Asymmetric division: a major cause of cyanobacteria cell death

Cyanobacteria have a significant role in global primary productivity. Forms Prochlorococcus and Synechococcus alone contribute up to 50% of total fixed carbon in low latitudes, a great ecological significance. An individual cell has four potential fates; division, eaten by predators, viral lysis or cell death. The cause of death will greatly influence the carbon flow within the food web. Cell death will lead to cell lysis and a release of dissolved organic carbon, as does viral lysis, but this is very difficult to measure. Many studies have classified cell death as programmed cell death (PCD) however this study promotes the idea of asymmetric division as the major cause.

Cyanobacteria colonies aim to optimize colony fitness therefore PCD would make evolutionary sense, where there is differentiation between cells, in order for different roles to be completed. However in some colonial forms, cell death and DNA fragmentation happens randomly, making PCD a less clear response. PCD is detected from increased caspase activity during cell death. It happens when the metabolism is forced beyond its limits, however, in Trichodesmium colonies approximately 30% of the colony survives during mass cell death. This proportion of the population is known as a “persister” fraction that is more resistant to environmental stress.

Asymmetric division occurs when daughter cells are morphologically or physiologically different which could lead to differing fitness. In Escherichia coli, one daughter cell has a reduced fitness where the parent cell has deposited all its metabolic waste, such as damaged proteins, resulting in an increased fitness and growth of the other daughter cell. Dead cells can also be regularly produced as a by-product and this reduces the burden of damaged proteins on the colony, thus improving overall fitness.

A large proportion of cyanobacteria occur in the top 50m of the water column where excess light can lead to cell death by protein damage. Cell death increases throughout the day and peaks in the late afternoon. Is it possible that the individuals which die in these situations have already accumulated metabolic damage from their parent cell and are therefore more susceptible to environmental conditions?

Protein damage can be discovered by measuring the protein carbonylation which needs to be tested in inclusion bodies produced asymmetrically by the parent cell and track their distribution. To conclude that asymmetric division is a major cause of cell death, determining the amount of oxidative damage along with the fate of a bacterial cell needs to be addressed. This knowledge will provide a better understanding of the whole food web structure and the carbon within it.

Franklin, D.J. (2014) Explaining the causes of cell death in cyanobacteria: what role for asymmetric division? Journal of Plankton Research. 36: 11-17

Bacterial heat shock proteins? Inducing invertebrate immune responses?!

   Previous posts discussing infection management in aquaculture have referred to the paradigm that invertebrates do not have acquired immune systems like us vertebrates. Without antibodies, how does the invertebrate immune system rely completely on innate (non-specific) defences? This study details an interesting mechanism of how the innate immune system responds to pathogens.

   Litopenaeus vannamei is a shrimp widely devoured by Homo sapiens and is assumed to have an innate immune system, defending the arthropod using melanisation, hemolymph clotting, foreign particle trapping and good old phagocytosis. The hemolymph enzyme transglutaminase (TGase) is instrumental in all of these processes and inhibiting its production in shrimp results in bacteria rich hemolymph and very sick crustaceans. Hemocytes produce prophenoloxidase (proPOase), the precursor to another important immune enzyme, phenoloxidase (POase). Once activated, PO facilitates phenol oxidation to antimicrobial quinones, which can also polymerize into melanin to encapsulate pathogens. Basically, TGase and proPO are what makes the crustacean innate immune system tick.

   As you know, heat shock proteins (HSPs) are a highly multifunctional family of proteins used by all organisms. The family HSP70 is the most highly conserved and studied; recently it has been flagged as a notable regulator in the initial innate immune system response, improving POase activity in Artemia infected with Vibrio. But such studies are controversial; many have used a recombinant HSP70 extracted from E. coli called DnaK, which may have been contaminated with endotoxins able to activate the immune system in a similar way.

