Phenotypic variation in hatchling Chinese ratsnakes (Zaocys dhumnades) from eggs incubated at constant temperatures

We incubated eggs of the Chinese ratsnake Zaocys dhumnades at four constant temperatures (24, 27, 30 and 30 °C) to examine the effects of incubation temperature on hatching success and hatchling phenotypes. Incubation length increased nonlinearly as temperature decreased, with the mean incubation length being 76.7 d at 24 °C, 57.4 d at 27 °C, 47.3 d at 30 °C, and 44.1 d at 33 °C. Hatching successes were lower at the two extreme temperatures (69% at 24 °C, and 44% at 33 °C) than at the other two moderate temperatures (96% at 27 °C, and 93% at 30 °C). Incubation temperature affected nearly all hatchling traits examined in this study. Incubation of Z. dhumnades eggs at 33 °C resulted in production of smaller hatchlings that characteristically had less-developed carcasses but contained more unutilized yolks. Hatchlings from eggs incubated at 27 and 30 °C did not differ in any examined traits. Taking the rate of embryonic development, hatching success and hatchling phenotypes into account, we conclude that the temperature range optimal for incubation of Z. dhumnades eggs is narrower than the range of 24−33 °C but should be wider than the range of 27−30 °C.     

Thermostable DNA helicase improves the sensitivity of digital PCR

DNA/RNA helicases, which catalyze the unwinding of duplex nucleic acids using the energy of ATP hydrolysis, contribute to various biological functions involving DNA or RNA. Euryarchaeota-specific helicase Tk-EshA (superfamily 2) from the hyperthermophilic archaeon Thermococcus kodakarensis has been used to decrease generation of mis-amplified products (noise DNAs) during PCR. In this study, we focused on another type (superfamily 1B) of helicase, Tk-Upf1 (TK0178) from T. kodakarensis, and compared its effectiveness in PCR and digital PCR with that of Tk-EshA. For this purpose, we obtained Tk-Upf1 as a recombinant protein and assessed its enzymatic characteristics. Among various double-stranded DNA (dsDNA) substrates (forked, 5′ overhung, 3′ overhung, and blunt-ended duplex), Tk-Upf1 had the highest unwinding activity toward 5′ overhung DNAs. Noise DNAs were also eliminated in the presence of Tk-Upf1 at concentrations 10-fold lower than those required to yield a comparable reduction with Tk-EshA. When a 5′ or 3′ overhung mis-annealed primer was included as a competitive primer along with specific primers, noise DNAs derived from the mis-annealed primer were eliminated in the presence of Tk-Upf1. In digital PCR, addition of Tk-EshA or Tk-Upf1 increased fluorescent intensities and improved separation between common and risk allele clusters, indicating that both helicases functioned as signal enhancers. In comparison with Tk-EshA, a smaller amount of Tk-Upf1 was required to improve the performance of digital PCR.     

Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca²⁺ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca²⁺ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca²⁺ but was activated upon incubation with Ca²⁺ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ~5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a K_i of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.     

Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly⁻⁸² to Gly³¹⁶), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca²⁺ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.     

Predation activity of two winter-active spiders (Araneae: Anyphaenidae,Philodromidae)

Anyphaena accentuata and Philodromus spp. are cold adapted and winter-active spider species. Their predation activity was investigated at constant temperatures between –4 and 30 °C. The lower temperature threshold for Anyphaena was –3.7 °C, while that of Philodromus was –1.2 °C. At 1 °C the latency to capture and prey consumption was significantly shorter in Anyphaena than in Philodromus. The capture rate increased with temperature and was maximal at 15 °C in Anyphaena and at 30 °C in Philodromus. At 30 °C, the latency to the capture was significantly shorter in Philodromus than in Anyphaena whose mortality significantly increased.     

Crystal structure of Tk-subtilisin folded without propeptide: requirement of propeptide for acceleration of folding

Tk-subtilisin (a subtilisin homologue from Thermococcus kodakaraensis) is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of Tk-propeptide. To analyze the folding mechanism of Tk-subtilisin, the crystal structure of the active site mutant of Tk-subtilisin (S324A-subtilisin^∗), which was refolded in the presence of Ca²⁺ and absence of Tk-propeptide, was determined at 2.16 Å resolution. This structure is essentially the same as that of Tk-subtilisin matured from Pro-Tk-subtilisin. S324A-subtilisin^∗ was refolded with a rate constant of 0.17 and 1.8 min⁻¹ at 30 °C in the absence and presence of Tk-propeptide, respectively, indicating that Tk-subtilisin does not require Tk-propeptide for folding but requires it for acceleration of folding.     

