Changes in Chemical Composition and Accumulation of Cryoprotectants as the Adaptation of Anholocyclic Aphid Cinara tujafilina to Overwintering.

One of the consequences of climate change is the expansion of insects' ranges. Colonization of new habitats forces insects to adapt to new conditions, such as low temperatures in winter. Cinara tujafilina is a thermophilic anholocyclic aphid species, which reproduce exclusively parthenogenetic throughout the year, including winter. On the areas where the populations of C. tujafilina had expanded, it demonstrated its adaptation for surviving colder winters. Based on analyses of changes in body chemical composition using Fourier transform infrared (FTIR) and changes in cryoprotectant content using high performance liquid chromatography (HPLC), we showed how aphid C. tujafilina adapted to overwintering as an active stage. In the FTIR spectrum of the winter type of C. tujafilina, higher peak values originating from the carbohydrates, proteins and lipids, were observed. Glucose, trehalose, mannitol, myo-inositol and glycerol were identified in the aphid body in winter as main putative cryoprotectants to increase the insects' tolerance to cold. The complex sugar-polyol cryoprotectant system facilitates aphids' survival in unfavorable low temperatures.

Two distinct aphid diapause strategies: slow development or development arrest.

Aphids adapt to unfavourable environmental conditions, such as low temperatures in winter, by laying diapausing eggs that overwinter. Diapause is a stress-resistant and developmentally arrested stage that can be adopted in order to increase the chance of survival in adverse environmental conditions. The diapause process of aphids is still very poorly understood. We followed the development of two species of aphids, Brachycorynella asparagi and Appendiseta robiniae, using the immunostained embryos of the aphids to identify mitotic cell divisions. Two different models of aphid diapause were demonstrated for the first time. In the first strategy, the embryo developed continuously during winter diapause, while in the second case, there was an embryonic arrest. The possibility of slow development of the whole body during diapause is a characteristic feature of aphids. The link to the plant's phenology appears to be a key factor in determining the diapause strategy in aphids.

Highly Efficient Use of Infrared Spectroscopy (ATR-FTIR) to Identify Aphid Species.

Aphids are commonly considered to be serious pests for trees, herbaceous and cultivated plants. Recognition and identification of individual species is very difficult and is based mainly on morphological features. The aims of the study were to suggest the possibility of identifying aphids through the use of Fourier-transform infrared (FTIR) spectroscopy, and to determine which absorption peaks are the most useful to separate aphid species. Using FTIR spectroscopy, based on the chemical composition of the body, we were able to distinguish 12 species of aphid. We have shown that using nine distinct peaks corresponding to the molecular vibrations from carbohydrates, lipids, amides I and II, it is possible to accurately identify aphid species with an efficiency of 98%.

A Zebrafish/Drosophila Dual System Model for Investigating Human Microcephaly.

Microcephaly presents in neurodevelopmental disorders with multiple aetiologies, including bi-allelic mutation in TUBGCP2, a component of the biologically fundamental and conserved microtubule-nucleation complex, γ-TuRC. Elucidating underlying principles driving microcephaly requires clear phenotype recapitulation and assay reproducibility, areas where go-to experimental models fall short. We present an alternative simple vertebrate/invertebrate dual system to investigate fundamental TUBGCP2-related processes driving human microcephaly and associated developmental traits. We show that antisense morpholino knockdown (KD) of the Danio rerio homolog, tubgcp2, recapitulates human TUBGCP2-associated microcephaly. Co-injection of wild type mRNA pre-empts microcephaly in 55% of KD zebrafish larvae, confirming causality. Body shortening observed in morphants is also rescued. Mitotic marker (pH3) staining further reveals aberrantly accumulated dividing brain cells in microcephalic tubgcp2 KD morphants, indicating that tubgcp2 depletion disrupts normal mitosis and/or proliferation in zebrafish neural progenitor brain cells. Drosophila melanogaster double knockouts (KO) for TUBGCP2 homologs Grip84/cg7716 also develop microcephalic brains with general microsomia. Exacerbated Grip84/cg7716-linked developmental aberration versus single mutations strongly suggests interactive or coinciding gene functions. We infer that tubgcp2 and Grip84/cg7716 affect brain size similarly to TUBGCP2 and recapitulate both microcephaly and microcephaly-associated developmental impact, validating the zebrafish/fly research model for human microcephaly. Given the conserved cross-phyla homolog function, the data also strongly support mitotic and/or proliferative disruption linked to aberrant microtubule nucleation in progenitor brain cells as key mechanistic defects for human microcephaly.

