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Contribution of larval nutrition to adult reproduction in Drosophila melanogaster
Jerell R. Aguila, Deborah K. Hoshizaki, Allen G. Gibbs
Journal of Experimental Biology 2013 216: 399-406; doi: 10.1242/jeb.078311
Jerell R. Aguila
School of Life Sciences, University of Nevada, 4505 S. Maryland Parkway, Las Vegas, NV 89154, USA
Deborah K. Hoshizaki
Allen G. Gibbs
For correspondence: allen.gibbs@unlv.edu
Within the complex life cycle of holometabolous insects, nutritional resources acquired during larval feeding are utilized by the pupa and the adult. The broad features of the transfer of larval resources to the pupae and the allocation of larval resources in the adult have been described by studies measuring and tracking macronutrients at different developmental stages. However, the mechanisms of resource transfer from the larva and the factors regulating the allocation of these resources in the adult between growth, reproduction and somatic maintenance are unknown. Drosophila melanogaster presents a tractable system in which to test cellular and tissue mechanisms of resource acquisition and allocation because of the detailed understanding of D. melanogaster development and the experimental tools to manipulate its tissues across developmental stages. In previous work, we demonstrated that the fat body of D. melanogaster larvae is important for survival of starvation stress in the young adult, and suggested that programmed cell death of the larval fat cells in the adult is important for allocation of resources for female reproduction. Here, we describe the temporal uptake of larval-derived carbon by the ovaries, and demonstrate the importance of larval fat-cell death in the maturation of the ovary and in fecundity. Larvae and adults were fed stable carbon isotopes to follow the acquisition of larval-derived carbon by the adult ovaries. We determined that over half of the nutrients acquired by the ovaries in 2-day-old adult females are dependent upon the death of the fat cells. Furthermore, when programmed cell death is inhibited in the larval fat cells, ovarian development was depressed and fecundity was reduced.
The acquisition and allocation of resources across life stages to support growth, somatic tissue maintenance and reproduction are central to life history evolution. In holometabolous insects, distinct developmental stages are tightly linked to feeding and non-feeding periods, thus the different phases of the life cycle can potentially act upon each other. The larval stage is characterized by feeding, while metamorphosis is a non-feeding stage during which the larva is transformed into the adult, and the adult itself may not feed. Holometabolous insects eclose to adulthood containing energy acquired during larval stages that often represents a significant fraction of the adult energy budget (Zera and Harshman, 2001; Fischer et al., 2004; Boggs and Freeman, 2005; Boggs, 2009). In saturniid moths and the Ephemeroptera, adult metabolism and reproductive effort must be supported entirely by energy acquired as larvae. A less extreme example includes autogenous mosquitoes, in which the first clutch of eggs is laid before the female takes her first blood meal (Telang and Wells, 2004).
The mechanisms by which larval energy reservoirs are tagged and then released for use in later developmental stages, and by which they are allocated for somatic maintenance and reproduction, are not well understood, but programmed cell death (PCD) or autophagy of the larval fat cells appear to be involved. In the silkworm moth Bombyx mori, PCD begins in the early pupal stage (Kaneko et al., 2011), when egg maturation occurs. A different process occurs during metamorphosis of the skipper butterfly, Calpodes ethlius. Individual fat cells reorganize into nodular clumps surrounding the tracheoles, and then undergo autophagy, with intracellular remodeling taking place. Mitochondria, rough endoplasmic reticulum and other structures are sequestered in organelle-specific autophagic vacuoles and are destroyed by hydrolytic enzymes, but the cells retain their integrity (reviewed by Hoshizaki, 2005).
The fat body has a complex life history. During the last 3 days of larval development, Drosophila melanogaster increases 200-fold in mass (Church and Robertson, 1966), with nutrient reserves primarily accumulated in the larval fat body. Before metamorphosis, the larva stops feeding and a 12–24 h 'wandering' phase begins, during which the animal searches for a suitable pupariation site (Riddiford, 1993). During metamorphosis, the imaginal cells proliferate to give rise to the adult tissues, while the larval tissues undergo PCD (Lee and Baehrecke, 2001). A striking exception to the loss of larval tissues is the fat body, which undergoes tissue remodeling into individual cells during the pre-pupal stages of metamorphosis (Nelliot et al., 2006). These cells survive metamorphosis (Aguila et al., 2007) and are present in the newly eclosed adult. The larval fat cells presumably support the D. melanogaster pupae, which do not feed, and the newly eclosed adult, which remains inactive for approximately 8 h until the wings expand and the cuticle tans (Chiang, 1963; Edgecomb et al., 1994). Thus, larvae must acquire enough nutrients not only to fuel the developmental re-organization of the pupa but also to survive the late larval and early adult periods.
In the adult, the larval fat cells undergo PCD. Within 24 h of eclosion, 85% of the fat cells are absent, and by 3 days the larval fat cells have been replaced by the adult fat cells. Surprisingly, newly eclosed adult females are three times more starvation resistant than mature females (3 or 10 days old). This difference is not age dependent per se, because retention of the larval fat cells by inhibition of PCD increases the starvation resistance of older adults (Aguila et al., 2007). Thus, larval fat cells represent an important and accessible energy reservoir in the adult female, yet once these reserves are released (and presumably taken up by adult tissues) they are not available to maintain the adult during starvation.
In this study, we tracked transfer of larval- and adult-derived sugar into the ovaries to directly measure the contribution of these nutrients to the ovaries. Furthermore, we tested whether PCD of the larval fat cells is important for the allocation of larval-derived energy reserves and whether these reserves are important for the maturation of the ovaries. We took advantage of natural differences in stable carbon isotopes of cane and beet sugar to assess the relative contributions of larval- and adult-derived sugar carbon to the developing ovaries. The sugar beet undergoes C3 photosynthesis and therefore contains a lower ratio of 13C relative to 12C than sugar cane, which utilizes C4 photosynthesis (Farquhar et al., 1989). By measuring carbon isotope ratios in ovaries of flies fed different sugar sources as larvae and adults, we were able to determine the contributions of larval- versus adult-derived sugar carbon to the ovaries of adult females over a 10day period. Over half of the sugar carbon in ovaries of 2-day-old females was derived from larval feeding, but this was replaced rapidly and completely by adult-derived sugar carbon by day 7. When PCD was genetically inhibited in the larval fat cells, ovaries of 2-day-old feeding adults contained approximately half of the normal larval-derived sugar carbon. Furthermore, overall ovarian development was delayed, and fecundity was depressed. Our data demonstrate a novel use of PCD to mobilize larval resources for uptake and maturation of the ovaries.
Drosophila melanogaster husbandry and genetic crosses
Drosophila melanogaster Meigen 1830 were raised at 25°C on a corn-meal–sugar–yeast medium (42.6 g l−1 corn meal, 68.2 g l−1 beet or cane sugar, 23.9 g l−1 baker's yeast, 7.9 g l−1 agar and 4.5 ml l−1 propionic acid), supplemented with dry yeast.
Genetically constructed flies were used in assays of energy content, ovary mass, fecundity and stable isotope ratios. Briefly, ectopic expression of the cell death inhibitor gene, Drosophila inhibitor of apoptosis 1 (diap1), in the larval fat body was achieved using the GAL4/UAS system (Brand and Perrimon, 1993). Construction of flies with the genotype y w; P{Lsp2-GAL4.H}, P{w+mc=UAS-n-syb.eGFP}3 (abbreviated Lsp2-GAL4) is described by Aguila et al. (Aguila et al., 2007). A stock carrying the UAS-diap1 transgene, P{w[+mC]={UAS-DIAP1.H}1 (abbreviated UAS-diap), was obtained from the Bloomington Stock Center (Bloomington, IN, USA) and crossed to Lsp2-GAL4 to drive ectopic expression of diap1 specifically in the larval fat cells, thereby inhibiting PCD in these cells (Aguila et al., 2007). Because fat-cell death was inhibited, these experimental flies are abbreviated FCDI. In all experiments, the two parental stocks (UAS-diap and Lsp2-GAL4), in which normal PCD occurs, were used as controls.
Triglyceride, carbohydrate and protein assays
Energetic substrates (lipids, carbohydrates and proteins) were assayed in triplicate for individual flies. Adult female flies were homogenized in a solution containing detergent to solubilize lipids (1% NP-40, 0.5% deoxycholic acid, 0.1% Triton-X 100, 100 mmol l−1 NaCl, 0.1 mmol l−1 CaCl2, 2 mmol l−1 MgCl2, pH 7.6). The homogenates were heated for 5 min at 75°C to inactivate lipases. Triacylglyceride levels were measured using a commercial serum triglyceride kit (no. TR0100-1KT; Sigma-Aldrich, St Louis, MO, USA), and protein content was quantified using the bicinchoninic acid method (Smith et al., 1985). Carbohydrates (glycogen and trehalose) were digested with amyloglucosidase and quantified with a blood glucose kit (no. G7521; Pointe Scientific, Canton, MI, USA), as described by Marron et al. (Marron et al., 2003).
Ovaries were dissected from female flies in Dulbecco's phosphate buffered saline (DPBS) (52 mmol l−1 NaCl, 40 mmol l−1 KCl, 10 mmol l−1 Hepes, 1.2 mmol l−1 MgSO4, 1.2 mmol l−1 MgCl2, 2 mmol l−1 Na2HPO4, 0.4 mmol l−1 KH2PO4, 1 mmol l−1 CaCl2, 45 mmol l−1 sucrose, 5 mmol l−1 glucose, pH 7.2). Ovaries were examined by light microscopy, and photographs were taken using a Canon A620 digital camera coupled to a Zeiss Stemi 2000-C microscope and imported using Canon Zoom Browser EX photo software.
Fecundity assays
To establish when egg laying began and to determine total egg laying capacity, newly eclosed females (0–2 h) were collected and placed in individual wells of a Fly Condo (no. 59-110; Genesee Scientific, San Diego, CA, USA) containing grape agar and supplemented with yeast paste. Two males were included to ensure mating. The number of eggs laid in each 24 h period was recorded for 10 days.
