DISCUSSION

Functional value of fermented L. leucocephala as a fishmeal substitute

L. leucocephala leaves are widely recognized as a rich source of protein and essential minerals, making them a promising alternative feed ingredient for livestock and aquaculture species in tropical regions. Their high protein content is a key nutritional advantage, along with the presence of 17 flavonoids known for biological activities, including antioxidant and anti-inflammatory effects [14]. In the present study, L. plantarum TISTR1284 was used to improve the bioavailability and functional properties of L. leucocephala leaves. Fermentation is known to enhance food safety and nutritional quality by producing organic acids, including lactic, acetic, propionic, and formic acids, which help eliminate harmful bacteria and toxins while preserving sensory qualities [17]. However, the present experimental design does not allow separation of the individual contributions of fermentation, plant substitution, and probiotic effects; therefore, the outcomes observed here should be interpreted as the combined effect of the fermented ingredient rather than being attributed to a single factor.

_A. _hydrophila, a pathogenic Gram-negative bacterium, poses a serious threat to _Macrobrachium _nipponense and _M. _rosenbergii [34, 35]. Plant extracts from Withania somnifera and _Melaleuca _cajuputi have been shown to enhance immune responses and increase shrimp survival during _A. _hydrophila infection [36, 37]. L. leucocephala seed protein has also been shown to enhance bacterial disease resistance in the catfish _Clarias _gariepinus [12]. In the present study, dietary supplementation with fermented L. leucocephala leaves enhanced immune parameters and improved survival of _M. _rosenbergii following _A. _hydrophila challenge.

Figure 6: Effects of dietary fermented Leucaena leucocephala on IMD/Relish pathway and immune-related gene expression in the intestine of Macrobrachiumrosenbergii infected with Aeromonashydrophila. (A) Relative mRNA expression of IMD. (B) Relative mRNA expression of Relish. (C) Relative mRNA expression of HSP70. (D) Relative mRNA expression of Cu/Zn-SOD. (E) Relative mRNA expression of ALF. (F) Relative mRNA expression of C-lectin. Data are expressed as mean ± SD. For gene expression analyses, n = 4 (cDNA pooled from two prawns per sample). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7: Survival rate of Macrobrachiumrosenbergii following Aeromonashydrophila infection. Data are expressed as mean ± SD (n = 3 replicate tanks per group). Survival analysis was performed using the log-rank (Mantel–Cox) test. No significant differences were observed between the control and 10% replacement groups (p = 0.167) or between the control and 20% replacement groups (p = 0.204).

Hemocyte proliferation and hematopoietic activity

Crustacean resistance to pathogens relies primarily on the innate immune system. Hemocytes are crucial cells that mediate physiological responses to infection and environmental stress. Probiotics and medicinal plant extracts have been applied to enhance immune parameters in _M. _rosenbergii [38, 39]. In the present study, increased circulating hemocytes and a higher number of mitotic cells in hematopoietic tissue were observed when fermented L. leucocephala leaves were added to the diet of _M. _rosenbergii. This response may contribute to accelerated maturation of hemocyte precursors in hematopoietic tissue, thereby maintaining hemocyte population and functionality during bacterial challenge [40].

L. leucocephala contains apigenin, kaempferol, and juglanin, which induce the transcriptional activity of pHRE-Luc before promoting erythropoietin expression and stimulating red blood cell production [14]. Upregulation of CHF is a biological indicator associated with hemocyte and hematopoietic cell proliferation in _M. _rosenbergii [27]. The increase in hematopoietic proliferation, reflected by CHF expression in hemocytes and hematopoietic tissue of the 10% and 20% L. leucocephala groups, suggests that fermented L. leucocephala promotes hematopoietic activity and hemocyte homeostasis during bacterial infection.

PO activity and proPO expression

PO is an important enzyme that promotes humoral defense mechanisms and the elimination of pathogens in invertebrates [41]. Plant extracts have been shown to induce PO activity and improve immunocompetence against pathogens in crustaceans [38, 42, 43]. In the present study, _M. _rosenbergii fed fermented L. leucocephala showed increased PO activity in hemolymph and significant upregulation of proPO. Antimicrobial and antioxidative activities induced by phytochemicals in L. leucocephala have been previously reported [44]. These findings suggest that key bioactive properties of L. leucocephala were retained after fermentation, although the specific compounds responsible for the observed effects were not directly quantified in the present study.