Effect of water temperature on dietary protein requirement,growth and body composition of Asian catfish, Clarias batrachus fry

This study aimed to evaluate the protein requirement of Clarias batrachus fry, were estimated at two different water temperatures, 28 and 32 °C. The influence of dietary protein level and water temperature on body composition, weight gain, food and nutrient utilization were estimated. The Asian catfish, C. batrachus fry were fed four diets containing 28% (diet 1), 32% (diet 2), 36% (diet 3) and 40% (diet 4) protein levels and reared at two water temperatures 28 and 32 °C for 60 days. Fry fed with diet 3 containing 36% protein showed the highest mean final body weight at 32 °C. Final body weight was significantly (P<0.05) affected by dietary treatments and temperatures. Clarias batrachus fry raised at 28 °C had higher feed efficiency (93.20%) than the fry reared at 32 °C (87.58%) with 28% dietary protein level. Further, feed efficiency decreased with increase in dietary protein level. Higher daily protein retention (0.089%) observed at lower (0.0217 g) daily protein intake at 28 °C than 0.0283 g at 32 °C. While, optimal (0.0282 g) daily protein intake showed higher daily weight gain at 32 °C. Productive protein value (% PPV) was maximum (1.76%) at 32 °C than at 28 °C (0.76%). Final body lipid recorded higher value than initial body lipid at both the temperatures. Hepatosomatic index (HSI) observed to have been influenced (P<0.05) by diets and temperatures, while viscerosomatic index (VSI) affected (P<0.05) by only diets and not (P>0.05) by temperatures. The study concluded that the diet 3 containing 36% protein was optimal for growth of C. batrachus fry at both the temperatures.     

Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae)

We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18 °C under a 60 ± 5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C and 25 °C under a 60 ± 5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10 °C, and 12.2, 48.8 and 39.0% at 25 °C, respectively. On the other hand, when mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C, 18 °C and 25 °C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10 °C, 14.5, 26.9 and 58.6% at 18 °C, and 8.3, 21.2 and 70.5% at 25 °C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues.     

Thermogenic characteristics and evaporative water loss in the tree shrew (Tupaia belangeri)

Thermogenic characteristics and evaporative water loss were measured at different temperatures in Tupaia belangeri. The thermal neutral zone (TNZ) of T. belangeri was 30–35 °C. Mean body temperature was 39.76±0.27 °C and mean body mass was 100.86±9.09 g. Basal metabolic rate (BMR) was 1.38±0.03 ml O₂/g h. Average minimum thermal conductance (C_m) was 0.13±0.01 ml O₂/g h °C. Evaporative water loss in T. belangeri increased when the temperature rose; the maximal evaporative water loss was 3.88±0.41 mg H₂O/g h at 37.5 °C. The results may reflect features of small mammals in the sub-tropical plateau region: T. belangeri had high basal metabolic rate and high total thermal conductance, compared with the predicted values based on their body mass whilst their body temperatures are relatively high; T. belangeri has high levels of evaporative water loss and poor water-retention capacity. Evaporative water loss plays an important role in temperature regulation.     

The effect of temperature, water level and burial depth on seed germination of Myriophyllum spicatum and Potamogeton malaianus

The effects of temperature, water level and burial depth on seed germination of two submerged species, Myriophyllum spicatum and Potamogeton malaianus, were investigated under controlled laboratory conditions. There was no significant difference in final germination of M. spicatum among water level treatments, but P. malaianus germinations at 1 cm and 12 cm water levels were better than at 0 cm water level at temperatures of 20 °C and 30 °C. Little to no germination was observed for either species at the temperature of 10 °C. At 15 °C, however, germination increased significantly to 66.3-70.6% for M. spicatum and to 29.4-48.1% for P. malaianus under all three water level treatments. Increased temperature from 15 °C to 30 °C had no significant effect on the final germination of M. spicatum except at the 1 cm water level, but enhanced significantly the germination of P. malaianus. Analysis of the mean time to germination revealed that M. spicatum was a faster germinator relative to P. malaianus. The two species’ germination differed markedly in response to burial depth. Germination percentage of M. spicatum was 71.3% at 0 cm burial depth, but decreased to 5.0% and to 2.5% at depths of 1 cm and 2 cm, respectively; whereas germination percentages of P. malaianus were 40.0%, 23.8%, 12.5%, 7.5% and 1.3% at depths of 0 cm, 1 cm, 2 cm, 3 cm and 5 cm, respectively. We concluded that the two species respond differently to germination strategies. The findings provided further insight into how germination strategy contributes to the seed bank formation and species invasion.     

