Assessment of body condition of harp seals

We investigated the relationships between blubber content and a suite of condition indices in harp seals (Phoca groenlandica). Although blubber content was significantly correlated with xiphosternal blubber depth and Condition index they were insensitive indicators of condition. Blubber depth was most variable at a site ca. 60–70% down the length of the body and a more recent index incorporating length, mass and blubber depth devised for phocids was an improved indicator of blubbler mass. Blubber volume was calculated in three ways based on a geometric approximation of a cylinder and also via truncated cones. The cone method accounted for more variance than the other methods although it overestimated blubber mass. Surface area was ascertained using the girth measurements of serial cones and was significantly correlated with total body mass and total body water. We suggest that estimates of total body water generated from surface area could be used to calculate total fat mass and lean body mass     

Factors influencing the radiative surface temperature of grey seal (<Emphasis Type="Italic">Halichoerus grypus</Emphasis>) pups during early and late lactation

The aim of this study was to examine the variation in body surface temperature of grey seal (Halichoerus grypus) pups throughout lactation in response to different environmental conditions. Radiative surface temperatures (T _r, °C) of pups were measured on the Isle of May (56°11′N, 02°33′W), southeast Scotland from 29 October to 25 November 2003. Records were obtained from a total of 60 pups (32 female and 28 male) from three different pupping sites during early and late lactation. Pups were sheltered from high wind speeds but air temperature, humidity and solar radiation at pupping sites were similar to general meteorological conditions. The mean T _r of all pups was 15.8°C (range 7.7–29.7°C) at an average air temperature of 10.2°C (range 6.5–13.8°C). There was no difference in the mean T _r of pups between early and late lactation. However, the T _r varied between different regions of the body with hind flippers on average 2–6°C warmer than all other areas measured. There was no difference in mean T _r of male and female pups and pup body mass did not account for the variation in T _r during early or late lactation. Throughout the day there was an increase in the T _r of pups and this explained 20–28% of the variation in T _r depending on stage of lactation. There was no difference in the mean T _r of pups between pupping sites or associated with different substrate types. Wind speed and substrate temperature had no effect on the T _r of pups. However, solar radiation, air temperature and relative humidity accounted for 48% of the variation in mean T _r of pups during early lactation. During late lactation air temperature and solar radiation alone accounted for 43% of the variation in T _r. These results indicate that environmental conditions explain only some of the variation in T _r of grey seal pups in natural conditions. Differences in T _r however indicate that the cost of thermoregulation for pups will vary throughout lactation. Further studies examining intrinsic factors such as blubber thickness and activity levels are necessary before developing reliable biophysical models for grey seals.     

Variation in the fatty acid composition of blubber in Cape fur seals (<Emphasis Type="Italic">Arctocephalus pusillus pusillus</Emphasis>) and the implications for dietary interpretation

Analysis of the fatty acid (FA) composition of blubber is a valuable tool in interpreting the diet of marine mammals. This technique is based on the principle that particular FA present in prey can be incorporated largely untransformed into predator adipose tissue stores, thereby providing biochemical signatures with which to identify prey species. Several studies of phocid seals and cetaceans have documented vertical stratification in the FA composition of blubber such that inferences about diet may vary greatly depending on the layer of the blubber that is analysed. It is not known whether blubber in otariid seals (fur seals and sea lions) also displays vertical stratification in FA composition. Furthermore, it is not known whether the FA composition of blubber is uniform in these species. In the present study, the vertical and regional variation in FA composition of blubber was investigated in seven adult female Cape fur seals (Arctocephalus pusillus pusillus). The proportion of monounsaturated fatty acids (MUFA) was greater in the outer (43.6±1.3%) than inner portion (40.9±1.2%; t₂₀=5.59, P<0.001) whereas the proportions were greater in the inner than outer portions for saturated fatty acids (23.6±0.5% and 21.9±0.6%, respectively, t₂₀ = 5.31, P<0.001) and polyunsaturated fatty acids (PUFA, 35.5±0.7% and 34.5±0.7%, respectively, t₂₀ = 3.81, P < 0.001). There was an inverse relationship between MUFA and PUFA in the blubber, independent of sampling location. In addition, with the exception of the inner portion from non-lactating females, blubber from the mammary area had the highest proportions of 18:19c and total MUFA, followed by blubber from the rump and neck, suggesting that the deposition and mobilisation of blubber lipids may not be uniform around the body in otariid seals. These results support the need for blubber tissue to be sampled from the same site on animals, and to the full depth of the blubber layer, to minimise variation in FA profiles that could occur if different sites and depths were sampled. Such standardisation of sampling will further aid in interpreting diet in otariid seals using the FA Signature Analysis approach.     

