Mitochondrial Haemoglobin Is Upregulated with Hypoxia in Skeletal Muscle and Has a Conserved Interaction with ATP Synthase and Inhibitory Factor 1.

The globin protein superfamily has diverse functions. Haemoglobin has been found in non-erythroid locations, including within the mitochondria. Using co-immunoprecipitation and in silico methods, we investigated the interaction of mitochondrial haemoglobin with ATP synthase and its associated proteins, including inhibitory factor 1 (IF1). We measured the expression of mitochondrial haemoglobin in response to hypoxia. In vitro and in silico evidence of interactions between mitochondrial haemoglobin and ATP synthase were found, and we report upregulated mitochondrial haemoglobin expression in response to hypoxia within skeletal muscle tissue. Our observations indicate that mitochondrial pH and ATP synthase activity are implicated in the mitochondrial haemoglobin response to hypoxia.

Positive and Relaxed Selective Pressures Have Both Strongly Influenced the Evolution of Cryonotothenioid Fishes during Their Radiation in the Freezing Southern Ocean.

Evolution in the chronic cold of the Southern Ocean has had a profound influence on the physiology of cryonotothenioid fishes. However, the suite of genetic changes underlying the physiological gains and losses in these fishes is still poorly surveyed. By identifying the genomic signatures of selection, this study aims to identify the functional classes of genes that have been changed following two major physiological transitions: the onset of freezing temperatures and the loss of hemoproteins. Looking at the changes that followed the onset of freezing temperatures, positive selective pressure was found among a set of broadly acting gene regulatory factors, suggesting a route through which cryonotothenioid gene expression has been retooled for life in the cold. Further, genes related to the cell cycle and cellular adhesion were found under positive selection suggesting that both present key challenges to life in freezing waters. By contrast, genes showing signatures of the relaxation of selective pressure showed a narrower biological impact, acting on genes related to mitochondrial function. Finally, although chronic cold-water temperatures appear correlated with substantial genetic change, the loss of hemoproteins resulted in little observable change in protein-coding genes relative to their red-blooded relatives. Combined, the influence of positive and relaxed selection shows that long-term exposure to cold has led to profound changes in cryonotothenioid genomes that may make it challenging for them to adapt to a rapidly changing climate.

Quantitative Proteomics and Network Analysis of Differentially Expressed Proteins in Proteomes of Icefish Muscle Mitochondria Compared with Closely Related Red-Blooded Species.

Antarctic icefish are extraordinary in their ability to thrive without haemoglobin. We wanted to understand how the mitochondrial proteome has adapted to the loss of this protein. Metabolic pathways that utilise oxygen are most likely to be rearranged in these species. Here, we have defined the mitochondrial proteomes of both the red and white muscle of two different icefish species (Champsocephalus gunnari and Chionodraco rastrospinosus) and compared these with two related red-blooded Notothenioids (Notothenia rossii, Trematomus bernacchii). Liquid Chromatography-Mass spectrometry (LC-MS/MS) was used to generate and examine the proteomic profiles of the two groups. We recorded a total of 91 differentially expressed proteins in the icefish red muscle mitochondria and 89 in the white muscle mitochondria when compared with the red-blooded related species. The icefish have a relatively higher abundance of proteins involved with Complex V of oxidative phosphorylation, RNA metabolism, and homeostasis, and fewer proteins for striated muscle contraction, haem, iron, creatine, and carbohydrate metabolism. Enrichment analyses showed that many important pathways were different in both red muscle and white muscle, including the citric acid cycle, ribosome machinery and fatty acid degradation. Life in the Antarctic waters poses extra challenges to the organisms that reside within them. Icefish have successfully inhabited this environment and we surmise that species without haemoglobin uniquely maintain their physiology. Our study highlights the mitochondrial protein pathway differences between similar fish species according to their specific tissue oxygenation idiosyncrasies.

Proteomic analysis of the ATP synthase interactome in notothenioids highlights a pathway that inhibits ceruloplasmin production.

