Polymorphism in Gag Gene Cleavage Sites of HIV-1 Non-B Subtype and Virological Outcome of a First-Line Lopinavir/Ritonavir Single Drug Regimen

Virological failure on a boosted-protease inhibitor (PI/r) first-line triple combination is usually not associated with the detection of resistance mutations in the protease gene. Thus, other resistance pathways are being investigated. First-line PI/r monotherapy is the best model to investigate in vivo if the presence of mutations in the cleavage sites (CS) of gag gene prior to any antiretroviral treatment might influence PI/r efficacy. 83 patients were assigned to initiate antiretroviral treatment with first-line lopinavir/r monotherapy in the randomised Monark trial. We compared baseline sequence of gag CS between patients harbouring B or non-B HIV-1 subtype, and between those who achieved viral suppression and those who experienced virological failure while on LPV/r monotherapy up to Week 96. Baseline sequence of gag CS was available for 82/83 isolates; 81/82 carried at least one substitution in gag CS compared to HXB2 sequence. At baseline, non-B subtype isolates were significantly more likely to harbour mutations in gag CS than B subtype isolates (p<0.0001). Twenty-three patients experienced virological failure while on lopinavir/r monotherapy. The presence of more than two substitutions in p2/NC site at baseline significantly predicted virological failure (p = 0.0479), non-B subtype isolates being more likely to harbour more than two substitutions in this specific site. In conclusion, gag cleavage site was highly polymorphic in antiretroviral-naive patients harbouring a non-B HIV-1 strain. We show that pre-therapy mutations in gag cleavage site sequence were significantly associated with the virological outcome of a first-line LPV/r single drug regimen in the Monark trial

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Evolutionary-ecotoxicological study of the effects of daily temperature fluctuations on pesticide toxicity in damselfly larvae

In natural ecosystems animals encounter several environmental stressors that furthermore can interact with pollutants and potentially increase their toxicity. This is considered one of the underlying reasons why current ecological risk assessment is possibly failing to protect natural ecosystems as it may be lacking realism by being based on pesticide toxicity testing under standard laboratory conditions. While it is widely known that an increase in mean temperature increases the toxicity of pollutants, studies largely ignored the effects of daily temperature fluctuations (DTFs) on pesticide toxicity and never tested pollutant toxicity under both global warming stressors (the more realistic scenario in which both the mean temperature and the DTF increase). Furthermore, how DTFs shape pesticide toxicity is mostly unknown, which asks for an integrated approach including life history and physiology. In this thesis, I tested for the single and combined effects of exposure to the pesticide chlorpyrifos (CPF) and warming (+ 4 °C increase in mean temperature and a + 5 °C increase in DTF) in Ischnura elegans damselfly larvae by executing a series of common-garden experiments. I studied effects on life history traits, heat tolerance and candidate underlying physiological mechanisms. Thereby, I used damselfly larvae across a latitudinal gradient (European low- and high-latitude populations) to study the role of thermal adaptation in shaping the sensitivity to pesticides under global warming. The mean summer water temperatures and maximum summer DTFs in shallow freshwater ponds located in southern France (low latitude) are 24 °C with 10 °C DTF, and in southern Sweden (high latitude) are 20 °C with 5 °C DTF. This 4 °C difference in mean temperature and 5 °C difference in maximum DTF also matches the predicted increase in both factors by 2100 under the IPCC RCP 8.5 scenario, which allows for applying a space-for-time substitution to test if gradual thermal evolution in high-latitude populations may buffer for the increased pesticide toxicity under global warming (increase in both mean temperature and DTF). I reviewed the strengths and weaknesses of this approach in chapter I. In the second chapter, I studied the effects of global warming in the absence of the pesticide and I found that DTFs only had negative effects on growth rate under the 4 °C warming treatment. While 4 °C warming was beneficial for larvae of both latitudes, this changed in a negative effect in the presence of high DTF. These negative effects of DTF were stronger in high-latitude larvae and already occurred at low DTF, indicating local thermal adaptation. This also suggests that if high-latitude populations are able to gradually thermally evolve into 'low-latitude' populations, they would no longer suffer a growth reduction in the presence of DTF under 4 °C warming. When studying the effects of DTFs on pesticide toxicity in chapter III, I found the striking result that while the used chlorpyrifos concentration was not affecting the damselfly larvae's life history at a constant temperature of 20 °C, it did strongly decrease survival and growth in the presence of DTFs around 20 °C. These results suggest that in standard pesticide toxicity tests, which are carried out by current risk assessment, this concentration would have been regarded as safe. Thereby, this highlights it is crucial to integrate DTFs in current risk assessment to reach more realistic predictions about pesticide toxicity in natural systems. In the last three chapters, I studied the effects of pesticides under global warming, thereby including the predicted increases in both mean temperature and DTF. The toxicity of chlorpyrifos was magnified by the increase in both mean temperature and DTF, but especially at their combination. Furthermore, I described in chapter V a novel, likely general mechanism that contributes to the higher chlorpyrifos toxicity under global warming by coupling two general principles: the widespread temperature-size rule and the size-pesticide sensitivity pattern. Larvae got smaller under DTFs, and these smaller larvae were more vulnerable to the pesticide, hence the higher chlorpyrifos toxicity under DTFs was partly mediated through DTF-induced reductions in body size. In terms of physiological variables, I found evidence of chlorpyrifos-induced effects being stronger under DTFs in terms of oxidative damage to lipids, which may contribute to the mortality patterns. Further, in my last chapter, I observed that the chlorpyrifos-induced reductions in bioenergetic response variables (energy availability and net energy budget) were stronger when the high mean temperature was combined with the high DTF. Moreover, I also showed that the bioenergetic responses contributed to the higher chlorpyrifos toxicity under global warming as treatment combinations with lower net energy budgets showed higher mortality and lower growth rates. Although I did not always find evidence that possible gradual thermal evolution in high-latitude damselflies would buffer for the increased chlorpyrifos toxicity under global warming, I did find a strong signal in chapter IV. Latitude-specific thermal adaptation to both mean temperature and DTF buffered for the chlorpyrifos-induced reduction in heat tolerance, meaning that possible gradual thermal evolution in high-latitude populations may buffer for the negative effects of chlorpyrifos on heat tolerance under warming, unless the DTF increase is taken into account. My results indicate that it is crucial to not only consider DTFs, but also their interaction with increasing mean temperatures, and to integrate gradual thermal evolution to make more realistic predictions of pesticide toxicity in the current climate and in a warming world.status: publishe

