Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata

The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 °C) followed by adult flies and late embryos (100% survival at 39 °C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37-39 °C increased thermotolerance at higher temperatures by approximately 1 °C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.     

Novel means for variety breeding and sporeling production in the brown seaweed Undaria pinnatifida (Phaeophyceae): Crossing female gametophytes from parthenosporophytes with male gametophyte clones

The unialgal haploid gametophyte clones are frequently used for variety breeding and sporeling production in the brown seaweed Undaria pinnatifida because a single crossing of a pair of the selected male and female gametophyte clones can generate sporophytic offspring with identical genotype and phenotype. As the seeds to be sprayed on the collectors, the detachment rate of the filamentous gametophytes is high in comparison to the seeded spores. In this investigation, we report the use of parthenogenesis to achieve the same goal in variety selection and sporeling production but with higher efficiency. The selected female unialgal gametophyte clone (Code: 06‐8‐1F) was induced to produce parthenosporophytes. These sporophytes were grown up in a controlled system and used to release zoospores. All zoospores generated into female gametophytes. These female gametophytes were allowed to go through parthenogenesis for the second year to confirm the applicability of this means. In the third year, the zoospores released from the parthenosporophytes were seeded on collectors over summer in female gametophyte form. In the early autumn, a selected male unialgal gametophyte clone (code: 5#F1‐2‐5M) was used to cross the seeded female gametophytes on the collectors. When the sporelings reached a mean length of 780 μm, they were transplanted to open sea on longlines for growing up. At harvest, the average length, width and wet weight of the adult sporophytes were 211 cm, 48.8 cm and 373 g, respectively. The sporophytic blades were uniformly smooth without wrinkles on both sides of the midrib, indicating top quality of the products. Amplified fragment length polymorphism analyses confirmed the identical genotypes of sporophytic offspring. These results suggested that this novel variety breeding and sporeling production method could serve as an efficient alternative to the traditional breeding technique for U. pinnatifida and possibly other commercial kelps that have identical life cycles.     

Effect of pre-incubation irradiance on survival of cryopreserved gametophytes of Undaria pinnatifida (Phaeophyta) and morphology of sporophytes formed from the gametophytes

Gametophyte strains originating from indigenous sporophytes of Undaria pinnatifida (Harvey) Suringar in Iwate Prefecture, Northeast Japan, were maintained for 9–10 months at 45 μmol photons m⁻² s⁻¹. Before cryopreservation in liquid nitrogen for more than 12 h (1–14 days) using a two-step cooling method with a mixture of cryoprotectants (10% l-proline and 10% glycerol), these were pre-incubated for 2, 4 and 8 months at 15 μmol photons m⁻² s⁻¹. After 1 week of thawing, no surviving gametophytes were detected in the strains without pre-incubation, but both the female and male gametophytes, pre-incubated for more than 4 months, showed high survival rates (43–60% for females and 64–100% for males). This revealed the induction of freezing tolerance by incubation at low irradiance. Thereafter, sporophytes derived from cryopreserved gametophytes and subcultured gametophytes, stored under pre-incubation conditions, were formed from the strain, and a morphological comparison was conducted with 10 characters (stipe length, stipe wet weight, blade length, blade wet weight, blade width, incision depth, blade thickness, sporophyll length, sporophyll wet weight, and sporophyll width). The morphology of the sporophytes formed from the cryopreserved gametophytes corresponded well with that of the subcultured gametophytes from the same strain. The results suggest that the cryopreservation method is applicable for preserving culture stocks of U. pinnatifida to be used in mariculture.     