Stable isotope studies
Flies for stable isotope analysis were reared on either a cane-sugar- or beet-sugar-based diet as larvae. Note that corn meal, baker's yeast and agar were also potential carbon sources for developing larvae and adult flies. Newly eclosed (0–2 h old) adult females were collected and maintained on the same diet or switched to the other sugar-based diet. Thus four different feeding regimes were used in this study (larval diet/adult diet): cane/cane, cane/beet, beet/beet and beet/cane. We performed stable isotope analyses of ovaries from 1- to 7-day-old virgin female adults to prevent transfer of male carbon sources to the ovaries. Ovaries were dissected on a 25×75 mm glass slide in a drop of DPBS. Approximately 0.5 mg of ovaries (six to 36 ovaries per sample) were placed in 5×9 mm pressed-tin capsules (no. 041061; Costech Analytical Technologies, Valencia, CA, USA) and dried at 50°C for 48 h. The dry mass for each sample was then determined using a Cahn C-30 microbalance (Cahn Instruments, Cerritos, CA, USA) with a precision of 1 μg. These data were used to calculate dry mass per ovary. Carbon isotope ratios were determined by the Las Vegas Isotope Science Laboratory (LVIS; University of Nevada, Las Vegas, NV, USA) using a Costech NA 2000 Elemental Analyzer coupled with a Delta V Plus mass spectrometer (Thermo Scientific, Waltham, MA, USA). Isotope ratios (δ13C) are reported in permil (parts per thousand) relative to Peedee Belemnite (Werner and Brand, 2001). Isotope ratios for internal standards and dietary carbon sources are provided in Table 1.
Contributions of larval and adult feeding to ovarian carbon
For each sample used in stable isotope analyses, total carbon per ovary was calculated as the product of the dry mass per ovary and the fraction of dry mass consisting of carbon, as indicated by mass spectrometry (mean=50.43% across all samples). To estimate the relative contribution of cane- and beet-sugar diets to ovarian carbon, we calculated the fraction (F) of carbon from each source as the weighted average of stable isotope ratios for ovaries from females fed a consistent sugar source as larvae and adults:
(2) where δ13Ccane:cane (−12.4‰) and δ13Cbeet:beet (−20.3‰) are mean stable carbon isotope ratios for ovaries from all females fed cane sugar or beet sugar, respectively, throughout their lifespan, and δ13Csample equals the carbon isotope ratio for a given sample.
Stable carbon isotope ratios of internal standards and media components
Absolute contributions of cane- and beet-derived carbon were calculated as the product of carbon per ovary (see above) and the fraction of cane- and beet-derived carbon, respectively, from Eqns 1 and 2.
Data were analyzed using Statistica 7.0 (SAS Institute, Cary, NC, USA). In most cases, the cutoff for accepting statistical significance was set at P<0.05. In the case of the stable isotope experiments, we performed a total of 12 ANOVAs. We therefore used a Bonferroni correction to minimize the possibility of Type I error. Means ± s.e.m. are reported unless otherwise indicated.
Disruption of PCD of the fat cells does not affect energy storage
Tissue-specific expression of the PCD inhibitor gene, Drosophila inhibitor of apoptosis, in the larval fat cells results in increased starvation resistance in adults (Aguila et al., 2007). It is possible that increased starvation resistance is not due to the retention of the larval fat cells, but rather to increased energy storage in the fat body or other tissues. To test this possibility, we measured the triacylglyceride, glycogen and protein content of 1- to 10-day-old adults in which fat-cell death was inhibited (FCDI) and in parental controls (Lsp2-GAL4 and UAS-diap) (Fig. 1). At eclosion, the initial macronutrient content was unaffected by genotype (i.e. no difference between FCDI and parental controls); thus inhibition of larval fat-cell death did not alter the energy stores brought forward into the adults. Triacylglyceride levels were unaffected by genotype and age (P>0.5 for both factors; Fig. 1). Protein levels were also unaffected by genotype or age, although a statistically significant genotype–age interaction effect was noted (F8,101=2.58, P<0.02). Furthermore, glycogen content increased in all genotypes as flies matured, and then declined (genotype–age interaction, F4,101=3.96, P<0.005). To summarize, the inhibition of larval fat-cell death did not affect the initial energy stores in the adult, and the overall pattern of macronutrient content during the first 10 days of adult life was similar in FCDI flies and parental controls.
Effects of inhibiting larval fat-cell death on energy stores in adult female Drosophila melanogaster. Lsp2-GAL4 and UAS-diap are female parental controls, and the FCDI flies are F1 females from the Lsp2-GAL4 and UAS-diap cross, in which larval fat-cell death was inhibited. Data are means ± s.d.; N=24 for 0-, 2- and 3-day-old adult females; N=20 for 10-day-old adult females. Zero-day-old adult females were collected within 30 min of eclosion.
Ovary mass and egg number
Inhibition of PCD of the larval fat cells did not affect the macronutrient content of the adults (Fig. 1), yet it has been shown to increase starvation resistance (Aguila et al., 2007). The increase is likely due to changes in the allocation pattern of the larval resources within the adult. Qualitative differences in ovary size between FCDI and parental control flies were readily apparent (Fig. 2). Ovarian dry mass in control flies tripled from day 1 to day 2, and then declined and stabilized by day 4 (Fig. 3). In contrast, ovaries from experimental (FCDI) females attained only half the dry mass of ovaries from the parental lines on day 2, but eventually achieved the same dry mass as ovaries from parental controls after day 4 (Fig. 3). These data suggest that PCD of the larval fat cells contributes to the rapid maturation of the ovaries.
Effects of age on ovary appearance in adult female Drosophila melanogaster. Ovaries from FCDI females (A–E) appeared smaller than ovaries from the Lsp2-GAL4 control females (F–J) during the first 1–2 days. Scale bar, 10 μm.
To test whether the apparent delay in ovarian maturation affected fecundity, we determined the number of eggs laid by mated FCDI and parental control females. Egg laying commenced by day 2 post-eclosion for all females (Fig. 4A), but overall fecundity of the FCDI females was lower. FCDI females produced ~200 eggs within the first 10 days of adult life, compared with ~300 eggs for the parental control females (F2,27=158, P<10−6; Fig. 4A). The daily rate of egg laying was reduced in young FCDI females but approached the control rate during the latter half of the egg laying period (days 6–10; Fig. 4B). Thus, PCD is associated with both rapid maturation of the ovaries and efficient egg production.
Larval nutrient uptake by the ovaries
Our data are consistent with a model in which PCD serves as a cellular mechanism to allow the rapid release of nutrients from the larval fat cells for uptake by the ovaries. To test this idea, we first measured the uptake of larval-derived carbon by the ovaries, using stable carbon isotopes, and then tested whether uptake was dependent upon PCD. Larvae were raised on a cane- or beet-sugar based diet, and newly eclosed females were either maintained on the larval diet (control treatment) or switched to a diet containing the other sugar (experimental treatment). Because these sugars contain different 13C:12C ratios, we were able to estimate the relative contributions of ovarian carbon assimilated from larval and adult sugar sources.
Effects of age on ovary dry mass in Drosophila melanogaster. Data are means ± s.e.m.; N=3. Six to 36 ovaries were combined for each measurement, depending on the mass of the individual ovaries for each genotype.
One day after eclosion, ovaries from flies maintained on the cane/cane diet had a carbon isotope ratio of δ13C=−12.2±0.2‰ (Fig. 5), similar to the carbon signature of the cane sugar used in our experiments (−11.2±0.3‰). Ovaries from flies reared on the beet/beet diet had a δ13C value of −20.4±0.5‰ (Fig. 5), similar to that of the beet sugar used to prepare media (−23.6±0.5‰). The differences between the δ13C values of the pure sugars and the ovaries were primarily caused by the presence of additional carbon sources in the diet, especially corn meal and yeast (Table 1).
As expected, stable isotope ratios in the ovaries remained consistent over 7 days when flies were reared on the same larval and adult diets. In contrast, carbon isotope δ13C ratios of the ovaries changed when adults were switched to a different adult sugar source (Fig. 5). An initial ANOVA, using genotype, age, and larval and adult diets as factors, revealed highly significant differences for all main effects and most interactions (P<10−6). To simplify analysis, at each age we performed an ANOVA for flies fed the same adult diet. We also compared 1-day-old females fed different larval diets but the same adult diet. Because of the large number of ANOVAs performed, we used a Bonferroni correction, with P<0.004 required to reject the null hypothesis. This analysis allowed us to address our main hypothesis, that PCD is a primary driver of larval nutrient uptake by the adult ovaries.
Larval cane to adult beet diet
Ovaries from 1-day-old females from parental control lines reared on a larval cane-sugar diet and switched to an adult beet-sugar diet (cane/beet) initially had a cane-sugar-like carbon isotope ratio of δ13C=−12.9±0.3‰ (Fig. 5A). As expected, ovaries from cane/beet adults gradually acquired a carbon isotope δ13C ratio similar to that of beet sugar. In contrast, when programmed fat-cell death was blocked, the ovaries more quickly acquired an adult diet signature, as reflected by a more beet-like signature at day 1 (δ13C=−14.3±0.2‰; Fig. 5A). An ANOVA for all 1-day-old flies reared on a larval cane-sugar diet revealed significant effects of genotype (F2,12=15.49, P<0.0005), adult diet (F1,12=87.27, P<10−6) and their interaction (F2,12=13.7, P<0.001) in establishing the carbon isotope δ13C ratio of the ovaries. Tukey's post hoc tests indicated that these differences were driven by the FCDI flies that had been switched to a beet diet (P<0.0005 for all pairwise comparisons between this and other experimental treatments on day 1; no other comparisons had P-values less than 0.035). Thus, ovaries from 1-day-old FCDI females on a cane/beet diet contained a more adult-like carbon isotope signature, indicative of a significant contribution from adult feeding, relative to control flies.