   This study aimed to determine whether or not HSP70 homologue DnaK induces immune response in L. vannamei, by measuring changes in the expression of proPOase and TGase genes in response to an injection of either recombinant HSP70 (DnaK) from E. coli or a contaminant-free control of chemically synthesised HSP70 (synDnaK). All shrimp used were specific pathogen free (SPF) and given a high and a low dose injection. Gene expression was measured by synthesising cDNA from isolated RNA, then amplifying it by quantitative PCR with TGase and proPOase primers. TGase gene expression increased very significantly in response to low and high doses of DnaK, low doses of synDnaK, but did not change for high dose synDnaK. Expression of a proPOase gene increased significantly for low and high doses of DnaK; a similar pattern was observed for synDnaK. DnaK contamination was measured also, showing endotoxin levels much lower than concentrations found to induce crustacean immune response in other studies.

Recombinant DnaK and synthetic DnaK induced similar patterns of immune response, so contaminants are not the only contributors to stimulation of proPOase and TGase up-regulation. A novel mechanism for TGase and proPOase increase in response to pathogens has been demonstrated. Bizarrely, lower injections of both treatments induced stronger responses; this inverted dose-response relationship points to an unknown mechanism of immune system inhibition by DnaK. In conclusion, this study proposes the hypothesis that bacterial derived HSP70 acts as an alarm signal, activating host TGase and proPOase genes. I wonder how this response compares between organisms with only innate immune system and those with both acquired and innate immune system; is it stronger in the former because it is the main immune response? When pathogens initially colonise a host, they are probably exposed to a sudden range of stresses and therefore increase HSP production. This could be why the innate immune system has evolved to respond to HSP, because high bacterial HSP levels coincide with early infection.

Hu, B., Phuoc, L. H., Sorgeloos, P., & Bossier, P. (2014). Bacterial HSP70 (DnaK) is an efficient immune stimulator in Litopenaeus vannamei. Aquaculture, 418, 87-93.

Dormant V.cholerae cells are resuscitated by quorum sensing molecules

Detection of Vibrio cholerae in environmental samples is made difficult by its ability to enter a dormant ‘viable but nonculturable’ (VBNC) state. VBNC cells will persist in an environment but will not grow on traditional growth media, allowing them to go undetected by conventional techniques. Recently, fluorescent antibody based microscopy has allowed identification of V.cholerae in samples from which it would previous have gone unnoticed. Antibiotic selection techniques, using knowledge of the antibiotic resistance of previously cultured strains from pandemics, also allows small numbers of VBNC cells to be identified, by suppressing the growth of other bacteria.

The danger of VBNC cells is not just that they are difficult to detect, they can be ‘resuscitated’, switching from dormancy to an active state that is capable of infection. The researches had in a previous study identified two autoinducer molecules that were upregulated in conditions of large cells densities to promote the production of Vibrio extracellular polysaccharide by active cells. Because autoinducers are only produced under conditions of high cell density, they reasoned that they might also signal to dormant VBNC cells that conditions are favourable for growth, causing resuscitation.

They therefore proceeded to investigate whether biologically or synthetically produced autoinducer molecules would induce resuscitation of VBNC V.cholerae in environmental samples from Bangladesh.

Antibiotic selection techniques were used to identify samples that contained active V.cholerae, only samples that did not already contain active cells were used. They found that samples containing no culturable V.cholerae, when treated with spent media from either autoinducer-producing V.cholerae or E.coli, produced resuscitated cells within only a few hours of treatment. Water from the same samples tested negative for culturable V.cholerae following treatment with spent media from the controls: a V.cholerae strain that had had its autoinducer genes deleted and E.coli that contained the cloning vector used to induce autoinducer production but without the genes required for their production. Furthermore, profiling of the culturable V.cholerae strains produced via this resuscitation showed their close similarity to strains that had caused recent epidemics in the area.

Having established the link between autoinducers and resuscitation, they proceeded to investigate whether a wild type strain of V.cholerae could initiate resuscitation of VBNC cells. It could not without the addition of a recombinant plasmid containing synthase genes. Wild strains that already contain this plasmid do show resuscitation activity.

Chemically synthesized versions of the two autoinducer molecules caused resuscitation when provided together and individually, showing that the presence of only one of them is required for dormant cells to become active. One of the autoinducers is narrowly produced by Vibrios only, however the other is produced by many bacterial groups. This study therefore suggests that resuscitation of VBNC V.cholerae could occur either outside or inside the human host as a result of high concentrations of autoinducer molecules produced by environmental bacteria or those of the human microbiome. They suggest that this may explain the seasonality of cholera, as the seasonal occurrence of heterologous bacteria that produce the same autoinducer molecule may trigger resuscitation of large numbers of dormant V.cholerae. Another model would be that the bacteria of the human microbiome cause resuscitation upon ingestion, or that faecal bacteria that enter the environment are favoured by seasonal conditions and so induce resuscitation in the environment.

