RNA interference offers promising antiviral methods to minimise nodovirus outbreaks in aquaculture

RNA interference offers promising antiviral methods to minimise nodovirus outbreaks in aquaculture

Following on from the talk by Leigh Owens and the recent complementary lecture by Colin, this research used interfering RNA (RNAi) to assess its effectiveness of reducing mortality from white tail disease (WTD) in giant freshwater prawns, Macrobrachium rosenbergii, using redclaw crayfish, Cherax quadricarinatus as a model organism.

Global demand for seafood is increasing and with intensive farming comes disease.  WTD has lead to mass mortalities in prawn hatcheries the world over, causing economic problems and loss in production.  M. rosenbergii nodavirus (MrNV) has been identified as the causative agent of WTD and has caused 100% mortality in hatcheries within one week of the first clinical signs in the hosts.

RNAi is the sequence-specific degradation of complementary mRNA using small, functional pieces of double stranded RNA (dsRNA) to target specific genes.  It has been used as an antiviral mechanism, making it possible to knock-down specific viral genes and limiting any off-target effects.   This experiment targeted protein B2 of MrNV, which inhibits the degradation of viral RNA that would normally occur in the host cells.  Sequence specific RNAi was therefore used to silence the production of protein B2 by degrading viral mRNA – basically an arms race between virus and host cell.

MrNV from infected M. rosenbergii was homogenised with phosphate buffered saline (PBS) and injected into select C. quadricarinatus.  Sequence specific RNAi (targeting protein B2), along with control RNAi (non-specific), was designed and injected into select animals in a cross-over experimental design, also controlling for exposure to MrNV and RNAi by using placebo treatments and PBS.  All experimental animals, unless mortality occurred during the trial, were sacrificed at the end of 60 days.  RNA was extracted from C. quadricarinatus muscle for qPCR analyses and tissue sections prepared for histopathological analysis.

Invertebrates have no acquired immunity and RNAi is important for antiviral innate immunity. Only specific dsRNA to protein B2 was effective in affording increased antiviral defence against MrNV infection in focal C. quadricarinatus (10% mortality) compared with high mortality (60%) and clinical signs consistent with MrNV infection reported in the control RNAi group (see Fig. 1).  

 

The authors note that injection of RNAi + MrNV did not reduce the average viral titre in comparison to the control RNAi + MrNV group, contrasting their results to a similar study in crickets that reported a 10-fold reduction in viral titres (La Fauce & Owens, 2009).  An alternative method of delivering sequence specific dsRNA may yield lower viral counts, for example including it in feeds via a bacterial plasmid.  This may also allow viable scale up for commercial use and offer an alternative to phage therapy (see Sanket’s recent posts).  In Leigh’s talk, he stated that his group has had difficulty in scaling up quantities for phage therapy to be effective outside of laboratory conditions and considers RNAi a more promising viral control technique.  Inclusion of plasmid contained sequence specific dsRNA in commercial feeds may still offer antiviral protection to the prawns, whilst maintaining its select target (eg. the virus) and minimising other undesirable, off target effects.  It will be interesting to see where this promising technique leads, although further long-term, transgenerational research would benefit here, to gain a better understanding of potential long-term impacts this may have.

Hayakijkosol, O., & Owens, L. (2012). B2 or not B2: RNA interference reduces (Macrobrachium rosenbergii) nodavirus replication in redclaw crayfish (Cherax quadricarinatus). Aquaculture, 326, 40-45.

 

Controlled, safe, clean hatcheries actually enhance salmon disease

 Selective fish hatchery breeding rarely uses quantitative genetic techniques to maintain genetic diversity as they require 10-20 years to affect production and are therefore expensive. Most salmonids are bred by artificial random mating with selection of desirable traits; the resulting production increases associate with genetic diversity decreases. Whilst the genetic changes contrived by aquaculture may benefit artificial rearing of salmonids, they may be maladaptive in natural conditions. Such selection may have counterproductive effects when the salmon are moved to ocean net pens and partially explain mass mortalities from bacterial and viral infections. Some semi-wild breeding practises have been implemented, providing a breeding habitat that exposes salmonids to wild microbe communities and sexual selection. The role of major histocompatibility (MH) genes in vertebrate sexual selection is well known; these genes are central to the adaptive immune system, allowing T cells to produce a range of very polymorphic immune proteins. Heterozygous MH individuals are therefore better able to identify a wider array of pathogenic particles and be favoured in nature; particular alleles conferring resistance to a specific disease may also be favoured and amplified within a population. The diversity of MH genes is likely generated by sexual selection and has important influence on fitness.

This study attempted to quantify the advantages of outdoor spawning of Chinook salmon (Oncorhynchus tshawytscha) for aquaculture by testing captive bred versus semi-wild bred fish immune systems, challenged by Vibrio anguillarum. Mortality, antibody-mediated (humoral) immune response and MH genotypes were assessed in survivors and mortalities.