The combined influence of sub-optimal temperature and salinity on the in vitro viability of Perkinsus marinus, a protistan parasite of the eastern oyster Crassostrea virginica

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25 ppt) to 10 °C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 °C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7 ppt cultures acclimated to each temperature and then transferred to 3.5 ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30 days (3.5 ppt, 2 °C: 0% viability), 60 days (3.5 ppt, 10 °C: 0% viability) and 90 days (7 ppt, 2 °C: 0.6 ± 0.7%; 7 ppt, 10 °C: 0.2 ± 0.2%).     

Duration tenacity: A method for assessing acclimatory capacity of the Antarctic limpet, Nacella concinna

The limpet, Nacella concinna, collected from the Antarctic Peninsula (67°S), was incubated at − 0.3 °C and 2.9 °C for 9 months to test if the previously reported absence of acclimation capacity in Antarctic marine ectotherms could be due to the extended time it takes for them to adjust their physiology to a new stable state. Acclimation was tested through acute measurements of upper lethal limit and a modified measure of tenacity, that tested muscle capacity by measuring the length of time that N. concinna were able to remain attached to the substratum at different temperatures. Both measures acclimated in response to incubation to the higher temperature. Lethal limits were elevated in N. concinna incubated at 2.9 °C (8.1 ± 0.3 °C) compared to those incubated at − 0.3 °C (6.9 ± 0.4 °C). 2.9 °C incubated N. concinna also had a maximum tenacity at 2.1 °C, a higher temperature than the maximum tenacity of those incubated at − 0.3 °C, which occurred at − 1.0 °C. This study is the first to show that the Antarctic limpet can acclimate its physiology, but that it requires a greater period of time for acclimation to occur than previous studies have allowed for.     

Synergistic effects of high temperature and sulfide on tropical seagrass

To examine the synergism of high temperature and sulfide on two dominant tropical seagrass species, a large-scale mesocosm experiment was conducted in which sulfide accumulation rates (SAR) were increased by adding labile carbon (glucose) to intact seagrass sediment cores across a range of temperatures. During the initial 10 d of the 38 d experiment, porewater SAR in cores increased 2- to 3-fold from 44 and 136 μmol L^− 1 d^− 1 at 28-29 °C to 80 and 308 μmol L^− 1 d^− 1 at 34-35 °C in Halodule wrightii and Thalassia testudinum cores, respectively. Labile C additions to the sediment resulted in SAR of 443 and 601 μmol L^− 1 d^− 1 at 28-29 °C and 758 to 1,557 μmol L^− 1 d^− 1 at 34-35 °C in H. wrightii and T. testudinum cores, respectively. Both T. testudinum and H. wrightii were highly thermal tolerant, demonstrating their tropical affinities and potential to adapt to high temperatures. While plants survived the 38 d temperature treatments, there was a clear thermal threshold above 33 °C where T. testudinum growth declined and leaf quantum efficiencies (Fv/Fm) fell below 0.7. At this threshold temperature, H. wrightii maintained shoot densities and leaf quantum efficiencies. Although H. wrightii showed a greater tolerance to high temperature, T. testudinum had a greater capacity to sustain biomass and short shoots under thermal stress with labile C enrichment, regardless of the fact that sulfide levels in the T. testudinum cores were 2 times higher than in the H. wrightii cores. Tropical seagrass tolerance to elevated temperatures, predicted in the future with global warming, should be considered in the context of the sediment-plant complex which incorporates the synergism of plant physiological responses and shifts in sulfur biogeochemistry leading to increased plant exposure to sulfides, a known toxin.     

