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Living in high latitudes and altitudes sets specific requirements on species’ ability to forecast seasonal changes and to respond to them in an appropriate way. Adaptation into diverse environmental conditions can also lead to ecological speciation through habitat isolation or by inducing changes in traits that influence assortative mating. In this review, we explain how the unique time-measuring systems of Drosophila virilis group species have enabled the species to occupy high latitudes and how the traits involved in species reproduction and survival exhibit strong linkage with latitudinally varying photoperiodic and climatic conditions. We also describe variation in reproductive barriers between the populations of two species with overlapping distributions and show how local adaptation and the reinforcement of prezygotic barriers have created partial reproductive isolation between conspecific populations. Finally, we consider the role of species-specific chromosomal inversions and the X chromosome in the development of reproductive barriers between diverging lineages. peerReviewed

Late embryogenesis abundant (LEA) proteins are important players in the management of responses to stressful conditions, such as drought, high salinity, and changes in temperature. Many LEA proteins do not have defined three-dimensional structures, so they are intrinsically disordered proteins (IDPs) and are often highly hydrophilic. Although LEA-like sequences have been identified in bacterial genomes, the functions of bacterial LEA proteins have been studied only recently. Sequence analysis of outer membrane interleukin receptor I (BilRI) from the oral pathogen Aggregatibacter actinomycetemcomitans indicated that it shared sequence similarity with group 3/3b/4 LEA proteins. Comprehensive nuclearcgq magnetic resonance (NMR) studies confirmed its IDP nature, and expression studies in A. actinomycetemcomitans harboring a red fluorescence reporter protein-encoding gene revealed that bilRI promoter expression was increased at decreased temperatures. The amino acid backbone of BilRI did not stimulate either the production of reactive oxygen species from human leukocytes or the production of interleukin-6 from human macrophages. Moreover, BilRI-specific IgG antibodies could not be detected in the sera of A. actinomycetemcomitans culture-positive periodontitis patients. Since the bilRI gene is located near genes involved in natural competence (i.e., genes associated with the uptake of extracellular (eDNA) and its incorporation into the genome), we also investigated the role of BilRI in these events. Compared to wild-type cells, the ΔbilRI mutants showed a lower transformation efficiency, which indicates either a direct or indirect role in natural competence. In conclusion, A. actinomycetemcomitans might express BilRI, especially outside the host, to survive under stressful conditions and improve its transmission potential. peerReviewed

BackgroundTracing the association between insect cold tolerance and latitudinally and locally varying environmental conditions, as well as key morphological traits and molecular mechanisms, is essential for understanding the processes involved in adaptation. We explored these issues in two closely-related species, Drosophila montana and Drosophila flavomontana, originating from diverse climatic locations across several latitudes on the coastal and mountainous regions of North America. We also investigated the association between sequence variation in one of the key circadian clock genes, vrille, and cold tolerance in both species. Finally, we studied the impact of vrille on fly cold tolerance and cold acclimation ability by silencing it with RNA interference in D. montana.ResultsWe performed a principal component analysis (PCA) on variables representing bioclimatic conditions on the study sites and used latitude as a proxy of photoperiod. PC1 separated the mountainous continental sites from the coastal ones based on temperature variability and precipitation, while PC2 arranged the sites based on summer and annual mean temperatures. Cold tolerance tests showed D. montana to be more cold-tolerant than D. flavomontana and chill coma resistance (CTmin) of this species showed an association with PC2. Chill coma recovery time (CCRT) of both species improved towards northern latitudes, and in D. flavomontana this trait was also associated with PC1. D. flavomontana flies were darkest in the coast and in the northern mountainous populations, but coloration showed no linkage with cold tolerance. Body size decreased towards cold environments in both species, but only within D. montana populations largest flies showed fastest recovery from cold. Finally, both the sequence analysis and RNAi study on vrille suggested this gene to play an essential role in D. montana cold resistance and acclimation, but not in recovery time.ConclusionsOur study demonstrates the complexity of insect cold tolerance and emphasizes the need to trace its association with multiple environmental variables and morphological traits to identify potential agents of natural selection. It also shows that a circadian clock gene vrille is essential both for short- and long-term cold acclimation, potentially elucidating the connection between circadian clock system and cold tolerance.

