Establishment,growth kinetics,and susceptibility to AcMNPV of heat tolerant lepidopteran cell lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33°C and 35°C was carried out by shifting the culture temperature between 28°C and higher temperatures by a gradual stepwise increase in temperature. The process of adaption to a higher culture temperature was accomplished over a period of 2 months. The cell lines with the temperature adaption were designated as sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, MG1-ht35. These cell lines have been subcultured over 70 passages. Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines. The population doubling time of heat adapted cell lines were 1–4 h less than these of parental cell lines. Cell shapes did not show obvious change, however, the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption. When the cell lines were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) at 28°C, 33°C, 35°C and 37°C, production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.     

Adaptation of an insect cell line of Spodoptera frugiperda to grow at 37°C: Characterization of an endodiploid clone

Summary Sf21 and Sf9 cell lines established from the lepidoptera Spodoptera frugiperda do not display major induction of heat shock proteins when exposed to a temperature of 37°C. After some months of adaptation at 37°C we obtained two new cell lines, Sf21-HT and SF9-HT, which have now been established for several years in our laboratory. The Sf9-HT line displays a slightly shorter doubling time at 37°C than the wild type at 28°C, but cell lethality gives rise to an earlier growth arrest. The composition of total lipid extract from heat-adapted cells reveals a higher sphingomyelin to phosphatidylcholine ratio and a higher percentage of saturated fatty acids, which are expected for the lower membrane fluidity, required for thermotolerance. The cell volume of Sf9-HT is doubled, and by flow cytometry we showed that the DNA content is twice that in the parental cell line. Karyotypic examination of metaphasic cells achieved under epifluorescence microscopy revealed a doubled chromosome number in Sf9-HT.     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

Effect of temperature on the life history and demographic parameters of Spalgis epius (Lepidoptera: Lycaenidae), a candidate biological control agent of mealybugs (Hemiptera: Pseudococcidae)

Many species of mealybugs (Hemiptera: Pseudococcidae) are serious pests of economically important crops worldwide. We evaluated the influence of constant temperatures: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 34°C on the life history and demographic parameters of Spalgis epius (Lepidoptera: Lycaenidae), a candidate biological control agent of various species of mealybugs. No eggs completed their development at 14 and 34°C. Egg-to-adult developmental time significantly decreased from 89.9 days at 16°C to 20.4 days at 32°C. The estimated lower temperature threshold of 10.2°C and 416.6 degree-days were required to complete egg-to-adult development. The mortality of immature stages was maximum at 16 and 32°C and minimum at 28°C. The highest lifetime fecundity was recorded at 28°C and it significantly decreased at 32°C. The longevity of adults was about three times more at 16°C than at 30 and 32°C. The net reproductive rate (R ₀) significantly increased with increased temperatures up to 28°C and significantly decreased at 32°C. The mean generation time (T) significantly decreased with increased temperature up to 30°C, but it significantly increased at 32°C. The intrinsic rate of population increase (r _m ) was highest at 30°C. The finite rate of increase (λ) was significantly greater at 30°C than at other temperatures. These data suggest that S. epius can develop, reproduce and survive in a wide range of temperatures and thus could be regarded a potential biological control agent of mealybugs.     

EXPERIMENTAL EVOLUTION OF HSP70 EXPRESSION AND THERMOTOLERANCE IN DROSOPHILA MELANOGASTER

To examine whether recent evolutionary history affects the expression of Hsp70, the major heat-induced-heat shock protein in Drosophila melanogaster, we measured Hsp70 expression, thermotolerance, and hsp70 gene number in replicate populations undergoing laboratory evolution at different temperatures. Despite Hsp70's ancient and highly conserved nature, experimental evolution effectively and replicably modified its expression and phenotype (thermotolerance). Among five D. melanogaster populations founded from a common ancestral population and raised at three different temperatures (one at 18°C, two each at 25°C and 28°C) for twenty years, Hsp70 expression varies in a consistent pattern: the replicate 28°C lines expressed 30–50% less Hsp70 than the other lines at a range of inducing temperatures. This modification was refractory to acclimation, and correlated with thermotolerance: the 28°C lines had significantly lower inducible tolerance of 38.5°C and 39°C. We verified the presence of five hsp70 genes in the genome of each line, excluding copy number variation as a candidate molecular basis of the evolved difference in expression. These findings support the ability of Hsp70 levels in D. melanogaster populations to change over microevolutionary time scales and implicate constancy of environmental temperature as a potentially important selective agent.     

