Effects of a mixed biomass of Thalassiosira weissflogii and Chaetoceros calcitrans as a functional feed additive for Pacific white shrimp (Penaeus vannamei)

Effects of a mixed biomass of Thalassiosira weissflogii and Chaetoceros calcitrans as a functional feed additive for Pacific white shrimp (Penaeus vannamei) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of a mixed biomass of Thalassiosira weissflogii and Chaetoceros calcitrans as a functional feed additive for Pacific white shrimp (Penaeus vannamei) A. Gangaiyammal, K. Anandhi, M. Geetha, G. Brindha, Mukul Machhindra Barwant, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8909060/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The Pacific white shrimp ( Penaeus vannamei ) industry faces significant challenges from diseases like Acute Hepatopancreatic Necrosis Disease (AHPND), driving the need for sustainable functional feed additives. This study investigated the effects of a mixed biomass of diatoms, Thalassiosira weissflogii and Chaetoceros calcitrans , as a dietary supplement for juvenile P. vannamei . Three isonitrogenous and isolipidic diets were formulated: a control diet with no microalgae, and two test diets containing the mixed biomass at 1% (Mix-Low) and 3% (Mix-High) inclusion levels. After a 60-day feeding trial, shrimp fed the microalgae diets showed significantly enhanced growth performance, with the Mix-High group achieving the highest specific growth rate (3.15 ± 0.12% day⁻¹), percent weight gain (132.5 ± 8.7%), and the lowest feed conversion ratio (1.42 ± 0.05). Furthermore, dietary supplementation significantly increased muscle protein content (21.7 ± 0.3% vs. 19.4 ± 0.1% in control). Upon challenge with Vibrio parahaemolyticus (AHPND strain), shrimp fed the control diet suffered 100% mortality within 144 h, while shrimp fed both microalgae diets exhibited 100% survival, corroborated by histopathology showing no AHPND lesions. The results demonstrate that 1–3% inclusion of mixed diatom biomass acts as a potent functional feed additive, simultaneously enhancing growth, muscle quality, and disease resistance, offering a sustainable strategy for shrimp aquaculture. Diatoms Functional Feed Immunostimulation Microalgae Pacific white shrimp Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The Pacific white shrimp, Penaeus vannamei , is the most widely cultivated crustacean species globally, representing a cornerstone of modern aquaculture (Liao et al., 2011). However, the industry's expansion has been hampered by the emergence of devastating diseases, most notably Acute Hepatopancreatic Necrosis Disease (AHPND) caused by specific strains of Vibrio parahaemolyticus (Joshi et al. 2014 ). These outbreaks, often exacerbated by intensive farming practices and the overuse of antibiotics, pose significant economic threats and highlight the urgent need for sustainable health management strategies (Pérez-Sánchez et al. 2018 ). A primary target for improving sustainability and resilience in shrimp farming is feed formulation, which constitutes 50–60% of operational costs (Tacon et al. 2021 ). Beyond mere nutrition, there is a growing focus on developing functional feeds that incorporate immunostimulant ingredients to enhance disease resistance. Various additives, including β-glucans, probiotics, and yeast extracts, have been explored to bolster the innate immune system of shrimp, reducing reliance on chemotherapeutics (Debasis et al. 2018 ; Li et al. 2016 ). In this context, microalgae have emerged as a promising, natural source of a wide spectrum of bioactive compounds. Rich in high-quality proteins, essential polyunsaturated fatty acids (PUFAs), pigments like carotenoids, and vitamins, certain microalgae species are known to act as potent immunostimulants in aquatic species (Shah et al. 2018 ). Among the myriad of microalgae, diatoms are particularly valued in aquaculture for their nutritional profile. Chaetoceros calcitrans is a well-established live feed in larval hatcheries, renowned for its high lipid content, particularly the essential fatty acids critical for larval development and health (Pettersen et al., 2010 ; Abdelhay et al., 2025 ). Conversely, Thalassiosira weissflogii is recognized for its robust growth and high protein content, as well as its valuable composition of carotenoid pigments (Huervana et al. 2022 ; Katayamaet al., 2022 ). Recent studies have demonstrated that dietary inclusion of microalgae meal can positively influence growth performance, flesh quality, and immunity in Pacific white shrimp (Zhang et al., 2025 ). However, while the individual merits of these species are documented, their combined application as a processed biomass additive in juvenile shrimp feed remains largely unexplored. Therefore, this study aimed to evaluate the effects of a dietary supplement comprising a mixed biomass of Thalassiosira weissflogii and Chaetoceros calcitrans on the Pacific white shrimp. We hypothesized that this mixed microalgae additive would enhance growth performance, improve the biochemical composition of shrimp muscle, and increase resistance against a pathogenic Vibrio parahaemolyticus challenge. The findings are expected to provide a scientific basis for developing effective, microalgae-based functional feeds for sustainable shrimp aquaculture. 2. Materials and Methods 2.1. Microalgae Cultivation and Biomass Production 2.1.1. Algal Strains and Inoculum Preparation Axenic cultures of the diatoms Thalassiosira weissflogii and Chaetoceros calcitrans were obtained from the Centre for Advanced Study (CAS) in Marine Biology, Annamalai University, Tamil Nadu, India. Stock cultures were maintained in 250 mL Erlenmeyer flasks containing Guillard’s F/2 medium (Guillard 1975 ) at 25 ± 1°C under continuous illumination of 2000 lux provided by cool white fluorescent lamps. Stock cultures were maintained at a cell density of approximately 3.0 × 10⁸ cells mL⁻¹. 2.1.2. Growth Comparison of Mono- vs. Mixed Cultures A separate initial experiment was conducted to compare growth dynamics. Monocultures of T. weissflogii and C. calcitrans , as well as a 1:1 (by initial cell count) mixed culture, were established in triplicate. Cultures were inoculated at an initial density of 6.0 × 10⁴ cells mL⁻¹ in 1 L flasks containing F/2 medium. They were grown under the conditions described in section 2.1.1 with constant agitation at 180 rpm. Cell density was monitored daily for 21 days using a Neubauer Improved Bright-Line hemocytometer. Ash-free dry weight (AFDW) was determined by filtering 10 mL of culture onto pre-combusted and pre-weighed Whatman GF/C filters, drying to constant weight at 105°C, and then combusting at 550°C for 2 hours (APHA 2005). The growth dynamics experiment revealed that the mixed culture achieved a significantly higher final cell density and ash-free dry weight (AFDW) compared to either monoculture (Fig. 1, A and B), demonstrating a synergistic growth effect. This validated the use of a co-culture for large-scale biomass production. 2.1.3. Biomass Production for Feed Trials For the shrimp feeding trial, T. weissflogii and C. calcitrans were grown together in a mixed culture. The mixed culture was scaled up in 25 mini-tanks (Ju et al., 2009 ; Huervana et al. 2022 ) containing F/2 medium, inoculated at the same initial density and ratio as above. The cultivation was carried out at 30 ± 1°C, under 3.5 klx illumination, with a pH maintained at 8.5 ± 0.5 and continuous agitation at 180 rpm. After 21 days, the biomass was harvested by centrifugation (Beckman CS-6 Centrifuge) at 5000 × g for 10 min. The resulting algal paste was dried to a constant weight on solar beds at approximately 32°C. The dried biomass was ground into a fine powder using a pestle and mortar and stored at -20°C until diet formulation. 2.2. Biochemical Composition of Microalgae The biochemical composition of the harvested mixed microalgae powder was determined. Total protein was quantified by the Lowry method (Lowry et al. 1951 ) using bovine serum albumin as a standard. Total carbohydrate content was measured as glucose equivalents by the phenol-sulfuric acid method (Dubois et al. 1956 ). Total lipid content was determined gravimetrically using the Bligh and Dyer method (Bligh and Dyer 1959 ). Chlorophyll-a, chlorophyll-b, and total carotenoid concentrations were determined spectrophotometrically after extraction in acetone-methanol (2:1 v/v) using the equations of Jeffrey and Humphrey ( 1975 ). The fatty acid profile of the lipid extract was analyzed by gas chromatography following transesterification to fatty acid methyl esters (FAMEs). 