Time-course for attainment and reversal of acclimation to constant temperature in two Ceratitis species

Acclimation in the thermal tolerance range of insects occurs when they are exposed to novel temperatures in the laboratory. In contrast to the large number of studies that have tested for the ability of insects to acclimate, relatively few have sought to determine the time-course for attainment and reversal of thermal acclimation. In this study the time required for the Mediterranean fruit fly, Ceratitis capitata Wiedemann, and the Natal fruit fly, Ceratitis rosa Karsch, to acclimate to a range of constant temperatures was tested by determining the chill-coma recovery time and heat knock-down time of flies that had been exposed to novel benign temperatures for different durations. The time required for reversal of acclimation for both Ceratitis species was also determined after flies had been returned to the control temperature. Acclimation to 31 °C for only one day significantly improved the heat knock-down time of C. capitata, but also led to slower recovery from chill-coma. Heat knock-down time indicated that acclimation was achieved after only one day in C. rosa, but it took three days for C. rosa to exhibit a significant acclimation response to a novel temperature of 33 °C when measured using chill-coma recovery time. Reversal of acclimation after return to initial temperature conditions was achieved after only one day in both C. capitata and C. rosa. Adult C. capitata held at 31.5 °C initially exhibited improved heat knock-down times but after 9 days the heat knock-down time of these flies had declined to levels not significantly different from that of control flies held at the baseline temperature of 24 °C. In both Ceratitis species, heat knock-down time declined with age whereas chill-coma recovery time increased with age, indicating an increased susceptibility to high and low temperatures, respectively.     

Postingestive sorting of living and heat-killed Chlorella within the sea scallop, Placopecten magellanicus (Gmelin)

Suspension-feeding bivalves may enhance the energy value of their food supply by sorting particles both before and after ingestion. Previous research has indicated that the sea scallop (Placopecten magellanicus (Gmelin) (Mollusca: Bivalvia)) is capable of sorting particles within the gut both on the basis of physical properties (particle size and density) as well as chemical properties. In this study, the ability of the sea scallop to sort living from dead material solely on the basis of chemical properties was tested. The microalga Chlorella (Chlorophyta: Chlorophyceae) was chosen as the test particle because its thick cell wall remains physically intact following heat treatment, while its carbon, nitrogen, and chlorophyll a content declines. Scallops were fed a mixture of radiolabelled live and heat-killed Chlorella. We demonstrate that P. magellanicus can distinguish between living and dead algae, retaining live Chlorella cells longer than heat-killed cells. This ability to detect the subtle chemical differences between living algal material and detrital material would enhance the digestive efficiency of this species by reducing the amount of energy expended, digesting poor-quality materials. This paper presents the first study of the ability of a bivalve to distinguish between two physically identical but nutritionally different forms of the same species of microalgae.     

Arabidopsis GIGANTEA protein is post-transcriptionally regulated by light and dark

GIGANTEA (GI) is a key regulator of photoperiodic flowering in Arabidopsis and encodes a protein with no domains of known biochemical function. Expression of GI mRNA is controlled by the circadian clock, but GI protein accumulation has not been previously investigated. We generated plants that produced functional epitope-tagged GI to enable us to track the protein through the daily cycle. Here we show that GI protein levels oscillate when either constitutively overexpressed or driven by its promoter and that its accumulation is modulated by day length as well as by phase-specific factors. Also, we demonstrate that one of the mechanisms underlying GI protein oscillation occurs post-translationally via dark-induced proteolysis by the 26S proteasome.     

Hydrothermal and thermal time models for the invasive grass, Arundo donax

Controlled laboratory and field experiments were performed to determine the developmental response to temperature and moisture of Arundo donax, a riparian invasive grass and potential bioenergy crop. A logistic function was parameterized and used to predict thermal times to sprouting and the nine-leaf stage. Consistent estimates of the base temperature (T_b) and base water potential (ψ_b) below which development ceases were obtained from various statistical and mathematical analyses. Estimates of T_b and ψ_b were 12.7 ± 1.7 °C and −1.56 ± 0.43 MPa, respectively, for the median fraction of sprouting rhizomes. Median hydrothermal time to sprouting was 124.1 MPa °Cd under laboratory conditions and median thermal times, or degree-day (°Cd), to sprouting and nine-leaf stage was estimated to be 94 and 129 °Cd under field conditions, respectively. A degree-day is defined as one day (24 h) spent one degree above T_b. Results demonstrated that thermal time alone is sufficient to accurately predict time to sprouting under field conditions. Further, there may be a fixed moisture threshold of about 6% volumetric water content above which sprouting rate was constant. This threshold corresponded very closely to the −1.5 MPa for ψ_b that was estimated under laboratory conditions for the soil typically infested by A. donax. This information is crucial for assessing risk of invasive spread for A. donax.     

Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum

In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.     

Inflorescence abnormalities occur with overexpression of Arabidopsis lyrata FT in the fwa mutant of Arabidopsis thaliana

Arabidopsis thaliana is a quantitative long-day plant with the timing of the floral transition being regulated by both endogenous signals and multiple environmental factors. fwa is a late-flowering mutant, and this phenotype is due to ectopic FWA expression caused by hypomethylation at the FWA locus. The floral transition results in the activation of the floral development process, the key regulators being the floral meristem identity genes, AP1 (APETALA1) and LFY (LEAFY). In this study, we describe inflorescence abnormalities in plants overexpressing the Arabidopsis lyrata FT (AlFT) and A. thaliana FWA (AtFWA) genes simultaneously. The inflorescence abnormality phenotype was present in only a proportion of plants. All plants overexpressing both AlFT and AtFWA flowered earlier than fwa, suggesting that the inflorescence abnormality and earlier flowering time are caused independently. The inflorescence abnormality phenotype was similar to that of the double mutant of ap1 and lfy, and AP1 and LFY genes were down-regulated in the abnormal inflorescences. From these results, we suggest that not only does ectopic AtFWA expression inhibit AtFT/AlFT function to delay flowering but that overexpression of AtFWA and AlFT together inhibits AP1 and LFY function to produce abnormal inflorescences.     

More reliable estimates of divergence times in Pan using complete mtDNA sequences and accounting for population structure

Here, we report the sequencing and analysis of eight complete mitochondrial genomes of chimpanzees (Pan troglodytes) from each of the three established subspecies (P. t. troglodytes, P. t. schweinfurthii and P. t. verus) and the proposed fourth subspecies (P. t. ellioti). Our population genetic analyses are consistent with neutral patterns of evolution that have been shaped by demography. The high levels of mtDNA diversity in western chimpanzees are unlike those seen at nuclear loci, which may reflect a demographic history of greater female to male effective population sizes possibly owing to the characteristics of the founding population. By using relaxed-clock methods, we have inferred a timetree of chimpanzee species and subspecies. The absolute divergence times vary based on the methods and calibration used, but relative divergence times show extensive uniformity. Overall, mtDNA produces consistently older times than those known from nuclear markers, a discrepancy that is reduced significantly by explicitly accounting for chimpanzee population structures in time estimation. Assuming the human–chimpanzee split to be between 7 and 5 Ma, chimpanzee time estimates are 2.1–1.5, 1.1–0.76 and 0.25–0.18 Ma for the chimpanzee/bonobo, western/(eastern + central) and eastern/central chimpanzee divergences, respectively.     

内生真菌角担子菌B6对连作西瓜土壤尖孢镰刀菌的影响

建立了快速定量检测土壤中角担子菌(Ceratobasidum stevensii)B6的实时定量PCR方法,同时跟踪了土壤中尖孢镰刀菌数量的动态变化,以及不同的发酵组分对开花期西瓜土壤微生物区系的影响。PCR扩增分析表明引物Cf1/Cr1有很好的特异性,能对角担子菌B6特异性扩增得到371bp的条带,对其它10株真菌不能有效扩增。使用荧光定量PCR对施加角担子菌B6的土壤总DNA扩增,结果表明将活菌B6固体发酵物施加到土壤1周后,B6的数量有一定的增殖,达到7.4 log(pg DNA/g干土),随着时间推移,数量逐渐减少,在第5周的时候低于检测限;而液体发酵液处理从一开始施加到土壤后,B6的数量就开始逐渐减少,在第4周的时候就低于检测限。施加活的B6菌4周内能够有效地控制尖孢镰刀菌的数量(尖孢镰刀菌数量维持在5×103 CFU/g干土左右),之后随着B6数量的减少尖孢镰刀菌数量大量增加。活菌B6在土壤中能够存活1个月左右,不会过度影响土著微生物区系,是一株环境友好型菌株,对土壤微环境的干扰较小。     

