Chytrids cannot survive at high temperatures in liquid growth media: implications for soil ecosystems

Sixteen isolates in the orders Blastocladiales, Chytridiales, Rhizophydiales and Spizellomycetales were incubated for 2 d in liquid PYG growth medium at 33, 37, 40, 45 and 50 °C. These fungi could not resume growth when returned to 20 °C if the temperature of incubation was more than a few degrees above the maximum temperature for growth on solid PYG growth medium. The maximum temperatures for survival of some chytrids are probably close to the temperatures reached periodically on the surface of moist soils in warm climates. Desiccated thalli of chytrids in the Blastocladiales and Spizellomycetales can survive much higher temperatures than chytrids in the Chytridiales and Rhizophydiales. Therefore, the maximum temperature and moisture content of the soil could significantly affect the diversity of chytrids in the ecosystem.     

Size-dependence of reproductive allocation of Sargassum thunbergii (Sargassaceae, Phaeophyta) in Bohai Bay, China

Reproductive allocation (the proportion of biomass allocated to reproductive tissue, RA) in Sargassum thunbergii was studied in Yantai, Shandong Peninsula, southern coast of Bohai Bay, China. Annual reproduction initiated in mid-June and peaked in mid-July (90 ± 8% fertile thalli and 75 ± 6% RA mean). Both RA and percentage of fertile thalli exhibited significant temporal variations during reproduction. Sterile thalli were only observed in small length hierarchies at peak reproduction and mean values of RA showed a significantly hierarchical variation, suggesting that the size of thalli played an important role in reproduction and RA were size-dependent. Numerous receptacles were produced along the lateral branches during the reproductive period. A distinct seasonal pattern was observed wherein the presence of lateral branches was followed by the onset of reproduction. RA was positively correlated with the number of lateral branches, as well as the total length of lateral branches. In addition, fertile thalli decayed quickly after peak reproduction. The probability of decay was evidently higher for fertile than for sterile thalli, because all surviving thalli were sterile and short during July–August. So, a trade-off between reproduction and survival may exist at individual levels in S. thunbergii.     

Selection of Beauveria bassiana isolates for control of the whiteflies Bemisia tabaci and Trialeurodes vaporariorum on the basis of their virulence, thermal requirements, and toxicogenic activity

As part of a 3-fold approach to select potential mycoinsecticides for whitefly control, we evaluated infectivity, thermal requirements, and toxicogenic activity of the entomopathogenic fungus Beauveria bassiana (Ascomycota: Clavicipitaceae) under laboratory conditions. Twenty-five native B. bassiana isolates and a commercially available mycoinsecticide (based on B. bassiana) were evaluated for virulence to fourth instar nymphs of sweetpotato whitefly, Bemisia tabaci, and greenhouse whitefly, Trialeurodes vaporariorum, at a concentration of 1 × 10⁷ conidia/ml. All isolates were pathogenic for both whitefly species, whereas mortality rates varied from 3 to 85%. A second series of bioassays was conducted on 10 selected isolates using four 10-fold concentrations ranging from 1 × 10⁵ to 1 × 10⁸ conidia/ml. Median lethal concentrations (LC₅₀) of the four most virulent isolates varied from 1.1 × 10⁵ to 6.2 × 10⁶ conidia/ml and average survival time (AST) of treated nymphs from 5.9 to 7.4 days. T. vaporariorum were significantly more susceptible to all B. bassiana isolates than B. tabaci. The thermal biology of the eight most virulent isolates to both whitefly species was investigated at six temperatures (10–35 °C). The colony radial growth rate was estimated from the slope of the linear regression of colony radius on time and data were then fitted to a modified generalized β function that accounted for 90.5–99.3% of the data variance. Optimum temperatures for extension rate ranged from 23.1 to 27.1 °C, whereas maximum temperatures for fungal growth varied from 31.8 to 36.6 °C. On the basis of their virulence and thermal requirements, three isolates showed promise as candidates for whitefly management in Mediterranean greenhouses. Whilst in vitro production of macromolecular compounds toxic to Galleria mellonella larvae was not a requisite for virulence, ASTs of larvae injected with Sephadex G-25 fractions from candidate isolates ranged from 1.4 to 3.7 days compared with 5–6 days for non-toxic G-25 fractions. In addition, proteinase K treatment significantly reduced their toxic activity suggesting that they were proteins and revealing the potential of these isolates to be further improved through biotechnology to kill the pest more quickly.     

Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms

Pythium porphyrae is a fungal pathogen responsible for red rot disease of the seaweed Porphyra (Rhodophyta). Infection forecasts of Porphyra by P. porphyrae were estimated from the epidemiological observations of Porphyra thalli and numbers of zoospore of P. porphyrae in laboratory and cultivation areas. Four features of forecasting infections were determined by relating zoospore concentrations to the incidence of thallus infection; infection (in more than 1000 zoospores L⁻¹), microscopic infection [less than 2 mm in diameter of lesion (in from 2000 to 3000 zoospores L⁻¹)], macroscopic infection [more than 2 mm in diameter of lesion (in from 3000 to 4000 zoospores L⁻¹), and thallus disintegration (in more than 4000 zoospores L⁻¹). High zoospore concentrations led to more infection. The tendency that zoospore concentration of P. porphyrae increased with the rate of infection of Porphyra thalli was generally observed in forecasting infections in both the laboratory and in cultivation areas. Based on the Porphyra cultivation areas, the accuracy and consistency of forecasting infections suggest that this method could be employed to manage and control red rot disease.     

Hatchling painted turtles (Chrysemys picta) survive exposure to subzero temperatures during hibernation by avoiding freezing

Hatchling painted turtles (Chrysemys picta) were placed individually into artificial nests constructed in jars of damp soil and then were cooled slowly to temperatures between-7.7 and-12.7 °C. Distinct exotherms were recorded in all jars when water in the soil began to freeze at temperatures between-0.9 and-2.4 °C. A second (animal) exotherm was subsequently detected in some of the jars when water in hatchlings also began to freeze. An animal exotherm occurred in the temperature records for all 23 hatchlings that died in tests terminating at temperatures between-7.7 and-10.8 °C, but no such exotherm was apparent in the temperature records for the 23 turtles that survived these treatments. Moreover, the 4 hatchlings that produced exotherms in tests terminating between-11.5 and-12.7 °C failed to survive, but 5 of 7 hatchlings that produced no exotherm in these tests also died. Thus, turtles that die at subzero temperatures above-11 °C apparently succumb to freezing when ice propagates across their integument from the frozen soil, but animals that die at temperatures below-11 °C generally perish from some other cause. These findings indicate that hatchling painted turtles overwintering inside their shallow, subterranean nests survive exposure to subzero temperatures by avoiding freezing instead of by tolerating freezing.     

Cryosurgical technique: assessment of the fundamental variables using human prostate cancer model systems

Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoablation of cancerous tissues sometimes fails due to technical challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (−5 to −40 °C), and cells were thawed followed by return to 37 °C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlue™ (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20 min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At −15 °C, PC-3 yielded 55% viability versus 20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze–thaw cycle. Both cell types experienced increased cell death when exposed to freezing temperatures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10 min freeze. Finally, the results demonstrate that just reaching −40 °C in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze–thaw cycles, and passive thawing to provide maximum cell destruction.     

Oviposition and reproductive performance of a generalist parasitoid (Trichogramma pretiosum) exposed to host species that differ in their physical characteristics