Effects of temperature and nutrients on microscopic stages of the bull kelp (Nereocystis luetkeana,Phaeophyceae)

Warming ocean temperatures have been linked to kelp forest declines worldwide, and elevated temperatures can act synergistically with other local stressors to exacerbate kelp loss. The bull kelp Nereocystis luetkeana is the primary canopy-forming kelp species in the Salish Sea, where it is declining in areas with elevated summer water temperatures and low nutrient concentrations. To determine the interactive effects of these two stressors on microscopic stages of N. luetkeana, we cultured gametophytes and microscopic sporophytes from seven different Salish Sea populations across seven different temperatures (10–22°C) and two nitrogen concentrations. The thermal tolerance of microscopic gametophytes and sporophytes was similar across populations, and high temperatures were more stressful than low nitrogen levels. Additional nitrogen did not improve gametophyte or sporophyte survival at high temperatures. Gametophyte densities were highest between 10 and 16°C and declined sharply at 18°C, and temperatures of 20 and 22°C were lethal. The window for successful sporophyte production was narrower, peaking at 10–14°C. Across all populations, the warmest temperature at which sporophytes were produced was 16 or 18°C, but sporophyte densities were 78% lower at 16°C and 95% lower at 18°C compared to cooler temperatures. In the field, bottom temperatures revealed that the thermal limits of gametophyte growth (18°C) and sporophyte production (16–18°C) were reached during the summer at multiple sites. Prolonged exposure of bull kelp gametophytes to temperatures of 16°C and above could limit reproduction, and therefore recruitment, of adult kelp sporophytes.     

Phenology and wintering capacity of sporophytes and gametophytes of ferns native to northern Japan

Summary To investigate life history adaptations to cold climates, the leaf development, sporulation period, growing stage of gametophytes, and the frost and drought resistance of sporophytes and gametophytes of 67 fern species native to Kokkaido were studied. Most ferns common in Hokkaido are summer-green with leaves developing during late May to June and decaying during October. Most of the ferns in Hokkaido sporulate during August to early September. Spores dispersed from June to September germinate before winter begins, forming vegetative prothallia. Gametophytes mature only in the following summer. Thus in Hokkaido the gametophytes as well as perennial sporophytes are exposed to severe winter conditions. In order to correlate the life cycles of temperate ferns with winter cold stress, frost resistance of gametophytes, rhizomes, and leaves of sporophytes were determined. Maximal frost resistance of rhizomes reflects the stress conditions of their habitats: rhizomes of forest understory ferns are damaged at-5°to -17.5°C, epiphytic ferns and ferns of habitats exposed to severe frost sustained temperatures of -20° to-40°C. The leaves of winter-green and evergreen ferns resist frost ranging from -25° to -40°C. The leaves of summer-green ferns are killed by late frost below -5°C. With some exceptions, gametophytes of ferns growing on the forest floor resist frost to -40°C and are much hardier than sporophytes. These results suggest the possible restrictive effects of cold climate on the life span of leaves as well as on the sporulation period. If winter cold is one of the decisive factors for seasonality expression and habitat distribution of ferns, the sensitive generation must be the sporophyte rather than the gametophyte. The hardier gametophyte is therefore able to colonize habitats in which the sporophyte is excluded by frost if mechanisms of vegetative propagation are evolved.Contribution No. 2451 from The Institute of Low Temperature Science     

Effect of temperature and photon fluence rate on gametophytes and young sporophytes of Laminaria ochroleuca Pylaie

We analysed the effects of temperature and photon fluence rate on meiospore germination, growth and fertility of gametophytes, and growth of young sporophytes of Laminaria ochroleuca. Maximum percentages of germination (91–98%) were obtained at 15°C and 18°C, independent of photon fluence rate. Optimal development of female gametophyte and maximum fecundity and reproductive success of gametophytes occurred at 15°C and 18°C and at 20 and 40 μmol m^–2 s^–1. Maximum relative growth rate of young sporophytes after 2 weeks of culture was achieved under the same conditions. L. ochroleuca gametophytes cannot reproduce and growth of its sporophytes is not competitive at temperatures close to 10°C. Received in revised form: 31 August 2001 Electronic Publication     

Ocean acidification and kelp development: Reduced pH has no negative effects on meiospore germination and gametophyte development of Macrocystis pyrifera and Undaria pinnatifida

