High resolution mass spectrometric suspect screening, wide-scope target analysis of emerging contaminants and determination of legacy pollutants in adult black-tailed godwit Limosa limosa limosa in the Netherlands - A pilot study.

The Dutch breeding population of the black-tailed godwit Limosa limosa limosa has declined substantially over recent decades; the role of contaminants is unknown. We analysed liver samples from 11 adult birds found dead on their breeding grounds in SW Friesland 2016-2020, six from extensive, herb-rich grasslands, five from intensive grasslands. We carried out LC and GC wide-scope target analysis of more than 2400 substances, LC suspect screening for more than 60,000 substances, target analysis for Cd, Hg, Ni and Pb, organo-phosphate flame retardants (OPFRs), dechlorane plus compounds and selected polybrominated diphenyl ether flame retardants (PBDEs), and bioassay for polybrominated dibenzo-p-dioxins and dibenzofurans (PBDDs/PDBFs) and dioxin-like polychlorinated biphenyls (dl-PCBs). Residues of 29 emerging contaminants (ECs) were determined through wide-scope target analysis. Another 20 were tentatively identified through suspect screening. These contaminants include industrial chemicals (personal care products, surfactants, PAHs and others), plant protection products (PPPs) and pharmaceuticals and their transformation products. Total contaminant load detected by wide-scope target analysis ranged from c. 155 to c. 1400 ng g-1 and was generally lower in birds from extensive grasslands. Heatmaps suggest that birds from intensive grasslands have a greater mix and higher residue concentrations of PPPs, while birds from extensive grasslands have a greater mix and higher residue concentrations of per- and polyfluoroalkyl substances (PFAS). All four metals and two OPFRs were detected. All tested PBDEs were below the respective LODs. Bioassay revealed presence of PBDDs, PBDFs and dl-PCBs. Further research is required to elucidate potential health risks to godwits and contaminant sources.

Benthic primary producers are key to sustain the Wadden Sea food web: stable carbon isotope analysis at landscape scale.

Coastal food webs can be supported by local benthic or pelagic primary producers and by the import of organic matter. Distinguishing between these energy sources is essential for our understanding of ecosystem functioning. However, the relative contribution of these components to the food web at the landscape scale is often unclear, as many studies lack good taxonomic and spatial resolution across large areas. Here, using stable carbon isotopes, we report on the primary carbon sources for consumers and their spatial variability across one of the world's largest intertidal ecosystems (Dutch Wadden Sea; 1460 km2 intertidal surface area), at an exceptionally high taxonomic (178 species) and spatial resolution (9,165 samples from 839 locations). The absence of overlap in δ13 C values between consumers and terrestrial organic matter suggests that benthic and pelagic producers dominate carbon input into this food web. In combination with the consistent enrichment of benthic primary producers (δ13 C -16.3‰) relative to pelagic primary producers (δ13 C -18.8) across the landscape, this allowed the use of a two-food-source isotope-mixing model. This spatially resolved modelling revealed that benthic primary producers (microphytobenthos) are the most important energy source for the majority of consumers at higher trophic levels (worms, molluscs, crustaceans, fish, and birds), and thus to the whole food web. In addition, we found large spatial heterogeneity in the δ13 C values of benthic primary producers (δ13 C -19.2 to -11.5‰) and primary consumers (δ13 C -25.5 to -9.9‰), emphasizing the need for spatially explicit sampling of benthic and pelagic primary producers in coastal ecosystems. Our findings have important implications for our understanding of the functioning of ecological networks and for the management of coastal ecosystems.

Independence among physiological traits suggests flexibility in the face of ecological demands on phenotypes.