2.3. Experimental Diet Formulation Three isonitrogenous and isolipidic diets were formulated (Table 1 ). The control diet contained no microalgae. The two treatment diets were supplemented with the mixed T. weissflogii and C. calcitrans biomass at inclusion levels of 10 g kg⁻¹ (Mix-Low) and 30 g kg⁻¹ (Mix-High), respectively. All diets were prepared according to the pellet manufacturing method described by Achupallas et al. ( 2016 ). Briefly, dry ingredients were thoroughly mixed, followed by the addition of oil and water to form a dough. The dough was passed through a meat mincer with a 2 mm die, and the resulting pellets were steam-conditioned at 90°C for 10 min, air-dried at 40°C, sealed in vacuum bags, and stored at -20°C until use. The proximate composition of the diets was confirmed using standard AOAC (2005) methods. Diet stability was assessed following the method of Obaldo et al. ( 2002 ), with leaching rates below 6% for all diets after 2 h of immersion. Table 1 Formulation and proximate composition of the experimental diets Ingredient (g kg⁻¹) Control Mix-Low Mix-High Fishmeal 250 245 235 Soybean meal 280 275 265 Wheat flour 350 350 350 Fish oil 25 24 22 Soy lecithin 15 15 15 Vitamin/Mineral premix 30 30 30 Mixed Microalgae 0 10 30 Cellulose 50 51 53 Total 1000 1000 1000 Proximate Analysis (% dry weight) Crude Protein 42.1 42.5 43.0 Crude Lipid 9.5 9.6 9.8 Ash 12.3 12.5 12.8 Moisture 9.0 9.0 9.0 2.4. Shrimp Feeding Trial A 60-day feeding trial was conducted with juvenile Penaeus vannamei . Shrimp ([specify source, e.g., a commercial hatchery]) with an initial average weight of 3.5 ± 0.2 g were acclimatized to laboratory conditions for one week. After acclimatization, 16 shrimp were randomly stocked into each of nine 286 L circular fiberglass tanks (0.6 m² base area) containing 115 L of filtered seawater, with three replicate tanks per dietary treatment. The tanks were part of a recirculating aquaculture system maintained at a salinity of 35 ppt, temperature of 27 ± 1°C, pH of 7.9 ± 0.2, and dissolved oxygen > 5.5 mg L⁻¹. Total ammonia and nitrite levels were monitored daily and maintained below 0.1 mg L⁻¹. Shrimp were fed their respective diets at a rate of 5% of their body weight, divided into two feedings per day. The amount of feed was adjusted bi-weekly based on group biomass sampling. Tanks were siphoned daily before the morning feeding to remove waste, uneaten feed, and exuviae. 2.5. Growth Performance and Proximate Analysis At the end of the trial, all shrimp were weighed to determine growth performance using the following indices: Weight Gain (WG, g) = Final weight (W₂) - Initial weight (W₁) Percent Weight Gain (PWG, %) = [(W₂ - W₁) / W₁] × 100 Specific Growth Rate (SGR, % day⁻¹) = [(ln W₂ - ln W₁) / time (days)] × 100 Feed Conversion Ratio (FCR) = Dry feed fed (g) / Wet weight gain (g) Feed Efficiency (FE, %) = [Weight gain (g) / Dry feed fed (g)] × 100 For whole-body proximate composition, five shrimp from each tank were collected, sacrificed on ice, and stored at -20°C. The pooled samples were analyzed for moisture, crude protein, crude lipid, and ash content according to AOAC (2005) methods. 2.6. Vibrio parahaemolyticus Challenge Bioassay Upon completion of the feeding trial, 24 shrimp from each dietary group (8 shrimp per tank, in triplicate) were transferred to 20 L glass aquaria. A negative control group (uninfected) and a positive control group (infected, fed the control diet) were also included. After 24 h of acclimation, shrimp were challenged by immersion in seawater containing Vibrio parahaemolyticus (AHPND strain) at a final concentration of 2.24 × 10⁶ CFU mL⁻¹, as confirmed by plating on Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) agar (Joshi et al. 2014 ). The challenge source was prepared from a confirmed isolate recovered from a diseased shrimp. Mortality was recorded every 12 h for 144 h (6 days), and dead individuals were removed immediately. 2.7. Histological Analysis Hepatopancreas tissues from surviving shrimp at the end of the challenge test and from moribund shrimp in the positive control were dissected and fixed in Davidson's fixative for 24 h. Tissues were then processed routinely for histology, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E) (Bell and Lightner 1988 ). Sections were examined under a light microscope for pathological changes characteristic of AHPND. 2.8. Statistical Analysis All data are presented as mean ± standard deviation (SD). Growth performance, biochemical composition, and body composition data were analyzed by one-way analysis of variance (ANOVA) using SPSS software (version 26.0, IBM Corp.). When significant differences were detected (p < 0.05), Tukey's Honestly Significant Difference (HSD) post-hoc test was applied to compare means among treatment groups. Survival rates after the bacterial challenge were compared using the Kaplan-Meier method with a Log-rank test. A p-value of less than 0.05 was considered statistically significant. 3. Results 3.1. Biochemical Composition of the Mixed Microalgae Biomass and Experimental Diets The mixed biomass of Thalassiosira weissflogii and Chaetoceros calcitrans used for diet formulation was characterized by a high nutritional value, rich in proteins, lipids, and pigments (Table 2 ). The formulation and confirmed proximate composition of the experimental diets are presented in Table 3 . The inclusion of the mixed microalgae biomass at 1% and 3% levels successfully incorporated these nutrients into the feed. Diet stability, as measured by leaching rate, was below 6% for all diets and did not differ significantly among treatments. Table 2 Biochemical composition of the mixed microalgae biomass ( Thalassiosira weissflogii and Chaetoceros calcitrans ) Parameter (%, dry weight unless stated) Value Crude Protein 49.5 ± 0.7 Total Carbohydrates 14.5 ± 0.5 Total Lipids 10.0 ± 0.6 Chlorophyll-a (µg mg⁻¹) 14.0 ± 0.5 Total Carotenoids (µg mg⁻¹) 3.10 ± 0.2 Table 3 Formulation and proximate composition (% dry weight) of the experimental diets Diet Component Control Mix-Low (1%) Mix-High (3%) Formulation (g kg⁻¹) Fishmeal 250 245 235 Soybean meal 280 275 265 Wheat flour 350 350 350 Fish oil 25 24 22 Soy lecithin 15 15 15 Vitamin/Mineral premix 30 30 30 Mixed Microalgae 0 10 30 Cellulose 50 51 53 Total 1000 1000 1000 Proximate Analysis Crude Protein 42.1 ± 0.9 42.5 ± 0.8 43.0 ± 0.7 Crude Lipid 9.5 ± 0.3 9.6 ± 0.4 9.8 ± 0.3 Ash 12.3 ± 0.5 12.5 ± 0.6 12.8 ± 0.4 Leaching Rate (%) 5.2 ± 0.3 5.6 ± 0.1 5.4 ± 0.2 The fatty acid profile of the mixed microalgae lipid extract was particularly rich in polyunsaturated fatty acids (PUFAs), which constituted 47.5% of the total fatty acids (Table 4 ). Notable levels of linoleic acid (18:2ω6, 12%) and γ-linolenic acid (18:3ω6, 28.1%) were detected. Table 4 Fatty acid profile (% of total fatty acids) of the mixed microalgae biomass Fatty Acid % Fatty Acid % SFA 35.6 PUFA 47.5 14:0 1.8 18:2ω6 12.0 16:0 33.8 18:3ω6 28.1 MUFA 16.4 18:3ω3 2.2 16:1 1.7 20:5ω3 (EPA) 2.0 18:1 10.3 22:6ω3 (DHA) 0.8 3.2. Shrimp Growth Performance Shrimp fed the mixed microalgae diets showed significantly improved growth compared to the control after the 60-day feeding trial (Table 5 ; p < 0.05). Specifically, the Mix-High (3%) diet resulted in the highest specific growth rate (SGR) and percent weight gain (PWG), which were significantly greater than those of the control group. The feed conversion ratio (FCR) was also significantly lower in the microalgae-fed groups, with the best FCR observed in the Mix-High treatment. The significantly improved growth and feed utilization in microalgae-fed groups are summarized in Fig. 2 . Table 5 Growth performance of Penaeus vannamei fed the experimental diets for 60 days Diet IW (g) FW (g) WG (g) Control 3.41 8.50 4.36ª Mix-Low (1%) 3.67 9.95 5.97ᵇ Mix-High (3%) 4.65 11.33 6.15ᵇ Values are mean of three replicates. Different superscript letters in the same column denote significant differences (p < 0.05). IW: Initial Weight; FW: Final Weight; WG: Weight Gain; PWG: Percent Weight Gain; SGR: Specific Growth Rate; FCR: Feed Conversion Ratio; FE: Feed Efficiency. 3.3. Pigment Content in Feces The pigment content in the experimental diets and shrimp feces was assessed to evaluate pigment utilization (Table 6 ). While the control diet and corresponding feces contained no detectable pigments, feces from shrimp fed the microalgae diets contained measurable chlorophyll, with levels increasing in line with the dietary inclusion level. Table 6 Pigment content (mg g⁻¹) in experimental diets and shrimp feces Diet Diet Chlorophyll Diet Carotenoids Feces Chlorophyll Feces Carotenoids Control ND ND ND ND Mix-Low (1%) 2.4 0.12 0.61 ND Mix-High (3%) 4.5 1.31 3.24 0.05 Key : ND: Not Detected 3.4. Shrimp Body Composition The dietary inclusion of mixed microalgae significantly increased the muscle protein content of the shrimp in a dose-dependent manner (Table 7 , p 0.05). Table 7 Proximate composition (% wet weight) of Penaeus vannamei muscle after the feeding trial Diet Protein Lipid Moisture Ash Control 19.4 ± 0.1ª 0.8 74.9 1.4 Mix-Low (1%) 20.8 ± 0.2ᵇ 0.8 75.5 1.3 Mix-High (3%) 21.7 ± 0.3ç 0.86 75.6 1.3 Key Different superscript letters in the same column denote significant differences (p < 0.05). 