Ontogenetic Changes in Azoxyglycoside Levels in the Leaves of Dioon edule Lindl.

Plants have multiple strategies, including phytochemicals that protect their vulnerable tissues against pathogens and herbivores. Dioon edule, like all cycads, possess unique azoxy-type compounds, azoxyglycosides (AZGs) as a chemical defense; however, the ontogenetic variability of these compounds in this long-lived cycad is unknown. Here, we investigated the effects of plant age, sex, genotype and individual heterozygosity on AZG levels in mature leaves of wild D. edule populations from eastern Mexico. Individuals were divided into three ontogenetic stages: seedlings, juveniles and adults. We established overall leaf quality by quantifying pigments associated with photosynthesis; chlorophylla, chlorophyllb and lutein. Leaf chlorophylla levels were higher in seedlings compared to adult cycads. Plants were genotyped using 11 microsatellite markers and foliar AZG levels were quantified by HPLC. AZG levels do not correlate with plant genotype or the individual's heterozygosity. Genetic analysis identified a distinction between lowland and highland individuals; foliar AZG levels were higher in lowland adult cycads compared to highland individuals. In both populations, the highest AZG levels were found in seedlings compared to adult cycads. These young cycads are highly reliant on their few leaves since seedlings bear one or two leaves for the first years of their life and, thus, are unlikely to recover from defoliation. The results suggest that cycad leaves with a greater nutritive content and a higher value for long-term survival are better protected with higher AZG levels. Female adult cycads have higher AZG levels compared to males, suggesting that the benefits of defense could also be linked to reproductive costs.

Leaf traits and herbivory levels in a tropical gymnosperm, Zamia stevensonii (Zamiaceae).

PREMISE OF THE STUDY: Slow-growing understory cycads invest heavily in defenses to protect the few leaves they produce annually. The Neotropical cycad Zamia stevensonii has chemical and mechanical barriers against insect herbivores. Mechanical barriers, such as leaf toughness, can be established only after the leaf has expanded. Therefore, chemical defenses may be important during leaf expansion. How changes in leaf traits affect the feeding activity of cycad specialist insects is unknown. We investigated leaf defenses and incidence of specialist herbivores on Z. stevensonii during the first year after leaf flush. METHODS: Herbivore incidence, leaf production, and leaf traits that might affect herbivory-including leaf age, lamina thickness, resistance-to-fracture, work-to-fracture, trichome density, and chlorophyll, water, and toxic azoxyglycoside (AZG) content-were measured throughout leaf development. Principal component analysis and generalized linear models identified characteristics that may explain herbivore incidence. KEY RESULTS: Synchronized leaf development in Z. stevensonii is characterized by quick leaf expansion and delayed greening. Specialist herbivores feed on leaves between 10 and 100 d after flush and damage ∼37% of all leaflets produced. Young leaves are protected by AZGs, but these defenses rapidly decrease as leaves expand. Leaves older than 100 d are protected by toughness. CONCLUSIONS: Because AZG concentrations drop before leaves become sufficiently tough, there is a vulnerable period during which leaves are susceptible to herbivory by specialist insects. This slow-growing gymnosperm invests heavily in constitutive defenses against highly specialized herbivores, underlining the convergence in defensive syndromes by major plant lineages.

Warmer winters are associated with lower levels of the cryoprotectant glycerol, a slower decrease in vitellogenin expression and reduced virus infections in winter honeybees.

Within the context of climate change, winter temperatures at high latitudes are predicted to rise faster than summer temperatures. This phenomenon is expected to negatively affect the diapause performance and survival of insects, since they largely rely on low temperatures to lower their metabolism and preserve energy. However, some insects like honeybees, remain relatively active during the winter and elevate their metabolic rate to produce endothermic heat when temperatures drop. Warming winters are thus expected to improve overwintering performance of honeybees. In order to verify this hypothesis, for two consecutive years, we exposed honeybee colonies to either a mild or cold winter. We then monitored the influence of wintering conditions on several parameters of honeybee overwintering physiology, such as levels of the cryoprotectant glycerol, expression levels of immune and antioxidant genes, and genes encoding multifunctional proteins, including vitellogenin, which promotes bee longevity. Winter conditions had no effect on the expression of antioxidant genes, and genes related to immunity were not consistently affected. However, mild winters were consistently associated with a lower investment in glycerol synthesis and a higher expression of fat body genes, especially apidaecin and vitellogenin. Finally, while we found that viral loads generally decreased through the winter, this trend was more pronounced under mild winter conditions. In conclusion, and without considering how warming temperatures might affect other aspects of honeybee biology before overwintering, our data suggest that warming temperatures will likely benefit honeybee vitality by notably reducing their viral loads over the winter.

Two genera of Aulacoscelinae beetles reflexively bleed azoxyglycosides found in their host cycads.

