Insects and plants in a changing atmosphere
2001; Wiley; Volume: 89; Issue: 4 Linguagem: Inglês
10.1046/j.0022-0477.2001.00582.x
ISSN1365-2745
Autores Tópico(s)Botany and Plant Ecology Studies
Resumo'Clean the air! Clean the sky! Wash the wind!' T.S. Eliot, Murder in the Cathedral, 1935 This is the winter meeting for the year 2000, a year in which we all spent some time looking to the past as well as considering the opportunities and challenges of the future. For this Society, founded in 1913, the 20th century was the formative time. It progressed from its origins in the Vegetation Committee, through the initial rather separate growth of animal ecology and the launching of the Journal of Animal Ecology in 1932, to the present interdisciplinary view of our subject in which terms like molecular ecology are common place. Having had the privilege of being a member of the society for almost half of that period I found myself reflecting on the circumstances which encouraged me to go along my particular ecological track. Like most biologists of my generation I followed separate A-levels in botany and zoology with a specialist university training in one of these, in my case zoology. However, this was soon to change. The new universities of the 1960s were establishing departments of biological sciences and the oldest had begun to teach ecology in year one as an integrated subject (Varley et al. 1967). I was much influenced by these developments and, I suppose, also by my background from the then heavily polluted industrial East Lancashire uplands. I count myself lucky that the ecology I was taught was wide-ranging and had an interdisciplinary element, whilst the organisms I chose to work on (phytophagous insects) were better botanists than I was! I was fortunate to have the opportunity to participate in J.B. Cragg's programme of upland ecology at the Moor House National Nature Reserve (Cragg 1961) and later had the similar good fortune to work at Wytham Woods. The former is an area much admired and written about by W.H. Pearsall (1950) and regularly visited by Charles Elton, and the latter, of course, was made famous by the likes of Charles Elton, David Lack, Mick Southern, George Varley, Mike Hassell and Ian Newton. So past presidents of this society have had much to do with both of these sites. Moor House has been a site of two major ecological studies: the UK tundra component of the International Biological Programme, and with Wytham, the NERC-sponsored TIGER programme of climate change. Thus it is that I have chosen to discuss some interacting lines of enquiry which have occupied myself and my research group for some years: long-term population dynamics of some herbivorous insects; and consequences to these dynamics of atmospheric pollution, elevated carbon dioxide and raised temperatures. The field sites used include the Moor House NNR and Wytham Woods. The common thread is insect herbivory. The common objective was to try to understand what effects changes in these environmental parameters might have on insect populations. In discussing this I expect to touch on a number of issues which pervade the practice of ecology as a science. The most significant of these, to which I shall return, are: the importance of long-term data sets in the field; the role of experiments (in field and laboratory) in ecology and the dilemma between realism and accuracy; and the need to try to understand mechanisms at a number of levels and scales. Concentrations of gases which occur naturally in the earth's atmosphere have varied considerably over palaeo time-scales (Houghton et al. 1990). They were, however, relatively constant in ecological time up to the industrial revolution and period of rapid increase of human population. Collectively these gases have a 'greenhouse effect' which has been enhanced significantly over the last 150 years. Some of these gases, e.g. SO2 and NOx (discussed in detail by John Lee in his presidential address, Lee 1998) and O3, are known to affect physiological and ecological processes in plants, and might be supposed to have important consequences to herbivorous insects. It was suggested more than 150 years ago and observed in the field in the 1970s that plants downwind of pollution sources and close to busy roads appeared to support increased densities of some insect herbivores (including sawflies, moths and aphids) (e.g. Flückiger et al. 1978; Port & Thompson 1980). By 1981, it was demonstrated experimentally that bean plants grown in high levels of SO2 supported faster development and greater growth and survival of Mexican bean beetles (Hughes et al. 1981). These findings stimulated a decade of studies of this phenomenon, reviewed in Riemer & Whittaker (1989), Whittaker & Warrington (1990) and Brown (1995). Although emissions of sulphur dioxide in the UK are declining, it is still a major source of pollution in other countries, particularly the developing world (Rao et al. 1990), whilst vehicle exhaust pollutants are still an issue