Winter is coming: Interactions of multiple stressors in winter and implications for the natural world.

Winter is a key driver of ecological processes in freshwater, marine and terrestrial ecosystems, particularly in higher latitudes. Species have evolved various adaptive strategies to cope with food limitations and the cold and dark wintertime. However, human-induced climate change and other anthropogenic stressors are impacting organisms in winter in unpredictable ways. In this paper, we show that global change experiments investigating multiple stressors have predominantly been conducted during summer months. However, effects of anthropogenic stressors sometimes differ between winter and other seasons, necessitating comprehensive investigations. Here, we outline a framework for understanding the different effects of anthropogenic stressors in winter compared to other seasons and discuss the primary mechanisms that will alter ecological responses of organisms (microbes, animals and plants). For instance, while the magnitude of some anthropogenic stressors can be greater in winter than in other seasons (e.g. some pollutants), others may alleviate natural winter stress (e.g. warmer temperatures). These changes can have immediate, delayed or carry-over effects on organisms during winter or later seasons. Interactions between stressors may also vary with season. We call for a renewed research direction focusing on multiple stressor effects on winter ecology and evolution to fully understand, and predict, how ecosystems will fare under changing winters. We also argue the importance of incorporating the interactions of anthropogenic stressors with winter into ecological risk assessments, management and conservation efforts.

Transgenerational exposure to marine heatwaves ameliorates the lethal effect on tropical copepods regardless of predation stress.

Marine heatwaves (MHWs) are emerging as a severe stressor in marine ecosystems. Extreme warm sea surface temperatures during MHWs often exceed the optimal thermal range for more than one generation of tropical coastal zooplankton. However, it is relatively unknown whether transgenerational plasticity (TGP) to MHWs may shape the offspring's fitness, particularly in an ecologically relevant context with biotic interactions such as predation stress. We addressed these novel research questions by determining the survival, reproductive success, and grazing rate of the copepod Pseudodiaptomus incisus exposed to MHW and fish predator cues (FPC) for two generations (F1 and F2). The experiment was designed in a full orthogonal manner with 4 treatments in F1 and 16 treatments in F2 generation. In both generations, MHW reduced P. incisus survival, reproductive parameters, and grazing by 10%-62% in MHW, but these parameters increased by 2%-15% with exposure to FPC, particularly at control temperature. F2 reproductive success and grazing rate as indicated by cumulative fecal pellets were reduced by 20%-30% in F1-MHW, but increased by ~2% in F1-FPC. Strikingly, MHW exposure reduced 17%-18% survival, but transgenerational exposure to MHWs fully ameliorated its lethal effect and this transgenerational effect was independent of FPC. Increased survival came with a cost of reduced reproductive success, constrained by reduced grazing. The rapid transgenerational MHW acclimation and its associated costs are likely widespread and crucial mechanisms underlying the resilience of coastal tropical zooplankton to MHWs in tropical coastal marine ecosystems.

Toxicity of di-2-ethylhexyl phthalate and tris (2-butoxyethyl) phosphate to a tropical micro-crustacean (Ceriodaphnia cornuta) is higher in Mekong River water than in standard laboratory medium.

Plasticizers such as di(2-ethylhexyl) phthalate (DEHP) and tris (2-butoxyethyl) phosphate (TBOEP) are manufactured chemicals produced in high volumes. These chemicals are frequently detected in the aquatic environment and cause toxic effects on organisms. In this study, we assessed the chronic impacts of DEHP and TBOEP, respectively, at the concentration of 100 µg L-1 dissolved in the artificial medium (M4/4) and Mekong River water on life history traits of a tropical micro-crustacean, Ceriodaphnia cornuta, for 14 days. DEHP and TBOEP substantially reduced the survival of C. cornuta. In M4/4 medium, both plasticizers strongly enhanced reproduction but did not influence the growth of C. cornuta. Mekong River water, plasticizers-exposed C. cornuta produced less neonates than those in the control. The detrimental impacts of DEHP and TBOEP on the fitness of C. cornuta were much stronger in natural river water than in M4/4. Our results suggest that plasticizers can cause adverse effects on tropical freshwater cladocerans, particularly in natural water. These results are of a deep concern, as national and international regulatory guidelines which are based on ecotoxicological tests using standard media may not fully capture these effects.