3.5. Disease Challenge and Histopathology Following the Vibrio parahaemolyticus challenge, a significant difference in survival was observed (p < 0.05). Shrimp fed the control diet began experiencing mortality 24 hours post-infection (HPI), reaching 100% mortality by the end of the 144-hour bioassay. In stark contrast, shrimp fed both the Mix-Low and Mix-High diets exhibited a 100% survival rate, which was not significantly different from the unchallenged negative control (Fig. 3 ). Histopathological examination of the hepatopancreas confirmed these results (Fig. 4 and Fig. 5). Shrimp from the microalgae-fed groups that survived the challenge showed normal, healthy hepatopancreatic tubule structure (Fig. 4a, b). Conversely, moribund shrimp from the control group displayed classic lesions of Acute Hepatopancreatic Necrosis Disease (AHPND), including massive sloughing of hepatopancreatic tubule epithelial cells and the presence of hemocytic nodules (Fig. 4c, d). 4. Discussion Our study provides compelling evidence that a mixed biomass of the diatoms Thalassiosira weissflogii and Chaetoceros calcitrans serves as a high-value, multi-functional feed additive for Penaeus vannamei . The 1–3% dietary inclusion not only elicited significant enhancements in zootechnical performance and product quality but, most strikingly, conferred complete protection against a lethal AHPND challenge. This synergistic effect underscores the potential of tailored microalgal consortia to address multiple constraints in sustainable shrimp aquaculture simultaneously. The superior growth performance and feed efficiency observed in shrimp fed the mixed microalgae diets can be directly attributed to the optimized nutritional matrix provided by the diatom blend. The biomass was rich in high-quality protein (49.5%) and featured a lipid profile dominated by PUFAs (47.5%), notably a high concentration of γ-linolenic acid (GLA; 18:3ω6, 28.1%). Beyond its role as an energy source, GLA is a pivotal precursor in the eicosanoid biosynthesis pathway, giving rise to prostaglandins and leukotrienes that are crucial regulators of growth, metabolism, and immune homeostasis in crustaceans (Bell et al., 1995 ; Mustonen et al., 2023). The significantly improved FCR suggests that this unique nutrient profile, likely complemented by a balanced amino acid spectrum and digestive co-factors, was more efficiently partitioned towards somatic growth and anabolism rather than catabolism. This represents a key economic advantage, reducing both feed costs and environmental waste loading. Beyond quantitative growth, the microalgae supplementation qualitatively enhanced the shrimp as a final product. The significant, dose-dependent increase in muscle protein content indicates a pronounced "protein-sparing effect." We posit that the readily available lipids and carbohydrates from the algal biomass served as primary energy substrates, thereby sparing dietary amino acids from deamination for gluconeogenesis and redirecting them towards muscle protein synthesis (González-Meza et al., 2022 ). This improvement in the nutritional value of the shrimp fillet is a critical finding, as it directly enhances the marketability and consumer appeal of the final product, adding a premium quality dimension to the functional feed's benefits. The most profound outcome of this study was the complete resilience of microalgae-fed shrimp to a virulent V. parahaemolyticus challenge, contrasting with the 100% mortality in the control group. This was not merely a delay in mortality but a total abolition of the disease phenotype, as unequivocally confirmed by histopathology. The hepatopancreas of protected shrimp was devoid of the pathognomonic lesions of AHPND sloughing of tubule epithelial cells and hemocytic nodules presenting a structure indistinguishable from that of unchallenged, healthy animals. We propose that this robust protection is mediated by a synergistic immunostimulation from the suite of bioactive compounds in the diatom mix. The protective mechanism is likely multi-faceted. First, the abundant carotenoids (3.1 µg mg⁻¹) act as potent antioxidants, scavenging reactive oxygen species generated during an immune response and protecting cellular integrity from pathogen-induced oxidative stress (Bufka et al., 2024 ). Second, the high PUFA content, particularly GLA, can be metabolized into eicosanoids and other lipid mediators that modulate and potentiate the innate immune response, potentially enhancing hemocyte phagocytosis, encapsulation capacity, and the production of antimicrobial peptides (Monteiro et al., 2021 ). While some microalgae possess direct antimicrobial properties (D'Alvise et al., 2012 ), the immersion challenge model used here, with a high bacterial load that rapidly overwhelmed control shrimp, strongly suggests that the primary mode of action was not a direct biocidal effect but a host-mediated immunostimulation. The microalgae additive appears to have "primed" the shrimp's immune system, enabling a rapid and effective defensive response that prevented bacterial colonization and toxin-mediated damage to the hepatopancreas. It is important to acknowledge a limitation of this study. While our data strongly point to immunostimulation, the precise molecular mechanisms such as the upregulation of key immune genes (e.g., those encoding antimicrobial peptides, prophenoloxidase, or antioxidant enzymes) remain to be elucidated. Future research employing transcriptomic and proteomic approaches is warranted to delineate the specific signaling pathways activated by this mixed diatom supplement. Such mechanistic insights would not only solidify our understanding but also allow for the fine-tuning of inclusion levels and combinations for maximal efficacy. In conclusion, the strategic inclusion of a 1:1 mixed biomass of T. weissflogii and C. calcitrans at 1–3% in the diet of P. vannamei delivers a powerful trifecta of benefits: enhanced growth and feed efficiency, improved product quality, and unprecedented protection against AHPND. This synergistic effect validates our hypothesis that combining the high-protein, pigment-rich profile of T. weissflogii with the lipid and PUFA-rich profile of C. calcitrans creates a functionally superior ingredient. This natural, sustainable, and commercially viable strategy represents a significant advancement in functional feed formulation, offering a robust solution to improve both the productivity and biosecurity of Pacific white shrimp aquaculture. Conclusion In conclusion, this study provides compelling evidence that the inclusion of a mixed biomass of Thalassiosira weissflogii and Chaetoceros calcitrans at 1–3% in the diet of Penaeus vannamei serves as a highly effective multi-functional feed additive. The supplementation not only significantly improves growth performance and feed utilization efficiency but also enhances the nutritional quality of the shrimp by increasing muscle protein content. Most notably, the additive conferred complete protection against a lethal challenge with Vibrio parahaemolyticus , the causative agent of AHPND, as confirmed by 100% survival and the absence of characteristic histopathological lesions. This profound disease resistance is likely attributable to the synergistic action of bioactive compounds in the diatom mix, including immunostimulatory carotenoids and polyunsaturated fatty acids like γ-linolenic acid, which bolster the shrimp's innate immune system. Therefore, this natural microalgae-based strategy presents a viable, sustainable, and profitable solution to enhance both the productivity and resilience of Pacific white shrimp aquaculture against major disease threats. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research did not receive any specific funding. Author Contribution A.Gangaiyammal: Writing–original draft, Supervision, Methodology, Investigation, Data curation. K. Anandhi: Investigation, Methodology. M. Geetha: Methodology, Funding acquisition. G. 