throughout the world. At Lancaster I was extremely fortunate in having as colleagues Terry Mansfield and Alan Wellburn, who had been leaders in studies of effects of atmospheric pollutants on plants since the mid-1970s. A series of chambers from 2 m3 to hemispherical solardomes 4.4 m in diameter was available with extremely accurately controlled fumigation facilities. With these we were able to investigate interactions between atmospheric pollutants, elevated carbon dioxide and insect herbivore populations, with the following main conclusions. Response of a phloem-feeding aphid on pea plants to SO2 (Fig. 1) was found to be dose-dependent (Warrington 1987). Although increase in mean relative growth rate (MRGR) compared with clean air controls peaked at about 100 p.p.b. SO2, there was a detectable response at concentrations as low as 20 p.p.b. It is worth noting that at this concentration there is no visible damage to the plant, but as White (1984) points out, available nitrogen may already be increasing as a consequence of damage to the plant by these relatively low concentrations of SO2. Higher levels of damage lead to a plant eventually becoming unsuitable for herbivores. In our experiments this was beginning to occur above c. 300 p.p.b. when there was a negative effect on aphid growth. Relationship between sulphur dioxide concentration (p.p.b.) and percentage change in mean relative growth rate (MRGR) relative to controls of pea aphids feeding on pea plants. Reprinted with permission from Warrington (1987), copyright 1987 Elsevier Science. The 12% increase in MRGR seen at 120 p.p.b. of SO2 translated into a 19% increase in the rate of production of nymphs, a 4.6% increase in rm and population 1.8 times as great as on pea plants grown in clean air (Warrington et al. 1987). Complementary studies by S McNeill and colleagues at Silwood Park had shown that quite brief episodes of SO2 (4 h exposure) had as great an effect as long exposures (Fig. 2) (McNeill & Whittaker 1990). MRGR (with 95% confidence intervals) measured over 3 days of Elatobiumabietinum nymphs on Sitka spruce pre-fumigated with 100 p.p.b. SO2 at a range of fumigation times (h). Reprinted with permission from McNeill & Whittaker (1990), copyright Intercept, Andover. These studies inform us about the mechanisms involved, but the population consequences which follow are more difficult to determine. For example, an important discovery of the Mansfield group was that responses of plants to mixtures of pollutants was not simply additive. NO2, for example, can interact with SO2 to cause more damage to plants than the sum of the independent effects of the two gases (Mansfield & Freer-Smith 1981). This is reflected in the build-up of aphid populations (Elatobium abietinum (Walker)) on Sitka spruce saplings when exposed to SO2, NO2 or NO2 + SO2 over a 24-week period (Fig. 3) (Whittaker 1994a). Population growth rate is seen to be much greater in the gas mixture than in separate SO2 or NO2, neither of which differed from the control at the concentrations used. Numbers of Elatobium abietinum over 24 weeks on Picea sitchensis saplings exposed to SO2 (▾), NO2 (□) or NO2 + SO2 (▪) and on unfumigated controls (●). Reprinted with kind permission from Whittaker (1994a), copyright Kluwer Academic Publishers. In healthy plants, uptake of these gaseous pollutants is mainly by diffusion through stomata (Kersteins 1996; Mansfield, in press). However, the damage caused to the plant by insect feeding can itself significantly affect this uptake. Leaf hoppers (Homoptera: Typhlocybinae), such as Ossiannilssonolacallosa (Then) on sycamore, feed by penetrating the abaxial leaf surface with their stylet mouth-parts and removing the contents of mesophyll cells below the adaxial epidermis, leaving a stippled appearance of dead cells. Apart from the fact that even moderate damage reduces net photosynthesis by 20% (Whittaker 1984; Warrington et al. 1989), the plant does not immediately seal off the puncture holes and in an average infestation these occur with similar frequency and of similar size to stomata (Whittaker 1984). It is no surprise therefore to find that uptake of SO2 at night by a plant damaged in this way is increased (Warrington et al. 1989) (Fig. 4) as the feeding damage increases. If this also happens in the case of NOx, we may ask (Wellburn 1990) whether any increased N uptake in this way is useful as a nutrient or results in further damage (or both!). This question is further discussed by Mansfield, in press and includes the role of NO (one of the main constituents of car exhaust emissions) as a signalling agent in plants and animals. There is evidence that NO is a mediator of defence gene expression in plants (Durner et al. 1998) and so the picture with regard to insect herbivory may be even more complex than I have outlined. Relationship between rate of uptake of SO2 and the area of damage (stippling) on control and leafhopper-damaged seedlings of sycamore immediately after removal of the