Effects of inhibition of larval fat-cell death on (A) total and (B) daily fecundity in Drosophila melanogaster. The daily number of eggs produced from mated FCDI and control females was determined for 10 days. Data are means ± s.e.m.; N=9–10 adult females.
The acquisition of an adult carbon profile as flies aged was examined by separate ANOVAs on each day. All flies fed beet sugar as adults were included, with genotype and larval diet treated as fixed effects. On day 1, genotype (parental controls and FCDI) and larval diet (beet compared to cane) significantly affected δ13C (Fig. 5A). Ovaries from the cane/beet parental controls had δ13C values closer to that of cane sugar than the ovaries from the FCDI flies, which had a more beet-sugar-like isotope signature (Tukey's post hoc tests, P<0.001). Similar results were obtained on day 2 (P<0.001 for main effects of genotype and larval diet), and a significant genotype by larval diet interaction was detected. The same general pattern held on day 3, except that the effect of genotype was marginally significant (F2,12=8.8, P<0.0045), and FCDI flies differed significantly only from the Lsp2-GAL4 controls. By day 4, larval diet was the only factor affecting δ13C, and on day 7, δ13C values were statistically indistinguishable among experimental groups. In summary, ovaries from FCDI flies reared on a cane/beet diet acquired an adult carbon isotope signature more rapidly than the parental controls.
Stable isotope analysis of dissected ovaries from aged female Drosophila melanogaster. Flies were reared on one of four diet regiments (larval diet/adult diet): cane/cane, cane/beet, beet/beet and beet/cane. (A) Larvae reared on cane- (upper lines) or beet-sugar medium (lower lines), and then reared on beet-sugar medium as adults. (B) Larvae reared on beet- (lower lines) or cane-sugar (upper lines) medium, and then reared on cane-sugar medium as adults. Points represent mean ± s.e.m. δ13C values of dissected ovaries; N=3. Open circles, ovaries from UAS-diap controls; open triangles, ovaries from Lsp2-GAL4 controls; filled circles, ovaries from F1 females in which larval fat-cell death was inhibited (FCDI).
Larval beet to adult cane diet
In the reciprocal diet experiment (cane sugar as adults), similar results were obtained (Fig. 5B). For 1-day-old flies reared as larvae on beet-sugar medium, adult diet and genotype–diet interactions had significant effects on δ13C of the ovaries (P<0.004 for both factors). Ovaries from the FCDI flies tended to have higher δ13C values (i.e. more cane-like) than parental controls after exposure to cane-sugar medium for 1 day, although these differences were not statistically significant after Bonferroni correction (P >0.02; Fig. 5B). On days 2 and 3, genotype, larval diet and their interaction each significantly affected ovarian carbon isotope signature, with FCDI flies on the beet/cane diet having a more adult-derived carbon signature (less negative δ13C values) than parental controls. Genotypic differences were gone by day 4, although larval diet still had a significant effect (P<0.00002). On day 7, larval diet no longer affected δ13C (P>0.07).
The results of these experiments can be summarized as follows. Carbon isotope signatures of ovaries from 1-day-old flies closely resembled those of their larval diet, and then changed to match the signature of the adult diet as the flies aged. When PCD was inhibited in the larval fat cells, the ovarian isotope signature of 1-day-old females was more similar to the adult diet than the signature of ovaries from the parental controls. These differences in isotope signatures remained for nearly 3 days. In ovaries from 4-day-old flies, δ13C did not differ between the parental controls and FCDI flies, although ovaries still retained a significant contribution from larval sugar intake. By day 7, carbon isotope signatures indicated no significant contribution of larval sugar carbon in the ovaries.
Larval- and adult-derived carbon
Our results demonstrate that carbon acquired during larval feeding is transferred to the ovaries, and identifies PCD of the larval fat cells as an important determinant in the effective uptake of larval nutrients into the ovaries. However, because the isotopic analysis provides only a ratio of 13C to 12C, it is not possible to distinguish between a decrease in the uptake of larval-derived nutrients and a compensatory increase in the uptake of adult-derived nutrients. To address this problem, we calculated the absolute amount of carbon derived from the larval diet based on the δ13C value. We used the mean δ13C values for all flies (1–7 days) reared on either beet/beet or cane/cane diets to represent 100% beet diet contribution and 100% cane diet contribution, respectively (see Materials and methods). The actual δ13C values for flies on beet/cane and cane/beet represent a weighted average of contributions of carbon from larval and adult feeding (Min et al., 2006), allowing us to estimate the relative contributions of larval and adult carbon derived from sugar. From our measurements of ovary dry mass (Fig. 2) and carbon fraction (determined as part of the stable isotope analyses), we then calculated the mass of carbon derived from larval and adult diets (see Materials and methods). It is important to note that sugar is not the only carbon source in the diet, and differences in assimilation of yeast and cornmeal protein could affect these calculations.
Fig. 6 shows the calculated amounts of ovarian carbon from sugar derived from larval and adult feeding. ANOVAs for beet/corn and corn/beet flies revealed significant effects of genotype, age and their interaction on the amount of ovarian carbon derived from larval feeding (P<0.0001 for all comparisons), with ovaries from FDCI females having less larval carbon than parental controls. These differences were greatest in 2-day-old females, which is consistent with large differences in ovarian dry mass at this age (Fig. 2). In contrast, the quantity of sugar carbon derived from adult feeding did not differ among genotypes (Tukey's post hoc test, P>0.45 for all pairwise comparisons), suggesting that FCDI females did not engage in compensatory feeding to offset the reduced availability of nutrients from the larval fat cells. Approximately 50% of ovarian sugar carbon in 2-day-old parental control females was obtained from larval feeding in flies fed the beet/cane diet, compared with 25% in FCDI females. In 2-day-old cane/beet flies, we estimated that larval carbon accounted for 84 and 58% of ovarian carbon in control and FCDI females, respectively.
Energy reserves acquired during the larval stages of holometabolous insects are stored in the larval fat body to fuel the later re-architecture of the animal to the adult form during metamorphosis. Those larval nutrients that are not consumed during metamorphosis are allocated to somatic maintenance and reproduction in the adult (Fischer et al., 2004; Boggs and Freeman, 2005; Min et al., 2006; Boggs, 2009). Previously we reported that the larval fat cells are carried forward into the adult D. melanogaster, thus leading to the conclusion that the larval fat cells are the physical transporters of larval nutrients into the adult. Although D. melanogaster feed as adults, larval nutrients are assumed to be important for the support of the adult during the first 8 h post-eclosion before feeding commences (Chiang, 1963). The larval fat cells also serve as 'meals-ready-to-eat' in the face of starvation (Aguila et al., 2007), where newly eclosed females that still retain their larval fat cells are over twice as starvation resistant as older adults in which the larval fat cells have undergone PCD.
Carbon content of dissected ovaries from aged female Drosophila melanogaster. The carbon content of dissected ovaries was calculated from flies reared on beet/cane or cane/beet diet and based on ovary dry masses, carbon contents and carbon isotope ratios. (A) The amount of carbon in the ovary derived from larval-derived nutrients and from adult nutrients from females reared on a beet/cane diet. (B) The amount of carbon in the ovary derived from larval-derived nutrients and from adult nutrients from females reared on a cane/beet diet. Triangles, ovaries from parental control lines; circles, ovaries from F1 females where larval fat-cell death was inhibited (FCDI). Filled symbols, carbon derived from larval feeding; open symbols, adult-derived carbon.
Rapid maturation of the ovaries corresponds to the window during which larval fat cells normally undergo PCD (Nelliot et al., 2006; Aguila et al., 2007), suggesting a direct connection between these processes in which PCD allows for the rapid release of larval fat cell nutrients for uptake by the ovaries. To test this idea, we disrupted normal larval fat-cell death in the adult and determined the effects on ovarian maturation and fecundity by examining dissected ovaries and determining the number of eggs produced over a 10day period. Concomitant with this idea is the assumption that larval nutrients represent a major energy source for the maturation of the ovaries in the adult. Thus to determine the larval contribution, we used stable isotopes to establish the ratio of larval- and adult-derived nutrients into the ovaries of females when the larval fat cells underwent normal PCD or when it was inhibited.
Contribution of larval- and adult-derived nutrients to the ovaries
The stable isotope studies revealed that the δ13C values of ovaries from 1-day-old female flies resembled those of their larval sugar source (Fig. 5). This is not surprising, because ovarian development begins during metamorphosis (Bodenstein, 1950). In addition, newly eclosed adults do not feed for ~8 h after eclosion (Chiang, 1963), so gonadal growth and development occurring during very early adulthood must rely on larval nutrition. Most of the sugar carbon contained in the ovaries of young flies is derived from larval feeding, but this pattern reverses when PCD is inhibited in the larval fat body. When beet/cane and cane/beet treatments are combined, two-thirds of sugar carbon in ovaries from 2-day-old control flies was derived from the larval diet, compared with 42% in FCDI flies (Fig. 6). These values are only approximations of the relative contributions of larval and adult nutrition, as yeast and corn meal provide significant nutrition during all life stages, and their relative contributions to ovarian carbon may change as flies age. It is clear, however, that in the absence of PCD larval nutrient stores are not readily taken up by the ovaries.
