Kjell Sjöberg

Gunnar Gunnarsson
email: gunnar.gunnarsson@hkr.se

Hannu Pöysä

Johan Elmberg

Petri Nummi

C Gortázar

Arnold Chouinard

Born to cope with climate change? Experimentally manipulated hatching time does not affect duckling survival in the mallard Anas platyrhynchos

Keywords: Breeding biology, Duckling mortality, Mismatch hypothesis, Timing of breeding, Waterfowl

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Born to cope with climate change? Experimentally manipulated hatching time does not affect duckling survival in the mallard

Introduction

The timing of reproduction is a major fitness-affecting trait in species living in seasonal environments [START_REF] Th | Reproductive success[END_REF]. Considering birds, two general patterns prevail. First, in many species, females that lay later in the breeding season tend to have smaller clutches (e.g. [START_REF] Crick | Seasonal changes in clutch size in British birds[END_REF][START_REF] Hamann | Intra-seasonal decline of clutch size in lesser snow geese[END_REF][START_REF] Klomp | The determination of clutch-size in birds[END_REF][START_REF] Rohwer | The evolution of reproductive patterns in waterfowl[END_REF] and references therein), setting an upper limit to the number of offspring that single-brooded species can produce within a season. Second, young that hatch later in the season usually have lower survival (e.g. [START_REF] Norris | Seasonal variation in the reproductive success of blue tits: an experimental study[END_REF][START_REF] Rohwer | The evolution of reproductive patterns in waterfowl[END_REF]Verhulst et al. 1995 and references therein). Two general hypotheses have been proposed to explain the seasonal decline in reproductive performance. According to 'the timing hypothesis', seasonal decline in offspring survival is due to deterioration of environmental conditions, e.g. food abundance [START_REF] Verhulst | Experimental evidence for a causal relationship between timing and success of reproduction in the great tit Parus m. major[END_REF][START_REF] Verhulst | Seasonal decline in reproductive success of the great tit: variation in time or quality?[END_REF]. Alternatively, lower survival of late-hatched offspring may be due to lower parental quality of late breeders ('the quality hypothesis'; e.g. [START_REF] Price | Directional selection and the evolution of breeding date in birds[END_REF][START_REF] Verhulst | Seasonal decline in reproductive success of the great tit: variation in time or quality?[END_REF]. The validity of these hypotheses is difficult to assess because smaller late-season clutches may be laid by lower quality females. Experimental manipulations of hatching date have found support for both hypotheses, also within the same species (reviews in [START_REF] Arnold | Seasonal declines in reproductive success of the common tern Sterna hirundo: timing or parental quality?[END_REF][START_REF] Nilsson | Fitness consequences of timing of reproduction[END_REF][START_REF] Verhulst | The timing of birds' breeding season: a review of experiments that manipulated timing of breeding[END_REF].

Even though earlier experimental studies have found evidence for both environment-driven and parent-qualitydependent seasonal declines in offspring survival, those manipulations themselves have introduced confounding factors that may bias results (discussed in [START_REF] Arnold | Seasonal declines in reproductive success of the common tern Sterna hirundo: timing or parental quality?[END_REF][START_REF] Brinkhof | Timing of reproduction and fledging success in the coot Fulica atra: evidence for a causal relationship[END_REF][START_REF] Hansson | The quality and timing hypotheses evaluated using data on great reed warblers[END_REF][START_REF] Monaghan | Why don't birds lay more eggs?[END_REF]. First, experimental delay of hatching is often accomplished by removing the initial clutch to induce a second, thus invoking a re-laying cost in females [START_REF] Brinkhof | Timing of reproduction and fledging success in the coot Fulica atra: evidence for a causal relationship[END_REF][START_REF] Heaney | A within-clutch trade-off between egg production and rearing in birds[END_REF]. Second, manipulation of hatching date usually has involved shortening or lengthening of the incubation period, in which case also the cost of incubation has been manipulated [START_REF] Brinkhof | Timing of reproduction and fledging success in the coot Fulica atra: evidence for a causal relationship[END_REF]. Third, egg or chick swaps between nests, a manipulation that does not involve a relaying cost, may be confounded and biased by differences in egg quality, territory quality and parental characteristics (e.g. [START_REF] Arnold | Seasonal declines in reproductive success of the common tern Sterna hirundo: timing or parental quality?[END_REF][START_REF] Brinkhof | Timing of reproduction and fledging success in the coot Fulica atra: evidence for a causal relationship[END_REF]). Finally, although not explicitly discussed in earlier papers, differences in clutch or brood size may introduce additional noise because they usually show a seasonal decline (see above), and offspring mortality may depend on the number of offspring (e.g. [START_REF] Lessells | The evolution of life histories[END_REF][START_REF] Lindén | Cost of reproduction and covariation of lifehistory traits in birds[END_REF]; for precocial species, see [START_REF] Milonoff | Brood-size-dependent offspring mortality in common goldeneyes reconsidered: fact or artifact?[END_REF][START_REF] Paasivaara | Survival of common goldeneye Bucephala clangula ducklings in relation to weather, timing of breeding, brood size, and female condition[END_REF][START_REF] Rohwer | The evolution of reproductive patterns in waterfowl[END_REF]).

In the present study, we address the potentially confounding factors mentioned above (either as a part of the experimental design or in subsequent analyses; see "Materials and methods"). We thus provide a more clearcut test of the timing hypothesis by asking whether survival of mallard (Anas platyrhynchos) ducklings depends on the time of hatching. The mallard is an excellent model species in this context. It has uniparental (female) care and ducklings are precocial. Previous experimental studies involving pairs, hens and broods show that it is suited for manipulation, in captivity as well as under field conditions (e.g. [START_REF] Cunningham | Egg investment is influenced by male attractiveness in the mallard[END_REF]Elmberg et al. 2005a;[START_REF] Gunnarsson | Experimental evidence for density-dependent survival in mallard (Anas platyrhynchos) ducklings[END_REF][START_REF] Pöysä | Habitat selection rules in breeding mallards (Anas platyrhynchos): a test of two competing hypotheses[END_REF][START_REF] Rhymer | The effect of egg size variability on thermoregulation of mallard (Anas platyrhynchos) offspring and its implications for survival[END_REF][START_REF] Sjöberg | Response of mallard ducklings to variation in habitat quality-an experiment of food limitation[END_REF]. In addition, the relationship between hatching date and survival in mallard ducklings has been addressed in numerous descriptive studies, and the results are contradicting (see Table 1). The mallard is hence wellstudied when it comes to descriptive patterns of seasonal variation in reproductive success, but experimental work addressing this topic in the species is lacking. As recent reviews indicate [START_REF] Arnold | Seasonal declines in reproductive success of the common tern Sterna hirundo: timing or parental quality?[END_REF][START_REF] Nilsson | Fitness consequences of timing of reproduction[END_REF][START_REF] Verhulst | The timing of birds' breeding season: a review of experiments that manipulated timing of breeding[END_REF], experimental work testing the timing hypotheses in birds is strongly biased towards altricial species. We are aware of only one experimental study addressing chick survival in a truly precocial species, namely that by [START_REF] Lepage | Seasonal decline of growth and fledging success in snow geese Anser caerulescens: an effect of date or parental quality?[END_REF] on snow geese (Anser caerulescens).

Although research about the seasonal timing of reproduction and its fitness consequences has been ongoing since [START_REF] Perrins | The timing of birds' breeding season[END_REF] seminal work, the topic has enjoyed a renaissance due to the effects of global climate change on breeding phenology and other fitness-affecting features in birds and other taxa [START_REF] Both | Climatic effects on timing of spring migration and breeding in a long-distance migrant, the pied flycatcher Ficedula hypoleuca[END_REF][START_REF] Brown | Long-term trend toward earlier breeding in an American bird: a response to global warming?[END_REF][START_REF] Schoech | Latitude affects degree of advancement in laying by birds in response to food supplementation: a metaanalysis[END_REF][START_REF] Visser | Warmer springs lead to mistimed reproduction in great tits (Parus major)[END_REF][START_REF] Visser | Variable responses to large-scale climate change in European Parus populations[END_REF][START_REF] Weatherhead | Effects of climate variation on timing of nesting, reproductive success, and offspring sex ratios of redwinged blackbirds[END_REF]. A general pattern is that climate change advances rather than delays breeding phenology in birds [START_REF] Both | Large-scale geographical variation confirms that climate change causes birds to lay earlier[END_REF][START_REF] Dunn | Climate change has affected the breeding date of tree swallows throughout North America[END_REF][START_REF] Møller | Climate change and micro-geographic variation in laying date[END_REF][START_REF] Yalden | Warmer springs advance the breeding phenology of golden plovers Pluvialis apricaria and their prey (Tipulidae)[END_REF][START_REF] Torti | Variable effects of climate change on six species of North American birds[END_REF]. However, changes in laying date may go in either direction (i.e. advance or delay) even within a species depending on the geographical area (see [START_REF] Both | Climate change and timing of avian breeding and migration throughout Europe[END_REF]. It is therefore important to understand how flexible in terms of the width of the breeding time window species are to climate-driven changes in timing. In particular, fitness consequences of climate change are likely to depend on the ability to shift the timing of breeding and resource utilisation to match shifts in resource phenology (the 'mismatch hypothesis'; [START_REF] Drever | Spring temperature, clutch initiation date and duck nest success: a test of the mismatch hypothesis[END_REF][START_REF] Visser | Warmer springs lead to mistimed reproduction in great tits (Parus major)[END_REF]. Accordingly, we will discuss our findings also within the framework of climate change impact.