leafhoppers. R = 0.689, P < 0.05. Reprinted with permission from Warrington et al. (1989), copyright Springer-Verlag. In any case, the message is that since the majority of plants in the field are not in the healthy undamaged state of laboratory specimens, studies of eco-physiological responses of plants need to take account of insect and other damage when extrapolated to the field. Our evidence (Whittaker et al. 1989) and that of other investigators (e.g. Jones et al. 1994) shows that responses of insects to increases of ozone concentrations are difficult to predict. They can be positive or negative in the same species, depending on experimental conditions, or very often show no or non-significant responses (summarized by Heliõvaara & Väisänen 1993). In recent years attention has turned from atmospheric pollutants such as SO2, NOx and O3 to the consequences of elevated CO2 concentrations in the atmosphere. Although the key physiological responses of plants to increased CO2 are transpiration and photosynthesis (Long 1999), the proximal factor affecting insect herbivores is again probably nitrogen through changes in C : N ratios and in the concentrations of amino acids. Whereas pollution events are usually of an episodic nature and are therefore relatively easy to simulate in controlled conditions, the modern increase in CO2 concentration in the atmosphere has been gradual and is expected to continue rising. Realistic experiments on its effects are therefore difficult to do and interpret because they usually involve an artificial and abrupt increase from current levels (about 355 p.p.m.) to those expected by the mid-21st century (c. 650 p.p.m.). Furthermore the majority of experiments involving insects have utilized feeding and growth measures on insects presented with detached leaves in small containers or on very young plants exposed for relatively brief periods to CO2 and insect feeding. Nevertheless some patterns are emerging (reviewed by Watt et al. 1995; Bezemer & Jones 1998; Whittaker 1999). It is common to observe increased consumption by chewing insects to compensate for reduced nitrogen supply, and a tendency for growth rates, fecundity and survival to be depressed in insects feeding on plants grown in elevated CO2 (Watt et al. 1995). However, responses are variable and few studies have shown significant changes at population level of insect herbivores (Whittaker 1999). The only feeding guild in which some species have shown increases in population density are the phloem feeders; of 10 aphid species studied, two have shown a significant increase, two a non-significant increase, two faster reproduction or development times, and four show population decrease or no change (Whittaker 1999). Some of these points, and especially the different ways in which insects in different feeding guilds respond to the same perturbation, are illustrated by our studies of a leaf-mining fly, a leaf-chewing beetle and an aphid feeding on Rumex, and xylem-feeding spittlebugs on Rumex and Juncus squarrosus L. (Fig. 5). Damage to Rumex obtusifolius by a range of insects. Reprinted with permission from Salt & Whittaker (1998), copyright Michael J. Roberts. An important feature of these experiments is that they were carried out in as near natural conditions as possible over at least one (sometimes up to three) complete generations of the insects on intact plants. The physiological responses of the plant to elevated CO2 were already known (Pearson & Brooks 1995 for Rumex and Wolfenden & Diggle 1995 for Juncus squarrosus). In both cases the community of insects and population dynamics of the insects associated with the plants are also well known and in the case of Rumex, responses of the plant to insect attack are known (e.g. Salt & Whittaker 1998). Rumex obtusifolius L. plants grown in replicated solardomes at CO2 concentrations of 350 or 600 p.p.m., were infected with Aphis rumicis L. (phloem-feeding aphid), Gastrophysa viridula Degeer (leaf chewing chrysomelid beetle), Pegomya nigritarsis Zett. (leaf-mining fly) or Philaenus spumarius (L.) (xylem-feeding spittlebug). Changes in plant chemistry were simultaneously measured. The plants in elevated CO2 showed reduction in leaf nitrogen and total amino acid concentration, increase in C : N ratio, lower concentrations of calcium oxalate and greater leaf areas than in ambient air (see also Pearson & Brooks 1995). The tri-voltine G. viridula was reared for three successive generations on Rumexobtusifolius (Brooks & Whittaker 1998). Despite compensatory feeding there was no effect on larval weight of the beetle in generations one and two, and although in generation three larval weight was significantly reduced, weight of emerging adults was unaffected in any generation. Nevertheless generation two females laid 30% fewer and smaller eggs in elevated CO2 (in line with reduction in food quality) and thus larvae in generation three were consistently smaller. The increased C : N ratio of leaf tissue and greater leaf areas