Stage- and sex-specific heat tolerance in the yellow dung fly Scathophaga stercoraria

Thermal tolerance varies at all hierarchical levels of biological organization: among species, populations, individuals, and even within individuals. Age- or developmental stage- and sex-specific thermal effects have received relatively little attention in the literature, despite being crucial for understanding thermal adaptation in nature and responses to global warming. We document stage- and sex- specific heat tolerance in the yellow dung fly Scathophaga stercoraria (Diptera: Scathophagidae), a species common throughout the northern hemisphere that generally favours cool climates. Exposure of eggs to temperatures up to 32 °C did not affect larval hatching rate, but subsequent egg-to-adult survival at a benign temperature was reduced. Permanent transfer from benign (18 °C) to hot temperatures (up to 31 °C) at different larval and pupal stages strongly decreased egg-to-adult survival, though survival continuously improved the later the transfer occurred. Temporary transfer for only two days increased mortality more weakly, survival being lowest when temperature stress was imposed early during the larval or pupal stages. Adult flies provided with sugar and water tolerated 31 °C longer than previously thought (5 days in males to 9 days in females). Eggs were thus less susceptible to thermal stress than larvae, pupae or adults, in agreement with the hypothesis that more mobile stages require less physiological protection against heat because they can behaviourally thermoregulate. The probability of mating, of laying a clutch, and hatching success were generally independently reduced by exposure of females or males to warm temperatures (24 °C) during the juvenile or adult stages, with some interactions evident. High temperature stress thus affects survival differentially depending on when it occurs during the juvenile or the pre-reproductive adult life stage, and affects reproductive success via the mating behaviour of both sexes, female physiology in terms of oviposition, and fertility via sperm and/or egg quality. Our results illustrate that temperature stress, even when moderate and temporary, during early development can have profound lethal and non-lethal fitness-consequences later in life.     

Physiological and molecular mechanisms associated with cross tolerance between hypoxia and low temperature in Thaumatotibia leucotreta

Biochemical adaptations allow insects to withstand exposures to hypoxia and/or hypothermia. Exposure to hypoxia may interact either synergistically or antagonistically with standard low temperature stress responses yet this has not been systematically researched and no clear mechanism has been identified to date. Using larvae of false codling moth Thaumatotibia leucotreta, a pest of southern Africa, we investigated the physiological and molecular responses to hypoxia or temperature stress pre-treatments, followed by a standard low temperature exposure. Survival rates were significantly influenced by pre-treatment conditions, although T. leucotreta shows relatively high basal resistance to various stressors (4% variation in larval survival across all pre-treatments). Results showed that mild pre-treatments with chilling and hypoxia increased resistance to low temperatures and that these responses were correlated with increased membrane fluidity (increased UFA:SFA) and/or alterations in heat shock protein 70 (HSP70); while general mechanical stress (shaking) and heat (2 h at 35 °C) do not elicit cross tolerance (no change in survival or molecular responses). We therefore found support for some limited cold hardening and cross tolerance responses. Given that combined exposure to hypoxia and low temperature is used to sterilize commodities in post-harvest pest management programs, researchers can now exploit these mechanisms involved in cross tolerance to develop more targeted control methods.     

Combined effect of temperature and zinc on Caenorhabditis elegans wild type and daf-21 mutant strains

Heavy metal pollution in aquatic ecosystems is a far reaching environmental problem. The possible influences of heavy metal exposure and the potential harm to organisms when combined with other environmental stressors such as temperature have been largely unexplored. An aquatic toxicity test of Caenorhabditis elegans was performed to estimate the 24 h median lethal concentration (LC₅₀) of different zinc concentrations at different temperatures (15 °C, 20 °C, 25 °C, and 30 °C). We also examined the time course thermotolerance on wild type (N2) and daf-21 null (JT6130) adults exposed to 6.1 mM zinc at 37 °C. Hsp90 protein expression level in response to the combined effect of temperature and zinc toxicity was also investigated by both Western blots and ELISA. Our results show that C. elegans wild type nematodes exhibit severe lethal toxicity after a 24 h exposure to zinc at higher temperatures. In addition, the expression level of Hsp90 was highly inhibited in adult worms subjected to zinc stress. This toxicity assay at different temperatures provides insight into organism response to combined effects of temperature and zinc toxicity.     

