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The present study aimed to evaluate the effects of high carbohydrate diet in the absence of presence of dietary benfotiamine on growth and immune response against ammonia toxicity (50 mg NH4Cl L-1, average ambient temperature=27oC, pH 8.3) of post larval Penaeus monodon. A feeding trial involving three dietary treatments followed by an ammonia toxicity tests and a feed attractability test were conducted separately. During the feeding trial, shrimps were fed three experimental diets, namely, (1) a diet containing 15% starch (C), (2) a diet with 25% corn starch (high carbohydrate diet, HC) and (3) a diet containing both 25% starch supplemented with 0.2 g kg-1 benfotiamine (HCB). At the termination of the feeding trial, the shrimps were subjected to ammonia toxicity test to determine the effects of the diets on the immune response of the post larval shrimps. Attractability tests using customized aquaria showed that the HCB diet elicited significantly the highest attractability even after 10 min of feed placement in the feeding chamber. In the feeding trial that lasted for 60 days, results showed that shrimps fed with diet containing HCB exhibited significantly the highest growth rate (p<0.05) among all groups of shrimps in terms of final average body weight (FABW), weight gain (WG), and specific growth rate (SGR); they also exhibited significantly the highest feed intake (p<0.05). Upon challenge of the shrimps with 50 mg L-1 ammonia, shrimps fed with the HCB diet exhibited significantly the highest cumulative survival rate of 60% after 72 h while those fed with diet C and HC exhibited lower survival rates of 33% and 40%, respectively, and were not significantly different from each other. In conclusion, benfotiamine enhances attractability of the feed, growth performance and immune response against ammonia toxicity at the level of 0.2 g kg-1. 2 Nudalo, Tumbokon & Serrano Introduction In aquaculture, carbohydrates are the most economical dietary energy source due to their abundance, ready availability and relatively low cost. However, as an energy source, it cannot be fully and efficiently used by aquatic animals compared with poultry and mammals (Wilson and Poe, 1987). High dietary carbohydrate can cause decreased growth rate and other negative effects (Shiau et al., 1991b). Crustaceans have a limited ability to utilize carbohydrate and cannot adapt to high levels of dietary carbohydrate intake (Wang et al., 2016). Consumption of simple sugars in crustaceans can cause negative physiological effect since glucose has a high absorption rate across the digestive tract leading to prolonged hyperglycemia (Shiau et al., 1991; Rosas et al., 2001). Regardless of the carbohydrate source, it is generally accepted that the carbohydrate requirement for crustaceans is 20–30% of the total diet, and increased carbohydrate intake can lead to slow growth, low immunity, and a high mortality rate (Wang et al., 2016). Shrimps are diabetic-like animals with high post prandial hemolymph levels of glucose even hours after consumption of a meal containing carbohydrates (Shiau et al., 1991; Rosas et al., 2001). This implies that shrimp needs an energy source, a special requirement linked to the build-up of the new exoskeleton after each molt, or a difficulty to handle the large load that the hemolymph glucose brings (Niu et al., 2012). Thiamine (vitamin B1) plays a key role in the process of converting carbohydrates into energy (Balakumar et al. 2010). Benfotiamine (pro-vitamin B1) is a lipid-soluble thiamine derivative having much higher bioavailability in human than thiamine (Xie et al., 2014). It is commonly used for the treatment of type II diabetes to improve glucose homeostasis by enhancing glycolysis (Portari et al., 2013) as well as removing excess glycolytic intermediates and reducing hyperglycemic damage (Hammes et al., 2013). In mammals, pro-vitamin B1 stimulates glucose uptake by enhancing the glycolytic and glycogenetic capabilities, increasing mitochondrial glucose oxidation, promoting insulin synthesis, enhancing insulin-stimulated glucose disposal, and increasing the activity of GLUT-2, a glucose transporter (Seh-Hoon et al. 2009; Chung et al. 2014; Fraser et al. 2012). In aquatic animals, information regarding the beneficial