Identification of the soybean HyPRP family and specific gene response to Asian soybean rust disease

Soybean [Glycine max (L.) Merril], one of the most important crop species in the world, is very susceptible to abiotic and biotic stress. Soybean plants have developed a variety of molecular mechanisms that help them survive stressful conditions. Hybrid proline-rich proteins (HyPRPs) constitute a family of cell-wall proteins with a variable N-terminal domain and conserved C-terminal domain that is phylogenetically related to non-specific lipid transfer proteins. Members of the HyPRP family are involved in basic cellular processes and their expression and activity are modulated by environmental factors. In this study, microarray analysis and real time RT-qPCR were used to identify putative HyPRP genes in the soybean genome and to assess their expression in different plant tissues. Some of the genes were also analyzed by time-course real time RT-qPCR in response to infection by Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease. Our findings indicate that the time of induction of a defense pathway is crucial in triggering the soybean resistance response to P. pachyrhizi. This is the first study to identify the soybean HyPRP group B family and to analyze disease-responsive GmHyPRP during infection by P. pachyrhizi.     

Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues

Two evolutionary lineages, called Trypanosoma cruzi I and II, have been identified in T. cruzi, the etiologic agent of human Chagas disease. Here, we describe a molecular strategy for direct genetic typing of these major groups of T. cruzi directly in human tissues. The protocol is based on heminested PCR amplification of the D7 region of the 24Salpha ribosomal DNA (rDNA), followed by identification of the products using denaturation curves in real time PCR. The repetitive nature of the gene, and the heminested PCR format insured the high sensitivity necessary to detect the presence of the very scarce T. cruzi DNA present in the chronically infected human tissues. There is 80% DNA sequence homology between the two 24Salpha rDNA alleles that define the T. cruzi I and II groups, sufficient to produce different thermal denaturation curves with melting temperature (TM) values of 81.7+/-0.43 and 78.2+/-0.33 degrees C (mean+/-SEM). Using this technical approach, we analysed tissue samples (esophagi, hearts and colon) from 25 different patients with the gastrointestinal or cardiac forms of Chagas disease; in all of them we found only the presence of T cruzi II. Previous epidemiological and immunological findings had already led to the idea that chronic human infections occurring in Brazil and Argentina might be primarily due to T. cruzi II strains, but all the evidence available had been indirect. Our findings provide definitive proof of this hypothesis and will also allow the establishment of which group of T. cruzi is responsible for Chagas disease in other countries.     

Real Time PCR to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis

Bovine venereal campylobacter infection, caused by Campylobacter fetus venerealis, is of significant economic importance to the livestock industry. Unfortunately, the successful detection and discrimination of C. fetus venerealis from C. fetus fetus continue to be a limitation throughout the world. There are several publications warning of the problem with biotyping methods as well as with recent molecular based assays. In this study, assessed on 1071 isolates, we report on the successful development of two Real Time SYBR® Green PCR assays that will allow for the detection and discrimination of C. fetus fetus and C. fetus venerealis. The sensitivity reported here for the C. fetus (CampF4/R4) and the C. fetus venerealis (CampF7/R7) specific PCR assays are 100% and 98.7% respectively. The specificity for these same PCR assays are 99.6% and 99.8% respectively.     

Influence of Temperature on Multiplication and Egg Hatching of Longidorus africanus

Longidorus africanus multiplication on tomato was highest at 29 °C. Few nematodes were recovered after 6 weeks at soil temperatures of 35 °C or below 23 °C. The time to egg hatching was shortest and the percentage of eggs hatching was highest at 29 °C. The minimum temperature and the heat sum above this temperature required for egg development were calculated to be 14.3 °C and 94.08 degree-days, respectively. The thermal times required for egg development by L. africanus and L. elongatus were nearly identical. For both species the product of the base temperature and the heat sum was near constant, and at a temperature of 22.3 °C the rates of egg development were equal.     

