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Penaeus vannamei is an important shrimp species that is farmed globally, owing to its fast growth and good environmental adaptability. However, vibriosis outbreaks, such as acute hepatopancreatic necrosis disease and translucent post-larva disease, have become a considerable constraint on the development of the shrimp aquaculture industry globally in recent years. Due to the negative consequences associated with the use of antibiotics to control bacterial diseases, alternative technologies, such as improving shrimp immunity, the nutrition level of shrimp diets, and the ecological environment of shrimp ponds by using probiotics, have been developed. Owing to the limitations related to the sourcing and cost of fish meal, a large amount of vegetable protein is often used as a fish meal substitute in shrimp artificial feed, which is not only difficult for the shrimp to digest and absorb but can also lead to nutritional antagonism. In order to improve the health status of aquaculture animals by providing high-quality feed, the development and optimization of fermented feed have received extensive attention. Probiotics in fermented feed can produce exogenous cellulase, protease, amylase, and other digestive enzymes in the digestive tract of farmed animals, which can improve the feed utilization rate, promote growth performance, and enhance the immunity of shrimp.In this study, in order to explore the application effects of shrimp feed fermented by different probiotic bacteria, probiotic strains, including Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus acidophilus, were used individually and in combination for the fermentation of commercial feed and fed to shrimp (P. vannamei) for 28 days. The survival, body length growth, specific growth, and feed conversion rates of the shrimp were analyzed, and the density of Vibrio in vitro and in vivo, as well as relevant immune indicators in the serum, were analyzed. Furthermore, ammonia nitrogen and nitrite nitrogen concentrations in the water column were compared between the groups.The results demonstrated that the survival rates of shrimp in the fermented feed groups of B. subtilis, L. acidophilus, and complex bacteria were 8.54%, 8.54%, and 9.76%, respectively, which were significantly higher than that of the control group (P < 0.05). The body length growth rates in the B. subtilis and L. acidophilus groups were significantly higher than that of the control group (P < 0.05), while the feed conversion rate in the L. acidophilus group was significantly decreased compared with that of the control group (P < 0.05). There was no significant difference in the specific growth rate of shrimp between the experimental groups and the control group (P > 0.05). Fermented feed can affect the composition of bacteria in the hepatopancreas of shrimp. The density of Vibrio in the hepatopancreas of shrimp in each experimental group was significantly lower than that in the control group on days 14 and 21 of the experiment (P < 0.05). The feeding of different fermented feed in the short term (within 14 days) had no significant effect on reducing the total density of Vibrio in the water. However, feeding B. subtilis and L. acidophilus fermented feed significantly reduced the Vibrio density in the water body on day 21 (P < 0.05). In addition, the serum total protein concentration in each experimental group was significantly higher than that in the control group (P < 0.05). The activities of serum peroxidase, superoxide dismutase, and phenoloxidase in the L. acidophilus fermented feed group increased significantly compared with those of the other experimental and control groups (P < 0.05). The activities of serum peroxidase and phenoloxidase in the serum of shrimp fed S. cerevisiae fermented feed were significantly higher than those of the control group (P < 0.05). Complex bacteria-fermented feed could significantly increase the activities of serum lysozyme, peroxidase, and phenoloxidase (P < 0.05). However, the activity of the detected immune-related enzymes in the B. subtilis group did not increase significantly compared with that of the control group (P > 0.05), except for the activity of total serum protein, which was higher than that of the control group. The results also revealed that the concentration of ammonia nitrogen and nitrite nitrogen in the water group of each experimental group at the end of the experiment was significantly lower compared to that of the control group. S. cerevisiae and L. acidophilus groups had the best effect in reducing the concentration of ammonia nitrogen and nitrite nitrogen in the water, respectively.In summary, shrimp feed fermented by B. subtilis, S. cerevisiae, and L. acidophilus singly or in combination could improve the survival rate, growth performance, and immunity of shrimp to a degree, and reduce the concentration of ammonia nitrogen and nitrite nitrogen in aquaculture water. Moreover, L. acidophilus shows great potential as a candidate probiotic for shrimp feed fermentation. The results of this study provide a theoretical basis for the development of shrimp fermentation feed, which can improve the feed utilization and survival rates of shrimp in aquaculture.