The response of generalist egg parasitoids to alternative natural hosts that are present simultaneously is not well known. We investigated the behavior of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) in relation to two field hosts Helicoverpa armigera Hübner and Spodoptera litura Fabricius, in choice and no choice tests. We quantified the effects of natal host species and post-emergence adult age on the oviposition preference of the parasitoids. H. armigera eggs were consistently preferred over S. litura eggs, regardless of the natal host and adult age. When only S. litura eggs were available as hosts, they were parasitized at statistically similar rates to H. armigera eggs (average of 17 ± 2.7 vs. 13 ± 3.0, H. armigera to S. litura). The adult lifespan and lifetime fecundity of T. pretiosum were variable but were affected by natal host species and/or host species to which they were exposed. Mean lifespan and fecundity of parasitoids that had developed in H. armigera eggs and were exposed to H. armigera eggs for oviposition were 13.9 ± 1.8 days and 98.7 ± 11.0 adult offspring. By contrast, those that developed in S. litura eggs and were exposed to S. litura eggs for oviposition lived for 7 ± 0.9 days and produced 53.8 ± 8.0 adult offspring. The ovigeny index (OI) was significantly lower in the parasitoids exposed to H. armigera eggs than in those exposed to S. litura eggs, regardless of the natal host, indicating that H. armigera eggs sustain the adult parasitoids better than S. litura eggs. These results are used to predict parasitoid behavior in the field when both hosts are available.     

Phylogenetic Position of Parasitic Chytrids on Diatoms: Characterization of a Novel Clade in Chytridiomycota

Chytrids are true fungi that reproduce with posteriorly uniflagellate zoospores. In the last decade, environmental DNA surveys revealed a large number of uncultured chytrids as well as undescribed order‐level novel clades in Chytridiomycota. Although many species have been morphologically described, only some DNA sequence data of parasitic chytrids are available from the database. We herein discuss five cultures of parasitic chytrids on diatoms Aulacoseira spp. and Asterionella formosa. In order to identify the chytrids examined, thallus morphologies were observed using light microscopy. We also conducted a phylogenetic analysis using 18S, 5.8S, and 28S rDNA sequences to obtain their phylogenetic positions. Based on their morphological characteristics, two cultures parasitic on As. formosa were identified as Rhizophydium planktonicum and Zygorhizidium planktonicum. The other three cultures infecting Aulacoseira spp. (two on Aulacoseira ambigua and the other on Aulacoseira granulata) were regarded as Zygorhizidium aff. melosirae. The results of the molecular phylogenetic analysis revealed that R. planktonicum belonged to the known order Chytridiales, while the two species of Zygorhizidium were placed in a novel clade that was previously reported as an undescribed clade composed of only the environmental sequences of uncultured chytrids.     

Ultrastructural and molecular phylogenetic delineation of a new order,the Rhizophydiales (Chytridiomycota)

In the order Chytridiales, Rhizophydium is a morphologically defined genus based upon the production of a monocentric, inoperculate, epibiotic sporangium, an endobiotic rhizoidal axis which branches, and an epibiotic resting spore. Despite its simple morphology, over 220 species of Rhizophydium have been described. Recent phylogenetic analyses using nuLSU rRNA (28 S rRNA) gene sequences of a geographically diverse sampling of Rhizophydium cultures revealed that the classical genus Rhizophydium is genetically more variable than previously understood and actually represents multiple genera. In the present study, we use zoospore ultrastructural characters and 28 S rRNA and 5.8 S ribosomal gene sequences of 96 isolates in culture to circumscribe the monophyletic Rhizophydium clade as a new order, Rhizophydiales. Correspondingly, zoospores of members of the Rhizophydiales exhibit a unique suite of ultrastructural character states that further define the order and distinguish it from the order Chytridiales. Molecular analyses reveal several strongly supported clades within the Rhizophydiales. Three of those clades encompass a broad range of isolates and are defined as new families Rhizophydiaceae, Terramycetaceae, and Kappamycetaceae. To resolve close relationships within Terramycetaceae, combined 28 S rRNA and ITS1–5.8 S–ITS2 sequences were analysed and details of zoospore ultrastructural character states determined, with two new genera, Terramyces and Boothiomyces, described. Two species formerly classified in Rhizophydium are transferred to the new genera. This work provides a framework for additional taxonomic revisions within the new order Rhizophydiales and compares genetic variation useful in defining genera, species, and populations within this lineage of chytrids. A broader sampling of representatives is needed before taxonomic decisions can be made for remaining clades within the Rhizophydiales.     