Antarctic notothenioids have unique adaptations that allow them to thrive in subzero Antarctic waters. Within the suborder Notothenioidei, species of the family Channichthyidae (icefish) lack hemoglobin and in some instances myoglobin too. In studies of mitochondrial function of notothenioids, few have focused specifically on ATP synthase. In this study, we find that the icefish Champsocephalus gunnari has a significantly higher level of ATP synthase subunit α expression than the red-blooded Notothenia rossii, but a much smaller interactome than the other species. We characterize the interactome of ATP synthase subunit α in two red-blooded species Trematomus bernacchii, N. rossii, and in the icefish Chionodraco rastrospinosus and C. gunnari and find that, in comparison with the other species, reactome enrichment for C. gunnari lacks chaperonin-mediated protein folding, and fewer oxidative-stress-associated proteins are present in the identified interactome of C. gunnari. Reactome enrichment analysis also identifies a transcript-specific translational silencing pathway for the iron oxidase protein ceruloplasmin, which has previously been reported in studies of icefish as distinct from other red-blooded fish and vertebrates in its activity and RNA transcript expression. Ceruloplasmin protein expression is detected by Western blot in the liver of T. bernacchii, but not in N. rossii, C. rastrospinosus, and C. gunnari. We suggest that the translation of ceruloplasmin transcripts is silenced by the identified pathway in icefish notothenioids, which is indicative of altered iron metabolism and Fe(II) detoxification.

Strong and lasting impacts of past global warming on baleen whales and their prey.

Global warming is affecting the population dynamics and trophic interactions across a wide range of ecosystems and habitats. Translating these real-time effects into their long-term consequences remains a challenge. The rapid and extreme warming period that occurred after the Last Glacial Maximum (LGM) during the Pleistocene-Holocene transition (7-12 thousand years ago) provides an opportunity to gain insights into the long-term responses of natural populations to periods with global warming. The effects of this post-LGM warming period have been assessed in many terrestrial taxa, whereas insights into the impacts of rapid global warming on marine taxa remain limited, especially for megafauna. In order to understand how large-scale climate fluctuations during the post-LGM affected baleen whales and their prey, we conducted an extensive, large-scale analysis of the long-term effects of the post-LGM warming on abundance and inter-ocean connectivity in eight baleen whale and seven prey (fish and invertebrates) species across the Southern and the North Atlantic Ocean; two ocean basins that differ in key oceanographic features. The analysis was based upon 7032 mitochondrial DNA sequences as well as genome-wide DNA sequence variation in 100 individuals. The estimated temporal changes in genetic diversity during the last 30,000 years indicated that most baleen whale populations underwent post-LGM expansions in both ocean basins. The increase in baleen whale abundance during the Holocene was associated with simultaneous changes in their prey and climate. Highly correlated, synchronized and exponential increases in abundance in both baleen whales and their prey in the Southern Ocean were indicative of a dramatic increase in ocean productivity. In contrast, the demographic fluctuations observed in baleen whales and their prey in the North Atlantic Ocean were subtle, varying across taxa and time. Perhaps most important was the observation that the ocean-wide expansions and decreases in abundance that were initiated by the post-LGM global warming, continued for millennia after global temperatures stabilized, reflecting persistent, long-lasting impacts of global warming on marine fauna.

Sequence and structure comparison of ATP synthase F0 subunits 6 and 8 in notothenioid fish.

Mitochondrial changes such as tight coupling of the mitochondria have facilitated sustained oxygen and respiratory activity in haemoglobin-less icefish of the Channichthyidae family. We aimed to characterise features in the sequence and structure of the proteins directly involved in proton transport, which have potential physiological implications. ATP synthase subunit a (ATP6) and subunit 8 (ATP8) are proteins that function as part of the F0 component (proton pump) of the F0F1complex. Both proteins are encoded by the mitochondrial genome and involved in oxidative phosphorylation. To explore mitochondrial sequence variation for ATP6 and ATP8 we analysed sequences from C. gunnari and C. rastrospinosus and compared them with their closely related red-blooded species and eight other vertebrate species. Our comparison of the amino acid sequence of these proteins reveals important differences that could underlie aspects of the unique physiology of the icefish. In this study we find that changes in the sequence of subunit a of the icefish C. gunnari at position 35 where there is a hydrophobic alanine which is not seen in the other notothenioids we analysed. An amino acid change of this type is significant since it may have a structural impact. The biology of the haemoglobin-less icefish is necessarily unique and any insights about these animals will help to generate a better overall understanding of important physiological pathways.