It is possible that FCDI females perform compensatory feeding as adults to offset the lack of availability of energy from the larval fat cells. Ovaries in these flies were smaller than those of parental controls (Fig. 1), suggesting that compensatory feeding could not completely make up for reduced larval energy. More direct evidence against compensatory feeding is provided by the finding that ovaries of 2-day-old females of all genotypes contained similar amounts of adult-derived sugar carbon (Fig. 6B). Thus, we found no indication that FCDI females compensated by eating more or directing more adult energy intake to the ovaries.
It is important to note that nutrients can be mobilized from the larval fat cells, even when PCD is inhibited. Evidence for this is provided by the greater starvation resistance of FCDI females relative to controls (Aguila et al., 2007). It is possible that normal larval fat body energy storage is disrupted in FCDI flies, as expression of the driver we used, Lsp2-GAL4, begins early in the third larval instar and continues into adulthood, but we found no significant differences in whole-animal energy content between FCDI flies and parental controls (Fig. 1). Thus, it is unlikely that a reduced capacity of the larval fat body to store nutrients during the larval stages caused decreased fecundity and ovarian development in FCDI flies. We propose that mechanisms for macronutrient mobilization are active within the fat cells but may not be sufficiently rapid to support early ovarian development.
Larval contribution to adult fecundity
Our results demonstrate that PCD of the larval fat cells is an important mechanism for the reallocation of larval nutrients to the ovaries. Normally, wild-type females contain fully developed eggs ready to be laid approximately 2 days after eclosion, at which time their larval fat cells are absent. In our experiments, egg laying commenced by day 2 post-eclosion for all flies, indicating that some ovarian development occurred, but total fecundity over 10 days decreased by a third in experimental versus control flies (Fig. 4A). Even in FCDI flies, fat-cell loss occurs at a slow rate, and by 96 h larval fat cells are nearly gone (Aguila et al., 2007). By this time, larval energy stores have been released, and inhibition of fat-cell death should no longer affect ovarian function. Consistent with this idea, FCDI flies laid numbers of eggs close to parental controls by day 6 post-eclosion, but never quite attained the same daily fecundity (Fig. 4B). Our results support the idea that a critical period exists in early adulthood of female flies when the mobilization of nutrient stores from the larval fat body to the ovaries is necessary for robust gonadal development. If this window of opportunity is disrupted, then overall gonadal development is compromised.
Previous work has shown that early egg production is largely supported by larval nutrition, with half of the carbon content of the eggs derived from dietary sugar (Min et al., 2006; O'Brien et al., 2008). Within 7 days, however, larval-derived sugar carbon provided no detectable contribution to ovarian carbon. When larval fat-cell death was blocked, ovaries acquired an isotope signature similar to that of the adult sugar source even more quickly, because larval-derived nutrients were less available. Min et al. (Min et al., 2006) proposed the existence of a high-turnover 'fast' pool of carbon associated with reproductive effort, and a 'slow' pool of somatic carbon. Our results are consistent with this idea. In addition, we can identify the larval fat body as the primary carbon source for the 'fast' pool during the first few days of adult life. Thus, PCD represents a novel mechanism for the efficient redistribution of larval-derived energy stores to the ovaries.
The timing of PCD of larval fat cells differs among holometabolous insects. In B. mori, it begins in the early pupal stage (Kaneko et al., 2011), when egg maturation occurs. Fat-cell autophagy in the adult has also been linked to oogenic cycles in mosquitoes (Bryant and Raikhel, 2011). Although the timing and extent of PCD differ among these species, PCD or autophagy may be generally characteristic of rapid oogenesis in insects. It should be noted, however, that PCD of fat cells begins during the wandering period of the final larval instar in Manduca sexta (Müller et al., 2004) and has not been linked to oogenesis. The timing of PCD in M. sexta could suggest that energy must be mobilized to support metamorphosis, but this process requires relatively little energy (Merkey et al., 2011).
A remaining conundrum concerns the transport of macronutrients from the fat cells to the ovaries upon fat-cell death. It is not known whether unique mechanisms exist that modify and package macronutrients that are released by cell death for delivery and uptake to the ovaries, or whether circulating lipophorins are sufficient for transport. The utilization of nutrients once they enter the gonads is also unclear. We note that total energetic content of D. melanogaster does not change when fat body stores are released and taken up by the ovaries (Fig. 5), yet starvation resistance declines (Aguila et al., 2007). It appears that lipid and carbohydrates are not easily mobilized once incorporated into the ovaries. This is unexpected, as D. melanogaster females can reabsorb eggs during nutritional stress (Wilson, 1985).
Environmental stress, such as starvation, affects tradeoffs between reproduction and survival, but the physiological and molecular mechanisms underlying environmental mediation of these tradeoffs are largely unknown. In an earlier study, we showed that inhibiting normal PCD of the larval fat cells increased starvation resistance in adult D. melanogaster (Aguila et al., 2007). In this study, we found that inhibition of PCD reduced the amount of sugar carbon, derived from larval feeding, that entered the ovaries during the first few days of adulthood. Ovarian mass and early fecundity were significantly reduced. Thus, PCD is required for allocation of larval energy stores to the gonads. We propose that when normal fat-cell death is blocked, energy is not rapidly released and is available to other tissues during starvation. In contrast, energy that has entered the gonads following fat-cell death is not readily available to other tissues during starvation.
We thank Faris Tahsin and Dan Hamm for technical support and Diane O'Brien for many helpful comments on an earlier version of this study.
↵* Present address: Stony Brook University Medical Center, Department of Pathology, BST-9, Stony Brook, NY 11794-8691, USA
↵† Present address: National Institutes of Health, NIDDK, 6707 Democracy Boulevard, Two Democracy Plaza, Room 645, Bethesda, MD 20892, USA
This research was supported by National Science Foundation award IOS-0719551 and the HB Head Foundation. J.R.A. was supported by the UNLV Office of Research and Graduate Studies.
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Suszko, J.,
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Boggs, C. L. and
Freeman, K. D.
(2005). Larval food limitation in butterflies: effects on adult resource allocation and fitness. Oecologia 144, 353-361.
Bryant, B. and
Raikhel, A. S.
(2011). Programmed autophagy in the fat body of Aedes aegypti is required to maintain egg maturation cycles. PLoS ONE 6, e25502.
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Fischer, D.,
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(2004). Allocation of larval and adult resources to reproduction in a fruit-feeding butterfly. Funct. Ecol. 18, 656-663.
(2005). Fat-cell development. In Complete Molecular Insect Science, Vol. 2 (ed. Gilbert, L. I., Iatrou, K. and Gill, S.), pp. 315-345. Berlin: Elsevier.
Kaneko, Y.,
Yasanga, T.,
Suzuki, M. and
Sakurai, S.
(2011). Larval fat body cells die during the early pupal stage in the frame of metamorphosis remodelation in Bombyx mori. J. Insect Physiol. 57, 1715-1722.
Lee, C. Y. and
Baehrecke, E. H.
(2001). Steroid regulation of autophagic programmed cell death during development. Development 128, 1443-1455.
Marron, M. T.,
Markow, T. A.,
Kain, K. J. and
Gibbs, A. G.
(2003). Effects of starvation and desiccation on energy metabolism in desert and mesic Drosophila. J. Insect Physiol. 49, 261-270.
Merkey, A. B.,
Wong, C. K.,
Hoshizaki, D. K. and
(2011). Energetics of metamorphosis in Drosophila melanogaster. J. Insect Physiol. 57, 1437-1445.
Min, K. J.,
Hogan, M. F.,
Tatar, M. and
O'Brien, D. M.
(2006). Resource allocation to reproduction and soma in Drosophila: a stable isotope analysis of carbon from dietary sugar. J. Insect Physiol. 52, 763-770.
Müller, F.,
Adori, C. and
Sass, M.
(2004). Autophagic and apoptotic features during programmed cell death in the fat body of the tobacco hornworm (Manduca sexta). Eur. J. Cell Biol. 83, 67-78.
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Bond, N. and
(2006). Fat-body remodeling in Drosophila melanogaster. Genesis 44, 396-400.
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Larsen, T. and
(2008). Use of stable isotopes to examine how dietary restriction extends Drosophila lifespan. Curr. Biol. 18, R155-R156.
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(1993). Hormones and Drosophila development. In The Development of Drosophila, Vol. 2 (ed. Bate, M. and Arias, A. M.), pp. 899-939. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
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(1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85.
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(2004). The effect of larval and adult nutrition on successful autogenous egg production by a mosquito. J. Insect Physiol. 50, 677-685.
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(2001). Referencing strategies and techniques in stable isotope ratio analysis. Rapid Commun. Mass Spectrom. 15, 501-519.
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(2001). The physiology of life history trade-offs in animals. Annu. Rev. Ecol. Syst. 32, 95-126.
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In this role, the successful candidate Position Summary: The principal function of this position is to perform data support to Departmental Projects as assigned by the Associate Director.
Provide desktop support for the various software programs and I.T. related issues; create helpdesk tickets if needed.
Promotes own professional growth and development in research role and maintains current expertise in area of practice.
Performs other assignments as needed.
College degree in Computer Science, Computer Engineering or related field with 4 years experience in a similar capacity with at least two years experience in healthcare.


