Towards direct evidence of the effects of salmon lice (<Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis> Krøyer) on sea trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> L.) in their natural habitat: proof of concept for a new combination of methods

Studies addressing the impact of salmon lice (Lepeophtheirus salmonis Krøyer) on sea trout in their natural habitat are scarce and mostly limited to prophylaxis-based experiments. The main drawbacks with this approach are that lice infestations on control fish are not known and the anti-parasitic treatment is of unknown efficacy and may have unwanted side-effects. We tested an innovative approach where prophylaxis is replaced with artificial infestation of the fish. Twenty-nine sea trout post-smolts were caught in a farming-free area in southern Norway and half the fish were artificially infested with lice. Survival and behavior of individual fish was investigated using acoustic telemetry. Furthermore, salinity values were extracted from an hydrodynamical model simulation and connected to individual 3D positions. Results from this pilot study show consistent trends in behavioral differences between artificially infested and control fish. All fish that died or prematurely returned to freshwater were artificially infested fish, although results were not statistically significant. Besides, power analysis confirmed the limitations of this small pilot study for delivering statistically significant inferences. We found also indications of artificially infested fish remaining in shallower waters and within shorter distance to low salinity habitats, but only differences in modelled salinity values were statistically significant. Methodological progress and limitations with this original approach are discussed, and we recommend further studies using this combination of methods and the lessons learnt from this pilot study to provide better quantitative evidence on the effect of salmon lice on sea trout in the wild.     

Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Krøyer) influences the physiological response to an acute stressor

The influence of infection with the juvenile stages of the sea louse, Lepeophtheirus salmonis (Kr?yer) on the response of rainbow trout Oncorhynchus mykiss (Walbaum) to a net confinement protocol was investigated. The experiment consisted of two groups of seawater-adapted rainbow trout, one which was exposed to a total of 4000 nauplii/copepodid stages of L. salmonis 30, 25 and 14 days prior to confinement. Confinement elicited a greater stress response in the lice-exposed fish, than in the controls, as seen by higher plasma cortisol and glucose levels. A reduced spleen somatic index in exposed fish following 6 h confinement coincided with increased erythrocyte and lymphocyte numbers in the blood. Circulating lymphocyte numbers were significantly reduced in both groups 24 h post-confinement, when a lower alternative complement activity was recorded in control fish. Prior to confinement, lice-exposed fish had an elevated serum lysozyme activity and reduced oxygen radical production by blood leukocytes. Following confinement, lysozyme activity was gradually reduced in lice-exposed trout. During confinement, oxygen radical production decreased in control fish and increased in infested fish. Overall, transient exposure to juvenile lice altered the response to a second stressor, which has implications for management procedures of L. salmonis exposed fish.     

Effects of salmon lice infection and salmon lice protection on fjord migrating Atlantic salmon and brown trout post-smolts

Effects of artificial salmon lice infection and pharmaceutical salmon lice prophylaxis on survival and rate of progression of Atlantic salmon (n = 72) and brown trout post-smolts (n = 72) during their fjord migration, were studied by telemetry. The infected groups were artificially exposed to infective salmon lice larvae in the laboratory immediately before release in the inner part of the fjord to simulate a naturally high infection pressure. Groups of infected Atlantic salmon (n = 20) and brown trout (n = 12) were also retained in the hatchery to control the infection intensity and lice development during the study period. Neither salmon lice infection nor pharmaceutical prophylaxis had any effects on survival and rate of progression of fjord migrating Atlantic salmon post-smolts compared to control fish. Atlantic salmon spent on average only 151.2 h (maximum 207.3 h) in passing the 80 km fjord system and had, thus, entered the ocean when the more pathogenic pre-adult and adult lice stages developed. The brown trout, in comparison to Atlantic salmon, remained to a larger extent than Atlantic salmon in the inner part of the fjord system. No effect of salmon lice infection, or protection, was found in brown trout during the first weeks of their fjord migration. Brown trout will, to a larger extent than Atlantic salmon, stay in the fjord areas when salmon lice infections reach the more pathogenic pre-adult and adult stages. In contrast to Atlantic salmon, they will thereby possess the practical capability of returning to freshwater when encountering severe salmon lice attacks.     