The absorption of anthropogenic CO₂ by the oceans is causing a reduction in the pH of the surface waters termed ocean acidification (OA). This could have substantial effects on marine coastal environments where fleshy (non‐calcareous) macroalgae are dominant primary producers and ecosystem engineers. Few OA studies have focused on the early life stages of large macroalgae such as kelps. This study evaluated the effects of seawater pH on the ontogenic development of meiospores of the native kelp Macrocystis pyrifera and the invasive kelp Undaria pinnatifida, in south‐eastern New Zealand. Meiospores of both kelps were released into four seawater pH treatments (pH_T 7.20, extreme OA predicted for 2300; pH_T 7.65, OA predicted for 2100; pH_T 8.01, ambient pH; and pH_T 8.40, pre‐industrial pH) and cultured for 15 d. Meiospore germination, germling growth rate, and gametophyte size and sex ratio were monitored and measured. Exposure to reduced pH_T (7.20 and 7.65) had positive effects on germling growth rate and gametophyte size in both M. pyrifera and U. pinnatifida, whereas, higher pH_T (8.01 and 8.40) reduced the gametophyte size in both kelps. Sex ratio of gametophytes of both kelps was biased toward females under all pH_T treatments, except for U. pinnatifida at pH_T 7.65. Germling growth rate under OA was significantly higher in M. pyrifera compared to U. pinnatifida but gametophyte development was equal for both kelps under all seawater pH_T treatments, indicating that the microscopic stages of the native M. pyrifera and the invasive U. pinnatifida will respond similarly to OA.     

Growth and reproductive responses of Laminaria longicruris (Laminariales,Phaeophyta) to nutrient enrichment

A series of comparative culture experiments were conducted in order to determine responses of Laminaria longicruris male and female gametophytes and juvenile sporophytes to several temperatures (5, 10, 15, 20 °C), light levels (10, 35, 75 µmol m^–2 s^–1) and media nitrogen concentrations (0, 20, 100 µM ammonium-nitrogen). Responses were measured as numbers of male and female gametophytes producing gametangia and number of sporophytes produced following fertilization. Both male and female gametogenesis was reduced at 5 and 20 °C versus 10 and 15 °C. At 20 °C gametogenesis inhibition was greater with higher levels of ammonium-nitrogen concentration (100 µM). Sporophyte production was more sensitive to light, temperature and nitrogen concentration than gametogenesis. Production of sporophytes was inhibited completely at 20 °C. At lower temperatures, increasingly higher nutrient concentrations produced greater inhibition of production of sporophytes.     

Genetic differentiation of high-temperature tolerance in the kelp Undaria pinnatifida sporophytes from geographically separated populations along the Pacific coast of Japan

The kelp Undaria pinnatifida has a widespread latitudinal range in Japan, with populations exposed to very different temperature regimes. To test the hypothesis that U. pinnatifida exhibits genetic differentiation in its temperature response, juvenile sporophytes from a warmer location (Naruto, southern Japan) and two colder locations (Okirai Bay and Matsushima Bay, northern Japan) were collected and transplanted to long lines, cultivated under the environmental conditions in Matsushima Bay. These plants were bred using successive self-crossing methods for three generations and the characteristics of photosynthesis, growth, survival, and nitrogen contents of the third-generation juvenile sporophytes (2–3 cm) then were measured and compared. The plants from Naruto showed significantly higher photosynthetic activities and respiration than those from the northern populations at warmer temperatures of 20–35°C. The juvenile sporophytes from all three locations had similar growth rates below 18°C, but significant differences were observed at 18–24°C. The optimum temperatures for growth were 14–16°C in plants that originated from Okirai Bay and Matsushima Bay and 18°C in plants that originated from Naruto. These results reflected the differences in latitude. Dead plants were observed at high temperatures of 22 and 24°C in the northern population plants, whereas no plants from Naruto died. Juvenile sporophytes from Naruto exhibited the greatest capacity to accumulate high nitrogen reserves. These results suggest that the differences in high-temperature tolerance in juvenile U. pinnatifida sporophytes from geographically separated populations are due to genetic differentiation rather than phenotypic plasticity.     

