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Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long‐ and short‐read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC‐FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post‐fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y‐linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X‐linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis. [ABSTRACT FROM AUTHOR] more...

Chemical communication is better understood in freshwater fish than marine fish. The Senegalese sole (Solea senegalensis) is a marine flatfish wherein one of the problems in aquaculture is the poor reproductive performance of hatchery-bred males. Is chemical communication involved in the reproduction of this species? Urine, intestinal fluid and mucus samples were taken from adult fish (wildcaught and hatchery-bred) over the spawning season (March-May), and assessed for olfactory potency using the electro-olfactogram (EOG). The effect of stimulation of the olfactory system with adult female urine on circulating luteinizing hormone (LH) levels was also tested in males. Intestinal fluid and urine were potent olfactory stimuli for both juvenile and adult conspecifics, evoking large-amplitude, concentration-dependent EOG responses, with thresholds of detection at approximately 1:106. However, the amplitude of the response to urine depended on the sex and state of maturity of both the donor and the receiver. Most olfactory activity could be extracted by C18 solid-phase cartridges. Urine from mature females evoked a slight, but significant, increase in circulating LH levels in mature males 30 min after exposure. Furthermore, the olfactory potency of urine differed between wild-caught and hatchery-bred fish; however, contrary to expectations, urine from wild-caught females was less potent than that from hatchery-bred females. Taken together, these results strongly suggest that faeces- and urine-released odorants are involved in reproduction in the Senegalese sole, and establish a basis for further investigation into pheromonal communication in marine teleosts. [ABSTRACT FROM AUTHOR] more...

Senegalese sole was one of the earliest identified candidate species with high potential for aquaculture diversification in the south of Europe. Its culture has been possible, and commercially attempted, for several decades, but intensive production has been slow to take off. This has been explained mostly by serious disease problems, high mortality at weaning, variable growth and poor juvenile quality. However, a strong and sustained research investment that started in the eighties has led to a better understanding of the requirements and particularities of this species. More recently, better management and technical improvements have been introduced, which have led to important progress in productivity and given a new impetus to the cultivation of Senegalese sole. As a result, the last 5 years have marked a probable turning point in the culture of sole towards the development of a knowledge-driven, competitive and sustainable industry. This review will focus on the main technical improvements and advances in the state of knowledge that have been made in the last decade in areas as diverse as reproductive biology, behaviour, physiology, nutritional requirements, modulation of the immune system in response to environmental parameters and stress, and characterization and mitigation of the main disease threats. It is now clear that Senegalese sole has important particularities that differentiate it from other current and candidate marine aquaculture species, which bring about important challenges, some still unsolved, but also notable opportunities (e.g. a nutritional physiology that is better adapted to dietary vegetable ingredients), as will be discussed here. [ABSTRACT FROM AUTHOR] more...