Not Frozen in the Ice: Large and Dynamic Rearrangements in the Mitochondrial Genomes of the Antarctic Fish.

The vertebrate mitochondrial genomes generally present a typical gene order. Exceptions are uncommon and important to study the genetic mechanisms of gene order rearrangements and their consequences on phylogenetic output and mitochondrial function. Antarctic notothenioid fish carry some peculiar rearrangements of the mitochondrial gene order. In this first systematic study of 28 species, we analyzed known and undescribed mitochondrial genome rearrangements for a total of eight different gene orders within the notothenioid fish. Our reconstructions suggest that transpositions, duplications, and inversion of multiple genes are the most likely mechanisms of rearrangement in notothenioid mitochondrial genomes. In Trematominae, we documented an extremely rare inversion of a large genomic segment of 5,300 bp that partially affected the gene compositional bias but not the phylogenetic output. The genomic region delimited by nad5 and trnF, close to the area of the Control Region, was identified as the hot spot of variation in Antarctic fish mitochondrial genomes. Analyzing the sequence of several intergenic spacers and mapping the arrangements on a newly generated phylogeny showed that the entire history of the Antarctic notothenioids is characterized by multiple, relatively rapid, events of disruption of the gene order. We hypothesized that a pre-existing genomic flexibility of the ancestor of the Antarctic notothenioids may have generated a precondition for gene order rearrangement, and the pressure of purifying selection could have worked for a rapid restoration of the mitochondrial functionality and compactness after each event of rearrangement.

Draft genome assembly and transcriptome data of the icefish Chionodraco myersi reveal the key role of mitochondria for a life without hemoglobin at subzero temperatures.

Antarctic fish belonging to Notothenioidei represent an extraordinary example of radiation in the cold. In addition to the absence of hemoglobin, icefish show a number of other striking peculiarities including large-diameter blood vessels, high vascular densities, mitochondria-rich muscle cells, and unusual mitochondrial architecture. In order to investigate the bases of icefish adaptation to the extreme Southern Ocean conditions we sequenced the complete genome of the icefish Chionodraco myersi. Comparative analyses of the icefish genome with those of other teleost species, including two additional white-blooded and five red-blooded notothenioids, provided a new perspective on the evolutionary loss of globin genes. Muscle transcriptome comparative analyses against red-blooded notothenioids as well as temperate fish revealed the peculiar regulation of genes involved in mitochondrial function in icefish. Gene duplication and promoter sequence divergence were identified as genome-wide patterns that likely contributed to the broad transcriptional program underlying the unique features of icefish mitochondria.

Host-microbiota interactions shed light on mortality events in the striped venus clam Chamelea gallina.

Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate- and pollution-induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA-sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over-expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down-regulation of several genes involved in immune and stress response, and the over-representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment.

Along-shelf connectivity and circumpolar gene flow in Antarctic silverfish (Pleuragramma antarctica).

The Antarctic silverfish (Pleuragramma antarctica) is a critically important forage species with a circumpolar distribution and is unique among other notothenioid species for its wholly pelagic life cycle. Previous studies have provided mixed evidence of population structure over regional and circumpolar scales. The aim of the present study was to test the recent population hypothesis for Antarctic silverfish, which emphasizes the interplay between life history and hydrography in shaping connectivity. A total of 1067 individuals were collected over 25 years from different locations on a circumpolar scale. Samples were genotyped at fifteen microsatellites to assess population differentiation and genetic structuring using clustering methods, F-statistics, and hierarchical analysis of variance. A lack of differentiation was found between locations connected by the Antarctic Slope Front Current (ASF), indicative of high levels of gene flow. However, gene flow was significantly reduced at the South Orkney Islands and the western Antarctic Peninsula where the ASF is absent. This pattern of gene flow emphasized the relevance of large-scale circulation as a mechanism for circumpolar connectivity. Chaotic genetic patchiness characterized population structure over time, with varying patterns of differentiation observed between years, accompanied by heterogeneous standard length distributions. The present study supports a more nuanced version of the genetic panmixia hypothesis that reflects physical-biological interactions over the life history.

The population genomics of yellowfin tuna (Thunnus albacares) at global geographic scale challenges current stock delineation.