Phenotypic flexibility allows animals to adjust their physiology to diverse environmental conditions encountered over the year. Examining how these varying traits covary gives insights into potential constraints or freedoms that may shape evolutionary trajectories. In this study, we examined relationships among haematocrit, baseline corticosterone concentration, constitutive immune function and basal metabolic rate in red knot Calidris canutus islandica individuals subjected to experimentally manipulated temperature treatments over an entire annual cycle. If covariation among traits is constrained, we predict consistent covariation within and among individuals. We further predict consistent correlations between physiological and metabolic traits if constraints underlie species-level patterns found along the slow-fast pace-of-life continuum. We found no consistent correlations among haematocrit, baseline corticosterone concentration, immune function and basal metabolic rate either within or among individuals. This provides no evidence for constraints limiting relationships among these measures of the cardiovascular, endocrine, immune and metabolic systems in individual red knots. Rather, our data suggest that knots are free to adjust individual parts of their physiology independently. This makes good sense if one places the animal within its ecological context where different aspects of the environment might put different pressures on different aspects of physiology.

Spatially structured genetic variation in a broadcast spawning bivalve: quantitative vs. molecular traits.

Understanding the origin, maintenance and significance of phenotypic variation is one of the central issues in evolutionary biology. An ongoing discussion focuses on the relative roles of isolation and selection as being at the heart of genetically based spatial variation. We address this issue in a representative of a taxon group in which isolation is unlikely: a marine broadcast spawning invertebrate. During the free-swimming larval phase, dispersal is potentially very large. For such taxa, small-scale population genetic structuring in neutral molecular markers tends to be limited, conform expectations. Small-scale differentiation of selective traits is expected to be hindered by the putatively high gene flow. We determined the geographical distribution of molecular markers and of variation in a shell shape measure, globosity, for the bivalve Macoma balthica (L.) in the western Dutch Wadden Sea and adjacent North Sea in three subsequent years, and found that shells of this clam are more globose in the Wadden Sea. By rearing clams in a common garden in the laboratory starting from the gamete phase, we show that the ecotypes are genetically different; heritability is estimated at 23%. The proportion of total genetic variation that is between sites is much larger for the morphological additive genetic variation (QST = 0.416) than for allozyme (FST = 0.000-0.022) and mitochondrial DNA cytochrome-c-oxidase-1 sequence variation (phiST = 0.017). Divergent selection must be involved and intraspecific spatial genetic differentiation in marine broadcast spawners is apparently not constrained by low levels of isolation.

Carrying large fuel loads during sustained bird flight is cheaper than expected.

Birds on migration alternate between consuming fuel stores during flights and accumulating fuel stores during stopovers. The optimal timing and length of flights and stopovers for successful migration depend heavily on the extra metabolic power input (fuel use) required to carry the fuel stores during flight. The effect of large fuel loads on metabolic power input has never been empirically determined. We measured the total metabolic power input of a long-distance migrant, the red knot (Calidris canutus), flying for 6 to 10 h in a wind tunnel, using the doubly labelled water technique. Here we show that total metabolic power input increased with fuel load, but proportionally less than the predicted mechanical power output from the flight muscles. The most likely explanation is that the efficiency with which metabolic power input is converted into mechanical output by the flight muscles increases with fuel load. This will influence current models of bird flight and bird migration. It may also help to explain why some shorebirds, despite the high metabolic power input required to fly, routinely make nonstop flights of 4,000 km longer.

A sport-physiological perspective on bird migration: evidence for flight-induced muscle damage.

Exercise-induced muscle damage is a well-described consequence of strenuous exercise, but its potential importance in the evolution of animal activity patterns is unknown. We used plasma creatine kinase (CK) activity as an indicator of muscle damage to investigate whether the high intensity, long-duration flights of two migratory shorebird species cause muscle damage that must be repaired during stopover. In two years of study, plasma CK activity was significantly higher in migrating western sandpipers (a non-synchronous, short-hop migrant), than in non-migrants. Similarly, in the bar-tailed godwit (a synchronous, long-jump migrant), plasma CK activity was highest immediately after arrival from a 4000-5000km flight from West Africa to The Netherlands, and declined before departure for the arctic breeding areas. Late-arriving godwits had higher plasma CK activity than birds that had been at the stopover site longer. Juvenile western sandpipers making their first southward migration had higher plasma CK activity than adults. These results indicate that muscle damage occurs during migration, and that it is exacerbated in young, relatively untrained birds. However, the magnitude of the increases in plasma CK activity associated with migratory flight were relatively small, suggesting that the level of muscle damage is moderate. Migrants may avoid damage behaviourally, or have efficient biochemical and physiological defences against muscle injury.