Funding: This work was supported by Academy of Finland projects 268214 and 322980 to MK and a NERC (UK) grant NE/P000592/1 to MGR. Organisms can plastically alter resource allocation in response to changing environmental factors. For example, in harsh conditions, organisms are expected to shift investment from reproduction toward survival; however, the factors and mechanisms that govern the magnitude of such shifts are relatively poorly studied. Here we compared the impact of cold on males and females of the highly cold-tolerant species Drosophila montana at the phenotypic and transcriptomic levels. Although both sexes showed similar changes in cold tolerance and gene expression in response to cold treatment, indicating that the majority of changes are concordant between the sexes, we identified a clear reduction in sexually dimorphic gene expression, suggesting that preparing for the colder season involves reducing investment in sex-specific traits. This reduction was larger in males than females, as expected if male sexual traits are more condition-dependent than female traits, as predicted by theory. Gene expression changes were primarily associated with shifts in metabolic profile, which likely play a role in increasing cold tolerance. Finally, we found that the expression of immune genes was reduced following cold treatment, suggesting that reduced investment in costly immune function may be important in helping flies survive colder periods. Publisher PDF

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During winter insects face energetic stress driven by lack of food, and thermal stress due to sub-optimal and even lethal temperatures. To survive, most insects living in seasonal environments such as high latitudes, enter diapause, a deep resting stage characterized by a cessation of development, metabolic suppression and increased stress tolerance. The current study explores physiological adaptations related to diapause in three beetle species at high latitudes in Europe. From an ecological perspective, the comparison is interesting since one species (Leptinotarsa decemlineata) is an invasive pest that has recently expanded its range into northern Europe, where a retardation in range expansion is seen. By comparing its physiological toolkit to that of two closely related native beetles (Agelastica alni and Chrysolina polita) with similar overwintering ecology and collected from similar latitude, we can study if harsh winters might be constraining further expansion. Our results suggest all species suppress metabolism during diapause and build large lipid stores before diapause, which then are used sparingly. In all species diapause is associated with temporal shifts in storage and membrane lipid profiles, mostly in accordance with the homeoviscous adaptation hypothesis, stating that low temperatures necessitate acclimation responses that increase fluidity of storage lipids, allowing their enzymatic hydrolysis, and ensure integral protein functions. Overall, the two native species had similar lipidomic profiles when compared to the invasive species, but all species showed specific shifts in their lipid profiles after entering diapause. Taken together, all three species show adaptations that improve energy saving and storage and membrane lipid fluidity during overwintering diapause. While the three species differed in the specific strategies used to increase lipid viscosity, the two native beetle species showed a more canalized lipidomic response, than the recent invader. Since close relatives with similar winter ecology can have different winter ecophysiology, extrapolations among species should be done with care. Still, range expansion of the recent invader into high latitude habitats might indeed be retarded by lack of physiological tools to manage especially thermal stress during winter, but conversely species adapted to long cold winters may face these stressors as a consequence of ongoing climate warming. peerReviewed

Funding: UK Natural Environment Research Council (Grant Number(s): NE/L501852/1, NE/P000592/1); Academy of Finland (GrantNumber(s): 267244, 268214, 322980), Ella ja Georg Ehrnroothin Säätiö. Detecting signatures of ecological adaptation in comparative genomics is challenging, but analysing population samples with characterised geographic distributions, such as clinal variation, can help identify genes showing covariation with important ecological variation. Here, we analysed patterns of geographic variation in the cold-adapted species Drosophila montana across phenotypes, genotypes and environmental conditions and tested for signatures of cold adaptation in population genomic divergence. We first derived the climatic variables associated with the geographic distribution of 24 populations across two continents to trace the scale of environmental variation experienced by the species, and measured variation in the cold tolerance of the flies of six populations from different geographic contexts. We then performed pooled whole genome sequencing of these six populations, and used Bayesian methods to identify SNPs where genetic differentiation is associated with both climatic variables and the population phenotypic measurements, while controlling for effects of demography and population structure. The top candidate SNPs were enriched on the X and fourth chromosomes, and they also lay near genes implicated in other studies of cold tolerance and population divergence in this species and its close relatives. We conclude that ecological adaptation has contributed to the divergence of D. montana populations throughout the genome and in particular on the X and fourth chromosomes, which also showed highest interpopulation FST. This study demonstrates that ecological selection can drive genomic divergence at different scales, from candidate genes to chromosome-wide effects. Publisher PDF