Yellowfin tuna, Thunnus albacares, is one of the most important seafood commodities in the world. Despite its great biological and economic importance, conflicting evidence arises from classical genetic and tagging studies concerning the yellowfin tuna population structure at local and global oceanic scales. Access to more powerful and cost effective genetic tools would represent the first step towards resolving the population structure of yellowfin tuna across its distribution range. Using a panel of 939 neutral Single Nucleotide Polymorphisms (SNPs), and the most comprehensive data set of yellowfin samples available so far, we found genetic differentiation among the Atlantic, Indian and Pacific oceans. The genetic stock structure analysis carried out with 33 outlier SNPs, putatively under selection, identified discrete populations within the Pacific Ocean and, for the first time, also within the Atlantic Ocean. Stock assessment approaches that consider genetic differences at neutral and adaptive genomic loci should be routinely implemented to check the status of the yellowfin tuna, prevent illegal trade, and develop more sustainable management measures.

Population genomics meet Lagrangian simulations: Oceanographic patterns and long larval duration ensure connectivity among Paracentrotus lividus populations in the Adriatic and Ionian seas.

Connectivity between populations influences both their dynamics and the genetic structuring of species. In this study, we explored connectivity patterns of a marine species with long-distance dispersal, the edible common sea urchin Paracentrotus lividus, focusing mainly on the Adriatic-Ionian basins (Central Mediterranean). We applied a multidisciplinary approach integrating population genomics, based on 1,122 single nucleotide polymorphisms (SNPs) obtained from 2b-RAD in 275 samples, with Lagrangian simulations performed with a biophysical model of larval dispersal. We detected genetic homogeneity among eight population samples collected in the focal Adriatic-Ionian area, whereas weak but significant differentiation was found with respect to two samples from the Western Mediterranean (France and Tunisia). This result was not affected by the few putative outlier loci identified in our dataset. Lagrangian simulations found a significant potential for larval exchange among the eight Adriatic-Ionian locations, supporting the hypothesis of connectivity of P. lividus populations in this area. A peculiar pattern emerged from the comparison of our results with those obtained from published P. lividus cytochrome b (cytb) sequences, the latter revealing genetic differentiation in the same geographic area despite a smaller sample size and a lower power to detect differences. The comparison with studies conducted using nuclear markers on other species with similar pelagic larval durations in the same Adriatic-Ionian locations indicates species-specific differences in genetic connectivity patterns and warns against generalizing single-species results to the entire community of rocky shore habitats.

An integrated genomic approach for the study of mandibular prognathism in the European seabass (Dicentrarchus labrax).

Skeletal anomalies in farmed fish are a relevant issue affecting animal welfare and health and causing significant economic losses. Here, a high-density genetic map of European seabass for QTL mapping of jaw deformity was constructed and a genome-wide association study (GWAS) was carried out on a total of 298 juveniles, 148 of which belonged to four full-sib families. Out of 298 fish, 107 were affected by mandibular prognathism (MP). Three significant QTLs and two candidate SNPs associated with MP were identified. The two GWAS candidate markers were located on ChrX and Chr17, both in close proximity with the peaks of the two most significant QTLs. Notably, the SNP marker on Chr17 was positioned within the Sobp gene coding region, which plays a pivotal role in craniofacial development. The analysis of differentially expressed genes in jaw-deformed animals highlighted the "nervous system development" as a crucial pathway in MP. In particular, Zic2, a key gene for craniofacial morphogenesis in model species, was significantly down-regulated in MP-affected animals. Gene expression data revealed also a significant down-regulation of Sobp in deformed larvae. Our analyses, integrating transcriptomic and GWA methods, provide evidence for putative mechanisms underlying seabass jaw deformity.

Integrated studies of organismal plasticity through physiological and transcriptomic approaches: examples from marine polar regions.

