Tilapia lake virus resistance breakthrough announced

Certain tilapia families have been shown to be completely resistant to tilapia lake virus (TiLV), offering hope that breeding programmes can combat one of the worst diseases to hit the global fish farming industry in recent years.

The GIFT strain of Nile tilapia was developed by WorldFish

In a study published in the journal Aquaculture, researchers from University of Edinburgh’s Roslin Institute and WorldFish analysed the genes of 1,821 genetically improved farmed tilapia (GIFT), which were tagged and placed in a pond that had an outbreak of TiLV.

The fish used in this experiment were members of 124 families, and the team discovered that there was a large variation in family survival. Some family groups had no deaths, whereas others found to have a 100 percent death rate.

The team then used statistical models to show that resistance to the virus was very heritable, which means that selective breeding to produce more resistant tilapia strains is likely to be effective.

The variations in TiLV resistance were found to be independent of genetic variation in growth, meaning that any future breeding programmes for GIFT that produce fish resistant to TiLV will not adversely affect the growth of the fish, and will benefit farmers’ yields.

The GIFT strain has been selectively bred to be fast-growing and adaptable to a wide range of environments. The strain is produced in at least 14 countries, helping to reduce poverty and hunger.

Professor Ross Houston, lead author and personal chair of aquaculture genetics at the Roslin Institute, said: “Tilapia lake virus poses a real problem to fish farmers worldwide, impacting on the livelihoods and food security of millions of people. This research is the result of a long-term collaboration between Roslin and WorldFish, and is the first step to breeding tilapia strains with improved resistance to the virus.”

Dr Michael Phillips, director of aquaculture and fisheries sciences at WorldFish and the CGIAR Research Program on Fish Agri-Food Systems, said: “This is a truly exciting finding at the frontier of fish genetics. WorldFish will build on this research, with our partners in the research, donor and investment community, to accelerate the further development of resilient TiLV resistant tilapia strains and their wide accessibility to small scale fish farmers.”

Further information on TiLV

• Tilapia are the most farmed variety of fish after carps, and are worth nearly $10 billion to the global economy. They are also a vital protein source in Africa, Asia and South America

• Since its detection in 2014, tilapia lake virus (TiLV) has ravaged tilapia populations – causing mortalities of up to 90 percent – in 16 countries across three continents.

• Clinical signs of the virus observed in representative tilapia include behavioural changes, skin damage such as skin erosion, discoloration, skin haemorrhages and loss of scales, eyeball protrusion (exophthalmia) and abdominal swelling. There are currently no treatments or vaccines for TiLV.

• The full study can be accessed here:

Genetic parameters for resistance to Tilapia Lake Virus (TiLV) in Nile tilapia (Oreochromis niloticus)

Abstract

Tilapia Lake Virus (TiLV) is one of the primary disease concerns for tilapia farming, with mass mortality events and biosecurity restrictions threating aquaculture in several continents. Selective breeding for improved host resistance to TiLV may help to mitigate this problematic disease, but the extent of genetic variation in resistance is not yet known. The objective of the current study was to estimate genetic parameters for host resistance to TiLV in a Nile tilapia breeding population of the Genetically Improved Farmed Tilapia (GIFT) strain. Using data from 1821 pedigreed fish (from 124 full-sibling families) collected during and after a pond ‘field’ outbreak, resistance was defined using both binary survival (BS) and days to death (TD) traits. Animal and sire-dam linear mixed models were fitted for BS and TD, and BS was also evaluated with using two sire-dam threshold models with either probit (Pro-SD) or logit-link (Log-SD) functions. Cumulative mortality was 39.6% at the end of the outbreak, with family survival rates ranging from 0 to 100%. Moderate to high heritability values were estimated for resistance to TiLV using all models. Significant heritabilities were estimated on the binary scale (0.40 for both animal and sire-dam models) which equates to 0.63 on the underlying liability scale. Using threshold models, heritabilities of 0.56 and 0.48 were estimated for Pro-SD and Log-SD, respectively. Correlation among the full-sib families EBVs predicted by the different models ranged from 0.912 to 0.999, suggesting a low re-ranking of the families and a high consistency of the results obtained using the different models. In addition, significant and moderate heritability of 0.41 (0.06) was estimated for harvest weight (HW), and the genetic correlation between this trait and resistance to TILV was not statistically different from zero. These results demonstrate that host resistance to TiLV is highly heritable in a Nile tilapia breeding population with GIFT origin. Therefore, selective breeding to increase resistance and reduce mortalities due to TiLV is a feasible and promising approach.