Metabolic Response of Aphid Cinara tujafilina to Cold Stress.

Climate changes enable thermophilic insect species to expand their ranges, but also force them to adapt to unfavourable environmental conditions in new habitats. Focusing on Cinara tujafilina, we investigated the metabolic changes in the body of the aphid that enabled it to survive the low temperatures of winter. Using GC-MS analysis, differences in the chemical composition of the aphids in summer and winter were found. The metabolic changes were mainly related to the increased activity of the pathways of carbohydrate metabolism, such as glycolysis and the pentose phosphate pathway; a decrease in tricarboxylic acid cycle (TCA); accumulation of polyols; and increased levels of proline, tyrosine, and fatty acids.

Changes in Aphid-Plant Interactions under Increased Temperature.

Thermal stress in living organisms causes an imbalance between the processes of creating and neutralizing reactive oxygen species (ROS). The work aims to explain changes in the aphid-host plant interaction due to an increase in temperature. Tests were carried out at three constant temperatures (20, 25, or 28 °C). Firstly, changes in development of Macrosiphum rosae were determined. Secondly, the activity of enzymatic markers (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), ß-glucosidase, polyphenol oxidase (PPO), and peroxidase (POD)) in aphid M. rosae tissues and host plant were analyzed at all temperatures. An increase in temperature to 28 °C had a negative effect on the biology of M. rosae by shortening the period of reproduction and longevity, thus reducing the demographic parameters and fecundity. Two stages of the aphid's defensive response to short-term (24-96 h) and long-term (2 weeks) thermal stress were observed. Aphid defense responses varied considerably with temperature and were highest at 28 °C. In turn, for the plants, which were exposed to both abiotic stress caused by elevated temperature and biotic stress caused by aphid feeding, their enzymatic defense was more effective at 20 °C, when enzyme activities at their highest were observed.

Impact of Forest Management on the Temporal Dynamics of Herbaceous Plant Diversity in the Carpathian Beech Forests over 40 Years.

In recent years, there has been a growing awareness of the complex dependence of herbaceous plant diversity on forest structure and management. However, among the studies presented so far, those in which the chronosequence (approach based on the assumption of space-for-time substitution) was used, dominate. On the other hand, it is rare to find results based on long-term research on permanent or semi-permanent sampling plots. The aim of this study was to recognize the changes in the vegetation composition and dynamics of various indices of herbaceous plant diversity over 40 years of forest development, and their dependence on forest structure and management. Here we analyzed the temporal dynamics of herbaceous plant diversity in Carpathian fertile beech forests, based on datasets recorded on semi-permanent plots in three censuses (the 1970s, 2000s and 2010s). We checked the temporal changes in alpha, beta, and gamma diversity. Analyses of the plant diversity were performed on the background of changes in forest structure and management systems. We found that the within-plot (alfa diversity) and between-plot (beta diversity) herbaceous plant diversity metrics showed inconsistent patterns along with changes in the forest structure, management systems, and intensity of forest management, during the last 40 years. Temporal changes in the gamma diversity followed the changes in alpha diversity. Although the beta diversity after 40 years is greater than in the past, we argue that the conservation status of habitats typical for well-preserved fertile mountain beech forests has deteriorated due to a decline in the sharing of the diagnostic species of these forests. We showed the importance of the different temporal interactions between the forest structure and management for herbaceous plant diversity. We argue that, in view of the complexity of these processes, it would be a mistake to reject or prioritize alpha or beta diversity measurements to determine the real course of long-term changes in herbaceous plant diversity and to properly assess the state of the forest biodiversity, their conservation status, or conservation action plans. In addition, we need far more data from long-term observations to fully understand the possible relationship patterns between the factors controlling the forest structure and plant diversity.

Changes in Antioxidative, Oxidoreductive and Detoxification Enzymes during Development of Aphids and Temperature Increase.