Temperature requirements for the growth of young sporophytes of Undaria pinnatifida and Undaria undarioides (Laminariales, Phaeophyceae)

The relative growth rate of young sporophytes of Undaria pinnatifida (Harvey) Suringar and Undaria undarioides (Yendo) Okamura was examined in order to understand the difference in distribution of these two species around the coast of Japan. The optimal temperature for growth of both species was similar at 20°C and the upper critical temperature for growth was also similar, at 27°C for U. pinnatifida and 26°C for U. undarioides. Therefore, the optimal and upper critical temperatures for growth of the young sporophytes are not the main factors determining the distribution of each species. Next, the lower critical temperatures for growth were examined. For the young sporophytes of U. pinnatifida, the lower limit was less than 5°C while for those of U. undarioides it was 15°C. Thus, the difference in the lower critical temperature for growth between the two species was approximately 10°C. During the period of young sporophyte growth in the field, the temperature at the mouth of Ise Bay, Japan, where U. pinnatifida occurs, ranges from 12.7°C in December to 13.1°C in April, with a minimum of 7.9°C in February. Our experiments indicate that young sporophytes are able to grow throughout this period. The temperature off Hamajima, Japan, where U. undarioides occurs, ranges from 19.1°C to 14.8°C during the same time period. Again, young sporophytes are able to growth throughout this period, although minimum winter temperatures are only just high enough for growth. These natural temperature ranges during the growth season of the sporophytes agree well with the experimentally determined temperature requirements for growth of each species. Therefore, the difference between the two species in the critical temperature required for growth of the young sporophytes, especially in the low temperature range, is one of the major factors determining the distribution pattern of each species.     

Three heat shock proteins are essential for rotifer thermotolerance

Heat shock proteins (HSPs) are important molecules in the stress response of organisms from prokaryotes to mammals, and thus may be useful biomarkers for environmental stress. Here we characterize the functional roles of genes belonging to four distinct families of HSPs (hsp40, hsp60, hsp70, and hsp90) in the monogonont rotifer Brachionus manjavacas. Because B. manjavacas inhabits ponds of varying thermal regimes, including ephemeral ponds that may experience temperature fluctuations, HSP-mediated thermotolerance likely is important to its survival and adaptation. Using interference RNA (RNAi), we provide the first conclusive evidence that HSPs are required for rotifer survival following heat stress. Effective RNAi-mediated suppression of all hsp genes except hsp90 was verified via quantitative PCR. Hsp40, hsp60, and hsp70 are required for rotifer thermotolerance (P < 0.05); however, our data do not indicate hsp90 is essential. Quantitative PCR further revealed immediate up-regulation of hsp40 mRNA following heat stress. Additionally, we demonstrated expression of hsp40 mRNA in multiple tissues using fluorescent in situ hybridization. Our characterization of mRNA expression and functional roles for four distinct hsp genes provides a baseline for molecular-level comparisons of the stress response of rotifers with other taxonomic groups, and the technique for in-depth studies of the role of specific genes in rotifer stress responses. Considering the potential for ambient temperatures to impact species survival, competitive interactions, and body size of individuals, thermotolerance may be an important influence on zooplankton community structure.     

Gametophyte contribution to sporophyte growth on the basis of carbon gain in the fern <Emphasis Type="Italic">Thelypteris palustris</Emphasis>: effect of gametophyte organic-matter production on sporophytes

At an early stage of growth gametophytes support the sporophytes of ferns. Young sporophytes become independent of gametophytes when the first leaves develop. Although large fern gametophytes produce multiple archegonia simultaneously, only one sporophyte is typically established on one gametophyte. The number of sporophytes is believed to be controlled in two possible directions, from gametophyte to sporophyte or from preceding sporophyte to another sporophyte. To investigate the effects of gametophytes on their sporophytes, we studied the relationship between organic matter production by gametophytes and the growth of young sporophytes of Thelypteris palustris. We cut gametophytes in half (CGs) to reduce the gametophytes’ production of matter. There was no significant difference between the growth of sporophytes on intact gametophytes (IGs) and that on CGs. According to our estimates, based on the rate of organic matter production, the large gametophyte was able to produce two or more sporophytes. The resources required for CGs to make similar-sized sporophytes was twice that for IGs. In polyembryony each of the multiple sporophytes was similar in size to the single sporophytes. Resource limitation does not seem to explain why fern gametophytes establish single sporophytes.     