in elevated CO2 had no effect on development time from egg to pupation nor on numbers reaching pupation of the leaf-mining fly P. nigritarsis. There was, however, compensatory feeding resulting in 20% larger mined areas of the leaves (Salt et al. 1995). This example illustrates just how complicated the consequences of this to the plant can become. In the absence of Pegomya, elevated CO2 led to an increase in total leaf area. Also these leaves showed an average increase of 7% in photosynthetic rate per unit area (Whittaker 1994b). However, in elevated CO2, if Pegomya was present, there was 20% more damage by the insect than in ambient CO2, and a reduction of 80% in photosynthesis of mined leaves compared with healthy leaves. Also, increased leaf surface temperature in elevated CO2 could lead to faster growth of the Pegomya larvae and there is a likelihood of larger clutch sizes of Pegomya on larger leaves (Godfray 1986), both leading to greater damage. These interacting effects are summarized in Fig. 6. Some features of damage of Rumex obtusifolius by Pegomya nigritarsis in elevated carbon dioxide. Over a 3-week period when Aphis rumicis was allowed to feed on the plants, we found that six out of 16 amino acids were at significantly lower concentrations (Fig. 7) in elevated CO2 plants, and the aphids achieved three times greater abundance on these plants than on the controls (Fig. 8) (Whittaker 1999). Concentrations of total amino acids in Rumex obtusifolius leaves grown in elevated CO2 (▪) and ambient CO2 (□) (Brooks & Whittaker, unpublished). Mean number ± 1 SE of aphids (Aphis rumicis) on Rumex obtusifolius plants in ambient (○) and elevated (●) CO2. Reprinted with permission from Whittaker (1999). Although the meadow spittlebug, Philaenus spumarius, a xylem feeder, was not significantly affected by elevated CO2 on Rumex, its survival during the nymphal stages was lower than on control plants. The same effect was seen in each of three generations (up to 20% significantly lower survival) of its close relative Neophilaenuslineatus L. feeding on Juncus squarrosus (Fig. 9) (Brooks & Whittaker 1999). Development rates were not affected in either species. These responses were coincident with increased C : N ratio and reduced transpiration rates in the food plant. Survival (%) of Neophilaenus lineatus on Juncus squarrosus in ambient (□) and elevated (▪) CO2. Vertical bars 1 SE. Reprinted with permission from Brooks & Whittaker (1998), copyright Blackwell Science Ltd. This last experiment on N. lineatus feeding on Juncus squarrosus was conducted in solardomes on large (450 × 450 × 250 mm) vegetation monoliths brought into the chambers from 550 m a.s.l. at the Moor House National Nature Reserve, and cooled to temperatures comparable with those found at that location in the field. It therefore offers a comparison of the response of N. lineatus to elevated CO2 with its response to associated temperature changes which I shall discuss later. A criticism of all such experiments in which plants and animals are exposed to modified environmental conditions in closed chambers is that although they may lead to considerable advances in understanding underlying mechanisms of responses, they do not necessarily permit predictions of real ecological changes in the open field, where expected responses may be buffered by additional species interactions and other factors. Leaving aside the point reiterated by Sam Berry that 'prediction is not an essential part of science' (Berry 1989), a major objective of the TIGER programme on Climate Change was to try to develop generalizations and models with predictive value. Use of open-top chambers, accessible to more natural weather variation and to natural enemies, is one way forward. Our approach was to repeat some of the observations made in closed chambers, in FACE (free air CO2 enrichment) facilities at Zurich (Norton et al. 1999) and similar open fumigation sites at Liphook, Hampshire (courtesy of the then Central Electricity Generating Board), where S. McNeill and colleagues have also conducted open access experiments. As responses of insect herbivores to many atmospheric changes (at least as normally experienced in the field) are not direct, but mediated by changes in the host plant, it is unlikely that predators and parasitoids would respond in the same way. Aminu-Kano et al. (1991) used an open air system in which winter cereals were exposed to increased SO2 without excluding other environmental variables. They could not detect any effect of the fumigation on specialist predators of the aphids colonizing the cereals (which were increasing in SO2 fumigated plots) nor on four other predatory groups, though a further two predator species decreased in high SO2. Rates of parasitism of the aphids were inversely proportional to SO2 concentration, presumably because the aphids in high SO2 treatments were escaping from control by these natural enemies. An unexpected, but interesting finding of our closed chamber experiments