Brown trout Salmo trutta released to support recreational fishing in a Norwegian fjord

One- and two-year-old hatchery reared juveniles of seven freshwater resident and anadromous populations of Scandinavian brown trout were released in the outer and inner part of the Oslofjord and in the River Akerselva, flowing through the city of Oslo. Recapture rates were highest (mean 20.3%) for river released fish and lowest for those released in the outer (16.8%) and inner (12.1%) fjord. In general, recapture rate increased with fish size at release (r=0.76). When released in fresh water, most of the recaptures were from fresh water and when released in the fjord, most recaptures were from the fjord. In general, freshwater resident stocks showed a higher degree of freshwater residency than anadromous stocks. However, mean migratory distance was longer for freshwater resident than anadromous fish. Trout moved longer distances at sea when released in the outer than in the inner fjord. Specific growth rate and size at recapture varied among release sites and stocks; they were highest for fish released in the outer fjord and lowest for river-released trout. There was no consistent difference in sea growth between freshwater resident and anadromous stocks. Estimated total yield was highest for fish released in the outer fjord, whereas there was no significant difference in yields between trout released in the river and the inner Oslofjord.     

Differences in susceptibility to sea lice infection between a sea run and a freshwater resident population of brown trout

Two experimental groups comprising mixed Norwegian sea run and freshwater resident brown trout Salmo trutta were infected with sea lice Lepeophtheirus salmonis in replicate tanks. Comparison of mean abundance and louse development between the sea run and resident groups revealed highly significant differences in lice abundance. The resident trout had an average abundance ± of 6·3±0·37 and 6·6±0·43 lice whilst the sea trout had an average abundance of 3·5 ±0·25 and 3·3 ±0·28 lice 29 days post infection at 9° C. No differences in development of lice, of either sex, were detected between the groups. As host groups were naive to sea lice at the start of the experiment, this suggests that there was a significant difference in susceptibility to sea lice infection between them, which may be genetically determined.     

Seasonal occurrence of sea lice Lepeophtheirus salmonis on sea trout in two north Norwegian fjords

Seasonal occurrence of the parasitic copepod Lepeophtheirus salmonis (sea lice) was studied from March to December 2001 in two large north Norwegian sill fjords without fish farming activity, the Ranafjord and the Balsfjord. Anadromous brown trout Salmo trutta (sea trout) in both fjords had a low infestation rate during all sampling periods, but followed a seasonal pattern. During early and late winter (November to December and March to April) and spring (May to June), the prevalence varied from 0 to 25% and the abundance was <0·5 sea lice. Adults dominated (92%) during this period, particularly gravid females. In both fjords, the highest prevalence was during September (80–81%, all stages represented). In Ranafjord, the abundance and mean intensity during this month was 6·8 and 8·6 sea lice, respectively, while in Balsfjord it was 3·6 and 4·5 sea lice, respectively. Fish were captured at temperatures down to 1° C and at full strength sea water which is supposed to cause osmoregulatory problems for the fish. This observation has implications for the understanding of high‐latitude sea trout behaviour and can also make the fish more vulnerable to heavy sea lice infestation during this period. It is suggested that winter running sea trout help to maintain a self replicating local population of sea lice within such fjord systems where other possible hosts ( e.g . farmed Atlantic salmon Salmo salar ) are not present during a whole year cycle.     

Genetic differentiation of populations of the copepod sea louse Lepeophtheirus salmonis (Krøyer) ectoparasitic on wild and farmed salmonids around the coasts of Scotland: Evidence from RAPD markers