Seasonal Synechococcus and Thaumarchaeal population dynamics examined with high resolution with remote in situ instrumentation

Monterey Bay, CA is an Eastern boundary upwelling system that is nitrogen limited much of the year. In order to resolve population dynamics of microorganisms important for nutrient cycling in this region, we deployed the Environmental Sample Processor with quantitative PCR assays targeting both ribosomal RNA genes and functional genes for subclades of cyanobacteria (Synechococcus) and ammonia-oxidizing Archaea (Thaumarchaeota) populations. Results showed a strong correlation between Thaumarchaea abundances and nitrate during the spring upwelling but not the fall sampling period. In relatively stratified fall waters, the Thaumarchaeota community reached higher numbers than in the spring, and an unexpected positive correlation with chlorophyll concentration was observed. Further, we detected drops in Synechococcus abundance that occurred on short (that is, daily) time scales. Upwelling intensity and blooms of eukaryotic phytoplankton strongly influenced Synechococcus distributions in the spring and fall, revealing what appear to be the environmental limitations of Synechococcus populations in this region. Each of these findings has implications for Monterey Bay biogeochemistry. High-resolution sampling provides a better-resolved framework within which to observe changes in the plankton community. We conclude that controls on these ecosystems change on smaller scales than are routinely assessed, and that more predictable trends will be uncovered if they are evaluated within seasonal (monthly), rather than on annual or interannual scales.     

Infection density of Wolbachia and level of cytoplasmic incompatibility in the Mediterranean flour moth,Ephestia kuehniella

Wolbachia, a causative agent of various reproductive changes in arthropods, induces cytoplasmic incompatibility (CI) in the Mediterranean flour moth, Ephestia kuehniella. Two strains of E. kuehniella, Yokohama and Tsuchiura, harbor closely related Wolbachia, but the Yokohama strain expresses stronger CI than the Tsuchiura strain. A transinfected E. kuehniella strain that harbors the Wolbachia derived from the almond moth Cadra cautella, expresses weak CI at a similar level to the Tsuchiura strain. In the present study, we measured the Wolbachia density in the testis of the three E. kuehniella strains in order to examine the effects of bacterial strain and infection load on the expression of CI. When individuals of the same strain were compared, a correlation of bacterial density to CI level was observed. In addition, the Wolbachia density was higher in the Yokohama strain than the Tsuchiura strain in agreement with the CI levels expressed. However, this relationship did not hold in the comparison between the naturally infected and transinfected strains that carried phylogenetically distant Wolbachia.     

鳙Sox基因克隆及序列进化分析

利用简并引物SoxN和Sox9在鳙基因组DNA中进行PCR扩增和产物克隆测序,并对序列进行同源性比较和系统进化分析。结果表明本文鉴定出鳙15个Sox基因HMG盒序列,分别属于SoxB、SoxC和SoxE组,依据斑马鱼同源基因将其分别命名为Sox1a、Sox1b、Sox2、Sox3、Sox4a、Sox4b、Sox9a、Sox9b、Sox10、Sox11b、Sox12、Sox14a、Sox14b、Sox19和Sox21a。基于鳙和斑马鱼 Sox1、Sox4和Sox9 基因核苷酸序列构建的系统进化树显示这3个Sox基因的复制时间发生在鳙和斑马鱼的分化之前,结果支持了鱼类特异的基因组复制假说。以Sox1a、Sox1b和Sox4基因为分子钟标记构建系统进化树探讨鳙和斑马鱼的分化时间,结果显示,同属于鲤科鱼类的鳙（鲤亚科）和斑马鱼（鱼丹亚科）在原始的鱼丹亚科鱼类中存在一个共同祖先,大约出现在63.7百万年前。研究结果为进一步研究鱼类Sox基因复制和基因组进化等问题提供了重要参考资料。       

Effects of egg-to-adult development time and adult age on olfactory neuron response to semiochemicals in European corn borers

We used the cut-sensillum technique to assess the effect of both adult age and egg-to-adult development time on olfactory neuron responses of Z strain moths of the European corn borer, Ostrinia nubilalis. Compounds tested included the pheromone components, (Z)-11-tetradecenyl acetate and (E)-11-tetradecenyl acetate, the behavioral antagonist, (Z)-9-tetradecenyl acetate, and components of the O. furnicalis (Asian corn borer) sex pheromone, (Z)-12-tetradecenyl acetate and (E)-12-tetradecenyl acetate. The proportion of moths having neurons responding to the two O. nubilalis sex pheromone components and antagonist increased with longer development time and age. The spike frequency of neurons responding to (E)-11-tetradecenyl acetate and the antagonist increased with longer development time. Fourteen of 45 moths with neurons sensitive to either of the O. nubilalis pheromone components responded to (Z)-12-tetradecenyl acetate or (E)-12-tetradecenyl acetate. The likelihood of (Z)-12-tetradecenyl acetate stimulating a neuron similar in spike shape and waveform to that responding to (E)-11-tetradecenyl acetate increased with development time.     