Comparisons and Uncertainty in Fat and Adipose Tissue Estimation Techniques: The Northern Elephant Seal as a Case Study

Fat mass and body condition are important metrics in bioenergetics and physiological studies. They can also link foraging success with demographic rates, making them key components of models that predict population-level outcomes of environmental change. Therefore, it is important to incorporate uncertainty in physiological indicators if results will lead to species management decisions. Maternal fat mass in elephant seals (Mirounga spp) can predict reproductive rate and pup survival, but no one has quantified or identified the sources of uncertainty for the two fat mass estimation techniques (labeled-water and truncated cones). The current cones method can provide estimates of proportion adipose tissue in adult females and proportion fat of juveniles in northern elephant seals (M. angustirostris) comparable to labeled-water methods, but it does not work for all cases or species. We reviewed components and assumptions of the technique via measurements of seven early-molt and seven late-molt adult females. We show that seals are elliptical on land, rather than the assumed circular shape, and skin may account for a high proportion of what is often defined as blubber. Also, blubber extends past the neck-to-pelvis region, and comparisons of new and old ultrasound instrumentation indicate previous measurements of sculp thickness may be biased low. Accounting for such differences, and incorporating new measurements of blubber density and proportion of fat in blubber, we propose a modified cones method that can isolate blubber from non-blubber adipose tissue and separate fat into skin, blubber, and core compartments. Lastly, we found that adipose tissue and fat estimates using tritiated water may be biased high during the early molt. Both the tritiated water and modified cones methods had high, but reducible, uncertainty. The improved cones method for estimating body condition allows for more accurate quantification of the various tissue masses and may also be transferrable to other species.     

Experimental evidence of seawater drinking in juvenile hooded (Cystophora cristata) and harp seals (Phoca groenlandica)

This study was undertaken to measure whether young harp seals (Phoca groenlandica) and hooded seals (Cystophora cristata) drink seawater and, if so, to investigate how the excess salt load is handled. Blood and urine samples were collected from hooded seal pups (n=3) and harp seal pups (n=3) after 2 weeks of freshwater exposure, at intervals during 3 weeks of seawater exposure and, finally, after 2 weeks of re-exposure to fresh water. Total water turnover, as measured by injection of tritiated water, was 2200 ml · day⁻¹ and 3300 ml · day⁻¹ in hooded seals and harp seals, respectively. The extent of mariposia was taken as the difference between total water turnover and influx of water through food (free and metabolic water) and respiratory water exchange. Seawater drinking amounted to 14% and 27% of total water turnover (r_H2O) for the hooded seals and harp seals, respectively. Further evidence of mariposia was obtained from an increase in the excretion rate of the urine osmolytes Na⁺, Cl⁻ and Mg²⁺, during the period of seawater exposure. It is concluded that water influx due to seawater drinking can not be excluded as a source of error when estimating food consumption of free-ranging harp seals and hooded seals, by use of labeled water techniques. Accepted: 11 May 2000     

Investigating Annual Diving Behaviour by Hooded Seals (Cystophora cristata) within the Northwest Atlantic Ocean

With the exception of relatively brief periods when they reproduce and moult, hooded seals, Cystophora cristata, spend most of the year in the open ocean where they undergo feeding migrations to either recover or prepare for the next fasting period. Valuable insights into habitat use and diving behaviour during these periods have been obtained by attaching Satellite Relay Data Loggers (SRDLs) to 51 Northwest (NW) Atlantic hooded seals (33 females and 18 males) during ice-bound fasting periods (2004−2008). Using General Additive Models (GAMs) we describe habitat use in terms of First Passage Time (FPT) and analyse how bathymetry, seasonality and FPT influence the hooded seals’ diving behaviour described by maximum dive depth, dive duration and surface duration. Adult NW Atlantic hooded seals exhibit a change in diving activity in areas where they spend >20 h by increasing maximum dive depth, dive duration and surface duration, indicating a restricted search behaviour. We found that male and female hooded seals are spatially segregated and that diving behaviour varies between sexes in relation to habitat properties and seasonality. Migration periods are described by increased dive duration for both sexes with a peak in May, October and January. Males demonstrated an increase in dive depth and dive duration towards May (post-breeding/pre-moult) and August–October (post-moult/pre-breeding) but did not show any pronounced increase in surface duration. Females dived deepest and had the highest surface duration between December and January (post-moult/pre-breeding). Our results suggest that the smaller females may have a greater need to recover from dives than that of the larger males. Horizontal segregation could have evolved as a result of a resource partitioning strategy to avoid sexual competition or that the energy requirements of males and females are different due to different energy expenditure during fasting periods.     