Materials and methods

Study lakes

This study was done in 2004-2005 in Västerbotten province, northern Sweden (64°N, 20°E). We selected 20 lakes typical for the boreal forest biome, i.e. they were oligo-to mesotrophic and surrounded by mixed coniferous forest and bogs. All lakes freeze over in winter and their breeding waterbirds are long-distance migrants [START_REF] Fransson | Svensk ringmärkningsatlas[END_REF]. Lakes were selected using the following criteria: (1) no regular human disturbance from cabins, fishing and other outdoor activities during the experiment, (2) roundish or elliptical outline and limited size (mean 3.5 ha, range 1.1-8.2), (3) an easily accessible vantage point from which waterbirds could be observed and surveyed without disturbing them and (4) documented or potential breeding sites for wild dabbling ducks (cf. [START_REF] Kauppinen | Densities and habitat distribution of breeding waterfowl in boreal lakes in Finland[END_REF].

Eggs, ducklings and introductions

We used a sequence of procedures to standardise the properties of ducklings and broods in the introduction experiment. In both years, mallard eggs were purchased on one occasion in mid-May from one of Sweden's largest game farms, hosting several hundreds of egg-laying wildstrain mallards. All eggs had been laid on the day of purchase, and they were randomly chosen from the farm's daily production. Hence, all eggs were the same age and had been produced by many different females. In order to standardise duckling quality among subsequent broods and treatments, we randomly picked half of the eggs and put them in a cool storage room upon arrival at the Boda Game Station (62°N, 17°E, some 250 km NNW from the purchase site). These eggs comprised 'the late treatment' (see below).

At the game station, the remaining half of the eggs were randomly assigned to ten clutches of ten eggs each and put in the nest of an incubating mallard, whose own first clutch (ranging from nine to 13 eggs; i.e. the normal span for wild mallards) was taken away at the same time. Each 'foster mother' had been incubating for 5-14 days when the swap occurred, and they had built their own nest in a large fenced outdoor area with natural vegetation and wetlands. These females were locally caught wing-clipped wild-strain birds paired with captive and/or free-flying males. Hence, there was no genetic connection between foster mothers and their subsequent brood. All females accepted their new brood, which hatched 27-28 days later, i.e. the normal incubation time in the wild [START_REF] Cramp | The birds of the western Palearctic[END_REF]. Consequently, females incubated for a longer time than wild females would have done unless re-nesting, but this potential bias was the same in early and late treatments. Moreover, incubating females had access to natural animal and plant food in the wetlands of the pen, but also continuously to commercial fodder. Females therefore had the opportunity to compensate for greater incubation costs, which also was the case if the size of the own (pre-swap) clutch was different from the added clutch, by supplemental feeding. Meanwhile, 'the late treatment eggs' were stored at 10°C (but only for 12 days in order not to reduce viability) and rotated twice daily according to standard game farming procedures. These late treatment eggs were assigned to ten broods of ten eggs each and put in the nest of ten other female mallards in the same outdoor pen. Just like females in the early treatment group, late treatment females had incubated their own first clutch for 5-14 days when the swap occurred. Thus, late treatment females were naturally late-nesting individuals, whereas females in the early treatment were natural early nesters. In both years, this second and artificially delayed batch of broods subsequently hatched 12 days after the early batch. Hence, there was not any reason to assume a consistent difference between early and late-brooding females when it comes to laying and incubation costs, i.e. reproductive costs that have been particularly difficult to control for in earlier experiments [START_REF] Arnold | Seasonal declines in reproductive success of the common tern Sterna hirundo: timing or parental quality?[END_REF][START_REF] Monaghan | Why don't birds lay more eggs?[END_REF][START_REF] Verhulst | The timing of birds' breeding season: a review of experiments that manipulated timing of breeding[END_REF]. We cannot rule out that there might have been some kind of quality difference between the groups of females related to them being either early or late nesters (e.g. [START_REF] Arnold | Seasonal declines in reproductive success of the common tern Sterna hirundo: timing or parental quality?[END_REF]), but it seems unlikely. [START_REF] Batt | Laying dates, clutch size and egg weight of captive mallards[END_REF] found in a 3-year study with captive mallards (originally reared from eggs taken from wild nests) that the starting date of the first clutch varied considerably between females within a season (43-58 days) and that the repeatability of an individual's clutch initiation date is high from 1 year to the next. This may have several explanations, and one is that female mallards may have a genetically determined propensity for laying earlier or later than the population mean, and if so, differences in natural laying dates may not necessarily reflect differences in quality. Genetically determined inclination for laying earlier or later than the population mean has been found in other waterfowl too, for example, in the common eider (Somateria mollissima; [START_REF] Laurila | Environmental and genetic factors influencing clutch size, egg volume, date of laying and female weight in the common eider Somateria mollissima[END_REF]. Most importantly, even if there was a quality difference between early and late females, this potential bias apparently did not affect our results (see "Discussion").

In both treatments in 2004 and in the early treatment in 2005, all ducklings hatched successfully and all broods thus comprised ten ducklings. In the late treatment in 2005, however, one female deserted her brood at the farm. In order to obtain a balanced design (i.e. the same number of ducklings in all broods released) for this treatment, brood size was standardised to eight ducklings by reducing the full broods by two arbitrarily chosen ducklings each. These ducklings were then pooled to make up a new full brood (eight ducklings) for the female that deserted her previous brood. All females were weighed and measured (wing and tarsus), and all ducklings were weighed individually within 24 h of hatching. Body mass was found to differ between early and late treatment females in both years (general linear model, 2004: F 1, 11 =14.74, p=0.003; 2005: F 1, 11 =4.99, p=0.047; PC1 scores in a principal component analysis of wing and tarsus lengths were used as a covariate to control for body size differences). However, these differences were in opposite directions in 2004 and 2005, with leaner females in the early treatment than in the late in 2004 (887.7 g [SE=13.0] 3]; independent t=-5.04, df=266, p<0.001). These analyses of body mass are based on ducklings and hens eventually included in the survival analysis (i.e. data from six lakes were omitted due to unsuccessful introductions; see below).

After weighing, each brood was transported separately with its foster mother from the game station to a randomly assigned study lake in Västerbotten (ca. 350 km NE of Boda Game Station), where they were released at dawn the day after, i.e. when ducklings were 24-36 h old. All broods were released successfully in the sense that the foster female and her brood swam out on the lake together in a tight brood formation. In 2004, the early treatment broods were released on June 17 and the late broods on June 29. In 2005, the corresponding dates were June 19 and July 1, respectively. Hence, in both years, early as well as late treatment ducklings hatched within the natural time window of back-calculated hatching dates of local wild mallard broods observed on study lakes and other lakes in the region (2004: range June 3-July 1, n=8; 2005: range May 22-July 3, n=7; Sjöberg et al., unpublished data).

To account for possible effects of consistent lake-level differences in, e.g. depth and shoreline vegetation, we used a between-year cross-over design, i.e. lakes receiving an early brood in 2004 had a late brood in 2005 and vice versa. In terms of our modelling requirements, the experimental introduction failed in one or both years on six of the lakes in the sense that neither the hen nor the ducklings were observed after the day of introduction. This left us with full data sets from 14 lakes, i.e. 60 ducklings (six lakes) in the early treatment and 80 ducklings (eight lakes) in the late treatment in 2004 and 80 ducklings (eight lakes) in the early treatment and 48 ducklings (six lakes) in the late treatment in 2005. These duckling data were used in the subsequent survival analyses (see "Modelling analysis" below).

All handling of ducks, ducklings and eggs was in compliance with Swedish law. Introductions were done in accordance with permit A19-04 from the Research Animals Ethics Board at the Court of Appeal for North Sweden, Umeå, Sweden.