Temperature, being the main factor that has an influence on insects, causes changes in their development, reproduction, winter survival, life cycles, migration timing, and population dynamics. The effects of stress caused by a temperature increase on insects may depend on many factors, such as the frequency, amplitude, duration of the stress, sex, or the developmental stage of the insect. The aim of the study was to determine the differences in the enzymatic activity of nymphs and adult aphids Aphis pomi, Macrosiphum rosae and Cinara cupressi, and changes in their response to a temperature increase from 20 to 28 °C. The activity of enzymatic markers (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), ß-glucosidase, polyphenol oxidase (PPO) and peroxidase (POD)) in aphid tissues was analysed for three constant temperatures. The results of our research showed that the enzymatic activity of aphids (measured as the activity of antioxidant, detoxifying and oxidoreductive enzymes) was mainly determined by the type of morph. We observed a strong positive correlation between the activity of the detoxifying and oxidoreductive enzymes and aphids' development, and a negative correlation between the activity of the antioxidant enzymes and aphids' development. Moreover, the study showed that an increase in temperature caused changes in enzyme activity (especially SOD, CAT and ß-glucosidase), which was highest at 28 °C, in both nymphs and adults. Additionally, a strong positive correlation between metabolic activity (heat flow measured by microcalorimeter) and longevity was observed, which confirmed the relationship between these characteristics of aphids. The antioxidant enzyme system is more efficient in aphid nymphs, and during aphid development the activity of antioxidant enzymes decreases. The antioxidant enzyme system in aphids appears to deliver effective protection for nymphs and adults under stressful conditions, such as high temperatures.

Mild Abiotic Stress Affects Development and Stimulates Hormesis of Hemp Aphid Phorodon cannabis.

The hemp aphid Phorodon cannabis Passerini is a well- known (Asia, Europe) or newly emerging (North America) insect. It is a monophagous insect pest causing considerable damage in field and glasshouse cultivations. The aim of this work was to study the effects of meteorological (temperature) and agronomical (herbicide) factors on the biology of the hemp aphid. In one experiment, hemp plants were kept at constant temperatures ranging from 20 to 30 °C, and aphid survival and fecundity were measured. In a related experiment conducted at 20 °C, plants were treated with field-appropriate rates of a selective graminicide containing quizalofop-P-tefuryl (40 gL-1, 4.38%, HRAC group 1), commonly used to control weeds in hemp, and aphid enzyme activity was measured in addition to population parameters. We found that hemp aphids could live, feed and reproduce within the whole studied range of temperatures, demonstrating its great evolutionary plasticity. However, the optimal temperature for development was 25 °C, at which the insect lived and reproduced for 25 and 15 days, respectively, with an average fecundity of 7.5 nymphs per reproduction day. The herbicide treatment increased the activity of superoxide dismutase (SOD), catalase (CAT), ß-glucosidase, S-glutathione transferase (GST), oxidoreductive peroxidase (POD), and polyphenol oxidase (PPO) in the aphids, but only on certain days after treatment, which indicates a mild stress in aphid tissues, related to a higher reproduction and changed feeding behavior; aphids moved from the actively growing tips compared to untreated plants. The results of these experiments are discussed in terms of the impact on the future management of this pest.

Biotic and abiotic factors causing the collapse of Robinia pseudoacacia L. veteran trees in urban environments.

Robinia pseudoacacia L. is an interesting example of how one plant species can be considered invasive or useful depending on its environment. In the past this tree species was planted for decorative purposes and for wood in Poland. For many years it was recommended in poor and degraded habitats because it facilitated late-successional plant species. The aim of this study was to verify if black locust can still be regarded as a resistant tree species in urban greenery. The health condition of old tree specimens growing along streets and in parks was compared. The occurrence of pests and pathogens on R. pseudoacacia trees was assessed and the content of mineral elements in leaves was examined. The research results showed that the health of black locust trees growing in the urban environment in Polish cities, especially along streets (in comparison to park sites), deteriorated significantly due to the interaction of harmful biotic and abiotic factors. Increased level of toxic metals (Fe, Zn, Pb, Mn and Cd) in plant tissues and the accumulation of pests and pathogens negatively influenced the health of R. pseudoacacia.