Fat transfer and energetics during lactation in the hooded seal: the roles of tissue lipoprotein lipase in milk fat secretion and pup blubber deposition

Hooded seals (Cystophora cristata) lactate for 3.6 days during which females simultaneously fast and transfer large amounts of energy to their pups through fat-rich milk. Pups grow rapidly, principally due to blubber deposition. Lipoprotein lipase (LPL), the primary enzyme responsible for tissue uptake of triglyceride fatty acids, may strongly influence both maternal milk fat secretion and pup blubber deposition. We measured the energetic costs of lactation (using hydrogen isotope dilution, ³H₂0), milk composition, prolactin, and LPL activity (post-heparin plasma LPL [PH LPL], blubber, mammary gland and milk; U) in six females. PH LPL and blubber LPL were measured in their pups. Females depleted 216.3 MJ · day⁻¹ of body energy and fat accounted for 59% of maternal mass loss and 90% of postpartum body energy loss, but maternal body composition changed little. Maternal blubber LPL was negligible (0.0–0.2 U), while mammary LPL was elevated (1.8–2.5 U) and was paralleled by changes in prolactin. Estimated total mammary LPL activity was high (up to 20,000 U · animal⁻¹) effectively favoring the mammary gland for lipid uptake. Levels of total blubber LPL in pups increased seven-fold over lactation. Pups with higher PH LPL at birth had greater relative growth rates (P = 0.025). Pups with greater blubber stores and total blubber LPL activity had elevated rates of fat deposition (P = 0.035). Accepted: 4 May 1999     

Ecological Characterisation of the Colonic Microbiota in Arctic and Sub-Arctic Seals

Dominant colonic bacteria in wild hooded (n = 9), harbour (n = 1) and grey (n = 1) seals were identified using 16S rRNA gene clone libraries (313 clones), revealing 52.7% Bacteroidetes, 41.5% Firmicutes, 4.5% Proteobacteria and 1.0% Fusobacteria. Thirty (77%) of the 39 phylotypes identified were novel, showing <97% sequence similarity to their nearest cultivated relatives. Mean colonic bacterial cell density, determined by real-time PCR, was high (12.8 log₁₀ cells/g wet wt) for the hooded seals, while the number of methanogenic Archea was low (4.0 log₁₀ cells/g wet wt). The level of ampicillin (amp^r) and tetracycline-resistant (tet^r) isolates was investigated by cultivation. Aerobic amp^r isolates were only detected in colon contents from four hooded seals, whereas aerobic tet^r isolates were found in seven of the nine hooded seals. These data provide novel insight to the gut microbiota of Arctic and sub-Arctic seals living in the wild.     

Seasonal variation in energy intake and condition of harp seals: Is there a harp seal morph? Problems for bioenergetic modelling

Seasonal changes in mass, fat depth, condition index and energy intake were measured in eight captive harp seals (Phoca groenlandica) over a 15-month period. Two of the five adult females were pregnant but lost their pups before term. There were large shifts in all variables measured. The range of these changes in the individual adult animals were for mass, 15–89%, blubber depth 56–240%, and condition index 13–23% (girth x 100)/length. Daily energy consumption varied from as little as 1.16 MJ to as much as 70.97 MJ. The two young males showed larger seasonal changes than the adults. Consistent with our earlier studies of harbour seals (Phoca vitulina) , there was either a significantly negative or no correlation between energy intake and changes in mass or fat. Mass and blubber thickness were positively related in the male seals, however, this was true of the females only during spring and summer. Water and air temperature varied indirectly with changes in blubber thickness in all but two seals. It is proposed that metabolic rate varies seasonally in seals. The implications of these findings are discussed in the light of their impact on bioenergetic modelling.     