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The UK's most punctual airport
April 16th, 2014 by elisa
The UK's most punctual airport is London City, according to the latest figures from the Civil Aviation Authority.
Across the 10 airports monitored last year, 80% of flights were on time. At London City, however, 89% were on time. Heathrow is at the bottom of the list with 24% of flights delayed.
The average delay of all scheduled flights monitored in the last quarter was the same as last year, at 12 minutes.
Iain Osborne, group director for regulatory policy at the CAA, said: "Whilst it is pleasing to see the majority of passengers arrived at their destination in good time at the end of last year, our figures show that too many people still had their flights disrupted by delays.
"We also know there were a number of cancellations during December that will have disrupted other passengers.
"Some severe weather clearly played a part in this, but there is still room for improvement. Airports, airlines and air traffic control service providers all have a role to play in delivering that improvement and it is vital they work together to make sure fewer passengers have their journeys disrupted by delays and cancellations".
Discount airlines

redpajama_set_name: RedPajamaCommonCrawl


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Institute for Chemistry and Technology of Materials
ICTM/Research/Daniel Rettenwander (reseacher, Wilkening group)
Wilkening Group
Publications in 2018/19
Ilie Hanzu (senior researcher, Wilkening group)
Daniel Rettenwander (reseacher, Wilkening group)
Trimmel Group
Thomas Rath
Members of the group
Gollas Group
Functional coatings
Electrochem. energy storage
Reaction mechanisms
Slugovc Group
Olefin Metathesis
Click chemistry
Porous polymers
Organocatalysis
Stefan Freunberger (ERC junior researcher of ICTM)
Single crystals as a model system for materials for advanced energy storage technologies
Flux grown single crystals of NASICON-based electrode materials
Orientated single Crystals of Ga-stabilized LLZO made by Czochralski method
One of the main research areas of my group is the design of ceramics with tunable functionality by crystal chemical engineering. In particular the understanding of the underlying mechanism of dynamic processes in solid electrolytes for future Li- and Na-ion battery technologies. This information is also important for industry to provide guidelines to improve their materials to enable future micro-electronic devices. In order to investigate those materials, we use a wide spectrum of methods, such as powder and single crystal (X-ray and neutron) diffraction, Raman/IR spectroscopy, impedance spectroscopy, and electrochemistry in general to study the material properties over different length scales.
We use of single crystals grown by different methods, such as flux growth (by our group and Prof. Günther Redhammer, University of Salzburg), floating zone (Prof. Junji Akiomot, AIST, Japan) and Czochralski method (Dr. Steffen Ganschow, IKZ, Berlin) in combination with impedance spectroscopy and electrochemical methods for battery testing.
In order to study small single crystals grown by flux (50-100 micron) I deposit microelectrodes (15-30 micron) on top polished surfaces. The electrodes are contacted by a metal tip using a micromanipulator and an optical microscope to investigate the Li-ion dynamic as a function of temperature using impedance spectroscopy. The data are correlated afterwards with single-crystal diffraction (SCXRD) data and DSC data to understand the interplay of Li-ion dynamics, phase behavior and structure. The main benefit of this method is that chemical, structural, and microstructural inhomogeneities can be excluded, which provides the most accurate description of structure-property relations.
Moreover, we study single crystalline electrode materials, in particular the structural changes which occur during charge and discharge as well as their impact on kinetics (e.g., exchange current densities, diffusion). Therefore, I glue small single crystals (10 - 100 micron) on top of metal tip (see Figure 2), put it into a liquid electrolyte with counter and reference electrodes (Na or Li) and perform electrochemical tests. The advantage is compared to standard methods that volume, and surface area is very well known makes it the most accurate way to determine the material properties.
Both techniques can be applied to all battery materials of interest. This makes it a huge research area for potential new discoveries with high impact.
Despite small single crystals large single crystals provide even more benefits. It is obvious, if large single crystals are available many techniques can be applied on one and the same sample. This providing a great chance to gain a fundamental understanding of the interplay of structure and properties. Unfortunately, such single crystals are rarely available. As an example we use Li6La3ZrTaO12 single crystal, one of the most promising solid electrolytes, grown by Czochralski method (see Figure 3). Afterwards a broad portfolio is applied to understand structural and mechanical properties, as well as electrochemical properties giving new insights into materials, which clearly push the field forward.
List of Conference Contributions
Single Crystals as Modelsystem
Interfaces in Solid-State Batteries
Degradation of Ionic Conductors
Synthesis of New and Better Materials
Ceramic-Polymer Composites
InterBatt (FWF)


Papal legate to Constantinople for Pope Saint Gregory the Great. Chosen 66th pope in 607. He decreed, and Emperor Phocas confirmed that Rome was the head of all churches, and its bishop was "Universal Bishop".


I haven't written anything new for a couple of weeks because, honestly, I don't want to fake it to make it. I see bloggers who share everyday, but this spreads the content very thin. I am still trying to decide how often I can expect anyone besides my own family to want to hear what I have to say. In the meantime, I will try to be regular with the Friday link-sharing feature. Here is what you missed this week while you were cleaning up biohazards and keeping really cute people from harming themselves or others.
There are no Words – If you are a word geek like me, this is fun. this is a list of words from other languages that have no English equivalent. You will wonder why some of them are necessary and how we manage without the others.
Breast Ironing – Yes, I did a double-take on that one too. Just when you thought you have heard everystrange practice of women around the world, here is another. This story also shows us the strong women of the area who are trying to stop it.
The Apostle Paul getting worse and worse – Paul is my go-to guy when I am feeling sorry for myself or even proud of myself. He tells us to imitate him in his walk with Christ. That sounds like some serious swagger for an apostle. This article will show you how humility was a developing characteristic for him and a great goal for us.
This is just for fun – Stick with this video and you will either see something you are all too familiar with, or you will learn about some pop-culture that will catch you up to speed….or catch you up to about 2005 anyway. That's about the standard time warp for busy moms.
I remember sitting on the edge of the bed in the hotel room after our first night as man and wife. It was time to finally get up and get on with our first day together. For the past few months, my morning routine included taking a birth control pill. For some reason I paused and we prayed about what to do with the rest of those pills. We felt complete peace with leaving them there in that wastebasket. I can still see them laying there in their pastel case, still open and alone. It was one of the top three decisions we ever made.
Little did we know (a lot of stories from newlywed days start with that phrase) that we would be expecting a baby so soon. By September, this June bride was queasy and green and pregnant. I was only 18, just out of high school. All I had ever wanted to be was a mom. I never even thought about being a teacher or a nurse or even a rock star. Well, maybe for a few months when I was little, I wanted to be one of Barker's Beauties – dodged a bullet there. Other than that, when adults asked me what I wanted to be when I grew up I would say "pregnant". Needless to say, I was thrilled. I had achieved my career goals ahead of all those suckers who were just starting college.
It would take many posts to cover all that happened to bring us where we are now. We are the parents of six beautiful children who make our lives better, contrary to what most people think. We all make each others lives better and that is the bonus of it all. We decided for our family that we would follow our desire to have babies, even when it went against modern thinking and even common sense. I would pretend for a while after each baby that I was done, but I knew I was lying to myself.
I used to tell people we were letting God decide when we would have another child. I now believe that He decides this whether you "let" Him or not. He can override your birth control method, He can heal a vasectomy, He can change a heart and He can tell you when it is time to move on to Phase II of family growth. During this time of arrogance when I believed I had "let" God do something others had not, I heard numerous stories of regret from women who had done something permanent to prevent babies in their family. I would feel sad for them or angry at their selfish husbands or fed-up with the world and its ways. Now I believe that all the babies that are supposed to be here are here. God will not be thwarted. There are no unused souls sitting around sad in heaven because someone took her pill today. This is just not what the Bible teaches us.
Now, before you start using all CAPS in the comment boxes, I do believe that the decisions to have babies or not to have babies should be taken very seriously. My point is that God leads in so many subtle and mysterious ways. Every woman should educate themselves and do whatever they do in an informed manner. For instance, I do not take birth control pills because they come with the chance that a fertilized egg will be spontaneously aborted. Once I knew this information, I could not take them in good conscience.
The best thing to ask when deciding about children is are your reasons for attempting to prevent a baby purely selfish? So many times this is the case. I believe any couple trying to make this important call does best to stay close to God, search His Word, listening for His guidance. In my experience, it will not be subtle. No blog can tell you what to do or let you off the hook from what you are called to do. You must know in the deepest way possible that you and your God are clear on this.
My heart hurts for women who want children or more children and their husbands do not. (or vice versa) However, I can't imagine having a child with a man who had simply lost this argument. I would much rather watch for your hearts to line up on this issue, one way or the other. I have a friend who prayed earnestly for a year and watched her husband's heart soften to the plan of a second child. This joint decision-making comes in handy in the middle of the night for feedings, puddle cleaning during potty training and when that first car insurance bill comes after they pass that driving test. Then you can happily think, no regrets. None.
Seriously now, these things are deeply personal. What happens with your family is ultimately up to an Almighty God who does not make a mistake while His head is turned. He goes ahead of you, He has your back, and He takes care of your regrets as well. The child you are imagining could just be a future, flesh-and-blood child – His child.
I am republishing this post today as a reminder to myself. Anyone out there need some grace today?
My six-year-old is known for saying some dinner-stopping stuff, but this past week she let one fly that I will need to remember forever. She started by saying that she loves her daddy more than she loves me. Now that may sound awful, but I was glad to hear that one. Her daddy would have been gratified to hear it, if he had been there. He is always having to be the heavy around here so he gets the lower flow of the warm fuzzies. The reason she gave is what echoed in my head and still stirs around uncomfortably in my gut. She said, "Daddy loves God more than you." …Ouch. Wow. Really?
Of course I had a ton of questions for her. Why did she think this? He prays with them every night before bed and I have them pray. To quote her, "He prays more than you." Then my ten-year-old piped in with, "But Mommy reads her Bible more than Daddy." Oh dear.
I realized I was avoiding talking to them about spiritual things because I am afraid. I am afraid of doing the classic talk-the-talk without walking the walk. I know too many who have been burned by this hypocrisy. There is no way I could live up to all the ideals I want to teach them. It is one of the hardest dichotomies of parenthood. My children see me for who I really am. They see me get angry and tired and complain and make jokes at the expense of others. They see me dance like a fool to a Kesha song. They see me laugh my annoying laugh at less-than-spiritual things on 30 Rock. This is why I don't want to preach to my children. I'm very prone to give up doing something if I can't get it just right.
So God in His great timing has been teaching me about grace. Not just the work of grace that He did on the cross by reconciling us to God, but the constant state of grace that we live in every moment. I realized, through the work of the Spirit, and my great ladies group, that I have been trying to reach a place where I no longer need grace. I was striving for the ultimate sweet spot where I would live consistently and be transformed into some other kind of Christian who didn't need to feel failure or darkness or doubt or defeat. This place is one we are all to look forward to, while at the same time realizing our inability to achieve anything but human-ness this side of heaven. Until that great day, we rely on Jesus as our righteousness. Not to teach it to us or help us achieve it but to BE it for us. It is our only peace.
So now my job is to be human and spiritual with my children. I will try to do this more each day, keeping grace in mind as the motto for me and for them. I can demonstrate love for them even when they fail. I can explain to them how this is possible through what God has done for me. I'm only sad that I learned this so late in the game. The beauty part is, His great grace will cover that too.