Temperature requirements for the growth and maturation of the gametophytes of Undaria pinnatifida and U. undarioides (Laminariales, Phaeophyceae)

Gametophytes of two Undaria species, U. pinnatifida and U. undarioides (Laminariales, Phaeophyceae), were studied to determine their water temperature requirements in order to understand their different distributions in Mie Prefecture, Japan. The optimal temperature for growth was 20°C for gametophytes of both species, and the upper critical temperature for growth was also the same for both species at 28°C. Therefore, the optimal and critical temperatures for growth of the gametophytes are not the main factors determining distribution. The optimal temperature for maturation of U. pinnatifida was approximately 10–15°C, whereas it was closer to 20–21°C for U. undarioides, a difference between these species of at least 5°C. In autumn and early winter, the seawater temperature at the mouth of Ise Bay, where U. pinnatifida is distributed, ranges from 21.6°C (October) to 12.7°C (December), and off Hamajima, where U. undarioides is found, the range is from 22.7°C (October) to 19.1°C (December). The seawater temperatures from October to December, which is the maturation season for the gametophytes, agreed well with the optimal temperature requirements for maturation of the gametophytes of both species. Thus the difference in the maturation temperature range of the gametophytes is a major factor determining distribution of these Undaria species along the Japanese coast.     

Environmental Influences on Kelp Performance across the Reproductive Period: An Ecological Trade-Off between Gametophyte Survival and Growth?

Most kelps (order Laminariales) exhibit distinct temporal patterns in zoospore production, gametogenesis and gametophyte reproduction. Natural fluctuations in ambient environmental conditions influence the intrinsic characteristics of gametes, which define their ability to tolerate varied conditions. The aim of this work was to document seasonal patterns in reproduction and gametophyte growth and survival of Ecklonia radiata (C. Agardh) J. Agardh in south-western Australia. These results were related to patterns in local environmental conditions in an attempt to ascertain which factors explain variation throughout the season. E. radiata was fertile (produced zoospores) for three and a half months over summer and autumn. Every two weeks during this time, gametophytes were grown in a range of temperatures (16–22°C) in the laboratory. Zoospore densities were highly variable among sample periods; however, zoospores released early in the season produced gametophytes which had greater rates of growth and survival, and these rates declined towards the end of the reproductive season. Growth rates of gametophytes were positively related to day length, with the fastest growing recruits released when the days were longest. Gametophytes consistently survived best in the lowest temperature (16°C), yet exhibited optimum growth in higher culture temperatures (20–22°C). These results suggest that E. radiata releases gametes when conditions are favourable for growth, and E. radiata gametophytes are tolerant of the range of temperatures observed at this location. E. radiata releases the healthiest gametophytes when day length and temperature conditions are optimal for better germination, growth, and sporophyte production, perhaps as a mechanism to help compete against other species for space and other resources.     

The effect of irradiance and temperature responses and the phenology of a native alga,Undaria pinnatifida (Laminariales), at the southern limit of its natural distribution in Japan

Phenology, irradiance, and temperature characteristics of an edible brown alga, Undaria pinnatifida (Laminariales), were examined from the southernmost natural population in Japan, both by culturing gametophytes and examining the photosynthetic activity of sporophytes using dissolved oxygen sensors and pulse amplitude-modulated chlorophyll fluorometer (IMAGING-PAM). Our surveys confirmed that sporophytes were present between winter and early summer, but absent by July. IMAGING-PAM experiments were used to measure maximum effective quantum yield (ΦII at 0 μmol photons m^?2 s^?1) for each of 14 temperatures (8–36 °C). Oxygen production was also determined over a coarser temperature gradient. Net photosynthesis and ΦII (at 0 μmol photons m^?2 s^?1) were observed to be temperature-dependent; the maximum ΦII was estimated to be 0.67, occurred at 21.2 °C, and was nearly identical to the optimal temperature of the net photosynthetic rate (21.7 °C). A net photosynthesis–irradiance (P–E) model revealed that saturation irradiance (E _k) was 119.5 μmol photons m^?1 s^?1, and the compensation irradiance (E _c) was 17.4 μmol photons m^?1 s^?1. Culture experiments on the gametophytes revealed that most individuals could not survive temperatures over 28 °C and that growth rates were severely inhibited. Based on our observations, temperatures greater than 20 °C are likely to influence photosynthetic activity and gametophyte survival, and therefore, it is possible that this species might become locally extinct if seawater temperatures in this region continue to rise.     