All salmon were obtained from a farm which had previously lost stock to vibriosis and bacterial kidney disease from Renibacterium salmoninarum and V. anguillarum, so were probably selectively bred for resistance to these diseases. Fish were bred in an exposed, semi-wild channel and left to spawn non-randomly (CH) or in a traditional hatchery with random mating (H); in the CH group the fertilised eggs were left buried until they hatched into fry. After 6 months the H and CH groups were reciprocally transplanted for 5 months, creating four groups. Afterwards, all four groups were exposed to V. anguillarum.

Mortality did not significantly differ between CH/CH and CH/H groups, but H/CH fish had much higher mortality than H/H fish. 12 different MH alleles were identified, a low amount compared to wild populations; H fish more frequently had low allelic diversity than CH fish, however this was not significant and nor was there a significant difference in allelic diversity between the 31 survivors and the 31 mortalities.

Temperature, photoperiods and other abiotic factors are known to affect fish immune systems, which are altered from natural levels in hatcheries. The vibriosis susceptibility of hatchery raised fish transplanted to the channels may be a product of relaxed selection for robust immune systems; genotype is stated to outweigh the effects of the environment, which is why

transplanted CH fish had only moderate mortality. The MH genotypes findings align with the theory that heterozygous MH individuals can cope better with disease. The increased genetic diversity from sexually selective breeding in semi-wild conditions is recommended as a broodstock management technique in aquaculture.

Note that pathogen exposure in semi-wild is not controlled for; meaning that selective pressure or disease from pathogens other than V. anguillarum could have been influencing mortality and genotypes. Another coinciding factor could be that natural microbe levels may stimulate immune systems, affecting development and preparing the fish for later infection, possibly explaining the inferior immune systems of the more microbe-naive H fish. Also, allowing eggs and juveniles to develop in natural conditions may allow colonisation of fish gut or skin with beneficial bacteria able to exclude pathogens, but this study did not control for this. If we could identify advantageous heterozygous MH combinations then quantitative genetic techniques (which are getting cheaper and more efficient), could allow us to combine hatchery breeding with artificial sexual selection, which would probably be more efficient than semi-wild breeding. Hatcheries could also use less sterile conditions to imitate the effects of the environment on wild fish immune systems. This study focused on freshwater rearing, but it relates to the marine environment, because when salmonids are transported to the sea to mature in sea pens, they are exposed to a sudden new range of microbial threats, so a strong immune system is crucial for this stage of salmonid aquaculture. The implications are worse for salmon hatcheries used for replenishing wild populations.

Becker, L. A., Kirkland, M., Heath, J. W., Heath, D. D., & Dixon, B. (2014). Breeding strategy and rearing environment effects on the disease resistance of cultured Chinook salmon (Oncorhynchus tshawytscha). Aquaculture,422, 160-166.

Shrimp Farm Logistics Responsible for Virus Infections?

In the last lectures we talked about viral and bacterial diseases in marine animals, and this paper in particular focuses on the occurrence of the white spot syndrome virus (WSSV) which was mentioned briefly in the lecture, and infectious hypodermal and hematopoidetic necrosis virus (IHHNV) in wild organisms which live close to shrimp farms around the Pacific coast of Mexico. The WSSV has a broad range of host animals, and especially crustaceans appear to be most affected. Since the start of shrimp farming in Mexico, WSSV and IHHNV have been identified as major threat to the industry and much effort has been put into studying the dynamics of these pathogens in order to prevent disease reoccurring disease outbreaks.

The viruses spread horizontally (food/water intake by animals) or vertically (infected brood stock to developing larvae), and as the use of non-infected larvae did not solve the problem, it is now generally accepted that wild animals in the surrounding environment of the farms are the sources and carriers of the viruses. In this study, the authors wanted to address the question of how prevalent WSSV and IHHNV are in wild populations of animals around these shrimp farms.

Methods involved the collection of 1736 organisms (11 species) in total over a period of 10 months, before, during and after the shrimp cultivation cycle. The total DNA was extracted and a nested PCR was conducted with the DNA samples with primer sets for WSSV and IHHNV. The amplicon sequences obtained from this were compared to WSSV and IHHNV sequences from databanks in order to establish a similarity profile.

The viruses were found in various species, including fish, shrimps and crabs throughout sample period with a prevalence of 19.5% for IHHNV and 3.6% for WSSV. Moreover, the occurrence of these viruses before cultivation was discovered in 1.2% of animals (WSSV) and 2.3% (IHHNV). After cultivation, IHHNV had a prevalence of over 30% (5.7% for WSSV) and it becomes obvious that the cultivation has a major influence on the spread of the viruses. During cultivation, the water used in the farms is constantly exchanged with water from the surrounding environment, which could potentially be the cause of an increase in virus prevalence in wild organisms. 