1. Introduction

Nile tilapia (Oreochromis niloticus) is among the most important aquaculture species farmed worldwide. According to the Food and Agriculture Organization of the United States (FAO), the production of tilapia reached approximately 6.2 million tons during 2016, representing one of the major sources of animal protein for human consumption (FAO, 2018), particularly in developing countries in Asia, South America, and Africa (Shelton and Popma, 2006).

However, as with other intensive production systems, infectious disease is one of the main issues threatening the success and sustainability of tilapia production. A relatively new pathogen, the orthomyxovirus-like Tilapia Lake Virus (TiLV) has emerged as a major threat for Nile tilapia (Eyngor et al., 2014; Fathi et al., 2017; Mugimba et al., 2018; Pulido et al., 2019), and also for other farmed tilapias, including red tilapia (Oreochromis sp.) and hybrid strains (O. niloticus x O. aureus) (Eyngor et al., 2014; Surachetpong et al., 2017). Although the virus was discovered in 2014, it may have been responsible for mortalities since 2008–2009 (Bacharach et al., 2016; Eyngor et al., 2014). To date, it has been identified in countries from different geographical regions and continents, including Peru (Pulido et al., 2019), Ecuador (Bacharach et al., 2016), Malaysia (Amal et al., 2018), India (Behera et al., 2018), Thailand (Dong et al., 2017), Egypt (Fathi et al., 2017) and Uganda (Mugimba et al., 2018).

The agent is a novel single-stranded orthomyxo-like RNA enveloped virus, with a diameter ranging from 55 to 100 nm (Bacharach et al., 2016; Del-Pozo et al., 2017; Eyngor et al., 2014). The virus can cause disease in several stages of the tilapia life-cycle, from fingerlings to adults (Ferguson et al., 2014; Senapin et al., 2018), and in multiple organs such as spleen, heart and brain, and even in the reproductive organs, and can be transmitted vertically (Dong et al., 2020). However, some studies suggest a higher prevalence of the virus in kidney, gills and liver (Bacharach et al., 2016; Dong et al., 2017; Mugimba et al., 2018). The clinical signs may vary depending on the geographical origin, and include skin erosion and darkening, gill pallor, anemia and swollen abdomen (Dong et al., 2017; Ferguson et al., 2014), with subclinical infections also being reported (Senapin et al., 2018).

TiLV can cause high levels of mortalities, but these can vary substantially (ranging from 5 to 90%) in disease outbreaks, which are usually observed within the subsequent weeks post-transfer from hatcheries to growth out ponds (Dong et al., 2017; Fathi et al., 2017). After these outbreaks, it has been shown that surviving fish have a higher resistance to this infection to subsequent outbreaks suggesting some degree of resistance via acquired immunity (Eyngor et al., 2014). For a detailed review about TiLV diagnosis, mitigation and control measurements, please see Jansen et al. (2018).

Selective breeding for genetic improvement of tilapia is increasingly used to improve production traits. To date, tilapia breeding programs have included growth-related traits as an objective of selection, reaching genetic gains ranging between 10 and 15% per generation (Ponzoni et al., 2011), highlighting the feasibility of improving production traits by means of selective breeding (Gjedrem and Rye, 2018). In case of infectious diseases, selective breeding is a sustainable strategy to reduce the mortality rate, enhance disease resistance and increase welfare and productivity (Stear et al., 2001).

One approach to improve disease resistance is based on controlled challenge tests. This methodology allows for control of environmental variables and evaluation of one specific pathogen at a time. Generally, the trait of resistance is assessed by infecting the host by cohabitation, immersion or intraperitoneal injection, and ideally selecting a pathogen strain identical to the observed in the field (Houston, 2017). A second alternative, is to collect samples and data from outbreaks of disease in production environments (i.e. field outbreak). While the latter is potentially the most relevant source of data to quantify genetic resistance, is often difficult to obtain high quality samples and confirm the cause of death. However, both approaches have their own advantages and drawbacks, and both can be successfully used to improve disease resistance, and a high genetic correlation between disease resistance traits measured using the two methods has previously been shown for some diseases. A wide range of studies have showed the viability of improving disease resistance to specific pathogens via selective breeding in a variety of aquaculture species, including European sea bass (Dicentrarchus labrax) (Palaiokostas et al., 2018), Pacific oyster (Crassostrea gigas) (Gutierrez et al., 2018a), Pacific white shrimp (Penaeus vannamei), and in the three main farmed salmonids species i.e. Atlantic salmon (Salmo salar), (Correa et al., 2015) rainbow trout (Oncorhynchus mykiss) (Vallejo et al., 2017) and coho salmon (Oncorhynchus kisutch) (Barria et al., 2019). For a reviews of genetic improvement of disease resistance in aquaculture species, please see Houston (2017) and Yanez et al. (2014).