SEASONAL CHANGES IN BLUBBER DISTRIBUTION IN ATLANTIC HARBOR SEALS: INDICATIONS OF THERMODYNAMIC CONSIDERATIONS

Among its functions, the hypodermal blubber layer of pinnipeds serves as both an energy reserve and insulation. This study examined seasonal changes in blubber distribution and body morphology in a group of captive changesharbor seals to test whether these changes were designed to maximize insulative effectiveness. Seasonal changes were found in girth, blubber volume, mean blubber depth, and the ratio of blubber depth to body radius (d/r ratio). These changes were more evident in older seals. The d/r ratio demonstrated a smaller relative annual change than mean blubber depth. The d/r ratio also exhibited less variation along the length of the seal than blubber depth at any given time. Similar to reports for ringed seals, and contrary to those for southern elephant seals, the harbor seals preferentially lost blubber from overinsulated areas of the body. These results suggest that core tissue and blubber mass are lost in a manner that maximizes insulative effectiveness.     

SEASONAL CHANGES IN FOOD INTAKE OF HARP SEALS (PHOCA GROENLANDICA) AT 69°N

Food intake (PI), body mass ( BM ), and compartmental growth were recorded for 12 mo in four captive 2–4-yr-old male harp seals ( Phoca groenlandica ), exposed to an artificial light regime that closely resembled natural day length at 69°. In early May before molting, both FZ and BM decreased in all four animals. Total body fat ( TBF ) declined from 51% of BM in March (n = 4) to 30% in August (n = 2), while total body water ( TBW ) concomitantly increased from 37% to 51% and total body protein ( TBP ) from 11% to 17%. In July FI started to increase, while BM started to increase in August. TBF increased while TBW and TBP decreased from August, all three parameters reaching a stable level in October at 47%, 39%, and 12%, respectively. Thereafter, body composition was maintained rather constant until May. Between October and March/April FI fluctuated for all animals, while BM showed a fairly steady increase. Average daily amount of capelin consumed was 2.67 kg·d⁻¹, equivalent to 25,600 kJ·d⁻¹, or 343 kJ·kg⁻¹·d⁻¹.     

Health and condition in the adult Weddell seal of McMurdo Sound, Antarctica

Weddell seals (Leptonychotes weddellii) are large-bodied, long-lived mammals that live only in the circumpolar Antarctic. As such, they represent an ideal sentinel species for the impacts of environmental change on polar species. However, longitudinal physiological studies of marine mammals are notoriously difficult due to their largely aquatic lifestyle, such that many baseline indicators of health and condition are not well defined. Fifty known-age, adult seals (9-27 years, 24 males, 26 females) in McMurdo Sound were sampled for mass, total body fat, blubber depth and a suite of blood parameters (21 variables) to assess hydration state, nutritional plane, reproductive hormones (females only), organ function and immune status. Total body mass increased with age, whereas variation in blubber depth was best described by mass and/or sex, with an overall greater blubber thickness in female seals (p < 0.001). Ten blood parameters showed a significant effect of one or more model variables, most prominently between the sexes. Serum chemistry suggested that females were better nourished and displayed lower indicators of acute stress compared to males. We noted limited age-related declines that might indicate impaired organ function. Overall, seals demonstrated clinically normal values for large mammals, even at the upper end of the age continuum. Future physiological studies in this species are strongly encouraged to incorporate potential sex effects in experimental design and analysis.     

THERMAL FUNCTION OF PHOCID SEAL FUR

In phocid seals, blubber serves as the main thermal insulation instead of fur. The thermal function of fur, at least in adult phocid seals, has therefore been questioned. We measured the relative contribution of fur to the combined thermal resistance (insulation) offered by blubber, skin, and fur in newborn and adult harp ( Pagophilus groenlandicus ) and hooded ( Cystophora cristata ) seals, in air and water, to elucidate the role of fur as insulation in phocid seals. In air the fur contributed 90% of the combined thermal resistance of blubber, skin, and fur in newborn harp seal pups and 29% in adulrs, whereas in hooded seals the fur contributed 73% in newborn pups and 34% in adults. When submerged the thermal resistance of the fur was reduced by 84%-92%, and contributed 65% to the total insulation in newborn harp seal pups and 3% in adults, and 26% in newborn hooded seal pups and 5% in adults. We conclude that in air the fur of phocid seals makes an important contribution to the insulation of pups, and also contributes considerably to the insulation of adult animals. In water, even though the thermal resistance of the fur is dramatically reduced, the fur still contributes substantially to the insulation of pups, but its contribution in adults is negligible.     