Genomic approaches to detecting thermal stress in Calanus finmarchicus (Copepoda: Calanoida)

Zooplankton may be subjected to physiological stress as they encounter rapid and large changes in temperature through vertical migration or transfer into different water masses. Induction of one or more heat shock proteins (hsp) is a common protective response to thermal stress in organisms. We looked for evidence for such a response in Calanus finmarchicus. We compared hsp70 expression in copepods exposed to temperature stress with that for non-stressed controls. Partial sequences of the amplified cDNA product were obtained and aligned with known hsp70 sequences to establish the identity of the heat shock protein. In one experiment, animals were transferred from their collection temperature (8 °C) to 20 °C for 30 min, and then returned to 8 °C for 4 h before sampling for gene expression levels. In another, the animals were exposed to 18 °C over 48 h before sampling for molecular analysis. A four-fold induction of hsp70 was measured in both groups of heat shocked animals using quantitative real time polymerase chain reaction (PCR). The experimental temperatures, although high for C. finmarchicus, are within the range of temperatures experienced by this species in their habitat. In addition to confirming an hsp70-mediated response in C. finmarchicus, the findings suggest that a recent history of thermal stress may be assessed in natural populations through a routine molecular assay.     

Sex‐linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta)

In our microsatellite analysis of three male and three female gametophytes of Undaria pinnatifida (Harv.) Suringar, a microsatellite marker (part of the locus Up‐AC‐2A8, GenBank accession no. AY738602.1) was only polymerase chain reaction‐amplified in three female gametophytes. This putative female‐specific marker was further tested by the use of 32 male and 21 female gametophytes maintained in the Marine Biological Culture Collection Centre, China. In addition, three sporophytes were included for confirmation. Results showed that the marker was present in all of the female gametophytes and sporophyte cultures, but absent in all of the male gametophytes. To our knowledge, this is the first sex‐related marker ever reported in U. pinnatifida. The discovery of this marker will accelerate gender identification and shed light on our understanding of the mechanisms of sex determination at a molecular level in this commercially important seaweed.     

Practical methodology for gametophyte proliferation and sporophyte production in green penny fern (Lemmaphyllum microphyllum C. Presl) using mechanical fragmentation

Propagation of gametophytes and sporophytes using mechanical fragmentation has been considered a suitable method for mass production of ferns. This study aimed to develop a practical propagation method for Lemmaphyllum microphyllum C. Presl, which is a fern of significant ornamental and medicinal value. Gametophytes were obtained through in vitro spore germination and used for propagation experiments. The gametophyte was mechanically fragmented using a scalpel into small fragments, which were then used to investigate gametophyte proliferation. In addition, the gametophyte was fragmented using a blender and then used to study sporophyte formation. Optimal proliferation conditions of the gametophyte were determined using Murashige and Skoog (MS) basal medium (double-, full-, half-, quarter-strength), Knop medium, and medium components (sucrose, nitrogen sources, activated charcoal), at various concentrations. The fresh weight of the gametophyte was 14-fold higher than that of gametophytes (300 mg) used as culture material, when cultured on double-strength MS. Moreover, 1 g of the gametophyte fragmented in 25 mL of distilled water formed more than 430 sporophytes in a soil mixture in an area of 7.5 cm². The sporophytes were successfully cultivated in the greenhouse after acclimation. A large-scale production method for L. microphyllum that can be easily implemented in a fern production farm is outlined.