Time course and reversibility of changes in the gizzards of red knots alternately eating hard and soft food.

The ability to change organ size reversibly can be advantageous to birds that perform long migrations. During winter, red knots (Calidris canutus) feed on shellfish and carry a muscular gizzard that weighs 10% of their body mass. Gizzard size decreases when these birds eat soft foods, e.g. while breeding in the tundra. We studied the reversibility and time course of such changes using ultrasonography. Two groups of shellfish-adapted knots (N=9 and N=10) were fed alternately a hard and a soft food type. Diet switches elicited rapid reversible changes. Switches from hard to soft food induced decreases to 60% of initial gizzard mass within 8.5 days, while switches to hard food induced increases in gizzard mass to 147% within 6.2 days. A third group of knots (N=11), adapted to soft food for more than 1 year, initially had very small gizzards (25% of the mass of shellfish-adapted gizzards), but showed a similar capacity to increase gizzard size when fed shellfish. This is the first non-invasive study showing rapid digestive organ adjustments in non-domesticated birds.

Is long-distance bird flight equivalent to a high-energy fast? Body composition changes in freely migrating and captive fasting great knots.

We studied changes in body composition in great knots, Calidris tenuirostris, before and after a migratory flight of 5,400 km from northwest Australia to eastern China. We also took premigratory birds into captivity and fasted them down to their equivalent arrival mass after migration to compare organ changes and nutrient use in a low-energy-turnover fast with a high-energy-turnover fast (migratory flight). Migrated birds were as economical as any fasting animal measured yet at conserving protein: their estimated relative protein contribution (RPC) to the energy used was 4.0%. Fasted birds had an estimated RPC of 6.8% and, consequently, a much lower lean mass and higher fat content for an equivalent body mass than migrated birds. Lean tissue was catabolized from most organs in both groups, except the brain. Furthermore, a principal components biplot showed that individuals were grouped primarily on the basis of overall organ fat or lean tissue content rather than by the size of specific organs. This indicates that organ changes during migratory flight are similar to those of a low-energy fast, although the length of the fast in this study probably accentuated organ reductions in some functional groups. Whether the metabolic characteristics of a flying migratory fast follow the three-phase model described in many inactive fasting animals is unclear. We have some evidence for skeletal fat being catabolized without phase 3 of a fast having been reached.

Breeding plumage honestly signals likelihood of tapeworm infestation in females of a long-distance migrating shorebird, the bar-tailed godwit.

The indicator mechanism for sexual selection proposed by Hamilton and Zuk (i.e. that sexually selected ornaments signal parasite resistance) has received rather little observational support, and none in the case of long-distance migrant birds. Here we present a test by examining the association between helminth infestations and breeding plumage quality in bar-tailed godwits Limosa lapponica taymyrensis during their spring staging period in the Wadden Sea, The Netherlands. After a non-stop flight from West Africa, these shorebirds refuel in the Wadden Sea in preparation for a second flight to the central Siberian Arctic breeding grounds. Earlier studies have shown that only relatively heavy and well ornamented birds carry out a "top-up" moult during stopover, in which part of the contour feathers recently grown in West Africa are replaced by even fresher ones. Active body moult was therefore taken as the primary indicator of ornament quality. Of 78 birds collected between 1992 and 1997, 42% carried helminths, including four species of digenean trematodes (flukes), three species of cestodes (tapeworms) and an acanthocephalan (spiny-headed worm). Faecal samples examined for helminth eggs in another 92 birds in 1998 and 2000 showed similar rates of infestation. Actively moulting bar-tailed godwits were confirmed to be heavier and to show more extensive breeding plumage than non-moulting birds. In females, but not in males, active moult was associated with fewer cestodes and acanthocephalans. Also, breeding plumage and presence of cestodes were negatively associated in females. We argue that the quality of the breeding plumage reliably indicates parasite resistance in female godwits. The repeatability of plumage scores of females between years is consistent with such resistance having a heritable component. In contrast, male ornaments may demonstrate other qualities, e.g. an ability to combine adequate fuelling and flight performances with moult during the time-stress of migration.

