Daphnia magna modifies its gene expression extensively in response to caloric restriction revealing a novel effect on haemoglobin isoform preference.

Caloric restriction (CR) produces clear phenotypic effects within and between generations of the model crustacean Daphnia magna. We have previously established that micro-RNAs and cytosine methylation change in response to CR in this organism, and we demonstrate here that CR has a dramatic effect on gene expression. Over 6,000 genes were differentially expressed between CR and well-fed D. magna, with a bias towards up-regulation of genes under caloric restriction. We identified a highly expressed haemoglobin gene that responds to CR by changing isoform proportions. Specifically, a transcript containing three haem-binding erythrocruorin domains was strongly down-regulated under CR in favour of transcripts containing fewer or no such domains. This change in the haemoglobin mix is similar to the response to hypoxia in Daphnia, which is mediated through the transcription factor hypoxia-inducible factor 1, and ultimately the mTOR signalling pathway. This is the first report of a role for haemoglobin in the response to CR. We also observed high absolute expression of superoxide dismutase (SOD) in normally fed individuals, which contrasts with observations of high SOD levels under CR in other taxa. However, key differentially expressed genes, like SOD, were not targeted by differentially expressed micro-RNAs. Whether the link between haemoglobin and CR occurs in other organisms, or is related to the aquatic lifestyle, remains to be tested. It suggests that one response to CR may be to simply transport less oxygen and lower respiration.

Genome-wide methylation is modified by caloric restriction in Daphnia magna.

BACKGROUND: The degradation of epigenetic control with age is associated with progressive diseases of ageing, including cancers, immunodeficiency and diabetes. Reduced caloric intake slows the effects of ageing and age-related disease in vertebrates and invertebrates, a process potentially mediated by the impact of caloric restriction on epigenetic factors such as DNA methylation. We used whole genome bisulphite sequencing to study how DNA methylation patterns change with diet in a small invertebrate, the crustacean Daphnia magna. Daphnia show the classic response of longer life under caloric restriction (CR), and they reproduce clonally, which permits the study of epigenetic changes in the absence of genetic variation. RESULTS: Global cytosine followed by guanine (CpG) methylation was 0.7-0.9%, and there was no difference in overall methylation levels between normal and calorie restricted replicates. However, 333 differentially methylated regions (DMRs) were evident between the normally fed and CR replicates post-filtering. Of these 65% were hypomethylated in the CR group, and 35% were hypermethylated in the CR group. CONCLUSIONS: Our results demonstrate an effect of CR on the genome-wide methylation profile. This adds to a growing body of research in Daphnia magna that demonstrate an epigenomic response to environmental stimuli. Specifically, gene Ontology (GO) term enrichment of genes associated with hyper and hypo-methylated DMRs showed significant enrichment for methylation and acyl-CoA dehydrogenase activity, which are linked to current understanding of their roles in CR in invertebrate model organisms.

Daphnia magna microRNAs respond to nutritional stress and ageing but are not transgenerational.

Maternal effects, where the performance of offspring is determined by the condition of their mother, are widespread and may in some cases be adaptive. The crustacean Daphnia magna shows strong maternal effects: offspring size at birth and other proxies for fitness are altered when their mothers are older or when mothers have experienced dietary restriction. The mechanisms for this transgenerational transmission of maternal experience are unknown, but could include changes in epigenetic patterning. MicroRNAs (miRNAs) are regulators of gene expression that have been shown to play roles in intergenerational information transfer, and here, we test whether miRNAs are involved in D. magna maternal effects. We found that miRNAs were differentially expressed in mothers of different ages or nutritional state. We then examined miRNA expression in their eggs, their adult daughters and great granddaughters, which did not experience any treatments. The maternal (treatment) generation exhibited differential expression of miRNAs, as did their eggs, but this was reduced in adult daughters and lost by great granddaughters. Thus, miRNAs are a component of maternal provisioning, but do not appear to be the cause of transgenerational responses under these experimental conditions. MicroRNAs may act in tandem with egg provisioning (e.g., with carbohydrates or fats), and possibly other small RNAs or epigenetic modifications.

Blockade of KCa3.1: A novel target to treat TGF-ß1 induced conjunctival fibrosis.

Postoperative conjunctival fibrosis is common in patients after glaucoma filtration surgery. The calcium activated potassium (KCa3.1) channel has been shown to inhibit fibrosis in many non-ocular tissues. However, its potential in treating ocular fibrosis remains unknown. We tested the anti-fibrotic potential of TRAM34, a selective blocker of KCa3.1 channel, in treating conjunctival fibrosis. Primary human conjunctival fibroblast (HCF) cultures derived from donor tissues. Myofibroblasts causing conjunctival fibrosis were generated by growing HCFs in the presence of TGFß1 for 72 h. KCa3.1 mRNA and protein expression in HCF was examined with PCR and western blot. The anti-fibrotic potential of TRAM34 was examined by measuring fibrotic gene expression with quantitative PCR (qPCR), immunofluorescence, and western blotting in HCFs in ±â€¯TGFß1 (5 ng/ml) and TRAM34 (0-25 µM). The cytotoxicity of Tram34 was analyzed with trypan blue assay and its role in Smad signaling was studied with immunofluorescence. Expression of KCa3.1 mRNA and protein was detected in HCFs and TGFß1 treatment to HCFs significantly increased expression of KCa3.1. TRAM34 treatment attenuated transcription of fibrotic markers, αSMA (p < .001), fibronectin (p < .05), collagen I (p < .001) and collagen IV (p < .001) in TGFß1-induced HCFs. Further, TRAM34 significantly inhibited TGFß1-stimulated αSMA protein expression (p < .01) and nuclear translocation of fibrotic Smad2/3 in HCFs and showed no significant cytotoxicity (p < .05). The KCa3.1 potassium channel plays a significant role in the prevention of conjunctival fibrosis and TRAM34 has potential to control post surgical bleb fibrosis in patients. In vivo studies are warranted.