H.646, An Act improving recycling in the Commonwealth- For the past three sessions, Senator Moore has been a primary sponsor of comprehensive recycling reform for Massachusetts. The Commonwealth's existing recycling infrastructure is outdated and inefficient. The primary state recycling program remains the bottle deposit law, a dated policy targeting litter prevention, not recycling. Moving Massachusetts' recycling forward will offer significant savings for taxpayers and communities. Senator Moore's bill would phase out the bottle deposits paid by residents, and institute a one cent fee on beverage manufacturers and distributors. Under the current system, the Commonwealth relies on people not returning their cans through the deposit system. By collecting a fee on every bottle, this program will bring in millions more than the deposit system, despite the smaller fee. The funds collected from the beverage industry would go directly into a newly created Municipal Recycling Enhancement Fund. The Fund would distribute the money to municipalities to develop better recycling policies, including single-stream, curbside pick-up, and pay as you throw programs. You can find the bill text and monitor its progress on the Legislature's website: https://malegislature.gov/Bills/189/House/H646.
S.446, An Act enhancing the enforcement of illegal hunting practices- Poaching—the illegal harming or killing of wildlife—is a serious problem across the country and in the Commonwealth. Law enforcement officials report that poachers are rarely, if ever, breaking the law to put food on the table. This legislation elevates Massachusetts' penalties for poaching to bring them in line with other states. This will provide adequate deterrence for poachers who illegally exploit wildlife and cheat ethical hunters and other residents. The bill elevates fines, jail time, and license suspensions for existing laws, some of which have remained unchanged since the 1930s. It also creates an elevated penalty for chronic poachers with repeat violations, to give the Massachusetts Environmental Police an additional tool to target those who blatantly disregard our laws. Additionally, this legislation would bring Massachusetts into a nationwide law enforcement network known as the Interstate Wildlife Violator Compact. Massachusetts is one of just 7 states that is not a member or in the process of joining, and the only state in New England. Membership in the compact will end our state's status as a "safe haven" for out-of-state poachers whose licenses have been suspended in other states and it prevents resident violators from evading punishment by applying for a license in a state other than where the violator lives. You can find the bill text and monitor its progress on the Legislature's website: https://malegislature.gov/Bills/189/Senate/S446.
S.447, An Act relative to hunting with Unmanned Aerial Systems- This legislation makes it illegal for Unmanned Arial Systems, or drones, to be used for the purpose of hunting. Recent advancements in technology have made drones notably cheaper and easier to use, especially for individual citizens. One issue that has manifested in other states is hunters using UAS to track their prey, harass hunters, and disturb wildlife. Several states have made regulatory changes to ban these methods, which is in direct opposition to the concept of fair-chase hunting. While the Massachusetts General Laws already outlaws the use of motor vehicles during hunting, UAS do not qualify as 'aircraft' under the existing definition. Similar efforts in other states have been met with approval from hunting, animal, and environmental advocates alike. Updating the statute to include Unmanned Arial Systems maintains the intent of the existing law and brings our hunting regulations into line with technological advancements. You can find the bill text and monitor its progress on the Legislature's website: https://malegislature.gov/Bills/189/Senate/S447.


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Wilton es una ciudad ubicada en el condado de McLean en el estado estadounidense de Dakota del Norte. En el Censo de 2010 tenía una población de 711 habitantes y una densidad poblacional de 426,27 personas por km².

Geografía
Wilton se encuentra ubicada en las coordenadas . Según la Oficina del Censo de los Estados Unidos, Wilton tiene una superficie total de 1.67 km², de la cual 1.64 km² corresponden a tierra firme y (1.86%) 0.03 km² es agua.

Demografía
Según el censo de 2010, había 711 personas residiendo en Wilton. La densidad de población era de 426,27 hab./km². De los 711 habitantes, Wilton estaba compuesto por el 96.34% blancos, el 0% eran afroamericanos, el 2.11% eran amerindios, el 0.14% eran asiáticos, el 0% eran isleños del Pacífico, el 0.56% eran de otras razas y el 0.84% pertenecían a dos o más razas. Del total de la población el 1.27% eran hispanos o latinos de cualquier raza.

Referencias

Enlaces externos

Ciudades de Dakota del Norte
Localidades del condado de McLean (Dakota del Norte)

redpajama_set_name: RedPajamaWikipedia


Female Widespread throughout the British Isles, this is typically a woodland insect but at higher altitudes inhabits moorland and damp grassland. Although usually found singly or in pairs under logs or bark where it is damp, Cychrus can be gregarious; in Whippendell wood logs only a few metres apart often host several pairs. Larvae occur during autumn and winter from autumn breeding. The narrow and elongate forebody is adapted to entering and feeding on snails, especially the small Oxychilis spp.
14-19mm. Very distinctive and unlikely to be confused with any other species. Entirely black. Head and mandibles produced, palpi long with terminal segment dilated, especially in the male. Appendages long. Pronotum flat, rugosely punctate. Elytra granulate and fused, oval and strongly convex.