Uninterrupted Development of Two Aphid Species Belonging to Cinara Genus during Winter Diapause.

Aphids are herbivores carrying the status of major pests for crops and ornamental plants. The many specific biological features of aphids allow them to survive unfavorable environmental conditions. As for other insects, a predominant strategy for aphids surviving winter, is laying diapausing eggs. During diapause, the expression of development may vary between species. Most of the insects stop growing during diapause; however, there is limited information about this process. We immunostained the embryos of aphids in order to detect cell division during diapause. Here, for the first time, we present that two species of aphids belonging to Cinara grow and develop throughout the duration of the winter diapause. Our results showed that the "resting stage" does not occur in embryos of these aphid species. The embryo of C. cupressi and C. juniperi undergoes a type of diapause, with slow growth. It seems that this feature is conducive to the rapid development of embryos in the egg, which may be another specific feature for aphid biology of overwintering.

Enzymatic Defense Response of Apple Aphid Aphis pomi to Increased Temperature.

Climate change, and in particular the increase in temperature we are currently observing, can affect herbivorous insects. Aphids, as poikilothermic organisms, are directly exposed to temperature increases that influence their metabolism. Heat stress causes disturbances between the generations and the neutralization of reactive oxygen species (ROS). The aim of this work is focused on explaining how the aphid, using the example of Aphis pomi, responds to abiotic stress caused by temperature increase. The experiment was carried out under controlled conditions at three temperatures: 20, 25, and 28 °C. In the first stage, changes in the activity of enzymatic markers (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), ß-glucosidase, polyphenol oxidase (PPO), and peroxidase (POD)) were determined in aphid tissues, at each temperature. In the second stage, microcalorimetry monitored changes in heat emitted by aphids, at each temperature. Our results showed that A. pomi defense responses varied depending on temperature and were highest at 28 °C. The flexible activity of enzymes and increase in the metabolic rate played the role of adaptive mechanisms and ran more effectively at higher temperatures. The A. pomi thus protected itself against ROS excessive induction and the aphids were able to respond quickly to environmental stress.

Effects of Temperature on Lifespan of Drosophila melanogaster from Different Genetic Backgrounds: Links between Metabolic Rate and Longevity.

Despite many studies of the aging process, questions about key factors ensuring longevity have not yet found clear answers. Temperature seems to be one of the most important factors regulating lifespan. However, the genetic background may also play a key role in determining longevity. The aim of this study was to investigate the relationship between the temperature, genetic background (fruit fly origin), and metabolic rate on lifespan. Experiments were performed with the use of the wild type Drosophila melanogaster fruit flies originating from Australia, Canada, and Benin and the reference OregonR strain. The metabolic rate of D. melanogaster was measured at 20 °C, 25 °C, and 28 °C in an isothermal calorimeter. We found a strong negative relationship between the total heat flow and longevity. A high metabolic rate leads to increased aging in males and females in all strains. Furthermore, our results showed that temperature has a significant effect on fecundity and body weight. We also showed the usefulness of the isothermal calorimetry method to study the effect of environmental stress conditions on the metabolic activity of insects. This may be particularly important for the forecasting of impact of global warming on metabolic activity and lifespan of various insects.

Defense responses of Thuja orientalis to infestation of anholocyclic species aphid Cinara tujafilina.