Alternative splicing, in which one gene produce multiple transcripts, may influence how adaptive genes respond to specific environments. A newly produced transcriptome of Drosophila montana shows the Gs1-like (Gs1l) gene to express multiple splice variants and to be down regulated in cold acclimated flies with increased cold tolerance. Gs1l’s effect on cold tolerance was further tested by injecting cold acclimated and non-acclimated flies from two distantly located northern and southern fly populations with double stranded RNA (dsRNA) targeting Gs1l. While both populations had similar cold acclimation responses, dsRNA injections only effected the northern population. The nature of splicing expression was then investigated in the northern population by confirming which Gs1l variants are present, by comparing the expression of different gene regions and by predicting the protein structures of splices using homology modelling. We find different splices of Gs1l not only appear to have independent impacts on cold acclimation but also elicit different effects in populations originating from two very different environments. Also, at the protein level, Gs1l appears homologous to the human HDHD1A protein and some splices might produce functionally different proteins though this needs to be verified in future studies by measuring the particular protein levels. Taken together, Gs1l appears to be an interesting new candidate to test how splicing influences adaptations.

The work has been supported by the Academyof Finland to A.H. (project 267244) and Natural Environment Research Council (NERC) funding (NE/J020818/1 to M.G.R.; NE/L501852/1 to R.A.W.W.). The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are inter-related is not well understood. We performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and/or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence from the control line in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled those detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, our study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity. Publisher PDF

Biocatalysts are the backbone of bioprocessing industries that are going through a phase of transition with reference to the requirement of extraordinary enzymes for various biochemical processes. This transition is well reported and documented by various researchers through elucidation of different features and applications of mesophilic and thermophilic enzymes. However, there is little information available about psychrophilic enzymes and their involvement in industrial processes. Therefore, understanding the features and functions of psychrophilic enzymes could suggest some of their novel applications in various industries such as food, agriculture, chemicals, pharmaceuticals, and waste management etc. Currently, different industries are looking for such novel psychrophilic enzymes to develop efficient biochemical processes that will help to reduce the reaction time, lower the energy inputs and as well as be eco-friendly. These bioprocesses will help to increase the profit margin by reducing the overall cost of the final products. This review article will provide new insights in technical and scientific analysis of psychrophilic microbes, their enzymes and low energy biochemical processes that are useful in various industries for the production of valuable products. It will also further strengthen the understanding of academia and industry about these ubiquitous biocatalysts. peerReviewed

Selection experiments offer an efficient way to study the evolvability of traits that play an important role in insects’ reproduction and/or survival and to trace correlations and trade-offs between them. We have exercised bi-directional selection on Drosophila montana flies’ pre-adult development time under constant light and temperature conditions for 10 generations and traced the indirect effects of this selection on females’ diapause induction under different day lengths, as well as on the body weight and cold tolerance of both sexes. Overall, selection was successful towards slow, but not towards fast development. However, all fast selection line replicates showed at the end of selection increased variance in females’ photoperiodic diapause response and about one hour increase in the critical day (CDL), where more than 50% of emerging females enter diapause. Indirect effects of selection on flies’ body weight and cold-tolerance were less clear, as the flies of the slow selection line were significantly heavier and less cold-tolerant than the control line flies after five generations of selection, but lighter and more cold-tolerant at the end of selection. Changes in females’ diapause induction resulting from selection for fast development could be due to common metabolic pathways underlying these traits, collaboration of circadian clock and photoperiodic timer and/or by the interaction between the endocrine and circadian systems. peerReviewed

This work was supported by the Academy of Finland to AH (projects 132619 and 267244) and to MK (projects 268214 and 272927) and NERC (UK) funding to MGR (grants NE/E015255/1 and NE/J020818/1) and PhD studentship to DJP (NE/I528634/1). The genomes of species that are ecological specialists will likely contain signatures of genomic adaptation to their niche. However, distinguishing genes related to ecological specialism from other sources of selection and more random changes is a challenge. Here we describe the genome of Drosophila montana, which is the most extremely cold-adapted Drosophila species. We use branch tests to identify genes showing accelerated divergence in contrasts between cold- and warm adapted species and identify about 250 genes that show differences, possibly driven by a lower synonymous substitution rate in cold-adapted species. We look for evidence of accelerated divergence between D. montana and D. virilis, a previously sequenced relative, and do not find strong evidence for divergent selection on coding sequence variation. Divergent genes are involved in a variety of functions, including cuticular and olfactory processes. We also re-sequenced three populations of D. montana from its ecological and geographic range. Outlier loci were more likely to be found on the X chromosome and there was a greater than expected overlap between population outliers and those genes implicated in cold adaptation between Drosophila species, implying some continuity of selective process at these different evolutionary scales. Publisher PDF