Variation in blubber fatty acid composition among marine mammals in the Canadian Arctic

The composition of predator adipose stores can provide important insights into foraging patterns and the ecological relationships among species. We determined the fatty acid (FA) composition of 843 blubber samples from 80 bearded seals (Erignathus barbatus), 33 harbor seals (Phoca vitulina), 239 harp seals (Pagophilus groenlandicus), 32 hooded seals (Cystophora cristata), 281 ringed seals (Phoca hispida), 53 walruses (Odobenus rosmarus rosmarus), 105 beluga whales (Delphinapterus leucas), and 20 narwhals (Monodon monoceros) across the Canadian Arctic to examine patterns of variability among and within species. FA signatures accurately distinguished phocid seals, walruses, and whales. Belugas and narwhals had the most similar FA signatures of any two species, suggesting substantial overlap in their diets, especially in the narwhal‐wintering area off eastern Baffin Island. Among phocid seals, harp and hooded seals had the most similar FA signatures. Bearded seals were most similar to walruses, which was consistent with the benthic feeding habits of both species. Within species, geographic differences in FA signatures were found over both large (>4,000 km) and small (<100 km) spatial scales. Overall, within‐species differences were smaller than among‐species differences. In general, FA signature patterns were consistent with previous studies of the ecology and diets of arctic marine mammals.     

Paternity exclusion by DNA fingerprinting,and mate guarding in the hooded seal Cystophora cristata

Hooded seal Cystophora cristata trios consist of an adult female, her pup, and an attending adult male. Using DNA fingerprinting, we excluded the possibility that the attending males within hooded seal trios were the fathers of the pups, proving that these hooded seals did not remain paired from one breeding season to the next. Behavioural observations of the trios after capture and release revealed that male hooded seals displace one another in attending nursing females. Mate guarding appears to be the preferred mating strategy available to male hooded seals given intense competition for females, a very brief nursing period, and oestrus occuring soon after weaning, but its effectiveness remains unclear.     

Morphological and thermal properties of mammalian insulation: the evolutionary transition to blubber in pinnipeds

Carnivora includes three independent evolutionary transitions to the marine environment: pinnipeds (seals, sea lions, and walruses), sea otters, and polar bears. Among these, only the pinnipeds have retained two forms of insulation, an external fur layer and an internal blubber layer for keeping warm in water. In this study we investigated key factors associated with the transition to the use of blubber, by comparing blubber characteristics among the pinnipeds. Characteristics included gross morphology (blubber thickness), fat composition (fatty acid profiles, percentage lipid, and water), and thermal conductivity. Sea lions, phocids, and walrus, which have lower fur densities than fur seals, have thicker blubber layers than fur seals (P < 0.001). Comparisons of lipid content, water content, and fatty acid composition indicated significant differences in the composition of the inner and outer regions of the blubber between groups (P < 0.001), consistent with the hypothesis that phocids and sea lions utilize the outer layer of their blubber primarily for thermal insulation, and the inner layer for energy storage. Fur seals, by contrast, rely more on their fur for thermal insulation, and utilize their blubber layer primarily for energy storage. Comparing across carnivore species, differences in total insulation (fur and/or blubber) are influenced substantially by body size and habitat, and to a lesser extent by latitudinal climate. Overall, these results indicate consistent evolutionary trends in the transition to blubber and evidence for convergent evolution of thermal traits across lineages. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ??, ??–??.     