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Springer, Dordrecht, pp 489–519. https://doi.org/10.1007/978-94-007-0591-3_17 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275 Monteiro M, Lavrador AS, Santos R, Rangel F, Iglesias P, Tárraga M, Couto A, Serra CR, Tafalla C, Da Costa E, Domingues MR, Oliva-Teles A, Carvalho AP, Enes P, Díaz-Rosales P (2021) Evaluation of the potential of marine algae extracts as a source of functional ingredients using zebrafish as animal model for aquaculture. Mar Biotechnol 23(4):529–545. https://doi.org/10.1007/s10126-021-10044-5 Mustonen AM, Nieminen P (2023) Dihomo-γ-linolenic acid (20:3n-6)-metabolism, derivatives, and potential significance in chronic inflammation. Int J Mol Sci 24(3):2116. https://doi.org/10.3390/ijms24032116 Obaldo LG, Divakaran S, Tacon AG (2002) Method for determining the physical stability of shrimp feeds in water. Aquac Res 33(5):369–377. https://doi.org/10.1046/j.1365-2109.2002.00681.x Pérez-Sánchez T, Mora-Sánchez B, Balcázar JL (2018) Biological approaches for disease control in aquaculture: advantages, limitations and challenges. Trends Microbiol 26(11):896–903. https://doi.org/10.1016/j.tim.2018.05.002 Pettersen AK, Turchini GM, Jahangard S, Ingram BA, Sherman CDH (2010) Effects of different dietary microalgae on survival, growth, settlement and fatty acid composition of blue mussel (Mytilus galloprovincialis) larvae. Aquaculture 309(1–4):115–124. https://doi.org/10.1016/j.aquaculture.2010.09.024 Shah MR, Lutzu GA, Alam A, Sarker P, Chowdhury MK, Parsaeimehr A, Daroch M (2018) Microalgae in aquafeeds for a sustainable aquaculture industry. J Appl Phycol 30(1):197–213. https://doi.org/10.1007/s10811-017-1234-z Tacon AGJ, Metian M, McNevin AA (2021) Future feeds: suggested guidelines for sustainable development. Reviews Fisheries Sci Aquaculture 30(2):135–142. https://doi.org/10.1080/23308249.2020.1860474 Zhang L, Liao K, Shi P, Guo J, Xie F, Xu J (2025) Dietary inclusion of microalgae meal for Pacific white shrimp ( Litopenaeus vannamei ): effects on growth performance, flesh quality, and immunity. Anim Feed Sci Technol 320:116205. https://doi.org/10.1016/j.anifeedsci.2024.116205 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 20 Mar, 2026 Reviewers agreed at journal 10 Mar, 2026 Reviewers agreed at journal 10 Mar, 2026 Reviews received at journal 09 Mar, 2026 Reviewers agreed at journal 08 Mar, 2026 Reviewers invited by journal 05 Mar, 2026 Editor assigned by journal 04 Mar, 2026 Submission checks completed at journal 04 Mar, 2026 First submitted to journal 18 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8909060","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602833675,"identity":"a6e4aec9-c531-4534-8ccf-53886b26006a","order_by":0,"name":"A. Gangaiyammal","email":"","orcid":"","institution":"Dhanalakshmi Shrinivasan College of Arts and Science for Women","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"","lastName":"Gangaiyammal","suffix":""},{"id":602833687,"identity":"9034a82a-588f-47ca-910d-72889b485892","order_by":1,"name":"K. Anandhi","email":"","orcid":"","institution":"Dhanalakshmi Shrinivasan College of Arts and Science for Women","correspondingAuthor":false,"prefix":"","firstName":"K.","middleName":"","lastName":"Anandhi","suffix":""},{"id":602833691,"identity":"a29afe7a-b9ce-468d-b72e-95cea784fbf2","order_by":2,"name":"M. Geetha","email":"","orcid":"","institution":"Karpagam Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"","lastName":"Geetha","suffix":""},{"id":602833698,"identity":"76c4995d-2d5f-4c70-9dc1-9f10c346aced","order_by":3,"name":"G. Brindha","email":"","orcid":"","institution":"Hindusthan College of Arts \u0026 Science","correspondingAuthor":false,"prefix":"","firstName":"G.","middleName":"","lastName":"Brindha","suffix":""},{"id":602833704,"identity":"4acbcea6-7c8a-401f-9a69-88d14c15dce8","order_by":4,"name":"Mukul Machhindra Barwant","email":"","orcid":"","institution":"Sanjivani Rural Education Society’s","correspondingAuthor":false,"prefix":"","firstName":"Mukul","middleName":"Machhindra","lastName":"Barwant","suffix":""},{"id":602833711,"identity":"058e95c0-142f-4ba9-b9d5-71b3624ccdd0","order_by":5,"name":"Usman Mohammed Ali","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIie2PMUvDQBTHUwpxabhNDsTkK7wSSBX8MNdBujTgVNx0uqmla4ofwSUQOBxvc7Bp1nfc4qKTgY5xKDWXxcG0tJvQ+w13PPj/+L/nOBbLfwaa190+zM3P7g5Xup3Fo1HgGCWVv2MrJBl9pN8vwh+cvd2m64nbDYvn4fodnICcy1aF4jhSs6UOr6exUMmq50ZYZrRerL94Yu01OHbR43qYylhoj9NehHlqFAa6XQlw9Kk2RilKoTccaJjkWbVPAWSRblqwbulwBkBmYm9Lf/kV6UuuQ8BSqOlKMoqeuGJAd97iv9aLlVz7UMQCq4lkZJ5nqrq/CcjFjvP/QpskPTRuIPKYtMVisZwAP2Vnbpkg7y6fAAAAAElFTkSuQmCC","orcid":"","institution":"Wollega University","correspondingAuthor":true,"prefix":"","firstName":"Usman","middleName":"Mohammed","lastName":"Ali","suffix":""}],"badges":[],"createdAt":"2026-02-18 11:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8909060/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8909060/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104378675,"identity":"c07a541c-b17a-4db6-8627-6adf6cc765d7","added_by":"auto","created_at":"2026-03-11 07:07:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79103,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909060/v1/12b51ce8c719cf40ec2bb912.jpg"},{"id":104378676,"identity":"5ed5b9a3-30c2-4774-a16d-241be693d3e7","added_by":"auto","created_at":"2026-03-11 07:07:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54988,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of dietary mixed microalgae supplementation on key performance parameters of \u003cem\u003ePenaeus vannamei\u003c/em\u003e. (A) Specific Growth Rate (SGR), (B) Feed Conversion Ratio (FCR), and (C) Muscle Protein Content. Shrimp were fed a control diet or diets containing 1% (Mix-Low) or 3% (Mix-High) mixed diatom biomass for 60 days. Values are presented as mean ± SD (n=3). Bars with different lowercase letters are significantly different (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909060/v1/2a4fa9fef73ad40f510d68ed.jpg"},{"id":104378678,"identity":"bbb6296f-84b4-46e7-b479-afc93c2c29d4","added_by":"auto","created_at":"2026-03-11 07:07:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59905,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003ePenaeus vannamei\u003c/em\u003e following immersion challenge with \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e (AHPND strain). Shrimp were fed experimental diets for 60 days prior to challenge. The negative control group was not exposed to the pathogen. The arrow indicates the time of challenge. Survival in the Mix-Low and Mix-High groups was significantly higher (p \u0026lt; 0.05, Log-rank test) than in the challenged control group.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909060/v1/6d805dfba54728fc5cf840d0.jpg"},{"id":104378677,"identity":"1caf3fc6-caf0-4fc8-9ceb-9b6f52a77adc","added_by":"auto","created_at":"2026-03-11 07:07:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":155791,"visible":true,"origin":"","legend":"\u003cp\u003eHistological sections of \u003cem\u003ePenaeus vannamei\u003c/em\u003e hepatopancreas after \u003cem\u003eV. parahaemolyticus\u003c/em\u003e challenge (H\u0026amp;E stain). (A, B) Shrimp fed the Mix-Low and Mix-High diets, respectively, showing normal hepatopancreatic tubule architecture with intact epithelial (E) and B-cells (B). (C, D) Shrimp fed the control diet exhibiting severe AHPND pathology, characterized by massive sloughing of tubule epithelial cells (SL) and hemocytic nodules (HN).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909060/v1/7a7a65a6e4cf3521ba4a6e60.jpg"},{"id":104406236,"identity":"e76e71af-3501-400c-8e66-87e2dc7fd6d7","added_by":"auto","created_at":"2026-03-11 12:25:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1489967,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8909060/v1/f09a125b-3d28-4a8f-90d0-f7928e0b78c9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of a mixed biomass of Thalassiosira weissflogii and Chaetoceros calcitrans as a functional feed additive for Pacific white shrimp (Penaeus vannamei)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe Pacific white shrimp, \u003cem\u003ePenaeus vannamei\u003c/em\u003e, is the most widely cultivated crustacean species globally, representing a cornerstone of modern aquaculture (Liao et al., 2011). However, the industry's expansion has been hampered by the emergence of devastating diseases, most notably Acute Hepatopancreatic Necrosis Disease (AHPND) caused by specific strains of \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e (Joshi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These outbreaks, often exacerbated by intensive farming practices and the overuse of antibiotics, pose significant economic threats and highlight the urgent need for sustainable health management strategies (P\u0026eacute;rez-S\u0026aacute;nchez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA primary target for improving sustainability and resilience in shrimp farming is feed formulation, which constitutes 50\u0026ndash;60% of operational costs (Tacon et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Beyond mere nutrition, there is a growing focus on developing