The life histories of ectoparasites are shaped by both host and off-host environment. A suitable host is primarily needed during reproduction, whereas juvenile stages outside the host are directly exposed to environmental variability. Viviparity, i.e. the development of an embryo inside the body of the mother resulting in large offspring size, increases offspring survival. The production of large offspring has its consequences in terms of high variation in offspring age and in the environment that each young individual will face. I used a viviparous ectoparasite, the deer ked (Lipoptena cervi), to investigate the consequences of long reproductive lifespan and varying offspring environment on offspring life-histories and seasonal adaptations. Offspring life-histories varied seasonally. I showed that the resources provided by the deer ked females determine offspring performance throughout its off-host period. Offspring size increased towards the spring and the end of the reproductive period, and simultaneously offspring survival and cold tolerance increased. Seasonal variation in offspring size did not reflect the resources that would guarantee offspring survival during the longest diapause or the highest cold tolerance during the harshest winter period. Diapause intensity varies with birth time according to the expected length of the winter ahead. However, the deer ked pupae, regardless of their age, overwinter at an opportunistic diapause, which may be terminated rapidly only by an exposure to high temperature. Contrary to general observations, photoperiod has no role in regulating the seasonal shifts of the deer ked. Neither is high cold tolerance associated only with diapause, but it remains high through four seasons, also in the active developmental and adult stages. I also evaluated the effects of life-history variation on the invasion potential of the deer ked. I conducted a large-scale transplant experiment to test the survival and pupal development at and beyond the current range. I found that the lower spring and summer temperatures and the shorter growth season in the north cause a deterioration in pupal performance and shorten the flight period. However, the colder climate may not totally prevent further spread. A more important factor that will affect deer ked invasion is host availability, and especially in Finland, the density of the moose population. Seasonal variation in offspring life histories in viviparous ectoparasites differs from the variation patterns reported in most invertebrates. This may be due to the extremely large offspring size and to the fact that maternally derived resources determine offspring performance through the entire off-host period. Variation in offspring performance is thus determined by maternal resources and seasonal variation in the condition of the moose. Tiivistelmä Useimpien ulkoloisten elinkierto on riippuvainen isännästä ja isännän ulkopuolella kasvavien jälkeläisten kohtaamista ympäristöoloista. Viviparia eli jälkeläisen kehitys naaraan sisällä ja siitä usein seuraava suuri jälkeläiskoko parantavat jälkeläisten selviytymistä. Suurten jälkeläisten tuottaminen pitkällä aikavälillä johtaa siihen, että eri-ikäiset jälkeläiset kohtaavat vuodenajasta riippuen hyvin erilaiset olosuhteet. Väitöstyössäni tarkastelin Suomessa nopeasti yleistyneen hirvieläinten ulkoloisen, hirvikärpäsen (Lipoptena cervi), avulla, mitä seurauksia viviparialla, pitkällä lisääntymiskaudella ja ympäristön vuodenaikaisvaihtelulla on jälkeläisten elinkierto-ominaisuuksiin. Väitöskirjatyössäni havaitsin, että hirvikärpäsjälkeläisten elinkiertopiirteet vaihtelevat jälkeläisen syntymäajan mukaan. Osoitin myös, että hirvikärpäsnaaraan jälkeläisilleen tarjoamat ravintovarat määrittelevät jälkeläisten isännästä riippumattoman elinkierron aikaisen menestyksen. Jälkeläisten keskimääräinen koko kasvoi lisääntymiskauden edetessä talvesta kohti kevättä, jolloin myös selviytyminen ja kylmänsietokyky paranivat. Jälkeläiskoon vuodenaikaisvaihtelu ei näin ollen vastaa jälkeläisten tarvitsemia resursseja suhteessa talvehtimisen pituuteen eikä korreloi koville talvipakkasille altistumisen todennäköisyyden kanssa. Lepotilan keston määrittelevä diapaussin syvyys vaihteli syntymävuodenaikaa vastaavasti. Diapaussin havaitsin kuitenkin olevan ensisijaisesti opportunistinen, jolloin pelkästään korkea lämpötila voi nopeasti päättää lepotilan kaikenikäisillä yksilöillä. Vastoin yleisiä käsityksiä valorytmi ei vaikuta diapaussin säätelyyn. Vastoin ennakko-odotuksia kylmänsietokyky säilyy korkeana vuoden ympäri ja kaikissa tutkituissa elinkierron vaiheissa. Sovelsin tutkimieni elinkiertopiirteiden vaikutusta myös lajin invaasiokykyyn, ja tutkin istutuskokeen avulla koteloiden selviytymistä ja kehitystä nykyisellä esiintymisalueella ja sen pohjoispuolella. Alhaisemmat kevät- ja kesälämpötilat sekä lyhyempi kasvukausi vähentävät aikuiseksi selviytymistä ja lyhentävät lentoaikaa syksyllä. Ilmastotekijöiden suhteen hirvikärpänen voisi esiintyä nykyistä pohjoisempana. Tärkein tekijä hirvikärpäsen leviämistä tarkasteltaessa on kuitenkin sopivien isäntäeläimien saatavuus ja Suomen oloissa erityisesti hirven eli hirvikärpäsen pääisännän kannan tiheys. Tutkimukseni perusteella ulkoloisten vivipariasta seuraava jälkeläisten elinkiertopiirteiden ajallinen vaihtelu eroaa muiden selkärangattomien vastaavasta vaihtelusta. Yhtenä syynä eroihin lienee se, että hirvikärpäsnaaras tuottaa erityisen suuria jälkeläisiä ja että jälkeläiset puolestaan ovat täysin riippuvaisia emon antamista resursseista. Emon lisääntymisresurssit ja hirven kunnon vuodenaikaisvaihtelu vaikuttavat mahdollisesti siihen, minkälaisia jälkeläisiä hirvikärpänen milloinkin kykenee tuottamaan.