Thermal variation and factors influencing vertical migration behavior in Daphnia populations

The antipredator behavior diel vertical migration (DVM), common in aquatic keystone species Daphnia, involves daily migration from warmer surface waters before dawn to cooler deeper waters after dusk. Plasticity in Daphnia DVM behavior optimizes fitness via trade-offs between growth, reproduction, and predator avoidance. Migration behavior is affected by co-varying biotic and abiotic factors, including light, predator cues, and anthropogenic stressors making it difficult to determine each factor's individual contribution to the variation in this behavior. This study aims to better understand this ecologically significant behavior in Daphnia by: (1) determining how Daphnia pulicaria thermal preferences vary within and among natural populations; (2) distinguishing the role of temperature verses depth in Daphnia vertical migration; and (3) defining how two anthropogenic stressors (copper and nickel) impact Daphnia migratory behavior.Simulated natural lake stratification were constructed in 8 L (0.5 m tall, 14.5 cm wide) water columns to monitor under controlled laboratory conditions the individual effects of temperature gradients, depth, and metal stressors on Daphnia vertical migration. Three major findings are reported. First, while no difference in thermal preference was found among the four populations studied, within lake populations variability among isolates was high. Second, decoupling temperature and depth revealed that depth was a better predictor of Daphnia migratory patterns over temperature. Third, exposure to environmentally relevant concentrations of copper or nickel inhibited classic DVM behavior. These findings revealed the high variability in thermal preference found within Daphnia populations, elucidated the individual roles that depth and temperature have on migratory behavior, and showed how copper and nickel can interfere with the natural response of Daphnia to fish predator cues. Thus contributing to the body of knowledge necessary to predict how natural populations of Daphnia will be affected by climate related changes in lake temperatures and increased presence of anthropogenic stressors.     

Effects of a neonicotinoid pesticide on thermoregulation of African honey bees (Apis mellifera scutellata)

Thiamethoxam is a widely used neonicotinoid pesticide that, as agonist of the nicotinic acetylcholine receptors, has been shown to elicit a variety of sublethal effects in honey bees. However, information concerning neonicotinoid effects on honey bee thermoregulation is lacking. Thermoregulation is an essential ability for the honey bee that guarantees the success of foraging and many in-hive tasks, especially brood rearing. We tested the effects of acute exposure to thiamethoxam (0.2, 1, 2 ng/bee) on the thorax temperatures of foragers exposed to low (22 °C) and high (33 °C) temperature environments. Thiamethoxam significantly altered honey bee thorax temperature at all doses tested; the effects elicited varied depending on the environmental temperature and pesticide dose to which individuals were exposed. When bees were exposed to the high temperature environment, the high dose of thiamethoxam increased their thorax temperature 1–2 h after exposure. When bees were exposed to the low temperature, the higher doses of the neonicotinoid reduced bee thorax temperatures 60–90 min after treatment. In both experiments, the neonicotinoid decreased the temperature of bees the day following the exposure. After a cold shock (5 min at 4 °C), the two higher doses elicited a decrease of the thorax temperature, while the lower dose caused an increase, compared to the control. These alterations in thermoregulation caused by thiamethoxam may affect bee foraging activity and a variety of in-hive tasks, likely leading to negative consequences at the colony level. Our results shed light on sublethal effect of pesticides which our bees have to deal with.     