Observation of introduced birds

Generally, most duckling mortality occurs the first 2 weeks after hatching (e.g. [START_REF] Orthmeyer | Survival of mallard broods on Benton Lake National Wildlife Refuge in northcentral Montana[END_REF]Rotella and Ratti 1992a), and each lake was therefore visited daily for 12 days after the release of ducks to count remaining introduced birds.

We used the waterfowl point count method in [START_REF] Koskimies | Monitoring bird populations[END_REF], i.e. 20 min of still observation and scans from a fixed point. Introduced foster females were readily told from wild females due to their lack of primary projection (wing tip) on one of the wings. Most counts were made in the morning or in the evening, when duckling foraging activity normally peaks [START_REF] Ringelman | Diurnal visibility and activity of blue-winged teal and mallard broods[END_REF]. No counts were made at night. Considerable effort was made to approach lakes without alarming the ducks. For practical reasons, it was not feasible to totally randomise the visiting order of lakes. The latter comprised a northern and a southern cluster, between which visit order was alternated. To minimise the distance driven, lakes were visited in a specified sequence within each cluster, the starting end of which was alternated between study days.

Invertebrate trapping

Previous research has demonstrated a positive correlation between duckling survival and invertebrate prey abundance in breeding wetlands [START_REF] Cox | Mallard duckling growth and survival in relation to aquatic invertebrates[END_REF][START_REF] Danell | Seasonal emergence of chironomids in relation to egglaying and hatching in a restored lake (northern Sweden)[END_REF][START_REF] Hill | Survival of mallard ducklings Anas platyrhynchos and competition with fish for invertebrates on a flooded gravel quarry in England[END_REF][START_REF] Sedinger | Ecology of prefledgling waterfowl[END_REF]). Since dabbling ducks consume benthic, nektonic as well as hatching metamorphosing invertebrates, we used two trap types in each lake: (1) six floating emergence traps catching insects leaving their aquatic phase were deployed on shallow water close to the shore and (2) six submerged activity traps catching benthic and free-swimming invertebrates were placed on the bottom in shallow water according to standard procedures (e.g. [START_REF] Elmberg | Factors affecting species number and density of dabbling duck guilds in North Europe[END_REF]). Both trap types were used continuously from day 0 (introduction day) until day 12 at all lakes, and they were emptied every second day (48 h). Hence, for both trap types, there were six trapping periods with six traps each, i.e. 36 invertebrate samples from each trap type and study lake. The content of each trap was analysed separately, and all prey were identified to taxonomic order and size class (cf. [START_REF] Elmberg | Factors affecting species number and density of dabbling duck guilds in North Europe[END_REF]Elmberg et al. , 2005a;;[START_REF] Nudds | Predator-prey size relationships in North American dabbling ducks[END_REF]. Amphibians and typical terrestrial invertebrates found in traps were left out from analysis. Catches from traps containing fish were also omitted, as intruding fish may bias invertebrate samples [START_REF] Elmberg | Do intruding predators and trap position affect the reliability of catches in activity traps?[END_REF]. Catches were weighted by body length class, and their partial weights were summed and finally standardised to "per 100 trap days" to obtain an invertebrate abundance index value for each trap (for details, see Elmberg et al. 2005a).

Modelling analysis

The modelling input file included an encounter history for each individual duckling for its 13-day study period, i.e. days 0-12. In order to allow lake-specific covariates in the subsequent modelling (see below), input data were categorised in 28 different groups (i.e. 14 lakes in 2 years).

We used program MARK [START_REF] White | Program MARK: survival estimation from populations of marked animals[END_REF]) and the Cormack-Jolly-Seber (CJS) modelling option with "live recaptures only" [START_REF] Lebreton | Modelling survival and testing biological hypotheses using marked animals: a unified approach with case studies[END_REF]) to analyse duckling survival. Estimates for apparent survival (Ф; i.e. mortality cannot be distinguished from permanent emigration) as well as for the probability that a duckling was re-sighted (p) were generated by modelling. Ducklings were not individually marked, but the CJS approach was still considered valid because (1) introduced broods were always successfully identified based on the presence of the introduced hen lacking primary feathers, (2) wild mallard broods were seldom present and brood mixing between wild and introduced broods was never suspected and (3) on only a very few (5.4%) occasions more ducklings were observed than on the preceding visits at the same lake (indicating that ducklings had been missed-not counted-on previous visits). We consider the latter bias on survival and resighting estimations as minor.

The global model included the dummy variables treatment (i.e. early vs. late introduction) and year, as well as their interaction. Potential lake-level effects could not be evaluated due to over-parameterised models, which was also true for models with full time dependence. However, earlier research in the same study region shows that duckling age is important for survival the first 2 weeks after hatching [START_REF] Gunnarsson | Why are there so many empty lakes? Food limits survival of mallard ducklings[END_REF][START_REF] Gunnarsson | Experimental evidence for density-dependent survival in mallard (Anas platyrhynchos) ducklings[END_REF], and to include such a constraint, the logit-link function was used to be able to add age effects as covariates in the design matrix. Two different effects of duckling age were considered: (1) a linear (this was done by entering the values 0-11 for the study days) and (2) a quadratic (i.e. the squared products of the linear effect values; cf. [START_REF] Gunnarsson | Why are there so many empty lakes? Food limits survival of mallard ducklings[END_REF][START_REF] Gunnarsson | Experimental evidence for density-dependent survival in mallard (Anas platyrhynchos) ducklings[END_REF].

Goodness-of-fit of the global model was evaluated with a bootstrap approach. This was done by first running a simulation of the most parameterised model (covariates excluded; i.e. model #10 in Table 2) with 500 iterations. The deviance for each iteration was then ranked and compared to the observed deviance for the global model. The observed model deviance was finally divided by the average deviance from the bootstrap simulation, and this quotient was used to adjust the variance inflation factor (ĉ) in order to control for overdispersion (i.e. lack of fit; [START_REF] Burnham | Model selection and multi-model inference: a practical information-theoretic approach[END_REF].

To find more parsimonious models, variables of minor importance were dropped consecutively from the global model, as judged by the change of the quasi-likelihood Akaike's information criterion with adjustment for low sample size (QAICc). Competing models within two QAICc units were considered as potential best models, unless higher-ranked models were a hierarchical subset of a lower-ranked model. If the latter was true, the more complex and lower-ranked model was excluded [START_REF] Burnham | Model selection and multi-model inference: a practical information-theoretic approach[END_REF]; see also [START_REF] Arnold | Uninformative parameters and model selection using Akaike's information criterion[END_REF]. The Ф parameters were considered first, followed by the p parameters (cf. [START_REF] Doherty Pf | Sources of variation in breeding-ground fidelity of mallards (Anas platyrhynchos)[END_REF]. The two age covariates were added as constraints to the highest ranked model in the candidate set.

In Table 2, we report the global model (used for the bootstrap simulation), the model including the intercept only, the highest ranked model and finally the models needed to validate the variables not included in the highest ranked model. This validation was done by including variables to the highest ranked model one at a time. For evaluation of the variables among the highest ranked models, β values with SEs were considered. Modelaveraged real estimates are presented for survival and resighting probabilities in Fig. 2.

Results

Duckling survival

Goodness-of-fit testing revealed that the global model did not adequately fit the data, i.e. deviances from the bootstrap simulation were all ranked lower (highest deviance was 390.92) than the observed deviance (924.74). Overdispersion was controlled accordingly by adjusting ĉ to 3.70, i.e. the deviance from the global model was divided by the average deviance from the bootstrap simulation (i.e. 924.74/250.23).

Excluding parameters of minor importance from the global model resulted in more parsimonious models. For the survival (Φ) parameters, the highest ranked model included the year effect only. Evaluation of the beta estimates revealed that survival probability was higher in 2005 than in 2004 (β=0.70; SE=0.33). The other variables were included in lower-ranked models, which despite relatively high model weights confirm that all variables except year were of minor importance. This was based on the high variation relative to the β values: treatment (β = -0.47; SE = 0.34), linear effect of duckling age (β=0.12; SE=0.09) and quadratic effect of duckling age ) (additional to the linear effect). A multiplication sign indicates the interaction term between two variables along with their separate effects. See footnotes for model details and "Modelling analysis" for a more thorough description of the modelling procedure (β=-0.02; SE=0.03). Moreover, model-averaged estimates for survival (Fig. 1) confirmed that year was the only variable of importance to survival.