Parental exposures increase the vulnerability of copepod offspring to copper and a simulated marine heatwave.

Extreme temperatures from marine heatwaves (MHWs) and pollution are dominant stressors in tropical marine ecosystems. However, we know little about the role of transgenerational effects of metals and MHWs in shaping the offspring's vulnerability to these stressors. We addressed this fundamental knowledge gap by exposing the planktonic copepod Pseudodiaptomus incisus to copper (Cu: control, 15 and 60 µg L-1) under 2 temperatures (30 and a simulated marine heatwave at 34 °C) in the first generation (F1) and 16 treatments in F2: offspring from each of 4 F1 conditions (control or 15 µg Cu L-1 × 30 or 34 °C) was reared in 4 F2 conditions (control or 15 µg Cu L-1 × 30 or 34 °C). We assessed changes in copepod performance, particularly survival, adult size, grazing, and reproduction. In F1, Cu or marine heatwave (MHW) exposures reduced all fitness traits of F1; the effects were particularly strong when both stressors were present. Transgenerational effects of Cu or MHW also strongly reduced F2 performance. Direct Cu and MHW effects on the offspring were further strengthened by transgenerational effects, resulting in more substantial reductions in F2 performance when both generations were exposed to these stressors. As copepods are major food resources for corals, shrimps, or fish larvae and juveniles, strong transgenerational and direct effects of Cu and MHW can have a cascading effect on entire coastal food webs. These results highlight the importance of considering the interaction of transgenerational and direct effects of multiple stressors, particularly relevant for short-lived organisms in tropical marine ecosystems.

Reversible and irreversible transgenerational effects of metal exposure on nine generations of a tropical micro-crustacean.

Micro-crustaceans are important grazers that control the algal blooms in eutrophic lakes. However, we know little about how these key species may be affected by long-term exposure to contaminants and when the transgenerational effects are reversible and irreversible. To address this, we investigated the effects of lead (Pb, 100 µg L-1) exposure on morphology and reproduction of Moina dubia for nine consecutive generations (F1-F9) in three treatments: control, Pb, and pPb (M. dubia from Pb-exposed parents returned to the control condition). In F1-F2, Pb did not affect morphology, and reproduction of M. dubia. In all later generations, Pb-exposed M. dubia had a smaller body and shorter antennae than those in control. In F3-F6, pPb-exposed animals showed no differences in body size and antennae compared to the control, suggesting recoverable effects. In F7-F9, the body size and antennae of pPb-exposed animals did not differ compared to Pb-exposed ones, and both were smaller than the control animals, suggesting irreversible effects. Pb exposure reduced the brood size, number of broods and total neonates per female in F3-F9, yet the reproduction could recover in pPb treatment until F7. No recovery of the brood size and number of broods per female was observed in pPb-exposed animals in the F8-F9. Our study suggests that long-term exposure to metals, here Pb, may cause irreversible impairments in morphology and reproduction of tropical urban micro-crustaceans that may lower the top-down control on algal blooms and functioning of eutrophic urban lakes.

Biodiversity and Enzyme Activity of Marine Fungi with 28 New Records from the Tropical Coastal Ecosystems in Vietnam.