effects of benfotiamine is scarce. Studies on the effects of benfotiamine on the growth performance of shrimps have not been conducted so far. In fish such as the blunt snout bream Megalobrama amblycephala (Xu et al. 2017), it has been demonstrated that the fish fed a high carbohydrate diet supplemented with benfotiamine exhibited higher protein efficiency ratio, nitrogen efficiency ratio and energy retention efficiency. In addition, fish showed relatively high values of whole-body lipid contents, tissue glycogen and lipid contents. In our laboratory, the Nile tilapia fed high carbohydrates diet supplemented with benfotiamine showed significantly better growth than those fish fed high carbohydrate diet without benfotiamine (Lauzon et al. 2019). The present study aimed to evaluate the effects of high carbohydrate diet alone or supplemented with benfotiamine on the growth performance and immune response of the black tiger shrimp. In addition, we also aimed to determine whether or not it is also an immune-enhancing feed additive. Ammonia and ammonium ion are two of the toxic components in the water medium in aquaculture media since these are produced by the shrimps themselves as a metabolic waste; they are the end products of protein utilization of aquatic animals. The toxicities that result from deteriorating water quality, such as that from ammonia stress, have lethal effects on juvenile shrimp and can increase their susceptibility to pathogens. The toxicity of ammonia plays an important role in the frequent high mortality occurrence during the early stage on shrimp farms (Lu et al., 2017). It is common knowledge that shrimps possess only the innate immune system and not the adaptive immune system; thus, the response to environmental stress such as an elevated ammonia/ammonium ion, or whether to a pathogenic microorganism will elicit similar mechanism of immune response from the shrimp. Thus, an additional objective of determining whether or not dietary benfotiamine enhances the immune response of the black tiger shrimp post larvae was done by challenging it with an elevated ammonia/ammonium level. Effects of dietary benfotiamine on P. monodon 3 Materials and Methods This study was conducted at the University of the Philippines Visayas-National Institute of Molecular Biology and Biotechnology (UPV-NIMBB), Miagao, Iloilo, Philippines. Experimental Shrimps Penaeus monodon (PL20) were purchased from a private hatchery in the nearby town (Guimbal, Iloilo). Prior to the feeding experiment, P. monodon post larvae were acclimatized to the experimental conditions and control diet for a week. During the feeding trial, apparent satiation was measured within the first three days of the week and was used as the basis for the feeding rate for the rest of the week, and the same cycle was observed until termination. After acclimatization, 270 P. monodon post larvae of similar size (average weight: 0.01g) were randomly distributed among nine indoor plastic containers (40 L volume) at 30 shrimps per container. Experimental diet preparation Three experimental diets were prepared for the first feeding trial namely: (1) control diet containing 15% corn starch; (2) 25% corn starch (high carbohydrate diet, HC); and (3) 25% corn starch (i.e. high carbohydrate diet, HCB) with benfotiamine (0.2 g kg-1; Table 1). Dry ingredients were mixed first followed by the liquid components; the mixture was thoroughly blended manually with water until a soft dough was obtained. The mixture was steam-cooked and oven-dried at 600C to a moisture content of 10% or less. An appropriate amount was allocated for proximate analysis. The dry feeds were stored at 4OC until use; they were ground and sieved to appropriate size before feeding. Table 1 Composition of experimental diets to determine the effects of High Carbohydrates and High Carbohydrates supplemented with benfotiamine Feed composition in (g) Feed ingredients Control HC HCB Shrimp (acetes) meal Soybean meal Corn starch Cod liver oil Carboxymethyl cellulose (CMC) Lecithin – Soy (70%) Trace mineral premix Vitamin premix Peruvian Fishmeal BHT benfotiamine TOTAL 200.0 200.0 130.0 40.0 104.8 5.0 10.0 10.0

Benfotiamine counteracts the negative effects of a high dietary carbohydrate on growth and ammonia toxicity resistance in post larval Penaeus monodon