For the re-sighting probability (p), none of the variables was among the highest ranked models. beta values from lower-ranked models made it clear that no variable was of great importance to re-sighting probability: treatment (β=-0.36; SE=0.39), year (β=-0.28; SE=0.43), linear effect of duckling age (β=0.06; SE=0.07) and quadratic effect of duckling age (β < -0.01; SE= 0.02). Modelaveraged real estimates for re-sighting probabilities confirm these findings (Fig. 1).

Invertebrate abundance

Invertebrate abundance varied markedly among the 14 lakes (Fig. 2). The abundance indices of emergence trap catches did neither vary between treatments in either of the years nor between years in the two treatments (independent t test: p>0.49), and the overall mean for 100 trap days was 648.3 (SE=105.8). The abundance indices of invertebrates caught in activity traps were higher in the early treatment (mean=1,232.6 [SE=189.2]) than in the late (mean=658.1 [SE=173.6]) in 2004 (independent t=2.22, df=12, p=0.05), but not in 2005 (independent t=-1.69, df=5.27 [equal variances not assumed], p=0.15; overall mean in 2005= 1,653.6 [SE=527.8]). Abundance indices from activity traps did not differ between years in either treatment (independent t test: p>0.12).

Discussion

Our manipulation of hatching time did not corroborate the timing hypothesis predicting that survival in late-hatched mallard ducklings should be lower than in those hatched early. Some previous observational mallard studies have found lower duckling survival in late broods than in early, but a lack of a seasonal effect and even opposite trends have also been reported (see Table 1). Although there are no pertinent previous mallard studies from the study region, there are indeed inconsistent patterns in other species that have been studied there. Elmberg et al. (2005b; observational study) reported higher breeding success in early nesting Eurasian teal (Anas crecca crecca), whereas [START_REF] Paasivaara | Survival of common goldeneye Bucephala clangula ducklings in relation to weather, timing of breeding, brood size, and female condition[END_REF]observational study) find any support for hatching date effects on survival in common goldeneye (Bucephala clangula) ducklings. However, seasonal effects (i.e. timing of breeding) have been studied in other stages of the mallard's breeding cycle in Europe. For example, in an experimental study contrasting Mediterranean and boreal biomes, [START_REF] Elmberg | Putting density dependence in perspective: nest density, nesting phenology, and biome, all matter to survival of simulated mallard Anas platyrhynchos nests[END_REF] found that nest survival was higher in early treatments than in nests deployed later. For overall breeding success, though, it is final duckling production that is the most relevant aspect to study in a 'mismatch' context as driven by climate change, since, e.g. weather and food conditions are crucial for the survival of young ducklings in particular [START_REF] Cox | Mallard duckling growth and survival in relation to aquatic invertebrates[END_REF][START_REF] Gunnarsson | Why are there so many empty lakes? Food limits survival of mallard ducklings[END_REF][START_REF] Gunnarsson | Experimental evidence for density-dependent survival in mallard (Anas platyrhynchos) ducklings[END_REF][START_REF] Koskimies | Cold-hardiness of the newly hatched young in relation to ecology and distribution in ten species of European ducks[END_REF][START_REF] Nummi | Individual foraging behaviour indicates resource limitation: an experiment with mallard ducklings[END_REF][START_REF] Sjöberg | Response of mallard ducklings to variation in habitat quality-an experiment of food limitation[END_REF]; see also further discussion below).

Mechanisms of season-dependent duckling survival, as described in many earlier studies, are not well understood, but seasonal declines have often been attributed to concomitant deterioration of wetland conditions and abundance (e.g. [START_REF] Dzus | Brood survival and recruitment of mallards in relation to wetland density and hatching date[END_REF][START_REF] Hoekman | Factors affecting survival of mallard ducklings in southern Ontario[END_REF][START_REF] Krapu | Factors limiting mallard brood survival in prairie pothole landscapes[END_REF]Rotella and Ratti 1992a). Indeed, most of the observational studies come from the North American prairies, where seasonal wetlands (i.e. which dry up as the breeding season progresses) are common and where overall deterioration of habitat conditions during the breeding season is frequent. In line with this, [START_REF] Dzus | Brood survival and recruitment of mallards in relation to wetland density and hatching date[END_REF] reported an increase in mallard duckling survival in 1 year when abundance of seasonal wetlands increased exceptionally over the season. [START_REF] Gendron | Survival of gadwall and mallard ducklings in southcentral Saskatchewan[END_REF] concluded that the absence of season-dependent duckling survival in their study may have been related to stable wetland conditions in the two study years. In addition, [START_REF] Simpson | Mallard duckling survival in the Great Lakes region[END_REF] did not find a seasonal decline in mallard duckling survival in the Great Lakes region, and these authors, too, attributed this pattern to relatively stable wetland conditions. Our experimental results suggest that season-dependent duckling survival found in many observational studies is likely due to deteriorating habitat conditions. We thus find support for the 'declining habitat condition' hypothesis, in the sense that the boreal lakes used by us do not undergo such seasonal changes in habitat condition (i.e. they do not dry up and do not even vary much in water level during the breeding season) and neither was duckling survival season dependent.

Did our study design introduce any confounding factor that prevented us from detecting a seasonal effect on duckling survival? As declared above (see "Eggs, ducklings and introductions"), body condition (i.e. body mass corrected for structural size) of females and body mass of ducklings differed between treatments (only in 2004 for ducklings), which, one could argue, might affect the outcome. This seems not to be the case, though, since Abundance of emergence trap invertebrates did not differ between treatments and years, in contrast to abundance of activity trap invertebrates, which differed between treatments in 2004 (see "Results") these differences did not relate consistently to the absence of a treatment effect on duckling survival. However, the overall higher body mass of ducklings in 2005 as compared to 2004 may have led to the observed higher survival rates in the same year.

Our experiment took advantage of the natural variation in clutch initiation date among female mallards. We delayed hatching 12 days, which may not seem much compared to the natural variation [START_REF] Batt | Laying dates, clutch size and egg weight of captive mallards[END_REF][START_REF] Hill | Laying date, clutch size and egg size of the mallard Anas platyrhynchos and tufted duck Aythya fuligula[END_REF][START_REF] Oja | Spring phenology, latitude, and the timing of breeding in two migratory ducks: implications of climate change impacts[END_REF]. However, in terms of predicted climate change, this is a truly significant difference in phenology. For example, [START_REF] Both | Climate change and timing of avian breeding and migration throughout Europe[END_REF] modelled trends in laying date in relation to recent climate change for the European starling (Sturnus vulgaris) and the pied flycatcher (Ficedula hypoleuca) across Europe. The predicted laying date changes ranged, depending on location, from 4 days delay to 7 days advance over a 25-year period (1984 to 2004). It should be noted that the length of the period during which seasonal effects on duckling survival was addressed varied much more in two observational studies that, like our experiment, did not find a seasonal decline in survival-59-80 days (within a season) in [START_REF] Gendron | Survival of gadwall and mallard ducklings in southcentral Saskatchewan[END_REF] and 33-96 days in [START_REF] Simpson | Mallard duckling survival in the Great Lakes region[END_REF]. These observational studies evidently included re-nesting attempts, but still did not find lower survival in late-hatched broods. Hence, we argue that the possibility of detecting season-dependent survival in our experiment most probably was not limited by the time difference between treatments. Nor did the storing of late-hatched eggs affect their quality, since we did not find any differences in hatching success between the treatments.

As demonstrated in previous experiments [START_REF] Gunnarsson | Why are there so many empty lakes? Food limits survival of mallard ducklings[END_REF]; see also [START_REF] Nummi | Individual foraging behaviour indicates resource limitation: an experiment with mallard ducklings[END_REF][START_REF] Sjöberg | Response of mallard ducklings to variation in habitat quality-an experiment of food limitation[END_REF], food abundance may limit duckling survival on lakes of this type (i.e. same region, similar nutrient status and lake size). We therefore argue that our study design permits resource-related seasonal effects on duckling survival to be detected, if such indeed exist. Likewise, we hypothesise that the amount of available invertebrate prey, together with higher overall body mass of ducklings (see above), possibly had a positive effect on duckling survival, since both were higher in 2005 than in 2004. That food abundance is crucial to mallard duckling survival has previously been demonstrated by others in different study systems. For example, [START_REF] Cox | Mallard duckling growth and survival in relation to aquatic invertebrates[END_REF] found that growth of young mallard ducklings was sensitive to variation in food invertebrate abundance and that duckling survival, in turn, was positively related to growth. In addition to food limitation, oligotrophic lakes in boreal environments have sparse emergent vegetation [START_REF] Kauppinen | Ordination and classification of waterfowl communities in south boreal lakes[END_REF], and this may adversely affect mallard duckling survival by decreased thermoregulation during inclement weather or by providing less cover from predators [START_REF] Gunnarsson | Experimental evidence for density-dependent survival in mallard (Anas platyrhynchos) ducklings[END_REF][START_REF] Koskimies | Cold-hardiness of the newly hatched young in relation to ecology and distribution in ten species of European ducks[END_REF][START_REF] Rhymer | The effect of egg size variability on thermoregulation of mallard (Anas platyrhynchos) offspring and its implications for survival[END_REF][START_REF] Simpson | Effects of habitat on mallard duckling survival in the Great Lakes region[END_REF][START_REF] Stafford | Survival of radio-marked mallard ducklings in South Dakota[END_REF]). Although we do not know the ultimate fate of the ducklings in our study, predation likely had an additive effect on mortality (cf. [START_REF] Gunnarsson | Why are there so many empty lakes? Food limits survival of mallard ducklings[END_REF].