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BBDO president and CEO discusses the competition for consumers‰' attention in a rapidly changing world
11.4.2009 Faculty News, Global, News Release
Click here to watch the Wake Forest Leadership Speaker Series presentation by
Andrew Robertson, President and CEO of BBDO Worldwide.
When Andrew Robertson became president and chief executive officer of BBDO Worldwide Inc. in June 2004, he decided that a focused strategy for the organization was essential.
He turned to a photograph, and the simple image served as his inspiration. The photo showed his adolescent daughter, a computer on her lap and a cell phone to her ear. She was watching TV, though it did not appear in the image.
"It struck me that this, in a single frame, captured both the challenge and the opportunity facing those of us working in marketing and communications," said Robertson, who spoke to a packed room in the Worrell Professional Center at Wake Forest University on Thursday, Oct. 29.
"The challenge," he said, "was that it made crystal clear just how difficult it was to hold somebody's attention, to change the way that they behave. Because, the world she grew up in … had moved on so much than the one I grew up in."
Today Robertson, a native of Zimbabwe and a member of the Wake Forest Board of Visitors, leads the world's second largest advertising network, overseeing 16,000 employees in 79 countries. In introducing him, Schools of Business Dean Steve Reinemund called Robertson one of the "true greats in the advertising industry."
BBDO Worldwide and its subsidiaries develop and execute marketing and communications plans for international brands and companies as well as provide campaign planning, management and brand-promotion services. It has 287 offices and some 1,200 clients throughout the world. In 2008, The Gunn Report named BBDO Worldwide the "Most Awarded Agency Network in the World."
The photo, Robertson said, does not depict multi-tasking.
"She was moving from something that was no longer (offering) any real reward for the use of her time to something that was. What technology had made possible was the she could switch, literally at the touch of a button … to any one of a millions of options."
Media, he said, must not be viewed as a means of distribution but as a means of access.
"We are competing with every other option she has at the time for her attention."
That's the problem, Robertson said. But opportunity exists as well. "In this digital age, many of the things people used to pay for – time, distance, space – are limitless and free."
The ability to earn time with a consumer changes the rules of the game, Robertson continued. The competitive advantage no longer lies in how much money can be spent to buy distribution. Rather, it's about creating good content and earning consumers' attention.
Robertson's son is just 18 months younger than his sister depicted in the photo. But, as she pointed out to her father, he's from a completely different generation when considering the advances in technology and digital communication.
"That's what we're dealing with," Robertson said. And this must be dealt with, he said, through the magic of creativity. At BBDO, the mission is to create and deliver the world's most compelling commercial content.
That goal is realized by three guiding principles: securing an unfair share of a limited pool of exceptional talent, which is defined by 10 characteristics; by leveraging that talent as widely as possible across brands, borders and cultures; and by utilizing the worldwide network to capitalize on the value of diversity.
Achieving those principles can be accelerated, he said, by spending time actually solving problems instead of working on ways to solve them; by observing behavior as opposed to relying solely on listening to clients and consumers; and by defining a "big idea" in a phrase as short as a text message, created by what Robertson calls "the workout" and involves co-creating, crafting and engineering of specific ideas across cultures.
Robertson illustrated the results of the workout process by showing campaigns for the 2009 Dodge Ram pickup, which included five Webisodes showing teams of contractors, firefighters, military members and cowboys taking the truck through an obstacle course, showcasing fierce competition and the elements of a hard-core reality show.
"How do you launch a full-size truck in the most difficult economy conceivable?" an announcer asks, citing a nearly 40 percent drop in sales for Ford trucks in September 2008.
The answer: "We had to create a program so cool, so different and so exciting, it would convince whoever was still in the truck market to say, 'No doubt about it, I'm buying that frickin' Ram," the announcer says.
The five Webisodes, which were six minutes each, boasted a 90 percent completion rate by viewers who spent an average of 12 minutes on the site. The program ranked in the top 10 of IAG's most memorable ads and has attracted more than 2 million visitors.
Robertson also showed ads for AT&T in which five spots were measured for return on investment; Wrigley's Orbit gum; and a campaign for HBO, which featured an apartment building that gave viewers of "HBO voyeur" an intimate look at the lives of the residents who lived there.
An announcer says: "HBO wanted a piece of communication that said, 'We're the greatest storytellers in the world and we're going to tell those stories not just on TV, but in a wide variety of mediums. So instead of creating a piece that just said those things, we created a media program that demonstrated those things."
The program began with an image projected on a building in New York City, giving the illusion that an entire wall had been removed. It was then adapted for the Web, as viewers could follow the story on HBO's site. More than a million unique viewers visited the site during the first week of its launch, and more than half of the network's OnDemand viewers turned to the site during the first week of the program's release.
"Everything is connected; everything is driven by a single idea," Robertson said.
In the AT&T commercials the difference between the best- and worst-performing spots, according to the ROI, was 65 percent. "So, that's why modeling this stuff and measuring the worth is that important," Robertson said.
For Orbit, a hip-hop star called Big Pak was invented to promote a package that included 35 pieces of gum – "big enough to clean up the dirtiest mouths." The "cleans dirty mouths" ad campaign featured a Web site in which Big Pak would clean up any word typed in by viewers; he also helped to host the 2009 MTV Movie Awards show, sponsored by Orbit, that incorporated the campaign theme.
"That's what I mean by an integrated big idea," said Robertson, citing "fabulous" results from the campaign. For instance, repeat purchases were up 24 percent vs. the norm, TV awareness was up 105 percent and digital awareness was up 81 percent.
He discards the idea of a revolution in favor of evolution, defined by "a very clear purpose, a very clear set of operating principles. And then progress that you make literally job by job, client by client, office by office, day by day."
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This year we celebrated eight years of marriage and we went with more of a "go big or go home" 24 hours. We checked into the Intercontinental Chicago around 5:00 p.m. and promptly popped a bottle of champagne. We had dinner reservations set for 7:30, so there was plenty of time to get ready (and of course take some photos).
I also added a sweater vest over because I knew we were going out after dinner, but this felt more appropriate for the first part of the night. It was also perfect for the two bars we went to after because boy was it warm on the dance floor!
We had the most amazing dinner at The Kitchen. We kicked our night off with this gorgeously delicious seafood platter and from there we shared soup and a steak entree. The food here was beyond amazing. We were truly blown away at how good everything tasted down to the seasoning on the potatoes that came with the steak. We can't wait to go there again!
This would have been more then a great anniversary had we ended our night here and headed back to the hotel. However, we had a kid free night and we were already out in the city, so when in Rome!
Up next we headed to Vertigo Sky Lounge and had a drink outside. Yes, it was cold, but oh was it a beautiful view up there. Next up, we headed to Untitled and sat for a drink before hitting the dance floor. I don't think we have danced that much ever! Ryan is not a big dance guy, but if it's just us and no one we know is around (plus a few drinks in the system), he is a dancing machine. I'm talking fast songs folks. He is my dream man!
We headed back to the hotel around 1:00 a.m. and found ourselves back in our day clothes headed to the hotel bar complete with a piggy back ride. What? That's standard for 1:00 am, no? We wised up soon after and called room service around 2:30 a.m. for this amazing meal below!
How do you celebrate your anniversaries? Check out how we've celebrated in the past here.


As you scroll down this page, you'll find interesting links in the following categories: Fun Mystery Sites, Police Research Sites, Equestrian Sites, and Writer's Resources.
Fun Mystery Sites . . .
BookLoons - columns, contests, excerpts, reviews, teen section. Romance and other genres.
Crescent Blues - interesting editorials on all things mysterious, book reviews.
Deadly Pleasures - articles, reviews of some of the best and current mysteries, interviews, author sites, links.
Futures Mystery Anthology - Futures Mystery Anthology Magazine's home on the web. Reviews.
January Magazine - crime fiction reviews and author interviews.
Murder.com - a collection of links. Everything from mystery cons to mystery publications and organizations to mystery bookstores.
Mystery Ink - reviews, interviews, reference guide, award information and tributes, author links.
Mystery Net.com - an incredible wealth of information on this site, from data on tv mysteries and movies to organizations to bookstores to discussion groups.
Poisoned Pen Press - the second largest mystery publisher in the country.
Police Research Sites . . .
Bartholomew County Sheriff's Office, Columbus, Indiana - my local hometown Sheriff's department where I attended a Citizens' Academy, toured the jail's control room, took part in a ride-along, and role playing.
Columbus Police Department - my local hometown police department where I attended a Citizens' Academy, took part in a ride-along, role playing, and I got to shoot an officer's gun on their firing range.
Fauquier County Sheriff's Office - the "real" Sheriff's department in COLD BURN.
How to Become a Police Officer - this website provides information on how to become a police officer.
Laurel Police Department - the police department featured in DEAD MAN'S TOUCH. I was able to tour the station which was very cool.
Warrenton, VA Police Department - The "real" PD in COLD BURN.
Equestrian Sites . . .
Carter Performance Horses - web home of AQHA stallion, Peppy Dry Bonanza. Great source of equestrian links.
Churchill Downs - home of the famed Kentucky Derby.
Cowboy.com where you'll find all kinds of equestrian products.
Visit HorsesRing.com - a stable meeting place.
Horses for Sale - where you can buy and sell horses online. Browse through tens of thousands of horses for sale, all displayed in full color photos . . .
Horse Jumps - the largest manufacturer of horse jumps in the world. Online ordering, fast shipping and the lowest prices around . . .
Ride Magazine - a Hunter / Jumper / Dressage / Eventing magazine.
Sidelines - a wonderful magazine about horse people, for horse people. Polo and equestrian magazine.
Tim Woolley Racing has a nice website.
Tom Chapman - wonderful equine artist. Tom used to be a jockey.
Writer's Resources . . .
Kentucky Romance Writers - take them anywhere, and they'll take you everywhere!
Great information from Lee Lofland, a police procedural expert, found on his blog: The Graveyard Shift.
Sadly, gallant Barbaro, the 2006 Kentucky Derby winner, was euthan-ized on Monday, January 29, 2007.
The photo shown below is by Barbara D. Livingston.
TRIPLE CROSS FUN LINKS . . .
Laurel Park Race-course is the "real-life" Washington Park in DEAD MAN'S TOUCH.


9:30am, board a private wooden motor boat to Unicorn Island. On this boat along the river's bank, you'll be passing through natural creeks, quiet villages and watch rural people going to work. You will see an interesting fishermen's port, stilt houses (half on land, half on water). One of the boat's stops during this trip is at a large orchard that has many different types of tasty tropical fruits for your enjoyments.
After breakfast at hotel, boarding a motor boat for a long distance trip to discover the area's largest marketplace…on water! (the biggest Cai Rang floating market).
7:30 – 8:00am: Free complimentary breakfast at the resort's restaurant.
8:30am: checking out some secrets of a pearl farm.
11:00 am: boarding a wooden motor boat enjoy the busy activities of an ocean-front seafood marketplace and the fantastic scene of hundreds of colorful fishing boats cluttering the area. After 1 hr being out sea, boat anchors in a quiet, fish-laden bay for your fun fishing activities… Lunch served with special local dishes. Bring your swimsuit to check out sea urchin colonies.
Have a lunch. Return to your hotel. Free at leisure in the afternoon. Overnight in hotel.
Accommodation (Please see details at "Hotel" Part).
Print your voucher; locate the voucher in your travel confirmation e-mail or in "show ticket" link on the site and show the voucher to guide.
This entry was posted in Phu Quoc Island Tours and tagged Tour Sai Gon - My Tho - Ben Tre - Can Tho - Phu Quoc Island in 5 Days.


text:
Alban Butler (13 October 171015 May 1773) was an English Roman Catholic priest and hagiographer.