The coastal marine ecosystems of Vietnam are one of the global biodiversity hotspots, but the biodiversity of marine fungi is not well known. To fill this major gap of knowledge, we assessed the genetic diversity (ITS sequence) of 75 fungal strains isolated from 11 surface coastal marine and deeper waters in Nha Trang Bay and Van Phong Bay using a culture-dependent approach and 5 OTUs (Operational Taxonomic Units) of fungi in three representative sampling sites using next-generation sequencing. The results from both approaches shared similar fungal taxonomy to the most abundant phylum (Ascomycota), genera (Candida and Aspergillus) and species (Candida blankii) but were different at less common taxa. Culturable fungal strains in this study belong to 3 phyla, 5 subdivisions, 7 classes, 12 orders, 17 families, 22 genera and at least 40 species, of which 29 species have been identified and several species are likely novel. Among identified species, 12 and 28 are new records in global and Vietnamese marine areas, respectively. The analysis of enzyme activity and the checklist of trophic mode and guild assignment provided valuable additional biological information and suggested the ecological function of planktonic fungi in the marine food web. This is the largest dataset of marine fungal biodiversity on morphology, phylogeny and enzyme activity in the tropical coastal ecosystems of Vietnam and Southeast Asia. Biogeographic aspects, ecological factors and human impact may structure mycoplankton communities in such aquatic habitats.

Heat tolerance and thermal preference of the copepod Tigriopus californicus are insensitive to ecologically relevant dissolved oxygen levels.

Shifting climate patterns may impose novel combinations of abiotic conditions on animals, yet understanding of the present-day interactive effects of multiple stressors remains under-developed. We tested the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis and quantified environmental preference of the copepod Tigriopus californicus, which inhabits rocky-shore splashpools where diel fluctuations of temperature and dissolved oxygen (DO) are substantial. Egg-mass bearing females were exposed to a 5 h heat ramp to peak temperatures of 34.1-38.0 °C crossed with each of four oxygen levels: 22, 30, 100 and 250% saturation (4.7-5.3, 5.3-6.4, 21.2-21.3, and 50.7-53.3 kPa). Survival decreased at higher temperatures but was independent of DO. The behavioral preference of females was quantified in seven combinations of gradients of both temperature (11-37 °C) and oxygen saturation (17-206% or 3.6-43.6 kPa). Females avoided high temperatures regardless of DO levels. This pattern was more pronounced when low DO coincided with high temperature. In uniform temperature treatments, the distribution shifted toward high DO levels, especially in uniform high temperature, confirming that Tigriopus can sense environmental pO2. These results question the ecological relevance of OCLTT for Tigriopus and raise the possibility of microhabitat selection being used within splashpool environments to avoid physiologically stressful combinations of conditions.

Changes in the Magnitude of the Individual and Combined Effects of Contaminants, Warming, and Predators on Tropical Cladocerans across 11 Generations.

A massive challenge in ecotoxicology is assessing how the interaction of contaminants, climate change, and biotic stressors shapes the structure and functions of natural populations. Furthermore, it is not known whether contemporary evolutionary responses to multiple stressors across multigenerations may alter the interaction of these stressors. To address these issues, we exposed Moina dubia to lead (Pb, 50 µg/L) under two temperatures (25 and 28 °C) with/without predator cues from climbing perch (Anabas testudineus) for 11 generations (F1-F11). We assessed changes in M. dubia fitness, including development time, adult size, lifespan, fecundity, and neonate production. We found strong negative effects of Pb, elevated temperature, and predator cues on the fitness of M. dubia. Strikingly, Pb-induced reduction in the performance of M. dubia was stronger at 25 °C and in the absence of predator cues. The individual and interactive effects of Pb, temperature, and predator cues on M. dubia were stronger across F1-F9 and generally leveled off in F10-F11. Our results highlight the high vulnerability of M. dubia to multiple stressors, thus weakening top-down control on algal blooms in eutrophic lakes. Our study underscores the importance of integrating evolutionary responses in realistic ecotoxicological risk assessments of contaminants interacting with climatic and biotic stressors.

Population- and sex-specific sensitivity of the marine amphipod Allorchestes compressa to metal exposure.