Transcriptomic methods are now widely used in functional genomic research. The vast amount of information received from these studies comes along with the challenge of developing a precise picture of the functional consequences and the characteristic regulatory mechanisms. Here we assess recent studies in marine species and their adaptation to polar (and seasonal) cold and explore how they have been able to draw reliable conclusions from transcriptomic patterns on functional consequences in the organisms. Our analysis indicates that the interpretation of transcriptomic data suffers from insufficient understanding of the consequences for whole organism performance and fitness and comes with the risk of supporting only preliminary and superficial statements.We propose that the functional understanding of transcriptomic data may be improved by their tighter integration into overarching physiological concepts that support the more specific interpretation of the 'omics' data and, at the same time, can be developed further through embedding the transcriptomic phenomena observed. Such possibilities have not been fully exploited.In the context of thermal adaptation and limitation, we explore preliminary evidence that the concept of oxygen and capacity limited thermal tolerance (OCLTT) may provide sufficient complexity to guide the integration of such data and the development of associated functional hypotheses. At the same time, we identify a lack of methodological approaches linking genes and function to higher levels of integration, in terms of organism and ecosystem functioning, at temporal and geographical scales, to support more reliable conclusions and be predictive with respect to the effects of global changes.

Microsatellite markers for the notothenioid fish Lepidonotothen nudifrons and two congeneric species.

BACKGROUND: Loss of genetic variability due to environmental changes, limitation of gene flow between pools of individuals or putative selective pressure at specific markers, were previously documented for Antarctic notothenioid fish species. However, so far no studies were performed for the Gaudy notothen Lepidonotothen nudifrons. Starting from a species-specific spleen transcriptome library, we aimed at isolating polymorphic microsatellites (Type I; i.e. derived from coding sequences) suitable to quantify the genetic variability in this species, and additionally to assess the population genetic structure and demography in nototheniids. RESULTS: We selected 43,269 transcripts resulting from a MiSeq sequencer run, out of which we developed 19 primer pairs for sequences containing microsatellite repeats. Sixteen loci were successfully amplified in L. nudifrons. Eleven microsatellites were polymorphic and allele numbers per locus ranged from 2 to 17. In addition, we amplified loci identified from L. nudifrons in two other congeneric species (L. squamifrons and L. larseni). Thirteen loci were highly transferable to the two congeneric species. Differences in polymorphism among species were detected. CONCLUSIONS: Starting from a transcriptome of a non-model organism, we were able to identify promising polymorphic nuclear markers that are easily transferable to other closely related species. These markers can be a key instrument to monitor the genetic structure of the three Lepidonotothen species if genotyped in larger population samples. When compared with anonymous loci isolated in other notothenioids, i.e. Type II (isolated from genomic libraries), they offer the possibility to test how the effects of occurring environmental change influence the population genetic structure in each species and subsequently the composition of the entire ecosystem.

Non-Antarctic notothenioids: Past phylogenetic history and contemporary phylogeographic implications in the face of environmental changes.

The non-Antarctic Notothenioidei families, Bovichtidae, Pseudaphritidae and Eleginopsidae, diverged early from the main notothenioid lineage. They are important in clarifying the early evolutionary processes that triggered notothenioid evolution in the Antarctic. The early-diverged group represents 8% of all notothenioid species and never established themselves on the Antarctic shelf. Most attention has been paid to the Antarctic notothenioids and their limited physiological tolerance to climate change and increased temperatures. In this review, we discuss key life history traits that are characteristic of the non-Antarctic early-diverged notothenioid taxa as well as the genetic resources and population differentiation information available for this group. We emphasise the population fitness and dynamics of these species and indicate how resource management and conservation of the group can be strengthened through an integrative approach. Both Antarctic waters and the non-Antarctic regions face rapid temperature rises combined with strong anthropogenic exploitation. While it is expected that early-diverged notothenioid species may have physiological advantages over high Antarctic species, it is difficult to predict how climate changes might alter the geographic range, behaviour, phenology and ultimately genetic variability of these species. It is possible, however, that their high degree of endemism and dependence on local environmental specificities to complete their life cycles might enhance their vulnerability.

Methodological assessment of 2b-RAD genotyping technique for population structure inferences in yellowfin tuna (Thunnus albacares).