Flinders technology associates (FTA) card (Whatman®) is a paper-based matrix designed to fix, purify, and store genetic material from various biological sources. It can conveniently and quickly preserve nucleic acids and may fulfil the requirements of long-distance and cross-border sample transportation. The FTA card can store and transport tissue, nucleic acid, and other sample types at room temperature (20–25 ℃). Nucleic acid can be extracted directly for detection and be sent by express as an ordinary parcel without being treated as dangerous or as special goods, eliminating tedious processes, saving time, and ensuring sample quality. It is widely used in the human and animal medicine field. It has been successfully used for the storage and transportation of livestock pathogens and viral nucleic acids. In terms of aquatic animals, the FTA card has been used by researchers to store white spot syndrome virus (WSSV) and the shrimp Enterocytozoon hepatopenaei (EHP): However, there is no relevant research report on the elution effect of the nucleic acid stored in the FTA card, which affects the application of the FTA card. The infectious hypodermal and haematopoietic necrosis virus (IHHNV) is an important shrimp pathogen and severely impacts the shrimp culture industry. It was first found in Hawaii, United States, in 1981 and then spread to several countries, including to Australia, Singapore, Malaysia, South Korea, Brazil, and China. The IHHNV infecting Penaeus vannamei does not cause high mortality, but growth would become slow and deformed, resulting in great economic losses. Early detection and prevention management are particularly important in current situations, which lack effective control measures for the disease. Several IHHNV detection methods have been established that use molecular biological methods, including conventional polymerase chain reaction (PCR) and real-time PCR (these are recommended in the aquatic animal disease diagnosis Manual of the World Organization for Animal Health). Nucleic acid extraction by the above methods meets the requirements for samples, usually frozen, ethanol, or other nucleic acid preservation reagents. Low temperature preservation conditions and composition restrictions of preservation solutions present certain difficulties in disease investigation, surveillance, and monitoring of shrimp farming. It is particularly important to address this dilemma. To find a fast method for the preservation and separation of IHHNV DNA and provide complete nucleic acid materials for subsequent research, we selected FTA cards as the preservation medium, and designed seven kinds of FTA cards with attached DNA elution methods based on the FTA purification reagent, TE buffer, and deionized water. We evaluated the elution and separation effects of different nucleic acids and the minimum amount of dot FTA card nucleic acids through real-time PCR detection. The appropriate solution was spotted onto FTA cards according to the manufacturer′s protocol, labeled, and air-dried for 1 day at room temperature. The result shows that on the 4 mm2 FTA card, the sample volume was 2.5 μL. When the eluent is used as the template, the minimum FTA card nucleic acid concentration needs to be 1.47×104 copies/μL above the best detection sensitivity, and 100% detection rate can be obtained by washing the FTA card with 50 μL TE buffer solution at 95 ℃ for 5 min. Using the FTA card as the template, the nucleic acid concentration of the dot FTA card needs to be above 1.82×103 copies/μL, eluted with FTA purified reagent thrice at room temperature, and then eluted with TE buffer twice. Each elution time is 5 min as this can obtain the best detection sensitivity and demonstrates 100% accuracy. The elution effect of the above two schemes was better than that of the other five schemes. The nucleic acids of WSSV, EHP, decapod iridescent virus 1, covert mobility Noda virus, and Vibrio parahaemolyticus causing acute hepatopancreatic necrosis were preserved using FTA card to test the efficiency of the established elution method. It is assumed that this method is universal for the elution of other shrimp pathogenic nucleic acids. At present, research on the application of FTA card is mostly seen in the nucleic acid effect of its preservation and transportation of tissue samples. There are few reports on the relationship between the amount of preserved nucleic acid, separation methods, and detection effect. This study shows that FTA cards used to preserve pathogenic nucleic acid requires a specific amount of nucleic acid in the sample and directly affects the detection results of the sample with different FTA card elution methods. This study provides a feasible scheme for the preservation and elution of IHHNV DNA with FTA cards. The application of this technology has potential use as storage and transport strategy for surveillance programs and can enhance biosecurity in shrimp culture, which provides a scientific basis for the preservation and transportation conditions for the collection of wild shrimp samples and the regional transmission of viral nucleic acid samples.