CHROMOSOMAL ANALYSIS OF HEAT-SHOCK TOLERANCE IN DROSOPHILA MELANOGASTER EVOLVING AT DIFFERENT TEMPERATURES IN THE LABORATORY

We investigated the heat tolerance of adults of three replicated lines of Drosophila melanogaster that have been evolving independently by laboratory natural selection for 15 yr at “nonextreme” temperatures (18°C, 25°C, or 28°C). These lines are known to have diverged in body size and in the thermal dependence of several life-history traits. Here we show that they differ also in tolerance of extreme high temperature as well as in induced thermotolerance (“heat hardening”). For example, the 28°C flies had the highest probability of surviving a heat shock, whereas the 18°C flies generally had the lowest probability. A short heat pretreatment increased the heat tolerance of the 18°C and 25°C lines, and the threshold temperature necessary to induce thermotolerance was lower for the 18°C line than for the 25°C line. However, neither heat pretreatment nor acclimation to different temperatures influenced heat tolerance of the 28°C line, suggesting the loss of capacity for induced thermotolerance and for acclimation. Thus, patterns of tolerance of extreme heat, of acclimation, and of induced thermotolerance have evolved as correlated responses to natural selection at nonextreme temperatures. A genetic analysis of heat tolerance of a representative replicate population each from the 18°C and 28°C lines indicates that chromosomes 1, 2, and 3 have significant effects on heat tolerance. However, the cytoplasm has little influence, contrary to findings in an earlier study of other stocks that had been evolving for 7 yr at 14°C versus 25°C. Because genes for heat stress proteins (hsps) are concentrated on chromosome 3, the potential role of hsps in the heat tolerance and of induced thermotolerance in these naturally selected lines is currently unclear. In any case, species of Drosophila possess considerable genetic variation in thermal sensitivity and thus have the potential to evolve rapidly in response to climate change; but predicting that response may be difficult.     

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

Summary A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.     

EFFECTS OF LIGHT INTENSITY AND TEMPERATURE INTERACTIONS ON GROWTH CHARACTERISTICS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cell division rate, carbon fixation per cell, cell width and chloroplast length of Phaeodactylum tricornutum Bohlin were determined at 30 different combinations of light intensity and temperature. Division rate peaked at 23° C or less depending on light intensity. For each light intensity studied, carbon fixation increased directly with growth temperature from 14 to 25° C. The slope of this relationship was modified by light intensity. Cells grown at 23–25° C tended to be larger than those grown at lower temperatures, possibly due to increased carbon fixation per cell coupled with lower division rates. Chloroplasts were largest at a combination of temperatures above 21° C and low light intensities. This effect could cause cells to sink at a higher than normal rate due to reduced vacuole size and is presented as a possible mechanism affecting the distribution of P. tricornutum.     

Production of ginkgolides and bilobalide from optimized the<Emphasis Type="Italic">Ginkgo biloba</Emphasis> cell culture

The influence of various culture conditions on growth and ginkgolides (GKA and GKB), and bilobalide formation in callus and suspension cultures ofGinkgo biloba were investigated. Callus induced from the leaf petioles exhibited distinct morphological and physiological responses. The cell biomass and ginkgolides content varied among the cell lines brownish callus lines produced high levels of ginkgolides and bilobalide in spite of poor cell growth. Among the culture media used, MS medium showed significant effect on cell growth and ginkgolides production. Low concentration of sucrose (3%) improved cell growth, while higher sucrose levels (5 and 7%) improved ginkgolides production. Cultivation of callus cultures above 28°C dramatically reduced their growth rate; however the cell lines grown at 36°C showed increased levels of bilobalide content. A 2.5-L balloon type bubble bioreactor (BTBB) was successfully developed for the cell growth and ginkgolides production.     

