The locomotor behavior of Drosophila melanogaster was quantified in a large population of inbred lines derived from a single natural population, showing that many pleiotropic genes show correlated transcriptional responses to multiple behaviors. Locomotion is an integral component of most animal behaviors, and many human health problems are associated with locomotor deficits. Locomotor behavior is a complex trait, with population variation attributable to many interacting loci with small effects that are sensitive to environmental conditions. However, the genetic basis of this complex behavior is largely uncharacterized.Drosophila melanogaster in a large population of inbred lines derived from a single natural population, and derived replicated selection lines with different levels of locomotion. Estimates of broad-sense and narrow-sense heritabilities were 0.52 and 0.16, respectively, indicating substantial non-additive genetic variance for locomotor behavior. We used whole genome expression analysis to identify 1,790 probe sets with different expression levels between the selection lines when pooled across replicates, at a false discovery rate of 0.001. The transcriptional responses to selection for locomotor, aggressive and mating behavior from the same base population were highly overlapping, but the magnitude of the expression differences between selection lines for increased and decreased levels of behavior was uncorrelated. We assessed the locomotor behavior of ten mutations in candidate genes with altered transcript abundance between selection lines, and identified seven novel genes affecting this trait.We quantified locomotor behavior of Expression profiling of genetically divergent lines is an effective strategy for identifying genes affecting complex behaviors, and reveals that a large number of pleiotropic genes exhibit correlated transcriptional responses to multiple behaviors. Locomotion is required for localization of food and mates, escape from predators, defense of territory, and response to stress, and is, therefore, an integral component of most animal behaviors. In humans, Parkinson's disease, Huntington's disease, activity disorders and depression are associated with deficits in locomotion. Thus, understanding the genetic architecture of locomotor behavior is important from the dual perspectives of evolutionary biology and human health.Drosophila melanogaster, where one can assess the effects of mutations to infer what genes are required for the manifestation of the behavior, and map QTL affecting naturally occurring variation with high resolution [Drosophila locomotion may well be relevant in humans. For example, Parkinson's disease is associated with progressive degeneration of nigrostriatal dopaminergic neurons [Drosophila [Locomotion is a complex behavior, with variation in nature attributable to multiple interacting quantitative trait loci (QTL) with individually small effects, whose expression is sensitive to the environment . Dissectsolution . Generalsolution . Thus, o neurons ,5, and d neurons  and Drososophila ,7-12.Drosophila. The neurotransmitters serotonin (5-hydroxytryptamine) [Drosophila locomotion; as do genes required for the proper neuroanatomical development of the mushroom bodies and components of the central complex, brain regions required for normal locomotion [Several studies reveal the underlying genetic complexity of locomotor behavior in ptamine) , octopamptamine) , and γ-aptamine)  affect Dcomotion -21. Rececomotion , but wasAn alternative strategy to discover genes affecting complex behaviors is to combine artificial selection for divergent phenotypes with whole genome expression profiling -28. The 326,25736 = 41.84, P < 0.0001; Figure H2) of locomotor reactivity in this population was high: H2 = 0.519. The line by sex interaction term was not significant , indicating that magnitude and/or rank order of the sexual dimorphism does not vary among the lines in this population. The correlation in locomotor reactivity between the sexes (rGS = 0.973 ± 0.015) was correspondingly high and positive, and not significantly different from unity.We quantified the magnitude of variation in locomotor activity among a panel of 340 inbred lines derived from the Raleigh natural population. We observed substantial naturally segregating variation in locomotor reactivity behavior  and low (L) activity levels, as well as replicate unselected  lines. At generation 25, the H and L lines diverged by 27.6 seconds, or 61% of the total 45 s assay period Figure .h2 ± standard error of the regression coefficient) of locomotor reactivity from the regressions of the cumulated response on cumulated selection differential [h2 = 0.147 ± 0.008 (P < 0.0001) for replicate 1 and h2 = 0.170 ± 0.010 (P < 0.0001) for replicate 2  and h2 = 0.074 ± 0.0265 (P = 0.01) for H line replicates 1 and 2, respectively; and h2 = 0.181 ± 0.0093 (P < 0.0001) and h2 = 0.201 ± 0.011 (P < 0.0001) for L line replicates 1 and 2, respectively. There was no inbreeding depression for locomotor reactivity: the regression of locomotor behavior in the control lines over 25 generations was b = 0.0006 ± 0.053 (P = 0.98) and b = -0.012 ± 0.044 (P = 0.78) for C line replicates 1 and 2, respectively.We estimated realized heritability . All 42 probe sets that were significant for the interaction term were also significant for the main effect of line.We assessed transcript abundance in the H, L, and C selection lines using Affymetrix high density oligonucleotide whole genome microarrays, for flies of the same age and physiological state as selected individuals. The raw microarray data are given in Additional data file 1, and have been deposited in the GEO database  under seWe used ANOVA contrast statements on the 1,825 probe sets with differences in transcript abundance between selection lines to detect probe sets that