Sea trout are the sea-going migratory form of the freshwater brown trout (Salmo trutta L.) and since 1989 there have been marked declines in their stocks on the west coasts both of Scotland and Ireland. Various factors have been attributed as possible causal agents in these stock declines, including fresh water acidification, overfishing, climatic fluctuations, habitat degradation and sea lice parasitic burdens. The putative impact of infestations of sea trout by the ectoparasitic copepod sea louse, Lepeophtheirus salmonis (Krøyer), has featured prominently in the controversy, especially with regard to the role of inshore commercial salmon farms as a possible source of infestation of wild salmonids by sea lice. This study focused on the population genetics of L. salmonis around the coasts of Scotland: We sampled fish from wild and cultured stocks and included salmon (Salmo salar L.), rainbow trout (Oncorhynchus mykiss Walbaum) and sea trout as host species. Analyses of allozyme variation of sea lice were confined to data for two polymorphic loci (Fum, Got-2) and conformed to our initial expectation — that the inclusion of a planktonic larval phase in the life cycle of the copepod, in addition to the high mobility of the host fish, would enhance gene flow and preclude genetic differentiation of L. salmonis populations as a result of random drift alone. DNA polymorphism was quantified by means of PCR and RAPD analysis. Six primers were screened for 16 samples (from wild and farmed salmon, wild sea trout and farmed rainbow trout) — including the east, north and west coasts of Scotland — and the data analyzed by AMOVA (Analysis of Molecular Variance). In contrast to the allozyme results, the RAPD analysis showed striking patterns of genetic differentiation around the coasts of Scotland. The overall pattern was one of genetic homogeneity of L. salmonis populations sampled from wild salmon and sea trout. All of the L. salmonis samples taken from farmed salmon and rainbow trout did, however, show highly significant levels of genetic differentiation, both between wild and farmed salmonids and among the various farms themselves. Evidence of high levels of small-scale spatial or temporal heterogeneity of RAPD marker band frequencies was shown for the one farm from which repeat samples (July and November, 1995) were analysed. Samples of sea lice taken from west coast wild sea trout subjected to RAPD analysis also revealed the occurrence of putative “farm markers” in some individual parasites, indicating that they had possibly originated from salmon farms.     

Factors influencing return rate and marine residence duration in sea trout populations in Central Norway

Brown trout (Salmo trutta) display extensive plasticity in marine migratory behaviours, with marine migrations considered to be an adaptive strategy which enables sea trout to maximize growth and reproductive potential. However, marine migrations are not without associated costs, including threats posed by ever-increasing salmon lice (Lepeophtheirus salmonis) infestations. In the present study, we used passive integrated transponder technology to characterize variability in sea trout migration behaviour amongst three catchments situated in a region of intensive salmon farming in central Norway. Specifically, we investigate how lice infestation, out-migration date and body size alter sea trout return rate and marine residence duration during the first out-migration to sea from each catchment. Distinct catchment-specific differences in sea trout out-migration size and the number of cohorts were observed, but larger body size did not guarantee the successful return of migrating trout. The marine residence duration of individuals that successfully returned to freshwater was positively correlated with lice infestation risk, suggesting for these individuals the lethal infestation threshold had not been reached. Our results also suggest that sea trout populations from lotic-dominated catchments are potentially at greater risk from size-related threats to their survival encountered during their marine migrations than sea trout from lentic-dominated catchments. The variability in sea trout migratory behaviour amongst catchments observed here emphasizes the challenges fisheries managers face when deciding the best actions to take to protect the anadromous portion of brown trout populations.     

Uptake and accumulation of zinc in juvenile rainbow trout,Salmo gairdneri

Synopsis The toxicity of zinc to rainbow trout was determined and the 72 h median lethal concentration was found to be 2.00 mg l^–1 in freshwater, hardness 7.50 mg l^–1 as calcium. An insignificant increase in zinc concentration of internal tissues occurred in fish exposed to 1.52 mg l^–1 in freshwater for 72 h. However, there was a significant uptake of zinc by gills and the body surface. Fish exposed to 10 mg l^–1 zinc for 72 h in two-thirds sea water showed significant zinc uptake by liver, rectum and muscle, when compared to control fish. Drinking rate decreased from 1.43 to 0.26 ml kg^–1 h^–1 when zinc sulphate was added to freshwater. Trout adapted to two-thirds sea water showed no decrease in drinking, about 7 ml kg^–1 h^–1 when zinc was added to the water.     

Glochidia of the freshwater mussel,Anodonta anatina, affecting the anadromous European smelt (Osmerus eperlanus) from the Eider estuary, Germany

During a 2-year survey on external disease conditions of fish between January 1988 and December 1989, 0.2% of 40011 smelt, Osmerus eperlanus , from 30 stations in the German Wadden Sea and estuaries of tributary rivers, were infected with glochidia of the freshwater mussel Anodonta anatina . Seasonality was marked, with glochidia only being observed in March and April 1989, when prevalences were 37% and 15% respectively, in fish 10–20cm long at the most heavily infected site. A marked increase in prevalence in fish 15cm in length and longer, suggested that only temporarily resident spawning fish (as opposed to resident juveniles) were infected. Infection was almost exclusively restricted to the Eider estuary, where prevalence decreased with increasing salinity. It is concluded that the glochidia carried by smelt returning from their freshwater spawning sites in the Eider River to the sea would have died, thus representing a loss to the 1989 cohort of A. anatina from the Eider. The potential importance of the smelt– A. anatina relationship as indicator for the detection of (a) spawning mussel populations and of (b) possible ecological effects of climate changes is noted.     