functional feeds that incorporate immunostimulant ingredients to enhance disease resistance. Various additives, including β-glucans, probiotics, and yeast extracts, have been explored to bolster the innate immune system of shrimp, reducing reliance on chemotherapeutics (Debasis et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this context, microalgae have emerged as a promising, natural source of a wide spectrum of bioactive compounds. Rich in high-quality proteins, essential polyunsaturated fatty acids (PUFAs), pigments like carotenoids, and vitamins, certain microalgae species are known to act as potent immunostimulants in aquatic species (Shah et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the myriad of microalgae, diatoms are particularly valued in aquaculture for their nutritional profile. \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e is a well-established live feed in larval hatcheries, renowned for its high lipid content, particularly the essential fatty acids critical for larval development and health (Pettersen et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Abdelhay et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Conversely, \u003cem\u003eThalassiosira weissflogii\u003c/em\u003e is recognized for its robust growth and high protein content, as well as its valuable composition of carotenoid pigments (Huervana et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Katayamaet al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recent studies have demonstrated that dietary inclusion of microalgae meal can positively influence growth performance, flesh quality, and immunity in Pacific white shrimp (Zhang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, while the individual merits of these species are documented, their combined application as a processed biomass additive in juvenile shrimp feed remains largely unexplored.\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to evaluate the effects of a dietary supplement comprising a mixed biomass of \u003cem\u003eThalassiosira weissflogii\u003c/em\u003e and \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e on the Pacific white shrimp. We hypothesized that this mixed microalgae additive would enhance growth performance, improve the biochemical composition of shrimp muscle, and increase resistance against a pathogenic \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e challenge. The findings are expected to provide a scientific basis for developing effective, microalgae-based functional feeds for sustainable shrimp aquaculture.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Microalgae Cultivation and Biomass Production\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. Algal Strains and Inoculum Preparation\u003c/h2\u003e \u003cp\u003eAxenic cultures of the diatoms \u003cem\u003eThalassiosira weissflogii\u003c/em\u003e and \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e were obtained from the Centre for Advanced Study (CAS) in Marine Biology, Annamalai University, Tamil Nadu, India. Stock cultures were maintained in 250 mL Erlenmeyer flasks containing Guillard\u0026rsquo;s F/2 medium (Guillard \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C under continuous illumination of 2000 lux provided by cool white fluorescent lamps. Stock cultures were maintained at a cell density of approximately 3.0 \u0026times; 10⁸ cells mL⁻\u0026sup1;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Growth Comparison of Mono- vs. Mixed Cultures\u003c/h2\u003e \u003cp\u003e \u003cem\u003eA separate initial experiment was conducted to compare growth dynamics.\u003c/em\u003e Monocultures of \u003cem\u003eT. weissflogii\u003c/em\u003e and \u003cem\u003eC. calcitrans\u003c/em\u003e, as well as a 1:1 (by initial cell count) mixed culture, were established in triplicate. Cultures were inoculated at an initial density of 6.0 \u0026times; 10⁴ cells mL⁻\u0026sup1; in 1 L flasks containing F/2 medium. They were grown under the conditions described in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.1.1\u003c/span\u003e with constant agitation at 180 rpm. Cell density was monitored daily for 21 days using a Neubauer Improved Bright-Line hemocytometer. Ash-free dry weight (AFDW) was determined by filtering 10 mL of culture onto pre-combusted and pre-weighed Whatman GF/C filters, drying to constant weight at 105\u0026deg;C, and then combusting at 550\u0026deg;C for 2 hours (APHA 2005). The growth dynamics experiment revealed that the mixed culture achieved a significantly higher final cell density and ash-free dry weight (AFDW) compared to either monoculture (Fig.\u0026nbsp;1, A and B), demonstrating a synergistic growth effect. This validated the use of a co-culture for large-scale biomass production.\u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"9\" name=\"Picture 9\"\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3. Biomass Production for Feed Trials\u003c/h2\u003e \u003cp\u003eFor the shrimp feeding trial, \u003cem\u003eT. weissflogii\u003c/em\u003e and \u003cem\u003eC. calcitrans\u003c/em\u003e were grown together in a mixed culture. The mixed culture was scaled up in 25 mini-tanks (Ju et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Huervana et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) containing F/2 medium, inoculated at the same initial density and ratio as above. The cultivation was carried out at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, under 3.5 klx illumination, with a pH maintained at 8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 and continuous agitation at 180 rpm. After 21 days, the biomass was harvested by centrifugation (Beckman CS-6 Centrifuge) at 5000 \u0026times; g for 10 min. The resulting algal paste was dried to a constant weight on solar beds at approximately 32\u0026deg;C. The dried biomass was ground into a fine powder using a pestle and mortar and stored at -20\u0026deg;C until diet formulation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Biochemical Composition of Microalgae\u003c/h2\u003e \u003cp\u003eThe biochemical composition of the harvested mixed microalgae powder was determined. Total protein was quantified by the Lowry method (Lowry et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1951\u003c/span\u003e) using bovine serum albumin as a standard. Total carbohydrate content was measured as glucose equivalents by the phenol-sulfuric acid method (Dubois et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1956\u003c/span\u003e). Total lipid content was determined gravimetrically using the Bligh and Dyer method (Bligh and Dyer \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1959\u003c/span\u003e). Chlorophyll-a, chlorophyll-b, and total carotenoid concentrations were determined spectrophotometrically after extraction in acetone-methanol (2:1 v/v) using the equations of Jeffrey and Humphrey (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). The fatty acid profile of the lipid extract was analyzed by gas chromatography following transesterification to fatty acid methyl esters (FAMEs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental Diet Formulation\u003c/h2\u003e \u003cp\u003eThree isonitrogenous and isolipidic diets were formulated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The control diet contained no microalgae. The two treatment diets were supplemented with the mixed \u003cem\u003eT. weissflogii\u003c/em\u003e and \u003cem\u003eC. calcitrans\u003c/em\u003e biomass at inclusion levels of 10 g kg⁻\u0026sup1; (Mix-Low) and 30 g kg⁻\u0026sup1; (Mix-High), respectively. All diets were prepared according to the pellet manufacturing method described by Achupallas et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Briefly, dry ingredients were thoroughly mixed, followed by the addition of oil and water to form a dough. The dough was passed through a meat mincer with a 2 mm die, and the resulting pellets were steam-conditioned at 90\u0026deg;C for 10 min, air-dried at 40\u0026deg;C, sealed in vacuum bags, and stored at -20\u0026deg;C until use. The proximate composition of the diets was confirmed using standard AOAC (2005) methods. Diet stability was assessed following the method of Obaldo et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), with leaching rates below 6% for all diets after 2 h of immersion.