Effects of temperature on embryonic and early larval growth and development in the rough-skinned newt (Taricha granulosa)

We investigated the effects of temperature on the growth and development of embryonic and early larval stages of a western North American amphibian, the rough-skinned newt (Taricha granulosa). We assigned newt eggs to different temperatures (7, 14, or 21 °C); after hatching, we re-assigned the newt larvae into the three different temperatures. Over the course of three to four weeks, we measured total length and developmental stage of the larvae. Our results indicated a strong positive relationship over time between temperature and both length and developmental stage. Importantly, individuals assigned to cooler embryonic temperatures did not achieve the larval sizes of individuals from the warmer embryonic treatments, regardless of larval temperature. Our investigation of growth and development at different temperatures demonstrates carry-over effects and provides a more comprehensive understanding of how organisms respond to temperature changes during early development.     

Effect of thermal stress on metabolic and oxidative stress biomarkers of Hoplosternum littorale (Teleostei,Callichthyidae)

The present study aimed to investigate in Hoplosternum littorale (Hancock, 1828) the effects of different water temperatures (10 °C, 25 °C-control group- and 33 °C) on physiologic and metabolic traits following acute (1 day) and chronic (21 days) exposures. We analyzed several biomarker responses in order to achieve a comprehensive survey of fish physiology and metabolism under the effect of this natural stressor. We measured morphological indices, biochemical and hematological parameters as well as oxidative stress markers. To evaluate energy consumption, muscle and hepatic total lipid, protein and glycogen concentrations were also quantified. Extreme temperatures exposures clearly resulted in metabolic adjustments, being liver energy reserves and plasma metabolites the most sensitive parameters detecting those changes. We observed reduced hepatosomatic index after acute and chronic exposure to 33 °C while glycogen levels decreased at both temperatures and time of exposure tested. Additionally, acute and chronic exposures to 10 °C increased liver lipid content and plasma triglycerides. Total protein concentration was higher in liver and lower in plasma after chronic exposures to 10 °C and 33 °C. Acute exposition at both temperatures caused significant changes in antioxidant enzymes tested in the different tissues without oxidative damage to lipids. Antioxidant defenses in fish failed to protect them when they were exposed for 21 days to 10 °C, promoting higher lipid peroxidation in liver, kidney and gills. According to multivariate analysis, oxidative stress and metabolic biomarkers clearly differentiated fish exposed chronically to 10 °C. Taken together, these results demonstrated that cold exposure was more stressful for H. littorale than heat stress. However, this species could cope with variations in temperature, allowing physiological processes and biochemical reactions to proceed efficiently at different temperatures and times of exposure. Our study showed the ability of H. littorale to resist a wide range of environmental temperatures and contributes for the understanding of how this species is adapted to environments with highly variable physicochemical conditions.     

Linking vertebral number to performance of aquatic escape responses in the axolotl (Ambystoma mexicanum)

Environmental conditions during early development in ectothermic vertebrates can lead to variation in vertebral number among individuals of the same species. It is often seen that individuals of a species raised at cooler temperatures have more vertebrae than individuals raised at warmer temperatures, although the functional consequences of this variation in vertebral number on swimming performance are relatively unclear. To investigate this relationship, we tested how vertebral number in axolotls (Ambystoma mexicanum) affected performance of aquatic escape responses (C-starts). Axolotls were reared at four temperatures (12–24 °C) encompassing their natural thermal range and then transitioned to a mean temperature (18 °C) three months before C-starts were recorded. Our results showed variation in vertebral number, but that variation was not significantly affected by developmental temperature. C-start performance among axolotls was significantly correlated with caudal vertebral number, and individuals with more caudal vertebrae were able to achieve greater curvature more quickly during their responses than individuals with fewer vertebrae. However, our results show that these individuals did not achieve greater displacements or velocities, and that developmental temperature did not have any effect on C-start performance. We highlight that the most important aspects of escape swim performance (i.e., how far individuals get from a threat and how quickly they move the most important parts of the body away from that threat) are consistent across individuals regardless of developmental temperature and morphological variation.     