Seed germination and dormancy in the medicinal woodland herbs Collinsonia canadensis L. (Lamiaceae) and Dioscorea villosa L. (Dioscoreaceae)

We used a double germination phenology or “move-along” experiment (sensu Baskin and Baskin, 2003) to characterize seed dormancy in two medicinal woodland herbs, Collinsonia canadensis L. (Lamiaceae) and Dioscorea villosa L. (Dioscoreaceae). Imbibed seeds of both species were moved through the following two sequences of simulated thermoperiods: (a) 30/15 °C→20/10 °C→15/6 °C→5 °C→15/6 °C→20/10 °C→30/15 °C, and (b) 5 °C→15/6 °C→20/10 °C→30/15 °C→20/10 °C→15/6 °C→5 °C. In each sequence, seeds of both species germinated to high rates (>85%) at cool temperatures (15/6 and 20/10 °C) only if seeds were previously exposed to cold temperatures (5 °C). Seeds kept at four control thermoperiods (5, 15/6, 20/10, 30/15 °C) for 30 d showed little or no germination. Seeds of both species, therefore, have physiological dormancy that is broken by 12 weeks of cold (5 °C) stratification. Morphological studies indicated that embryos of C. canadensis have “investing” embryos at maturity (morphological dormancy absent), whereas embryos of D. villosa are undeveloped at maturity (morphological dormancy present). Because warm temperatures are required for embryo growth and cold stratification breaks physiological dormancy, D. villosa seeds have non-deep simple morphophysiological dormancy (MPD). Neither species afterripened in a 6-month dry storage treatment. Cold stratification treatments of 4 and 8 weeks alleviated dormancy in both species but C. canadensis seeds germinated at slower speeds and lower rates compared to seeds given 12 weeks of cold stratification. In their natural habitat, both species disperse seeds in mid- to late autumn and germinate in the spring after cold winter temperatures alleviate endogenous dormancy.     

Laboratory evaluation of two diatomaceous earth formulations against Blattisocius keegani fox (Mesostigmata, Ascidae) and Cheyletus malaccensis oudemans (Prostigmata, Cheyletidae)

Diatomaceous earths (DEs) are very promising natural-origin pesticides against stored-product pests, but there is still inadequate information about the effect of DEs against stored-product mites. For this purpose, laboratory bioassays were conducted to assess the effects of DEs against the predatory mites Blattisocius keegani Fox (Mesostigmata, Ascidae) and Cheyletus malaccensis Oudemans (Prostigmata, Cheyletidae). Two DEs were used: SilicoSec, which contains 92% SiO₂, and PyriSec which contains 95.7% SilicoSec, 1.2% natural pyrethrum and 3.1% piperonyl butoxide. As prey, eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) were used. The tests were conducted at three temperatures, 20, 25 and 30 °C, on wheat treated with DEs at two dose rates, 500 and 1000 ppm and mortality of mite individuals was measured after 7 days of exposure. For B. keegani, protonymphs were proved significantly less susceptible in comparison with adults, in most temperature/DE combinations examined. Also, for both DEs, significantly more B. keegani adults were dead at 30 °C than at the other two temperatures. C. malaccensis protonymphs were less susceptible than adults, for both DEs tested, with the exception of PyriSec at 30 °C. In the case of adults, in SilicoSec-treated wheat, significantly fewer individuals were dead at 30 °C in comparison with the other two temperatures, but this was reversed for PyriSec. The results of the present work indicate that both species are susceptible to the two DEs tested, but this susceptibility is highly determined by several factors such as formulation, dose rate and temperature.     

Meristem clusters in cortical layer of thallus cells of the cultured red alga Palmaria palmata

For the first time somatic spotlike formations of 1.5 mm in diameter and height up to 0.5 mm were revealed in thalli of the red alga Palmaria palmata reared in an aerated stirred culture. It was determined that some cortical cells of the thallus are able to divide in the periclinal and anticlinal directions forming spots. Deep freezing and subsequent thawing of thalli showed that the cortical cells of spots were meristematic cells. In certain conditions they enabled the formation of prolifications, which germinated plantlets. These clusters of meristem tissue in the cortical layer of cells of the thallus of P. palmata, which were formed in the culture as in nature, function as growth cells facilitating growth of thalli in thick and natural “planting material” formed upon thalli fragmenting after their freezing in the winter season.     