The importance of escaped farmed rainbow trout (Oncorhynchus mykiss) as a vector for the salmon louse (Lepeophtheirus salmonis) depends on the hydrological conditions in the fjord

The objectives of the study were to see if escaped rainbow trout (Oncorhynchus mykiss) spread rapidly or not from fish farms, and to test whether the hydrological conditions in a fjord influence their vertical distribution and importance as vector for the salmon lice (Lepeophtheirus salmonis). Fifty farmed rainbow trout were tagged with acoustic transmitters including depth sensors and released from two of 11 fish farms in the fjord system. In addition, unintentionally escaped rainbow trout were recaptured for analysis of salmon lice and stomach content. Dispersal out of the fjord system was limited. Most fish stayed in the vicinity of and moved between the fish farms but fed primarily on a variety of indigestible items. They moved in the warm relatively fresh surface layer from late spring until early autumn where the risk of being infested with salmon lice was low. They swam gradually deeper and became much more infested with salmon lice as the surface layers cooled and salinity and temperature gradients became less distinct over the course of the winter. The observed post-escapement behavior may challenge the control of the spread of diseases and parasites between neighboring farms and to wild fish, but also increases opportunities for recapture.     

Experimental sea ranching of brook trout, Salvelinus fontinalis Mitchill

An experiment to induce anadromy in a population of wild brook trout, Salvelinus fontinalis , was conducted near Sept-Iles, Quebec, in 1978–1979. Brook trout were captured from the Matamek River, tagged and transported to the Matamek River estuary during late spring and early summer, and allowed free movement between an impassable waterfall 0.7 km upstream and the sea. Fish were recaptured in autumn as they returned to fresh water. Over two years, 34.0% of the released fish were recaptured. Best returns were in the 2+ and 3+ age classes with 38.0 and 62.1% recaptured, respectively. Straying of transplanted fish appeared to be <1%. All age classes included sea run brook trout (sea trout) but the largest percentages of sea trout occurred in older fish. Growth was better in sea trout than in fish which did not develop anadromy, presumably a function of an increased food supply at sea. Severe tagging effects stunted growth and probably suppressed anadromy, especially among younger fish. Sexual characteristics of recaptured fish indicated suppressed maturation of gonads in sea trout compared to fish remaining in fresh water and there was a shift to a larger percentage of females in the sea trout. Comparisons between our results and data on other anadromous Salvelinus species underscore the potential for sea-ranching of trout and char as a moderate effort, high yield aquaculture technique.     

And the Last Shall Be First: Heterochrony and Compensatory Marine Growth in Sea Trout (Salmo trutta)

Early juvenile growth is a good indicator of growth later in life in many species because larger than average juveniles tend to have a competitive advantage. However, for migratory species the relationship between juvenile and adult growth remains obscure. We used scale analysis to reconstruct growth trajectories of migratory sea trout (Salmo trutta) from six neighbouring populations, and compared the size individuals attained in freshwater (before migration) with their subsequent growth at sea (after migration). We also calculated the coefficient of variation (CV) to examine how much body size varied across populations and life stages. Specifically, we tested the hypothesis that the CV on body size would differ between freshwater and marine environment, perhaps reflecting different trade-offs during ontogeny. Neighbouring sea trout populations differed significantly in time spent at sea and in age-adjusted size of returning adults, but not on size of seaward migration, which was surprisingly uniform and may be indicative of strong selection pressures. The CV on body size decreased significantly over time and was highest during the first 8 months of life (when juvenile mortality is highest) and lowest during the marine phase. Size attained in freshwater was negatively related to growth during the first marine growing season, suggesting the existence of compensatory growth, whereby individuals that grow poorly in freshwater are able to catch up later at sea. Analysis of 61 datasets indicates that negative or no associations between pre- and post-migratory growth are common amongst migratory salmonids. We suggest that despite a widespread selective advantage of large body size in freshwater, freshwater growth is a poor predictor of final body size amongst migratory fish because selection may favour growth heterochrony during transitions to a novel environment, and marine compensatory growth may negate any initial size advantage acquired in freshwater.     

