Kim Wong

Jackie Y C Lam

J H Liu

Y K Yung

Alle A Y Lie
email: alie@usc.edu

C Kim Wong

Changes in the nutrient ratios and phytoplankton community after declines in nutrient concentrations in an eutrophic semi-enclosed bay

Keywords: Nutrients, Nutrient ratios, Phytoplankton, Eutrophication, Coastal waters, Tolo Harbour

Changes in the nutrient ratios and phytoplankton community after declines in nutrient concentrations in an eutrophic semi-enclosed bay in Hong Kong

Introduction

Eutrophication is a common consequence of anthropogenic activities on water bodies, and is widely regarded as the most wide-spread water quality problem globally (Carpenter et al., 1999;[START_REF] Smith | Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems[END_REF][START_REF] Smith | Eutrophication of freshwater and coastal marine ecosystems. A global problem[END_REF]. The most common effects of eutrophication are increases in phytoplankton productivity and biomass, which in turn produce other adverse effects such as decreases in water transparency, increases in the frequency of bottom hypoxia or anoxia and loss of various aquatic organisms [START_REF] Nixon | Coastal marine eutrophication: A definition, social causes, and future concerns[END_REF][START_REF] Smith | Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems[END_REF][START_REF] Smith | Eutrophication of freshwater and coastal marine ecosystems. A global problem[END_REF]. Numerous recent studies have linked eutrophication to algal blooms which cause ecological and economical damages (e.g. [START_REF] Anderson | Harmful algal blooms and eutrophication: Examining linkages from selected coastal regions of the United States[END_REF][START_REF] Cloern | Our evolving conceptual model of the coastal eutrophication problem[END_REF][START_REF] Smith | Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems[END_REF]. Nutrient loadings may cause changes in the nutrient ratios [START_REF] Justić | Stoichiometric nutrient balance and origin of coastal eutrophication[END_REF][START_REF] Turner | Element ratios and aquatic food webs[END_REF], and such changes may affect the species composition of the phytoplankton community and higher trophic levels [START_REF] Officer | The possible importance of silicon in marine eutrophication[END_REF][START_REF] Philippart | Long-term phytoplankton-nutrient budgets, and denitrification potential[END_REF][START_REF] Smith | Low nitrogen to phosphorus ratios favor dominance by blue-green algae in lake phytoplankton[END_REF][START_REF] Turner | Fluctuating silicate:nitrate ratios and coastal plankton food webs[END_REF][START_REF] Turner | Element ratios and aquatic food webs[END_REF]. Although eutrophication in freshwater systems has been studied extensively and intensively during the past few decades, with many studies reporting on the effects of increased or decreased nutrient concentrations in lakes (e.g. [START_REF] Battarbee | Combining palaeolimnological and limnological approaches in assessing lake ecosystem[END_REF]Carvalho and Kirika, 2003;[START_REF] Jeppesen | Lake responses to reduced nutrient loading -an analysis of contemporary long-term data from 35 case studies[END_REF][START_REF] Jeppesen | Restoration of shallow lakes by nutrient control and biomanipulatioin -the successful strategy varies with lake size and climate[END_REF][START_REF] Mehner | Restoration of a stratified lake (Feldberger Haussee, Germany) by a combination of nutrient load reduction and long-term biomanipulation[END_REF], similar kinds of studies, with long-term monitoring data, are relatively scarce in marine coastal ecosystems (Examples summarized by [START_REF] Smith | Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems[END_REF].

Hong Kong is one of the most densely populated cities in the world. Intensive urbanization and industrialization during the past few decades has dramatically increased nutrient input into Hong Kong's coastal seas [START_REF] Lee | Eutrophication dynamics in Hong kong Coastal Waters: Physical and Biological Interactions[END_REF]. The concern on the coastal water quality led to the initiation of a marine water quality monitoring program by the Hong Kong Environmental Protection Department in the 1980s [START_REF] Lee | Eutrophication dynamics in Hong kong Coastal Waters: Physical and Biological Interactions[END_REF]. One of the intensely monitored zones is Tolo Harbour, a landlocked bay in the northeastern corner of Hong Kong.

Tolo Harbour consists of a shallow inner bay, with a mean depth of 6-7 m, and a narrow tidal channel which opens into the neighboring Mirs Bay. Urban development and large-scale reclamation around Tolo Harbour began in the 1970s. Sewage discharge from the expanding population caused a sharp increase in nutrient input [START_REF] Lam | Phytoplankton characteristics of Tolo Harbour[END_REF][START_REF] Wear | Hydrography, nutrients and plankton in Tolo Harbour, Hong Kong[END_REF]. [START_REF] Hodgkiss | A case study of Tolo Harbour, Hong Kong[END_REF] estimated a daily nitrate and phosphate loading of 32,460 kg and 3,190 kg respectively into Tolo Harbour. In addition to nutrient inputs, the bottleneck topography of the Tolo Harbour leads to poor tidal exchange rates and persistence of high nutrient concentrations [START_REF] Lee | Eutrophication dynamics in Hong kong Coastal Waters: Physical and Biological Interactions[END_REF]. Increased nutrient concentrations produced algal blooms, with dinoflagellates such as Noctiluca scintillans being the most common causative species during the 1980s and 1990s [START_REF] Lam | Phytoplankton characteristics of Tolo Harbour[END_REF][START_REF] Liu | Seasonal and spatial dynamics of Noctiluca scintillans in a semi-enclosed bay in the northeastern part of Hong Kong[END_REF]. Although Noctiluca are generally considered as heterotrophic [START_REF] Kiørboe | Feeding, prey selection and prey encounter meachanisms in the heterotrophic dinoflagellate Noctiluca scintillans[END_REF], strains of Noctiluca miliaris with phytosynthetic symbionts have been found in Southeast Asia [START_REF] Sweeney | Laboratory studies of a green Noctiluca from New Guinea[END_REF][START_REF] Hansen | Green Noctiluca scintillans: a dinoflagellate with its own greenhouse[END_REF], which may benefit from increased nutrient concentrations [START_REF] Sriwoon | Population dynamics of green Noctiluca scintillans (Dinophyceae) associated with the monsoon cycle in the upper gulf of Thailand[END_REF]. In addition, the increased nutrient concentrations would also favor the growth of diatoms and other prey for N.

scintillans [START_REF] Liu | Seasonal and spatial dynamics of Noctiluca scintillans in a semi-enclosed bay in the northeastern part of Hong Kong[END_REF]. [START_REF] Wear | Hydrography, nutrients and plankton in Tolo Harbour, Hong Kong[END_REF] reported mean concentrations of NO x -N and PO 4 -P in the inner Tolo Harbour to be 0.05 µM and 0.01 µM respectively during 1974-1976. By early 1990s, annual mean concentrations have increased to 19.99 µM for inorganic nitrogen and 1.94 µM for PO 4 -P (Hong Kong Environmental Protection Department, 1991). Moreover, PO 4 -P values as high as 10.65 µM have been reported in Tolo Harbour in 1978-1979[START_REF] Hodgkiss | Pollution studies in Tolo Harbour, Hong Kong[END_REF]. Decreasing trends in N:P ratio were recorded during the 1980s and early 1990s (Hodgkiss and Chan, 1997;Yung et al., 1997), and was considered to be caused by excessive P input (Hodgkiss and[START_REF] Hodgkiss | Pollution studies in Tolo Harbour, Hong Kong[END_REF]Yim,1995). Some researchers noted that the observed changes in nutrient parameters had caused a shift in the species composition of the phytoplankton community, with diatoms gradually replaced by dinoflagellates, (Chan and Hodgkiss, 1987;[START_REF] Hodgkiss | Are changes in N:P ratios in coastal waters the key to increased red tide blooms?[END_REF][START_REF] Lam | Phytoplankton characteristics of Tolo Harbour[END_REF], although a review of the data collected by the Hong Kong Environmental Protection Department did not reveal such a shift in the phytoplankton during 1982-1992(Yung et al., 1997)).

The Hong Kong Government initiated the Tolo Harbour Action Plan in 1986 to halt the declining water quality of Tolo Harbour. Two of the most important components of the action plan were the modification of existing sewage treatment works processes (implemented in 1992) and the Tolo Harbour Effluent Export Scheme (Chau, 2007). Commissioned in the mid-1990s and became fully operational in 1998, the Tolo Harbour Effluent Export Scheme allows treated sewage from two large sewage treatment works to be exported via pipelines for discharge outside of Tolo Harbour. Nitrogen input into Tolo Harbour was reduced to ~1000 kg day -1 by 2000 (Hong Kong Environmental Protection Department, 2006).

In this study, data collected from four sampling stations in Tolo Harbour during the period from 1986-2008 were analyzed to provide information on the long-term changes in the hydrography and phytoplankton dynamics. Conditions before (1986-1997) and after (1998-2008) the full implementation of the Tolo Harbour Effleunt Export Scheme were compared to: (1) determine whether declines in nutrient concentrations had led to changes in the nutrient ratios, and (2) identify shifts in the phytoplankton community that may be related to changes in the nutrient concentrations and ratios. Overall changes of the hydrography and phytoplankton community in Tolo Harbour after declines in nutrient concentrations were also compared to those reported in other ecosystems after recovery from eutrophication.