Membrane and calcium clock mechanisms contribute variably as a function of temperature to setting cardiac pacemaker rate in zebrafish Danio rerio

Here, we show that heart rate in zebrafish Danio rerio is dependent upon two pacemaking mechanisms and it possesses a limited ability to reset the cardiac pacemaker with temperature acclimation. Electrocardiogram recordings, taken from individual, anaesthetised zebrafish that had been acclimated to 18, 23 or 28°C were used to follow the response of maximum heart rate (f_Hmax) to acute warming from 18°C until signs of cardiac failure appeared (up to c. 40°C). Because f_Hmax was similar across the acclimation groups at almost all equivalent test temperatures, warm acclimation was limited to one significant effect, the 23°C acclimated zebrafish had a significantly higher (21%) peak f_Hmax and reached a higher (3°C) test temperature than the 18°C acclimated zebrafish. Using zatebradine to block the membrane hyperpolarisation-activated cyclic nucleotide–gated channels (HCN) and examine the contribution of the membrane clock mechanisms to cardiac pacemaking, f _Hmax was significantly reduced (by at least 40%) at all acute test temperatures and significantly more so at most test temperatures for zebrafish acclimated to 28°C vs. 23°C. Thus, HCN channels and the membrane clock were not only important, but could be modified by thermal acclimation. Using a combination of ryanodine (to block sarcoplasmic calcium release) and thapsigargin (to block sarcoplasmic calcium reuptake) to examine the contribution of sarcoplasmic reticular handling of calcium and the calcium clock, f _Hmax was again consistently reduced independent of the test temperature and acclimation temperature, but to a significantly lesser degree than zatebradine for zebrafish acclimated to both 28 and 18°C. Thus, the calcium clock mechanism plays an additional role in setting pacemaker activity that was independent of temperature. In conclusion, the zebrafish cardiac pacemaker has a limited temperature acclimation ability compared with known effects for other fishes and involves two pacemaking mechanisms, one of which was independent of temperature.     

Isolation and Identification of α,α-Trehalose and Glycerol from an Arctic Psychrotolerant Streptomyces sp. SB9 and their Possible Role in the Strain's Survival

Abstract

Streptomyces sp. strain SB9 was isolated from perm frost soil samples in Spitsbergen, Arctic Ocean; it grows in a temperature range between 4°C and 28°C. During the survey of biologically active metabolites biosynthesized by this strain, significant amounts of α,α-trehalose (1) and glycerol (2) were detected. The compounds were isolated from the mycelium, were chromatographically separated, and the structures were elucidated on the basis of MS and NMR measurements. A possible role of trehalose in cold adaptation of the strain was examined. It was determined that the mycelium of the strain cultivated at 4°C accumulated 5-fold higher amounts of trehalose in comparison with the cells cultivated at 28°C. The mesofilic reference strains, Streptomyces spectabilis NRRL 2494 and Streptomyces lividans TK64, accumulated 100-fold less trahalose than the psychrotolerant Streptomyces sp. SB9. High amounts of trehalose in the cells could be a reason for adaptation of the strain to life at Arctic conditions.     

Oviposition pattern of the predator Podisus nigrispinus (Heteroptera: Pentatomidae) under different temperatures

The daily reproductive rate of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) fed with Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) larvae was studied at constant temperatures of 20, 23, 25, 28, 30 and 33±0.2°C, relative humidity of 60±10% and photoperiod of L:D 14:10. Daily reproductive rate of P. nigripinus was affected by age of this predator. Each P. nigrispinus female laid 5.3 (20°C) to 19.9 eggs/day (28°C) which developed into 4.3–16.5 nymphs, respectively. Highest daily reproductive rate of P. nigrispinus was recorded at 28 and 30°C for 5–30-day-old females. This predator showed higher daily reproductive rate than its prey A. argillacea at 25°C. It was also able to reproduce at temperatures from 20 to 33°C with maximum daily reproductive rate between 25 and 30°C. These results are important for optimizing mass rearing of P. nigrispinus in the laboratory.     

Biology ofCryptolaemus montrouzieri Mulsant [Coccinellidae: Coleoptera] in relation with temperature

The effect of temperature on the developmental duration ofCryptolaemus montrouzieri Mulsant was quantified by deriving a regression equation for each developmental stage as well as the total life cycle. While the duration of life stages was shorter during summer and longer during winter, the optimum constant temperature for maximal development was found to be 30°C. The adult longevity was extended when reared at 20°C than at 30°C and ambient temperature. The longevity of adults was longer when maintained on grape mealybugMaconellicoccus hirsutus (Green) than on honey and when maintained at 20°C. The fecundity of the predator was higher at 30°C than at 20°C. Eventhough the adults could survive at 10°C, the productive capacity was impaired.      