The sensitivity to contaminants of natural populations varies greatly depending on their historical exposure and on the sex of the individual. These factors result in great uncertainty in ecotoxicological risk assessments and challenge the protection of marine biodiversity. This study investigated the role of background pollution in the environment in shaping the sensitivity of males and females of the common marine amphipod Allorchestes compressa to the common trace marine pollutant, copper (Cu). Female and male amphipods were collected from two sites: Geelong (the polluted site) and Clifton Springs (the clean site). Amphipods were exposed to Cu treatments of 0, 50, 100, and 250 µg/L for 10 days, followed by a 10-day recovery period. Cu-exposed males from Geelong showed a reduction in feeding rate at a higher Cu concentration than males from Clifton Springs, suggesting that they have a higher tolerance to Cu than males from Clifton Springs. This can be explained by their higher base level of metallothioneins (MTs) and glutathione-S-transferase (GST), the key physiological responses for detoxification and defence against damages from Cu toxicity. Males showed a higher tolerance to Cu than females. This pattern was similar in both populations, which may be associated with a higher level of GST. During the recovery period, only males from Geelong fully recovered to the control level. Our results emphasize the importance of considering population- and sex-specific sensitivity of invertebrates to contaminants in ecotoxicological risk assessments.

Interactive effects of extreme temperature and a widespread coastal metal contaminant reduce the fitness of a common tropical copepod across generations.

Tropical coastal areas are increasingly exposed to temperature extremes from marine heatwaves and contaminants from anthropogenic activities. The interactive effects of these environmental changes on marine life are understudied. We investigated the direct and cross-generational effects of copper (Cu) on F0 and F1 generations of the common tropical copepod Pseudodiaptomus annandalei under extreme temperatures (30 and 34 °C). In F0, Cu exposure reduced survival and nauplii production; these patterns were more pronounced at 34 °C and in females. F0 Copepods produced more faecal pellets at 34 °C than 30 °C, indicating a higher energetic demand. In F1, the number of F1 adults was lower in CuF0 and at 34 °C. Cu-exposed F0 produced larger adult F1, while exposure to 34 °C resulted in smaller adult F1. Our results show that tropical copepods are highly vulnerable to the interactive effects of contaminants and extreme temperatures.

Development of metal adaptation in a tropical marine zooplankton.

Tropical marine ecosystems are highly vulnerable to pollution and climate change. It is relatively unknown how tropical species may develop an increased tolerance to these stressors and the cost of adaptations. We addressed these issues by exposing a keystone tropical marine copepod, Pseudodiaptomus annandalei, to copper (Cu) for 7 generations (F1-F7) during three treatments: control, Cu and pCu (the recovery treatment). In F7, we tested the "contaminant-induced climate change sensitivity" hypothesis (TICS) by exposing copepods to Cu and extreme temperature. We tracked fitness and productivity of all generations. In F1, Cu did not affect survival and grazing but decreased nauplii production. In F2-F4, male survival, grazing, and nauplii production were lower in Cu, but recovered in pCu, indicating transgenerational plasticity. Strikingly, in F5-F6 nauplii production of Cu-exposed females increased, and did not recover in pCu. The earlier result suggests an increased Cu tolerance while the latter result revealed its cost. In F7, extreme temperature resulted in more pronounced reductions in grazing, and nauplii production of Cu or pCu than in control, supporting TICS. The results suggest that widespread pollution in tropical regions may result in high vulnerability of species in these regions to climate change.

Author Correction: Extreme temperature impairs growth and productivity in a common tropical marine copepod.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Delayed effects of pyrene exposure during overwintering on the Arctic copepod Calanus hyperboreus.

Calanus hyperboreus is the largest copepod and a key species in the Arctic food web. During the spring bloom, C. hyperboreus builds up large lipid reserves, which enable it to survive and produce eggs during overwintering. The ecological effects of oil exposure on overwintering C. hyperboreus are unknown. The present study empirically tested if exposure to the polycyclic aromatic hydrocarbon (PAH) pyrene from crude oil affects the survival, egg production, and hatching success of overwintering C. hyperboreus. We also tested the delayed effects on faecal pellet production and lipid recovery in clean seawater. Direct exposure did not reduce survival and egg production, but reduced hatching success 3-18 times by the end of the exposure period. Remarkably, we documented strong delayed effects of pyrene on faecal pellet production and the recovery of lipid reserves. The current study reveals a high vulnerability of this key species of Arctic zooplankton to oil exposure during winter. Together with our previous study on C. glacialis, we complete the picture of the impact of oil on the largest and most lipid-rich copepod C. hyperboreus, which potentially can have huge ecological consequences for the fragile Arctic marine food web.

