Functional analysis of the DNA methylation system of Nasonia vitripennisSteffan Llewellyn, The Centre for Interdisciplinary ScienceDr. Eran Tauber, Department of Genetics

ABSTRACTThe presence of 5-hydroxymethylcytosine (5hmC) within the mammalian ‘epigenome’, and the discovery of the ten-eleven translocation (TET) enzyme family suggests that methylation patterns may be dynamic, reversing the methylated Cytosine state (demethylation). The parasitioid wasp Nasonia vitripennis demonstrates a full DNA methylation kit and may exhibit a TET homologue. Gene knockdown by RNA interference (RNAi) was performed on a TET candidate, and evaluated using quantitative PCR (qPCR). Wasps were incubated in contrasting photoperiodic conditions, and the phenotypic plasticity of the progeny was analysed, to observe possible epigenetic changes as a molecular basis for photoperiodism. Results suggest a knockdown in the gene expression of the protein, albeit statistically insignificant. The expected wild-type diapause state of the specimen's progeny was not observed in control samples.

Nasonia vitripennis

Image - West LaboratoryDepartment of Zoology

University of Oxfordwww.zoo.ox.ac.uk/group/west/precision.html

REFERENCES[1] Bird, A., 2002. DNA methylation patterns and epigenetic memory. Genes & Development, 16(1), 6–21. [2] Bhutani, N., Burns, D.M. & Blau, H.M. 2011. DNA demethylation dynamics. Cell, 146(6), 866–72. [3] Werren, J.H. et al., 2010. Functional and evolutionary insights from the genomes of three parasitoid Nasonia species. Science 327(5963), 343–8. [4] Tauber, E., Pegoraro, M. & Banfa, A. 2014. The role of DNA methylation in the photoperiodic effect of N. vitripennis. Unpublished results.[5] Lynch, J.A. & Desplan, C., 2006. A method for parental RNA interference in the wasp Nasonia vitripennis. Nature Protocols, 1(1), 486–94.

INTRODUCTION Epigenetic modifications are classified as heritable chemical modifications to the eukaryotic genome, which affects gene expression without altering the DNA sequence [1]. Recently, the stability of the epigenetic modification DNA methylation, involving the addition of the methyl group (CH3) to a cytosine base, has been questioned, as the enzyme family TET within the mammalian genome can actively oxidise the methylated cytosine base, producing 5-hydroxymethylcytosine,with the potential to revert back to cytosine [2]. The role of DNA methylation within invertebrates is poorly understood, as the necessary methylating enzyme family (Dnmt) in classic invertebrate lab models, such as the fruit fly (D. melanogaster), are absent. Advancements in genome sequencing have identified invertebrates with full DNA methylation kits, including the parasitioid wasp, Nasonia vitripennis [3]. This raises the question whether invertebrates may similarly possess TET homologues for active demethylation.

A N. vitripennis TET homologue candidate was recently identified, and wasps deficient in this protein may demonstrate aberrant development where DNA methylation is crucial, if the process is indeed dynamic. Research suggests that methylation patterns may provide the molecular basis for N. vitripennis’ photoperiodic response [4]. Female wasps produce offspring which develop normally, or remain dormant (diapause), depending on whether the female has been exposed to long day (LD) or short day (SD) conditions, respectively.

METHODSThe protein of interest (TET candidate) was targeted for gene knockdown via RNA interference. Female pupae were harvested and injected with a microinjection procedure, [5] using 500ng/µl of the double stranded RNA template (dsTET). Wasps which survived the injection procedure and eclosed were incubated at either long-day (LD) or short-day (SD) photoperiodic conditions for 5 days with hosts, to produce progeny. Subsequent to incubation, the adult wasps were frozen and homogenised. RNA was extracted, purified, and transcribed for quantitative PCR (qPCR). The cDNA of ribosomal protein (Rpl32) was used to normalise both sample and control TET expression.

After 15 days, progeny within the hosts exposed to different photoperiodic conditions (LD or SD) were classified as either larval (diapause state) or pupal stage (non-diapause state).

DISCUSSION Quantitative PCR suggests a positive knockdown of TET gene expression within dsTET wasps (see fig 2), as the TET expression of LD and SD dsTET wasps decreased by 60% and 53% respectively, compared to their control counterparts. Results were inconclusive as they were not statistically viable (ANOVA p > 0.1), most likely due to a limited sample size. The standard deviation of the normalised TET expression was greater than the average observed, as a result of a restricted sample size, from high injection morality, and low concentration of the injected dsRNA, decreasing the potency of the knockdown. Copies of DNA observed in some qPCR negative controls, indicate either contamination of samples, or possible primer dimer amplification.

The expected LD and SD wild-type diapause/non-diapause ratio was not observed in control samples. The low probability of dsGFP’s off-target knockdown indicates a technical issue. Reliable conclusions involving TET knockdown and phenotypic plasticity from dsTET specimens was not possible.

CONCLUSIONSInitial findings suggest that N. vitripennis is an emerging arthropod lab model for the study of dynamic methylation and demethylation, and the initial protocol merits potential improvements for future repeat experiments. The protein of interest is likely a TET homologue, and its gene expression can be decreased through the RNAi protocol. Determining the importance of epigenetics with regards to photoperiodism remains inconclusive.

Figure 2: Expression of TET from samples injected with dsTET RNAi and control (dsGFP RNAi) samples, incubated in either Long Day (LD) or Short Day (SD) conditions. As shown, wasps exposed to the dsTET RNAi template expressed lower TET mRNA levels.

Figure 1: (a) Developmental stages of N.vitripennis. Microinjection procedure is only viable during stage 1 and two of the wasp, when the dsRNA can spread throughout the body. Image - Lynch and Desplan (2006). (b) RNA gel electrophoresis of dsTET and the control dsRNA strand, which consists of the RNAi sequence for GFP.

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