Materials and Methods

All data were provided by the Hong Kong Environmental Department. Four sampling stations in Tolo Harbour, TM3, TM4, TM6 and TM8 (Fig. 1), were sampled frequently, mostly once each month, but irregularly from 1982-1992. Monthly means were calculated for each station. Physico-chemical parameters, including temperature, salinity, pH and dissolved oxygen concentration (DO), were recorded at 1 m below the water surface, mid-depth in the water column and 1 m above the bottom by a SEACAT 19plus CTD water profiler (Sea-BIRD Electronics). Water samples were also collected at the three depths using Niskin-type water bottles for the determination of BOD 5 , chlorophyll-a concentration, suspended solids concentration, and the concentrations of NO 3 -N, NH 4 -N, total inorganic nitrogen (TIN), total nitrogen (TN), PO 4 -P, total phosphorus (TP) and SiO 2 . The data from the three depths were averaged into depth-averaged data. Values from the bottom depth were also analyzed for DO and BOD 5 to provide information on hypoxia at the water bottom. Measurement of BOD 5 , nutrient concentrations, chlorophyll-a concentration and suspended solids concentration were carried out by the Hong Kong Government Laboratory using procedures described by APHA (American Public Health Association et al. 1998).

Data on algal bloom occurrences were obtained from the Hong Kong Agriculture, Fisheries and Conservation Department, and were independent from our data. Such data on algal bloom occurrences was based on sighting reports of seawater discoloration, and may therefore underestimate algal bloom occurrences. It also does not contain taxonomic information regarding the algal bloom.

(Insert Fig. 1 here)

Water samples from 1 m below the water surface were preserved in Lugol's iodine solution (1: 100 ml). In the laboratory, phytoplankton were examined in an Utermöhl counting chamber under an inverted microscope, and the densities of diatoms, dinoflagellates and other algal groups (Algae that were neither diatoms nor dinoflagellate) were counted. Phytoplankton data for TM6 was only available after 1993, while data collection for other stations have began in 1986 (TM8) and 1988 (TM3 and TM4). Two-way ANOVA was used to test for temporal differences before and after nutrient loading reduction, as well as the spatial differences among sampling stations in various water quality and phytoplankton parameters. Tukey's test was used for multiple comparison between sites. Kendall's correlation was used to test the correlation between phytoplankton and water quality variables.

Results

In general, physico-chemical, biological and nutrient parameters showed similar changes among the four sampling stations after nutrient loading reduction (Table 1).

Temperature, salinity, bottom DO and TIN/PO 4 increased at all stations after nutrient loading reduction, while pH, BOD 5 , chlorophyll-a concentrations, all nutrient concentrations and TIN/SiO 2 decreased. DO and SiO 2 concentration did not differ significantly between the two time periods and among the stations respectively (p > 0.05). All other parameters showed significant differences both temporally and spatially, with most p values < 0.001. Comparison among sampling stations revealed a general trend of improvement in water quality, marked by changes such as decreasing nutrient concentrations and increasing DO and secchi depth, from TM3 to TM8.

(Insert Table 1 here) N (including NO 3 -N, NH 4 -N, total inorganic N and total N) and P (including PO 4 -P and total P) concentrations were high, especially at TM3 and TM4, prior to nutrient loading reduction (Fig. 2). Concentrations decreased noticeably within a year after the full implementation of the sewage export scheme, and have remained at relatively low levels in recent years. N and P concentrations differed markedly among stations before nutrient loading reduction, but the differences became less apparent afterwards. SiO 2 concentrations did not appear to differ among stations before nutrient loading reduction. NO 3 /NH 4 fluctuated widely during the entire study period, but the value was generally lowest at TM3 and highest at TM8 (Fig. 3). In contrast, TIN/PO 4 and TIN/SiO 2 showed much clearer increasing and decreasing trends respectively (Fig. 3). TIN/PO 4 decreased gradually from between 1986 and 1996, but have returned to approximately the 16:1 Redfield N:P ratio after declines in nutrient concentrations.

Declines in nutrient concentrations reduced the among-station differences in TIN/SiO 2 and the values at all stations were well below the 1:1 Redfield ratio for N:Si.

(Insert Fig. 2 and Fig. 3 here)

Chlorophyll-a concentrations and densities of different phytoplankton groups exhibited varying trends temporally (Figs. 4 and 5 respectively). The lack of apparent similar trends between chlorophyll-a concentrations and phytoplankton densities may be due to the fact that chlorophyll-a data were depth-averaged, while phytoplankton data were from the surface only. In general, the concentrations of chlorophyll-a and densities of all phytoplankton groups were highest at TM3 and lowest at TM8. Dinoflagellate density at TM3 increased to two major peaks of > 10,000 cells ml -1 in 1996 and 1998, and decreased to < 1,000 cells ml -1 in recent years. Other algal groups occurred at densities of < 5000 cells ml -1 before 1996, but increased rapidly and considerably after 1996. Density at TM3 remained > 6,000 cells ml -1 after declines in nutrient concentrations. As diatoms were the most abundant group, the density of total phytoplankton exhibited temporal and spatial patterns more similar to those of diatoms than those of dinoflagellates and other algal groups. Declines in nutrient concentrations did not affect the density of dinoflagellates significantly, but densities of diatoms, other algal groups and total phytoplankton all showed significant increase after declines in nutrient concentrations. Despite a significant increase in total phytoplankton density, the number of algal blooms recorded in Tolo Harbour dropped significantly from a mean of 15.8 before nutrient loading reduction to 7.6 (Paired t-test, p = 0.02) afterwards. No specific phytoplankton group or genus identified in the study could be concluded to be restricted to a particular station. Diatom genera common in all four stations and with average densities > 1,000 cells ml -1 in TM3 and > 600 ml -1 in TM8 included Cerataulina, Chaetoceros, Pseudo-nitzschia, Leptocylindrus and Thalassiosira. Prorocentrum and Scrippsiella were the most abundant dinoflagellate genera in all stations. Average density was > 1,000 cells ml -1 in TM3 and > 250 cells ml -1 in TM8 for Prorocentrum, and > 700 cells ml -1 in TM3 and > 100 cells ml -1 in TM8 for Scrippsiella. The dinoflagellates Gymnodinium, Gyrodinium and Protoperidinium were also frequently found at all stations, but their average densities were much lower. Small flagellates represented the most abundant and frequently recorded other algal group in the samples.

(Insert Fig. 4 and Fig. 5 here)

Hong Kong is characterized by a warm wet season from April to September, and a relatively dry and cool season for the rest of the year. However, when plots of seasonal means of nutrient concentrations and phytoplankton densities were made, no apparent and general trend could be identified (Data not shown).

Kendall's correlation did not reveal any strong or obvious trends between phytoplankton variables and various parameters (Table 2). Results for TM3 and TM4 seemed to oppose those for TM6 and TM8 at times. For instance, correlation between phytoplankton variables and DO and BOD 5 were usually positive at TM3 and TM4, but negative at TM6 and TM8, and vice versa with the results for SiO 2 . It is also not uncommon to find contradicting results between phytoplankton cell densities and chlorophyll-a concentrations due to the different trends exhibited by these two parameters. Correlation between phytoplankton variables and nutrient variables were mostly negative in all stations except TM6. Most phytoplankton variables at TM3 and TM4 correlated significantly and negatively with nutrient concentrations. In TM6, chlorophyll-a concentrations correlated negatively with NO 3 -N and NH 4 -N concentrations, while dinoflagellate densities correlated positively with total N, PO 4 -P and total P concentrations. NO 3 /NH 4 correlated negatively with the densities of all groups of phytoplankton at TM3, and positively with diatom densities at TM6 and TM8. TIN/PO 4 correlated positively with other algal group densities in all stations. TIN/SiO 2 correlated negatively with phytoplankton densities in all stations, but exhibited especially strong negative correlations with the densities of dinoflagellates, other algal groups and total phytoplankton at TM3 and TM4. Kendall's correlations between the number of algal blooms and all parameters were also determined.

Surprisingly, the number of algal blooms did not correlate significantly with phytoplankton densities, nutrient concentrations and nutrient ratios. It correlated weakly with pH (r = 0.313) and bottom DO (r = 0.305) (p < 0.05), and strongly with BOD 5 (r = 0.513) and chlorophyll-a concentration (r = 0.417) (p < 0.01).

(Insert Table 2 here)

Discussion

Temporal and spatial variations in marine water quality and phytoplankton densities

While spatially there is an apparent pattern of better water quality in TM6 and TM8, all parameters show high temporal variations and do not exhibit an apparent trend except changes due to nutrient loading reduction measures. Our data set is unable to explain the cause of specific events such as the relatively lower nutrient concentrations during 1989 (Fig. 2), and the rise in phytoplankton densities in 1995 (Fig. 5), which may be due to isolated algal bloom events that inflated the annual mean value. Both of these parameters may also be affected by factors such as water current, weather conditions and anthropogenic activities that are not available in our data set.