• Reared breeders of Solea senegalensis show plasticity in observational learning. • Housing conditions from the juvenile stage contribute to reproductive behaviour success. • Social learning from experienced congeners offers opportunities for courtship success. • A lack of consistency in reproductive behaviour and spawning occurs over time. • A system of stimulus reinforcement may be necessary for reactivation of the behaviour. The reproductive failure of Senegalese sole (Solea senegalensis) cultured males (reared entirely in captivity from egg through to adult) that do not participate in reproductive behaviours to fertilise spawns, results in a problem to achieve reproductive control in captivity. However, cohabitation with wild males has led to an increase in the involvement of cultured males in reproductive behaviour, although their contribution to fertilised spawning is still lower than that of wild breeders. This study aimed to examine the effect of different social conditions, on the reproductive behaviour and spawning success of cultured breeders over three reproductive seasons. Before starting this study, different social learning opportunities were provided to the breeders from the juvenile to the pubertal stages of the individuals. Behaviour and spawning were evaluated in four experimental groups of cultured breeders: two groups (W1 and W2) that prior to this study were reared during the juvenile stage with wild breeders that fertilized spawns, a Culture breeder group (CB) that was previously reared with cultured breeders that spawned unfertile eggs, and a negative control group (CN) that was reared in isolation from adult fish. During the three reproductive seasons, spawning was obtained from all groups. Generally, the first year had the highest egg production and the third year the lowest. However, fertilised eggs were only obtained from W1 in the first year. A total of eight fertilised spawns were collected with a fertilisation rate of 28.02 ± 13.80 % and a hatching rate of 15.04 ± 10.40 %. The mean number of larvae obtained per spawn was 7,683 ± 5,947 and the total number of larvae from all eight spawns was 61,468. The paternity analysis assigned 64.3 % of larvae to a single couple of breeders, while 34.3 % of larvae were not assigned to any single family, but inconclusively to more than three parents. The highest locomotor activity was observed in W1, while no significant differences were observed in the number of movements within W2, CB and CN. In all groups, during the peak of locomotor activity (19 h 00-20 h 00), the main reproductive behaviours observed were Rest the Head and Follow , while the Guardian behaviour was low and Coupled behaviour was only observed in W1. Over time, the reproductive behaviours decreased, except for Follow. The social learning opportunities provided by cohabitation with wild fish during juvenile stages prior to spawning in W1, increased activity and fertilised spawning. However, the number of successful spawns was low and over time stopped in association with a decrease in reproductive behaviour. This suggests that other mechanisms of behavioural learning could be involved in reproductive success, such as reproductive dominance, environmental conditions or hormonal interactions that could affect physiological processes in the reproduction of captive breeders. [ABSTRACT FROM AUTHOR] more...

The intensive culture of the Senegalese sole ( Solea senegalensis ) is hampered by the low or null fertilization rates exhibited by the first generation (F1) of reared males. To investigate the regulation of the reproductive processes in this species by the pituitary gonadotropins follicle-stimulating and luteinizing hormones (Fsh and Lh, respectively), we developed a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for Lh measurements. Quantification of the Fsh and Lh plasma levels in cultured sole using the Lh ELISA developed here, and a previously developed ELISA for Fsh, indicated that in both males and females circulating Fsh steadily increased during autumn and winter and prior to the major spawning in spring, whereas an Lh surge occurred specifically during spawning. The increase in Fsh was associated with a rise of plasma levels of the steroid hormones testosterone (T), 11-ketotestosterone (11-KT) and estradiol-17β (E2), but that of Lh was concomitant with a strong decline of the levels of E2 in females and of 11-KT in males, possibly reflecting a rapid steroidogenic shift promoting the final maturation of gametes. Comparison of the plasma levels of gonadotropins and steroids between wild and F1 fish during autumn and spring revealed that F1 males showed significantly lower plasma Lh titres compared to wild males, whereas the levels of T and 11-KT were similar or more elevated in the F1 fish. These data suggest that an impaired Lh secretion during spawning, and perhaps altered Lh-mediated mechanisms in the testis, may be underlying causes for the low reproductive performance of Senegalese sole F1 males. [ABSTRACT FROM AUTHOR] more...

Consecutive treatments with recombinant follicle-stimulating and luteinizing hormones (rFsh and rLh, respectively) stimulate spermatogenesis and potentiate sperm production in pubescent specimens of the oligospermic Senegalese sole ( Solea senegalensis ). However, sperm production in response to the hormones is highly variable, and the steroidogenic potential of the testis may be diminished due to sustained hormone supply. Here, we compared the effectiveness of low (9 μg/kg) and high (18 μg/kg) doses of rFsh and rLh to improve sperm production in adult sole during late winter-early spring (onset of the natural spawning period), and in autumn under a controlled temperature of 12 °C (period of testicular recrudescence). Treatment with rFsh over six weeks during spring, followed by a single rLh injection, did not enhance sperm production, possibly because of an advanced stage of sexual maturation of the males, as reflected by high Lh plasma levels (~17 ng/ml) before rFsh treatment. In contrast, in autumn, when the Lh circulating levels were much lower (~3 ng/ml), the low doses of rFsh and rLh generated a four-times increase in sperm production, whereas the high doses of the hormones were ineffective. However, treatment with rLh, regardless of the effect of rFsh, improved the motility of spermatozoa during both spring and autumn. These data confirm that consecutive rFsh and rLh treatments increase sperm production and quality in adult sole males, although they seem to be highly sensitive to the rFsh dose. The efficiency of recombinant gonadotropins also appears to be season-dependent despite the asynchronous nature of the sole testis. [ABSTRACT FROM AUTHOR] more...