Extreme temperature impairs growth and productivity in a common tropical marine copepod.

Shallow, tropical marine ecosystems provide essential ecosystem goods and services, but it is unknown how these ecosystems will respond to the increased exposure to the temperature extremes that are likely to become more common as climate change progresses. To address this issue, we tracked the fitness and productivity of a key zooplankton species, the copepod Pseudodiaptomus annandalei, acclimated at two temperatures (30 and 34 °C) over three generations. 30 °C is the mean temperature in the shallow water of the coastal regions in Southeast Asia, while 34 °C simulated a temperature extreme that occurs frequently during the summer period. For each generation, we measured the size at maturity and reproductive success of individuals. In all three generations, we found strong negative effects of warming on all measured fitness-related parameters, including prolonged development time, reduced size at maturity, smaller clutch sizes, lower hatching success, and reduced naupliar production. Our results suggest that P. annandalei are already exposed to temperatures that exceed their upper thermal optimum. Increased exposure to extreme temperatures may reduce the abundance of these tropical marine copepods, and thus reduce the availability of resources to higher trophic levels.

An adaptive transgenerational effect of warming but not of pesticide exposure determines how a pesticide and warming interact for antipredator behaviour.

The impact of pesticides on organisms may strongly depend on temperature. While many species will be exposed to pesticides and warming both in the parental and offspring generations, transgenerational effects of pesticides under warming are still poorly studied, particularly for behaviour. We therefore studied the single and combined effects of exposure to the pesticide chlorpyrifos (CPF) and warming both within and across generations on antipredator behaviour of larvae of the vector mosquito Culex pipiens. Within each generation pesticide exposure and warming reduced the escape diving time, making the larvae more susceptible to predation. Pesticide exposure of the parents did not affect offspring antipredator behaviour. Yet, parental exposure to warming determined how warming and the pesticide interacted in the offspring generation. When parents were reared at 24 °C, warming no longer reduced offspring diving times in the solvent control, suggesting an adaptive transgenerational effect to prepare the offspring to better deal with a higher predation risk under warming. Related to this, the CPF-induced reduction in diving time was stronger at 20 °C than at 24 °C, except in the offspring whose parents had been exposed to 24 °C. This dependency of the widespread interaction between warming and pesticide exposure on an adaptive transgenerational effect of warming is an important finding at the interface of global change ecology and ecotoxicology.

Transgenerational effects of cyanobacterial toxins on a tropical micro-crustacean Daphnia lumholtzi across three generations.

Climate change and human activities induce an increased frequency and intensity of cyanobacterial blooms which could release toxins to aquatic ecosystems. Zooplankton communities belong to the first affected organisms, but in tropical freshwater ecosystems, this issue has yet been poorly investigated. We tested two questions (i) if the tropical Daphnia lumholtzi is capable to develop tolerance to an ecologically relevant concentration of purified microcystin-LR and microcystins from cyanobacterial extract transferable to F1 and F2 generations? And (ii) would F1 and F2 generations recover if reared in toxin-free medium? To answer these questions, we conducted two full factorial mutigenerational experiments, in which D. lumholtzi was exposed to MC-LR and cyanobacterial extract at the concentration of 1 µg L-1 microcystin continuously for three generations. After each generation, each treatment was spit into two: one reared in the control (toxin free) while the other continued in the respective exposure. Fitness-related traits including survival, maturity age, body length, and fecundity of each D. lumholtzi generation were quantified. Though there were only some weak negative effects of the toxins on the first generation (F0), we found strong direct, accumulated and carried-over impacts of the toxins on life history traits of D. lumholtzi on the F1 and F2, including reductions of survival, and reproduction. The maturity age and body length showed some inconsistent patterns between generations and need further investigations. The survival, maturity age (for extract), and body length (for MC-LR) were only recovered when offspring from toxin exposed mothers were raised in clean medium for two generations. Chronic exposure to long lasting blooms, even at low density, evidently reduces survival of D. lumholtzi in tropical lakes and reservoirs with ecological consequences.

