Protoplast isolation and regeneration in the marine red alga Porphyra nereocystis

We have developed a method for isolating viable protoplasts from the blade phase of the epiphytic marine red alga Porphyra nereocystis Anderson, using a two-step enzymatic digestion with commercially available enzymes. The first step uses papain, the second step uses abalone acetone powder. The method is rapid and gives a high yield of viable protoplasts. In liquid culture in enriched seawater medium, the protoplasts can undergo regeneration along three pathways: they directly form filaments resembling the conchocelis phase of Porphyra; they form calli with relatively thick-walled, pigmented cells; and they indirectly form blades from the edges of these calli. Porphyra nereocystis protoplasts also may serve as an alternative propagation method in aquaculture and be useful for studies of cell-wall formation, cell division, and thallus differentiation. They may also be used in somatic selection, somatic hybridization and gene-transfection experiments.Abbreviations AAP abalone acetone powder - PAP papain - FDA fluorescein diacetate This paper is dedicated to the memory of the late Dr. Munenao Kurogi (1921–1988), Professor Emeritus of Hokkaido UniversityThis research was supported by the Washington Sea Grant Program (National Oceanic and Atmospheric Administration). We thank Professor Y. Fujita (Nagasaki University, Japan), Professor S.-J. Wang (Shanghai University of Fisheries, P.R. China) and Dr. H. Kito (Seikai Regional Fisheries Research Laboratory, Nagasaki, Japan) for sharing their experience with Porphyra protoplast production with us. We thank J.S. Charleston for expert technical assistance in preparation of the electron-microscopy specimens. We also thank Dr. S.K. Herbert and John Carrier (Friday Harbor Laboratories) and Dr. John Merrill and D. Gillingham (American Sea Vegetable Co. and Applied Algal Research, Seattle) for collections of P. nereocystis.     

How photoperiod influences body temperature selection in Lacerta viridis

Summary European green lizards, Lacerta viridis, show a distinct annual cycle in their day and nighttime selected body temperature (T _b) levels when monitored under natural photoperiod. The amplitude between daily photophase and scotophase temperatures varies throughout the year. Highest body temperatures with smallest day/night variation are selected from May through July. Throughout fall, the difference between day and nighttime selected T _b levels increases. Lizards inevitably enter a state of winter dormancy which terminates daily rhythmicity patterns. Under natural photoperiodic conditions, cessation of dormancy occurs spontaneously by mid-March, regardless whether high temperatures are available or not. Lacerta viridis respond to an artificial long photoperiod (16 h light, 8 h dark) at all times of the year with modifications in both diel patterns and levels of selected T _b to summer-like conditions. When, however, the natural photoperiod at different phases in the annual cycle is held constant for six to eight weeks, T _b selection of Lacerta viridis also remains stable at the level corresponding to the prevailing photoperiod. These results implicate that the photoperiod is a more prominent Zeitgeber for seasonal cueing of temperature selection than has been surmised in the past. Further, we suggest that the large variations recorded in daily T _b cycles do not imply that this lizard is an imprecise thermoregulator, but rather indicates an important integral process necessary for seasonal acclimatization.     

Kelp gulls Larus dominicanus as predators in kelp Macrocystis pyrifera beds

Summary Minimum daily consumption of bivalves Gaimardia sp by kelp gulls in a Macrocystis kelp bed at Porvenir, Tierra del Fuego, was calculated to be ca. 525 000 individuals, corresponding to an energy removal of ca. 21 kJ·m^-2·d^-1. This is far in excess of energy removal by any other higher-trophic-level predator in southern Chilean Macrocystis beds, but previous work has ignored kelp gulls as a component of the kelp bed community.     