Typical hosts of these viruses have been previously considered to be crustaceans, however this study could detect the viruses in fish as well, and they are now considered as virus vectors; susceptibility of fish to these viruses is yet to be established. Persistence of WSSV and IHHNV in animals surrounding the farms has been revealed which explains repeated disease outbreaks when wild fish enter the farm ponds. The solution to this problem seems to be fairly logical to me; moving and isolating the farm ponds away from the natural environment could prevent future outbreaks, however I guess this would be generally difficult to realise. I think this was a very neat study and the results fill important gaps about the dynamics of the viruses affecting the aquaculture industry so greatly.

Macías-Rodríguez, et al., 2014. Prevalence of viral pathogens WSSV and IHHNV in wild organisms at the Pacific Coast of Mexico. Journal of Invertebrate Pathology, Vol. 116, pp. 8-12.

PS.: I hope the authors noticed their several spelling mistakes, it was very irritating to read some parts. They also managed to calculate their percentages wrong. Nevermind, still nice work ;-).

 

Black band disease – don’t blame it all on bacteria!

Black Band Disease (BBD) is an infection, hosted in corals, that is characterised by a darkly pigmented microbial consortium. It causes necrosis to underlying coral tissues exposing the bare skeleton as it migrates across colonies contributing to global coral decline.

The development of BBD was studied over time in situ on Montipora corals. Lesions dominated by cyanobacteria and aptly named ‘cyanobacterial patches’ (CP) precede the development of BBD lesions. Profiles of the microbial communities within both CP and subsequent BBD lesions were studied using two indepentant amplicon-pyrosequencing assays of 16S rRNA coding genes.

Overall, analyses consistently found both taxanomic composition and diversity of bacteria shifted within all coral colonies sampled with the progression from CP lesions to BBD. A shift from Trichodesmium spp. dominance of the cyanobacteria in CP to Oscillatoria spp. dominated BBD cyanobacteria  was indicated as the largest driver of alterations in community composition between the two disease stages.

Alphaproteobacteria were in significantly lower proportions in BBD samples that CP samples, though the opposite trend was true of Arcobacter sp. (within Epsilonproteobacteria).  Other bacterial species were found to be present in both BBD and CP lesions with no distinctive variations in relative abundances.

Successional changes in microbial communities during the disease transition from CP to BBD were concurrent with the shift to a typically anoxic, sulphide-rich microenvironment. Intensification of such conditions accounts for the increased pathogenicity of BBD and highlight the importance of the microbial consortium. Whilst these findings corroborate with previous studies, the potential contribution of non-bacterial groups to BBD has never previously been investigated.

Archaea are ubiquitous throughout many aquatic habitats, and account for large proportions of the biomass in extreme conditions. They are known to inhabit anoxic and sulphide-rich environments, a niche present within a BBD mat.

It was found that archaeal communities are not only present in the microbial consortia in both CP and BBD lesions, but that a similar shift in phylogeny to bacteria occurs with development of the disease. CP lesions were typically diverse assemblages whereas BBD lesions were found to be dominated by a novel archaeon, distantly related to other archaea usually associated with sulphurous and low-oxygen environments.

The dominance of such an archaeon makes it highly interesting for future study. Whilst its metabolic modes cannot be inferred from other archaea due to phylogenetic distance, its capacity to inhabit such environment could infer a potential role in methanogenesis, chemo-organotrophy and anerobic methane oxidation. Testing these and identifying the biological capabilities of the novel archaeon warrants further study.

The distinct shift in bacterial assemblages with the progression of Black Band Disease was evidenced, and has been noted for other coral species and diseases alike (see Rachel, Roberto, and Ellie's blog posts). However it is clear that further research is required to assess both the significance and the potential functional roles of the archaea.

The findings of this focal study have enhanced our understanding of Black Band Disease and may aid in understanding other diseases contributing to coral decline. The application of these findings to other diseases, where archaea may not have previously been tested for could perhaps highlight the importance of archaea in influencing disease virulence and the associated microbial ecology.

Y. Sato, B. Willis and D. Bourne (2013) Pyrosequencing-based profiling of archaeal and bacterial 16S rRNA genes identifies a novel archaeon associated with black band disease in corals. Environmental Microbiology, 15(11), 2994-3007

Previously Unknown Dinoflagellates Diversity in Global Cultured Strain. All Thanks to A New Barcoding Marker.