Baseline corticosterone peaks in shorebirds with maximal energy stores for migration: a general preparatory mechanism for rapid behavioral and metabolic transitions?

In captive red knots (Calidris canutus, Scolopacidae) showing a regulated body mass increase of 50% related to their migration from temperate staging sites to tundra breeding grounds, plasma corticosterone concentrations increased from less than 10 ng. ml(-1) to levels as high as 30 ng. ml(-1) when the energy storage for migration was complete. These birds did not fly, but concentrations dropped to very low levels (<5 ng. ml(-1)) as soon as the birds started their voluntary fasts to the low body masses preceding the early wing and body molts normally occurring after an unsuccessful breeding season. As the elevated levels of corticosterone are associated with stable body mass rather than with the preceding increase or subsequent decrease, it is suggested that a major role of corticosterone during the final stages just before departure may be to prepare birds for long-distance flights. Birds heading into the Arctic to breed face potentially arduous flights into unpredictable environmental and social conditions. Activation of the hypothalamic-pituitary-adrenal axis, as measured by elevated levels of corticosterone, may induce the suite of behavioral and metabolic changes necessary to negotiate these challenges successfully.

Ingested water equilibrates isotopically with the body water pool of a shorebird with unrivaled water fluxes.

We investigated the applicability of (2)H to measure the amount of body water (TBW) and water fluxes in relation to diet type and level of food intake in a mollusk-eating shorebird, the Red Knot (Calidris canutus). Six birds were exposed to eight experimental indoor conditions. Average fractional (2)H turnover rates ranged between 0. 182 day(-1) (SD = 0.0219) for fasting birds and 7.759 day(-1) (SD = 0.4535) for birds feeding on cockles (Cerastoderma edule). Average TBW estimates obtained with the plateau method were within the narrow range of 75.9-85.4 g (or between 64.6 and 70.1% of the body mass). Those obtained with the extrapolation method showed strong day-to-day variations (range 55.7-83.7 g, or between 49.7 and 65.5%). Average difference between the two calculation methods ranged between 0.6% and 36.3%, and this difference was strongly negatively correlated with water flux rate. Average water influx rates ranged between 15.5 g/day (fasting) and 624.5 g/day (feeding on cockles). The latter value is at 26.6 times the allometrically predicted value and is the highest reported to date. Differences in (2)H concentrations between the blood and feces (i.e., biological fractionation) were small but significant (-3.4% when fed a pellet diet, and -1.1% for all the other diets), and did not relate to the rate of water flux (chi(2)(1) = 0.058, P < 0.81). We conclude that the ingested water equilibrated rapidly with the body water pool even in an avian species that shows record water flux rates when living on ingested marine bivalves.

Molecular analysis of intact preen waxes of Calidris canutus (Aves: Scolopacidae) by gas chromatography/mass spectrometry.

The intact preen wax esters of the red knot Calidris canutus were studied with gas chromatography/mass spectrometry (GC/MS) and GC/MS/MS. In this latter technique, transitions from the molecular ion to fragment ions representing the fatty acid moiety of the wax esters were measured, providing additional resolution to the analysis of wax esters. The C21-C32 wax esters are composed of complex mixtures of hundreds of individual isomers. The odd carbon-numbered wax esters are predominantly composed of even carbon-numbered n-alcohols (C14, C16, and C18) esterified predominantly with odd carbon-numbered 2-methyl fatty acids (C7, C9, C11, and C13), resulting in relatively simple distributions. The even carbon-numbered wax esters show a far more complex distribution due to a number of factors: (i) Their n-alcohol moieties are not dominated by even carbon-numbered n-alcohols esterified with odd carbon-numbered 2-methyl fatty acids, but odd and even carbon-numbered n-alcohols participate in approximately equal amounts; (ii) odd carbon-numbered methyl-branched alcohols participate abundantly in these wax ester clusters; and (iii) with increasing molecular weight, various isomers of the 2,6-, 2,8-, and 2,10-dimethyl branched fatty acids also participate in the even carbon-numbered wax esters. The data demonstrate that there is a clear biosynthetic control on the wax ester composition although the reasons for the complex chemistry of the waxes are not yet understood.