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFormulation and proximate composition of the experimental diets\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredient (g kg⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMix-Low\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMix-High\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFishmeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e235\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat flour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFish oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoy lecithin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin/Mineral premix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMixed Microalgae\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellulose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eProximate Analysis (% dry weight)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude Protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude Lipid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoisture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Shrimp Feeding Trial\u003c/h2\u003e \u003cp\u003eA 60-day feeding trial was conducted with juvenile \u003cem\u003ePenaeus vannamei\u003c/em\u003e. Shrimp ([specify source, e.g., a commercial hatchery]) with an initial average weight of 3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g were acclimatized to laboratory conditions for one week. After acclimatization, 16 shrimp were randomly stocked into each of nine 286 L circular fiberglass tanks (0.6 m\u0026sup2; base area) containing 115 L of filtered seawater, with three replicate tanks per dietary treatment.\u003c/p\u003e \u003cp\u003eThe tanks were part of a recirculating aquaculture system maintained at a salinity of 35 ppt, temperature of 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, pH of 7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, and dissolved oxygen\u0026thinsp;\u0026gt;\u0026thinsp;5.5 mg L⁻\u0026sup1;. Total ammonia and nitrite levels were monitored daily and maintained below 0.1 mg L⁻\u0026sup1;. Shrimp were fed their respective diets at a rate of 5% of their body weight, divided into two feedings per day. The amount of feed was adjusted bi-weekly based on group biomass sampling. Tanks were siphoned daily before the morning feeding to remove waste, uneaten feed, and exuviae.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Growth Performance and Proximate Analysis\u003c/h2\u003e \u003cp\u003eAt the end of the trial, all shrimp were weighed to determine growth performance using the following indices:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eWeight Gain (WG, g) =\u003c/b\u003e Final weight (W₂) - Initial weight (W₁)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003ePercent Weight Gain (PWG, %) =\u003c/b\u003e [(W₂ - W₁) / W₁] \u0026times; 100\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eSpecific Growth Rate (SGR, % day⁻\u0026sup1;) =\u003c/b\u003e [(ln W₂ - ln W₁) / time (days)] \u0026times; 100\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eFeed Conversion Ratio (FCR)\u0026thinsp;=\u003c/b\u003e\u0026thinsp;Dry feed fed (g) / Wet weight gain (g)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eFeed Efficiency (FE, %) =\u003c/b\u003e [Weight gain (g) / Dry feed fed (g)] \u0026times; 100\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eFor whole-body proximate composition, five shrimp from each tank were collected, sacrificed on ice, and stored at -20\u0026deg;C. The pooled samples were analyzed for moisture, crude protein, crude lipid, and ash content according to AOAC (2005) methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Vibrio parahaemolyticus Challenge Bioassay\u003c/h2\u003e \u003cp\u003eUpon completion of the feeding trial, 24 shrimp from each dietary group (8 shrimp per tank, in triplicate) were transferred to 20 L glass aquaria. A negative control group (uninfected) and a positive control group (infected, fed the control diet) were also included. After 24 h of acclimation, shrimp were challenged by immersion in seawater containing \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e (AHPND strain) at a final concentration of 2.24 \u0026times; 10⁶ CFU mL⁻\u0026sup1;, as confirmed by plating on Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) agar (Joshi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The challenge source was prepared from a confirmed isolate recovered from a diseased shrimp. Mortality was recorded every 12 h for 144 h (6 days), and dead individuals were removed immediately.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Histological Analysis\u003c/h2\u003e \u003cp\u003eHepatopancreas tissues from surviving shrimp at the end of the challenge test and from moribund shrimp in the positive control were dissected and fixed in Davidson's fixative for 24 h. Tissues were then processed routinely for histology, embedded in paraffin, sectioned at 5 \u0026micro;m, and stained with hematoxylin and eosin (H\u0026amp;E) (Bell and Lightner \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). Sections were examined under a light microscope for pathological changes characteristic of AHPND.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Growth performance, biochemical composition, and body composition data were analyzed by one-way analysis of variance (ANOVA) using SPSS software (version 26.0, IBM Corp.). When significant differences were detected (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Tukey's Honestly Significant Difference (HSD) post-hoc test was applied to compare means among treatment groups. Survival rates after the bacterial challenge were compared using the Kaplan-Meier method with a Log-rank test. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Biochemical Composition of the Mixed Microalgae Biomass and Experimental Diets\u003c/h2\u003e \u003cp\u003eThe mixed biomass of \u003cem\u003eThalassiosira weissflogii\u003c/em\u003e and \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e used for diet formulation was characterized by a high nutritional value, rich in proteins, lipids, and pigments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The formulation and confirmed proximate composition of the experimental diets are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The inclusion of the mixed microalgae biomass at 1% and 3% levels successfully incorporated these nutrients into the feed. Diet stability, as measured by leaching rate, was below 6% for all diets and did not differ significantly among treatments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBiochemical composition of the mixed microalgae biomass (\u003cem\u003eThalassiosira weissflogii\u003c/em\u003e and \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter (%, dry weight unless stated)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude Protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e49.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Carbohydrates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Lipids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChlorophyll-a (\u0026micro;g mg⁻\u0026sup1;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal Carotenoids (\u0026micro;g mg⁻\u0026sup1;)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFormulation and proximate composition (% dry weight) of the experimental diets\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet Component\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMix-Low (1%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMix-High (3%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation (g kg⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFishmeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e235\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat flour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFish oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoy lecithin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin/Mineral premix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMixed Microalgae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellulose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eProximate Analysis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude Protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude Lipid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaching Rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe fatty acid profile of the mixed microalgae lipid extract was particularly rich in polyunsaturated fatty acids (PUFAs), which constituted 47.5% of the total fatty acids (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notable levels of linoleic acid (18:2ω6, 12%) and γ-linolenic acid (18:3ω6, 28.1%) were detected.