization, validation. Caitlin E. Older: software, formal analysis, data curation, writing – review and editing. Lester H. Khoo: writing – review and editing, methodology, investigation. Nicholas Romano: investigation, writing – review and editing. Bradley M. Richardson: writing – review and editing, methodology. Brian D. Ott: writing – review and editing, methodology. David J. Wise: funding acquisition, writing – review and editing, supervision, resources. Cynthia Ware: methodology, writing – review and editing. Penelope M. Goodman: methodology, visualization, writing – review and editing. J. Grant Reifers: methodology, visualization, writing – review and editing. Matt J. Griffin: methodology, visualization, writing – review and editing, resources. This study was performed at the National Warmwater Aquaculture Center following the guidelines outlined by the Institutional Animal Care and Use Committee (IACUC 22-060), and funded by the Mississippi Agriculture and Forestry Experiment Station (MAFES) through the USDA production grant CRIS project MIS-523010. This research used resources provided by the SCINet project and/or the AI Center of Excellence of the USDA Agricultural Research Service, ARS project numbers 0201-88888-003-000D and 0201-88888-002-000D. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. Ingredients for the experimental diets were kindly donated by Delta Western Grains Inc., and by Fish Belt Feeds Inc. The assistance provided by Mrs. Sandra K. Phillips throughout the feeding trial is greatly appreciated. The authors declare no conflicts of interest. The feeding trial data can be made available by the corresponding author upon reasonable request. Sequencing data from the catfish digesta is available at the NCBI database under the bioproject PRJNA1064105. Supplementary Table 1 Feed formulation for the experimental diets fed to channel catfish (Ictalurus punctatus) juveniles. Proximate composition data is expressed as average between the two dietary treatments and as g/100 g on a dry-matter basis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.