Sylvain Forêt

Nutritional Control of Reproductive Status in Honeybees via DNA Methylation

Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly. We show that silencing the expression of DNA methyltransferase Dnmt3, a key driver of epigenetic global reprogramming, in newly hatched larvae led to a royal jelly–like effect on the larval developmental trajectory; the majority of Dnmt3 small interfering RNA–treated individuals emerged as queens with fully developed ovaries. Our results suggest that DNA methylation in Apis is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifications underpins, profound shifts in developmental fates, with massive implications for reproductive and behavioral status.

Whole Transcriptome Analysis of the Coral Acropora millepora Reveals Complex Responses to CO2‐driven Acidification during the Initiation of Calcification

The impact of ocean acidification (OA) on coral calcification, a subject of intense current interest, is poorly understood in part because of the presence of symbionts in adult corals. Early life history stages of Acropora spp. provide an opportunity to study the effects of elevated CO2 on coral calcification without the complication of symbiont metabolism. Therefore, we used the Illumina RNAseq approach to study the effects of acute exposure to elevated CO2 on gene expression in primary polyps of Acropora millepora, using as reference a novel comprehensive transcriptome assembly developed for this study. Gene ontology analysis of this whole transcriptome data set indicated that CO2‐driven acidification strongly suppressed metabolism but enhanced extracellular organic matrix synthesis, whereas targeted analyses revealed complex effects on genes implicated in calcification. Unexpectedly, expression of most ion transport proteins was unaffected, while many membrane‐associated or secreted carbonic anhydrases were expressed at lower levels. The most dramatic effect of CO2‐driven acidification, however, was on genes encoding candidate and known components of the skeletal organic matrix that controls CaCO3 deposition. The skeletal organic matrix effects included elevated expression of adult‐type galaxins and some secreted acidic proteins, but down‐regulation of other galaxins, secreted acidic proteins, SCRiPs and other coral‐specific genes, suggesting specialized roles for the members of these protein families and complex impacts of OA on mineral deposition. This study is the first exhaustive exploration of the transcriptomic response of a scleractinian coral to acidification and provides an unbiased perspective on its effects during the early stages of calcification.

DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees

In honey bees (Apis mellifera), the development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA methyltransferases. To understand the role of DNA methylation in this process we sequenced the larval methylomes in both queens and workers. We show that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination. We also tie methylation data to expressional profiling and describe a distinct role for one of the DMGs encoding anaplastic lymphoma kinase (ALK), an important regulator of metabolism. We show that alk is not only differentially methylated and alternatively spliced in Apis, but also seems to be regulated by a cis-acting, anti-sense non–protein-coding transcript. The unusually complex regulation of ALK in Apis suggests that this protein could represent a previously unknown node in a process that activates downstream signaling according to a nutritional context. The correlation between methylation and alternative splicing of alk is consistent with the recently described mechanism involving RNA polymerase II pausing. Our study offers insights into diet-controlled development in Apis.

RNAi-induced phenotypes suggest a novel role for a chemosensory protein CSP5 in the development of embryonic integument in the honeybee (Apis mellifera)

Small chemosensory proteins (CSPs) belong to a conserved, but poorly understood, protein family found in insects and other arthropods. They exhibit both broad and restricted expression patterns during development. In this paper, we used a combination of genome annotation, transcriptional profiling and RNA interference to unravel the functional significance of a honeybee gene (csp5) belonging to the CSP family. We show that csp5 expression resembles the maternal-zygotic pattern that is characterized by the initiation of transcription in the ovary and the replacement of maternal mRNA with embryonic mRNA. Blocking the embryonic expression of csp5 with double-stranded RNA causes abnormalities in all body parts where csp5 is highly expressed. The treated embryos show a “diffuse”, often grotesque morphology, and the head skeleton appears to be severely affected. They are ‘unable-to-hatch’ and cannot progress to the larval stages. Our findings reveal a novel, essential role for this gene family and suggest that csp5 (unable-to-hatch) is an ectodermal gene involved in embryonic integument formation. Our study confirms the utility of an RNAi approach to functional characterization of novel developmental genes uncovered by the honeybee genome project and provides a starting point for further studies on embryonic integument formation in this insect.

DMSP biosynthesis by an animal and its role in coral thermal stress response

Globally, reef-building corals are the most prolific producers of dimethylsulphoniopropionate (DMSP), a central molecule in the marine sulphur cycle and precursor of the climate-active gas dimethylsulphide. At present, DMSP production by corals is attributed entirely to their algal endosymbiont, Symbiodinium. Combining chemical, genomic and molecular approaches, we show that coral juveniles produce DMSP in the absence of algal symbionts. DMSP levels increased up to 54% over time in newly settled coral juveniles lacking algal endosymbionts, and further increases, up to 76%, were recorded when juveniles were subjected to thermal stress. We uncovered coral orthologues of two algal genes recently identified in DMSP biosynthesis, strongly indicating that corals possess the enzymatic machinery necessary for DMSP production. Our results overturn the paradigm that photosynthetic organisms are the sole biological source of DMSP, and highlight the double jeopardy represented by worldwide declining coral cover, as the potential to alleviate thermal stress through coral-produced DMSP declines correspondingly.

Carbohydrate metabolism genes and pathways in insects: insights from the honey bee genome.

Carbohydrate‐metabolizing enzymes may have particularly interesting roles in the honey bee, Apis mellifera, because this social insect has an extremely carbohydrate‐rich diet, and nutrition plays important roles in caste determination and socially mediated behavioural plasticity. We annotated a total of 174 genes encoding carbohydrate‐metabolizing enzymes and 28 genes encoding lipid‐metabolizing enzymes, based on orthology to their counterparts in the fly, Drosophila melanogaster, and the mosquito, Anopheles gambiae. We found that the number of genes for carbohydrate metabolism appears to be more evolutionarily labile than for lipid metabolism. In particular, we identified striking changes in gene number or genomic organization for genes encoding glycolytic enzymes, cellulase, glucose oxidase and glucose dehydrogenases, glucose‐methanol‐choline (GMC) oxidoreductases, fucosyltransferases, and lysozymes.