were consistently up- or down-regulated in replicate lines . We founcytochrome P-450 and Glutathione S tranferase gene families, genes affecting lipid metabolism, and genes encoding immune/defense molecules.The probe sets with altered transcript abundance between the selection lines fell into all major biological process and molecular function Gene Ontology (GO) categories . We assessed which categories were represented more frequently than expected by chance, based on representation on the microarray, since the over-represented GO categories are likely to contain probe sets for which transcript abundance has responded to artificial selection. Highlights of the transcriptional response to artificial selection for locomotor reactivity are given in Table P-element insertional mutations in ten candidate genes that were implicated by the analysis of differential transcript abundance. All of the P-element insertions were derived in a common isogenic background, and are viable and fertile as homozygotes [P-elements are inserted either in the coding region or approximately 100 bp upstream of the start of transcription of each candidate gene. The candidate genes are involved in diverse biological processes, including signal transduction , neurotransmitter secretion , nervous system and muscle development (muscleblind), chromosome segregation (nebbish), and copulation (ken and barbie). Three of the mutations are in computationally predicted genes . Six of the mutations exhibited significant differences in locomotor reactivity from the co-isogenic control line, after Bonferroni correction for multiple tests , but not at the conservative Bonferroni threshold of P = 0.005. There was no clear relationship between the pattern of transcriptional response to selection of the candidate genes and the results of the functional tests. The significant genes belonged to categories 1 , 2  and 3  . All of the non-significant candidate genes belonged to category 1. From these data, we infer that transcripts in category 3 do not solely represent instances of changes in expression as a consequence of reduced fitness of the selection lines relative to the control, as in this case one would not expect the genes to affect the selected trait.To assess the extent to which transcript profiling of divergent selection lines accurately predicts genes that directly affect the selected trait, we evaluated the locomotor reactivity of lines containing ozygotes ,35. The muscleblind mutants are paralytic [Amphiphysin [Cysteine string protein [nebbish mutants are not well coordinated [Mutations in each significant gene had lower levels of locomotor reactivity than the control line. Of these genes, four have been previously implicated to affect activity: aralytic ; Amphiphhiphysin  and Cyst protein  mutants rdinated .D. melanogaster exhibits a strong response to artificial selection for high and low levels of locomotor reactivity. The heritability of locomotor reactivity is fairly high for a behavioral trait (approximately 0.16). However, the genetic response to selection, as inferred from the realized heritability, was asymmetrical. Responses were much greater in the direction of decreased locomotor reactivity (heritabilities approximately 0.20) than for increased activity. Asymmetrical responses to selection are often observed for traits that are major components of fitness [ fitness ,40. HoweThe phenotypic response to selection appears to be specific for locomotor reactivity. In particular, we did not observe correlated responses to selection for locomotor reactivity for responses to different stressors, nor for other traits involving locomotion.H2 = 0.52) greatly exceeds the narrow sense heritability estimated from response to selection (h2 = 0.16), we infer that considerable non-additive genetic variance due to dominance and/or epistasis affects natural variation for this trait. We estimate the additive genetic variance (VA) as VA = h2VP = 3.74, where h2 is the narrow sense heritability from divergent response to artificial selection, averaged over both replicate lines, and VP is the total phenotypic variance for the first 10 generations averaged over all 6 selection lines (VP = 23.58). If only additive genetic variance affected locomotor reactivity, we would predict the total genetic variance among the inbred lines to be 2FVA = 7.48, for an expected F = 1 after 20 generations of full sib inbreeding [VG = 28.14. The difference, therefore, must be due to dominance and/or epistasis.Since the broad sense heritability estimated from the variation among inbred lines  between the selection lines, using a stringent false discovery rate of 0.001. Previously, we selected replicate lines for increased and decreased copulation latency  and incrQ < 0.001 were common between the selection lines for locomotor reactivity (this study), aggression [2 tests. We found 908 probe sets in common between copulation latency and locomotor reactivity , 474 probe sets in common between aggressive behavior and locomotor reactivity , and 878 probe sets in common between copulation latency and aggressive behavior . The transcript abundance of 311 genes  was altered as a correlated response to selection for all three behaviors .The large number of genes exhibiting changes in transcript abundance among replicate selection lines implies that genes affecting complex behaviors are highly pleiotropic: if approximately 10% of the genome affects any one trait, the same genes must affect multiple traits. Thus, genes affecting behavior are also likely to be involved in neurogenesis, metabolism, development and general cellular processes, and many of the same genes may affect multiple behaviors. Indeed, 1,638 probe sets that were significant at a false discovery rate of gression , and copgression  that werr = 0.059 ± 0.060 between mating and locomotor behavior; r = 0.025 ± 0.012 between mating and aggressive behavior; and r = 0.034 ± 0.081 between aggressive and locomotor behavior  for common probe sets that had significant H ≠ L contrast statements for all pairwise combinations of behaviors. The data set consisted of 655 probe sets for the comparison of copulation latency and locomotor reactivity, 580 probe sets for the comparison of mating and aggressive behavior, and 270 probe sets for the comparison of locomotion and aggressive behavior. The correlations of the mean difference in expression between H and L lines were: cytochrome P-450 and Glutathione S tranferase gene families, genes affecting lipid metabolism, genes encoding immune/defense molecules, and genes affecting circadian rhythm and sleep (data not shown). Pleiotropic effects of genes affecting circadian rhythm, cytochrome P450s and glutathione S transferases on multiple behaviors in Drosophila have been implicated by other studies. Expression levels of Pigment dispersing factor and cryptochrome, both of which affect circadian rhythm, were up-regulated in lines selected for positive geotaxis, and confirmed to affect geotaxis behavior in functional tests [GstE5 and GstE1, were among those that were differentially expressed between lines selected for locomotor behavior (this study), aggressive behavior [Drosophila parkin mutants is enhanced by loss-of-function mutants of GstS1 and reduced when GstS1 is over-expressed [The probe sets with altered transcriptional responses to selection that were in common to at least two behaviors were enriched for genes in the al tests . Similaral tests , and anoal tests . The expal tests . Glutathal tests . Glutathal tests . Two of behavior  and matibehavior . FunctioThe transcriptional response to selection that we observed is attributable to genes that have causally responded to selection, and genes that are co-regulated by these genes. Since the transcriptional response to single mutations with subtle phenotypic effects can involve over 100 co-regulated genes , the nummuscleblind encodes a protein with a zinc-finger domain involved in muscle development, and mutants are paralytic [muscleblind that had reduced locomotor reactivity in this study was also associated with increased aggressive behavior [Cysteine string protein is involved in stress response and neurotransmitter secretion, and mutants are sluggish and hypoactive [nebbish is involved in mitotic chromosome segregation, and mutants in this gene have been characterized as not well coordinated [Amphiphysin, which did not surpass the conservative estimate of significance, but is formally significant at the P < 0.05 significance level, is involved in neurotransmitter secretion and regulation of muscle contraction, and mutants have been shown to be flightless and hypoactive [CG10990, CG33523, and ken and barbie are novel genes affecting locomotor behavior. CG10990 and CG33523 are computationally predicted genes with unknown functions, and ken and barbie is involved in copulation and insemination [Regardless of whether or not the observed changes in gene expression are causally associated with genetic divergence in locomotor behavior between the selection lines, the genes exhibiting altered expression levels are candidate genes affecting locomotor behavior. We quantified locomotor reactivity for ten mutations in candidate genes that were generated in a common co-isogenic background, and identified seven genes with mutational effects on locomotor reactivity, six of which are significant using a conservative Bonferroni correction for multiple tests. Locomotor defects have been described previously for mutations of four of these genes. aralytic . The mutbehavior , consistpoactive . nebbishrdinated . Amphiphpoactive . The remmination .P values that did not meet our conservative false discovery rate criterion, and 13 genes  were differentially expressed at Q < 0.001 . We identified 13 positional candidate genes affecting locomotor reactivity in our previous QTL mapping study [Drop and turtle showed significant differences in transcript abundance between the selected lines. These results highlight the utility of applying multiple complementary approaches to understand the genetic basis of complex traits.The success of these functional tests validates using expression profiling on genetically divergent lines in directed mutagenesis screens to identify genes affecting complex traits. This strategy is complementary to traditional strategies and cannot supplant them, since many key genes will not be detected as differentially expressed. Specifically, we will not detect genes that are differentially expressed at a different developmental period or if the magnitude of the difference in transcript abundance is too small to be detected, or genes affecting the trait that are not regulated at the level of transcription. Of the 82 genes with known effects on some aspect of locomotor behavior, 20 were not present on the microarray, and eight did not give a statistically significant signal . Of the 54 remaining genes, 16 were statistically differentially expressed between the selection lines at nominally significant muscleblind is orthologous to Muscleblind-like Protein 1, which encodes a triplet repeat expansion, similar to the one that causes muscular dystrophy [Drosophila will elucidate corresponding mechanisms in other organisms, including humans.Many of the genes with mutational effects on locomotor behavior are evolutionarily conserved and have human orthologs Table . For exaystrophy . It is tFlies were reared on cornmeal/molasses/agar medium under standard culture conditions . Behavioral assays were conducted in a behavioral chamber  between 8 am and 12 pm (2-6 h after lights on).2 exposure, into vials containing 5 ml of standard cornmeal/agar/molasses media, and left them overnight to acclimate to their new environment. To