Influence of diet and photoperiod on development and reproduction of European populations of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae)

The current study examines the effect of photoperiod (16:08 or 12:12 h L:D) and diet (eggs of Ephestia kuehniella Zeller (Lepidoptera: Phycitidae) or the pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae)) on the development and reproduction of the multicoloured Asian lady beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). A long-term laboratory population of H. axyridis (since 1998) and a melanic and non-melanic population originating from field collected individuals of H. axyridis in Belgium were used in this study. Long day conditions (16 h photoperiod) shortened development of the field populations with 2–3 days when compared with short day conditions (12 h photoperiod). Oviposition in the field populations was delayed by 1–3 months when reared at a 12 h photoperiod. Dissections indicated that the females were in reproductive diapause. As compared with live pea aphids, a diet consisting of E. kuehniella eggs yielded heavier adult body weights (up to 12%) and increased the number of egg laying days (by 45–169%) for both field populations at a 16 h photoperiod and lengthened adult life span (by 45–92%) under both light regimens. The morph types differed in their response to the foods offered in terms of developmental rate, pre-oviposition period and number of oviposition days. The laboratory and field strains responded differentially to regimens of food and photoperiod. The study indicated a greater nutritional plasticity of the non-melanic morphs which may offer them a competitive advantage that may in part explain the predominance of non-melanic morphs in newly colonized areas.     

Effect of Photoperiod on the Regulation of Wheat Vernalization Genes VRN1 and VRN2

Wheat is usually classified as a long day (LD) plant because most varieties flower earlier when exposed to longer days. In addition to LD, winter wheats require a long exposure to low temperatures (vernalization) to become competent for flowering. Here we show that in some genotypes this vernalization requirement can be replaced by interrupting the LD treatment by 6 weeks of short day (SD), and that this replacement is associated with the SD down-regulation of the VRN2 flowering repressor. In addition, we found that SD down-regulation of VRN2 at room temperature is not followed by the up-regulation of the meristem identity gene VRN1 until plants are transferred to LD. This result contrasts with the VRN1 up-regulation observed after the VRN2 down-regulation by vernalization, suggesting the existence of a second VRN1 repressor. Analysis of natural VRN1 mutants indicated that a CArG-box located in the VRN1 promoter is the most likely regulatory site for the interaction with this second repressor. Up-regulation of VRN1 under SD in accessions carrying mutations in the CArG-box resulted in an earlier initiation of spike development, compared to other genotypes. However, even the genotypes with CArG box mutations required LD for a normal and timely spike development. The SD acceleration of flowering was observed in photoperiod sensitive winter varieties. Since vernalization requirement and photoperiod sensitivity are ancestral traits in Triticeae species we suggest that wheat was initially a SD–LD plant and that strong selection pressures during domestication and breeding resulted in the modification of this dual regulation. The down-regulation of the VRN2 repressor by SD is likely part of the mechanism associated with the SD–LD regulation of flowering in photoperiod sensitive winter wheat. These authors contributed equally to this work     

Identification and quantification of indole-3-acetic acid in the kelp Laminaria japonica Areschoug and its effect on growth of marine microalgae

A method was established for the identification and quantification of indole-3-acetic acid (IAA) in extracts of the kelp Laminaria japonica. An IAA content of 90–95 μg kg⁻¹ fresh weight in kelp extract was determined by high performance liquid chromatography (HPLC). IAA identification was based on a combination of co-chromatography and comparative chromatography with a standard, analysis of UV spectra, and atmospheric pressure electrospray mass spectrometry (APESI-MS). IAA was isolated by silica gel chromatography and HPLC. The effect on the growth of four marine microalgae of the pure IAA isolated from kelp extract was investigated. Exogenously added IAA from kelp enhanced the growth of Chlorella sp., Dunaliella salina and Porphyridium cruentum, but not that of Chaetoceros muelleri. IAA from kelp significantly inhibited the accumulation of soluble cellular proteins in Chlorella sp. and P. cruentum, and had a very significant effect on chlorophyll biosynthesis in Chlorella sp. However, there was no obvious effect of IAA on the regulation of biosynthesis of cellular polysaccharides in these four marine microalgae.     