The effect of water temperature and velocity on barnacle growth: Quantifying the impact of multiple environmental stressors

Organisms employ a wide array of physiological and behavioral responses in an effort to endure stressful environmental conditions. For many marine invertebrates, physiological and/or behavioral performance is dependent on physical conditions in the fluid environment. Although factors such as water temperature and velocity can elicit changes in respiration and feeding, the manner in which these processes integrate to shape growth remains unclear. In a growth experiment, juvenile barnacles (Balanus glandula) were raised in dockside, once-through flow chambers at water velocities of 2 versus 19 cm s⁻¹ and temperatures of 11.5 versus 14 °C. Over 37 days, growth rates (i.e., shell basal area) increased with faster water velocities and higher temperatures. Barnacles at high flows had shorter feeding appendages (i.e., cirri), suggesting that growth patterns are unlikely related to plastic responses in cirral length. A separate experiment in the field confirmed patterns of temperature- and flow-dependent growth over 41 days. Outplanted juvenile barnacles exposed to the faster water velocities (32±1 and 34±1 cm s⁻¹; mean±SE) and warm temperatures (16.81±0.05 °C) experienced higher growth compared to individuals at low velocities (1±1 cm s⁻¹) and temperatures (13.67±0.02 °C). Growth data were consistent with estimates from a simple energy budget model based on previously measured feeding and respiration response curves that predicted peak growth at moderate temperatures (15 °C) and velocities (20–30 cm s⁻¹). Low growth is expected at both low and high velocities due to lower encounter rates with suspended food particles and lower capture efficiencies respectively. At high temperatures, growth is likely limited by high metabolic costs, whereas slow growth at low temperatures may be a consequence of low oxygen availability and/or slow cirral beating and low feeding rates. Moreover, these results advocate for approaches that consider the combined effects of multiple stressors and suggest that both increases and decreases in temperature or flow impact barnacle growth, but through different physiological and behavioral mechanisms.     

Effect of temperature on the larval stage of Tetanocera elata (Diptera: Sciomyzidae) – Potential biological control agent of pestiferous slugs

Due to the considerable losses caused by slugs in terms of agricultural production and revenue, there is an urgent need for a cost effective biological control agent. The malacophagous nature of the sciomyzid fly, Tetanocera elata (Fab.) makes it a possible contender to meet this demand. This study examined the effect of constant temperatures (14, 17, 20, 23, and 26 °C), in addition to ambient outdoor and laboratory temperatures on T. elata larval duration and predation. In general, the mean and median larval stage duration decreased as temperature increased with percentage survival for the overall larval stage (62%) greatest at 20 °C with a median duration of 44 days. There was no significant difference between temperatures with regard to the number of slugs killed per larva and while predation rate increased with increasing constant temperature, there was also no significant difference between the constant temperatures. Our results show that puparial weight can be used to predict the sex of adult flies prior to their emergence. The results are discussed in the context of the suitability of T. elata as a biological control agent of pestiferous slugs.     

Survival of rapidly fluctuating natural low winter temperatures by High Arctic soil invertebrates

The extreme polar environment creates challenges for its resident invertebrate communities and the stress tolerance of some of these animals has been examined over many years. However, although it is well appreciated that standard air temperature records often fail to describe accurately conditions experienced at microhabitat level, few studies have explicitly set out to link field conditions experienced by natural multispecies communities with the more detailed laboratory ecophysiological studies of a small number of ‘representative’ species. This is particularly the case during winter, when snow cover may insulate terrestrial habitats from extreme air temperature fluctuations. Further, climate projections suggest large changes in precipitation will occur in the polar regions, with the greatest changes expected during the winter period and, hence, implications for the insulation of overwintering microhabitats. To assess survival of natural High Arctic soil invertebrate communities contained in soil and vegetation cores to natural winter temperature variations, the overwintering temperatures they experienced were manipulated by deploying cores in locations with varying snow accumulation: No Snow, Shallow Snow (30 cm) and Deep Snow (120 cm). Air temperatures during the winter period fluctuated frequently between +3 and −24 °C, and the No Snow soil temperatures reflected this variation closely, with the extreme minimum being slightly lower. Under 30 cm of snow, soil temperatures varied less and did not decrease below −12 °C. Those under deep snow were even more stable and did not decline below −2 °C. Despite these striking differences in winter thermal regimes, there were no clear differences in survival of the invertebrate fauna between treatments, including oribatid, prostigmatid and mesostigmatid mites, Araneae, Collembola, Nematocera larvae or Coleoptera. This indicates widespread tolerance, previously undocumented for the Araneae, Nematocera or Coleoptera, of both direct exposure to at least −24 °C and the rapid and large temperature fluctuations. These results suggest that the studied polar soil invertebrate community may be robust to at least one important predicted consequence of projected climate change.     