Temperature variation makes an ectotherm more sensitive to global warming unless thermal evolution occurs

To assess long-term impacts of global warming on species, there is growing interest in latitudinal intraspecific patterns in thermal adaptation. Yet, while both mean temperatures and daily temperature fluctuations (DTFs) are expected to increase under global warming, latitudinal differences in the effects of DTFs have not been documented. We tested whether low-latitude populations of an ectotherm deal better with greater DTF than high-latitude populations, especially at a high mean temperature close to the optimal temperature for growth where DTF causes exposure to extreme high temperatures. We evaluated the impact of DTFs when assessing the effect of gradual thermal evolution at the high latitude with a space-for-time substitution. We compared effects of both mean temperatures (20 and 24°C) and DTFs (constant = 0°C, low = 5°C and high = 10°C) on growth rates between low-latitude and high-latitude populations of the damselfly Ischnura elegans in a common-garden experiment. DTFs, if anything, reduced growth and were generally stressful as indicated by reductions in body condition, antioxidant defence and metabolic rate, and increases in oxidative damage. Most negative effects of DTFs were only present at a mean of 24°C when too high temperatures were reached during a daily cycle. Notably, while 4°C warming was beneficial in terms of growth rate at both latitudes at a constant temperature regime, this changed in a negative effect at high DTF. Moreover, this modulating effect of the mean temperature by DTF differed between latitudes indicating local thermal adaptation. While 4°C warming at low DTF still caused faster growth in low-latitude larvae, it already slowed growth in high-latitude larvae. This supports the emerging insight that warming would increase growth in high-latitude larvae in the absence of DTF, yet would decrease growth in the more realistic scenarios with DTF. In contrast, a space-for-time substitution approach suggested that under gradual thermal evolution, the evolved high-latitude larvae would no longer suffer a growth reduction in the presence of DTF. Our study provided important proof-of-principle that jointly integrating gradual thermal evolution and the expected increase in DTF generates opposing predictions of effects of global warming on this ectotherm.status: publishe

Data used for the analyses; Verheyen and Stoks

DTF = Daily Temperature Fluctuation; PO = phenoloxidase; MDA = malondialdehyde; CAT = catalase; ETS = electron transport system; SOD = superoxide dismutas

Negative bioenergetic responses to pesticides in damselfly larvae are more likely when it is hotter and when temperatures fluctuate

To make more realistic predictions about the current and future effects of pesticides, we need to better understand physiological mechanisms associated with the widespread higher toxicity of many pesticides under increasing mean temperatures and daily temperature fluctuations (DTFs). One overlooked, yet insightful, mechanism are bioenergetic responses as these provide information about the balance between energy gains and costs. Therefore, we studied how the bioenergetic responses to the insecticide chlorpyrifos were affected by a higher mean temperature and a higher DTF in Ischnura elegans damselfly larvae. To quantify bioenergetic responses we measured energy availability (Ea), energy consumption (Ec) and total net energy budget (cellular energy allocation, CEA). Exposure to chlorpyrifos considerably reduced CEA values when a high mean temperature was combined with a high DTF (up to -18%). Notably, chlorpyrifos had little effect on CEA at a constant 20 °C, meaning that the bioenergetic impact of chlorpyrifos would have been underestimated if we had only tested under standard testing conditions. The chlorpyrifos-induced reductions in CEA under warming were driven by reductions in Ea (up to -16%, mainly through large reductions in sugar and fat contents) while Ec was unaffected by chlorpyrifos. Treatment groups with a lower CEA value showed a higher mortality and a lower growth rate, indicating bioenergetic responses are contributing to the higher toxicity of chlorpyrifos under warming. Our study highlights the importance of evaluating the effects of pesticides under an increase in both mean temperature and DTF to improve the ecological risk assessment of pesticides under global warming.status: Published onlin