Changes in various physico-chemical and biological parameters, especially nutrient concentrations, after the implementation of the Tolo Harbour Action Plan suggested that nutrient loading reduction measures had led to water quality improvement in Tolo Harbour. While [START_REF] Hu | Nutrient release and sediment oxygen demand in a eutrophic land-locked embayment in Hong Kong[END_REF] warned that recovery after nutrient loading reduction in Tolo Harbour may be slowed due to nutrients released into the water column from contaminated sediments, N and P concentrations appeared to decrease quite rapidly (Fig. 2) after the full implementation of the Tolo Harbour Effluent Export scheme. Surprisingly, although chlorophyll-a concentrations had decreased after declines in nutrient concentrations, the densities of both diatoms and other algal groups have increased. This was similar to the observation reported earlier by Yung et al. (1997) that there was an increase in the density of phytoplankton, especially that of other algal groups, when chlorophyll-a concentrations had not increased. As other algal groups in both Yung et al. (1997) and this study included mainly unidentified small flagellates and cryptomonads, an increase in the abundance of these small organisms may not be accompanied by an increase of chlorophyll-a concentrations. This is somewhat supported by the fact that correlation between density of other algal group and chlorophyll-a concentrations was recorded only at TM8, while diatoms and dinoflagellates correlated strongly and positively with chlorophyll-a concentrations at all stations.

Water quality measured in terms of nutrients and chlorophyll-a concentration has always been better in the channel region than in the inner part of the harbour [START_REF] Hu | Nutrient release and sediment oxygen demand in a eutrophic land-locked embayment in Hong Kong[END_REF][START_REF] Lam | Phytoplankton characteristics of Tolo Harbour[END_REF]Yung et al., 1997), and a seaward decrease in nutrient concentrations from the inner harbour to the channel was evident from our results. A similar spatial pattern, with water quality improving from TM3 to TM8, is recorded in other water quality indicators such as secchi depth, DO and BOD 5 . Due to its proximity to the open sea and the much lower pollution levels, TM8 was less affected by eutrophication and subsequent declines in nutrient concentrations compared to stations in the inner part of the harbour. An interesting observation was that the mean values of NO 3 /NH 4 decreased at TM3, TM4 and TM6, but increased at TM8 after declines in nutrient concentrations. This may be caused by a relatively higher decline of NH 4 -N concentrations than that of NO 3 -N in TM8. The maximum release rate of NH 4 -N from contaminated sediments into the water column in Tolo Harbour was found to be more than sixfolds higher than that of NO 3 -N [START_REF] Hu | Nutrient release and sediment oxygen demand in a eutrophic land-locked embayment in Hong Kong[END_REF], and although [START_REF] Hu | Nutrient release and sediment oxygen demand in a eutrophic land-locked embayment in Hong Kong[END_REF] did not specify specific nutrient release rates in various parts of Tolo Harbour, it may be possible that comparatively less NH 4 -N was released back into the water column from sediments in the less polluted outer parts of the Tolo Harbour.

Spatial differences in nutrient loading and tidal flushing may have led to the observed differences in how phytoplankton in different stations had responded to declines in nutrient concentrations. Correlation between phytoplankton densities and nutrient concentrations was poor and usually insignificant at TM6 and TM8 in the outer part of the harbour. In contrast, phytoplankton densities at TM3 and TM4 tended to correlate strongly and negatively with nutrient concentrations, suggesting that nutrients were removed rapidly by phytoplankton in the inner part of Tolo Harbour.

Changes in nutrient ratios and shifts in the phytoplankton community

Since it is believed that some phytoplankton prefer to use NH 4 over NO 3 [START_REF] Dortch | The interaction between ammonium and nitrate uptake in phytoplankton[END_REF]), a decrease in NO 3 /NH 4 may favor phytoplankton growth. However, the lack of consistent correlation between NO 3 /NH 4 and phytoplankton density suggests that the phytoplankton community in Tolo Harbour did not have specific preference for either NO 3 or NH 4 .

Decreases in nutrient concentrations following nutrient loading reduction measures had altered the values of TIN/PO 4 and TIN/SiO 2 significantly. [START_REF] Hodgkiss | Are changes in N:P ratios in coastal waters the key to increased red tide blooms?[END_REF] related the increased abundance of dinoflagellates in Tolo Harbour to a decrease in N:P ratios. It must be noted that no evidence of a decrease in dinoflagellate density has been detected in our data, despite a significant increase in TIN/PO 4 after declines in nutrient concentrations. In general, the density of dinoflagellates correlate neither with TIN/PO 4 nor with most physico-chemical parameters other than Secchi depth and BOD 5 , both of which may be dependent variables affected by the dinoflagellate density. Hence, the density of dinoflagellate in Tolo Harbour may not be easily predicted from nutrient concentrations or N:P ratios alone. While our analysis has not established a strong link between nutrient concentrations and dinoflagellate densities, several investigators have reported an increasing dominance of dinoflagellates during the 1980s and early 1990s when nutrient concentrations in Tolo Harbour were at their highest (Chan and Hodkiss, 1987;[START_REF] Hodgkiss | Pollution studies in Tolo Harbour, Hong Kong[END_REF][START_REF] Hodgkiss | Are changes in N:P ratios in coastal waters the key to increased red tide blooms?[END_REF][START_REF] Lam | Phytoplankton characteristics of Tolo Harbour[END_REF]. The density of dinoflagellates was at a peak in 1996 when TIN/PO 4 was very low (Figs. 3 &4). The density of dinoflagellates reached another peak in 1998, due primarily to an extremely dense population of Prorocentrum (122,467 cells ml -1 ) at TM3 in April.

But dinoflagellate densities have remained relatively low since 2005, while TIN/PO 4 increased. [START_REF] Hodgkiss | Are changes in N:P ratios in coastal waters the key to increased red tide blooms?[END_REF] also noted a decrease in N:P ratios and an increase in algal blooms from 1981-1990. Our data showed no significant correlation between these two variables, although an increase in TIN/PO 4 and a decrease in the number of algal blooms were found after declines in nutrient concentrations. Nevertheless, while our data did not contain information about Noctiluca blooms, [START_REF] Liu | Seasonal and spatial dynamics of Noctiluca scintillans in a semi-enclosed bay in the northeastern part of Hong Kong[END_REF] showed that Noctiluca scintillans were still common in Tolo Harbour after declines in nutrient concentrations, and Noctiluca blooms still persisted.

Compared to the decrease in N concentrations, the decrease in SiO 2 concentrations was less dramatic. Although a significant decrease in depth-averaged SiO 2 concentrations was recorded after declines in nutrient input (p = 0.045), surface SiO 2 concentrations did not changed significantly (p = 0.849). Overall, TIN/SiO 2 tended to decrease at all sampling stations. Diatoms have a physiological N:Si ratio of 1:1 [START_REF] Turner | Element ratios and aquatic food webs[END_REF], and an environmental ratio of > 1 has been reported to limit diatom growth and shift a diatom-dominated community to one dominated by non-diatoms, particularly flagellates [START_REF] Cloern | Our evolving conceptual model of the coastal eutrophication problem[END_REF][START_REF] Egge | Silicate as regulating nutrient in phytoplankton competition[END_REF][START_REF] Paul | Nutrient regimes and their effect on distribution of phytoplankton in the Bay of Bengal[END_REF][START_REF] Turner | Fluctuating silicate:nitrate ratios and coastal plankton food webs[END_REF]. While diatom densities did correlate weakly with TIN/SiO 2 in TM3 and TM4, diatoms were the most abundant algal group in these two stations and Tolo Harbour [START_REF] Lam | Phytoplankton characteristics of Tolo Harbour[END_REF]Yung et al., 1997) before declines in nutrient concentrations, when TIN/SiO 2 at TM3 and TM4 were frequently > 1. This suggests that although N:Si ratios may have some effect on diatom densities, it is not enough to shift diatom dominance in Tolo Harbour. In addition, the Si concentrations in Tolo Harbour may not be limiting to diatom growth. [START_REF] Egge | Silicate as regulating nutrient in phytoplankton competition[END_REF] reported that a Si concentration of > 2 µM was enough to induce consistent dominance by diatoms in Norway. While this low threshold concentration may not apply in the warm subtropical waters of Hong Kong, the lowest SiO 2 concentration in this study was ~ 10 µM at TM8, fivefolds higher than the threshold concentration reported in Norway, and may be sufficient to support diatom growth regardless of TIN/SiO 2 . It should be noted that the maintenance of dominance by diatoms may not necessarily be beneficial both ecologically and economically. Although centric diatoms have been suggested to be the more desirable phytoplankton group when compared to non-diatom groups, such as dinoflagellates, due to their higher nutritional value and low toxicity [START_REF] Hecky | Nutrient limitation of phytoplankton in freshwater and marine environments: A review of recent evidence on the effects of enrichment[END_REF][START_REF] Officer | The possible importance of silicon in marine eutrophication[END_REF], pennate diatoms, including those belonging to the genus Pseudo-nitzschia, are also common in Tolo Harbour [START_REF] Lam | Phytoplankton characteristics of Tolo Harbour[END_REF]. Some species of Pseudo-nitzschia have been known to release the toxin domoic acid [START_REF] Lefebvre | Domoic acid in planktivorous fish in relation to toxic Pseudo-nitzschia cell densities[END_REF][START_REF] Sekula-Wood | Rapid downward transport of the neurotoxin domoic acid in caostal waters[END_REF], and a connection between Pseudo-nitzschia blooms and eutrophication has been proposed [START_REF] Parsons | Sedimentological evidence of an increase in Pseudo-nitzschia (Bacillariophyceae) abundance in response to coastal eutrophication[END_REF][START_REF] Schnetzer | Blooms of Psuedo-nitzschia and domoic acid in the San Pedro Channel and Los Angeles harbor areas of the Southern California Bight, 2003-2004[END_REF]. Fortunately, Pseudo-nitzschia spp. may prefer high nitrate waters [START_REF] Paul | Nutrient regimes and their effect on distribution of phytoplankton in the Bay of Bengal[END_REF]Carter et al., 2005), so declines in nutrient concentrations in Tolo Harbour should discourage Pseudo-nitzschia blooms.