Global population genetic structure of yellowfin tuna (Thunnus albacares) is still poorly understood despite its relevance for the tuna fishery industry. Low levels of genetic differentiation among oceans speak in favour of the existence of a single panmictic population worldwide of this highly migratory fish. However, recent studies indicated genetic structuring at a much smaller geographic scales than previously considered, pointing out that YFT population genetic structure has not been properly assessed so far. In this study, we demonstrated for the first time, the utility of 2b-RAD genotyping technique for investigating population genetic diversity and differentiation in high gene-flow species. Running de novo pipeline in Stacks, a total of 6772 high-quality genome-wide SNPs were identified across Atlantic, Indian and Pacific population samples representing all major distribution areas. Preliminary analyses showed shallow but significant population structure among oceans (FST=0.0273; P-value<0.01). Discriminant Analysis of Principal Components endorsed the presence of genetically discrete yellowfin tuna populations among three oceanic pools. Although such evidence needs to be corroborated by increasing sample size, these results showed the efficiency of this genotyping technique in assessing genetic divergence in a marine fish with high dispersal potential.

A first insight into the spleen transcriptome of the notothenioid fish Lepidonotothen nudifrons: Resource description and functional overview.

In this study, we describe a de novo sequencing and assembly of the spleen transcriptome of Lepidonotothen nudifrons, a notothenioid fish widely distributed around the Antarctic Peninsula and the Scotia Arc. Sequences were generated on an Illumina MiSeq system and assembled to a total of 112,477 transcripts. Putative functional annotation was possible for more than 34% of the transcripts. This data will be relevant for future studies targeting the erythrocyte turnover, oxygen transport mechanism and immune system, which are key functional traits to investigate cold adaptation and thermal sensitivity of Antarctic notothenioids.

First evidence of inbreeding, relatedness and chaotic genetic patchiness in the holoplanktonic jellyfish Pelagia noctiluca (Scyphozoa, Cnidaria).

Genetic drift and non-random mating seldom influence species with large breeding populations and high dispersal potential, characterized by unstructured gene pool and panmixia at a scale lower than the minimum dispersal range of individuals. In the present study, a set of nine microsatellite markers was developed and used to investigate the spatio-temporal genetic patterns of the holoplanktonic jellyfish Pelagia noctiluca (Scyphozoa) in the Southern Tyrrhenian Sea. Homozygote excess was detected at eight loci, and individuals exhibited intra-population relatedness higher than expected by chance in at least three samples. This result was supported by the presence of siblings in at least 5 out 8 samples, 4 of which contained full-sib in addition to half-sib dyads. Having tested and ruled out alternative explanations as null alleles, our results suggest the influence of reproductive and behavioural features in shaping the genetic structure of P. noctiluca, as outcomes of population genetics analyses pointed out. Indeed, the genetic differentiation among populations was globally small but highlighted: a) a spatial genetic patchiness uncorrelated with distance between sampling locations, and b) a significant genetic heterogeneity between samples collected in the same locations in different years. Therefore, despite its extreme dispersal potential, P. noctiluca does not maintain a single homogenous population, but rather these jellyfish appear to have intra-bloom localized recruitment and/or individual cohesiveness, whereby siblings more likely swarm together as a single group and remain close after spawning events. These findings provide the first evidence of family structures and consequent genetic patchiness in a species with highly dispersive potential throughout its whole life cycle, contributing to understanding the patterns of dispersal and connectivity in marine environments.

Ecological and genetic differences between Cacopsylla melanoneura (Hemiptera, Psyllidae) populations reveal species host plant preference.

The psyllid Cacopsylla melanoneura is considered one of the vectors of 'Candidatus Phytoplasma mali', the causal agent of apple proliferation disease. In Northern Italy, overwintered C. melanoneura adults reach apple and hawthorn around the end of January. Nymph development takes place between March and the end of April. The new generation adults migrate onto conifers around mid-June and come back to the host plant species after overwintering. In this study we investigated behavioural differences, genetic differentiation and gene flow between samples of C. melanoneura collected from the two different host plants. Further analyses were performed on some samples collected from conifers. To assess the ecological differences, host-switching experiments were conducted on C. melanoneura samples collected from apple and hawthorn. Furthermore, the genetic structure of the samples was studied by genotyping microsatellite markers. The examined C. melanoneura samples performed better on their native host plant species. This was verified in terms of oviposition and development of the offspring. Data resulting from microsatellite analysis indicated a low, but statistically significant difference between collected-from-apple and hawthorn samples. In conclusion, both ecological and genetic results indicate a differentiation between C. melanoneura samples associated with the two host plants.