<Emphasis Type="Italic">Lacuna vincta</Emphasis> (Mollusca,Neotaenioglossa) herbivory on juvenile and adult <Emphasis Type="Italic">Nereocystis luetkeana</Emphasis> (Heterokontophyta,Laminariales)

Herbivory can be an important factor structuring coastal algal communities. Herbivores may preferentially graze particular algal species or tissue types. Mesograzers, despite their small size, can critically weaken kelp thalli and impact entire kelp beds. We propose that when kelp beds are composed of several kelp cohorts, mesograzers will selectively choose to inhabit younger plants and grazing activities will have a greater impact on younger plants. This study investigated the effects of grazing by the littorinid gastropod, Lacuna vincta, on different age classes of the bull kelp, Nereocystis luetkeana by (1) testing food preference of L. vincta on juvenile, first-year adult, and second-year adult Nereocystis blades in the laboratory, (2) determining substrate (blades of different ages) preference of L. vincta in the laboratory, and by (3) estimating in-situ herbivore abundances and densities on juvenile and adult Nereocystis. Results demonstrated that grazing by L. vincta produced greater damage on juvenile than older Nereocystis tissues. Although L. vincta did not select juvenile versus older kelps as substrate in the laboratory, in situ surveys showed that differences existed between age classes with higher L. vincta densities on juvenile than adult kelp. We conclude that at a local scale, L. vincta can be an important structuring factor in Nereocystis populations due to its high density and grazing ability. Handling editor: K. Martens     

Conchospore production and seasonal occurrence of some Porphyra species (Bangiales,Rhodophyta) in Washington State

The leafy thalli of species of the marine red algal genus Porphyra grow rapidly but persist for a relatively short time on rocky intertidal or subtidal substrata or as epiphytes on other marine plants. In most species, the large, short-lived leafy thalli alternate with small, presumably perennial, filamentous conchocelis plants. Depending on the species of northeastern Pacific Porphyra, photoperiod and temperature are important regulators of conchospore formation and release. Data from laboratory studies of conchospore formation and release in five Washington species of Porphyra (P. abottae, P. nereocystis, P. perforata, P. pseudolanceolata and P. torta) indicate that conchospores are most likely to be released at a time that precedes the appearance of the leafy thalli in the field.     

Maternal and direct effects of natural-like changes of photoperiod and food condition manipulation on life history parameters in <Emphasis Type="Italic">Daphnia</Emphasis>

This study analyzed changes of life history parameters of a Daphnia pulicaria clone in response to increasing (spring conditions) versus decreasing (fall conditions) photoperiod and low and high food concentrations in maternal and offspring environments. One treatment increased the photoperiod from 12 to 16 h at a rate of 3 min per day, while the other treatment decreased the photoperiod from 16 to 12 h at the same rate. We also used two food concentrations: low (0.2 mg C/l) and high (0.5 mg C/l). Maturation time, first clutch size, survivorship, and somatic growth in offspring depended on the direction of photoperiodic changes for Daphnia grown under high food concentration. Natural-like changes of photoperiod on life history parameters and abrupt changes resulted in similar effects on life history parameters of Daphnia (see Alekseev and Lampert 2004 Hydrobilogia 526:225–230).     

The potential for seaweed resource development in subarctic Canada; Nunavik,Ungava Bay

Ungava Bay is ice covered 6–7 months of the year and evidence of ice scouring of seaweeds is extensive in the intertidal and shallow subtidal. Maximum tidal amplitudes of 16 m, among the highest in Canadian waters, compound this impact. Despite this level of annual perturbation, very extensive and dense beds of fucoids in the intertidal and laminarians in the subtidal are common on the western shores of Ungava Bay. Ground surveys of 24 intertidal stations combined with satellite images delineated 82,000 tons standing crop of Fucus vesiculosus and Fucus evanescens in Payne Bay, of which 36,000 tons were considered harvestable. Subtidally, kelp cover reached peak biomass at 5–10 m consisting of three primary species, Saccharina longicruris, Laminaria digitata, and Laminaria solidungula. In the area of Payne Bay, kelp beds of 100 ha were common, averaging 9–12 kg m⁻² wet weight. The productivity of brown algae at these latitudes has been assumed to be low relative to southern latitudes. Direct measurement of lineal growth indicates productivity is intermediate between arctic and temperate populations. The potential for medium level industrial harvest exists under conservative management strategies within the constraints of subarctic logistics.     