Many potential models are suggested, however, the major contribution of this paper is not its speculations but the mechanism it has identified that links resuscitation of dormant V.cholerae cells to bacterial inter-species communication via autoinducer molecules.

Bari, S. N., Roky, M. K., Mohiuddin, M., Kamruzzaman, M., Mekalanos, J. J., & Faruque, S. M. (2013). Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples. Proceedings of the National Academy of Sciences, 110(24), 9926-9931.

FARMED SHRIMPS FROM ASIA COULD BE CONTAMINATED WITH PATHOGENS OF VIBRIO SPP.

Popularity of shrimps as seafood has encouraged shrimp farming in South Asia and Latin America. We have been frequently coming across Vibrio spp. in recent lectures as very diverse group of bacteria, very dominant in the marine environment, associated with a number of diseases of corals, fish, crustaceans, marine mammals etc. and also of humans. As Colin explained in the last lecture, three Vibrio species Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus are of prime importance as they are causative agents of various disease outbreaks of humans. In intensive shrimp farming, antibiotics are heavily used, stimulating development of resistance among bacteria, including Vibrio spp. Similarly, antibiotics are also used to treat human diseases; for example, antibiotic treatment becomes necessity for patients of septicaemia caused by V. vulnificus. Here, aquaculture induced problem of antibiotic resistance, especially among human pathogens is notable and studies have shown resistant Vibrio spp. isolates from farmed shrimps.

Thailand has a huge aquaculture industry. Shrimps are farmed both in marine and inland waters. Producing shrimps also in inland waters with the use of antibiotics have had many ecological impacts, including impacts on bacteria like Vibrio spp. and their susceptibility to antibiotics. This study examined occurrence and antibiotic susceptibility of Vibrio pathogens of humans (V. cholerae, V. parahaemolyticus & V. vulnificus) in farmed shrimps from intensive farms of inland waters of Thailand.

A method called three-tube most-probable-number was used for isolating Vibrio species. Agar plating technique was used to culture the Vibrio pathogens and then colonies of bacteria were selected for biochemical tests, species-specific PCR and 16S rRNA sequencing. Total, 12 different antibiotics were tested in this study. Antibiotic resistant isolates were screened for resistant genes and those genes were sequenced using PCR.

The authors found that 94% of shrimp samples had V. cholerae. But, remarkably, none of them had cholera toxin. Thus, all the V. cholera isolated from farmed shrimps were non-choleragenic. Non-choleragenic strains of V. cholerae are abundant in estuarine environments. This study proposes that same is true for inland waters with shrimp aquaculture activity. Nevertheless, high frequencies and densities of V. cholera were found to be associated with the shrimps, capable of causing sporadic diarrhoea in humans.

V. parahaemolyticus was isolated from 38% of samples, of which one was positive for thermostable direct hemolysin (TDH). As Colin mentioned in the last lecture, TDH is produced by human disease producing strains of V. parahaemolyticus. This pathogen is found commonly in seafood and its contamination is a big problem in Asia. The authors also suggested a link between non-native species of shrimp (grown on farms in Thailand) which grows faster but found to have higher affinity for this pathogen.

The authors did not find notable concentrations of V. vulnificus from their samples. Nevertheless, they reported a previous study finding high concentration of V. vulnificus in farmed shrimps, sold in a market in China.

The authors discussed that shrimp aquaculture expansion in inland waters (and related lower salinity) might be a limiting factor for V. parahaemolyticus and V. vulnificus whereas this would be favourable for growth of V. cholerae. Nevertheless, shrimps grown in inland waters are at high risk of being contaminated with V. cholerae and V. parahaemolyticus. Other thing I want to mention here is that it is perhaps not very surprising to see Vibrios associated with shrimps because, as Colin have mentioned many times in the lectures that Vibrios prefer to colonise on chitinous organisms/chitin and have enzymes to breakdown chitin.