quantify locomotor reactivity, we subjected each fly to a mechanical disturbance by tapping the vial twice against a table, and recorded the amount of time the fly is active in the 45 seconds immediately following the disturbance using a stopwatch to record movement, while a timer counts down the 45 s assay period. The measure of locomotor reactivity is a score ranging from 0 s to 45 s, denoting the total amount of activity during the assay period.Locomotor reactivity was assessed as described previously ,12. BrieN = 20 males and 20 females per replicate) for each inbred line. The replicates for each line were assessed on different days.Isofemale lines were established from wild-type gravid females collected at the Raleigh, NC Farmer's Market in 2003. The lines were inbred by 20 generations of full-sib inbreeding to create 340 inbred lines. Locomotor reactivity for each of the inbred lines was measured by randomly assigning the lines into blocks of approximately 25 lines; each block was tested over a 2 week period. We obtained 2 replicate measurements  was used to partition variance in locomotor activity among the inbred lines, according to the model:B is the random effect of block, S is the fixed main effect of sex, Line(Block) is the random main effect of line within each block, Sex × Line(Block) is the random effect of the sex by line interaction within each block, Rep(Line × Sex × Block) is the random effect of replicate, and E is the within-vial variance. Parentheses indicate nested effects. The total genotypic variance among lines was estimated as:where μ is the overall mean, σL(B)2 is the among-line variance component and σLS(B)2 is the variance attributable to the L ×S interaction. The total phenotypic variance was estimated as:where σE2 is the environmental variance component. We estimated broad sense heritabilities as:where rGS) across sexes was calculated as:We also ran reduced analyses for each sex separately. The genetic correlation .where The base population was generated from 60 isofemale lines established from flies collected in Raleigh, NC in 1999. The isofemale lines were crossed in a round robin design . Single fertilized females from each cross were placed in each of two culture bottles. The two initial bottles were split into three replicate bottles each that were arbitrarily assigned into H, L, and C groups. The six bottles were maintained by random mating for four generations to allow initial linkage disequilibrium to decay. After four generations of random mating, the following generation (G1) and all subsequent generations repeated the same procedure: 50 virgin females and males were scored from each line , and the 20 most active males and females from the H lines and the 20 least active males and females from the L lines were selected as parents for the next generation. The first 20 males and females scored from the C lines were used as the C line parents. Selection was continued for 25 generations.h2) were calculated by regression of the cumulative selection response (ΣR) on the cumulative selection differential (ΣS) [Estimates of realized heritability  .Starvation resistance was assessed as previously described . Single Chill-coma recovery was quantified as previously described . We tranEthanol sensitivity was measured using an inebriometer . We aspiCopulation latency was scored as previously described . For eacLocomotor senescence was measured at generation 25. Approximately 200 2- to 4-day-old same-sex flies from each line were placed in separate bottles. The flies were transferred to fresh bottles every 2-3 days, and each week , 25 flies of each line and sex were scored for locomotor reactivity.Differences between the selection lines for the correlated traits were assessed using a nested mixed model analysis of variance (ANOVA):Y is the phenotypic score, μ is the overall mean, Selection is the fixed effect of the selection treatment , Line(Selection) is the random effect of the replicate within each selection group, Sex is the fixed effect of sex, Gen is the fixed effect of generation, and ε is the error variance. A significant Selection term is indicative of a correlated response in the trait being tested to selection for locomotor behavior. Behavioral locomotor senescence was analyzed in the same manner, with the replacement of the Week term instead of Gen term.where Drosophila GeneChip 2.0 oligonucleotide microarrays ) and visualized with a streptavidin-phycoerythrin conjugate, as described in the Affymetrix GeneChip Expression Analysis Technical Manual (2000), using internal references for quantification. The quantitative estimate of expression of each probe set is the Signal (Sig) metric, as described in the Affymetrix Microarray Suite, Version 5.0.At generation 25, two replicates of 12 3- to 7-day-old virgin males and females were collected from each selection line, at the same time of day that the behavioral assays were performed. Total RNA was extracted from the 24 samples (6 lines × 2 sexes × 2 replicates) using the Trizol reagent . Biotinylated cRNA probes were hybridized to high density Drosophila GeneChip 2.0 are represented by 14 perfect-match (PM) and 14 mismatch (MM) pairs. The Sig metric is computed using the weighted log(PM - MM) intensity for each probe set, and was scaled to a median intensity of 500. A detection call of present, absent, or marginal is also reported for each probe set. We excluded probe sets with more than half of the samples called absent from the analysis, leaving 11,656 probe sets. This filter retained sex-specific transcripts but eliminated probe sets with very low and/or variable expression levels [The 18,800 probe sets on the Affymetrix n levels . On the Sex and Line are the fixed effects of sex and selection line and ε is the variance between replicate arrays. We corrected the P-values computed in these ANOVAs for multiple