Growth dynamics of non-toxic Pseudo-nitzschia delicatissima and toxic P. seriata (Bacillariophyceae) under simulated spring and summer photoperiods

Marine planktonic diatoms of the genus Pseudo-nitzschia Peragallo have been responsible for amnesic shellfish poisoning (ASP) events worldwide through the production of the neurotoxin domoic acid (DA). The appearance and toxicity of Pseudo-nitzschia species is variable throughout the year and potentially linked to changes in environmental parameters; many ASP events occur in relatively high latitudes where day length is particularly variable with season. In UK waters, shellfish monitoring has prevented any impact on human health but has led to long-term closures of fisheries, with severe economic consequences. Laboratory experiments on two Pseudo-nitzschia species typically found in Scottish West Coast waters during spring (short photoperiod (SP)) and summer (long photoperiod (LP)) conditions were conducted to determine the influence of photoperiod on their growth and toxicity. Results indicated that non-toxic P. delicatissima (Cleve) Heiden achieved a greater cell density under SP (9-h light:15-h dark (L:D) cycle). For toxin-producing P. seriata (Cleve) H. Peragallo, a LP (18-h L:6-h D cycle) resulted in an enhanced growth rate, cell yield and total toxin production, but it decreased the toxin production per cell. A better understanding of the response of Pseudo-nitzschia species to photoperiod and other foreseeable environmental variables may help predict the appearance of toxic strains.     

Influence des factuers externes sur l'activité reproductrice de l'hirudinée rhynchobdelle Piscicola geometra L.

The annual cycle of P. geometra breeding activity depends on light, temperature and nutrition. A photoperiod of about 12-12 delays the genital maturation and prevents the incidence of breeding behaviour. A high temperature speeds up the genital maturation and above a threshold ranging from 15 to 17.5 °C it prevents light from acting. Fasting inhibits egg laying because of its immediate action on the clitellar gland activity. It only acts on oogenesis later.

     

Plant Hormones Isolated from “Katahdin” Potato Plant Tissues and the Influence of Photoperiod and Temperature on Their Levels in Relation to Tuber Induction

Qualitative and quantitative analyses were carried out on vegetative tissues of potato (Solanum tuberosum cv. “Katahdin”) in search of natural products thought to play a role in tuber induction. Tissues were obtained from plants initially grown in a growth chamber under noninducing conditions (30°C day and 28°C night with an 18-h photoperiod), and then half of the plants were moved to inducing chambers (28°C day and 13°C night with a 10-h photoperiod) for 10 days prior to tissue harvest. Plants from each chamber were then harvested at 2-day intervals for 10 days, separated into above- and belowground portions, and the lyophilized tissues were extracted and subjected to rigorous purification and separation using high-performance liquid chromatography. This was followed by identification and quantification using combined gas chromatography-mass spectrometry. Compounds isolated and identified included gibberellic acid; cytokinins cis-zeatin riboside, trans-zeatin, trans-zeatin riboside, and isopentenyladenine; and jasmonates jasmonic acid, tuberonic acid and its methyl ester, methyl 7-isocucurbate, and 9,10-dihydromethyljasmonate. Methyl 7-isocucurbate and 9,10-dihydromethyljasmonate were detected for the first time in potato tissue as endogenous compounds. Cytokinin and jasmonate levels generally increased under inducing conditions, whereas gibberellic acid levels declined progressively during the 10-day sampling period. Only gibberellic acid, jasmonic acid, and cis-zeatin riboside levels were significantly influenced by induction.     

Sucrose increase during floral induction in the phloem sap collected at the apical part of the shoot of the long-day plant Sinapis alba L.

Sinapis alba L., a long-day plant, has been induced to flower either by a single 22-h-long photoperiod or by an 8-h short photoperiod displaced by 10 h in a 24 h cycle. The ehtylenediametetraacetate method previously used for leaf exudation was modified to collect phloem sap at the apical part of the shoot. Carbohydrates in the phloem sap have been analysed comparatively in vegetative and induced plants, using high-performance liquid chromatography and refractometry. Sucrose was the major sugar detected. A dramatic increase of its flux in the apical sap occurred early and transiently during the floral transition in plants induced by both long days and displaced short days. These results indicate a message-like role for sucrose since they fit nicely with previous observations indicating that an early event in the floral transition in S. alba is the accumulation of sucrose in the meristem.Abbreviations DSD displaced short day - LD long day - SD short day This work was supported by the Fonds pour la Recherche Fondamentale et Collective (FRFC) of Belgium (Grant n 2.9009.87) and the University of Liège (Action de Recherche Concertée 88/93-29). One of us (P.L.) is grateful to the I.R.S.I.A. for the award of a research fellowship.     