Mallard hens were wing-clipped for recognition, but also to discourage them from leaving the introduction lake with their brood. Most of the ducklings that were not observed after release hence most likely died on the lakes. In this respect, our manipulation does not resemble a natural situation, in which wild hens may take their broods to better foraging lakes (e.g. [START_REF] Ball | Survival of wood duck and mallard broods in north-central Minnesota[END_REF][START_REF] Dzus | Overland travel, food abundance, and wetland use by mallards: relationships with offspring survival[END_REF]Rotella and Ratti 1992b). Yet, this should not cause any bias in the comparison between early-hatched and latehatched ducklings in our experiment. On the contrary, brood movements and overland travel in particular may be a severe confounding factor in observational studies done in prairie areas dominated by seasonal wetlands; movements may increase as seasonal wetlands dry up with time, thereby increasing predation risk (see discussion in [START_REF] Hoekman | Factors affecting survival of mallard ducklings in southern Ontario[END_REF]. Our experiment was designed to control for this problem and helped in studying the timing hypothesis per se.

Our findings have important bearings on the assessment of global climate change impact on population processes in ducks. Timing of breeding is closely associated with spring phenology in the mallard and other duck species, clutch initiation being earlier in early than in late springs [START_REF] Drever | Spring temperature, clutch initiation date and duck nest success: a test of the mismatch hypothesis[END_REF]Oja and Pöysä 2007 and references therein). However, the capacity of mallards to track changes in spring phenology may be imperfect; [START_REF] Oja | Spring phenology, latitude, and the timing of breeding in two migratory ducks: implications of climate change impacts[END_REF] found that the time interval between hatching date and ice break-up date decreased with increasing lateness of the spring. This suggests that there is a potential for a 'mismatch' between hatching phenology and resource phenology also in ducks, as has been found in for example some passerine and grouse species [START_REF] Laaksonen | Climate change, migratory connectivity and changes in laying date and clutch size of the pied flycatcher[END_REF][START_REF] Ludwig | Short-and long-term population dynamical consequences of asymmetric climate change in black grouse[END_REF][START_REF] Visser | Warmer springs lead to mistimed reproduction in great tits (Parus major)[END_REF]. Peak emergence of chironomids, the main food of newly hatched ducklings, has traditionally been considered important to the timing of hatching in mallard and other ducks breeding in the boreal region [START_REF] Danell | Seasonal emergence of chironomids in relation to egglaying and hatching in a restored lake (northern Sweden)[END_REF][START_REF] Hill | Laying date, clutch size and egg size of the mallard Anas platyrhynchos and tufted duck Aythya fuligula[END_REF]. However, just like [START_REF] Dessborn | Hatching in dabbling ducks and emergence in chironomids: a case of predator-prey synchrony?[END_REF], the present study did not reveal any consistent peaks or other strong within-season trends in invertebrate abundance. In other words, mallards probably have a rather wide time window and a flexibility to tolerate a moderate mismatch between hatching phenology and resource phenology without fitness consequences, at least during the brood stage and in relatively stable boreal breeding environments. We are aware of only one study explicitly addressing the mismatch hypothesis in ducks. Using observational data of nest success, [START_REF] Drever | Spring temperature, clutch initiation date and duck nest success: a test of the mismatch hypothesis[END_REF] found that relative (i.e. within season) clutch initiation date generally did not affect nest success in five duck species, including the mallard, breeding in prairies in Saskatchewan, Canada. These authors found evidence consistent with the mismatch hypothesis in only one species, the northern pintail (Anas acuta), and even here it was weak. [START_REF] Drever | Spring temperature, clutch initiation date and duck nest success: a test of the mismatch hypothesis[END_REF] and our results together suggest that breeding ducks by and large have considerable flexibility to tolerate variation in the timing of spring and resource phenology.

Model simulations have shown that increased drought will be a major climate-driven threat to waterfowl habitats and populations in the North American Prairies [START_REF] Johnson | Vulnerability of northern prairie wetlands to climate change[END_REF][START_REF] Sorenson | Potential effects of global warming on waterfowl populations breeding in the Northern Great Plains[END_REF]. The conditions for these widespread species are, however, different in other parts of the northern hemisphere. For example, a drought scenario affecting waterfowl seems unlikely in large parts of Eurasia, where breeding waters are generally deeper and have more stable water levels than in North America, within as well as between years. However, as spring temperatures during the past two decades have risen faster in Eurasia than in North America [START_REF] Piao | Net carbon dioxide losses of northern ecosystems in response to autumn warming[END_REF], also other climate-driven constraints on reproductive performance of ducks may differ between the continents. Waterfowl are considered an important natural resource in North America, and recreational hunting of waterfowl is widespread worldwide [START_REF] Kanstrup | Sustainable harvest of waterbirds: a global review[END_REF][START_REF] Kear | Conservation and management[END_REF]. Sustainable management of waterfowl populations is thus a challenging task even without global climate change (e.g. [START_REF] Elmberg | The scientific basis for a new and sustainable management of migratory European ducks[END_REF][START_REF] Johnson | Uncertainty and the management of mallard harvests[END_REF][START_REF] Nichols | Managing North American waterfowl in the face of uncertainty[END_REF][START_REF] Pöysä | Ecological basis of sustainable harvesting: is the prevailing paradigm of compensatory mortality still valid?[END_REF]. Consequently, in order to better understand and predict climate change impact on duck populations, more research is needed, preferably manipulative experiments addressing the flexibility of breeding ducks to respond to climatedriven changes in resource phenology.

a

  Model including the intercepts only b Most general model (excluding covariates) with all parameters estimated with precision c Quasi-likelihood Akaike's information criterion adjusted for low sample size d Difference between QAICc of the current model and the minimum QAICc value e Normalised Akaike weight f Number of parameters g Difference in -2log(likelihood) of the current model and -2log(likelihood) of the saturated model (i.e. the model containing as many parameters as the sample size)

2005 Fig. 1

 20051 Fig. 1 Averaged real estimates (with SE) for introduced mallard ducklings in early and late treatments in 2004 (upper graph) and 2005 (lower graph). Black lines represent survival estimates and grey lines re-sighting estimates. Data from early and late treatment broods are

  Fig. 2 Invertebrate index (with SE) per 100 trap days (see "Material and methods" for index details) for emergence traps (grey lines) and activity traps (black lines) in 2004 (upper graph) and 2005 (lower graph). Data from early and late treatment broods are separated by a break in the respective lines around June 29 in 2004 and July 1 in

Table 1

 1 Descriptive studies addressing the effect of timing of hatching on survival of mallard ducklings or broods

	Reference	Site	Wetland type	Duckling survival
	Orthmeyer and Ball 1990	Montana, USA	Marshes divided into impoundments,	Lower in late broods
			water level manipulated
	Rotella and Ratti 1992a	Manitoba, Canada	Mixture of seasonal, semi-permanent	Lower in late broods
			wetlands
	Mauser et al. 1994	California, USA	Mixture of permanent and seasonally	No seasonal differences
			flooded marshes
	Sayler and Willms 1997	North Dakota, USA	Lakes, water level manipulated	Lower in late broods
	Dzus and Clark 1998	Saskatchewan, Canada	Wetlands of varying permanency	Lower (here: brood survival)
				in late broods in 3 out of
				4 years (higher in 1 year)
	Krapu et al. 2000	North Dakota and	Mixture of lakes and temporary,	Lower in late broods
		Minnesota, USA	seasonal and semi-permanent
			wetlands
	Gendron and Clark 2002	Saskatchewan, Canada	Wetlands of varying permanency	No seasonal difference
	Stafford et al. 2002	South Dakota, USA	Mixture of seasonal, semi-permanent,	Lower in late broods
			and permanent wetlands
	Hoekman et al. 2004	Ontario, USA	Mixture of seasonal, semi-	Lower in late broods
			permanent and permanent wetlands
	Pearse and Ratti 2004	Saskatchewan, Canada	Range from ephemeral to permanent
			wetlands

  vs. 1,014.5 g [SE=30.2]) and heavier females in the early treatment in 2005 (912.5 g [SE = 34.7] vs. 878.2 g [SE = 28.0]).