Few studies have examined the influence of diet on larval proactive–reactive behavioural dimension of stress coping style responses. The present study evaluated the influence of using different vegetables oils (Linseed; Soybean; Olive) and fish oil (Cod liver) for Artemia metanauplii nutritional enrichment on the proactive–reactive behavioural responses of Senegalese sole ( Solea senegalensis ) post-larvae (40 days post hatch). Forty-two Senegalese sole larvae from each of the four replicate tanks per treatment were tested. Two tests were performed: a new environment individual-based test, which evaluate the larvae latency time to move, total activity time and total distance moved; and a risk group-based test, which consisted in evaluating the larval capacity to cross from a “comfort” zone to a “risk” zone. In the group-based test, proactive, intermediate and reactive individuals were identified depending on the time taken to cross between two zones. Larvae fed with Artemia metanauplii enriched with the cod liver oil emulsion were significantly ( P = 0.01) larger and in the individual-based test presented significantly higher total activity time ( P = 0.08) and total distance moved ( P = 0.01) than larvae from the other dietary treatments. No significant correlations ( P > 0.05) were observed between larvae total length and latency time to move, total activity time or total distance moved across all treatments or within any dietary treatment. In the group-based test, fish fed with Artemia enriched with the cod liver oil emulsion presented a significantly higher proportion of proactive larvae ( P = 0.02) and the lower proportion of reactive larvae. The present study showed for the first time that (i) Senegalese sole presented a defined proactive–reactive behaviour from early ontogenesis and (ii) dietary fatty acid composition significantly influenced the proactive–reactive behavioural dimension of stress coping style of sole larvae. The current study has practical implications that open the possibility to produce organisms that have behavioural styles that could ultimately result in improved aquaculture productivity. [ABSTRACT FROM AUTHOR] more...

Highlights: [•] Dietary ARA levels affect the steroid production in cultured Senegalese sole. [•] Changes in dietary ARA content modified the fatty acid profile of fish blood. [•] Increased ARA levels in blood reduce the production of 3-series prostaglandins. [•] Increased ARA levels in blood increase the CHOL mobilization in the blood. [ABSTRACT FROM AUTHOR] more...

Abstract: Previous studies have shown higher levels of arachidonic acid (20:4n-6, ARA) in testis, liver, and muscle of wild Senegalese sole (Solea senegalensis) compared to fish reared in captivity (first generation, G1). The present study was conducted to establish the optimal level of dietary ARA for G1 Senegalese sole broodstock, using as a reference the fatty acid profile of wild broodstock (gonads, liver and muscle). A total of 120 Senegalese sole broodstock were randomly distributed into 12 tanks (1:1 male and female) and fed in duplicate with six experimental diets containing increasing amounts of ARA (0.7%, 1.6%, 2.3%, 3.2%, 5.0%, and 6.0 % of total fatty acids) for 9months. The relative ARA levels in liver, muscle and male and female gonads at the end of the feeding period increased in a dose dependent manner. Dietary ARA was mainly incorporated and stored in testis or ovary, followed by liver and muscle. Fish fed 2.3% and 3.2% ARA showed no differences in the ARA content of testis, ovary and liver when compared to wild fish. In male fish, a significant increase in the levels of 22:4n-6 and 22:5n-6 fatty acids was also observed, which was consistent with the up-regulation of fatty acyl elongase (elovl5) and desaturase (d4fad) transcript levels in the liver of fish fed 0.7%, 2.3% and 6% ARA. These results suggest that dietary inclusion of 3.2% ARA during periods shorter than 9months, or of 2.3% ARA for prolonged periods, can maintain optimal levels of tissue ARA in captive Senegalese sole broodstock. In addition, the data indicate that male Senegalese sole is able to elongate and desaturate ARA to 22:4n-6 and 22:5n-6, suggesting that these fatty acids may be important for male reproduction. [Copyright &y& Elsevier] more...