Influence of step increases in hydraulic retention time on (RS)-MCPP degradation using an anaerobic membrane bioreactor

The effects of different hydraulic retention time (HRT) on (RS)-MCPP utilisation was investigated by decreasing the feed flow rate in an anaerobic membrane bioreactor (AnMBR). Results showed an average COD removal efficiency of 91.4%, 96.9% and 94.4% when the reactor was operated at HRT 3, 7 and 17 d, respectively. However, when the HRT was reduced to 1d, the COD removal efficiency declined to just only 60%, confirming the AnMBR is stable to a large transient hydraulic shock loads. The (RS)-MCPP removal efficiency fluctuated from 6% to 39% at HRT 3 d, however when it was increased to 7 and 17 d, the removal efficiency increased to an average of 60% and 74.5%. In addition, (RS)-MCPP specific utilisation rates (SUR) were dependent on the HRT and gradually improved from 18 to 43 μg mg VSS(-1) d(-1) as flow rate increased.     

Photoperiodic control of diapause in Pseudopidorus fasciata (Lepidoptera: Zygaenidae) based on a qualitative time measurement

In the zygaenid moth, Pseudopidorus fasciata, both larval diapause induction and termination are under photoperiodic control. In this study, we investigated whether photoperiodic time measurement (with a 24-h light-dark cycle) in this moth is qualitative or quantitative. Photoperiodic response curves, at 22, 25, and 28 degrees C indicated that the incidence of diapause depended on whether the scotophases exceeded the critical night length (CNL) or not. All scotophases longer than the CNL-induced diapause; all scotophases shorter than the CNL-inhibited diapause. The CNL was 10.5h at 25 and 28 degrees C, and 10h at 22 degrees C. By transferring from various short photoperiods (LD 8:16, LD 9:15, LD 10:14, LD 11:13, LD 12:12, and LD 13:11) to a long photoperiod (LD 16:8) at different times, the number of light-dark cycles required for 50% diapause induction at 25 degrees C was 7.14 at LD 8:16, 7.2 at LD 9:15, 7.19 at LD 10:14, 7.16 at LD 11:13, and 7.13 at LD 12:12, without showing a significant difference between the treatments. Only at LD 13:11 (near the CNL), the number of light-dark cycles was significantly increased to 7.64. The intensity of diapause induced under different short photoperiods (LD 8:16, LD 9:15, LD 10:14, LD 11:13, and LD 12:12) at 25 degrees C was not significantly different with an average diapause duration of 36 days. The duration of diapause induced under LD 13:11 was significantly reduced to 32 days. All results indicate that the night-lengths are measured as either "long" or "short" compared with some critical value and suggest that photoperiodic time measurement for diapause induction in this moth is based on a qualitative principle.     

Influence of steaming explosion time on the physic-chemical properties of cellulose from Lespedeza stalks (Lespedeza crytobotrya)

The synergistic effect of steam explosion pretreatment and sodium hydroxide post-treatment of Lespedeza stalks (Lespedeza crytobotrya) has been investigated in this study. In this case, Lespedeza stalks were firstly exploded at a fixed steam pressure (22.5 kg/m²) for 2–10 min. Then the steam-exploded Lespedeza stalks was extracted with 1 M NaOH at 50 °C for 3 h with a shrub to water ratio of 1:20 (g/ml), which yielded 57.3%, 53.1%, 55.4%, 52.8%, 53.2%, and 56.4% (% dry weight) cellulose rich fractions, comparing to 68.0% from non-steam-exploded material. The content of glucose in cellulose rich residues increased with increment of the steaming time and reached to 94.10% at the most severity. The similar increasing trend occurred during the dissolution of hemicelluloses. It is evident that at shorter steam explosion time, autohydrolysis mainly occurred on the hemicelluloses and the amorphous area of cellulose. The crystalline region of cellulose was depolymerized under a prolonged incubation time. The characteristics of the cellulose rich fractions in terms of FT-IR and CP/MAS ¹³C NMR spectroscopy and thermal analysis were discussed, and the surface structure was also investigated by SEM.