Modulation of Toll pathway-related signaling

TRAF6 is a highly expressed gene that acts as a crucial signal transducer triggering downstream cascades mediated by TNF receptors and the IL-1 receptor/Toll-like receptor superfamily during immune responses [45]. In Fenneropenaeus penicillatus, higher TRAF6 expression was correlated with peroxinectin expression after infection by white spot syndrome virus and Vibrio alginolyticus [46]. TRAF6-like transcription was upregulated after Staphylococcus aureus and Edwardsiella ictaluri challenge in Procambarus clarkii [47]. Knockdown of TRAF6-like inhibited downstream effector genes, including Dorsal [47]. TRAF6 regulates immune responses during _A. _hydrophila infection because mannose-binding lectin and crustin are decreased after TRAF6 silencing [30].

In the present study, TRAF6 expression decreased within 48 h, and Dorsal expression declined at specific time points in the 10% and 20% groups. These findings indicate modulation of inflammatory signaling; however, bacterial burden or clearance was not quantified in this study. Therefore, improved survival should not be directly interpreted as increased bacterial clearance. Flavonoids in L. leucocephala have been reported to reduce pro-inflammatory cytokine secretion in RAW 264.7 macrophages [14], which may partly explain the moderated inflammatory profile observed here.

IMD pathway and immune-related gene responses

Two major pathways are involved in shrimp immunity: the Toll pathway and the IMD pathway. These pathways share downstream signaling components, and crosstalk between them contributes to protection against bacterial infection [48]. In the present study, C-lectin was downregulated, whereas IMD-related genes (IMD and Relish) and effector/stress genes (HSP70, Cu/Zn-SOD, and ALF) were upregulated, particularly in the 20% group. This pattern suggests coordinated immune modulation rather than simple immune suppression. Such modulation may reflect regulated immune adjustment to dietary bioactive compounds, as previously observed in crustaceans exposed to functional feed additives and plant-derived extracts [49, 50].

However, the precise molecular mechanism underlying the activation of the immune pathway by fermented L. leucocephala remains unclear and warrants further mechanistic investigation. In particular, this study did not identify the specific bioactive compounds responsible for modulating the IMD pathway, although phytochemicals such as phenolics have been reported to influence immune responses in _C. _gariepinus [51]. Nevertheless, the exact molecular mediators and their direct interactions with IMD pathway components remain unclear and require targeted metabolomic and functional studies.

Gut health, paraprobiotic effects, and postbiotic contributions

In addition to immunomodulation, L. leucocephala has been associated with gut health and nutrient utilization [52]. Replacing 25%–50% of fishmeal with L. leucocephala leaves in Nile tilapia diets did not adversely affect fish health, although some growth retardation was observed [11]. Improvements in digestibility and nutrient uptake have also been reported when L. leucocephala leaves are fermented with beneficial gut bacteria. For example, Bacillus subtilis and _Bacillus _circulans enhanced nutritional value and growth performance in Labeo rohita when included in diets containing 30%–40% L. leucocephala [53]. In crustaceans, host-associated probiotics such as Lactococcus lactis have shown similar benefits in _M. _rosenbergii, improving both growth and immune function [39].

Although viable LAB counts in the final pelleted feed and during storage were not quantified, the present findings suggest that the beneficial effects observed in _M. _rosenbergii were likely mediated not only by live probiotic cells but also by fermentation-derived paraprobiotics and postbiotics acting synergistically with enhanced plant bioactive compounds. Paraprobiotics, including inactivated microbial cells and cell-wall components such as peptidoglycans, teichoic acids, and surface proteins, are known to interact with crustacean pattern-recognition receptors, thereby stimulating hemocyte proliferation and activating downstream immune pathways, including the IMD–Relish signaling cascade observed in the present study. Concurrently, fermentation by L. plantarum may generate beneficial postbiotic metabolites, including organic acids, bacteriocins, and bioactive intracellular compounds, which may further contribute to immune modulation and disease resistance.

Detoxification and antioxidant-related benefits

The present findings suggest that L. plantarum fermentation may help mitigate one of the major limitations associated with plant protein utilization, namely ANFs. As shown in Table 1, fermentation reduced mimosine levels by 42.18%, decreasing from 92.46 to 53.46 mg/100 g. Mimosine is a toxic non-protein amino acid known to impair nutrient utilization and physiological performance in animals. Furthermore, the incorporation of fermented L. leucocephala enhanced the phytochemical and antioxidant properties of the diets, as evidenced by increased TPC and TFC, higher FRAP values, and improved radical-scavenging activity, as indicated by lower DPPH and ABTS IC50 values in the 20% inclusion group. Collectively, these findings suggest that fermented diets may function as promising functional feeds enriched with detoxified plant matrixes, fermentation-derived metabolites, and microbial-derived bioactive components that collectively support innate immune responses in prawns.