Effect of relative humidity and origin of isolates of Neozygites tanajoae (Zygomycetes: Entomophthorales) on production of conidia from cassava green mite, Mononychellus tanajoa (Acari: Tetranychidae), cadavers

Neozygites tanajoae is a very specialized fungus pathogenic to the cassava green mite (CGM), Mononychellus tanajoa, an important cassava pest introduced to Africa from the Neotropics. Conidial discharge from cadavers of CGM that died from infections with 14 isolates of N. tanajoae collected from diverse climates of Brazil was quantified to help select potential candidate strains for introduction to Africa. Studies aimed to identify isolates with lower requirements for relative humidity for sporulation and isolates that discharged more conidia during short periods of moisture. At 96 ± 0.5% RH, production of conidia was variable and even isolates from the Brazilian semi-arid region, e.g., Petrolina and Itaberaba, produced few conidia. Significant differences in the numbers of conidia produced by diverse Brazilian isolates were observed after 6, 9 and 12 h at 100% RH. At 100% RH, production of primary conidia increased considerably from an average of 57 ± 4 conidia at 6 h to 509 ± 37 conidia at 12 h. The isolate sporulating least (BIN21) discharged only 45.7% of the number of conidia produced by isolate BIN1, one of the isolates producing the most spores. Results from this study demonstrate that differences in production of conidia among isolates should be considered when selecting Neozygites isolates for new biological control introductions.     

Entomopathogenic fungi of Eurygaster integriceps Puton (Hemiptera: Scutelleridae): collection and characterization for development

Exploratory activities were done in Syria, Turkey, Iran, Uzbekistan, Kazakhstan, The Kyrghyz Republic, and Russia to locate entomopathogenic fungi of Eurygaster integriceps. Isolates from the entomopathogenic genera Beauveria, Paecilomyces, and Verticillium were collected. Beauveria bassiana was the most commonly recovered species. Thirty-one isolates of the 221 recovered were examined at 20, 25, 30, and 35 °C for 20 days for growth and sporulation. Growth and sporulation were generally highest at 25 °C. None of the isolates grew at 35 °C, and at 30 °C growth was retarded with no conidia being produced. Single- and multiple-concentration bioassays were conducted on greenhouse-grown wheat plants and in pine litter to evaluate virulence of fungi from several sources to E. integriceps. When tested at a single concentration, mortality after 15 days ranged from 66 to >95% in the litter assays and 50 to 91% in the plant assays. There was a distinct concentration response for most of the isolates tested in the multiple-concentration assay, particularly in the in-litter environment. In litter, mortality tended to develop earlier than in on-plant assays. Several isolates of B. bassiana and one Metarhizium anisopliae displayed consistently high virulence against E. integriceps and were more virulent than two commercial strains. Our results demonstrate the potential of entomopathogenic fungi for management of E. integriceps in overwintering sites and in wheat fields.     

Thermal requirements for the embryonic development of Periplaneta americana (L.) (Dictyoptera: Blattidae) with potential application in mass-rearing of egg parasitoids

The embryonic development of oothecae of Periplaneta americana was evaluated under four different constant temperatures (5, 10, 15, 20, 25, 30, and 35 °C) and also at different exposure times at <5 °C. Their suitability as hosts after the treatment for the parasitoids Evania appendigaster and Aprostocetus hagenowii was also assessed. Temperatures of 5, 10, 15, and 35 °C adversely affected the development of the cockroaches, and exposure times to <5 °C longer than 5 days sufficed to kill all the embryos in the oothecae. The lower thermal threshold for complete development of P. americana was estimated to be 6.8 °C, with a required total amount of 900.9 degree-days. Cold-killed oothecae were still fit for the development of parasitoids. Parasitism rates of A. hagenowii were higher than those of E. appendigaster, although with lower emergence rates. Our results can be useful in aiding mass-rearing of these parasitoids for biological control programmes of P. americana, and may help forecast the time of emergence of nymphs of American cockroaches in infested areas.     