Aulacoscelinae beetles have an ancient relationship with cycads (Cycadophyta: Zamiaceae), which contain highly toxic azoxyglycoside (AZG) compounds. How these "primitive" leaf beetles deal with such host-derived compounds remains largely unknown. Collections were made of adult Aulacoscelis appendiculata from Zamia cf. elegantissima in Panama, A. vogti from Dioon edule in Mexico, and Janbechynea paradoxa from Zamia boliviana in Bolivia. Total AZG levels were quantified in both cycad leaves and adult beetles by high performance liquid chromatography (HPLC). On average, cycad leaves contained between 0.5-0.8% AZG (frozen weight, FW), while adult beetles feeding on the same leaves contained even higher levels of the compounds (average 0.9-1.5% FW). High AZG levels were isolated from reflex bleeding secreted at the leg joints when beetles were disturbed. Nuclear magnetic resonance and mass spectroscopy identified two AZGs, cycasin and macrozamin, in the reflex bleeding; this is the first account of potentially plant-derived compounds in secretions of the Aulacoscelinae. These data as well as the basal phylogenetic position of the Aulacoscelinae suggest that sequestration of plant secondary metabolites appeared early in leaf beetle evolution.

Caterpillar- and salivary-specific modification of plant proteins.

Though there is overlap, plant responses to caterpillar herbivory show distinct variations from mechanical wounding. In particular, effectors in caterpillar oral secretions modify wound-associated plant responses. Previous studies have focused on transcriptional and protein abundance differences in response to caterpillar herbivory. This study investigated Spodoptera exigua caterpillar-specific post-translational modification of Arabidopsis thaliana soluble leaf proteins by liquid chromatography/electrospray ionization/mass spectroscopy/mass spectroscopy (LC/ESI/MS/MS). Given that caterpillar labial saliva contains oxidoreductases, such as glucose oxidase, particular attention was paid to redox-associated modifications, such as the oxidation of protein cysteine residues. Caterpillar- and saliva-specific protein modifications were observed. Differential phosphorylation of the jasmonic acid biosynthetic enzyme, lipoxygenase 2, and a chaperonin protein is seen in plants fed upon by caterpillars with intact salivary secretions compared to herbivory by larvae with impaired labial salivary secretions. Often a systemic suppression of photosynthesis is associated with caterpillar herbivory. Of the five proteins modified in a caterpillar-specific manner (a transcription repressor, a DNA-repair enzyme, PS I P700, Rubisco and Rubisco activase), three are associated with photosynthesis. Oxidative modifications are observed, such as caterpillar-specific denitrosylation of Rubisco activase and chaperonin, cysteine oxidation of Rubisco, DNA-repair enzyme, and chaperonin and caterpillar-specific 4-oxo-2-nonenal modification of the DNA-repair enzyme.

Honeybee lifespan: the critical role of pre-foraging stage.

Assessing the various anthropogenic pressures imposed on honeybees requires characterizing the patterns and drivers of natural mortality. Using automated lifelong individual monitoring devices, we monitored worker bees in different geographical, seasonal and colony contexts creating a broad range of hive conditions. We measured their life-history traits and notably assessed whether lifespan is influenced by pre-foraging flight experience. Our results show that the age at the first flight and onset of foraging are critical factors that determine, to a large extent, lifespan. Most importantly, our results indicate that a large proportion (40%) of the bees die during pre-foraging stage, and for those surviving, the elapsed time and flight experience between the first flight and the onset of foraging is of paramount importance to maximize the number of days spent foraging. Once in the foraging stage, individuals experience a constant mortality risk of 9% and 36% per hour of foraging and per foraging day, respectively. In conclusion, the pre-foraging stage during which bees perform orientation flights is a critical driver of bee lifespan. We believe these data on the natural mortality risks in honeybee workers will help assess the impact of anthropogenic pressures on bees.

Insect pollination: an ecological process involved in the assembly of the seed microbiota.

The assembly of the seed microbiota involves some early microbial seed colonizers that are transmitted from the maternal plant through the vascular system, while other microbes enter through the stigma. Thus, the seed microbiota consists of microbes not only recruited from the plant vascular tissues, but also from the flower. Flowers are known to be a hub for microbial transmission between plants and insects. This floral-insect exchange opens the possibility for insect-transmitted bacteria to colonize the ovule and, subsequently, the seed to pass then into the next plant generation. In this study, we evaluated the contribution of insect pollination to the seed microbiota through high-throughput sequencing. Oilseed rape (OSR) flowers were exposed to visits and pollination by honey bees (Apis mellifera), red mason bees (Osmia bicornis), hand pollinated or left for autonomous self-pollination (ASP). Sequence analyses revealed that honey bee visitation reduced bacterial richness and diversity in seeds, but increased the variability of seed microbial structure, and introduced bee-associated taxa. In contrast, mason bee pollination had minor effects on the seed microbiota. Our study provides the first evidence that insect pollination is an ecological process involved in the transmission of bacteria from flowers to seeds.