Although our data does not contain information on the size structure of the phytoplankton community, changes in nutrient concentration or ratios may cause a shift from larger phytoplankton species or cells to smaller ones. [START_REF] Stolte | Effect of phytoplankton cell size on transiet-state nitrate and ammoniam uptake kinetics[END_REF] had theorized that eutrophication favors large phytoplankton species because their larger storage capacity allows them to better compete under high nutrient concentrations. Indeed, a shift towards an eutrophic environment has been shown to favor large diatoms in the western Wadden Sea (Stolte el al., 1994;[START_REF] Philippart | Long-term phytoplankton-nutrient budgets, and denitrification potential[END_REF], and [START_REF] Qiu | Phytoplankton dynamics in and near the highly eutrophic Pearl River Estuary, South China Sea[END_REF] reported strong negative correlations between < 3 µm chlorophyll-a and nutrient concentrations. The half-saturation constant for P uptake has been reported to be as high as 4.85-6.67 µM at 20 o C for certain large diatom species [START_REF] Nishikawa | Nitrate and phosphate uptake kinetics of the harmful diatom Eucampia zodiacus Ehrenberg, a causative organism in the bleaching of aquacultured Porphyra thalli[END_REF]2010), much higher than the means of TP in Tolo Harbour after nutrient loading reduction (Table 2). Declines in nutrient concentrations in Tolo Harbour may therefore favor smaller cells and species that are more competitive in a less eutrophic condition. [START_REF] Qiu | Phytoplankton dynamics in and near the highly eutrophic Pearl River Estuary, South China Sea[END_REF] reported significant positive correlation between < 20 µm chlorophyll a concentrations and N/P ratios.

Our data also show a strong and positive correlation between TIN/PO 4 and other algal group densities, caused mainly by an increase in the density of small flagellates and Cryptomonas. Recent studies in Tolo Harbour have revealed that cells < 5 µm accounted for > 25% of the chlorophyll-a biomass [START_REF] Lie | Selectivity and grazing impact of microzooplankton on phytoplankton in two subtropical semi-enclosed bays with different chlorophyll concentrations[END_REF][START_REF] Wong | Size structure and taxonomic composition of phytoplankton in Hong Kong waters[END_REF]. In addition, phytoplankton size structures in many aquatic ecosystems have also been known to be affected by climate change, particularly by increases in water column stratification due to increased temperature [START_REF] Finkel | Does size matter? The evolution of modern marine food webs[END_REF][START_REF] Smol | Climate-drive regime shifts in the biological communities of arctic lakes[END_REF]Winder et al., 2010). If indeed such a shift to smaller phytoplankton is occurring in Tolo Harbour, it would help to explain the increase in phytoplankton density as chlorophyll-a concentrations decrease.

Comparison with the recovery from eutrophication of other ecosystems

The effects of declines in nutrient concentrations had been intensively studied in freshwater lakes (e.g. [START_REF] Jeppesen | Response of phytoplankton, zooplankton and fish to re-oligotrophication: an 11-year study of 23 Danish lakes[END_REF][START_REF] Jeppesen | Lake responses to reduced nutrient loading -an analysis of contemporary long-term data from 35 case studies[END_REF][START_REF] Søndergaard | Seasonal dynamics in the concentrations and retention of phosphorus in shallow Danish lakes after reduced loading[END_REF], and most have reported improvements in water quality. Water quality improvement is mainly achieved through the decrease in phytoplankton biomass. This decrease can occur as a direct response to declines in nutrient concentrations, but it can be further facilitated through increased grazing pressure from zooplankton grazers [START_REF] Edmondson | Sixty years of Lake Washington: a curriculum vitae[END_REF] and the recolonization of submerged macrophytes, which has been shown to substantially affect the success rates of water quality improvement in lakes [START_REF] Mehner | Biomanipulation of lake ecosystems: successful applications and expanding complexity in the underlying science[END_REF]. While there may be differences between freshwater and marine systems in their responses to declines in nutrient concentrations [START_REF] Cloern | Our evolving conceptual model of the coastal eutrophication problem[END_REF][START_REF] Smith | Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems[END_REF], examples of dramatic improvements in once eutrophicated marine ecosystems such as Hillsborough Bay, Florida [START_REF] Johansson | Recent improvements in water quality and biological indicators in Hillsborough Bay, a highly impacted subdivision of Tampa Bay, Florida, USA[END_REF] and the coastal waters of Denmark [START_REF] Rask | Response to lowered nutrient discharges in the coastal waters around the island of Funen, Denmark[END_REF] had both involved the recolonization of macrophytes such as seagrasses (Hillsborough Bay) and eelgrass (Denmark).

However, an early study in Tolo Harbour has shown that most of the harbour was covered by extremely fine sediments that support little plant life [START_REF] Trott | Marine ecology in Tolo Harbour, Hogn Kong[END_REF]. Therefore, even with the decline of nutrient concentrations and increase in water transparency, the spread of macrophytes in Tolo Harbour is unlikely.

In regards to the zooplankton grazers in Tolo Harbour, a study comparing the copepod community before and after declines in nutrient concentrations in Tolo Harbour revealed a decrease in copepod density, from 6,392 ind. m -3 in 1987-1991, to 4,810 ind. m -3 in 2003-2004 (Wong et al., 1993; Zhang and Wong, submitted). The mesozooplankton community in Tolo Harbour was found to be heavily preyed upon by larval and juvenile stages of the black seabream, Acanthopagrus schlegeli, and the Japanese seaperch, Lateolabrax japonicus, which use Tolo Harbour as a nursery habitat [START_REF] Nip | Feeding ecology of larval and juvenile black seabream (Acanthopagrus schlegeli) and Japanese seaperch (Lateolabrax japonicus) in Tolo Harbour[END_REF]. While presently there is no detailed description on the fish population in Tolo Harbour, the abundance of small planktivorous fishes will most likely increase when increased fishing pressure from overfishing of larger piscivorous fishes reduces the predation pressure on the small fishes. Such changes will in turn increase the predation pressure on mesozooplankton grazers, reducing their abundance and delaying any further decrease of phytoplankton abundance.

Although every water mass may have different responses to declines in nutrient concentrations (Carvalho et al., 1995;[START_REF] Smith | Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems[END_REF], it is obvious that trophic interactions play a significant role in determining the outcome (Carpenter et al., 1995, Manca and[START_REF] Manca | Consequences of pelagic food-web changes during a long-term lake oligotrophication process[END_REF]Proulex et al., 1996;[START_REF] Smith | Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems[END_REF]. Increase in macrophytes and zooplankton grazers can further facilitate the decrease in phytoplankton biomass [START_REF] Edmondson | Sixty years of Lake Washington: a curriculum vitae[END_REF][START_REF] Mehner | Biomanipulation of lake ecosystems: successful applications and expanding complexity in the underlying science[END_REF], but the presence of planktivores and piscivores can affect the abundance of zooplankton grazers.

Therefore, an appropriate balance between piscivores, planktivores and zooplankton grazers should be maintained to ensure the long-term stability of the recovered ecosystem [START_REF] Mehner | Biomanipulation of lake ecosystems: successful applications and expanding complexity in the underlying science[END_REF]. Unfortunately, there is a lack of long-term monitoring on other communities, such as zooplankton and nekton, in Tolo Harbour.

As there is insufficient information on the responses of various communities to changes in nutrient loading, the phytoplankton community alone is not a reliable indicator of the trophic status of the whole ecosystem [START_REF] Philippart | Impacts of nutrient reduction on coastal communities[END_REF]). Future efforts in water quality monitoring and eutrophic coastal restoration should therefore focus not only on nutrient concentrations and phytoplankton biomass and densities, but also other important components of the aquatic ecosystem such as zooplankton and nekton communities. Monitoring began in 1988 for TM3 and TM4, 1994 for TM6 and 1986 for TM8.

Research highlights

Our study investigates the long-term changes in physico-chemical parameters, including nutrient concentration, nutrient ratios, and phytoplankton before and after nutrient reduction in an eutrophic bay in Hong Kong. Such study with long-term data relating environmental factors and phytoplankton is rather uncommon in marine ecosystems. We believe that such study of how anthropogenic causes led to changes in the phytoplankton community fits the aims and scope of your journal.