Stratification and age-related differences in blubber fatty acids of the male harbour porpoise (Phocoena phocoena)

Fatty acid composition of blubber was determined at four body sites of 19 male harbour porpoises. A total of 65 fatty acids were quantified in each sample. The array of fatty acids contained in harbour porpoise blubber was similar to those found in other marine mammals. While chemical composition of total blubber was uniform over the body, with the exception of the caudal peduncle, vertical stratification was evident between the deep (inner) and superficial (outer) blubber layers. Fatty acids with chain lengths shorter than 18 carbons were present in significantly greater amounts in the outer blubber layer, while the longer-chain unsaturated fatty acids were more prevalent in the inner layer. This distribution suggests that the inner blubber layer is more active metabolically than the outer layer in terms of lipid deposition and mobilization. The degree of stratification between the two layers appears to increase with age, indicating a predictable turnover in the blubber layer of male porpoises. Harbour porpoise blubber contained high levels (2–27%) of isovaleric acid in the outer blubber layer, and these levels were positively correlated with age.Abbreviations Caud caudal dorsal body site - GC gas chromatograph - FA fatty acid(s) - IUPAC International Union of Pure and Applied Chemistry - PUFA polyunsaturated fatty acid(s) - II dor II dorsal body site - III dor III Dorsal body site - II Ven II ventral body site     

Evaluating morphometric and metabolic markers of body condition in a small cetacean,the harbor porpoise (Phocoena phocoena)

Mammalian body condition is an important individual fitness metric as it affects both survival and reproductive success. The ability to accurately measure condition has key implications for predicting individual and population health, and therefore monitoring the population‐level effects of changing environments. No consensus currently exists on the best measure to quantitatively estimate body condition in many species, including cetaceans. Here, two measures of body condition were investigated in the harbor porpoise (Phocoena phocoena). First, the most informative morphometric body condition index was identified. The mass/length² ratio was the most appropriate morphometric index of 10 indices tested, explaining 50% of the variation in condition in stranded, male porpoises with different causes of death and across age classes (n = 291). Mass/length² was then used to evaluate a second measure, blubber cortisol concentration, as a metabolic condition marker. Cortisol is the main glucocorticoid hormone involved in the regulation of lipolysis and overall energy balance in mammals, and concentrations could provide information on physiological state. Blubber cortisol concentrations did not significantly vary around the girth (n = 20), but there was significant vertical stratification through the blubber depth with highest concentrations in the innermost layer. Concentrations in the dorsal, outermost layer were representative of concentrations through the full blubber depth, showed variation by sex and age class, and were negatively correlated with mass/length². Using this species as a model for live cetaceans from which standard morphometric measurements cannot be taken, but from which blubber biopsy samples are routinely collected, cortisol concentrations in the dorsal, outermost blubber layer could potentially be used as a biomarker of condition in free‐ranging animals.     

New records of angular rough sharks Oxynotus centrina in the coastal waters of Malta,with observations on post‐capture resilience and release behaviour

One male and one female angular rough sharks Oxynotus centrina were caught south‐east of Malta between May and June at a depth of 60–100 m < 5 km from shore. The immature female (total length, L_T 565 mm) was landed dead but the male (535 mm L_T) was found alive. This communication presents important biological observations on post‐capture recovery and release behaviour of this species.     

Seasonal Influences on the Ecology of Testate Amoebae (Protozoa) in a Small <Emphasis Type="Italic">Sphagnum</Emphasis> peatland in Southern Ontario,Canada

Testate amoebae (Protozoa) were studied in spring, summer, and fall from the same microhabitats in a small Sphagnum-dominated peatland in southern Ontario, Canada. A total of 32 sampling stations were established in two wetland plant communities, 19 in an open Ericaceae low-shrub community and 13 in a closed Picea mariana and Larix laricina swamp community. Sphagnum was collected in each station for analysis of testate amoebae and measurement of soil water content parameters and water table depth in May, August, and October 2001. pH and dissolved oxygen of the groundwater under the Sphagnum were measured also. A total of 52 taxa including the rotifer, Habrotrocha angusticollis, were identified. Soil water content and water table variables emerged as the primary factors separating testate amoebae between the open bog/fen community and swamp community. Testate amoebae in the open bog/fen community showed a clear separation between the May sampling period and the August and October sampling periods. Sampling stations in May had much higher water table and were wetter than those in August and October. Conversely, testate amoebae in the swamp community did not show a clear difference between sampling periods. Soil moisture and water tables appear to be more constant in the swamp communities. Biological factors or other microscale environmental factors may need to be considered to explain seasonal changes in testate amoebae. A greater understanding of relationships between testate amoebae and microenvironmental factors is necessary to track seasonality in testate amoebae distributions.