Effects of low-intensity microwave radiation on Tribolium castaneum physiological and biochemical characteristics and survival

The red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae) is a widespread pest that lives in, and feeds on, wheat flour. Here, we studied the effects of low-intensity microwave radiation (LIMR; ≤2.0 kW/kg) on physiological and biochemical characteristics of T. castaneum, and compared them to the effects of heat conduction treatment, to provide a theoretical basis for using LIMR for pest control. Lethal model equations with respect to temperature were shown to provide acceptable fitting accuracy for the effects of LIMR treatment. Semi-lethal and lethal temperatures induced through LIMR (48 °C and 50 °C, respectively) for T. castaneum were lower than those induced through heat conduction (50 °C and 52 °C). When T. castaneum were subjected to LIMR, the insects’ moisture content, pH values, alkaline phosphatase and acetyl cholinesterase activity were all lower than when the insects were subjected to heat conduction. Peroxide values and total free amino acid content were higher, and protein subunits molecular weights were lower when T. castaneum were exposed to LIMR than to heat; moreover, after LIMR exposure, the amino acid composition of T. castaneum was changed and the insect's DNA was damaged.     

Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50 MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80 °C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were − 45 and − 53 ml/mol at 25 °C for mpDHFR, which were smaller (less negative) than the corresponding values of − 77 and − 85 ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.     

Seed germination of Lasia spinosa as a function of temperature,light, desiccation,and storage

Lasia spinosa seeds were not dormant at maturity in early spring. The most favorable temperatures for germination were between 25 and 30 °C, and final percentage and rate of germination decreased with an increase or decrease in temperature. When L. spinosa seeds were transferred to 25 °C, after 60 days at 10 °C (where none of the seeds germinated), final germination increased from 0% to 78%. Seeds germinated to high percentage both in light and in dark, although dark germination took more than twice as long as in the light. During desiccation of seeds at 15 °C and 45% relatively humidity, moisture loss decreased exponentially from 2.02 to 0.13 g H₂O g⁻¹ dry wt within 16 days, and only a few seeds (12%) survived 0.13 g H₂O g⁻¹ dry wt moisture content. Seeds stored at 0.58 g H₂O g⁻¹ dry wt moisture content at four constant temperatures (4, 10, 15, and −18 °C) for up to 6 months exhibited a well-defined trend of decreasing viability with decreasing temperature. Thus, we concluded that freshly harvested L. spinosa seeds are non-dormant and recalcitrant. Also, the seeds with 0.58 g H₂O g⁻¹ dry wt moisture content could be effectively stored for a few months between 10 and 15 °C although the most appropriate temperature for wet storage appears to be 10 °C, as it is close to the minimum temperature for germination and so there will be less pre-sprouting compared to 15 °C.     

Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores

Blue multicopper oxidases, laccases displayed on the surface of Bacillus spores were used to decolorize a widely used textile dyestuff, indigo carmine. The laccase-encoding gene of Bacillus subtilis, cotA, was cloned and expressed in B. subtilis DB104, and the expressed enzyme was spontaneously localized on Bacillus spores. B. subtilis spores expressing laccase exhibited maximal activity for the oxidation of 2,2′-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) at pH 4.0 and 80 °C, and for the decolorization of indigo carmine at pH 8.0 and 60 °C. The displayed enzyme retained 80% of its original activity after pre-treatment with organic solvents such as 50% acetonitrile and n-hexane for 2 h at 37 °C. The apparent K_m of the enzyme displayed on spores was 443 ± 124 μM for ABTS with a V_max of 150 ± 16 U/mg spores. Notably, 1 mg of spores displaying B. subtilis laccase (3.4 × 10² U for ABTS as a substrate) decolorized 44.6 μg indigo carmine in 2 h. The spore reactor (0.5 g of spores corresponding to 1.7 × 10⁵ U in 50 mL) in a consecutive batch recycling mode decolorized 223 mg indigo carmine/L to completion within 42 h at pH 8.0 and 60 °C. These results suggest that laccase displayed on B. subtilis spores can serve as a powerful environmental tool for the treatment of textile dye effluent.     

Effect of lignocellulosic inhibitory compounds on growth and ethanol fermentation of newly-isolated thermotolerant Issatchenkia orientalis

A newly isolated thermotolerant ethanologenic yeast strain, Issatchenkia orientalis IPE 100, was able to produce ethanol with a theoretical yield of 85% per g of glucose at 42 °C. Ethanol production was inhibited by furfural, hydroxymethylfurfural and vanillin concentrations above 5.56 g L⁻¹, 7.81 g L⁻¹, and 3.17 g L⁻¹, respectively, but the strain was able to produce ethanol from enzymatically hydrolyzed steam-exploded cornstalk with 93.8% of theoretical yield and 0.91 g L⁻¹ h⁻¹ of productivity at 42 °C. Therefore, I. orientalis IPE 100 is a potential candidate for commercial lignocelluloses-to-ethanol production.