Interactive effects of elevated temperature and CO2 levels on metabolism and oxidative stress in two common marine bivalves (Crassostrea virginica and Mercenaria mercenaria)

Marine bivalves such as the hard shell clams Mercenaria mercenaria and eastern oysters Crassostrea virginica are affected by multiple stressors, including fluctuations in temperature and CO₂ levels in estuaries, and these stresses are expected to be exacerbated by ongoing global climate change. Hypercapnia (elevated CO₂ levels) and temperature stress can affect survival, growth and development of marine bivalves, but the cellular mechanisms of these effects are not yet fully understood. In this study, we investigated whether oxidative stress is implicated in cellular responses to elevated temperature and CO₂ levels in marine bivalves. We measured the whole-organism standard metabolic rate (SMR), total antioxidant capacity (TAOC), and levels of oxidative stress biomarkers in the muscle tissues of clams and oysters exposed to different temperatures (22 and 27 °C) and CO₂ levels (the present day conditions of ~ 400 ppm CO₂ and 800 ppm CO₂ predicted by a consensus business-as-usual IPCC emission scenario for the year 2100). SMR was significantly higher and the antioxidant capacity was lower in oysters than in clams. Aerobic metabolism was largely temperature-independent in these two species in the studied temperature range (22–27 °C). However, the combined exposure to elevated temperature and hypercapnia led to elevated SMR in clams indicating elevated costs of basal maintenance. No persistent oxidative stress signal (measured by the levels of protein carbonyls, and protein conjugates with malondialdehyde and 4-hydroxynonenal) was observed during the long-term exposure to moderate warming (+ 5 °C) and hypercapnia (~ 800 ppm CO₂). This indicates that long-term exposure to moderately elevated CO₂ and temperature minimally affects the cellular redox status in these bivalve species and that the earlier observed negative physiological effects of elevated CO₂ and temperature must be explained by other cellular mechanisms.     

Entomotoxic properties of Dioclea violacea lectin and its effects on digestive enzymes of Anagasta kuehniella (Lepidoptera)

Entomotoxic plant lectins have been extensively studied in the past two decades, yet the exact mechanisms underlying their toxic effects remain unknown. This study investigated the effects of Dioclea violacea lectin (DVL) on larval development in Anagasta kuehniella. Chronic exposure of larvae (from neonates to the fourth instar) demonstrated that DVL interfered with larval growth, retarding development and decreasing larval mass without affecting survival. DVL decreased trypsin-like, chymotrypsin-like, and α-amylase activities and proved resistant to proteolysis by midgut proteases up to 24 h. Shorter exposures to dietary DVL had no effect on midgut enzyme activity. Feeding fourth-instar larvae with fluorescently-labeled DVL revealed lectin binding to the peritrophic membrane.     