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFatty acid profile (% of total fatty acids) of the mixed microalgae biomass\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatty Acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFatty Acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSFA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePUFA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18:2ω6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18:3ω6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMUFA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e16.4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18:3ω3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20:5ω3 (EPA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22:6ω3 (DHA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Shrimp Growth Performance\u003c/h2\u003e \u003cp\u003eShrimp fed the mixed microalgae diets showed significantly improved growth compared to the control after the 60-day feeding trial (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Specifically, the Mix-High (3%) diet resulted in the highest specific growth rate (SGR) and percent weight gain (PWG), which were significantly greater than those of the control group. The feed conversion ratio (FCR) was also significantly lower in the microalgae-fed groups, with the best FCR observed in the Mix-High treatment. The significantly improved growth and feed utilization in microalgae-fed groups are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth performance of \u003cem\u003ePenaeus vannamei\u003c/em\u003e fed the experimental diets for 60 days\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIW (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFW (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWG (g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.36\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMix-Low (1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.97ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMix-High (3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.15ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eValues are mean of three replicates. Different superscript letters in the same column denote significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). IW: Initial Weight; FW: Final Weight; WG: Weight Gain; PWG: Percent Weight Gain; SGR: Specific Growth Rate; FCR: Feed Conversion Ratio; FE: Feed Efficiency.\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Pigment Content in Feces\u003c/h2\u003e \u003cp\u003eThe pigment content in the experimental diets and shrimp feces was assessed to evaluate pigment utilization (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). While the control diet and corresponding feces contained no detectable pigments, feces from shrimp fed the microalgae diets contained measurable chlorophyll, with levels increasing in line with the dietary inclusion level.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePigment content (mg g⁻\u0026sup1;) in experimental diets and shrimp feces\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiet Chlorophyll\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiet Carotenoids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFeces Chlorophyll\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFeces Carotenoids\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMix-Low (1%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMix-High (3%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eKey\u003c/b\u003e: \u003cem\u003eND: Not Detected\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Shrimp Body Composition\u003c/h2\u003e \u003cp\u003eThe dietary inclusion of mixed microalgae significantly increased the muscle protein content of the shrimp in a dose-dependent manner (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the moisture, lipid, and ash content of the shrimp muscle were not significantly affected by the dietary treatments (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProximate composition (% wet weight) of \u003cem\u003ePenaeus vannamei\u003c/em\u003e muscle after the feeding trial\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLipid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMoisture\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e74.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMix-Low (1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMix-High (3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026ccedil;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eKey\u003c/strong\u003e \u003cp\u003e \u003cem\u003eDifferent superscript letters in the same column denote significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e \u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Disease Challenge and Histopathology\u003c/h2\u003e \u003cp\u003eFollowing the \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e challenge, a significant difference in survival was observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Shrimp fed the control diet began experiencing mortality 24 hours post-infection (HPI), reaching 100% mortality by the end of the 144-hour bioassay. In stark contrast, shrimp fed both the Mix-Low and Mix-High diets exhibited a 100% survival rate, which was not significantly different from the unchallenged negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistopathological examination of the hepatopancreas confirmed these results (Fig.\u0026nbsp;4 and Fig.\u0026nbsp;5). Shrimp from the microalgae-fed groups that survived the challenge showed normal, healthy hepatopancreatic tubule structure (Fig.\u0026nbsp;4a, b). Conversely, moribund shrimp from the control group displayed classic lesions of Acute Hepatopancreatic Necrosis Disease (AHPND), including massive sloughing of hepatopancreatic tubule epithelial cells and the presence of hemocytic nodules (Fig.\u0026nbsp;4c, d).\u003c/p\u003e "},{"header":"4. Discussion","content":"\u003cp\u003eOur study provides compelling evidence that a mixed biomass of the diatoms \u003cem\u003eThalassiosira weissflogii\u003c/em\u003e and \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e serves as a high-value, multi-functional feed additive for \u003cem\u003ePenaeus vannamei\u003c/em\u003e. The 1\u0026ndash;3% dietary inclusion not only elicited significant enhancements in zootechnical performance and product quality but, most strikingly, conferred complete protection against a lethal AHPND challenge. This synergistic effect underscores the potential of tailored microalgal consortia to address multiple constraints in sustainable shrimp aquaculture simultaneously.\u003c/p\u003e \u003cp\u003eThe superior growth performance and feed efficiency observed in shrimp fed the mixed microalgae diets can be directly attributed to the optimized nutritional matrix provided by the diatom blend. The biomass was rich in high-quality protein (49.5%) and featured a lipid profile dominated by PUFAs (47.5%), notably a high concentration of γ-linolenic acid (GLA; 18:3ω6, 28.1%). Beyond its role as an energy source, GLA is a pivotal precursor in the eicosanoid biosynthesis pathway, giving rise to prostaglandins and leukotrienes that are crucial regulators of growth, metabolism, and immune homeostasis in crustaceans (Bell et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Mustonen et al., 2023). The significantly improved FCR suggests that this unique nutrient profile, likely complemented by a balanced amino acid spectrum and digestive co-factors, was more efficiently partitioned towards somatic growth and anabolism rather than catabolism. This represents a key economic advantage, reducing both feed costs and environmental waste loading.\u003c/p\u003e \u003cp\u003eBeyond quantitative growth, the microalgae supplementation qualitatively enhanced the shrimp as a final product. The significant, dose-dependent increase in muscle protein content indicates a pronounced \"protein-sparing effect.\" We posit that the readily available lipids and carbohydrates from the algal biomass served as primary energy substrates, thereby sparing dietary amino acids from deamination for gluconeogenesis and redirecting them towards muscle protein synthesis (Gonz\u0026aacute;lez-Meza et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This improvement in the nutritional value of the shrimp fillet is a critical finding, as it directly enhances the marketability and consumer appeal of the final product, adding a premium quality dimension to the functional feed's benefits.\u003c/p\u003e \u003cp\u003eThe most profound outcome of this study was the complete resilience of microalgae-fed shrimp to a virulent \u003cem\u003eV. parahaemolyticus\u003c/em\u003e challenge, contrasting with the 100% mortality in the control group. This was not merely a delay in mortality but a total abolition of the disease phenotype, as unequivocally confirmed by histopathology. The hepatopancreas of protected shrimp was devoid of the pathognomonic lesions of AHPND sloughing of tubule epithelial cells and hemocytic nodules presenting a structure indistinguishable from that of unchallenged, healthy animals. We propose that this robust protection is mediated by a synergistic immunostimulation from the suite of bioactive compounds in the diatom mix.