Dinoflagellates are an ecologically significant group related to ciliates and apicomplexans. Like many phytoplankton, this taxa is identified using traditional morphological archetype and over 2000 species have been described this way to date. However, Dinoflagellates pose a particular challenge as there are many cryptic species which remain unidentified. Recent molecular phylogeny has shown that Dinoflagellates, once thought to be of the same genera, are in fact paraphyletic e.g Amphidinum and Gymnodinium. Some taxa such as Symbiodinium, instead of being a single species by morphological standards, are now known to contain hundreds of cryptic taxanomic units; most of which have not been identified. In order to standardize the method of Dinoflagellate classification, a DNA-based identification system should be implemented. DNA-barcoding may hold the key for this concept to be conceived. This technique involves using a short stretch of DNA sequencing to identify a species; known as a barcode marker. This technique has been applied to two mitochondrial markers known as the Cytochrome Oxidase I (COI) and Cytochrome Oxidase B genes (COB). However, these barcodes have limitations as both cannot always be amplified from all Dinoflagellates and both cannot specifically identify species from the same genera. One of the main objectives of DNA-barcoding is to accurately and quickly identify toxic HAB species, though the modern barcode markers already in existence are unable to resolve many issues surrounding these hazardous taxa.

The aims of this study were to test a third barcode marker, this being the internal transcribed spacer (ITS) units 1 and 2. These markers were used to identify various Dinoflagellate taxa from a range of global algal collections. The ITS markers have previously been used to identify Eukaryotes, Metazoans and some Dinoflagellates.

The limitations of the ITS markers were previously described as being that they are found in multiple distinct copies, with high intra/intergenomic variation. This can make sequencing challenging and alignment difficult. However, to test the international applicability of the ITS markers this paper assessed nearly 400 dinoflagellate strains.

By collecting 669 dinoflagellates and eliminating low-quality sequences, 151 ITS barcodes were left remaining. 242 ITS sequences were collected from GenBank and added to this, equalling 393 ITS barcodes from 78 identified species. Each strain was centrifuged, snap-frozen and thawed three times before DNA extraction was conducted. PCR sequencing was then carried out resulting in products ranging from 500-600bp. These were then DNA-barcoded on the premises or sent to the Canadian Centre of DNA Bar-coding to be analysed. The sequences were screened for contamination using BLAST.

These markers were heavily biased toward larger assemblages with Symbiodinium, Alexandrium and Pfiesteriaceae being two-thirds of those identified. Some members of the Pfiesterial and Oxyrrhis were poorly represented whereas Lingulodinium, Symbiodinium, Gymnodinium, Gyrodinium and Karenia were represented at an above average level.

The ITS markers could identify 93% of known strains and was able to resolve previously genera/species arguments in Lingulodinium and Protoceratiumand taxa. Unusually, Gymnodinium sp, CCMP 424, was re-identified as Heterocapsa niei, in conflict with COI barcode markers. However, the strain was re-sequenced using ITS and SSU sequencing, confirming that this was not a case of PCR contamination as originally believed.

The results indicated that there were 21 cases of misidentification which may have arisen from two cryptic species being thought as one. It was discovered that there were four Dinoflagellate strains that did not match their given taxa and two other strains that, although given the same name, were not of the same species.

32 strains were shown to have been previously unknown cryptic species revealing a higher diversity than originally thought. This included strains from the Gyrodinium, Karlodium and Cryptoperidinopsis taxa.

The success rate in these studies varied between 2% and 53% suggesting that the ITS markers are too technical to work as a generic DNA-barcoding marker for Dinoflagellates, with COB and COI being more broadly comparable. Although, ti should be noted that COI primers were shown to fail when sequencing Amphidinium, Heterocapsa and Oxyrrhis. The causes of failure in ITS, COI and COB sequencing were thought to be a mixture of issues. Failed amplification, failed sequencing and poor sequencing quality were all thought to contribute toward failed sequencing. The ITS markers were shown to lack sequencing efficiency with an observed success rate of 50% compared to 66% for COI. This was attributed to poor sequencing quality and the mis-representation of Pfiesteriaceae and Alexandrium were thought to skew this data set. However, despite this, the ITS markers successfully identified 96% of strains including 3 genus that had no identity.

This study was considered significant as it highlights the genetic instability in long-term cultured strains worldwide and focuses on the need for re-examination into Heterocapsa taxa. DNA-barcoding is important for this reason as it helps to identify stability of cultured strains, with ITS bar-coding being particularly important as it is capable of identifying cryptic species and speciation events in long-cultured strains. ITS markers were also considered good markers to identify harmful Dinoflagellate groups.

Stern, RF, Anderson RA, Jameson I, Kupper FC, Coffroth MA, Vaulot D, Le Gall F, Veron B, Brand JJ, Skelton H, Kasai F, Lily EL & Keeling PJ. 2012 Evaluating the Ribosomal Internal Transcriber Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker. PLOS One. 10.