Transgenerational interactions between pesticide exposure and warming in a vector mosquito.

While transgenerational plasticity may buffer ectotherms to warming and pesticides separately, it remains unknown how combined exposure to warming and pesticides in the parental generation shapes the vulnerability to these stressors in the offspring. We studied the transgenerational effects of single and combined exposure to warming (4°C increase) and the pesticide chlorpyrifos on life-history traits of the vector mosquito Culex pipiens. Parental exposure to a single stressor, either warming or the pesticide, had negative effects on the offspring: parental exposure to both warming and the pesticide resulted in an overall lower offspring survival, and a delayed offspring metamorphosis. Parental exposure to a single stressor did, however, not alter the vulnerability of the offspring to the same stressor in terms of survival. Parental pesticide exposure resulted in larger offspring when the offspring experienced the same stressor as the parents. Within both the parental and offspring generations, warming made the pesticide more toxic in terms of survival. Yet, this synergism disappeared in the offspring of parents exposed to both stressors simultaneously because in this condition, the pesticide was already more lethal at the lower temperature. Our results indicate that transgenerational effects will not increase the ability of this vector species to deal with pesticides in a warming world. Bifactorial transgenerational experiments are crucial to understand the combined impact of warming and pesticides across generations, hence to assess the efficacy of vector control in a warming world.

Microplastic does not magnify the acute effect of PAH pyrene on predatory performance of a tropical fish (Lates calcarifer).

Microplastic (MP) leads to widespread pollution in the marine ecosystem. In addition to the physical hazard posed by ingestion of microplastic particles, concern is also on their potential as vector for transport of hydrophobic contaminants. We experimentally studied the single and interactive effects of microplastic and pyrene, a polycyclic aromatic hydrocarbon, on the swimming behaviour and predatory performance of juvenile barramundi (Lates calcarifer). Juveniles (18+ days post hatch) were exposed to MPs, or pyrene (100 nM), or combination of both, and feeding rate and foraging activity (swimming) were analysed. Exposure to MPs alone did not significantly influence feeding performance of the juveniles, while a dose-effect series of pyrene showed strong effect on fish behaviour when concentrations were above 100 nM. In the test of combined MP and pyrene exposure, we observed no effect on feeding while swimming speed decreased significantly. Thus, our results confirm that short-time exposure to pyrene impacts the performance of fish juveniles, while additional exposure to microplastic at the given conditions influenced their activity only and not their feeding rate. Further studies of the combined effects of microplastics and pollutants on tropical fish behaviour are encouraged.

Increased tolerance to oil exposure by the cosmopolitan marine copepod Acartia tonsa.

Oil contamination is an environmental hazard to marine ecosystems, but marine organism tolerance to oil after many generations of exposure remains poorly known. We studied the effects of transgenerational oil exposure on fitness-related traits in a cosmopolitan neritic copepod, Acartia tonsa. Copepods were exposed to an oil compound, the PAH pyrene, at concentrations of 1, 10, 100 and 100+(the saturated pyrene concentration in seawater)nM over two generations and measured survival, sex ratio, size at maturity, grazing rate and reproductive success. Exposure to the pyrene concentration of 100+nM resulted in 100% mortality before adulthood in the first generation. At the pyrene concentration of 100nM, pyrene reduced grazing rate, increased mortality, reduced the size of females and caused lower egg production and hatching success. Importantly, we found strong evidence for increased tolerance to pyrene exposure in the second generation: the reduction in size at maturity of females was less pronounced in the second generation and survival, egg production and hatching success were recovered to control levels in the second generation. The increased tolerance of copepods to oil contamination may dampen the direct ecological consequences of a coastal oil spill, but it raises the concern whether a larger fraction of oil components accumulated in survived copepods, may be transferred up the food web.