	Regardless of treatment, females were generally heavier
	in 2004 (960.1 g [SE=24.7]) than in 2005 (897.8 g
	[SE=22.9]; general linear model, F 1, 25 =7.94, p=0.009).
	Duckling body mass differed between early and late
	treatment in 2004 (early ducklings 29.3 g [SE=0.4]; late
	ducklings 33.1 g [SE=0.3]; independent t=-7.94, df=138,
	p < 0.001) but not in 2005 (early ducklings 34.0 g
	(SE=0.4), late ducklings 33.3 g (SE=0.7); independent
	t=0.92, df=73.4 [equal variances not assumed], p=0.36).
	Duckling body mass was higher in 2005 (33.7 g
	[SE=0.4]) than in 2004 (31.5 g [SE=0.

Table 2

 2 Live recapture (Cormack-Jolly-Seber) models for apparent survival (Φ) and re-sighting (p) probabilities of mallard ducklings introduced onto lakes in northernSweden 2004 and[START_REF] Weatherhead | Effects of climate variation on timing of nesting, reproductive success, and offspring sex ratios of redwinged blackbirds[END_REF] Dummy variables in the models are treatment (treat) and year (year) and their interaction. Two additional continuous covariates were added in the design matrix: linear effect of duckling age (age[l]) and quadratic effect of duckling age (age[q]

	Model		QAICc c	ΔQAICc d	w i e	K f	Deviance g
	1. Φ (year) p (.)		357.57	0.00	0.18	3	255.15
	2. Φ (year+ age[l]) p (.)		357.60	0.03	0.18	4	253.15
	3. Φ (treat+ year) p (.)		357.66	0.09	0.18	4	253.21
	4. Φ (year) p (age[l])		358.76	1.18	0.10	4	254.30
	5. Φ (year) p (treat)		358.78	1.21	0.10	4	254.33
	6. Φ (year) p (year)		359.20	1.62	0.08	4	254.74
	7. Φ (year+ age[q]) p (.)		359.38	1.81	0.07	5	252.88
	8. Φ (.) p (.) a		360.09	2.52	0.05	2	259.70
	9. Φ (year) p (age[q])		360.80	3.22	0.04	5	254.30
	10. Φ (treat ×year) p (trear×year)	b	362.90	5.33	0.01	8	250.23

Acknowledgements We sincerely thank Göran Karlsson for skilful work with eggs and ducks at the Boda Game Station. Per Wedholm, Eric Andersson, Johan Nilsson and Åke Nordström are gratefully acknowledged for field work assistance. The research was supported by grant V 98-04 from The Swedish Environmental Protection Agency (Naturvårdsverket).


T Vainionpää

Heikkinen

MD Santtu Heinonen

MD Heli Silvennoinen

MD, PhD¹ Pasi Lehtinen

PhD,² Raija Vainionpää

MD, PhD¹ Terho Heikkinen

Feasibility of diagnosing influenza within 24 hours of symptom onset in children 1-3 years of age

Keywords:

Feasibility of diagnosing influenza within 24 hours of symptom onset in children 1-3 years of age

INTRODUCTION

The burden of influenza is substantial in children [START_REF] Heikkinen | Burden of influenza in children in the community[END_REF][START_REF] Poehling | for the New Vaccine Surveillance Network (2006) The underrecognized burden of influenza in young children[END_REF]. The attack rates are highest in this age group [START_REF] Monto | Acute respiratory illness in the community. Frequency of illness and the agents involved[END_REF], and young children are hospitalized at rates similar to those for adults with high risk conditions [START_REF] Neuzil | The effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children[END_REF]. Further, influenza predisposes children to bacterial complications such as acute otitis media [START_REF] Heikkinen | Burden of influenza in children in the community[END_REF]. Neuraminidase inhibitors oseltamivir and zanamivir can be used for treating influenza in children from 1 and 5 years of age, respectively [START_REF]Centers for Disease Control and Prevention: Updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the 2009-2010 season[END_REF]. These drugs reduce the duration and severity of symptoms when started within 48 hours of symptom onset [START_REF] Whitley | Oral oseltamivir treatment of influenza in children[END_REF][START_REF] Hedrick | Zanamivir for treatment of symptomatic influenza A and B infection in children five to twelve years of age: a randomized controlled trial[END_REF]. The mechanism of action of neuraminidase inhibitors and the results of a previous study in adults [START_REF] Aoki | on behalf of the IMPACT study group (2003) Early administration of oral oseltamivir increases the benefits of influenza treatment[END_REF] suggest that the clinical benefits could be greater if the treatment were initiated at an earlier stage of illness. According to CDC recommendations, the antiviral treatment should be started as quickly as possible after the onset of symptoms [START_REF]Centers for Disease Control and Prevention: Updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the 2009-2010 season[END_REF].

Distinguishing influenza from other viral respiratory infections is difficult in young children because of the nonspecific signs and symptoms of influenza [START_REF] Silvennoinen | Clinical presentation of Influenza in unselected children treated as outpatients[END_REF], children's inability to describe their subjective symptoms, and high frequency of other viral infections even during an influenza outbreak [START_REF] Poehling | for the New Vaccine Surveillance Network (2006) The underrecognized burden of influenza in young children[END_REF][START_REF] Heikkinen | Incidence of influenza in Finnish children[END_REF][START_REF] Zambon | Contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study[END_REF]. In a previous study among children, the overall sensitivity of the clinical diagnosis of influenza was only 38% and the positive predictive value 32% [START_REF] Peltola | Accuracy of clinical diagnosis of influenza in outpatient children[END_REF]. Therefore, virological methods are needed to optimize the use of antiviral drugs in children. For clinical decision-making, rapid point-of-care tests that provide almost immediate results are most useful. Previous studies have demonstrated that these tests are generally specific, but their sensitivity has varied greatly [START_REF] Cazacu | Comparison of lateral-flow immunoassay and enzyme immunoassay with viral culture for rapid detection of influenza virus in nasal wash specimens from children[END_REF][START_REF] Grijalva | Accuracy and interpretation of rapid influenza tests in children[END_REF][START_REF] Agoritsas | Evaluation of the Quidel QuickVue test for detection of influenza A and B viruses in the pediatric emergency medicine setting by use of three specimen collection methods[END_REF][START_REF] Ghebremedhin | Comparison of the performance of the rapid antigen detection actim Influenza A&B test and RT-PCR in different respiratory specimens[END_REF][START_REF] Steininger | Near-patient assays for diagnosis of influenza virus infection in adult patients[END_REF][START_REF] Uyeki | Low sensitivity of rapid diagnostic test for influenza[END_REF][START_REF] Cazacu | Comparison of the directigen flu A+B membrane enzyme immunoassay with viral culture for rapid detection of influenza A and B viruses in respiratory specimens[END_REF].

Infants and young children are frequently brought for clinical examination during an early stage of influenza illness. The clinician´s dilemma is that influenza often commences with an abrupt onset of fever and ill appearance, with full-blown respiratory symptoms developing only later.

Occasionally, febrile convulsion is the first sign of influenza in young children [START_REF] Chiu | Influenza A infection is an important cause of febrile seizures[END_REF]. There are scarce data on whether influenza in children can be reliably diagnosed from upper respiratory specimens during the early stage of the illness, and limited data exist on the performance of influenza rapid tests in this clinical situation. We determined the feasibility of diagnosing influenza in outpatient children 1-3 years of age who presented within 24 hours of the onset of fever. In addition, we evaluated the diagnostic accuracy of influenza rapid testing performed in this setting.

MATERIALS AND METHODS

Study design

This study was conducted as part of a randomized placebo-controlled trial assessing the effectiveness of oseltamivir treatment started within 24 hours of the onset of symptoms in children (ClinicalTrials.gov; NCT00593502) during the influenza seasons of 2007-08 (predominance of A/H1N1 strains) and 2008-09 (predominance of A/H3N2 strains) at a single site in Turku, Finland [START_REF] Heinonen | Early oseltamivir treatment of influenza in children 1-3 years of age: a randomized controlled trial[END_REF]. Before each influenza season, children 1-3 years of age were recruited into follow-up cohorts (N=631 in 2007-08 and N=554 in 2008-09). No exclusion criteria were employed for enrollment. The study was approved by the Ethics Committee of the Hospital District of Southwest Finland. Written informed consent was obtained from the parents of the participating children.