Table 1. Mean values (± standard deviation) of various physico-chemical, biological and nutrient ratios. "All stations" refers to the mean of all four sampling stations. "86-97" indicates the period before nutrient loading reduction (i.e. 1986-1997), and "98-08" indicates the period after nutrient loading reduction (i.e. 1998-2008). "Overall" refers to the entire study period of 1986-2008. Values are depth-averaged values except for "Bottom DO" and "Bottom BOD 5 ".

  response to nutrient reduction. Freshwater Biology 50, 1772-1780.Carpenter, S.R., Christensen, D.L., Cole, J.J., Cottingham, K.L., He, X., Hodgson, J.R., Kitchell, J.F., Knight, S.E., Pace, M.L., Post, D.M., Schindler, D.E., Voichick, N., 1995. Biological control of eutrophication. Envrionmental Science and Technology 29, 784-786. Carpenter, S.R., Ludwig, D., Brock, W.A., 1999. Management of eutrophication for lakes subject to potentially irreversible change. Ecological Applications 9, 751-771. Carter, C.M., Ross, A.H., Schiel, D.R., Howard-Williams, C., Hayden, B., 2005. In situ microcosm experiments on the influence of nitrate and light on phytoplankton community composition. Journal of Experimental Marine Biology and Ecology 326, 1-13. Carvalho, L., Beklioglu, M., Moss, B., 1995. Changes in a deep lake following sewage diversion -a challenge to the orthodoxy of external phosphorus control as a restoration strategy? Freshwater Biology 34, 399-410. Carvalho, L., Kirika, A., 2003. Changes in shallow lake functioning: response to climate change and nutrient reduction. Hydrobiologia 506-509, 789-796. Chau, K.W., 2007. Integrated water quality management in Tolo Harbour, Hong Kong: a case study. Journal of Cleaner Production 15, 1568-1572. Wu, R.S.S., 1988. Marine pollution in Hong kong: a review. Asian Marine Biology 5:1-23. Yung, Y.K., Wong, C.K., Broom, M.J., Ogden, J.A., Chan, S.C.M., Leung, Y., 1997. Long-term changes in hydrography, nutrients and phytoplankton in Tolo Harbour, Hong Kong. Hydrobiologia 352, 107-115. Zhang, G., Wong, C.K. Changes in the planktonic copepod community in a landlocked bay in the subtropical coastal waters of Hong Kong during recovery from eutrophication. Hydrobiologia submitted.

Fig. 1 .

 1 Fig. 1. A map of Hong Kong and Tolo Harbour showing the location of the four

Fig. 2 .

 2 Fig. 2. Temporal variations in the annual means of total inorganic nitrogen, total

Fig. 3 .Fig. 4 .

 34 Fig. 3. Temporal variations in the annual means of NO 3 /NH 4 , TIN/PO 4 and TIN/SiO 2

Fig. 5 .

 5 Fig. 5. Temporal variations in the annual means of diatoms, dinoflagllates, other algal

  Fig. 1.

Fig

  Fig. 2.

Fig

  Fig. 4.

Figure 5 .

 5 Figure 5.

Table 2 .

 2 Kendall's correlation between phytoplankton variables and various physico-chemical, biological and nutrient ratios parameters at four sampling stations in Tolo Harbour. "Dino" represents dinoflagellates, "Others" represents algal groups other than diatoms or dinoflagellates, "Total" represents total phytoplankton cell counts. "+/-" represents statistical significance at the 0.05 level; "++/--" represents statistical significance at the 0.01 level. Tested values were depth-averaged values except for "Bottom DO" and "Bottom BOD 5 ".

	Station

Acknowledgements

The authors would like to express their thanks to the staff of the Hong Kong Environmental Protection Department and the Government Chemists for collecting and analyzing the samples. We are also grateful to the Director of Environmental Protection for his permission to use the data. The opinions in this paper are those of the authors and do not necessarily reflect the views or policies of the Hong Kong Government. Suggestions and comments given by two anonymous reviewers are greatly appreciated.


Jörn Lötsch
email: j.loetsch@em.uni-frankfurt.de

Genetic variability of pain perception and treatmentclinical pharmaco-logical implications

Keywords: Pain, analgesics, pain genetics, pharmacogenetics, drug targets, new drugs, investiga-tional drugs, personalized medicine

Abstract

Evidence of a genetic control of pain has led to efforts to exploit genotyping information from pain patients for the development of analgesics and for the selection of pharmacological approaches to pain. Translating the genetic bases of familial insensitivity to pain research has contributed to the discovery of crucial molecular pathways of pain and to the identification of new analgesic targets (e.g., sodium channels Na v 1.7, neurotrophic tyrosine kinase receptors or the nerve growth factor).

Moreover, human genetic variants leading to enhanced or reduced function of molecular pathways are employed as substitutes for lacking modulator molecules usable in humans. This allows early studying of nociceptive or anti-nociceptive pathways in humans before drug development. Transla-tional approaches also served to verify the human importance of experimentally discovered pain pathways, such as in GTP cyclohydrolase 1 and the potassium channel K v 9.1. Besides these uses of genetics as a research tool, an individualized pharmacological therapy based on the patient's genotype has been attempted. For the actual analgesics, such guidance has only marginally become available. For upcoming analgesics targeting, for example, Na v 1.7 or TRPA1, the genotype may pro-vide a selective cure for syndromes caused by increased-function mutations in the coding genes. The implementation of human genetics may accelerate analgesic drug development while reducing its cost because the clinical success may be partly anticipated by including the information of func-tional genetic variants that mimic the action of future analgesics, showing that genotyping information from pain patients plays a role in the clinical pharmacology of pain.

Introduction

A relation between a patient's genotype and pain phenotype has been suggested about 50 years ago [START_REF] Godinova | Phenotypic manifestations and genetic features of migraine[END_REF]. In that early report, genetics was used for basic research to show that the risk of migraine is hereditarily transmitted, emphasizing underlying molecular mechanisms. With increasing sophistica-tion of genetic research, genotyping has become an effective scientific tool to identify the particular molecular causes underlying the susceptibility to pain, its intensity or clinical development to-ward either disappearance or chronification up to neuropathic pain. The risk or protective mecha-nisms provide primary candidate targets of new pharmacological approaches to pain treatment.

Besides concluding a molecular mechanism of pain from the identification of its genetic cause, translational approaches are being used to verify and quantitatively estimate the human importance of a molecular mechanism found in basic research. Functional genetic variants may serve as substi-tutes for unavailable drugs modulating the newly discovered molecular mechanism. This provides support for the successful development of pharmacological treatments targeting the identified mechanism.

While associating genotypes with phenotypes of pain patients has proved its scientific value in drug development, genotyping information is also expected to provide clinical guidance for the individual pharmacological approach to pain. However, this had so far only limited success [START_REF] Lötsch | A critical appraisal of human genotyping for pain therapy[END_REF] and a genotype based pain therapy has not yet emerged. Nevertheless, there is an imminent chance that this might improve with the approval of analgesics addressing new targets that are currently in the clinical phases of development.

Successful applications of genotyping pain patients are summarized in the following. The focus is on a key role in analgesic drug development or use. This excludes several observations of genetic modu-lations of pain or analgesia without this particular application, which are extensively summarized elsewhere [START_REF] Lötsch | Genetic modulation of the pharmacological treatment of pain[END_REF]. It further excludes candidate genes that from the analgesic's pharmacology are likely to contribute to the pharmacogenetics of the particular analgesic but for which neither evidence nor scientific utility has been proven, which has been previously covered [START_REF] Lötsch | Genetic predictors of the clinical response to opioid analgesics: clinical utility and future perspectives[END_REF].

Pharmacogenetic infor-mation obtained from pain patients has its main clinical pharmacological utility so far in drug de-velopment and in evidence based rather than merely possible modulations of drug therapy.

Genotyping and phenotyping in the development process of analgesics

Discovery of analgesic targets

The carriers are not pain patients but the opposite, but the molecular causes of their particular phe-notype characterized by absent pain provide primary candidate targets of analgesics. Six very rare hereditary sensory and autonomic neuropathy (HSAN) syndromes have been described [START_REF] Oertel | Genetic mutations that prevent pain: implications for future pain medication[END_REF]. They differ by their genetic causes, consisting of several loss-of-function mutations in the genes SPTLC1, WNK1, IKBKAP, NTRK1, NGFB and SCN9A, and by their association with neurological failures (Table 1). The molecular targets of the last three of these symptoms, i.e., TRK1 (tyrosine kinase 1), NGF (nerve growth factor) and Na v 1.7, are being employed as targets of analgesic drugs under develop-ment.