Interactions Between Thiamethoxam and Deformed Wing Virus Can Drastically Impair Flight Behavior of Honey Bees.

Exposure to multiple stress factors is believed to contribute to honey bee colony decline. However, little is known about how co-exposure to stress factors can alter the survival and behavior of free-living honey bees in colony conditions. We therefore studied the potential interaction between a neonicotinoid pesticide, thiamethoxam, and a highly prevalent honey bee pathogen, Deformed wing virus (DWV). For this purpose, tagged bees were exposed to DWV by feeding or injection, and/or to field-relevant doses of thiamethoxam, then left in colonies equipped with optical bee counters to monitor flight activity. DWV loads and the expression of immune genes were quantified. A reduction in vitellogenin expression level was observed in DWV-injected bees and was associated with precocious onset of foraging. Combined exposure to DWV and thiamethoxam did not result in higher DWV loads compared to bees only exposed to DWV, but induced precocious foraging, increased the risk of not returning to the hive after the first flight, and decreased survival when compared to single stress exposures. We therefore provided the first evidence for deleterious interactions between DWV and thiamethoxam in natural conditions.

Exposure to pollen-bound pesticide mixtures induces longer-lived but less efficient honey bees.

Due to the widespread use of pesticides and their persistence in the environment, non-target organisms are chronically exposed to mixtures of toxic residues. Fungicides, herbicides and insecticides are all found at low doses in the diet of pollinators such as honey bees, but due to the lack of data on the toxicological effects of these mixtures, determining their risk is difficult to assess. We therefore developed a study combining the identification of common pollen-bound pesticide mixtures associated with poor colony development and tested their effects on bee behavior and physiology. We exposed bees to the identified pesticide mixtures during the first days of their adult life, a crucial period for physiological development. Using optic bee counters we recorded the behavior of bees throughout their lives and identified two pesticide mixtures that delay the onset of foraging and slow-down foraging activity. Furthermore, one of these mixtures hampers pollen foraging. As bee longevity is strongly influenced by the time spent foraging, bees exposed to these pesticide mixtures outlived control bees. Physiological analysis revealed that perturbations of the energetic metabolism preceded the altered behavior. In conclusion, we found that early-life exposure to low doses of pesticide mixtures can have long-term effects that translate into longer-lived but slower and less efficient bees. These surprising findings contrast with the commonly reported increase in bee mortality upon pesticide exposure, and demonstrate that exposure that may seem harmless (e.g., very low doses, pesticides not intended to kill insects) can have undesirable effects on non-target organisms.

Measuring biological age to assess colony demographics in honeybees.

Honeybee colonies are increasingly exposed to environmental stress factors, which can lead to their decline or failure. However, there are major gaps in stressor risk assessment due to the difficulty of assessing the honeybee colony state and detecting abnormal events. Since stress factors usually induce a demographic disturbance in the colony (e.g. loss of foragers, early transition from nurse to forager state), we suggest that disturbances could be revealed indirectly by measuring the age- and task-related physiological state of bees, which can be referred to as biological age (an indicator of the changes in physiological state that occur throughout an individual lifespan). We therefore estimated the biological age of bees from the relationship between age and biomarkers of task specialization (vitellogenin and the adipokinetic hormone receptor). This relationship was determined from a calibrated sample set of known-age bees and mathematically modelled for biological age prediction. Then, we determined throughout the foraging season the evolution of the biological age of bees from colonies with low (conventional apiary) or high Varroa destructor infestation rates (organic apiary). We found that the biological age of bees from the conventional apiary progressively decreased from the spring (17 days) to the fall (6 days). However, in colonies from the organic apiary, the population aged from spring (13 days) to summer (18.5 days) and then rejuvenated in the fall (13 days) after Varroa treatment. Biological age was positively correlated with the amount of brood (open and closed cells) in the apiary with low Varroa pressure, and negatively correlated with Varroa infestation level in the apiary with high Varroa pressure. Altogether, these results show that the estimation of biological age is a useful and effective method for assessing colony demographic state and likely detrimental effects of stress factors.

[Use of lung ultrasound as a prognostic tool in outpatients with heart failure]. / Utilidad pronóstica de la ecografía pulmonar en el seguimiento ambulatorio de pacientes con insuficiencia cardiaca.