was that whereas shoot-feeding aphids (Elatobium) on Sitka spruce trees responded to elevated SO2 by increasing growth rate and reproduction (see above, Warrington & Whittaker 1990), root-feeding Pachypappa species on the same trees decreased in numbers (Salt & Whittaker 1995). The reason for this is not yet understood but might be linked to flux-rates of phloem in roots compared with leaves of spruce. However, it was reassuring to find the same response of Pachypappa on Sitka spruce roots in the Liphook open air fumigations (Salt & Whittaker 1995) (Fig. 10). In a similar comparison, we were able to show that leaf-mining Pegomya flies on Rumexobtusifolius responded in generally the same way to elevated CO2 in a FACE facility at Zurich as we had found in closed chambers at Lancaster (Whittaker 1999 and, see above, Salt & Whittaker 1995). Mean numbers of root aphids (Pachypappa spp.) on Norway spruce during and after fumigation treatments. □, 1990 (during fumigation) ▪, 1991 (after fumigation). Bars are 1 SE. Reprinted with permission from Salt & Whittaker (1995), copyright Blackwell Science Ltd. Perhaps the ultimate in open air experimentation with CO2 are the studies around natural vents such as those in north Italy (Tognetti et al. 2000). I am not aware of any studies of insect herbivores around these vents, but Tognetti et al. have found that trees growing for decades under elevated CO2 concentrations (and originating from parent trees growing in high CO2) showed few significant differences in radial growth from trees growing at a nearby control site. The authors surmise that other resource limitations may have overridden any positive effect of CO2 on productivity or the trees may have acclimated to the high CO2. Either conclusion raises doubts as to whether one would expect much response of insect herbivores to the long-term high CO2 conditions. Oxides of nitrogen, ozone and sulphur dioxide are all components of what are commonly known as 'greenhouse gases'. Global warming is likely to be an indirect effect of their enhancement in the atmosphere (largely by human activities). Indeed there is already evidence that these changes have begun. Current models vary in their predictions of the magnitude of climate change (Weaver & Zeiss 2000) but a minimum effect is an expectation of 1 °C rise in mean temperature. An additional effect was reported in our studies of elevated CO2. Leaf surface temperatures in the Rumex (and possibly Juncus squarrosus) experiments mentioned above were raised by about 1 °C as a consequence of reduced transpiration. What will be the effects of raised temperatures on populations of plant-feeding insects in the field? This is not a straightforward question to answer. Attempts to use 'climate envelopes' in which climate change models are mapped on to ecophysiological models have been criticized (Davis et al. 1998) on the grounds that species interactions such as competition, predators and parasitoids and dispersal, which will themselves be temperature-dependent, will obscure any simplistic relationship between an organism's range and abundance and climate per se. Hodkinson (1999) has responded by arguing that there are many studies in the literature of populations whose dynamics are largely determined by climatic factors. This debate is reminiscent of the long-standing one about the relative roles of density-dependent and density-independent factors in determining distribution and abundance (see Cherrett 1989). There are close parallels here with the study by Watkinson (1985) of the abundance of plants along an environmental gradient where the balance between effects of density-dependent and density-independent processes was shown to change along a comparatively short dune system gradient. Our approach was three-fold: To detect the relative roles, e.g. climate and natural enemies, in different parts of the range of a single species (the lined spittlebug, Neophilaenus lineatus) (Whittaker 1971; Whittaker & Tribe 1998) at two TIGER flagship sites. To relate population parameters to climate along altitudinal transects which represented a 1 °C change over each 300 m of altitude. To experimentally manipulate temperature in the field by the use of cloches (courtesy of Professor Jeff Bale) and soil warming cables (courtesy of Professor Valerie Brown and Professor Phil Grime). Our principal field site was the Moor House National Nature Reserve in the Pennine hills of northern England (Cragg 1961), which was the site of the classic studies of climate by Manley in the 1930s and described by Marren (1994) as 'probably the harshest climate in England'. This was the largest of the first seven National Nature Reserves to be declared in 1952 and, following the IBP programme, thought by Charles Elton some 20 years later (personal correspondence, 13 November 1978) to be 'probably the best description of any British ecosystem' at this time. Comparative studies of N. lineatus and another spittlebug, Philaenus spumarius, were made on