V. cholerae and V. parahaemolyticus mostly showed resistance to ampicillin and oxytetracyclin. V. vulnificus showed resistance to nalidixic acid. Farmed shrimps from inland waters can act as reservoirs of numerous oxytetracyclin-resistance genes, originating from both marine and inland waters. The resistance to oxytetracycline was related to plasmids which are passed from one bacteria to other in a process called horizontal gene transfer.

In summary, this study provides an experimental evidence for risk of contamination of Vibrio pathogens in farmed seafood. This paper attracted my attention because it links aquaculture, prevalence of bacteria (Vibrios) in these environments and seafood-borne bacterial diseases in humans. In this linkage, a worrisome factor is heavy use of antibiotics in aquaculture which is moving antibiotics in marine, freshwater and terrestrial environments and driving widespread antibiotic-resistance development among various bacteria (including many pathogens) from different environments to existing antibiotics.

Yano, Y., Hamano, K., Satomi, M., Tsutsui, I., Ban, M., & Aue-umneoy, D. (2014). Prevalence and antimicrobial susceptibility of Vibrio species related to food safety isolated from shrimp cultured at inland ponds in Thailand. Food Control, 38, 30-36.

Epidemiology and pathogenesis of Vibrio vulnificus.

Epidemiology and pathogenesis of Vibrio vulnificus.

The authors (Strom and Paranjpye) have presented a very detailed review, I am going to summarise the epidemiology of the virus to prevent an essay style 1,000 word summary. I would be happy to cover the pathogenesis separately.

Vibrio vulnificus was first isolated by the US Centres for Disease Control (CDC) in 1964 and was classified as a distinct species in the 1970s. Vibrio vulnificus is a motile, Gram-negative bacterium. It is present in both marine and estuarine environments. Vibrio vulnificus is responsible for a significant percentage of vibrio related illnesses, is the leading cause of seafood-associated fatalities in the US and causes massive economic harm to the entire shellfish industry.

V. vulnificus is taken up by filter feeders, including oysters and clams where the bacteria concentrate in the gut. It has also been found in fish, and it is believed that this is the method by which the bacteria are transported.

V. vulnificus is a naturally occurring and free-living inhabitant of estuarine and marine environments, predominately in tropical areas. This bacterium is known to have caused illness throughout Northern Europe, the US and Australia. It proliferates in summer months when the water exceeds 18 degrees Celsius. It is believed that an onset of both systemic and wound infections occurs primarily between the months of April and September in the US. When water drops below 10 degrees Celsius, V. vulnificus enters a viable but nonculturable state (VBNC) where following several morphological changes, it becomes more resistant to environmental hardship.

There are two biotypes of V. vulnificus. Biotype 1 strains are typically associated with shellfish colonisation and human illness. Biotype 2 strains are implicated in infections of marine vertebrates, particularly in cultured eels. They have, however, been shown to be virulent in mice and capable of causing opportunistic infections in humans.

This bacterium has the ability to cause serious and often fatal infections. These infections include an invasive septicaemia (through eating raw or undercooked shellfish - cooking shellfish is the only reliable method to completely destroy the bacterium), as well as wound infections acquired through contact with shellfish or waters where the bacteria are prevalent. Both of these acquisition methods are just as common as the other.

• Wound infections are classified as those where a patient incurred a wound before or during exposure to seawater or seafood drippings and where V. vulnificus was subsequently cultured from that wound.
• Primary Septicaemia was defined as a systematic illness characterised as fever and shock where V. vulnificus was isolated from blood but no wound infection preceded the illness.

422 infections reported between 1988 and 1996; 45% were wound infections and 43% were primary septicaemia. Of the 181 cases of primary septicaemia, 173 patients reported eating raw oysters in the seven days prior to the symptoms.  Many wound infections appear to be related to occupational exposure with 69% reporting either fishing or handling raw seafood during the seven days preceding illness.

The lethal dose of V. vulnificus is unknown in humans however it is known that host susceptibility is a key factor. People who are most susceptible usually suffer from chronic disease that affects either liver function or the immune system. 97% of those people who had primary septicaemia had pre-existing conditions, as opposed to 67% in those with wound infections. Of the risk factors, liver disease was most common, found in 80% of those with primary septicaemia. It was also a strong indicator for fatality, as liver disease was found in 80% of those who died from the infection.