The formation and degradation of cyanobacterium Aphanizomenon flos-aquae blooms: the importance of pH, water temperature, and day length

Investigation of annual changes in phytoplankton community structure in a small artificial eutrophic pond was carried out from May 2002 to April 2003. A heavy bloom of Aphanizomenon flos-aquae var. klebahnii Elenk. (Cyanobacteria) persisted in most of the water column from June to the end of October. In November, the A. flos-aquae bloom suddenly crashed and green algae were predominant until the end of spring. Weekly monitoring suggested strong involvement of the changes in abiotic factors in the cyanobacterial bloom degradation. To clarify the effects of pH, water temperature, and day length on the growth of A. flos-aquae, laboratory batch experiments were conducted. The results showed that A. flos-aquae could not grow below pH 7.1 and 11°C, and the growth tended to be suppressed under a 10L:14D photoperiod. pH, water temperature, and day length are vital factors in the growth of A. flos-aquae and, additionally, grazing by cyclopoid copepods also seemed important in bloom collapse.     

Harvesting of the Kelp Ecklonia Maxima in South Africa Affects Its Three Obligate, Red Algal Epiphytes

In South Africa, more than 7000 t (f wt) of kelp (Ecklonia maxima) fronds are harvested annually to feed cultured abalone. Carpoblepharis flaccida, Gelidium vittatum and Polysiphonia virgata are conspicuous red algal epiphytes on older kelps and provide habitat and food for numerous animals. Over 4.5 y, we examined the effects of one destructive harvest of E. maxima on these 3 epiphytes. Two 20 × 20 m plots of kelp with similar epiphyte loads were demarcated. In one, all E. maxima sporophytes with stipes longer than 50 cm were harvested. The other plot served as a control. After 2.5 y the biomass of E. maxima in the harvested plot had recovered to control levels, but the epiphyte load (g epiphytes. Kg kelp⁻¹) was statistically lower in the harvested plot after 2.5 and 3.5 y, and only recovered after 4.5 y. While most commercial harvesters cut through the “heads” (primary blades) of the kelp, effectively killing them, a new, non-lethal method removes secondary blades 20–30 cm from their bases, leaving the meristems and primary blades intact. At 5 sites studied, G. vittatum and P. virgata were found almost entirely on stipes and primary blades, and harvesting only distal parts of secondary blades limited losses to about 50% of C. flaccida biomass. To protect epiphytes, non-lethal harvesting is recommended and permanent non-harvest zones have been established in addition to limiting kelp yields and disallowing harvesting in Marine Protected Areas.     

Patch formation by herbivorous fish in a temperate Australian kelp forest

Summary The odacid fish Odax cyanomelas feeds on the kelp Ecklonia radiata, an important component of subtidal reef habitats on the central coast of New South Wales, Australia. Herbivory by Odax has a major impact on the structure and dynamics of discrete patches within larger stands of kelp at Cape Banks. This three-year study showed that each year, between August and October, approximately the same patches of kelp were denuded by preferential feeding on the meristem and primary laminae. This coincided with a variable pulse of Ecklonia recruits to the cleared patches, thereby generating patches of a single age-class of plants. Neighbouring areas of Ecklonia forest, non cleared by Odax, consisted of larger, perennial plants, which exhibited more gradual changes in abundance. The seasonal impact of Odax appeared to be due to a change in the behaviour of female Odax during their spawning period. Observations suggested that females aggregate at traditional sites prior to spawning with territorial males in adjacent areas of kelp forest. An alternative hypothesis, that Odax preferentially attacked stands of one-year old Ecklonia plants, was rejected by a field experiment; the establishment of experimental stands of one-year old plants did not lead to increased damage due to Odax or any change in the use of space by the fish. The generality of this effect of fish herbivory is unknown, but this and other Odax species are widely distributed throughout temperate Australia, where Ecklonia is the dominant laminarian alga. The effects of pulsed herbivory by Odax is contrasted to the more continuous grazing by sea urchins in the same system. The latter herbivore has been shown to maintain areas free of Ecklonia, the long-term effects of herbivory by Odax remain unclear.     