Structural identification of the diester preen-gland waxes of the red knot (Calidris canutus).

The intact C(32)-C(48) diester wax esters of the preen gland of the migrating bird Calidris canutus are shown, using synthesized standards, to comprise predominantly C(12)-C(16) alkane-1,2-diols esterified with octanoic, decanoic, and dodecanoic acid at one position, and with predominantly even-numbered carbon fatty acids at the other position.

Avian pectoral muscle size rapidly tracks body mass changes during flight, fasting and fuelling.

We used ultrasonic imaging to monitor short-term changes in the pectoral muscle size of captive red knots Calidris canutus. Pectoral muscle thickness changed rapidly and consistently in parallel with body mass changes caused by flight, fasting and fuelling. Four knots flew repeatedly for 10 h periods in a wind tunnel. Over this period, pectoral muscle thickness decreased in parallel with the decrease in body mass. The change in pectoral muscle thickness during flight was indistinguishable from that during periods of natural and experimental fasting and fuelling. The body-mass-related variation in pectoral muscle thickness between and within individuals was not related to the amount of flight, indicating that changes in avian muscle do not require power-training as in mammals. Our study suggests that it is possible for birds to consume and replace their flight muscles on a time scale short enough to allow these muscles to be used as part of the energy supply for migratory flight. The adaptive significance of the changes in pectoral muscle mass cannot be explained by reproductive needs since our knots were in the early winter phase of their annual cycle. Instead, pectoral muscle mass changes may reflect (i) the breakdown of protein during heavy exercise and its subsequent restoration, (ii) the regulation of flight capacity to maintain optimal flight performance when body mass varies, or (iii) the need for a particular protein:fat ratio in winter survival stores.

Empirical evidence for differential organ reductions during trans-oceanic bird flight.

Since the early 1960s it has been held that migrating birds deposit and use only fat as fuel during migratory flight, with the non-fat portion of the body remaining homeostatic. Recent evidence from field studies has shown large changes in organ sizes in fuelling birds, and theory on fuel use suggests protein may be a necessary fuel during flight. However, an absence of information on the body condition of migrants before and after a long flight has hampered understanding of the dynamics of organs during sustained flight. We studied body condition in a medium-sized shorebird, the great knot (Calidris tenuirostris), before and after a flight of 5400 km from Australia to China during northward migration. Not only did these birds show the expected large reduction in fat content after migration, there was also a decrease in lean tissue mass, with significant decreases in seven organs. The reduction in functional components is reflected in a lowering of the basal metabolic rate by 42% [corrected]. Recent flight models have tried to separate the 'flexible' part of the body from the constant portion. Our results suggest that apart from brains and lungs no organs are homeostatic during long-distance flight. Such organ reductions may be a crucial adaptation for long-distance flight in birds.