SUMMARY Coping with seasonal changes in temperature is an important factor underlying the ability of insects to survive over the harsh winter conditions in the northern temperate zone, and only a few drosophilids have been able to colonize sub-polar habitats. Information on their winter physiology is needed as it may shed light on the adaptive mechanisms of overwintering when compared with abundant data on the thermal physiology of more southern species, such as Drosophila melanogaster. Here we report the first seasonal metabolite analysis in a Drosophila species. We traced changes in the cold tolerance and metabolomic profiles in adult Drosophila montana flies that were exposed to thermoperiods and photoperiods similar to changes in environmental conditions of their natural habitat in northern Finland. The cold tolerance of diapausing flies increased noticeably towards the onset of winter; their chill coma recovery times showed a seasonal minimum between late autumn and early spring, whereas their survival after cold exposure remained high until late spring. The flies had already moderately accumulated glucose, trehalose and proline in autumn, but the single largest change occurred in myo-inositol concentrations. This increased up to 400-fold during the winter and peaked at 147 nmol mg−1 fresh mass, which is among the largest reported accumulations of this compound in insects.

Kylmyys on yksi tärkeimmistä lajien levinneisyyteen ja runsauteen vaikuttavista stressitekijöistä. Karaistuminen ja lisääntymisdiapaussi ovat esimerkkejä hyönteisten fysiologisesta ja biokemiallisesta sopeutumista mataliin lämpötiloihin. Karaistumisella tarkoitetaan lyhyen ajan sisällä tapahtuvaa kylmänkestävyyden kasvamista, ja sillä voi olla huomattava vaikutus kylmistä olosuhteista selviytymiseen. Lisääntymisdiapaussin laukaisee ympäristöstä tulevat ärsykkeet, kuten muutokset päivän pituudessa ja lämpötilassa. Se parantaa naaraiden mahdollisuuksia selvitä epäedullisista vuodenajoista ja siirtää jälkeläisten tuoton ajankohtaan jolloin munien ja jälkeläisten selviytyminen on parhaimmillaan. Tämän työn tavoitteena oli etsiä sopivat lämpötilat ja käsittelyajat Drosophila montana -mahlakärpästen kylmänkestävyyden mittaamiseen ja selvittää vaikuttavatko lyhytaikainen karaistuminen ja lisääntymisdiapaussi naaraiden ja koiraiden kylmänkestävyyteen. Tutkimuslajina käytettiin D. montana -lajia, joka on sopeutunut talvehtimaan pohjoisissa luonnonoloissa. Kärpästen kylmänkestävyyttä tutkittiin mittaamalla niiden kylmäkoomasta selviytymiseen kuluva aika. Tutkittavia yksilöitä pidettiin aluksi kaksi viikkoa kasvatuskaapeissa (+16 C), joista toisessa oli jatkuva valaistus ja toisessa lisääntymisdiapaussin laukaiseva valojakso 16:8 (16 tuntia valoa ja 8 tuntia pimeää). Ennen kylmänkestävyyskoetta puolet yksilöistä sai karaistumiskäsittelyn (1 tunti 0 C:ssa). Varsinaiset kylmänkestävyyskokeet kestivät 16 tuntia ja ne tehtiin kolmessa eri lämpötilassa (−6 C, −7 C ja −8 C). Tämän tutkimuksen perusteella voitiin päätellä, että Drosophila montanan kylmäkoomasta selviämiseen kuluva aika voidaan parhaiten mitata −6 ˚C:ssa altistusajan ollessa 16 tuntia. Tämä lämpötila oli riittävä käsittelyjen välisten erojen selvittämiseksi, mutta ei liian rankka kärpästen selviytymisen kannalta. Tutkimuksen perusteella lisääntymisdiapaussi parantaa naaraiden ja samoissa olosuhteissa kasvatettujen koiraiden kylmänkestävyyttä, karaistumisen vaikutuksen ollessa selkein naarailla, jotka eivät ole diapaussissa. Diapaussin ja karaistumisen aikaansaamat muutokset kärpäsen fysiologiassa voivat olla samankaltaisia, ja näin karaistumisen antama lisähyöty on lähes olematonta tai sattumanvaraista lisääntymisdiapaussissa oleville yksilöille.