In case of tilapia, several studies have estimated significant genetic variation for resistance to bacterial pathogens in controlled challenge experiments (LaFrentz et al., 2016; Shoemaker et al., 2017; Wonmongkol et al., 2018). However, despite the serious consequences of TiLV-related infections, there are no published estimates of quantitative genetic parameters for resistance to TiLV, and its potential to be improved by selective breeding, and this is likely to be due to the current lack of a well-established and effective TiLV challenge model, although these have begun to be established (Jaemwimol et al., 2018; Pierezan et al., 2019; Tattiyapong et al., 2017).

Therefore, the aim of the current study was to estimate the levels of genetic variation for resistance to Tilapia Lake Virus in a population of Nile tilapia from the GIFT strain, using data collected from a field outbreak of the disease. In addition to estimating heritability values under different statistical models, the genetic correlation with harvest weight was also assessed. The results will inform improvement of this trait by means of selective breeding to help develop more resistant tilapia strains which can help mitigate and potentially control this problematic disease.

2. Materials and method

2.1. Study population

The Nile tilapia population used in the current study was from a major breeding program established in Malaysia and managed by WorldFish. This population originated from the GIFT strain, and has been selected for improved growth rate for 15 generations. A total of 124 families were produced using 115 sires and 124 dams. To retain pedigree information, each individual was tagged with a Passive Integrated Transponder (PIT) tag at an average weight and age of 4.97 g and 110.5 days, respectively. Once individuals reached typical harvest weight, their weight was recorded and they were transferred to a single pond, after which a TiLV outbreak was observed.

2.2. Tilapia Lake Virus outbreak

This population experienced a natural TiLV outbreak in February 2018. Survival or mortality data were obtained from a total of 1821 fish from a single pond, and this formed the basis of the trait of TiLV resistance. An average of 14 fish (ranging from 2 to 21) per family were measured for TiLV resistance. Fish were collected until the mortality curve had stabilized, i.e. mortalities had returned to baseline levels. Sex was identified for all fish, with a male:female ratio of 0.74:1.00. Surviving fish were euthanized with clove oil (400 mg/l). Necropsy assays were performed on a number of randomly selected dead fish to evaluate the cause of death and corroborate with the observed clinical signs of the disease. To confirm the presence of TiLV, spleen samples were obtained from a random sample of 39 individuals. The spleen tissue was maintained in RNALater and kept at −20 °C until analysis.

2.3. Trait definitions

Resistance to TiLV was defined as binary survival (BS) and time to death (TD). For the former, survivors and dead fish were assigned values of 1 and 0, respectively. In case of TD the values ranged from 1 (first day of observed moralities) to the last collection day (19). Survivor fish were assumed as censored data and each assigned the value of 18 or 19 days based on the sampling day.

3. Results

3.1. TiLV mortalities

Throughout the TiLV outbreak, clinical signs typical of TiLV infection were observed by a qualified veterinary expert. These included skin erosion, hemorrhage, and damage on the base of the pectoral and anal fin. The presence of TiLV was confirmed in 73.5% of the analyzed samples (n = 25). All 16 mortalities tested were positive for TiLV, while 9 survivors were positive and 9 survivors were negative for the virus. An average mortality rate of 56 fish per day was observed during the first five days of mortalities due the outbreak. This mortality rate had a peak of 128 dead fish at day 10 after the first mortality was collected. After this, mortality rate declined to seven fish per day four days later. During the last two days of data and sample collection (18th and 19th after the first mortality was registered) no mortalities were observed. The total cumulative mortality in the entire naturally exposed population (n = 1821) at the end of the TiLV outbreak was 39.6%. Furthermore, following assignment of mortalities and survivors to family using the PIT tags, a high between-family variation in mortality level was observed, ranging from 0 to 100%. (Fig. 1), suggestive of additive genetic variation in resistance. A cox proportional hazard model estimates no significant difference in mortality rate between sexes (p = .529).

Fig. 1. Cumulative mortality for each of the 124 Nile tilapia families throughout a Tilapia lake virus (TiLV) field outbreak.

4. Discussion

Tilapia Lake Virus (TiLV) has been a significant source of morbidity and mortality in various farmed Nile tilapia populations around the world, and is a currently major barrier to sustainable and profitable tilapia aquaculture. In the current study, host resistance to TiLV was found to be significant and high in a Nile tilapia breeding population with GIFT origin, using data collected during a field outbreak. These results highlight the significant potential of harnessing selective breeding to improve host resistance to TiLV in farmed Nile tilapia populations.

