* By Daniel Aguilera-Pesantes, Doménica Vargas-Arias y Patricio Bucheli M.
In Ecuador’s aquaculture, intensification has emerged as a crucial strategy to meet the growing demand for shrimp. However significant challenges, mainly in terms of health risks. In this context, the presence of pathogenic bacteria responsible for production losses has been identified. Notably, Vibrio parahaemolyticus associated with Acute Hepatopancreatic Necrosis (AHPND), stands out, prominently. This study evaluates the effectiveness of bacteriophages as both preventive and curative tool in farms with recirculation and open exchange water flow, to reduce the presence of V. parahaemolyticus and its effects on shrimp health and production.
Introduction
The intensification of aquaculture production in open systems brings environmental impacts that require thorough evaluation and effective mitigation strategies. Any intensification initiative must incorporate measures to minimize the impacts and ensure a long-term sustainability. In this scheme, eutrophication is the most common, and probably the most underestimated, frequently leading to the proliferation of toxins or a significant rise in in bioavailable substrates that can foster the growth of opportunistic pathogenic bacterial species.
Currently, the most opportunistic bacterial group of pathogens in shrimp cultures is Vibrio sp. Among the most important species associated with significant losses in larvae, juveniles and adults are: V. parahaemolyticus, V. vulnificus, V. harveyii, V. anguillarum, V. splendidus, V. alginolyticus (Chatterjee & Haldar, 2012).
Vibrio species are opportunistic, they can benefit from nursery and grow-out ponds conditions and become aggressive hosts from the beginning of a cycle (Xiao et al., 2017). They have the potential to cause damage at any stage of development. Currently, acute hepatopancreatic necrosis disease (AHPND) is notorious (Saavedra-Olivos et al., 2018), resulting in mass mortalities of shrimp in China, Vietnam, Thailand, and the Philippines. In Mexico, Soto-Rodríguez and others (2015) identified the V. parahaemolyticus as the primary causal agent of AHPND.
Recently, bacteriophage therapies have been introduced in aquaculture as an alternative for the prevention and treatment of bacterial infections. Due to the increasing relevance that AHPND has acquired in recent years, protocols for the use of bacteriophages have been developed (Han, Tang & Corbin, 2018). Bacteriophages are viruses that infect and replicate in bacteria. They are characterized by their ability to be specific, self-replicating and self-limiting (Kasman, 2022). The lytic effect of Phages on bacterial populations is overwhelming, eliminating them fast and efficiently.
In Ecuador, significant efforts have been carried out focused on defining effective and safe conditions for the application of bacteriophages in aquaculture. This approach considers microbiological factors related to the target species, including the type and concentration of bacterial strains, the health status of the cultured organisms and the prevalence of bacteria within the ponds. Previous studies have highlighted the need to employ a combination of bacteriophages to improve treatment success rates for V. parahaemolyticus (Gonzales-Gomez et al., 2023). Aguilera et al. (2023) have demonstrated the effectiveness of using a combination of 18 specific bacteriophages for controlling V. parahaemolyticus in commercial aquaculture operations in Ecuador.
Methodology
Isolation and identification of Vibrio sp and lytic bacteriophages
Based on the study by Aguilera et al., 2023, Vibrio sp and lytic bacteriophages were sourced through bioprospecting from ecosystems related to shrimp production in Ecuador. The process involved isolating bacteria on Vibrio-specific culture media, followed by species level identification by biochemical tests and confirmation though16S sequencing using Oxford Nanopore technology. The activity of bacteriophages was evaluated according to the methodology described by Yang et al. (2020), a total of 18 bacteriophages, specific for V. parahaemolyticus, were selected with the aim of developing a cocktail composed of different strains, capable of covering a wide range of hosts.
Preventive field trials
A total of 11 nursery ponds were selected from recirculating farms situated in the Gulf of Guayaquil. These included both mainland and island-based open water exchange farms, with 6 units designated as the treatment group and 5 as the control group. In addition, 22 grow-out ponds were selected in various locations, including mainland recirculation farms and island-based open water exchange farms, with 15 ponds assigned to the treatment group and 7 to the control group.
In the treatment ponds, the bacteriophage cocktail was administered in the food at a concentration of 9 mL/kg. This application was carried out throughout the entire nursery ponds cycle, with an average duration of approximately 20 days. While for the grow-out ponds treatment was carried out for the first15 days after stocking. Twice a week, random samples of juveniles were taken for maceration (pre-juveniles) and HP maceration for juveniles. Sowing was carried out by the plate extension method in the TCBS and CAV culture medium. Additionally, the presence of Vibrio in the water was evaluated and the results were expressed in Colony Forming Units per Gram (CFU/g).
Curative field trials
Curative tests were carried out on farms where Vibriosis events were identified, as indicated by the presence of clinical signs (with an incidence greater than 2% in the population) and high levels of V. parahaemolyticus (greater than 1×104 CFU/g) evaluated by microbiological analysis of hepatopancreas samples on TCBS and CAV media. A total of 12 grow-out ponds were selected from farms located in the Taura and Yaguachi area in the province of Guayas. In the treatment ponds, the bacteriophage cocktail was administered in the feed at a dose of 9 mL/kg for 7 consecutive days in ponds where the concentration of V. parahaemolyticus exceeded 1×104 CFU/g. For ponds exhibiting concentrations greater than 1×10⁵ CFU/g, the dosage was administered for 14 consecutive days.
Results and Discussion