Study limitations and future research

One limitation of the present study is that the experimental diets were not strictly formulated to be isonitrogenous. As shown in Table 2, crude protein content decreased from 44.63% in the control diet to 36.75% in the 20% replacement group, representing a substantial nutritional variation that may have influenced the physiological and immunological responses of _M. _rosenbergii. Therefore, the effects cannot be attributed exclusively to the bioactive properties of fermented L. leucocephala, as differences in dietary protein levels may also have contributed to the observed responses. In addition, crystalline amino acids such as lysine and methionine were not supplemented to achieve precise amino acid balancing among the experimental diets. Consequently, variations in essential amino acid composition resulting from the inclusion of fermented L. leucocephala may have further influenced immune and physiological responses. Future studies using strictly isonitrogenous, isolipidic, and amino acid-balanced diets are required to better distinguish the specific functional effects of fermented L. leucocephala from potential nutritional confounding factors.

Another limitation is that the feeding trial duration was relatively short compared with conventional aquaculture nutrition studies, and growth performance parameters such as weight gain, specific growth rate, and feed conversion ratio were not evaluated. In addition, pellet water stability and nutrient leaching rates were not quantitatively assessed. Since feed stability in water may influence nutrient availability, feed intake, and feeding efficiency in prawns, these parameters should be evaluated in future studies to better characterize the physicochemical quality and practical applicability of the experimental diets under aquaculture conditions. Therefore, the long-term nutritional suitability and commercial applicability of the diets could not be fully determined in the present study. Furthermore, the current experimental design does not allow precise determination of the optimal inclusion level or dose-dependent immune responses associated with fermented L. leucocephala supplementation.

The present study also has limitations related to fermentation characterization and microbial analysis. Post-fermentation LAB viability, pH, lactic acid concentration, titratable acidity, and detailed biochemical characteristics of the fermented product were not directly quantified. In addition, the microbial composition of the fermented material and viability of L. plantarum in the final feed and during storage were not evaluated. Consequently, microbial dynamics and fermentation-derived metabolites associated with the fermented product could not be fully characterized. Therefore, the relative contributions of live probiotic cells, postbiotic metabolites, and paraprobiotic effects remain unclear. Moreover, microbial safety analyses, including assessments of pathogen and mycotoxin contamination, were not performed directly, although fermentation was conducted under sealed, anaerobic conditions to minimize contamination risk. Future studies integrating comprehensive fermentation profiling, microbial safety analyses, metabolite characterization, and gut microbiota analysis would provide stronger mechanistic insight into host–microbe interactions and the functional properties of fermented L. leucocephala.

Antioxidant capacity was not directly measured in hemolymph or tissues of _M. _rosenbergii. The present study primarily evaluated antioxidative responses through immune- and oxidative stress-related gene expression, particularly Cu/Zn-SOD expression in hemocytes. Therefore, the antioxidative effects of fermented L. leucocephala supplementation should be interpreted as indirect molecular evidence rather than direct biochemical confirmation of antioxidant activity.

Several limitations were also associated with the bacterial challenge experiment. Unchallenged control prawns were not sampled at all post-challenge time points (6–120 h), limiting the ability to distinguish the direct effects of dietary supplementation from diet × infection interaction effects. In addition, bacterial load in hemolymph or tissues was not quantified. Therefore, the improved survival and immune responses observed in the treatment groups cannot be directly interpreted as evidence of enhanced bacterial clearance capacity. Correlation analyses between immune parameters and survival outcomes were also not performed, preventing detailed evaluation of the quantitative relationship between immune modulation and disease resistance. Future studies incorporating time-matched unchallenged controls, quantification of bacterial load, and integrated multivariate statistical analyses would strengthen the mechanistic interpretation of immune responses and disease resistance in _M. _rosenbergii.

Despite these limitations, the consistent enhancement of several immune parameters and improved resistance to _A. _hydrophila observed in the treatment groups suggest that fermented L. leucocephala may contribute beneficial immunomodulatory effects in prawns. Importantly, no adverse effects on intestinal morphology were detected, supporting the safety of the dietary inclusion levels used in the present study. Nevertheless, the findings should be interpreted with caution, and further long-term studies using rigorously controlled diet formulations and comprehensive physiological, microbiological, and biochemical evaluations are warranted to clarify the specific contribution of fermented L. leucocephala to immune modulation and disease resistance in prawn aquaculture.