montane transects in Wales and at Ben Lomond Scotland, and at a lowland, south of England site at Wytham Woods, Berkshire. After some 10 years of study it was concluded that whereas population change at 500 m at Moor House was largely determined by climate, at Wytham Woods density-dependent factors, in particular the action of a parasitoid fly, Verrallia aucta Fallén, which is absent at Moor House, were responsible (Whittaker 1971, 1973). It followed that although it might be possible to predict changes in numbers in relation to climate at Moor House (the Hodkinson case), the buffering effect of species interactions at Wytham would make this difficult or impossible (the Davis et al. case) in the same species. Nor should we ignore the potential consequences of catastrophic events of which most climate models predict an increase. The dock beetle, Gastrophysa viridula, for example, was locally eliminated on river bank shingles as a consequence of flooding at critical times in the life cycle (Ellistone et al. 1979). Figure 11 shows how as mean temperature declines with altitude, hatching dates and subsequent emergence of adult female N. lineatus get later in the season, eventually to the point where females are no longer able to contribute eggs to the next generation (Fielding et al. 1999). A simple model of these relationships (open symbols Fig. 12), driven by climate parameters, explained 70% of the variation in numbers at Moor House over a 35-year period (solid symbols Fig. 12) and predicted that a 1 °C rise in mean temperature would increase population density by 50%. In line with this prediction, experimental raising of the temperature within cloches (Miles et al. 1997) resulted in more than doubling of N. lineatus, the overshoot compared with the model probably caused by the cloches inadvertently varying other environmental parameters such as wind speed, dispersal of the insects and changes in the vegetation. Date of appearance of adult Neophilaenus lineatus at different altitudes on Little Dun Fell (Moor House NNR) in 1993, 1995, 1996. R2 = 0.73, P < 0.001. Reprinted with permission from Fielding et al. (1999), copyright Blackwell Science Ltd. Population density of N. lineatus at Moor House from 1961 to 1997. ● measured numbers m−2, □ calculated numbers m−2. Reprinted with permission from Whittaker & Tribe (1998), copyright Blackwell Science Ltd. Distribution of this species is also affected by temperature. On the Ben Lomond transect, there is a strong relationship between maximum altitude reached by N. lineatus and spring temperatures (Fig. 13). A rise of 1 °C on this transect would lead to an estimated expansion of the upper limit of the range of N. lineatus by 300 m a.s.l. (Whittaker & Tribe 1996). Relationship between the difference from the 25 years mean average temperature from March to June in 1986–95, and the maximum altitude of N. lineatus larvae on Ben Lomond on or about 1 July. Reprinted with permission from Whittaker & Tribe (1996). By contrast, winter warming of quadrats on the Wytham experimental site, where there are strong density-dependent processes operating on spittlebugs, had little or no effect on their population density, though there was an effect on hatching and adult emergence comparable with that seen on the Moor House transect (Masters et al. 1998). Additionally it was recorded that the population of spittlebugs at Wytham fluctuated less violently than did that at Moor House (Whittaker 1971), a phenomenon subsequently also observed in butterfly populations towards the northern edges of their ranges (Thomas et al. 1994). I shall turn now to considering the usefulness of experiments such as these in providing understanding of events in the field. Most ecologists have experience of making observations or designing and conducting experiments at a variety of scales in the field as well as in the laboratory or greenhouse. The more reductionist the approach, the easier it is to detect and study 'mechanisms' and to have confidence in the results, but the greater the difficulty in understanding whether the mechanism studied has any real significance in explaining observations made in the field. As Brandon put it 'there is a fundamental tension between the amount of control the investigator has over an experiment and how informative the results of such an experiment are with regard to natural populations' (Brandon 1996). Schindler (1998) highlights the same concern when he says that small-scale experiments often give high replicability but spurious results. The investigator is torn between accuracy and realism (Körner 2001). Very often the former wins because small-scale experiments are easier to design and carry out, variability can be reduced, the results are easier to analyse and interpret, and the conclusions can be stated with more apparent conviction. Moreover, the whole thing can be done more quickly and published more readily and cited more easily as a 'punch line', and not least, fit funding convention
Referência(s)