Extending digital PCR analysis by modelling quantification cycle data.

BACKGROUND: Digital PCR (dPCR) is a technique for estimating the concentration of a target nucleic acid by loading a sample into a large number of partitions, amplifying the target and using a fluorescent marker to identify which partitions contain the target. The standard analysis uses only the proportion of partitions containing target to estimate the concentration and depends on the assumption that the initial distribution of molecules in partitions is Poisson. In this paper we describe a way to extend such analysis using the quantification cycle (Cq) data that may also be available, but rather than assuming the Poisson distribution the more general Conway-Maxwell-Poisson distribution is used instead. RESULTS: A software package for the open source language R has been created for performing the analysis. This was used to validate the method by analysing Cq data from dPCR experiments involving 3 types of DNA (attenuated, virulent and plasmid) at 3 concentrations. Results indicate some deviation from the Poisson distribution, which is strongest for the virulent DNA sample. Theoretical calculations indicate that the deviation from the Poisson distribution results in a bias of around 5 % for the analysed data if the standard analysis is used, but that it could be larger for higher concentrations. Compared to the estimates of subsequent efficiency, the estimates of 1st cycle efficiency are much lower for the virulent DNA, moderately lower for the attenuated DNA and close for the plasmid DNA. Further method validation using simulated data gave results closer to the true values and with lower standard deviations than the standard method, for concentrations up to approximately 2.5 copies/partition. CONCLUSIONS: The Cq-based method is effective at estimating DNA concentration and is not seriously affected by data issues such as outliers and moderately non-linear trends. The data analysis suggests that the Poisson assumption of the standard approach does lead to a bias that is fairly small, though more research is needed. Estimates of the 1st cycle efficiency being lower than estimates of the subsequent efficiency may indicate samples that are mixtures of single-stranded and double-stranded DNA. The model can reduce or eliminate the resulting bias.

The development of pathogen resistance in Daphnia magna: implications for disease spread in age-structured populations.

Immunity in vertebrates is well established to develop with time, but the ontogeny of defence in invertebrates is markedly less studied. Yet, age-specific capacity for defence against pathogens, coupled with age structure in populations, has widespread implications for disease spread. Thus, we sought to determine the susceptibility of hosts of different ages in an experimental invertebrate host-pathogen system. In a series of experiments, we show that the ability of Daphnia magna to resist its natural bacterial pathogen Pasteuria ramosa changes with host age. Clonal differences make it difficult to draw general conclusions, but the majority of observations indicate that resistance increases early in the life of D. magna, consistent with the idea that the defence system develops with time. Immediately following this, at about the time when a daphnid would be most heavily investing in reproduction, resistance tends to decline. Because many ecological factors influence the age structure of Daphnia populations, our results highlight a broad mechanism by which ecological context can affect disease epidemiology. We also show that a previously observed protective effect of restricted maternal food persists throughout the entire juvenile period, and that the protective effect of prior treatment with a small dose of the pathogen ('priming') persists for 7 days, observations that reinforce the idea that immunity in D. magna can change over time. Together, our experiments lead us to conclude that invertebrate defence capabilities have an ontogeny that merits consideration with respect to both their immune systems and the epidemic spread of infection.

Daphnia magna shows reduced infection upon secondary exposure to a pathogen.

Previous pathogen exposure is an important predictor of the probability of becoming infected. This is deeply understood for vertebrate hosts, and increasingly so for invertebrate hosts. Here, we test if an initial pathogen exposure changes the infection outcome to a secondary pathogen exposure in the natural host-pathogen system Daphnia magna and Pasteuria ramosa. Hosts were initially exposed to an infective pathogen strain, a non-infective pathogen strain or a control. The same hosts underwent a second exposure, this time to an infective pathogen strain, either immediately after the initial encounter or 48 h later. We observed that an initial encounter with a pathogen always conferred protection against infection compared with controls.

The bacterial parasite Pasteuria ramosa is not killed if it fails to infect: implications for coevolution.

Strong selection on parasites, as well as on hosts, is crucial for fueling coevolutionary dynamics. Selection will be especially strong if parasites that encounter resistant hosts are destroyed and diluted from the local environment. We tested whether spores of the bacterial parasite Pasteuria ramosa were passed through the gut (the route of infection) of their host, Daphnia magna, and whether passaged spores remained viable for a "second chance" at infecting a new host. In particular, we tested if this viability (estimated via infectivity) depended on host genotype, whether or not the genotype was susceptible, and on initial parasite dose. Our results show that Pasteuria spores generally remain viable after passage through both susceptible and resistant Daphnia. Furthermore, these spores remained infectious even after being frozen for several weeks. If parasites can get a second chance at infecting hosts in the wild, selection for infection success in the first instance will be reduced. This could also weaken reciprocal selection on hosts and slow the coevolutionary process.