δC, δN, carbon, nitrogen and phosphorus as indicators of plant ecophysiology and organic matter pathways in Everglades deep slough, Florida, USA

Historically, the Florida Everglades was characterized by a corrugated landscape of shorter hydroperiod, elevated sawgrass (Cladium jamaicense) ridges and longer hydroperiod, deep water slough communities. Drainage and compartmentalization of the Everglades have fundamentally altered this pattern, and sawgrass ridge communities have expanded at the expense of deep water slough communities throughout much of the landscape. In this study we provide a simple isotopic and nutrient characterization of major components of the slough ecosystem to elucidate physiological and nutrient differences among species and to suggest pathways for organic matter decomposition that contribute to peat development in deep water sloughs. We examined carbon (C) and nitrogen (N) isotopes and C, N and phosphorus (P) concentrations of the floating-leaved macrophytes Nymphaea odorata and Nymphoides aquatica, the emergent macrophyte Eleocharis elongata, and the submerged species Utricularia foliosa and Utricularia purpurea, as well as soil and flocculent material from the southern Water Conservation Area 3-A. Flocculent material and soils had the highest N content (4.5 ± 0.2%) and U. foliosa and N. odorata had the highest P content (0.13 ± 0.01% to 0.12 ± 0.01%). The range for δ¹⁵N average ± SE values was 5.81 ± 0.29‰ (U. foliosa) to −1.84 ± 0.63‰ (N. odorata), while the range for δ¹³C values was −23.83 ± 0.12‰ (N. odorata) to −29.28 ± 0.34‰ (U. purpurea). Differences of up to 10‰ in C isotopic values of U. foliosa and N. odorata suggest fundamental physiological differences between these species. Along a degradation continuum, enrichment of ¹³C and ¹⁵N and extent of decomposition was negatively related to phosphorus concentrations. A two end-member ¹³C mixing model suggested that Utricularia species were the primary organic source for flocculent materials, whereas organic matter derived from root decomposition of N. odorata contributed to the progressively enriched δ¹³C values found with depth in soils. These results illustrate the fundamentally important roles of Nymphaea and Utricularia species in ecosystem dynamics of deep water sloughs.     

Phenology of first and peak emergence of Aphthona lacertosa and A. nigriscutis: Two flea beetles introduced for biological control of leafy spurge, Euphorbia esula L.

Nonlinear models were used to estimate first emergence and peak abundance dates for Aphthona lacertosa Rosenhauer and A. nigriscutis Foudras, two flea beetles introduced to control leafy spurge, Euphorbia esula L., in North America. For model development, 26 field sites were sampled for flea beetle abundance at weekly intervals for eight weeks in three western Minnesota counties in 2000, 2001, and 2002. A three-parameter Weibull function, fit to observed cumulative probability distributions, were used to predict accumulated degree-days (ADD) to first emergence. Bias testing indicated the Weibull function provided a useful estimate of first emergence for A. lacertosa (304 ADD, lower developmental threshold 7.5 °C), but failed to produce a useful estimate for A. nigriscutis. A third-order polynomial was used to approximate seasonal abundance and predict peak abundance for each species. Estimated ADD to peak abundance of A. lacertosa was 594 ± 24 (DD > 7.5 °C) and 670 ± 15 (DD > 9.3 °C) for A. nigriscutis. Models were validated with additional data sets from Minnesota, Montana, and North Dakota. Estimated date of peak emergence provided useful predictions of peak emergence for Minnesota and North Dakota, but failed to predict peak emergence in Montana. We speculate that variation in climate and environmental conditions between Midwestern states and Montana were responsible for differing emergence patterns. We conclude that phenology models should be developed regionally to provide useful predictions of peak emergence for land managers. Maps were developed for Minnesota to spatially display predicted dates of peak abundance for A. lacertosa and A. nigriscutis.     