tests using a stringent false-discovery rate criterion [Q < 0.001. We used contrast statements [L and/or S × L terms at or below the Q = 0.001 threshold were significantly different between selection groups  at the P < 0.05 level, both within each sex and pooled across sexes. GO categories were annotated using DAVID [where riterion  of Q < 0atements ,27 to asng DAVID   [We tested whether mutations in ten of the candidate genes with altered transcript abundance between the selection lines affected locomotor behavior. The mutations were homozygous kground) . LocomotY is the phenotypic score, μ is the overall mean, Line is the fixed effect of the genotype (mutant or control), Block is the random effect of the replicate, Sex is the fixed effect of sex, Line×Sex (fixed), Line×Block (random), Block×Sex (random), and Line×Sex×Block (block) are the interaction terms, and ε is the variance within lines. A significant Line term suggests that the mutant is significantly different from the control.where P and Q values for all probe sets with significant (Q < 0.001) expression differences between selection lines. Additional data file P values of significant contrasts of mean expression differences between selection lines. Additional data file P values of significant contrasts for probe sets that are differentially expressed between lines independently selected for locomotor, aggressive and mating behavior. Additional data file The following additional data are available with the online version of this paper. Additional data file ANOVA = Analysis of Variance; C line = control selection line; GO = Gene Ontology; H line = high selection line; L line = low selection line; QTL = quantitative trait locus.KWJ and TFCM designed the experiment, KWJ conducted the selection experiment and assays, MAC extracted and labeled RNA for the microarray experiment, AY carried out the functional tests to putative candidate genes, TJM performed the statistical analysis of the microarray data, and KWJ and TFCM wrote the paper.Raw microarray data.Click here for fileP and Q values for all probe sets with significant (Q < 0.001) expression differences between selection lines.Click here for fileP values of significant contrasts of mean expression differences between selection lines.Click here for fileGO categories of differentially expressed genes.Click here for fileGO categories that are over-represented by differentially expressed genes.Click here for fileProbe sets common to multiple behaviors.Click here for fileP values of significant contrasts for probe sets that are differentially expressed between lines independently selected for locomotor, aggressive and mating behavior.Click here for fileKnown genes affecting locomotor behavior.Click here for file


Stygiocaris (Atyidae) using nuclear and mitochondrial genes. Stygiocaris is part of the unique cave biota of the coastal, anchialine, limestones of the Cape Range and Barrow Island, most of whose nearest evolutionary relations are found in coastal caves of the distant North Atlantic. The dominance of atyids in tropical waters and their food resources suggest they are pivotal in understanding these groundwater ecosystems.We investigated the large and small scale evolutionary relationships of the endemic Western Australian subterranean shrimp genus Typhlatya as the sister taxon of Stygiocaris, rather than any of the numerous surface and cave atyids from Australia or the Indo-Pacific region. The two described Stygiocaris species were recovered as monophyletic, and a third, cryptic, species was discovered at a single site, which has very different physiochemical properties from the sites hosting the two described species.Our nuclear and mitochondrial analyses all recovered the Mexican cave genus Stygiocaris and Typhlatya may descend from a common ancestor that lived in the coastal marine habitat of the ancient Tethys Sea, and were subsequently separated by plate tectonic movements. This vicariant process is commonly thought to explain the many disjunct anchialine faunas, but has rarely been demonstrated using phylogenetic techniques. The Cape Range's geological dynamism, which is probably responsible for the speciation of the various Stygiocaris species, has also led to geographic population structure within species. In particular, Stygiocaris lancifera is split into northern and southern groups, which correspond to population splits within other sympatric subterranean taxa.Our findings suggest that  By their very nature, caves and other subterranean environments are poorly known, and yet they are home to large numbers of endemic species and unique relictual taxa from an earlier age Australia was once thought to have few areas of interest for those with a bent towards the underworld A highly diverse and distinct subterranean fauna is also found on the western edge of the Pilbara Craton, in the younger limestones of the Cape Range peninsula and Barrow Island  [17]. ThToday a low, arid, mountain range (∼300 m high) runs north-south along the spine of the tropical Cape Range peninsula (80 kms by 20 kms) , fringedThe subterranean habitats of the Cape Range area are largely of two types Coastal waterbodies with subterranean connections to the sea and tidal influences are known as “anchialine” Stygiocaris Holthuis, 1960 (Atyidae), Halosbaena Stock, 1976 (Thermosbaenacea), Haptolana Bowman, 1966 (Cirolanidae), various amphipods The coastal plain habitat of Cape Range supports a fauna of a type unknown elsewhere in the southern hemisphere, also occurring in similar anchialine habitats on either side of the North Atlantic . Although most of this fauna is known only in Australia from the Bundera Sinkhole, the sole accessible deep anchialine system, elements of the fauna occur widely in the more superficial waters of the linear Cape Range coastal plain, Barrow Island and the Pilbara coast  