OBJECTIVES: To assess the prognostic value of lung ultrasound for patients with chronic heart failure. METHODS: Prospective observational cohort study, in which a lung ultrasound was performed on 54 patients at a heart failure outpatient consultation. Ultrasonography was classified as positive or negative for ultrasound interstitial syndrome depending on the number of B lines observed. Patients were followed up for six months; considering emergency visits, readmissions and deaths due to heart failure as markers of poor prognosis. RESULTS: 53.7% (29) of the patients had ultrasound interstitial syndrome. Among them, 48.3% (14) were readmitted, compared to 16% (4) of those without the syndrome (P=.012). Considering any of the events previously described as end points (readmissions, emergencies and deaths), we found that in the group of patients with ultrasound interstitial syndrome, 55.2% (16) had at least one of these complications, compared to 20% (5) of participants without the syndrome (P=.008). CONCLUSIONS: Lung ultrasound in the outpatient setting is useful in predicting which patients are at increased risk of heart failure decompensation in the mid-term.

Transcriptome-derived microsatellite markers for Dioon (Zamiaceae) cycad species.

PREMISE OF THE STUDY: Dioon (Zamiaceae) is an endangered North American cycad genus of evolutionary and ornamental value. We designed and validated a set of microsatellite markers from D. edule that can be used for population-level and conservation studies, and that transferred successfully to D. angustifolium, D. spinulosum, and D. holmgrenii. METHODS AND RESULTS: We tested 50 primers from 80 microsatellite candidate loci in the OneKP D. edule transcriptome. Genotypes from 21 loci in 20 D. edule individuals revealed up to 14 alleles per locus and observed heterozygosity from 0.15 to 0.92; one locus was monomorphic. Seven of those 21 loci were polymorphic in D. angustifolium, D. spinulosum, and D. holmgrenii, with up to seven alleles, and an observed heterozygosity up to 0.89. CONCLUSIONS: The transcriptome-derived microsatellites generated here will serve as tools to advance population genetic studies and inform conservation strategies of Dioon, including the identification and origin of illegal plants in the cycad trade.

Arabidopsis redox status in response to caterpillar herbivory.

Plant responses to insect herbivory are regulated through complex, hormone-mediated interactions. Some caterpillar species have evolved strategies to manipulate this system by inducing specific pathways that suppress plant defense responses. Effectors in the labial saliva (LS) secretions of Spodoptera exigua caterpillars are believed to induce the salicylic acid (SA) pathway to interfere with the jasmonic acid (JA) defense pathway; however, the mechanism underlying this subversion is unknown. Since noctuid caterpillar LS contains enzymes that may affect cellular redox balance, this study investigated rapid changes in cellular redox metabolites within 45 min after herbivory. Caterpillar LS is involved in suppressing the increase in oxidative stress that was observed in plants fed upon by caterpillars with impaired LS secretions. To further understand the link between cellular redox balance and plant defense responses, marker genes of SA, JA and ethylene (ET) pathways were compared in wildtype, the glutathione-compromised pad2-1 mutant and the tga2/5/6 triple mutant plants. AtPR1 and AtPDF1.2 showed LS-dependent expression that was alleviated in the pad2-1 and tga2/5/6 triple mutants. In comparison, the ET-dependent genes ERF1 expression showed LS-associated changes in both wildtype and pad2-1 mutant plants and the ORA 59 marker AtHEL had increased expression in response to herbivory, but a LS-dependent difference was not noted. These data support the model that there are SA/NPR1-, glutathione-dependent and ET-, glutathione-independent mechanisms leading to LS-associated suppression of plant induced defenses.

DNA barcodes for Mexican Cactaceae, plants under pressure from wild collecting.

DNA barcodes could be a useful tool for plant conservation. Of particular importance is the ability to identify unknown plant material, such as from customs seizures of illegally collected specimens. Mexican cacti are an example of a threatened group, under pressure because of wild collection for the xeriscaping trade and private collectors. Mexican cacti also provide a taxonomically and geographically coherent group with which to test DNA barcodes. Here, we sample the matK barcode for 528 species of Cactaceae including approximately 75% of Mexican species and test the utility of the matK region for species-level identification. We find that the matK DNA barcode can be used to identify uniquely 77% of species sampled, and 79-87% of species of particular conservation importance. However, this is far below the desired rate of 95% and there are significant issues for PCR amplification because of the variability of primer sites. Additionally, we test the nuclear ITS regions for the cactus subfamily Opuntioideae and for the genus Ariocarpus (subfamily Cactoideae). We observed higher rates of variation for ITS (86% unique for Opuntioideae sampled) but a much lower PCR success, encountering significant intra-individual polymorphism in Ariocarpus precluding the use of this marker in this taxon. We conclude that the matK region should provide useful information as a DNA barcode for Cactaceae if the problems with primers can be addressed, but matK alone is not sufficiently variable to achieve species-level identification. Additional complementary regions should be investigated as ITS is shown to be unsuitable.

Estudios con moléculas de la misma clase pero con diseños distintos ofrecen resultados diferentes: EMPA-REG, CANVAS y DECLARE / Studies with molecules within the same class, but with different designs yield different results. EMPAREG, CANVAS and DECLARE

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