Abstract: Senegalese sole (Solea senegalensis) failed to reproduce and spawn viable eggs when bred and reared in captivity (first generation G1 cultured broodstock). Cultured sole were also shown to have significantly lower levels of arachidonic fatty acid (20:4n−6, ARA) in reproductive tissues compared to wild counterparts. However, to date no information about ARA requirements of Senegalese sole broodstock (wild or cultured) has been available. The present study was designed to determine dietary ARA preferences of adult G1 Senegalese sole by the use of automated demand feeders. For this purpose, three groups of 16 (eight females and eight males) G1 fish were offered to select between two diets, one with high (6.0% of total fatty acids, TFA) and another with low (0.7% TFA) ARA content. The experiment was conducted for 16 consecutive months. The results revealed that Senegalese sole changed the preference for ARA during the year, in correlation with the seasonal water temperature (r2 =0.7), and with periods of high and low demand of total food ingested. Thus, a high ARA (3.9% TFA) selection was observed in summer and early-autumn, whereas low ARA (2.2% TFA) selection occurred in winter. The average ARA demanded during the whole year was 3.0% TFA, with a resultant high level of ARA in the tissues of the fish; especially in male testis (8.8% TFA). These results should be considered when formulating seasonal diets to suit sole broodstock preferences in ARA in intensive aquaculture. [Copyright &y& Elsevier] more...

Abstract: A complete reproductive failure to spawn viable eggs from G1 cultured Senegalese sole (Solea senegalensis) broodstock compared to successful spawning from wild caught captive broodstock has been described previously. The aim of the present study was to compare the proximate and fatty acid composition of recently caught mature wild versus mature G1 Senegalese sole, to identify possible factors that contribute to this reproductive problem. The composition of 22 wild fish, captured in the Ebro Delta (Tarragona, Spain), was compared with 25 cultured fish raised under natural captive conditions. Significantly higher levels of total lipid were found, in male liver from G1 fish compared to wild fish (3.0- and 2.5-fold higher in female and male respectively). Similarly, the neutral lipid class was significantly higher in liver (88 and 90% total lipids (TL) from female and male, respectively) and male muscle (64% TL) from cultured fish. The relative value of linoleic acid (18:2n−6) was significantly higher in all tissues from cultured fish (approximately 11, 9 and 7.4% total fatty acid (TFA) in liver, gonads and muscle respectively). However, significantly lower levels were observed in cultured fish for the essential fatty acids, arachidonic (20:4n−6, ARA) in the liver (4.6-fold down male and 2.4-fold in female), testes (1.4-fold down) and muscle (2.9- and 2.0-fold down in male and female respectively); docosahexaenoic (22:6n−3, DHA) in liver (1.6- and 1.3 fold down in G1 female and male) and male muscle (1.3-fold down); eicosapentaenoic (20:5n−3, EPA) in muscle of male (1.4-fold down) and for cholesterol (CHOL) in liver of female (22% in wild and 7.8% in G1) and male (20 in wild and 7.0% in G1). These differences resulted in significantly higher ratios of EPA/ARA in the liver and muscle of cultured fish. The imbalance in lipids and essential fatty acids (EFAs) of G1 fish appeared to be a reflection of the extruded diet used to feed the fish. Future research should consider the effect of the nutritional imbalance on reproductive physiology of G1 fish, specially the effects of ARA and CHOL on prostaglandins (2- and 3-series) and steroid production. [Copyright &y& Elsevier] more...