it is widespread within the coastal plain and Barrow Island Stygiocaris is endemic to the area, its hypothesized closest evolutionary relatives are all geographically remote, namely Typhlopatsa Holthuis, 1956 from Madagascar Antecaridina Edmondson, 1954: Indo-Pacific Typhlatya Creaser, 1936: Caribbean, Europe The Cape Range coastal plain and Barrow Island host two endemic species of atyid shrimp, is, 1960 . StygiocMolecular phylogenetic and clock analyses can help to cut through the confusion of morphological convergence to help to resolve the evolutionary and biogeographic history of subterranean taxa Stygiocaris in northwestern Australia. Therefore, we hypothesized that Stygiocaris will be an evolutionary offshoot of one of the many surface or anchialine species of the Indo-Pacific or Australia, particularly as coastal Western Australia hosts many undescribed surface atyids  have been recorded in favourable caves Stygiocaris are expected to be similarly pivotal to understanding the dynamics of the anchialine system.Dense populations of Stygiocaris species from throughout their distributions on the Cape Range peninsula and Barrow Island, Western Australia , the Western Australian species Caridina spelunca Choy, 1996, and the Northern Territory species Parisia gracilis Williams, 1964. Also included were a number of anchialine species from the Indo-Pacific , and various epigean and subterranean species from throughout the world. Where possible, we integrated published sequences of numerous atyid species into our datasets , which is effective at discriminating at the population and species-level 5′-CWA CMA AYC ATA AGA YAT TGG-3′) and CR-COI-R (5′-GCR GAN GTR AAR TAR GCT CG-3′) Stygiocaris spp. and any atyid species that fell within a higher-level 16S clade with it.Genomic DNA was extracted, amplified and sequenced as per Stygiocaris 16S”) include only 16S sequences. The Atyid 16S dataset includes 13 genera of atyids (10 of which have subterranean species) from throughout the world, and includes all eight genera found in Australia. The Stygiocaris 16S dataset includes sequences from all of our specimens of Stygiocaris from nine sampling sites. The 16S sequences from these two datasets were aligned using ClustalX version 1.81 We assembled a number of separate datasets with various combinations of genes and taxa to investigate different phylogenetic levels . SequencStygiocaris Combined” includes H3, 16S and COI sequences from the three Stygiocaris species (and major intraspecific groupings) with Antecaridina sp. East Timor as an outgroup. Each gene region was analysed separately by gene as well as combined. We aligned H3 and COI sequences without gaps. Two further datasets of all available Stygiocaris 16S and Stygiocaris COI sequences respectively were also assembled to create haplotype networks and to derive genetic divergence estimates within and between species.The “Histone” dataset incorporates conserved nuclear H3 sequences from eight atyid genera, which were also analysed in combination with the relevant 16S sequence (“Combined 16S/H3”). “Stygiocaris Combined and Combined 16S/H3 Datasets, an appropriate model was selected for each gene, as well as for all genes combined. Three forms of phylogenetic analysis were employed. We used PHYML version 2.4.4 We used Modeltest version 3.06 Stygiocaris in the Combined 16S/H3 dataset were investigated using the Shimodaira–Hasegawa (S–H) test in PAUP (1000 replicates of resampling of estimated log-likelihood test distribution) and Bayes Factors Phylogenetic hypotheses of the sister taxon of Stygiocaris 16S and COI sequences using TCS version 1.21 Stygiocaris species were calculated using a correction for within-group polymorphism Haplotype networks were constructed separately for all Stygiocaris lancifera and S. stylifera, leading to their speciation Because of the geological dynamism of the Cape Range area, there is likely a close relationship between geological and biological events S. lancifera and S. stylifera referred to just above. We assumed the S. lancifera/stylifera split occurred 7–10 MYA and applied the derived rates from this single node to the divergence between S. lancifera/stylifera and S. sp. Bundera. Secondly, we used a relaxed  molecular clock method Typhlatya/Stygiocaris spp. and S. lancifera/stylifera/sp. Bundera, by fixing the S. lancifera/stylifera tmrca to fall within the 7–10 million year range.As molecular clock calculations are often contentious, in particular for cave species Pycnisia bunyip Suzuki & Davie, 2003 did not produce usable sequences. The Modeltest-derived models and tree scores from maximum likelihood, Bayesian and parsimony analyses for all phylogenetic datasets appear in sensuStygiocaris, implying the presence of a cryptic species (Stygiocaris sp. Bundera). The sister to Stygiocaris was the Mexican cave shrimp Typhlatya pearsei Creaser, 1936. The anchialine Halocaridina and Antecaridina  were recovered within a clade containing both Stygiocaris and Typhlatya. Interestingly, all of the species in this “Typhlatyinae” clade sensuStyCaridina-like” group. As previously found Parisia unguis Williams, 1964 and Pycnisia raptor Bruce, 1992 form a strong clade. Similarly, the other species of Parisia Holthuis, 1956 in Australia, Parisia gracilis, forms a clade with another cave genus, Pycneus, but both sets of clades group with completely separate surface species of Caridina H. Milne Edwards, 1837. This implies an independent colonisation of the underworld in Australia and morphological convergence. The DNA sequence of the Western Australian cave species, Caridina spelunca, is nearly identical to a previously unidentified surface species from the same area  relative to Stygiocaris sp. Bundera, which is distinct. In this nuclear dataset, Typhlatya was again recovered as the sister to Stygiocaris, and both again formed a clade