The nerve growth factor (NGF) is a small protein belonging to the class of neurotrophins and identi-fied originally as a survival factor for sensory and sympathetic neurons in the developing nervous system [START_REF] Fiore | Nerve growth factor as a signaling molecule for nerve cells and also for the neuroendocrine-immune systems[END_REF]. NGF binds at the p75 low affinity nerve growth factor receptor and at the transmem-brane tyrosine kinase receptor, the latter being associated with neurodegenerative disorders. NGF is not required for survival in adults but plays a crucial role in the generation of pain and hyperalgesia [START_REF] Fiore | Nerve growth factor as a signaling molecule for nerve cells and also for the neuroendocrine-immune systems[END_REF]. The expression of NGF is high in injured and inflamed tissues, and activation of the NGF receptor tyrosine kinase A on nociceptive neurons triggers and enhances nociceptive signalling by multi-ple mechanisms [START_REF] Hefti | Novel class of pain drugs based on antagonism of NGF[END_REF]. Members of a Swedish family carrying a hereditary insensitivity to pain were homozygous for a coding 661C>T SNP (R211W) in the NGFB gene, which affects a highly conserved region of the protein [START_REF] Einarsdottir | A mutation in the nerve growth factor beta gene (NGFB) causes loss of pain perception[END_REF]. These findings have triggered the development of a new class of analgesics including the monoclonal NGF-inhibiting antibody tanezumab which is an effective analgesic as shown for ostheoarthritic pain [START_REF] Lane | Tanezumab for the treatment of pain from osteoarthritis of the knee[END_REF][START_REF] Schnitzer | Longterm open-label study of tanezumab for moderate to severe osteoarthritic knee pain[END_REF].

+

The tyrosine kinase, TRK, is the receptor for NGF. As mice lacking the gene encoding the NGF receptor tyrosine kinase (ntrk1) display similar phenotypic features as patients with congenital insensitivity to pain with anhidrosis [11], mutations in the human NTRK1 gene have been studied as candi-date causes. Three mutations (1726delC with a premature translational stop, IVS15+3A>C with al-tered splicing, 1795 G>C with G571R amino acid substitution) in three unrelated subjects were iden-tified as the molecular basis of HSAN-IV [START_REF] Indo | Mutations in the TRKA/NGF receptor gene in patients with congenital insensitivity to pain with anhidrosis[END_REF]. Up-to-date, 37 different variations of the NTRK1 gene in families affected by CIPA are known (for a comprehensive review see [13]).

Among the at least 10 known subtypes of voltage-gated sodium channels responsible for action po-tential creation and propagation [14], Na v 1.7, 1.8 and 1.9 are expressed almost exclusively at noci-ceptive neurons. The complete inability to sense pain in otherwise healthy members of three con-sanguineous families from Pakistan was mapped as an autosomal recessive trait caused by a loss-offunction variant in the SCN9A gene [15]. This gene encodes the -subunit of the voltage-gated so-dium channel, Na v 1.7. In the three families, three distinct homozygous SCN9A nonsense mutations (S459X, I767X and W897X) were identified. In whole-cell voltage clamp experiments in HEK293 cells expressing mutant Na v 1.7, voltage-gated Na currents were not greater than the background level.

Additional very rare SCN9A variants have been added to the causes of this extreme phenotype [16]. Also rare, but not completely insensitive to pain, are carriers of a C1719R Na v 1.7 variant channel

[17], i.e., a genotype conferring residual channel function and therefore leading to a less extreme pain phenotype.

Verification of analgesic targets

Genetics plays a key role in identifying analgesic targets in laboratory animals [18]. In a transla-tional approach, variants in identified pain associated genes can be employed to test whether a molecular pathway identified in laboratory animals is relevant in humans.

Moreover, human genetic variants leading to enhanced or reduced function of molecular pathways can be employed as substi-tutes for lacking modulator molecules applicable to humans. This allows early studying of nocicep-tive or anti-nociceptive pathways in humans before drug development.

A genetic association was used as a tool for a proof-of-concept in assessments where the mechanism had been already identified without a major human genetics approach (Table 2). For example, the role of the enzyme guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1) activity for pain was found in laboratory animals and subsequently, its relevance for human pain was verified using a genetic approach Because rate limiting for BH 4 synthesis, GCH1 is a key modulator of peripheral neuropathic and in-flammatory pain [19]. Human GCH1 genetic variants in non-coding and non-splicing regions of the GCH1 gene were found to decrease the risk or intensity of chronic pain. They have allelic frequencies of approximately 15 -20% in Caucasians and decrease enzyme up-regulation and tetrahydrobiopterin production at the molecular level. Specifically, a particular GCH1 haplotype comprising 15 GCH1 SNPs was associated with an improved outcome in patients with chronic neuropathic pain

[19], and with reduced sensitivity to experimental pain [22,23]. Moreover, in a crosssectional observational study, carriers of this GCH1 haplotype were found to have been shorter on specialized therapy of chronic pain of various etiologies than non-carriers [24].

We interpreted this as a prophylactic role of decreased GCH1 upregulation delaying the need for pain therapy. This hypothesis could subse-quently be verified by showing that indeed the interval between cancer diagnosis and opioid therapy initiation was significantly longer in homozygous carriers of these GCH1 variants (78 ± 65.2 months) than in heterozygous and non-carriers (< 40 months) [25]. This suggested a future possibility of using partial GCH1 blockade or BH 4 inhibition to prevent or delay cancer pain.

Considering the shown molecular relevance of GCH1 for pain, negative findings such as lack of asso-ciation with pain following dental surgery [26]) or in chronic pancreatitis [27] do not necessarily substantially jeopardize GCH1 as an analgesic target. However, a cardiovascular association of a GCH1 variant with similar molecular consequences has to be considered during drug development. Specifically, the c.*243C>T SNP (dbSNP rs841 in the 3'UTR) was associated with decreased nitric oxide excretion in urine, mildly increased blood pressure and heart rate and dysfunction of the baro-receptor reflex in homozygous carriers [28]. Moreover, this variant is carried by most people who also carry the variants associated with decreased or delayed pain [START_REF] Doehring | Clinical genetics of functionally mild non-coding GTP cyclohydrolase 1 (GCH1) polymorphisms modulating pain and cardiovascular risk[END_REF]. This draws the attention at potential cardiovascular problems due to inhibition of BH 4 production.

The involvement of potassium channels in pain pathology has also benefitted from a genetic sup-port. Specifically, the potassium voltage-gated channel, delayed-rectifier, subfamily S, member 1 (KCNS1, K v 9.1) has been found in rodents to be a putative pain gene down-regulated following nerve injury by that facilitating the transition to chronic neuropathic pain [START_REF] Costigan | Multiple chronic pain states are associated with a common amino acid-changing allele in KCNS1[END_REF]. These potassium channels are co-regulated in several pain models. K v 9.1 is expressed in naive rats at high levels in a subset of dorsal root ganglia neurons [START_REF] Costigan | Multiple chronic pain states are associated with a common amino acid-changing allele in KCNS1[END_REF]. The association with pain was subsequently verified in humans. In 151 lumbar discectomy patients, the KCNS1 coding variant rs734784 (Ile489Val) and the synony-mous variant rs13043825 were associated with greater pain and with a higher risk to fail 1-year pain improvement [START_REF] Costigan | Multiple chronic pain states are associated with a common amino acid-changing allele in KCNS1[END_REF]. The functional association of the first variant was also found in 199 amputees with phantom limb pain and in two further cohorts with neuropathic pain [START_REF] Costigan | Multiple chronic pain states are associated with a common amino acid-changing allele in KCNS1[END_REF]. These first results may suggest agonism at K v 9.1 as a possible pharmacological strategy to prevent the development of chronic pain.

Genotyping and phenotyping patients receiving analgesics

With the popularity of genotyping and first successes in other fields such as cancer therapy, where the FDA has considered [START_REF] Maitland | TPMT, UGT1A1 and DPYD: genotyping to ensure safer cancer therapy?[END_REF] advising UGT1A1 (uridine diphosphate glucuronosyltransferase) geno-typing for irinotecan treatment safety [START_REF] Innocenti | Genetic variants in the UDP-glucuronosyltransferase 1A1 gene predict the risk of severe neutropenia of irinotecan[END_REF] and TPMT (thiopurin methyl transferase) [START_REF] Relling | Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus[END_REF]genotyping for 6-mercaptopurine treatment safety [START_REF] Relling | Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus[END_REF]) [START_REF] Maitland | TPMT, UGT1A1 and DPYD: genotyping to ensure safer cancer therapy?[END_REF], genotyping information has been expected [START_REF] Lötsch | Genetic modulation of the pharmacological treatment of pain[END_REF][START_REF] Kim | Genetics, Pain, and Analgesia[END_REF][START_REF] Kim | Genetic contributions to clinical pain and analgesia: avoiding pitfalls in genetic research[END_REF][START_REF] Lötsch | Current evidence for a genetic modulation of the response to analgesics[END_REF] to also provide a major basis of personalized pain therapy. While in drug discovery the identifi-cation of the analgesics' pharmacodynamic targets plays the main role, in individualized therapy both pharmacodynamics and pharmacokinetics of available analgesics may be subject to individual genetically caused modulations.

Pharmacodynamics of analgesics

Pharmacodynamic genetic modulations have been identified for the few primary targets of current analgesics and their downstream signalling, i.e., for -opioid receptors [START_REF] Lötsch | Are mu-opioid receptor polymorphisms important for clinical opioid therapy?[END_REF] and Girk3.2 potassium channels [38], and for COX-2 [START_REF] Lee | Genetically mediated interindividual variation in analgesic responses to cyclooxygenase inhibitory drugs[END_REF].