Abstract: Prostaglandin levels in different tissues and cyclooxygenase (COX-2) gene expression were compared between wild and cultured Senegalese sole (Solea senegalensis) broodstock in which a significantly different fatty acid profile, particularly lower tissue levels of arachidonic acid (ARA, 20:4n-6) and higher levels of eicosapentaenoic acid (EPA, 20:5n-3) in the cultured fish compared to wild had already been described. This is the first report of the COX-2 mRNA expression in Senegalese sole. Cyclooxygenase (COX-2) mRNA expression and prostaglandin (2- and 3-series) levels were determined in tissues from 32 broodstock fish, 16 (8 males and 8 females) from each origin wild and cultured (G1). Transcripts of COX-2 were highly expressed in gills, sperm-duct (s-duct), testis, oviduct and spleen compared to liver, kidney and muscle. Differences in COX-2 transcripts expression were found in response to the origin of the fish and expression was significantly higher in s-duct and gills from wild fish compared to cultured. Wild fish showed significantly higher levels of total 2-series PGs and lower levels of 3-series compared to cultured fish. The significance of the lower COX-2 expression and lower PG 2-series production in some of the tissues of cultured fish was discussed in relation to the previously described differences in fatty acid profile (lower tissue levels of ARA and higher levels of EPA and EPA/ARA ratio in cultured fish) and the reproductive failure to spawn viable eggs from G1 cultured Senegalese sole compared to successful spawning from captive wild broodstock. [Copyright &y& Elsevier] more...

The life cycle of Senegalese sole (Solea senegalensis) is not closed in captivity due to a reproductive dysfunction related to the lack of participation of cultured male breeders in the courtship. To discern a possible solution to this social reproductive dysfunction, the main objective of this study was to determine the effect of the presence of spawning wild Senegalese sole breeders on the courtship and reproduction of cultured breeders. Three experimental groups were formed: Control group (n = 10) formed with only cultured sole; groups M1 and M2 constituted of mixed-origin sole (10 cultured and 8 or 9 wild breeders). All cultured breeders were from the same stock, which had never successfully reproduced, whilst the wild broodstock had spawned viable eggs in captivity. All groups were held in the same captive conditions and spawning, and behaviour were recorded for each group over four spawning seasons. All spawns were collected, and the parameters registered were floating and non-floating eggs, fertilization and hatching rates. In addition, parental analysis was made of larvae from viable spawns. Behaviour was studied with video recordings to compare locomotor activity and courtship behaviours including the "Follow" behaviours where sole swim after each other in a procession. Fertilized spawns were obtained from the mixed-origin groups (M1 and M2) including spawns involving a cultured male. The cultured males also participated actively in the "Follow" behaviours with the courting wild sole and this participation of cultured males increased significantly over the four years of the experiment. Male percentage participation in the "Follow" behaviours was positively correlated (R = 0.81) to participation in spawning to indicate the importance of increasing cultured male participation in the "Follow" behaviours. A total of seven spawns were obtained from a cultured male that fertilized eggs from one cultured female and two wild females. The same cultured female also spawned with a wild male. No viable spawns were obtained from the Control group and locomotor activity and courtship behaviour counts were significantly lower than in the experimental mixed-origin groups. This is the first report of cultured male breeders participating in reproductive behaviour and spawning, which could be associated with social learning processes, mate selection and dominance where cultured males copied the behaviour and spawning of wild Senegalese sole breeders. • First observations that cultured Senegalese sole breeders participated actively in the courtship. • Cultured sole male breeders can learn to perform the reproductive behaviour and their participation increased over time. • A cultured male participated in spawning with both wild and cultured females. • The mixed-origin (wild and cultured) cohabitation shows high important cue for the reproductive disorder of Senegalese sole. [ABSTRACT FROM AUTHOR] more...