The fatality rate for V. vulnificus is between 30-48%, however this varies depending on the mode of infection and host factors. For example, fatalities occur in 60-75% of patients with primary septicaemia but only 20-30% in those with wound infection. This may be due to the differing in timing and severity of the symptoms in wound infections when compared to those of primary septicaemia.

M.S. Strom and R.N. Paranjpye, (2000). Epidemiology and pathogenesis of Vibrio vulnificus. Microbes and Infection. Volume 2, Issue 2, Pages 177-188

Molecular and microscopic evidence for viruses in marine copepods

Viruses are ubiquitous in aquatic systems and are infectious agents of many organisms from bacteria up to fish and mammals, yet despite their huge ecological significance very few studies have been conducted on the effects of viruses on aquatic metazoans. Copepods are the most dominant members of such mesozooplankton communities and are responsible for biogeochemical cycling and fundamental to marine food webs. Whilst previous work has focussed on the ecology and population dynamics of such organisms, causes of mortality are still poorly understood.

In the lecture Colin delivered on ‘Diseases of mollusks, crustaceans and fish’ it was stated Drake and Dobbs (2005) had found no negative effects of viruses on the fecundity or survival of copepod Acartia tonsa. This, as mentioned within the lecture, seems rather strange as viruses are central to controlling algal (phytoplankton) blooms so could be predicted to act in a similar manner on zooplankton. Consistent with viral disease in phytoplankton, mortality rates of copepods coincide with population peaks, and with ~35% of total copepod mortality unexplained by predation, another underlying cause of mortality must be present.

Using molecular methods the presence of viruses within A. tonsa and Labidocera aestiva copepods was assessed.

Using metagenomic sequencing on extracted and purified viruses from the sampled copepod species, the presense of circo-like viruses was detected, named LaCopCV and AtCopCV in L. aestiva and A. tonsa respectively. Whilst these viral genomes shared characteristics with known circoviruses; namely a small genome, a stem-loop with a conserved nonanucleotide motif and two nonoverlapping ORF’s coding for replication initiatior (Rep) and capsid proteins, the genomic architecture varied slightly. Unlike the bi-directional orientation of the 2 ORF’s in known circoviruses, the ORF’s in copepod circo-like virus genomes are mono-directional, a characteristic previously observed in environmental viral communities and fecal metagenomes. Additionally, the nonanucleotide motif on the stem-loop in copepod viruses exhibited variations to the normal conserved motif present in circoviruses.

Using a phylogenetic tree based on Rep amino acid sequences, it was shown that both LaCopCV and AtCopCV are highly divergent from recognised and identified circoviruses, and are, unsurprisingly, more closely related to environmental assembled circo-like virus sequences.

Using qPCR of the capsid gene, a high prevalence of LaCopCV among wild L. aestiva populations was observed in the order of magnitude similar to that of white spot syndrome virus in postlarval crustaceans, and was found to be actively replicating, with low levels of transcription detected using a modified qRT-PCR assay. AtCopCV was also detected, though only in samples from 7 months of the year principally in spring and autumn during times of population changes. Despite being suggestive of lower prevalence in comparison to LaCopCV, the presence of AtCopCV coninciding with population peaks and previously stated nonpredatory mortality peaks could suggest that AtCopCV has a greater influence in mortality during zooplankton blooms and LaCopCV accounts for a steady rate of mortality throughout the year. This is supported by the frequent identification of LaCopCV in sediments from Tampa Bay, perhaps signifying a potential reservoir for the virus.

Contrary to the study by Drake and Dobss (2005) the results of this study provide evidence of not only the presence, but the prevalence and active replication of previously undocumented viruses within two numerically dominant species of natural zooplankton populations. Though further study is required to understand the pathology of the viruses and the larger ecological implications they may have on marine food webs, the study is an important breakthrough in understanding potential roles of viruses in the nonpredatory mortality of zooplankton. Whilst both viruses found were limited to and only detected in their respective zooplankton species, it’s not to say that other circo-like viruses don’t propagate in other zooplankton species. With the demonstrated capacity to test this, it is an area which warrants future study. 