Study procedures

When local surveillance indicated that influenza was circulating in the area, a study clinic was opened. The parents were asked to bring their child to the study clinic within 24 hours if the child developed fever or any signs of respiratory infection. During each visit (n=764), the child was examined by a study physician, and nasal swabs were obtained for virological analyses.

Rapid influenza tests (n=188) were routinely performed (according to the study protocol of the oseltamivir trial) during the first visit to all febrile children without respiratory symptoms, but also to any other children according to the decision by the attending physician who considered e.g. time constraints for performing the rapid tests during the busy hours of the influenza epidemic. A follow-up visit was scheduled on day 5-8 for all children who received the study medication. In addition, the parents were invited to bring their child to the study clinic whenever they deemed it necessary. Nasal swabs were obtained during all follow-up visits if the child was symptomatic. All visits were free of charge to the parents.

Virological methods

Three flocked swab specimens (Copan, Italy) were obtained from a depth of 2-3 cm in the nostrils in non-standardized order. The specimens were refrigerated at +6°C until analyzed in the virological laboratory next working day. The samples were subjected to viral culture in Madin-Darby canine kidney cells followed by immunoperoxidase staining with monoclonal antibodies [START_REF] Waris | Rapid detection of respiratory syncytial virus and influenza-A virus in cell-cultures by immunoperoxidase staining with monoclonal-antibodies[END_REF] and antigen detection using time-resolved fluoroimmunoassay (TR-FIA) [START_REF] Nikkari | One-incubation time-resolved fluoroimmunoassay based on monoclonal antibodies in detection of influenza A and B viruses directly in clinical specimens[END_REF]. All samples that remained negative for influenza viruses with these methods were further tested with reverse-transcriptase polymerase-chain-reaction (RT-PCR) assays for influenza A and B viruses.

After collection of specimens for the virological laboratory, another nasal swab specimen for rapid testing was obtained from a proportion of children (as described earlier in "Study procedures") using the polyester swabs provided with the kit (Actim Influenza A & B, Medix Biochemica, Finland). The manufacturer of these rapid tests was not involved in this study; the authors purchased the test kits at market price. The rapid test was performed at the point of care immediately after the sampling according to the manufacturer's instructions. All samples were obtained and the rapid tests performed by one of four trained study nurses who also interpreted the results. The personnel performing the other virological analyses were unaware of the rapid test results.

Data analysis

Detection of influenza virus within 24 hours of symptom onset

In this analysis, we included all children in the placebo arm of the treatment trial in whom influenza was eventually confirmed by any of the conventional laboratory methods (viral isolation, TR-FIA, or RT-PCR) at any time within 8 days of the onset of symptoms. Children who had received oseltamivir were excluded from this analysis because the treatment might have decreased the viral load in the children and consequently led to increased numbers of negative test results during the follow-up visits. The sensitivity of detecting influenza virus within 24 hours of symptom onset was determined as the percentage of children (among all influenzapositive children) in whom influenza could be diagnosed in the nasal swabs collected during the first 24 hours of symptoms.

Performance of the influenza rapid test

The sensitivity, specificity, and predictive values of the rapid tests were assessed by comparing the results of the rapid tests obtained within 24 hours of symptom onset (i.e. before the start of any study medication) to those of the simultaneously obtained reference test samples. A reference test was considered positive if any of the methods (viral isolation, TR-FIA, or RT-PCR) yielded a positive result for influenza viruses.

Statistical analysis

Sensitivities, specificities, and predictive values and their binomial 95% confidence intervals were calculated for any influenza virus and for influenza A and B viruses separately. Fisher's exact test was used to compare proportions. Two-sided P values <0.05 were considered statistically significant. All analyses were performed using StatsDirect (version 2.7.7) software. In 57 (93%) children, nasal swabs were collected also during the follow-up visits. The mean age of the children was 2.5 (SD±0.8) years, and the median time from the onset of fever to the collection of the first nasal swab specimen was 6.5 hours (interquartile range, 3.4-13.3 h). Influenza virus was detected within 24 hours of the onset of fever in 56 of the 61 children, yielding a sensitivity of 92% (95% CI, 82-97%). The sensitivity was 94% (95% CI, 83-99%) for influenza A virus and 82% (95% CI, 48-98 %) for influenza B virus (P=0.2). There were no statistically significant differences in the detection of the virus between children presenting <12 hours and 12-24 hours from the onset of symptoms (P>0.9); between vaccinated and unvaccinated children (P=0.5); or between children <2 years and 2-3 years of age (P>0.9).

RESULTS

Detection of influenza virus within 24 hours of symptom onset

Performance of the influenza rapid test

Among a total of 764 visits to the study clinic for new respiratory symptoms or fever, rapid tests were performed in 188 (25%) cases. The results of 30 rapid tests were excluded from this analysis because of the following reasons: absence of fever [START_REF] Uyeki | Low sensitivity of rapid diagnostic test for influenza[END_REF]; duration of fever >24 h (3); absence of reference samples (4); rapid test expired [START_REF]Centers for Disease Control and Prevention: Updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the 2009-2010 season[END_REF]. Among the remaining 158 children, influenza virus was detected with one or more of the reference methods in 39 (25%) children (Table 1). The rapid test was positive in 31 children (including one false positive), resulting in an overall sensitivity of 77% (95% CI, 61-89%) and specificity of 99% (95% CI, 95-100%) for any influenza virus. The sensitivity of the rapid test was 90% (95% CI, 74-98%) for influenza A virus and 25% (95% CI, 3-61%) for influenza B virus (P<0.001).

DISCUSSION

In children in whom influenza was eventually confirmed, 92% of nasal swab specimens that were obtained within 24 hours of the onset of fever were positive by at least one of the conventionally used laboratory methods. This suggests that children shed viruses in their nasal secretions in sufficient amounts to be detected already during the first day of illness. This finding is clinically important because accurate diagnosis of influenza during the early stage of illness enables the rational use of antiviral drugs [START_REF] Whitley | Oral oseltamivir treatment of influenza in children[END_REF][START_REF] Hedrick | Zanamivir for treatment of symptomatic influenza A and B infection in children five to twelve years of age: a randomized controlled trial[END_REF] and proper cohorting of hospitalized patients to prevent nosocomial infections [START_REF] Serwint | Why diagnose influenza infections in hospitalized pediatricpatients[END_REF]. Rapid influenza diagnosis has also been associated with reduced use of antibiotics and shorter duration of hospitalization [START_REF] Noyola | Effect of rapid diagnosis on management of influenza A infections[END_REF][START_REF] Woo | Cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients[END_REF].

The influenza rapid test that we used had an overall sensitivity of 77% and specificity of 99%.

Our results are concordant with several previous studies in children, in which the sensitivities of different rapid tests have ranged from 63% to 85% and the specificities from 97% to 99% [START_REF] Cazacu | Comparison of lateral-flow immunoassay and enzyme immunoassay with viral culture for rapid detection of influenza virus in nasal wash specimens from children[END_REF][START_REF] Grijalva | Accuracy and interpretation of rapid influenza tests in children[END_REF][START_REF] Agoritsas | Evaluation of the Quidel QuickVue test for detection of influenza A and B viruses in the pediatric emergency medicine setting by use of three specimen collection methods[END_REF][START_REF] Stripeli | Performance of rapid influenza testing in hospitalized children[END_REF]. Two previous studies assessing the performance of the same rapid test that we used found sensitivities of 65% [START_REF] Ghebremedhin | Comparison of the performance of the rapid antigen detection actim Influenza A&B test and RT-PCR in different respiratory specimens[END_REF] and 53% [START_REF] Steininger | Near-patient assays for diagnosis of influenza virus infection in adult patients[END_REF] in children. There are, however, reports of significantly lower sensitivities (ranging from 20% to 33%) for various rapid tests in populations consisting mainly of adults [START_REF] Rouleau | Field performance of a rapid diagnostic test for influenza in an ambulatory setting[END_REF][START_REF] Stein | Performance characteristics of clinical diagnosis, a clinical decision rule, and a rapid influenza test in the detection of influenza infection in a community sample of adults[END_REF]. Lower sensitivities (27-44 %) have been observed also in some studies with mainly pediatric populations [START_REF] Uyeki | Low sensitivity of rapid diagnostic test for influenza[END_REF][START_REF] Cazacu | Comparison of the directigen flu A+B membrane enzyme immunoassay with viral culture for rapid detection of influenza A and B viruses in respiratory specimens[END_REF].