The European Pine Sawfly (Neodiprion sertifer Geoffroy) is one of the most serious defoliators of Scots pine (Pinus sylvestris L.) in northern Europe. We studied the pattern in the regional occurrence of the outbreaks of N. sertifer in Finland in years 1961-90, and made predictions about the outbreak pattern to the year 2050 after predicted winter warming. We tested whether minimum winter temperatures and forest type and soil properties could explain the observed outbreak pattern. We analysed outbreak patterns at two different spatial levels: forest board- and municipal-level. The proportion of coniferous forests on damage-susceptible soils (dry and infertile sites) explained a significant part of the variation in outbreak frequency at small spatial scale (municipalities) but not at large spatial scale (forest boards). At the forest board level, the incidence of minimum temperatures below -36 °C (= the critical value for egg mortality) explains 33% of the variation in the outbreak pattern, and at the municipal level the incidence of cold winters was also the most significant explaining variable in northern Finland. Egg mortality due to cold winters seems to be the most parsimonious factor explaining why there have been so few N. sertifer outbreaks in northern and north-eastern Finland. We predict that climate change (increased winter temperatures) may increase the frequency of outbreaks in eastern and northern Finland in the future.

Seedlings from four Norway spruce ( (L.) H. Karst.) stands originating from areas with effective temperature sums ranging from 710 d.d. to 1,150 d.d. were raised under artificial light and temperature treatment. After a 10-week growing period the hardening process was started by subjecting the seedlings to +8°C night temperature and +15°C day temperature, and increasing the night length by 1.5 hour/week. Hardiness was measured by means of artificial freezing treatment (-10°C or -15°C), followed by visual estimation of the degree of needle injury. The stem height, lignification and bud development were measured before the freezing treatment. The amount of injury increased the more southern the origin of the tested material was. Furthermore, the proportion of non-lignified part of the seedling stem was negatively correlated with the latitude of the provenances. The proportion of seedlings with clearly visible buds was more than 90% in the northernmost entry and less than 1% in the southernmost one. The overall correlation coefficient between the needle injuries and the proportion of non-lignified part of the stem was rather high, but varied considerably from 0.3 in the northernmost material to over 0.6 in the southern provenances. According to the results, it seems to be possible to use growth characteristics as an indicator of frost hardiness at the provenance level. The PDF includes an abstract in Finnish.Picea abies

Electrical impedance characteristics of plant cells are dependent on such physiological factors as physiological condition, developmental stage, cell structure, nutrient status, water balance and temperature acclimation. In the measurements also such technical and physical factors as type of electrodes, frequency, geometry of the object, inter-electrode distance and temperature have an effect. These factors are discussed especially with respect to the impedance method in frost resistance studies of plants. The PDF includes an abstract in Finnish.