The effects of increased constant incubation temperature and cumulative acute heat shock exposures on morphology and survival of Lake Whitefish (Coregonus clupeaformis) embryos

Increasing incubation temperatures, caused by global climate change or thermal effluent from industrial processes, may influence embryonic development of fish. This study investigates the cumulative effects of increased incubation temperature and repeated heat shocks on developing Lake Whitefish (Coregonus clupeaformis) embryos. We studied the effects of three constant incubation temperatures (2 °C, 5 °C or 8 °C water) and weekly, 1-h heat shocks (+3 °C) on hatching time, survival and morphology of embryos, as these endpoints may be particularly susceptible to temperature changes. The constant temperatures represent the predicted magnitude of elevated water temperatures from climate change and industrial thermal plumes. Time to the pre-hatch stage decreased as constant incubation temperature increased (148 d at 2 °C, 92 d at 5 °C, 50 d at 8 °C), but weekly heat shocks did not affect time to hatch. Mean survival rates and embryo morphometrics were compared at specific developmental time-points (blastopore, eyed, fin flutter and pre-hatch) across all treatments. Constant incubation temperatures or +3 °C heat-shock exposures did not significantly alter cumulative survival percentage (~50% cumulative survival to pre-hatch stage). Constant warm incubation temperatures did result in differences in morphology in pre-hatch stage embryos. 8 °C and 5 °C embryos were significantly smaller and had larger yolks than 2 °C embryos, but heat-shocked embryos did not differ from their respective constant temperature treatment groups. Elevated incubation temperatures may adversely alter Lake Whitefish embryo size at hatch, but weekly 1-h heat shocks did not affect size or survival at hatch. These results suggest that intermittent bouts of warm water effluent (e.g., variable industrial emissions) are less likely to negatively affect Lake Whitefish embryonic development than warmer constant incubation temperatures that may occur due to climate change.     

The importance of relative humidity and trophic resources in governing ecological niche of the invasive carabid beetle Merizodus soledadinus in the Kerguelen archipelago

Comprehensive studies to identify species-specific drivers of survival to environmental stress, reproduction, growth, and recruitment are vital to gaining a better understanding of the main ecological factors shaping species habitat distribution and dispersal routes. The present study performed a field-based assessment of habitat distribution in the invasive carabid beetle Merizodus soledadinus for the Kerguelen archipelago. The results emphasised humid habitats as a key element of the insect’s realised niche. In addition, insects faced food and water stress during dispersal events. We evaluated quantitatively how water availability and trophic resources governed the spatial distribution of this invasive predatory insect at Îles Kerguelen. Food and water stress survival durations [in 100%, 70%, and 30% relative humidity (RH) conditions] and changes in a set of primary metabolic compounds (metabolomics) were determined. Adult M. soledadinus supplied with water ad libitum were highly tolerant to prolonged starvation (LT₅₀ = 51.7 ± 6.2 d). However, food-deprived insect survival decreased rapidly in moderate (70% RH, LT₅₀ = 30.37 ± 1.39 h) and low (30% RH, LT₅₀ = 13.03 ± 0.48 h) RH conditions. Consistently, body water content decreased rapidly in insects exposed to 70% and 30% RH. Metabolic variation evidenced the effects of food deprivation in control insects (exposed to 100% RH), which exhibited a progressive decline of most glycolytic sugars and tricarboxylic acid cycle intermediates. Most metabolite levels were elevated levels during the first few hours of exposure to 30% and 70% RH. Augmented alanine and lactate levels suggested a shift to anaerobic metabolism. Simultaneously, peaks in threonine and glycolytic sugars pointed to metabolic disruption and a progressive physiological breakdown in dehydrating individuals. Overall, the results of our study indicate that the geographic distribution of M. soledadinus populations is highly dependent on habitat RH and water accessibility.     

Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta)

The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34 °C and 36 °C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36 °C, 3 h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events.     