Dunlap D., Fei Fan Ng T., Rosario K., Barbosa J., Greco A., Breitbart M. and Hewson I. (2012) Molecular and microscopic evidence of viruses in marine copepods. Proceedings of the National Academy of Sciences, 11(4), 1375-1380

Drake L. and Dobbs F. (2005) Do viruses affect fecundity and survival of the copepod Acartia tonsa Dana? Journal of Plankton Research, 27(2), 167-174

Associations between toxic blooms of Pseudo-nitzschia and sea lion strandings

After the lecture on diseases in marine mammals, I was interested to see what the latest findings were coastal harmful algal blooms (HABs) (including diatoms such as the genus Pseudo-nitzschia) and the production of domoic acid (DA), stemming on from one of the referenced studies (Bargu et al., 2002). The Californian sea lion in Monterey Bay (a coastal area off California) is one of the top predators in this region, but also may be most affected by HABs, as they predate on intoxicated species, which results in them becoming distressed, and hence stranded, along the coastline. Acute and chronic DA toxicosis both result in different neurological and behavioural signs in sea lions, such as seizures and disorientation in acute poisoning cases. Previous literature has shown that these toxic blooms are increasing in abundance and frequency, and simultaneously sea lion strandings are now exceeding previous numbers from Monterey Bay. 

The authors aimed to find associations between the presence of blooms and sea lion strandings along the coastline over the years 2004 to 2007, looking for potential links between acute and chronic DA poisoning, the abundance of toxic cells and concentrations of particulate DA in Californian coast waters, as well as in the faeces of stranded animals. 

DA-producing Pseudo-nitzschia diatoms can be the predominant group within the phytoplankton at certain times during the year, but not always to the level of abundance that typically forms blooms. This study did succeed in determining some correlations between some of the different parameters. For example, the highest concentration of particulate DA in spring 2007 was linked to the highest number of sea lion strandings with acute signs, and to the highest concentration of DA in faeces. Sea lions are most likely being exposed to varying levels of DA in their prey throughout the year, often at sublethal amounts, but because there is sometimes a time lag between the accumulation of toxin in the animals and stranding events, not many links have been made between chronic signs and toxic blooms. The combination of multiple acute cases could contribute to this observed increase in the number of chronic cases. 

All of the sea lions that stranded in the spring of 2007 tended to either be adult or sub-adult males. This could be because the normal pre-set of their breeding season is in May, so the number of males may be higher in Monterey Bay during this time. Therefore it seems like males are the most affected gender, because they are found in the Bay at similar times to the blooms. I wondered when reading this whether the females are less affected because they are in different areas during the same period of time, and if they feed on different, uncontaminated prey species, as they mention that they have a wide distribution of 30-60 km when foraging for prey. 

The clinical signs of low and repeated doses of DA are less well known in younger sea lion classes (pups, yearlings and juveniles) than in adult and sub-adult populations. Although it is unknown why, younger age classes appear to need higher levels of toxin in their systems to exhibit the same signs and symptoms as affected adults, or be detectable in their faeces (82%), so adults tend to be more susceptible to the toxins than juveniles. Perhaps the effects are less obvious, as their nervous systems are less well developed, or they exhibit different clinical signs at a young age that are currently not linked to DA poisoning? In any case, as a result of these findings, it may mean that the number of affected sea lions is an underestimate, due to the lack of recognised signs of poisoning. 

Similarly to Bargu et al. (2002), the authors have looked at a single entity within a food chain than studied the transfer of the toxin between species. It would be interesting to determine the extent to which DA accumulates up this food chain, depending on prey the predators feed on. I think linking studies of species at different trophic levels could give a more holistic view of the negative effects these HABs have on certain food webs. In conclusion, the presence of the toxin-producing cells in Monterey Bay may result in toxins accumulating in local pelagic food webs, but the survivability of intoxicated individuals may vary depend on the duration of the bloom in this region, as well as how much toxin the cells produce. They state that it is crucial to look at the stranding trends related to the season, sex and age class of these animals, so that the true potential impacts of domoic acid on sea lion populations can be quantified and detected. 

Bargu, S., Goldstein, T., Roberts, K., Li, C., and Gulland, F. (2012) Pseudo-nitzschia blooms, domoic acid, and related California sea lion strandings in Monterey Bay, California. Marine Mammal Science, 28 (2): 237-253