Seven hundred one-year-old Roth seedlings were given different concentrations of potassium fertilizer. Over the study period seedlings were subjected to artificial growing and dormant phases. Frost resistance of the seedlings was assessed by artificial freezing tests and electrical impedance measurements on stem cuttings. In general, high concentrations of potassium fertilizer reflected a low tolerance to frost. Pre-freezing impedance readings decreased with increasing potassium fertilizer dosages. Results from pre-freezing impedance measurements were found to be in broad agreement with the hypothesis that high impedance readings indicate a frost hardy tissue whereas low readings imply the opposite.Betula pendula

Models concerning the effects of temperature on dormancy release in woody plants were tested using two-year old seedlings of Scots pine ( L.) and Norway spruce ( (L.) H. Karst.). Chilling experiments suggest that the rest period has a distinct end point. Before the attainment of this end point high temperatures do not promote bud development towards dormancy release, and after it further chilling does not affect the subsequent bud development. A new hypothesis of dormancy release is suggested on the basis of a comparison between present and earlier findings. No difference in the proportion of growth commencing seedlings were detected between the forcing temperatures of 17°C and 22°C. The rest break of 50% of Norway spruce and Scots pine seedlings required six and eight weeks of chilling, respectively. Great variation in the chilling requirement was found, especially for Scots pine.Pinus sylvestrisPicea abies

Nursery grown Norway spruce ( (L.) H. Karst.) seedlings from 12 different seed orchards were tested for early autumn frost hardiness using artificial freezing tests. Seed orchards containing grafted parent clones originating from high altitudes produced seedlings showing higher damage than commercial control seed lots of the commercial controls. A seed orchard containing both German and Norwegian clones produced seedlings showing high damage. The correlation between bud-set and frost damage was high at the provenance level, but lower at the half- and full-sib-levels. Families with good growth capacity in progeny field tests showed large between-family variation in frost damage in the artificial freezing tests. This indicates the possibility to combine high growth rate with acceptable autumn frost hardiness in the selection of parent trees.Picea abies

Frost resistance during shoot elongation in seedlings of Norway spruce ( (L.) H. Karst.) was studied in two experiments. The aim of the first study was to evaluate the effect of varying mineral nutrition. Except for potassium, only minor differences in mineral elements concentrations were established, presumably due to low levels of irradiance and thus a low rate of dry matter production. No significant differences in frost injuries were found between the treatments in the experimental series, but the control seedlings were significantly less injured. It is assumed that poor hardiness development at the end of one growth period resulting from low levels of irradiance may decrease the frost resistance during the next shoot elongation phase. Observations from the second experiment with Norway spruce nursery stocks representing different seedling ages and production systems, support this assumption.Picea abies

The red spruce ( sarg.) population in the Green Mountains in Vermont has showed foliar deterioration that has not been fully explained. The most characteristic needle injuries of the sensitive trees appear in late winter when the first-year needles turn brown. The cytopathological and external observations on the symptoms support the interpretation that winter stress triggers the damage. It is possible that some anthropogenic stress factors (components of acid deposition or ozone) and/or natural factors predispose the trees to the damage.Picea rubens

Seasonal changed in total nitrogen, protein, amino acid, ammonia, nitrate and nitrite concentrations, and nitrate reductase and γ-glutamyltransferase activities in the needles, buds and shoots of young Scots pine ( L.) were studied. A relationship between the variation in the nitrogen metabolism and both winter dormancy and its breaking was proposed. Pine tissues stored soluble nitrogen over the winter largely in the form of arginine which, in addition to a high nitrogen content, can neutralize acidic cytoplasmic constituents such as nitrates and nitrites. Specific nitrate reductase and γ-glutamyltransferase activities were highest in late summer or autumn, and is apparently connected to the mobilization of nitrogen reserves for the winter.Pinus sylvestris