Radiation damage effects at four specimen temperatures from 4 to 100 K

Radiation damage is the primary factor that limits resolution in electron cryo-microscopy (cryo-EM) of frozen-hydrated biological samples. Negative effects of radiation damage are attenuated by cooling specimens to cryogenic temperatures using liquid nitrogen or liquid helium. We have examined the relationship between specimen temperature and radiation damage across a broad spectrum of resolution by analyzing images of frozen-hydrated catalase crystal at four specimen temperatures: 4, 25, 42, and 100 K. For each temperature, “exposure series” were collected consisting of consecutive images of the same area of sample, each with 10 e^?/Ų exposure per image. Radiation damage effects were evaluated by examining the correlation between cumulative exposure and normalized amplitudes or IQ values of Bragg peaks across a broad range of resolution (4.0–173.5 Å). Results indicate that for sub-nanometer resolution, liquid nitrogen specimen temperature (100 K) provides the most consistent high-quality data while yielding statistically equivalent protection from radiation damage compared to the three lower temperatures. At lower resolution, suitable for tomography, intermediate temperatures (25 or 42 K) may provide a modest improvement in cryo-protection without introducing deleterious effects evident at 4 K.     

Survival of tissue balls from the coral Pocillopora damicornis L. exposed to cryoprotectant solutions

In this study, the tolerance of tissue balls (TBs, 100–300 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions was tested. TBs were treated for 20 min at room temperature with solutions of ethylene glycol (EG), methanol (Met), glycerol (Gly) or dimethyl sulfoxide (Me₂SO) at concentrations between 1.0 and 4.5 M. Two parameters were used to evaluate the survival of TBs following CPA treatment. The Undamaged Duration of Tissue Balls (expressed in h) corresponded to the time period during which the membrane surface of TBs remained smooth and their motility was preserved. Tissue Ball Regression (expressed in μm/h) corresponded to the size reduction of TBs over time. TBs tolerated exposure to all CPAs tested at the three lower concentrations employed (1.0 M, 1.5 M and 2.0 M). No survival was achieved following exposure to a 4.5 M CPA solution. At concentrations of 3.0 and 4.0 M, higher Undamaged Duration of Tissue Balls and lower Tissue Ball Regression were obtained following treatment with EG compared to the other three CPAs. Our experiments show that TBs constitute a good experimental material to evaluate CPA toxicity on corals using large numbers of samples. Performing preliminary experiments with TBs may allow reducing the number of tests carried out with less easily available coral forms such as planulae, thereby preserving larval stocks.     

Efficacy assessment of Pichia guilliermondii strain Z1, a new biocontrol agent,against citrus blue mould in Morocco under the influence of temperature and relative humidity

The interactions of Penicillium italicum, which causes blue mould, and antagonistic yeast Pichia guilliermondii strain Z1 were examined in controlled environments, to determine the influence of relative humidity (RH) (45%, 75%, 85%, 98%, and 100%) and temperature (T) (5, 10, 15, 20, and 25 °C). All main effects and interactions were significant (P ? 0.05), with the exception of interactions RH×T and strain Z1 (BCA)×RH×T. In the pathogen control, the lesion diameter of blue mould developed under all environmental conditions but was the largest at a RH range between 98% and 100%, independent of the temperature. The efficacy of strain Z1 appeared to be independent of the environment and reduced disease incidence by more than 85% in all environmental conditions. Rapid colonization of the antagonistic yeast strain Z1 on citrus wounded sites was recorded during the first week at 5 °C. Colonization then stabilized at ±6.9 × 10⁶ CFU/ml for 30 days. This indicates that P. guilliermondii is able to adapt itself and colonize the wound sites prior to the arrival of the pathogen, displaying greater efficiency than when colonizing wounds after pathogen. The antagonist was capable of growing in low concentrations of orange juice (0.1–5%), with greatest growth at 5%. Applying strain Z1 (1 × 10⁸ CFU/ml) as a formulated product significantly reduced the incidence of infected fruits and the percentage of infected wounds relative to the pathogen control. Disease control with formulated product (45%) was slightly lower than that obtained with thiabendazole (20%) or strain Z1 culturable cells (25%). These results suggest that strain Z1 may be a useful BCA for control of blue mould under varying environmental conditions, and control may be enhanced by combining with other eco-friendly post-harvest treatments or improved formulation.