A sperm cryopreservation protocol for the loach Misgurnus anguillicaudatus and its applicability for other related species

The aim of the present study was to establish a protocol of sperm cryopreservation in Misgurnus anguillicaudatus and verify the applicability of the obtained protocol in other loach species. We evaluated the following parameters: inseminating dose, thawing temperatures (20, 25 and 30 °C for 10 s), extenders (loach or cyprinid extenders), internal cryoprotectants (dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), glycerol (Gly), ethylene glycol (EG), and methanol (MeOH) at 0, 5, 10 and 15%), external cryoprotectants (bovine serum albumin 1 and 2%; sucrose 0.5 and 1%; glucose 0.5 and 1%; glycine 0.5 and 1%), activating solutions (distilled water, dechlorinated tap water, 25 mM NaCl and 50 mM NaCl), and hatchability of the eggs when fertilized with fresh or cryopreserved sperm. After the evaluation of these parameters, we optimized the cryopreservation using the following procedure: thawing temperature at 25 °C for 10 s; loach or cyprinid extenders; methanol at 10 or 15% as internal cryoprotectants; glycine 0.5% or bovine serum albumin 1% as external cryoprotectants and 50 mM NaCl for sperm activation. Using this procedure, the fertilizability of the post-thawed sperm was 47% in comparison to the fresh sperm, at the minimum inseminating dose (687.65 spermatozoa egg⁻¹ mL⁻¹). Based on this protocol, sperm from other loach species Lefua nikkonis, Misgurnus mizolepis and Barbatula toni were cryopreserved successfully.     

TWO SPECIFIC CAUSES OF CELL MORTALITY IN FREEZE‐THAW CYCLE OF YOUNG THALLI OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Porphyra yezoensis Ueda is an important marine aquaculture crop with single‐layered gametophytic thalli. In this work, the influences of thallus dehydration level, cold‐preservation (freezing) time, and thawing temperature on the photosynthetic recovery of young P. yezoensis thalli were investigated employing an imaging pulse‐amplitude‐modulation (PAM) fluorometer. The results showed that after 40 d of frozen storage when performing thallus thawing under 10°C, the water content of the thalli showed obvious effects on the photosynthetic recovery of the frozen thalli. The thalli with absolute water content (AWC) of 10%–40% manifested obvious superiority compared to the thalli with other AWCs, while the thalli thawed at 20°C showed very high survival rate (93.10%) and no obvious correlation between thallus AWCs and thallus viabilities. These results indicated that inappropriate thallus water content contributed to the cell damage during the freeze‐thaw cycle and that proper thawing temperature is very crucial. Therefore, AWC between 10% and 40% is the suitable thallus water content range for frozen storage, and the thawing process should be as short as possible. However, it is also shown that for short‐term cold storage the Porphyra thallus water content also showed no obvious effect on the photosynthetic recovery of the thalli, and the survival rate was extremely high (100%). These results indicated that freezing time is also a paramount contributor of the cell damage during the freeze‐thaw cycle. Therefore, the frozen nets should be used as soon as time permits.     

Effect of temperature on survival, development and reproduction of the predatory lacewing Dichochrysa prasina (Neuroptera: Chrysopidae) reared on Ephestia kuehniella eggs (Lepidoptera: Pyralidae)

Preimaginal development and adult longevity and reproduction of Dichochrysa prasina Burmeister were studied at six constant temperatures (15, 20, 25, 27, 30 and 33 °C) and a photoperiod of 16:8 (L:D). Eggs of the flour moth Ephestia kuehniella (Zeller) were used as food throughout preimaginal development, whereas the adults of D. prasina fed on a liquid diet of water, yeast hydrolysate, sugar and honey. At the highest tested temperature of 33 °C no larvae completed their development. At the rest of the tested temperatures the egg to adult developmental period ranged from approximately 92 days at 15 °C to 25 days at 30 °C. Percentages of adult emergence ranged from 36% at 15 °C to 84% at 30 °C. Both adult longevity and fecundity were significantly affected by temperature and the intrinsic rate of increase (r_m) reached its maximum value at 27 °C. These results could be useful for the establishment of a small scale rearing and mass production of D. prasina.