The initial contamination levels of Vibrio sp in shrimp farms under analysis were significant high, particularly in the farm employing water recirculation methods in the Gulf of Guayaquil. The practice of water reuse results in accumulation of organic matter and nutrients and can provide an ideal environment for bacterial growth.
According to the results in the field with prophylactic treatment, a significant reduction in the concentration of V. parahaemolyticus was evident consistently in the juveniles of all the farms.


Production parameters indicate positive effects in treated units. (Table 1). The impact was notable especially in the farm with recirculation in the Gulf of Guayaquil.

Results in prophylactic treatment in grow-out ponds
In the grow-out ponds farm with recirculation in the Gulf of Guayaquil, an evident average increase in the size of shrimp was observed. By week 8, the average weight in treatment ponds was 20 grams, while in the controls it was approximately 17 grams (Figure 4).

Results of bacteriophages therapeutic use protocol
Analysis of treated ponds revealed that the applied treatment contributed to improving shrimp health and survival, with a significant decrease in the number of sick shrimp from the fourth day of treatment (Figure 5). These findings agree with what was shown by Schulz et al. (2022) and (Lomelí-Ortega & Martínez- Díaz, 2014), who suggest that the sequential application of bacteriophages allows for effective control of the concentration of V. parahaemolyticus.

Results observed in field, coincide with the findings obtained through microbiological analyses. During periods of bacteriophage administration that varied between 7 and 14 days, a reduction of up to 1,000 times in V. parahaemolyticus concentration was recorded (Figure 6, 7 and 8).



Constant monitoring and precise compliance with the application dosage were essential factors to obtain satisfactory results. The findings not only suggest immediate effects in the prevention and treatment of vibriosis, but also point out its potential use in diseases caused by other types of bacteria, such as V. vulnificus, V. cholerae, V. harveyi, and other bacteria, such as Pseudomonas and Aeromonas, associated with shrimp losses, and which are currently part of ongoing field research.
Conclusions
- A significant reduction in the concentration of V. parahaemolyticus was evident consistently in Nursery and in grow-out ponds, both in prophylactic and curative treatments
- The positive impact of Vibrio sp control was evident in notable improvements in production parameters after the shrimp transfer from nursery to grow-out ponds. Improvements were observed across all evaluated areas, demonstrating the effectiveness of a phage therapy against bacterial diseases associated with Vibrio species.
- The decontamination effect on the environment can become more significant, especially in farms with recirculation, if bacteriophages are used in more ponds simultaneously.
Bibliographic references and supporting information on analytical methodologies, upon request to the author.
Doménica Vargas-Arias*, Daniel Aguilera-Pesantes*, Patricio Bucheli M.
* Research and Development Department, Applied Blue Biotechnology APB-BIO C.A., Km 4.5 Vía a Taura, Ecuador. info@apb-bio.com