The duration of sea and freshwater residence of the sea trout,Salmo trutta,from the Vardnes River in northern Norway

Synopsis The sea trout,Salmo trutta, from the Vardnes River generally stays in freshwater during winter. The results from 11 years of tagging experiments showed that the mean annual duration of the feeding migration in the sea was 68 ± 21 days (yearly means ranging between 54 and 88 days). A pronounced variation was found with size, sex and time of migration of the fish and between the different years. The males stayed for a shorter time in the sea (mean 66 days) than the females (mean 69 days) and they usually descended later and ascended earlier. Those fish that descended first stayed for the longest period in the sea. The mean annual duration of stay in the sea for first and second time migrants to the sea was positively correlated with the mean sea temperature in May (r² > 0.5). A positive correlation was also found between the annual duration of stay in the sea for the sea trout above 33 cm and the mean temperature in the river in July (r² > 0. 6). Prolonged mean yearly sea sojourn was generally correlated with higher sea temperatures. Water temperature-salinity effects are discussed.     

External attachment of data storage tags increases probability of being recaptured in nets compared to internal tagging

Sea running Arctic charr Salvelinus alpinus and anadromous brown trout (sea trout) Salmo trutta (420–2030 g) tagged with externally attached data storage tags (DST) had a higher total recapture rate (39 of 44, 89%) due to entanglement in bag nets at sea (90% of all recaptures), compared with internally tagged fish (12 of 18, 67%) that were mostly trapped when returning to their home river (75% of all recaptures). The internally tagged fishes therefore spent longer time at sea before recapture (median 33 days) than externally tagged fishes (median 8 days), and more DST‐data were collected. Therefore, in areas with high net fishing intensity, external tagging increases the chances of recapture, but less data may then be recorded by the tags due to a generally shorter period of data sampling.     

Large-scale use of fish traps for monitoring sea trout (Salmo trutta) smolts and sea lice (Lepeophtheirus salmonis) infestations: efficiency and reliability

Lice-infected sea trout populations were monitored using fish traps in the Romsdalsfjord (Norway). The reliability and efficiency of this capture technique, which allows estimation of lice infestation rates without killing the fish, was evaluated through a mark–recapture study. A total of 2447 sea trout smolts were captured, tagged and released over a three-year period. There was a considerable variation in capture rates (range: 0.4–17.7 fish per day) and sea lice numbers (number of lice per fish: 2.8–30.3; number of lice per gram body weight: 0.02–0.69) among localities, sampling times and years. Recapture rates of tagged fish with traps, which were low (2% or 0.11 fish per day), showed that the risk for pseudoreplication was minor, in terms of counting lice on the same fish several times. Most of the tagged sea trout (90%) were recaptured within the first two months after release, and no significant variations in lice numbers were found between tagging and recapture. The lack of differences in lice levels between tagging and recapture during the first week after tagging indicated that the method most likely would not significantly underestimate the lice infestations due to loss of lice during handling. Therefore, our results confirm that the use of fish traps is a suitable method for estimation of lice numbers on wild salmonids.     

Tolerance to gas supersaturation in fresh water and sea water by steelhead trout, Salmo gairdneri Richardson

The euryhaline status of steelhead trout, Salmo gairdneri , smolts was challenged in sea water for 2 weeks after which half of the total fish population was returned to fresh water. Acclimation continued and created two test populations in 29%osea water and fresh water. Subsequently these fish were exposed in fresh water or sea water to approximately equal hyperbaric dissolved total gas pressures (ΔP) of 190 mm Hg or about 125% of barometric pressure. Sea water was easier to supersaturate with air and required only about 10% of the entrained air which was required in fresh water at the same temperature and pressure. Mean time to first mortality was sooner in sea water. Mean times to mortality (10–50%) were not significantly different between fresh water and sea water, but there was a noticeable trend for longer survival in fresh water.     