There are several potential explanations for the great variability between the sensitivities observed in different studies. Children shed influenza viruses in larger amounts and for longer periods than adults [START_REF] Hall | Viral shedding patterns of children with influenza B infection[END_REF][START_REF] Frank | Patterns of shedding of myxoviruses and paramyxoviruses in children[END_REF][START_REF] Cheng | Factors affecting QuickVue Influenza A + B rapid test performance in the community setting[END_REF], which may account for the increased sensitivity observed in children [START_REF] Steininger | Near-patient assays for diagnosis of influenza virus infection in adult patients[END_REF][START_REF] Cheng | Factors affecting QuickVue Influenza A + B rapid test performance in the community setting[END_REF][START_REF] Steininger | Effectiveness of reverse transcription-PCR, virus isolation, and enzyme-linked immunosorbent assay for diagnosis of influenza A virus infection in different age groups[END_REF][START_REF] Ruest | Comparison of the directigen Flu A+B test, the QuickVue influenza test, and clinical case definition to viral culture and reverse transcription-PCR for rapid diagnosis of influenza virus infection[END_REF]. Also, the delay between the onset of symptoms and the collection of the specimen may affect the sensitivity of the rapid test. In experimental volunteer studies in adults, the viral load peaked on the second day after virus inoculation, and the shedding lasted for approximately 5 days [START_REF] Carrat | Time lines of infection and disease in human influenza: A review of volunteer challenge studies[END_REF]. In children, the viral shedding has been described to peak 1-3 days after the onset of illness and to last for 6-7 days [START_REF] Hall | Viral shedding patterns of children with influenza B infection[END_REF][START_REF] Frank | Patterns of shedding of myxoviruses and paramyxoviruses in children[END_REF][START_REF] Sato | Viral shedding in children with influenza virus infections treated with neuraminidase inhibitors[END_REF]. In a Japanese pediatric study in which most samples were obtained within 24 hours of the onset of fever, the overall sensitivity of an influenza rapid test was the same as in the present study (77%) [START_REF] Watanabe | Sensitivity of rapid immunoassay for influenza A and B in the early phase of the disease[END_REF]. In that study, the sensitivity of the rapid test was lower for specimens collected within 9 hours (64-69%) compared with specimens collected within 9-24 hours (83 -100%) of the onset of fever. A study conducted in Nicaraguan children found a higher sensitivity (74-75%) of influenza rapid tests for samples obtained 2-3 days after symptom onset, compared with samples obtained either 1 or 4 days after symptom onset (52-58%) [START_REF] Gordon | Performance of an influenza rapid test in children in a primary healthcare setting in Nicaragua[END_REF]. In a recent study from Greece, the sensitivity of a rapid test was 76% when performed within 2 days of symptom onset and 65% when performed later during the course of the illness [START_REF] Stripeli | Performance of rapid influenza testing in hospitalized children[END_REF].

Presumably the most important factor affecting the yield of influenza virus is the quality of the sample, which, in turn, is dependent on the site and method of collection. A nasopharyngeal aspirate is often considered as the specimen of choice for everyday clinical purposes in children. The sensitivity of a nasal swab is, however, 91-92% when compared with a nasopharyngeal aspirate for the detection of influenza viruses in children, and the method is more pleasant for the patient and easy to perform in any setting [START_REF] Heikkinen | Comparative study of nasopharyngeal aspirate and nasal swab specimens for detection of influenza[END_REF][START_REF] Heikkinen | Nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses[END_REF]. The sensitivity of nasal swabs may be further enhanced by using flocked swabs [START_REF] Abu-Diab | Comparison between pernasal flocked swabs and nasopharyngeal aspirates for detection of common respiratory viruses in samples from children[END_REF][START_REF] Chan | Comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children[END_REF] or collecting the swab from the nasopharynx [START_REF] Agoritsas | Evaluation of the Quidel QuickVue test for detection of influenza A and B viruses in the pediatric emergency medicine setting by use of three specimen collection methods[END_REF].

We found the sensitivity of the rapid test to be high for influenza A virus but significantly lower for influenza B virus. Such significant differences were not seen in another study with Actim Influenza A&B rapid test, in which the sensitivities for influenza A and B viruses were 69% and 60%, respectively [START_REF] Ghebremedhin | Comparison of the performance of the rapid antigen detection actim Influenza A&B test and RT-PCR in different respiratory specimens[END_REF]. There are, however, previous reports of a lower sensitivity for influenza B virus with other rapid tests performed at different stages of illness. [START_REF] Cazacu | Comparison of lateral-flow immunoassay and enzyme immunoassay with viral culture for rapid detection of influenza virus in nasal wash specimens from children[END_REF][START_REF] Hurt | Performance of six influenza rapid tests in detecting human influenza in clinical specimens[END_REF][START_REF] Cruz | Rapid assays for the diagnosis of influenza A and B viruses in patients evaluated at a large tertiary care children's hospital during two consecutive winter seasons[END_REF]. One potential explanation for the varying sensitivities of rapid tests for influenza A and B viruses was provided by a recent study from Hong Kong that demonstrated that viral shedding in naturally acquired influenza A infections peaked already at the time of symptom onset, whereas in influenza B infections the peak was reached only 3 days after the onset of symptoms [START_REF] Lau | Viral shedding and clinical illness in naturally acquired influenza virus infections[END_REF]. Besides varying rates of viral replication, different assay methods and immunological differences might potentially interfere with the detection of influenza B viruses. In our study, however, the total number of children infected with influenza B viruses was too low for any firm conclusions to be made, and further studies in this area are warranted.

The main strength of the present study is that it was performed in real-life conditions. The patients were unselected young children who were examined at a primary care clinic during the acute phase of their illness. The rapid tests were performed immediately at the point of care by trained nurses, which reflects the actual situation in most clinical settings. In many previous studies, the rapid tests have been evaluated by using stored specimens in controlled laboratory conditions.

Our study has some limitations. We used flocked swabs for obtaining samples for the virology laboratory but a polyester swab included in the kit for the rapid test. This might have resulted in better-quality reference samples and thus underestimation of the sensitivity of the rapid test.

However, using the exact swabs provided with the kit more likely represents the test´s true performance in the clinical practice. A total of three or four nasal swab specimens were obtained from the children during each visit, and consequently the representativeness of the samples may have varied. In addition, most of the follow-up samples were collected on study days 6-7. At that time, the viral load may have already been declining, and therefore we may have missed some children who actually had influenza. However, all samples that remained negative with viral culture and TR-FIA were further tested with RT-PCR and therefore we believe that potential variations in the quality of the samples or in the concentrations of viruses in the specimens are unlikely to have significantly affected our results. Finally, due to practical reasons, we obtained specimens for rapid testing only in approximately one-fourth of all cases.

However, we have no reason to believe that this would have resulted in any major selection bias because the decisions to perform the rapid tests were made independently by several study physicians who worked at varying times, and there were no recommendations about which children should be preferably sampled for rapid testing.

In conclusion, our results indicate that influenza can be reliably diagnosed in children 1-3 years of age already during the first 24 hours of illness. Further, the influenza rapid test had a high specificity and sensitivity for diagnosing influenza A viruses, but its sensitivity for influenza B viruses was poor.

  Of 409 children randomized to receive the study medication, influenza was eventually confirmed by any of the conventional laboratory methods (viral isolation, TR-FIA, or RT-PCR) in 98 children. Thirty-seven children who had received oseltamivir were excluded from this analysis. Of the remaining 61 placebo-treated children included in the analysis, 50 (82%) had influenza A and 11 (18%) influenza B. The diagnosis was based on viral culture in 54 (89%) children; antigen detection (TR-FIA) provided the diagnosis in 2 (3%) children with negative viral culture, and RT-PCR in 5 (8%) children in whom both viral culture and antigen detection were negative.

TABLE 1 .

 1 Sensitivity, specificity, and predictive values, according to influenza virus type, of 158 rapid influenza tests performed within 24 hours of the onset of symptoms.a Composite of viral culture, antigen detection and RT-PCR was used as the reference test. The reference test was considered positive if influenza was detected by any of these methods.b Including one false positive that remained negative with all reference methods.

ACKNOWLEDGEMENTS 15 F. Hoffmann-La Roche Ltd. (Basel, Switzerland) provided financial support to the Hospital District of Southwest Finland (Turku University Hospital) for carrying out this investigatorinitiated study. Dr Heinonen was supported by grants from Turku University Hospital Foundation and the Foundation for Paediatric Research. The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation of the manuscript. The authors declare that they have no conflict of interest.