The most often addressed -opioid receptor variant is OPRM1 118 A>G (rs1799971), leading to an amino acid exchange N40D. Molecular evidence suggests that it causes a decreased receptor signal-ling in the pain-relevant secondary somatosensory brain area (S2) [START_REF] Oertel | A Common Human mu-Opioid Receptor Genetic Variant Diminishes the Receptor Signaling Efficacy in Brain Regions Processing the Sensory Information of Pain[END_REF] and was also reported to decrease -opioid receptor expression throughout the human brain, [START_REF] Zhang | Allelic expression imbalance of human mu opioid receptor (OPRM1) caused by variant A118G[END_REF],

however, not in S2 [START_REF] Oertel | A Common Human mu-Opioid Receptor Genetic Variant Diminishes the Receptor Signaling Efficacy in Brain Regions Processing the Sensory Information of Pain[END_REF]. From the altered receptor function or density, a decreased opioid potency can be predicted. From the first in-vitro [START_REF] Bond | Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction[END_REF] and in-vivo [START_REF] Lötsch | Does the A118G polymorphism at the mu-opioid receptor gene protect against morphine-6-glucuronide toxicity?[END_REF] reports of its functionality, frequent reports from experi-mental settings controlling for many confounders and in various clinical studies [START_REF] Lötsch | Are mu-opioid receptor polymorphisms important for clinical opioid therapy?[END_REF] found such roles. A decreased net opioid potency was also repeatedly found in clinical settings. However, a general association of the rs1799971 variant with reduced opioid effects and increased opioid dos-ing demands did not withhold a meta-analysis [START_REF] Walter | Meta-analysis of the relevance of the OPRM1 118A>G genetic variant for pain treatment[END_REF]. A practical prospective guidance for opioid dosing by this variant has not been obtained. Testing pain patients, who require extremely high opioid doses, for the OPRM1 variant rs1799971 is supported by a case report about a patient carrying the OPRM1 118G variant allele and needing more than 2 g daily morphine for cancer pain relief [START_REF] Hirota | Sequence variability and candidate gene analysis in two cancer patients with complex clinical outcomes during morphine therapy[END_REF]. However, a systematic assessment has not yet been published. In addition, the protection from res-piratory depression conferred by homozygous presence of the OPRM1 rs1799971 variant [START_REF] Oertel | The mu-opioid receptor gene polymorphism 118A>G depletes alfentanil-induced analgesia and protects against respiratory depression in homozygous carriers[END_REF] Downstream signalling of opioid receptors involves potassium channels. Among others (see above) the potassium inwardly-rectifying channel, subfamily J, member 6, (Kir3.2, GIRK2) is listed in the PainGenes database [START_REF] Lacroix-Fralish | The Pain Genes Database: An interactive web browser of pain-related transgenic knockout studies[END_REF] at http://www.jbldesign.com/jmogil/enter.html, a collection of nocicep-tive and pharmacological genetic association information obtained in rodents, as modulating opioid effects. This G protein coupled channel is important for opioid receptor transmission and is involved in opioid effects on postsynaptic inhibition [START_REF] Mitrovic | Contribution of GIRK2-mediated postsynaptic signaling to opiate and alpha 2-adrenergic analgesia and analgesic sex differences[END_REF] and mediating a significant component of analgesia [START_REF] Kobayashi | Ethanol opens G-protein-activated inwardly rectifying K+ channels[END_REF][START_REF] Marker | Spinal G-protein-gated K+ channels formed by GIRK1 and GIRK2 subunits modulate thermal nociception and contribute to morphine analgesia[END_REF]. Two genetic variants in KCNJ6 (rs6517442 and rs2070995) have been shown to increase opioid requirements in Japanese patients after abdominal surgery [38]. The same tendency toward higher opioid doses in carriers of rs2070995 was seen in chronic pain patients in outpatient care. In former heroin addicts average daily methadone substitution doses during the first therapy year were comparatively larger in carriers than in non-carriers of rs2070995 [START_REF] Lötsch | A KCNJ6 (Kir3.2, GIRK2) gene polymorphism modulates opioid effects on analgesia and addiction but not on pupil size[END_REF].

However, these findings (Table 3) have not entered broad clinical practice. Genetic guidance of the choice of analgesics is so far only marginally possible, such as for the selection of -opioid agonists being more effective in women carrying certain variants in the melanocortin1 receptor gene confer-ring a red-hair-fair-skin phenotype [START_REF] Mogil | The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans[END_REF]. Selective cyclooxygenase (COX) 2 inhibitors might not be chosen for treatment of inflammatory pain in carriers of PTGS2 variants observed to be associated with decreased COX-2 upregulation following injury [START_REF] Lee | Genetically mediated interindividual variation in analgesic responses to cyclooxygenase inhibitory drugs[END_REF], although this requires reproduction due to a contrasting finding [START_REF] Skarke | The cyclooxygenase 2 genetic variant -765G>C does not modulate the effects of celecoxib on prostaglandin E2 production[END_REF].

Regarding the upcoming new analgesics (Table 3) involving many new targets [59],

pharmacogene-tics of pain and analgesia may be employed as a guidance for the choice of the optimum analgesic. Pharmacogenetic information may therefore be of great value in choosing the optimum analgesics, along non-genetic, for example disease-specific, guidance factors. Upcoming analgesics targeting Na v 1.7 sodium channels [START_REF] Clare | Targeting voltage-gated sodium channels for pain therapy[END_REF] and pungent chemicals such as horseradish, mustard, cinnamon and garlic [61] or cannabinoids [START_REF] Jordt | Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1[END_REF], TRPM8 channels are stimulated by cold between 8 and 28°C [72] and cooling compounds such as menthol [START_REF] Peier | A TRP channel that senses cold stimuli and menthol[END_REF]. TRPV1 mediates pain in-duced by noxious heat (>43°C), capsaicin or protons. TRPV3, activated at temperatures of 22 -40°C, is also expressed at sensory nerve endings [START_REF] Eid | Transient receptor potential channels on sensory nerves[END_REF]. Several new analgesics are being developed for TRP channels, and future discoveries of pain syndromes caused by increased-function variants may benefit from this upcoming selection of analgesics.

Pharmacokinetics of analgesics

Functional CYP2D6 variants alter the effects of codeine [START_REF] Poulsen | Codeine and morphine in extensive and poor metabolizers of sparteine: pharmacokinetics, analgesic effect and side effects[END_REF] and tramadol [START_REF] Collart | Duality of the analgesic effect of tramadol in humans[END_REF] via absent or exag-gerated production of their active metabolites. The latter have been presented in the worst cases as the causes of deaths following codeine administrations [77][78][START_REF] Ferreirós | Fatal and severe codeine intoxication in 3-year-old twins--interpretation of drug and metabolite concentrations[END_REF][START_REF] Voronov | Apnea in a child after oral codeine: a genetic variant -an ultra-rapid metabolizer[END_REF]. Single cases of exaggerated fatal opioid effects after codeine administration were explained by the CYP2D6 ultrafast metabolizer gen-otype (summarized in [START_REF] Lötsch | Can extremely low or high morphine formation from codeine be predicted prior to therapy initiation?[END_REF]), i.e., carrying three or more copies of the CYP2D6 gene leading to in-creased enzyme expression. For example, a 33-year old woman took 60 mg codeine prophylactically to avoid pain due to tooth extraction. Within 30 min she experienced euphoria, dizziness and diffi-culties in recognizing details in a TV program [START_REF] Dalen | Quick onset of severe abdominal pain after codeine in an ultrarapid metabolizer of debrisoquine[END_REF]. Twelve hours after a last codeine dose, con-sciousness of a 62-year old patient deteriorated and he became unresponsive [START_REF] Gasche | Codeine intoxication associated with ultrarapid CYP2D6 metabolism[END_REF]. Both patients were identified as carriers of the CYP2D6 ultra-rapid metabolizer types. Codeine is a weak opioid analgesic and the genotyping effort before therapy initiating has not become a clinical priority, although rare cases of codeine toxicity might benefit but not all of the patients at risk [START_REF] Lötsch | Can extremely low or high morphine formation from codeine be predicted prior to therapy initiation?[END_REF].

Besides drug metabolism, drug distributional changes may cause altered opioid effects. For example, variants in ABCB1, coding for P-glycoprotein, increase the brain concentrations of various opioids via altering their outward transport across the blood brain barrier [START_REF] Campa | Association of ABCB1/MDR1 and OPRM1 gene polymorphisms with morphine pain relief[END_REF][START_REF] Skarke | Effects of ABCB1 (multidrug resistance transporter) gene mutations on disposition and central nervous effects of loperamide in healthy volunteers[END_REF][START_REF] Coller | ABCB1 genetic variability and methadone dosage requirements in opioiddependent individuals[END_REF][87]. However, despite statisti-cally significant association, a clinical utility of these genotyping has not emerged (Table 3).

Addiction potential

Among classical analgesics, opioids are associated with potential addiction and clinical pharmaco-logical expertise may be clinically queried to estimate the risk for drug dependence in pain patients.