The effect of pH and/or calcium-enriched freshwater on gill Ca2+-ATPase activity and osmotic water inflow in rainbow trout (Salmo gairdneri)

Rainbow trout (Salmo gairdneri) were exposed to pH 5.0-5.1, 6.6 and/or calcium-enriched freshwater for 14 days. Hematocrit, gill Ca2+-ATPase enzyme activities, gill osmotic water inflow, plasma calcium and osmolarity were measured. No significant changes in plasma calcium ion levels were found. The typical increase in hematocrit usually associated with exposure of fish to acidified water was not found in the present study and is discussed. Plasma osmolarity decreased in fish exposed to calcium-enriched freshwater (60 mg Ca2+ X 1(-1) ) in comparison to fish exposed to control freshwater conditions (2 mg Ca2+ X 1(1) ), irrespective of the pH level. Gill Ca2+-ATPase enzyme activities were measured for both low affinity (3 mM Ca2+) and high affinity (100 microM) activity. Exposure of rainbow trout to low pH (pH 5.0-5.1) did not affect the specific activity of Ca2+-ATPase enzyme. However, low affinity Ca2+-ATPase activity in fish exposed to calcium-enriched freshwater did show a significant reduction. The increase in gill osmotic water permeability in fish exposed to calcium-enriched freshwater is interpreted as a result of the increase in osmolarity of the ambient media.     

Higher growth variability and stronger responses to temperature changes in wild than hatchery‐reared sea trout (Salmo trutta L.)

Each year, millions of hatchery‐reared sea‐run brown trout Salmo trutta L. (the sea trout) juveniles are released into the natural environment in the Atlantic region. The aim of this work was to investigate the growth responses of sea trout to changing temperature conditions and to compare the growth plasticity between wild and hatchery‐reared fish. Scales were collected from sea trout in a selected river flowing into the southern Baltic Sea. We analyzed the scale increment widths as a proxy of somatic growth and investigated the interannual variabilities and differences in growth between fish groups (wild and hatchery‐reared). We used mixed‐effects Bayesian modeling and ascribed the variances in growth to different sources. Furthermore, we developed indices of interannual (2003–2015) growth variation in the marine and freshwater phases of the life cycle of the fish and analyzed the relationships between trout growth and temperature. Temperature positively affects fish growth, regardless of the origin of the fish. We observed stronger relationships between fish growth and temperature conditions in the marine phase than in the freshwater phase. Additionally, wild sea trout are characterized by stronger responses to temperature variability and higher phenotypic plasticity of growth than those of the hatchery‐reared individuals. Therefore, wild sea trout might be better suited to changing environmental conditions than hatchery‐reared sea trout. This knowledge identifies possible threats in management actions for sea trout with an emphasis on ongoing climate change.     

Opportunistic use of estuarine habitat by juvenile bull trout, <Emphasis Type="Italic">Salvelinus confluentus</Emphasis>, from the Elwha River before,during, and after dam removal

Estuaries are used by anadromous fishes, either as the definitive marine habitat or as transition habitat as they move to fully marine waters, and extent of estuary use may vary with habitat conditions and fish attributes. Bull trout (Salvelinus confluentus) are commonly fluvial or adfluvial, though anadromous populations also exist. However, little is known about estuary use, especially by juveniles of this threatened species. We sampled the estuaries of the Elwha River, where a spawning population exists, and the nearby Salt Creek, where none exists, to reveal seasonal timing of estuarine use by juvenile bull trout, size of those using the estuary, and possible use of the non-natal estuary. We captured juvenile bull trout (all ≥100 mm FL, most <300 mm) in the Elwha River estuary in all months except August, but primarily December through May. None was captured in Salt Creek’s estuary despite comparable sampling effort. We also evaluated how dam removal on the Elwha River influenced bull trout estuarine occupancy by sampling before, during, and after dam removal, because this process enlarged the estuary but also increased turbidity and sediment transport in the lower river. Catches were low before dam removal, increased during and immediately after removal, and returned to low levels in recent years, suggesting that juveniles temporarily sought refuge from conditions associated with dam removal. Our findings indicate juvenile bull trout occupy estuarine habitat opportunistically; this information may aid conservation efforts as anadromous populations occur elsewhere in rivers with estuaries altered by human development.