Body-building without power training: endogenously regulated pectoral muscle hypertrophy in confined shorebirds

Shorebirds such as red knots Calidris canutus routinely make migratory flights of 3000 km or more. Previous studies on this species, based on compositional analyses, suggest extensive pectoral muscle hypertrophy in addition to fat storage before take-off. Such hypertrophy could be due to power training and/or be effected by an endogenous circannual rhythm. Red knots of two subspecies with contrasting migration patterns were placed in a climate-controlled aviary (12 h:12 h L:D photoperiod) where exercise was limited. Using ultrasonography, we measured pectoral muscle size as the birds stored fat in preparation for migration. At capture, there were no differences in body mass and pectoral muscle mass between the two subspecies. As they prepared for southward and northward migration, respectively, the tropically wintering subspecies (C. c. canutus) gained 31 g and the temperate wintering subspecies (C. c. islandica) gained 41 g. During this time, pectoral mass increased by 43-44 % of initial mass, representing 39 % (C. c. canutus) and 29 % (C. c. islandica) of the increase in body mass. The gizzard showed atrophy in conjunction with a diet change from molluscs to food pellets. Although we cannot exclude the possibility that the birds' limited movement may still be a prerequisite for pectoral muscle hypertrophy, extensive power training is certainly not a requirement. Muscle hypertrophy in the absence of photoperiod cues suggests the involvement of an endogenous circannual process.

Rapid changes in the size of different functional organ and muscle groups during refueling in a long-distance migrating shorebird.

The adaptive value of size changes in different organ and muscle groups was studied in red knots (Calidris canutus islandica) in relation to their migration. Birds were sampled on five occasions: at arrival in Iceland in May 1994, two times during subsequent refueling, at departure toward, and on return from, the high arctic breeding grounds. During their 24-d stopover in May, body mass increased from 144.3 to 214.5 g. Mass gains were lowest over the first week (0.85 g/d, only fat-free tissue deposited). Over the subsequent 10 d, average mass increased by 5.0 g/d (fat contributing 78%), and over the last week before takeoff, it increased by 2.0 g/d (fat contributing over 100% because of loss of lean components). There were no sex differences in body and fat mass gains. Over the first interval, lean masses of heart, stomach, and liver increased. During the middle 10 d, sizes of leg muscle, intestine, liver, and kidneys increased. Stomach mass decreased over the same interval. In the last interval before takeoff, the stomach atrophied further and the intestine, leg muscles, and liver became smaller too, but pectoral muscles and heart increased in size. Sizes of "exercise organs" such as pectoral muscle and heart were best correlated with body mass, whereas sizes of organs used during foraging (leg muscles) and nutrient extraction (intestine, liver) were best correlated with rate of mass gain. Kidneys changed little before takeoff, which suggests that they are needed as much during flight as during refueling.

Estimating organ size in small migrating shorebirds with ultrasonography: An intercalibration exercise.

Organs, even of fully grown adult birds, mammals, and reptiles, may show substantial size changes in relation to specific performances. These changes are difficult to study, because measurements usually can only be obtained following the death of the animal. We explored the use of ultrasonographic imaging, a relatively simple noninvasive technique, to measure size of pectoral muscles and stomach in two small shorebird species (red knots Calidris canutus and golden plovers Pluvialis apricaria). Accuracy of ultrasound measurements in estimating organ mass in red knots was reasonably high. Depending on the equipment used, the error of individual measurements was 20%-25% for the pectoral muscles and 26%-44% for the stomach. In plovers the technique was less accurate, probably because of the low variability of the organs involved. Ultrasound scanning is particularly suited to measure rapidly changing organ sizes over short time intervals. We demonstrate this with an example in which changes in individuals in size of pectoral muscle and stomach were monitored in captive red knots following a change in diet. Ultrasound measures will enable studies on the links between body composition and future behavior and physiology.

Rapid reversible changes in organ size as a component of adaptive behaviour.

Organ structures and correlated metabolic features (e.g. metabolic rate) have often taken as fixed attributes of fully grown individual vertebrates. When measurements of these attributes became available they were often used as representative values for the species, disregarding the specific conditions during which the mesurement were made. Evidence is accumulating that the functional size of organs and aspects of the metabolic physiology of an individual may show great flexibility over timescales of weeks and even days depending on physiological status, environmental conditions and behavioural goals. This flexibility is a way for animals to cope successfully with a much wider range of conditions occurring during various life-cycle events than fixed metabolic machinery would allow. Such phenotypic flexibility is likely to be a common adaptive syndrome, typical of vertebrates living in variable environments.