Temperature-sensitive photosynthesis-deficient mutants of the cyanobacteriumAnabœna variabilis impaired in nitrogen assimilation

Temperature-sensitive (Ts) mutants ofAnabœna variabilis exhibited differences in the rates of oxygen evolution at 28 and 40°C. They were unable to perform photosynthesis and nitrogen fixation at 40°C beyond a period of 3 d in a nitrogen-deficient medium. However, the addition of combined nitrogen sources enhanced the growth of all the Ts mutants at both temperatures. Studies on nitrate uptake ty Ts mutants revealed the existence of a low affinity system, whereK _m values were found to be lower than 1 mmol/L. The activity of nitrate reductase gradually increased with time at 28°C with the exception of Ts-161 where the activity decreased with time at 40°C. The rate of ammonia uptake byA. variabilis and its Ts mutants greatly differed and the results suggest the existence of a single phase of uptake. The activity of glutamate-ammonia ligase (GAL) of the parent and Ts mutants was slightly higher in cells from nitrogen-deficient medium when compared to nitrate grown cells at 28°C. At 40°C, the GAL activity decreased after 3 d. The inability of the Ts mutants to grow at 40°C appears to be due to an impairment in nitrogen asimilation.     

High temperature inactivation of cucumber and alfalfa mosaic viruses in Nicotiana rustica cultures

Cucumber mosaic (CMV) and alfalfa mosaic (AlfMV) viruses could not be detected in Nicotiana rustica tissues cultured at 32 °C for 16–18 days or at 40 °C for 5 days, but infectivity remained high in comparable tissue cultured at 22 °C. Incubation of infected cultures at 28–30 °C resulted in an initial reduction followed by a partial recovery in the infectivity of both viruses. The infectivity of CMV in tissues grown between 12 and 32 °C was highest in cultures grown at 12 °C. Although CMV infectivity was not detected in cultures after 16–18 days at 32 °C, virus was eliminated only after a further 30 days at 32 °C. When cultures were transferred from 32 to 22 °C after shorter treatment periods, infectivity rapidly increased to levels higher than those of infected tissues grown continuously at 22 °C. At 40 °C, CMV was eliminated from infected tissues after 9 days and AlfMV after 7 days. Cultures grown continuously at 40 °C deteriorated rapidly but, when grown under diurnal alternating periods of 8 h at 40 °C and 16 h at 22 °C, they remained viable and CMV was also inactivated.     

Temperature-dependent phosphate solubilization by cold- and pH-tolerant species of Aspergillus isolated from Himalayan soil

Ten species of Aspergillus isolated from soil samples collected from different locations in the Indian Himalayan region have been studied for their growth requirements and tricalcium phosphate solubilization at different temperatures. The Aspergillus species could grow at low temperature and tolerated a wide range of pH. Phosphate solubilization by various Aspergillus species ranged between 374 μg/ml (A. candidus) to 1394 μg/ml (A. niger) at 28°C, 33 μg/ml (A. fumigatus) to 2354 μg/ml (A. niger) at 21°C, 93 μg/ml (A. fumigatus) to 1452 μg/ml (A. niger) at 14°C, and 21 μg/ml (A. wentii) to 83 μg/ml (A. niger) at 9°C. At 21 and 28°C, phosphate solubilization showed a decrease within 4 weeks of incubation, whereas at 9°C and 14°C, it continued further up to 6 weeks of incubation. In general, phosphate solubilization by different Aspergillus species was recorded at a maximum of 28°C or 21°C; biomass production was favored at 21°C or 14°C. Conversely, A. nidulans and A. sydowii exhibited maximum phosphate solubilization at 14°C and produced maximum biomass at 21°C. Data suggest that suboptimal conditions (higher or lower temperature) for fungal growth and biomass production were optimal for the production of metabolites involved in phosphate solubilization. Significant negative correlations were obtained between pH and phosphate solubilization for eight species at 28°C, for seven at 21°C, and for nine at 14°C. Extracellular phosphatase activity was exhibited only in case of A. niger, whreas intracellular phosphatase activity was detected in all species, the maximum being in A. niger. Statistically significant positive or negative correlations were obtained between phosphate solubilization and other parameters in most cases at different temperatures.     