As the main target of opioid analgesics, the -opioid receptor has been primarily assessed for vari-ants that modulate the addiction to opioids. The OPRM1 118>G variant has been repeatedly reported to increase the risk, mostly based on its higher frequency in addicts than in non-addicted subjects [START_REF] Szeto | Association between mu opioid receptor gene polymorphisms and Chinese heroin addicts[END_REF][START_REF] Shi | Sequence variations in the muopioid receptor gene (OPRM1) associated with human addiction to heroin[END_REF][START_REF] Tan | Mu opioid receptor gene polymorphisms and heroin dependence in Asian populations[END_REF] but lack of such association was similarly often found [START_REF] Bond | Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction[END_REF][START_REF] Hoehe | Sequence variability and candidate gene analysis in complex disease: association of mu opioid receptor gene variation with substance dependence[END_REF][START_REF] Crowley | A genetic association study of the mu opioid receptor and severe opioid dependence[END_REF]. The OPRM1

17C>T SNP showed also a tendency toward higher frequency in substance abusers [START_REF] Bond | Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction[END_REF][START_REF] Berrettini | Human mu opioid receptor gene polymorphisms and vulnerability to substance abuse[END_REF], and OPRM1 haplo-types were further suggested to be associated with a modified addiction risk [START_REF] Szeto | Association between mu opioid receptor gene polymorphisms and Chinese heroin addicts[END_REF][START_REF] Shi | Sequence variations in the muopioid receptor gene (OPRM1) associated with human addiction to heroin[END_REF][START_REF] Hoehe | Sequence variability and candidate gene analysis in complex disease: association of mu opioid receptor gene variation with substance dependence[END_REF][START_REF] Luo | Haplotypes at the OPRM1 locus are associated with susceptibility to substance dependence in European-Americans[END_REF]. How-ever, none of the OPRM1 genetic findings in the context of addiction has achieved a clinical utility. This applies to variants in other genes such as a modulation of methadone demands in former heroin addicts by dopamine 2 receptor (DRD2) polymorphisms [START_REF] Doehring | Genetic variants altering dopamine D2 receptor expression or function modulate the risk of opiate addiction and the dosage requirements of methadone substitution[END_REF][START_REF] Barratt | Association between the DRD2 A1 allele and response to methadone and buprenorphine maintenance treatments[END_REF]. Furthermore, a protective effect from heroin addiction was found for European Americans carrying a HTR1B (5-Hydroxytryptamine (serotonin)-1B receptor) haplotype [START_REF] Proudnikov | Association analysis of polymorphisms in serotonin 1B receptor (HTR1B) gene with heroin addiction: a comparison of molecular and statistically estimated haplotypes[END_REF]. In Hispanics, an association of the minor allele of the MC2R (melanocortin receptor type 2) SNP rs2186944 and for an MC2R haplotype with a protective effect from the development of heroin addiction was shown [98]. In addition to a modulation of the risk for addiction [START_REF] Goldman | The genetics of addictions: uncovering the genes[END_REF], which is a multigenetic trait not specific to opioid analgesics, further phar-macogenetic influences on opioid addiction are modulating their pharmacokinetics by drug metabolism or transport such as P-glycoprotein variants [START_REF] Coller | ABCB1 genetic variability and methadone dosage requirements in opioiddependent individuals[END_REF].

Conclusions

In the clinical setting, where genotyping information was viewed with high expectations, genetics have so far only reluctantly been included, which led to a general questioning of Therefore, genotyping and phenotyping pain patients have their main utility as a research tool for discovery or proof-of-concept of analgesics' targets.

( # Genetically cased functional change: ---to +++: The degree of genetically caused changes in the net function of the gene product, achieved by either modulation of the expression or function of the protein, is gradually displayed from absent function, "---", to highly increased function, "+++".

COMT Catchechol-O-methyl transferase rs4680 # Genetically cased functional change: ---to +++: The degree of genetically caused changes in the net function of the gene product, achieved by either modu-lation of the expression or function of the protein, is gradually displayed from absent function, "---", to highly increased function, "+++". *: Analgesic drug target

  [19]. GCH1 catalyzes the conversion of GTP to dihydroneopterin triphosphate, which is further metabolized to tetrahydrobiopterin (BH 4 ) by the enzymes 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase [20]. BH 4 is an essential co-factor for the three isoforms of nitric oxide synthase catalyzing the synthesis of nitric oxide and citrulline from arginine, and for tyrosine-, tryptophan-and phenylalanine hydroxylases [21] that catalyze the production of biogenic amines (noradrenaline, dopamine, adrenaline), the production of serotonin, and the degradation of phenylalanine to tyrosine, respectively. Inhibiting the de novo BH 4 synthesis in rats attenuated neuropathic and inflammatory pain whereas administering BH 4 intrathecally exacerbated pain [19]. After peripheral inflammation, BH 4 concentrations also increased in dorsal root ganglia due to en-hanced GCH1 enzyme activity [19].

  cannot be used to neglect the supervision of patients under postoperative opioid analgesia although it might provide reassurance to clinicians to administer high opioid doses to these patients. Therefore, the clinical utility of rs1799971 is unclear. A recently discovered OPRM1 SNP, rs563649 C>T causing alternative splicing of opioid receptors with the appearance of 6-transmembrane segment receptors [47] that produce increased cAMP concentrations after opioid binding [48], is possibly involved in hyperalgesic opioid effects. This might open an opportunity to predict this unwanted therapy course; however, it is so far not more than a clinical extrapolation from an in-vitro observation of the SNP's functional consequence. The expression of -opioid receptors seems modulated by the variant V158M catechol-Omethyl transferase coded by the 472G>A polymorphism (rs4680) of the COMT gene [49, 50]. It causes de-creased enzyme activity leading to chronic dopaminergic upregulation, which in turn causes enkephalinergic down-regulation. Finally, this results in a compensatory upregulation of -opioid receptors due to downregulation of endorphins, and patients carrying the V158M variant needed less morphine than non-carriers carrying this variant [51].

  their utility[100]. Despite positive results such as CYP2D6 related codeine toxicity, PTGS2 related COX-2 inhibitor inef-fectiveness, or OPRM1 variant associated safety of high opioid doses, cross sectional assessments show that in average pain patients analgesic therapy shows only dim traces of a genetic influence[101]. Moreover, most of the here presented positive findings are contrasted by negative reports about the same variants. In addition, many genetic variants, summarized elsewhere[START_REF] Lötsch | Genetic modulation of the pharmacological treatment of pain[END_REF][START_REF] Kim | Genetics, Pain, and Analgesia[END_REF] 102], may influence analgesic therapy by modifying the disease rather than the pharmacology of the cure. However, with the future availability of new analgesics addressing new targets [103], the clinical utility of genotyping and phenotyping of pain patients may become equal with its scientific utility in the clinical pharmacological context up to providing specific cures for pain syndromes caused by particular enhanced-function mutations of targets that will be soon possible to be inhibited.From a clinical pharmacological point of view, genotyping and phenotyping of pain patients has proven its use so far mainly for drug discovery and development. Human genetics may serve both directions, i.e., identifying analgesic drug targets or, in the classical translational sense, verifying the human relevance of targets found in basic research. The intelligent implementation of human genetics may accelerate analgesic drug development while reducing its cost because the clinical success may be partly anticipated by including the information of functional genetic variants that mimic the action of future analgesics.

Table 1 :

 1 Familial syndromes with absent pain and their genetic basis.

		): 485-488
	13	Indo Y (2001) Molecular basis of congenital insensitivity to pain with anhidrosis
	(CIPA):
		mutations and polymorphisms in TRKA (NTRK1) gene encoding the receptor tyrosine
		kinase for nerve growth factor. Hum Mutat 18 (6): 462-471

Table 2 :

 2 Genes that played an important role in analgesic drug discovery.

	Gene Gene product	Functional Genetically	Pharmacological implica-Reference
				change #	caused pheno-tions
					type
	NTRK	Neurotrophic ty-	---	Absent pain	Identification of TRK1	[113]
	1	rosine	kinase			as
		receptor 1				an analgesic target
	NGF1 Nerve	growth	---	Absent pain	Identification of NGF	[8]
		factor				as
	SCN9	Na v 1.7	sodium	---	Absent pain	an analgesic target Identification and verifi-	[15, 112]
	A	channel		-	Reduced pain	cation of Na v 1.7 as
				+++	Increased pain	an analgesic target	[62, 63]
				++
	GCH1 GTP cyclohydro-	-	Reduced or	Verification of GCH1	[19]
		lase 1			de-	as
					layed pain	pain relevant and a
	KCNS	Kv1.9 potassium	-	Increased risk	pos-sible analgesic Verification of KCNS1	[30]
	1	channel			of	target as
					pain chronifica-	pain	relevant	and
					tion	ago-nists	being
						possible an-algesic

Table 3 :

 3 Genes and example variants that might provide comparatively important genotyping information to guide analgesic therapy.

	-

Madadi P, Koren G, Cairns J, Chitayat D, Gaedigk A, Leeder JS, Teitelbaum R,