The aim of this study was to determine an interdependence between generation of semiquinone radicals, superoxide anion (O2-), manganese ions (Mn2+) and phenolic content in leaves of Thuja orientalis in response to infestation by varying populations of Cinara tujafilina, i.e. 40 or 80 aphids per plant. Also, superoxide dismutase (SOD) and ß-d-glucosidase activities in leaves of T. orientalis in a defense response to C. tujafilina was recorded. Analyses of electron paramagnetic resonance (EPR) showed generally a higher concentration of semiquinone radicals with g-values of 2.0051 ± 0.0005 and 20032 ± 0.0005 after C. tujafilina infestation in leaves in comparison to the control. Up to 48 h post-infestation in leaves infested by 80 aphids the level of semiquinone radicals was significantly higher than in the control, while in leaves infested by 40 aphids the highest concentrations of these radicals were recorded at later time points (i.e. at 72 and 96 hpi). In parallel, the highest total generation of O2- and low activity of SOD were recorded in 24-h leaves infested by 80 aphids. Additionally, analysis of confocal images showed that the strongest yellow fluorescence indicating O2- generation was detected in epidermal cells of leaves up to 48 hpi. Significant reduction of Mn2+ ions detected by EPR spectroscopy in relation to the control was observed in 4-w leaves infested by 80 and 40 aphids and in 48-h leaves infested by 40 aphids. Phenolic contents in leaves infested by 80 and 40 aphids at all time points were higher than in the control. The greatest ß-d-glucosidase activity and phenolic contents were recorded at 96 h of feeding. These results indicate that the perception of C. tujafilina infestation by T. orientalis leaves induces a specified sequence of defense mechanisms in the course of time.

Role of phenolic compounds during antioxidative responses of winter triticale to aphid and beetle attack.

One of the earliest responses of plants to insects' attack is generation of reactive oxygen species. However, the elevated level of ROS can elicit oxidative burst within plant tissues, and plants employ antioxidant systems against these radicals. Due to their chemical structures, polyphenols are able to diminish the level of ROS. Thus, we investigated the role of phenolic compounds in oxidative stress within winter triticale caused by Sitobion avenae and Oulema melanopus. It was found, that infestation by insects induced a high increase in the content of hydrogen peroxide and superoxide anion radical within resistant Lamberto cv. 24 hpi, whereas in sensitive Marko cv., an increase in H2O2 content was found within two days of aphid feeding. Moreover, resistant plants showed earlier and much greater induction of l-phenylalanine and l-tyrosine ammonia lyases and chalcone synthase activities, as well as accumulation of phenolic compounds in response to insect feeding than susceptible Marko. On the other hand, strong positive influence of hydrogen peroxide and superoxide radical contents on chalcone synthase activity and furthermore flavonoid biosynthesis was detected in the susceptible cultivar. Negative relationships between level of o-coumaric acid or flavonoid compounds and content of hydrogen peroxide or superoxide radical suggest their antioxidant capacity. Luteolin and o-coumaric acid may attend in scavenging of hydrogen peroxide, whereas quercetin, apigenin and (+)-catechin probably participate in reduction of superoxide anion radical content.

Redescription of males of the aphid species Cinara (Cupressobium) tujafilina and Cinara (Cupressobium) cupressi (Hemiptera, Lachninae).

This paper presents the first comprehensive description of the winged males of Cinara tujafilina and Cinara cupressi (Hemiptera, Lachninae) and establishes the morphological characters enabling differentiation between them. We show that some populations of C. tujafilina, a species considered to be anholocyclic, may not have lost the genetic ability to produce males and under favourable conditions can develop in a holocyclic manner.

Relationships within aphids Cinara (Cupressobium) (Hemiptera) based on mitochondrial and nuclear DNA sequences.

The relationships between Cinara (Cupressobium) aphids inhabiting woody parts and leaves of conifers belonging to Cupressaceae have been studied using a mitochondrial gene (COI) and a nuclear gene (EF1-α). Based on the COI sequences, genetic distances between species ranged from 5.6 % between Cinara (C.) tujafilina (del Guercio) and Cinara (C.) juniperi (De Geer) to 10.5 % between C. (C.) tujafilina and Cinara (C.) mordvilkoi (Pasek). Genetic distances among EF1-α sequences were lower and showed from 0.1 % between C. cupressi and C. juniperi to 2.3 % between C. tujafilina and C. mordvilkoi. Molecular phylogenetic trees were constructed using the Bayesian inference (BI) phylogenetic analysis and maximum parsimony (MP) criterion. Phylogenetic trees obtained based on COI and EF1-α marker genes created two sister clades. Our results indicate that Cinara (Cupressobium) are a monophyletic group of aphids. Phylogenetic relationships amongst Cupressobium aphids do not result from the association with the host plant, but from the feeding site on the host plant or an ability to change the microhabitat on the plant. As closely related species inhabit similar microhabitats on different host plants, it suggests that the host switching is the main mode of speciation in this subgenus.