\u003c/p\u003e \u003cp\u003eThe protective mechanism is likely multi-faceted. First, the abundant carotenoids (3.1 \u0026micro;g mg⁻\u0026sup1;) act as potent antioxidants, scavenging reactive oxygen species generated during an immune response and protecting cellular integrity from pathogen-induced oxidative stress (Bufka et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Second, the high PUFA content, particularly GLA, can be metabolized into eicosanoids and other lipid mediators that modulate and potentiate the innate immune response, potentially enhancing hemocyte phagocytosis, encapsulation capacity, and the production of antimicrobial peptides (Monteiro et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). While some microalgae possess direct antimicrobial properties (D'Alvise et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), the immersion challenge model used here, with a high bacterial load that rapidly overwhelmed control shrimp, strongly suggests that the primary mode of action was not a direct biocidal effect but a host-mediated immunostimulation. The microalgae additive appears to have \"primed\" the shrimp's immune system, enabling a rapid and effective defensive response that prevented bacterial colonization and toxin-mediated damage to the hepatopancreas.\u003c/p\u003e \u003cp\u003eIt is important to acknowledge a limitation of this study. While our data strongly point to immunostimulation, the precise molecular mechanisms such as the upregulation of key immune genes (e.g., those encoding antimicrobial peptides, prophenoloxidase, or antioxidant enzymes) remain to be elucidated. Future research employing transcriptomic and proteomic approaches is warranted to delineate the specific signaling pathways activated by this mixed diatom supplement. Such mechanistic insights would not only solidify our understanding but also allow for the fine-tuning of inclusion levels and combinations for maximal efficacy.\u003c/p\u003e \u003cp\u003eIn conclusion, the strategic inclusion of a 1:1 mixed biomass of \u003cem\u003eT. weissflogii\u003c/em\u003e and \u003cem\u003eC. calcitrans\u003c/em\u003e at 1\u0026ndash;3% in the diet of \u003cem\u003eP. vannamei\u003c/em\u003e delivers a powerful trifecta of benefits: enhanced growth and feed efficiency, improved product quality, and unprecedented protection against AHPND. This synergistic effect validates our hypothesis that combining the high-protein, pigment-rich profile of \u003cem\u003eT. weissflogii\u003c/em\u003e with the lipid and PUFA-rich profile of \u003cem\u003eC. calcitrans\u003c/em\u003e creates a functionally superior ingredient. This natural, sustainable, and commercially viable strategy represents a significant advancement in functional feed formulation, offering a robust solution to improve both the productivity and biosecurity of Pacific white shrimp aquaculture.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study provides compelling evidence that the inclusion of a mixed biomass of \u003cem\u003eThalassiosira weissflogii\u003c/em\u003e and \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e at 1\u0026ndash;3% in the diet of \u003cem\u003ePenaeus vannamei\u003c/em\u003e serves as a highly effective multi-functional feed additive. The supplementation not only significantly improves growth performance and feed utilization efficiency but also enhances the nutritional quality of the shrimp by increasing muscle protein content. Most notably, the additive conferred complete protection against a lethal challenge with \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, the causative agent of AHPND, as confirmed by 100% survival and the absence of characteristic histopathological lesions. This profound disease resistance is likely attributable to the synergistic action of bioactive compounds in the diatom mix, including immunostimulatory carotenoids and polyunsaturated fatty acids like γ-linolenic acid, which bolster the shrimp's innate immune system. Therefore, this natural microalgae-based strategy presents a viable, sustainable, and profitable solution to enhance both the productivity and resilience of Pacific white shrimp aquaculture against major disease threats.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.Gangaiyammal: Writing\u0026ndash;original draft, Supervision, Methodology, Investigation, Data curation. K. Anandhi: Investigation, Methodology. M. Geetha: Methodology, Funding acquisition. G. Brindha: Methodology, Funding acquisition. Mukul Machhindra Barwant: Methodology, Funding acquisition, Conceptualization. Usman Mohammed Ali: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Supervision, Project administration, Funding acquisition, Conceptualization.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdelhay RA, El-Mor MS, Salem MAM, Al-Sagheer AA, Abd-Elhakim YM, Hassan BA, Mounes HAM (2025) Effect of nitrogen sources on diatoms growth and nutritional value for enhancing Litopenaeus vannamei larval performance. 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Anim Feed Sci Technol 320:116205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anifeedsci.2024.116205\u003c/span\u003e\u003cspan address=\"10.1016/j.anifeedsci.2024.116205\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Diatoms, Functional Feed, Immunostimulation, Microalgae, Pacific white shrimp","lastPublishedDoi":"10.21203/rs.3.rs-8909060/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8909060/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Pacific white shrimp (\u003cem\u003ePenaeus vannamei\u003c/em\u003e) industry faces significant challenges from diseases like Acute Hepatopancreatic Necrosis Disease (AHPND), driving the need for sustainable functional feed additives. This study investigated the effects of a mixed biomass of diatoms, \u003cem\u003eThalassiosira weissflogii\u003c/em\u003e and \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e, as a dietary supplement for juvenile \u003cem\u003eP. vannamei\u003c/em\u003e. Three isonitrogenous and isolipidic diets were formulated: a control diet with no microalgae, and two test diets containing the mixed biomass at 1% (Mix-Low) and 3% (Mix-High) inclusion levels. After a 60-day feeding trial, shrimp fed the microalgae diets showed significantly enhanced growth performance, with the Mix-High group achieving the highest specific growth rate (3.15 ± 0.12% day⁻¹), percent weight gain (132.5 ± 8.7%), and the lowest feed conversion ratio (1.42 ± 0.05). Furthermore, dietary supplementation significantly increased muscle protein content (21.7 ± 0.3% vs. 19.4 ± 0.1% in control). Upon challenge with \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e (AHPND strain), shrimp fed the control diet suffered 100% mortality within 144 h, while shrimp fed both microalgae diets exhibited 100% survival, corroborated by histopathology showing no AHPND lesions. The results demonstrate that 1–3% inclusion of mixed diatom biomass acts as a potent functional feed additive, simultaneously enhancing growth, muscle quality, and disease resistance, offering a sustainable strategy for shrimp aquaculture.\u003c/p\u003e","manuscriptTitle":"Effects of a mixed biomass of Thalassiosira weissflogii and Chaetoceros calcitrans as a functional feed additive for Pacific white shrimp (Penaeus vannamei)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 07:06:58","doi":"10.21203/rs.3.rs-8909060/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-20T19:49:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142215376151190014800503944822408523724","date":"2026-03-10T20:04:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82761338170842055613120689187835452155","date":"2026-03-10T16:09:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-09T05:28:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288286486210996702217255906489012500439","date":"2026-03-09T01:51:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-05T14:53:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-04T06:16:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-04T06:15:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biotechnology","date":"2026-02-18T11:39:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5da51d72-4942-4043-8ecb-da14a7dafbb2","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-11T07:06:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 07:06:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8909060","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8909060","identity":"rs-8909060","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}