The current study utilized a natural ‘field’ disease outbreak to assess genetic resistance to TiLV. Typically, data for the genetic improvement of disease resistance traits are derived from controlled experimental challenges, which allows control of environmental factors. However, the use of survival data from natural field outbreaks can be a feasible alternative in genetic programs for aquaculture species (Bangera et al., 2014; Dégremont et al., 2015; Houston et al., 2008; Lillehammer et al., 2013). There are advantages to using such field data, because it reflects the natural method of infection of the agent in terms of time of exposure and its spread within the population. For example, in contrast to experimental injection of fish with a pathogen, a field challenge also requires that the pathogen surpasses the host barrier function, and this may be an important component of host genetic variation. However, obtaining high quality data and samples from a field outbreak is challenging, in part due to the difficulty to be sure that mortality is due to the pathogen under study. With this in mind, the outbreak of TiLV in the fish used in the current study was first analyzed by an expert veterinarian examining clinical signs Secondly, necropsy assays on the lesions observed on the fish attributed to this viral infection process, strongly suggest that TiLV was the major reason for the mortalities. Finally, the presence of TILV in all of the tested moralities and a proportion of survivors was confirmed by qPCR. The reasons for the absence of TiLV in some of the survivors could be due to the fish being resistant to the virus, and therefore potentially able to remove or reduce viral particles to a level below the detection threshold of the assay.

The detection of significant additive genetic variation for resistance to TiLV and the estimation of high heritability values is consistent with findings from other important infectious diseases in aquaculture species . Furthermore, the higher heritability estimated with threshold models than with linear models are in accordance with previou findings as are the high genetic correlation (0.95) between both resistance trait definitions (e.g. Barria et al., 2019; Bassini et al., 2019) highlighting that both binary survival and days to death are genetically the same trait using the methodology of the current study. This is to be expected to some extent since the majority of fish were survivors, and they were assigned a single value (1) for binary survival, and one of two values (18 or 19) for days to death.

Differences in the analysis and the definition of resistance can impact on the heritability estimates and their interpretations. Despite the fact that the cumulative mortality throughout the TiLV outbreak was below 50%, at which the phenotypic variance for a binary trait is maximized, significant heritability was estimated using both probit and logit-link functions. As shown in other studies into genetics of disease resistance in aquaculture species, a high correlation among family ranking was estimated using the different models, revealing a low re-ranking of the families genetically more resistant to TiLV. Furthermore, and although heritabilities for BS-SD were slightly lower than the threshold models, selection accuracy remained high and similar breeding values were predicted among these approaches. In case of resistance measured as time to death, an identical heritability estimation and correlation between full-sib families' EBVs was found between TD-LAN and TD-SD. These results suggests that binary survival is an appropriate measure of resistance to TiLV and time to death does not add additional useful information in this context.

Although high selection accuracy was estimated for TiLV resistance, selection of individuals based on the pedigree data is less accurate than when genomic data is available, as has been demonstrated on other farmed aquaculture species for growth-related traits and for disease resistance by means of genomic selection (GS). Recently, high-density SNP arrays have been developed for different Nile tilapia, populations with GIFT origin. The use of these technologies will most likely help to increase the selection response for this disease in the current breeding population by increasing the accuracy of selection and therefore reduce the mortalities ascribed to TiLV, as has been recently shown for responses to growth and fillet yield in Nile tilapia (Yoshida et al., 2019). Furthermore, the genotype data obtained from these SNP arrays will allow investigation of the genetic architecture of TiLV resistance, and whether there are significant QTL contributing to the genetic variation in the trait.

Moderate and significant genetic variation was also identified for harvest weight in the current study, which is in agreement with previous results in tilapia populations (Bentsen et al., 2012; Joshi et al., 2018b; Khaw et al., 2016; Marjanovic et al., 2016). Previous studies have shown different results in terms of genetic correlation between growth-related traits and disease resistance. The genetic correlations vary from negative (Yáñez et al., 2016), not different from zero (Silverstein et al., 2009) to positively correlated (Barria et al., 2019), depending on the age of the fish and the growth trait under study (i.e. body length, early growth rate, weight at harvest). The fact that the genetic correlation between HW and resistance to TILV found in the current study is not different from zero, suggests the feasibility of improving both traits independently and that selective breeding for TiLV resistance will not have a negative impact on weight at harvest, or vice versa.

In conclusion, resistance to TiLV as measured by survival during a field outbreak has a significant and high heritability. These results highlight that genetic improvement of TiLV resistance is feasible in a Nile Tilapia breeding population. However, for this trait to be included routinely into breeding programs, a reliable disease challenge model would be very useful, and assessing the genetic correlation between survival in an experimental and a field challenge would be highly informative. Nonetheless, the results herein are highly encouraging for the use of selective breeding to help tackle one of the primary disease concerns for tilapia aquaculture globally. Future studies will be required to evaluate the genetic architecture of host resistance to TiLV, and to evaluate the possibility of marker-assisted or genomic selection to expedite the breeding of tilapia strains with improved resistance to the virus.