Standard methods for molecular research in Apis mellifera

Summary From studies of behaviour, chemical communication, genomics and developmental biology, among many others, honey bees have long been a key organism for fundamental breakthroughs in biology. With a genome sequence in hand, and much improved genetic tools, honey bees are now an even more appealing target for answering the major questions of evolutionary biology, population structure, and social organization. At the same time, agricultural incentives to understand how honey bees fall prey to disease, or evade and survive their many pests and pathogens, have pushed for a genetic understanding of individual and social immunity in this species. Below we describe and reference tools for using modern molecular-biology techniques to understand bee behaviour, health, and other aspects of their biology. We focus on DNA and RNA techniques, largely because techniques for assessing bee proteins are covered in detail in Hartfelder et al. (2013). We cover practical needs for bee sampling, transport, and storage, and then discuss a range of current techniques for genetic analysis. We then provide a roadmap for genomic resources and methods for studying bees, followed by specific statistical protocols for population genetics, quantitative genetics, and phylogenetics. Finally, we end with three important tools for predicting gene regulation and function in honey bees: Fluorescence in situ hybridization (FISH), RNA interference (RNAi), and the estimation of chromosomal methylation and its role in epigenetic gene regulation.

The biology of coral metamorphosis: Molecular responses of larvae to inducers of settlement and metamorphosis

Like many other cnidarians, corals undergo metamorphosis from a motile planula larva to a sedentary polyp. In some sea anemones such as Nematostella this process is a smooth transition requiring no extrinsic stimuli, but in many corals it is more complex and is cue-driven. To better understand the molecular events underlying coral metamorphosis, competent larvae were treated with either a natural inducer of settlement (crustose coralline algae chips/extract) or LWamide, which bypasses the settlement phase and drives larvae directly into metamorphosis. Microarrays featuring >8000 Acropora unigenes were used to follow gene expression changes during the 12h period after these treatments, and the expression patterns of specific genes, selected on the basis of the array experiments, were investigated by in situ hybridization. Three patterns of expression were common-an aboral pattern restricted to the searching/settlement phase, a second phase of aboral expression corresponding to the beginning of the development of the calicoblastic ectoderm and continuing after metamorphosis, and a later orally-restricted pattern.

Patterns of Gene Expression in a Scleractinian Coral Undergoing Natural Bleaching

Coral bleaching is a major threat to coral reefs worldwide and is predicted to intensify with increasing global temperature. This study represents the first investigation of gene expression in an Indo-Pacific coral species undergoing natural bleaching which involved the loss of algal symbionts. Quantitative real-time polymerase chain reaction experiments were conducted to select and evaluate coral internal control genes (ICGs), and to investigate selected coral genes of interest (GOIs) for changes in gene expression in nine colonies of the scleractinian coral Acropora millepora undergoing bleaching at Magnetic Island, Great Barrier Reef, Australia. Among the six ICGs tested, glyceraldehyde 3-phosphate dehydrogenase and the ribosomal protein genes S7 and L9 exhibited the most constant expression levels between samples from healthy-looking colonies and samples from the same colonies when severely bleached a year later. These ICGs were therefore utilised for normalisation of expression data for seven selected GOIs. Of the seven GOIs, homologues of catalase, C-type lectin and chromoprotein genes were significantly up-regulated as a result of bleaching by factors of 1.81, 1.46 and 1.61 (linear mixed models analysis of variance, P < 0.05), respectively. We present these genes as potential coral bleaching response genes. In contrast, three genes, including one putative ICG, showed highly variable levels of expression between coral colonies. Potential variation in microhabitat, gene function unrelated to the stress response and individualised stress responses may influence such differences between colonies and need to be better understood when designing and interpreting future studies of gene expression in natural coral populations.

Rapid acclimation of juvenile corals to CO2 -mediated acidification by upregulation of heat shock protein and Bcl-2 genes.

Corals play a key role in ocean ecosystems and carbonate balance, but their molecular response to ocean acidification remains unclear. The only previous whole‐transcriptome study (Moya et al. Molecular Ecology, 2012; 21, 2440) documented extensive disruption of gene expression, particularly of genes encoding skeletal organic matrix proteins, in juvenile corals (Acropora millepora) after short‐term (3 d) exposure to elevated pCO2. In this study, whole‐transcriptome analysis was used to compare the effects of such ‘acute’ (3 d) exposure to elevated pCO2 with a longer (‘prolonged’; 9 d) period of exposure beginning immediately post‐fertilization. Far fewer genes were differentially expressed under the 9‐d treatment, and although the transcriptome data implied wholesale disruption of metabolism and calcification genes in the acute treatment experiment, expression of most genes was at control levels after prolonged treatment. There was little overlap between the genes responding to the acute and prolonged treatments, but heat shock proteins (HSPs) and heat shock factors (HSFs) were over‐represented amongst the genes responding to both treatments. Amongst these was an HSP70 gene previously shown to be involved in acclimation to thermal stress in a field population of another acroporid coral. The most obvious feature of the molecular response in the 9‐d treatment experiment was the upregulation of five distinct Bcl‐2 family members, the majority predicted to be anti‐apoptotic. This suggests that an important component of the longer term response to elevated CO2 is suppression of apoptosis. It therefore appears that juvenile A. millepora have the capacity to rapidly acclimate to elevated pCO2, a process mediated by upregulation of specific HSPs and a suite of Bcl‐2 family members.