In situ expression of the GmNMH7 gene is photoperiod-dependent in a unique soybean (Glycine max [L.] Merr.) flowering reversion system

Soybean is a short-day plant and its flowering process can be reversed when switching from short-day to long-day conditions. Flowering reversion provides a useful system to study the flowering process in both forward and backward directions. In this study, we optimized a soybean flowering reversion system using a photoperiod-sensitive cultivar Zigongdongdou. Three types of terminal structures were found during flowering reversion: reversed terminal raceme (RTR), short terminal raceme (STR), and vegetative terminal (VT). The relative frequency of these terminal structures during flowering reversion under long day was dependent on the duration of the prior short day (SD) pretreatment. This process is phytochrome dependent and young plants were more susceptible to flowering reversion. Leaf removal increased the minimal SD period needed for the induction of STR. To demonstrate the application of this system, we studied the patterns of in situ expression of the GmNMH7 gene during flowering development and reversion. NMH7 family members encode MADS-box proteins and are unique in legume families since their expression can be detected in both developing flowers and nodules. In situ hybridization experiments using plants grown under different photoperiod cycles provided several lines of evidence supporting a close relationship between GmNMH7 gene expression and floral development in soybean. Furthermore, it seems that GmNMH7 may participate in flower development at different stages. Interestingly, the expression pattern of GmNMH7 in root nodules was also found to be regulated by photoperiod. These results support the notion that the photoperiod sensitive GmNMH7 gene may play multiple roles in growth and development in soybean.C. Wu and Q. Ma contributed equally to this work.     

Reproduction strategies of Macrocystis pyrifera (Phaeophyta) in Southern Chile: The importance of population dynamics

Macrocystis pyrifera is an ecologically dominant species along the temperate Northern and Southern Pacific Coast of America, showing some similarities and differences at population and community level. In general, this kelp is reported to be reproductive all year round. Annual populations present in wave-protected areas of southern Chile suggest that the reproductive strategies of this population can be different. In this study we explore the reproductive strategies of annual M. pyrifera present in wave-protected areas and perennial populations encountered in exposed areas of southern Chile (41^∘S). Our results show that M. pyrifera present in wave-exposed locations has a reproductive strategy that is similar to populations in the northern hemisphere. These populations reproduce all year round and their strategy is to produce high numbers of sporophylls and ensure that most of them (over 90%) become sporogenous. On the other hand, the protected populations with an annual life cycle, produce more spores per area of sorus. In a few months, they are able to produce sufficient propagules to recolonize areas before the adult plants disappear in autumn.     

光质和光周期对山白兰苗木生长、生理的影响

为筛选出山白兰(Michelia baillonii)苗木培育过程中适宜的光环境，该研究以一年生山白兰幼苗为试验材料，设置12、16 h·d^-1两个光周期，配合使用红蓝复合光(8R1B、6R1B)、红蓝紫绿复合光(8R1B1P1G、6R1B1P1G)4种光质和白光(W)对照，采用双因素随机区组试验设计和隶属函数法，探讨了山白兰苗木生长、光合色素、内源激素含量对不同光质和光周期处理的响应规律。结果表明：(1)光质、光周期及其交互作用对山白兰苗高增长量、叶面积、叶绿素a、玉米素(ZR)、脱落酸(ABA)的含量、内源激素比值[IAA/ABA、(IAA+GA₃+ZR)/ABA]等均有显著影响(P<0.05)。(2)16 h·d^-1光周期有利于苗高增长量、叶面积、苗木质量指数、生物量、叶绿素a、生长素(IAA)、ZR的含量、内源激素比值的提高。(3)当光周期为16 h·d^-1时，8R1B处理下的苗高增长量、叶面积和苗木质量指数均最大，分别为21.84 cm、158.39 cm²和2...