Methods are needed to manage the sperm of Senegalese sole (Solea senegalensis), which will enable the industry to use artificial fertilisation to reproduce hatchery raised sole and implement breeding programs. The present study aimed to (a) describe the male reproductive and urinary system, (b) describe the effects of urine contamination on sperm quality and (c) examine the use of extenders for short term chilled storage of sole sperm. Nine males were dissected to describe the male reproductive and urinary system. A total of 49 males were examined and 32 (65.3%) provided adequate sperm samples of the study. Initially the samples were described by appearance (colour, transparency and fluidity) and sub-samples analysed for sperm quality, urea concentration, osmolality, pH and protein concentration. Cell concentration and sperm quality parameters, percentage motility, curvilinear velocity (VCL) and average path velocity (VAP), were measured using ImageJ CASA. Control samples and samples diluted (1:3) in six different extender solutions (modified Leibovitz, Ringer, NAM, Sucrose, Stor Fish® and Marine Freeze®) were stored short-term (4 °C) and tested zero, three, six and 24 h after collection. The close proximity of the reproductive and the urinary systems, especially the sperm ducts being attached to the urinary bladder makes obtaining sperm without urine contamination appear difficult. All the samples appeared to be contaminated with urine. Samples that appeared to be contaminated with urine (yellow colour) had similar spermatozoa cell concentration and urea concentration as samples that appeared not to be contaminated with urine (whitish colour), although motility was significantly lower in yellow samples. Seminal plasma urea concentration was positively correlated with osmolality. Cluster analysis grouped samples with significantly higher sperm quality and pH and significantly lower urea concentration and osmolality to indicate that urine contamination negatively affected sperm quality by increasing osmolality and decreasing pH. Amongst the six extender solutions Leibovitz and Marine Freeze® preserved significantly higher percentage motility 24 h after collection. Ringer, NAM and Stor Fish® were intermediate and Sucrose was similar to control samples that significantly decreased motility 3 h after collection. Taken together all sole sperm samples probably had urine contamination, which is difficult or impossible to avoid especially if all the sperm available needs to be collected. The extenders, Leibovitz and Marine Freeze® were used to maintain sperm quality and mitigate the negative effects of urine contamination. The collection and short term chilled storage in extenders of sole sperm from the majority of males in a broodstock (65.3%) can provide a valid sperm management system for industrial application for artificial fertilisation, however, further work is needed. • Urine contamination of sperm samples appears inevitable due to the proximity of male reproductive and urinary systems. • Urine contamination increased seminal plasma osmolality, decreased pH and reduced sperm quality. • The spermatozoa cell concentration was similar in samples that appeared to be uncontaminated or contaminated with urine. • The dilution of sperm in modified Leibovitz or Marine Freeze®, preserved sperm quality for 24 h. [ABSTRACT FROM AUTHOR] more...

• Stress coping styles behavioural classification is linked with mRNA abundance in Senegalese sole juveniles. • Senegalese sole juvenile population in captive conditions could be classified in three different stress coping styles categories; proactive, intermediate and reactive. • Several transcripts associated with important biological aspects in Senegalese sole are differently expressed depending on the stress coping styles categories. • This study addressed an emerging field for the fish biology related to physiology, behaviour and individual variation. In fish, proactive and reactive individual stress copying styles (SCS) have been used to resolve variation in molecular expression data. Stress coping styles have been previously described in several stages of Solea senegalensis by validating for the species the use of standard behavioural screening tests. The present study aimed to link behavioural SCS tests with brain transcript abundance in early Senegalese sole juveniles in order to observe the natural variation in a molecular pathway in this species. A total of 50 juveniles were subjected to three individual behavioural (Restraining, New environment and Confinement) and one group (Risk-taking) screening tests. The fish were classified in SCS categories by applying a hierarchical cluster to the variable "Total activity" (the total activity time that the fish was moving in each individual test). Three categories were defined, proactive, intermediate and reactive sole. Six transcripts were chosen and tested, one related to basic metabolism (gapdh-2), three to feeding behaviour (per1, igf-Ia, pparß) and two to the stress response (crh-BP and hsp90aa) in 30 juveniles (10 individuals per SCS category) using rt -qPCR to observe differences in the abundance of those transcripts among SCS. Four transcripts were differentially expressed (DETs) among them. The transcript gapdh-2 showed up-regulation for proactive and intermediate SCS sole while reactive individuals showed down-regulation. Target mRNAs per1, igf-Ia and pparß , showed different levels of up-regulation for proactive and reactive fish while intermediates were highly down-regulated. Surprisingly no differences in stress related transcripts were observed. Correlations were found between variation in coping styles and variation in the abundance of mRNAs involved in important biological functions in Senegalese sole. These results are the first evidence of the relationship between the behavioural individual variation and the fluctuation in brain transcripts abundance in Senegalese sole. [ABSTRACT FROM AUTHOR] more...