Effect of temperature and diet on hind wing colouration development and elytral hardness of adult Colorado potato beetle (Coleoptera: Chrysomelidae)

The effects of food and temperature on the development of colour pigment in the hind wings of adult Colorado potato beetle, Leptinotarsa decemlineata (Say), were investigated. In a replicated study, adults were held at 18°C, 28°C and 18/28°C on potato foliage (Solanum tuberosum L.), potato tubers, or without food in controlled-environment chambers. Representative subsamples of wings were collected at two-day intervals, mounted on microscope slides, and photographed to document the progression of colour change. Observations were also made on elytral hardening over time. Hind wing colour developed more quickly at 28°C than at 18°C, and after three weeks had attained a deeper red colour at the higher temperature. Colour development was also more rapid when adult beetles were fed on foliage compared with tubers. In foliage-fed beetles, elytra hardened more quickly at 28°C than 18°C, and many tuber-fed beetles never developed hardened elytra, regardless of temperature treatment. Unfed beetles developed no hind wing colour pigment and their elytra remained soft for the duration of the experiment. Colour plates documenting wing colour development over time are presented; variation in colour development under the conditions tested, suggests that the use of hind wing colouration to estimate beetle age in the field may be problematic.     

Heat inactivation of cucumber mosaic virus in cultured tissues of Stellaria media

In vivo heat inactivation of cucumber mosaic virus (CMV) was studied in a low-temperature-tolerant species, Stellaria media. Infectivity remained high in infected 5. media cultures grown at 25 °C or less, but could not be detected in cultures incubated at 32 °C for 28 days. When infected tissues were grown at 12–32 °C for 24 days, virus was still detectable at a low level in cultures incubated at 30 °C. The highest level of infectivity was in cultures at 12 °C.     

Promotion of Autolysis in Lactobacilli

The conditions of promotion of autolysis of three strains of Lactobacilli were investigated. The autolysis of L. acidophilus, L. helveticus and L. casei at exponential phase was remarkably enhanced by freezing storage at ?20°C overnight.

The turbidity decrease of L. acidophilus’ cell suspension corresponds to the increase of cell free nitrogen compounds, glucosamine and DNA component. All these compounds were more rapidly released from the cells stored at ?20°C than those stored at 3°C. The cells which were harvested at the exponential phase had higher autolytic activity than those at stationary phase. The storage of the cells at ?20°C for 2 days or more could effectively promote the autolysis.

The activity was increased by Ca²⁺ or Mg²⁺. Optimum pH of the autolytic enzyme of L. acidophilus was 6~7.     

Effects of temperature on life histories of three endangered Japanese diving beetle species

To elucidate population-increasing factors in the diving beetle Cybister tripunctatus lateralis (Fabricius) (Coleoptera: Dytiscidae) in Japan in recent years, life histories and oviposition patterns were compared among three endangered diving beetle species, Cybister brevis Aubé (qualified by the Japanese Red Data List as ‘near threatened’), Cybister chinensis Motschulsky (vulnerable), and C. tripunctatus lateralis (vulnerable). Oviposition in C. brevis, C. chinensis, and C. tripunctatus lateralis was observed from late April to mid-June, from late April to early July, and from late May to mid-August, respectively, under semi-outdoor conditions. There were no interspecies differences in total hatchling production during the reproductive season. In rearing experiments at various temperatures (20, 23, 25, 28, and 30 °C), the mortality of C. tripunctatus lateralis larvae was higher at 20 °C, and gradually lower with increasing temperature up to 30 °C. Adult body size of females in C. tripunctatus lateralis is larger than that of males but there were no significant differences among temperatures (25–30 °C). Cybister brevis had a higher emergence rate at 23–28 °C than at 20 and 30 °C. In C. brevis, the body size of adults reared at 25 or 28 °C was significantly larger than at other temperatures. Cybister chinensis did not differ in emergence rate and adult body size among the five temperature conditions. The developmental zero (i.e., the lower developmental threshold) from the first instar to adult emergence was 11.1 °C for C. brevis, 8.7 °C for C. chinensis, and 16.8 °C for C. tripunctatus lateralis. We speculate how the influence of global warming may have a positive impact on the growth and survival of C. tripunctatus lateralis.