Shrinking Body Size and Physiology Contribute to Geographic Variation and the Higher Toxicity of Pesticides in a Warming World

To improve current and future risk assessment of pesticides under global warming, mechanistic insights and consideration of daily temperature fluctuations (DTFs) are needed. One overlooked mechanism how both higher mean temperatures and DTFs may increase toxicity is by reducing body size (temperature-size-rule). We studied whether a higher mean temperature and DTF magnified chlorpyrifos toxicity in Ischnura elegans damselfly larvae, and whether this was mediated by temperature-induced reductions in body size and/or physiological changes. The lethal effects of chlorpyrifos were magnified at the high mean temperature (up to ∼15%) and under DTF (up to ∼33%), and especially at their combination (up to ∼46%) indicating synergisms. This highlights that not only considering DTFs, but also their interaction with higher mean temperatures is pivotal for realistic predictions of pesticide toxicity. Both higher temperatures and DTFs resulted in smaller larvae, which were more sensitive to chlorpyrifos. Notably, the DTF-induced smaller body sizes, as well as the higher oxidative damage to lipids, contributed to the higher chlorpyrifos toxicity under DTF. By integrating the temperature-size rule and size-pesticide sensitivity pattern we provide proof-of-principle for a novel, likely general mechanism contributing to geographic variation and the higher toxicity of pesticides in a warming world.status: publishe

Current and future daily temperature fluctuations make a pesticide more toxic: Contrasting effects on life history and physiology

There is increasing concern that climate change may make organisms more sensitive to chemical pollution. Many pesticides are indeed more toxic at higher mean temperatures. Yet, we know next to nothing about the effect of another key component of climate change, the increase of daily temperature fluctuations (DTFs), on pesticide toxicity. Therefore, we tested the effect of the pesticide chlorpyrifos under different levels of DTF (constant = 0 °C, low = 5 °C (current maximum level) and high = 10 °C (predicted maximum level under global warming)) around the same mean temperature on key life history and physiological traits of Ischnura elegans damselfly larvae in a common-garden experiment. At all levels of DTF, chlorpyrifos exposure was stressful: it reduced energy storage (fat content) and the activity of its target enzyme acetylcholinesterase, while it increased the activity of the detoxification enzyme cytochrome P450 monooxygenase. Notably, chlorpyrifos did not cause mortality or reduced growth rate at the constant temperature (0 °C DTF), yet increased mortality 6x and reduced growth rate with ca. 115% in the presence of DTF. This indicates that daily short-term exposures to higher temperatures can increase pesticide toxicity. Our data suggest that when 5 °C DTF will become more common in the studied high-latitude populations, this will increase the toxicity of CPF, and that a further increase from 5° DTF to 10 °C DTF may not result in a further increase of pesticide toxicity. Our results highlight the biological importance of including daily temperature fluctuations in ecological risk assessment of pesticides and as an extra dimension in the climate-induced toxicant sensitivity concept.status: publishe

Increased Daily Temperature Fluctuations Overrule the Ability of Gradual Thermal Evolution to Offset the Increased Pesticide Toxicity under Global Warming

The widespread evidence that global warming can increase species sensitivities to chemical toxicants, and vice versa, and the recent insight that thermal evolution may mitigate these effects is crucial to predict the future impact of toxicants in a warming world. Nevertheless, a major component of global warming, the predicted increase in daily temperature fluctuations (DTFs), has been ignored at the interface of evolutionary ecotoxicology and global change biology. We studied whether 4 °C warming and a 5 °C DTF increase (to 10 °C DTF) magnified the negative impact of the insecticide chlorpyrifos (CPF) in larvae of low- and high-latitude populations of the damselfly Ischnura elegans. While 4 °C warming only increased CPF-induced mortality in high-latitude larvae, the high (10 °C) DTF increased CPF-induced larval mortality at both latitudes. CPF reduced the heat tolerance; however, this was buffered by latitude-specific thermal adaptation to both mean temperature and DTF. Integrating our results in a space-for-time substitution indicated that gradual thermal evolution in high-latitude larvae may offset the negative effects of CPF on heat tolerance under warming, unless the expected DTF increase is taken into account. Our results highlight the crucial importance of jointly integrating DTFs and thermal evolution to improve risk assessment of toxicants under global warming.status: publishe