Biography
Alban Butler was born in 1710, at Appletree, Aston le Walls, Northamptonshire, the second son of Simon Butler, Esq. His father died when he was young and he was sent to the Lancashire boarding school ran by Dame Alice. He went on to a Catholic further education at the English College, Douai, in France. In 1735 Butler was ordained a priest. At Douai, he was appointed professor of philosophy, and later professor of theology. It was at Douai that he began his principal work The Lives of the Fathers, Martyrs and Other Principal Saints. He also prepared material for Richard Challoner's Memoirs of Missionary Priests, a work on the martyrs of the reign of Elizabeth.

In 1745, Butler came to the attention of the Duke of Cumberland, younger son of King George II, for his devotion to the wounded English soldiers during the defeat at the Battle of Fontenoy.

Around 1746, Butler served as tutor and guide on the Grand Tour to James and Thomas Talbot, nephews of Gilbert Talbot, 13th Earl of Shrewsbury.  Their elder brother, George, succeeded their uncle as 14th Earl of Shrewsbury. Both James and Thomas Talbot later became Catholic bishops.

He laboured for some time as a missionary priest in Staffordshire, and was finally appointed president of the English seminary at Saint Omer in France, where he remained until his death.

Butler returned to England in 1749 and was made chaplain to the Duke of Norfolk, whose nephew and heir, the Hon. Edward Howard, Butler accompanied to Paris as tutor. While he was in Paris, Butler completed his Lives. During his term as President of the English seminary, Butler also served the bishops of Arras, Saint-Omer, Ypres, and Boulogne-Sur-Mer as their Vicar-General. Butler died in Saint-Omer in 1773 and was buried in the parish church of Saint-Denis.

See An Account of the Life of A. B. by C. B., i.e. by his nephew Charles Butler (London, 1799); and Joseph Gillow's Bibliographical Dictionary of English Catholics, vol. i.

The Lives of Saints

Butler's great work, The Lives of the Fathers, Martyrs, and Other Principal Saints ("Butler's Lives"), the result of thirty years' study, was first published in four volumes in London, 1756–1759. It is a popular and compendious reproduction of the Acta Sanctorum, exhibiting great industry and research, and is in all respects the best compendium of Acta in English. Butler's magnum opus has passed many editions and translations.

The first edition (1756–1759)
This edition was printed initially in 4 octavo volumes, with no stated publisher or author's name. However they were so thick that they were usually bound in more volumes. There were actually 6 title pages since Vol. 3 and Vol. 4 both have a "part 2" issued thus: vol. 1, vol. 2, vol. 3, vol. 3 part 2, vol. 4, and vol. 4 part 2. Each "volume" contained three months of the liturgical calendar's Saints' lives. Vol. 1 also had a copperplate engraving with figures of the Roman devices of torture used, and a 2-page explanation of their use.

Charles Butler's assertion that "all the notes" were left out of the first edition at the suggestion of Bishop Challoner is exaggerated. There are many useful, and even extended, notes in the first edition, but not to the extent that they appear in the second, and succeeding editions. According to Charles Knight, the 1847 edition published in twelve volumes is considered the best and most complete.

Modern editions
Since Fr. Butler published his original edition of his Lives, many successors have revised and updated the work. Father Herbert Thurston, SJ, edited and significantly rewrote the work; his 12-volume "Revised Edition" was published between 1926 and 1938.

References

External links

 "The Lives of the Fathers, Martyrs, and Other Principal Saints", Complete text of the 12 vol. ed. of 1866 at Bartleby.com
Alban Butler letter from Saint-Omer, France, 1770 at Pitts Theology Library, Candler School of Theology
 One modern abridged Concise Edition is:
 A modern complete edition (1995–2000) is also available in 12 volumes, organized by month and feast day: (The January volume is ; February  ; March  ; April  ; May  ; June  ; July  ; August  ; September  ; October  ; November  ; December  )

1710 births
1773 deaths
English College, Douai alumni
People from West Northamptonshire District
Christian hagiographers
18th-century English Roman Catholic priests


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text:
Major Wind Energy Project Approved in Wyoming
by Walter Wang October 11, 2012 1 comment
The pace of wind energy development on public lands is picking up. Interior Ken Salazar announced this week that the Department has reached its goal of authorizing 10,000 megawatts of renewable power on public lands with the approval of the Chokecherry and Sierra Madre Wind Energy Project site as suitable for wind energy development. The Project is a proposed complex that could generate up to 3,000 megawatts of power in southeastern Wyoming. The project developers expect the proposal to create an estimated 1,000 construction, operation and maintenance jobs and generate enough energy to power nearly 1 million homes.
The decision authorizes the BLM to proceed with site-specific environmental analyses for the Sierra Madre Wind Farm, the Chokecherry Wind Farm, the internal haul road, the internal 230 kilovolt transmission line, the rail distribution facility, and substations to connect the generated power to the electric grid. The Record of Decision also approves amendments to the BLM's Rawlins Resource Management Plan, identifying the project area as available for wind energy development. The BLM Rawlins Field Office oversees more than 3.5 million acres in Albany, Carbon, Laramie and Sweetwater counties.
Additional environmental reviews will be needed for the specific turbine layout. The BLM will continue to engage stakeholders as these additional reviews are carried out.
The proposed Chokecherry and Sierra Madre project would consist of two sites encompassing up to 1,000 wind turbines on approximately 219,707 acres of land. The project, to be located about 10 miles south of Rawlins in Carbon County, will be developed in phases and operated by Power Company of Wyoming LLC. When constructed, the wind complex is expected to have a footprint of less than 2,000 acres.
At peak construction, project developers believe the project will create an estimated 1,000 full-time jobs; when operational, the facility is expected to employ 114 permanent workers. The complex could generate between $291 and $437 million in annual property taxes to Carbon County over 20 years. The Power Company of Wyoming estimates that this project will contribute $232 million in sales and use taxes to Carbon County; and an estimated $149 million to the State of Wyoming over 20 years for electricity generation tax.
"The Bureau of Land Management is committed to responsibly developing renewable energy on our country's public lands," said BLM Acting Director Mike Pool. "That includes an extensive environmental review and making sure that we're mitigating the potential impacts of energy development on our wildlife and our lands."
The proposal was evaluated under Interior's "Priority Projects" approach to processing existing applications for renewable energy development on public lands in a coordinated, focused manner with extensive environmental analysis, public review and landscape-level planning.
Read more on this at BLM.
Map shows Alternative 1A viewscape analysis of the project. Credit BLM.
Article by Roger Greenway, appearing courtesy Environmental News Network.
BLMken salazarRenewable Energytransmission lineWindWyoming
Walter Wang
Walter's contributions to CleanTechies over the past 4 years have been instrumental in growing the publications social media channels via his ongoing editorial and data driven strategies. He is the founder and managing director of Sunflower Tax, a renewable energy tax and finance consultancy based in San Diego, California. Active in the San Diego clean technology community, participating in events sponsored by CleanTech San Diego, EcoTopics, and Cleantech Open San Diego, Walter has also been a presenter at numerous California Center for Sustainability (CCSE) programs. He currently serves as an adjunct professor at the University of San Diego School of Law where he teaches a course on energy taxation and policy.
Software Maps Carbon Emissions Down to Building and Street Levels
Class Action Charges Nissan with LEAF Greenwash
Sabrina Hill October 12, 2012 - 8:03 am
They could harness quite a bit ow wind in Texas, if the would copy the windfarms that are around Sweetwater, Texas, and place them here in hudspeth, and Culverson Counties in Far west Texas.


text:
<?php

use Illuminate\Support\Facades\Schema;
use Illuminate\Database\Schema\Blueprint;
use Illuminate\Database\Migrations\Migration;

class AddIsEnabledToFeeds extends Migration
{
    /**
     * Run the migrations.
     *
     * @return void
     */
    public function up()
    {
        Schema::table('feeds', function (Blueprint $table) {
            $table->boolean('is_enabled')->default(true);
        });
    }

    /**
     * Reverse the migrations.
     *
     * @return void
     */
    public function down()
    {
        Schema::table('feeds', function (Blueprint $table) {
            $table->dropColumn('is_enabled');
        });
    }
}

redpajama_set_name: RedPajamaGithub


Ilyes "Stephano" Satouri is a French StarCraft II player.
At present, Stephano is ranked #314 in highest overall earnings, and #11 in highest earnings for players from France.
Before Stephano turned 18 years old, he earned $426.36 in cash prizes from 5 tournaments. 0.14% of his total prize money was earned before March 12, 2011.
The most money that Stephano has won from a single tournament was $30,000.00 from NASL Season 3 (StarCraft II Playoffs) on July 15, 2012. He placed 1st, making up 10.16% of his total prize money won.
Stephano obtained $100,000 in total prize money earned on February 25, 2012 with a $6,000.00 cash prize from his 2nd place finish at Assembly Winter 2012 (StarCraft II). Prize money from 47 tournaments got him to that point.
In the last 90 days, Stephano has won $900.00 in cash prizes from a single tournament, none of which came in the last month.