with Halocaridina and Antecaridina. As in the 16S dataset, Parisia gracilis, Pycneus and Caridina sp. LE are very closely related (with P. gracilis and Pycneus sharing an H3 haplotype). When the H3 and 16S sequences were combined and analysed as above (not displayed), the topology was identical to the H3-only dataset (Stygiocaris/Typhlatya relationship (Maximum Likelihood/Bayesian/Parsimony: 78/79/75), and comparable for the Stygiocaris/Typhlatya/Halocaridina relationship (66/75/88).Alternative explanations for some of these mitochondrial relationships are long-branch attraction and mitochondrial introgression  dataset . SupportTyphlatya as the sister taxon to Stygiocaris. But, as Halocaridina or Antecaridina spp. are also potential sisters of Stygiocaris in place of Typhlatya . Using Bayes Factors, the evidence against a hypothesis of Halocaridina as a sister was “Substantial” . The evidence against an Antecaridina sister was “Very Strong” (2×logs = 15.10) (Antecaridina constrained likelihood = –4130.39). The S-H test could not reject either Halocaridina or Antecaridina as potential sisters to Stygiocaris at the 0.05 level but would reject them both at the 0.10 level .Every best-scoring topology from all three forms of analysis of the three relevant datasets  recovered atya see , 5, we cStygiocaris species were recovered as a clade. A third Stygiocaris taxon was only found at site C-28 (Bundera Sinkhole). The three species are ∼13% divergent from each other at COI (∼5% at 16S)(uncorrected). The relationship between the three Stygiocaris species is not clear in the two 16S datasets . One of these three groups was only found on the eastern side of the Cape Range in the Defence Bores. The second group was only found at C-25 on the western side of the Cape, and the third group at both C-25 and Barrow Island.There is significant intraspecfic diversity within datasets , 7. WithS. lancifera which are ∼2% divergent at COI (∼1% 16S). These groups are geographically structured . Applying these rates, the common ancestor of S. lancifera/stylifera and Stygiocaris sp. Bundera diverged 6.15–12.30 MYA (±S.E.) for COI and 9.39–16.61 MYA for 16S.There is a range of molecular clock calculations available, depending on whether one uses more modern relaxed clock methods or fairly simplistic distance estimates, which will vary based on whether one uses model-corrected or uncorrected distances for either COI or 16S sequences. When we marry the distance estimates with the geological estimates, we can derive rates of molecular evolution for particular nodes. These give a wide range of possible divergence rates per million years (±S.E.) for the S. lancifera/stylifera/sp. Bundera is comparable to that above, with a mean of 11.08 MYA . The mean tmrca for Stygiocaris spp./Typhlatya is 24.53 MYA .Using a relaxed clock method on the Atyid 16S dataset, the time to most recent common ancestor for S. lancifera/stylifera node. Commonly used general interspecific rates for crustaceans are 0.65% Halocaridina, ∼20% per million years The mean 16S divergence rate resulting from the relaxed method is 0.75% per million years , which is also comparable to the point estimates above for the Parisia unguis, Pycnsia raptor) descend from localised surface species rather than morphologically similar subterranean species from further afield, and that formal systematic classifications often do not agree with inferred evolutionary relationships. We found a similar pattern in the present study for two further cave species . However, importantly, we did not find this pattern for Stygiocaris spp., which formed no clades with any Australian taxa. Stygiocaris formed a clade with subterranean species  is congruent with a “Tethyan Track” explanation Interestingly the nearest relation to Stygiocaris is by no means the only Cape Range representative of this pattern, as the Bundera Sinkhole (site C-28) hosts species of remipede crustaceans Halosbaena from C-215, Poore & Humphreys 1992) found nowhere else in the Southern Hemisphere, whose sister taxa are instead in coastal caves of the North Atlantic  Stygiocaris colonising the coast of the nearby coast of Pilbara Craton first and later moving to the Cape Range area Stygiocaris's sister taxon, Typhlatya, that are hypothesised to have lived in marine caves in the Caribbean before the formation of their current anchialine habitats One interesting point is that the closure of the Tethys Seaway  Stygiocaris sp. Bundera (as well as the unique “Tethyan” community referred to above), which were only sampled well beneath the overlaying layer of brackish water, in water with a salinity similar to local seawater Stygiocaris's sister, Typhlatya, which hosts both fully freshwater and brackish species The >30 kms, , and theStygiocaris sp. Bundera may well represent the original lifestyle of the coastal marine Tethyan ancestor of Stygiocaris and Typhlatya, with some species subsequently invading and adapting to freshwater. The dispersal of freshwater species is limited by saline conditions, whereas Stygiocaris sp. Bundera may be limited by surrounding freshwater. There is considerable evidence, from the nature of the faunas and their distribution, that anchialine habitats are both geologically very old and persistent Stygiocaris  and thus liable to extinction S. lancifera, the remipede Lasionectes exleyi Yager & Humphreys 1996, and both species of cave fish, are specially protected under Commonwealth of Australian and/or Western Australian fauna legislation.Although Stygiocaris, may play a keystone role (and be a potential bioindicator Subterranean habitats tend to have truncated


Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumor of the gastrointestinal tract. Clinically, they are associated with nonspecific symptoms, but some patients can present gastrointestinal bleeding with shock. We report two cases of GIST of the small bowel, revelated by hemorrhagic shock secondary to acute bleeding, succesfully treated by emergency surgery. Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. They can occur anywhere along the gastrointestinal tract but are most common in the stomach and small bowel. They are characterized by a remarkable cellular variability and their malignant potential is sometimes difficult to predict. Clinically, GIST are associated with nonspecific symptoms, but some patients can present gastrointestinal bleeding with shock. Emergency surgery after resuscitation is the treatment of choice for severely hemorrhagic GIST. Prognosis of GIST after surgical treatment is influenced by completeness of primary resection and tumor malignant potential. Adjuvant treatment (Imatinib) should be advocated for patients with either a high-grade GIST or after incomplete primary surgical treatment.3 (79% neutrophils), blood urea was 0.25 g/L and the creatinine level was 10 mg/L. Liver enzymes and hemostasis laboratory data were normal. The patient was hospitalized in the gastroenterology department for etiological investigation. The upper digestive endoscopy and colonoscopy were normal. No gastric or colic lesions were found as an explanation of the melena. An ultrasound and abdominal computerized tomography (CT) were realized to explore the abdominal mass, which showed a large heterogeneous tumor of the small bowel (diameter of 12 × 11 × 8 cm) [A 54-year-old man was admitted to the emergency department because of acute abdominal pain. He had a 3-month history of abdominal pain, bloating, anemia and three episodes of melena that resolved spontaneously. He was not using any specific medication and his medical history did not suggest any major disease. The physical examination showed a conscious patient hemodynamically stable whose temperature was 37°C, whose blood pressure was 130/80 mmHg and who had a good nutritional status and slightly discolored conjunctives. Abdominal examination showed a tenderness and a mobile hypogastric mass. The hemoglobin level was 8.2 g/dl and reduced hematocrit (24.9%) and iron level suggested chronic bleeding. The platelet count was 260,000/μL, white blood cell count was 10200/mm × 8 cm) ; thus, t × 8 cm) , there w × 8 cm)  and b.3, a hematocrit of 19% and a serum hemoglobin concentration of 6.4 g/dl, with a normal blood platelet level . Hemostasis laboratory data was normal. The patient was admitted to the ICU and was provided, by taking a central venous line  introduction of a nasogastric tube and urinary catheter, initial resuscitation by physiological serum and macromolecules and then transfusion with 8 units of red blood cells. After hemodynamic stability, a gastroscopy was performed, which did not demonstrate any gastric lesion responsible for this hemorrhage. No lesions or points of bleeding were found at the colonoscopy. But, the abdominal ultrasonography (US) realized after endoscopic explorations revealed the presence of a tissular mass in the left iliac fossa and the CT confirmed results of US and showed a solid tumor measuring (6 × 4.5 cm), probably a GIST of the small bowel [A 63-year-old man was admitted in the emergency room of the University Hospital of Hassan II with acute abdominal pain, hypotension, melena and severe anemia. His medical history revealed recurrent melena and upper abdominal discomfort over the last 4 months and did not suggest any major disease except hypertension treated for 3 years. He had no prior history of abdominal surgery or trauma. The physical examination revealed a conscious man with severe cutaneous paleness, a blood pressure of 90/60 mmHg, profuse sweating and a tachycardia with a weak and rapid pulse rate of 120 beats/min. There was no fever and there was minimal tenderness in the hypogastria at the abdominal examination. Laboratory data on admission showed a white blood cell count of 7,900/mmll bowel .Even if hemodynamic parameters were normalized after resuscitation and transfusion, and the hemoglobin control level was 10.9 g/dl, and because of the persistence of the severe melena, it was indispensable to shift the patient to the operating room for an emergency surgery to control the source of bleeding. A laparotomy was performed. Exploration noted a tumor of the terminal ileum  and no oGIST are the most common mesenchymal tumors of the gastrointestinal tract. The comm9GIST are a subset of gastrointestinal mesenchymal tumors, although relatively rare in absolute terms. The main manifestation of GIST is acute or chronic gastrointestinal hemorrhage, especially in the small bowel localizations, and sometimes this bleeding can be associated with hemorrhagic shock. Emergency surgery after resuscitation, fluid management and blood transfusion is the treatment of choice in these cases. Complete surgical resection of the primary tumor is the most definitive treatment. Prognosis of GIST is influenced by completeness of primary resection and tumor malignant potential. Adjuvant treatment (imatinib) should be advocated for patients with either a high-grade GIST or after incomplete primary surgical treatment.


Hydrostatic pressure (HP) is a significant factor in the function of many tissues, including cartilage, knee meniscus, temporomandibular joint disc, intervertebral disc, bone, bladder, and vasculature. Though studies have been performed in assessing the role of HP in tissue biochemistry, to the best of our knowledge, no studies have demonstrated enhanced mechanical properties from HP application in any tissue.A), with corresponding increases in GAG content. These regimens also resulted in significant 101% and 92% increases in Young's modulus (EY), with corresponding increases in collagen content. Phase II employed a 3×3 full-factorial design of HP  and growth factor application . The combination of 10 MPa static HP and TGF-β1 treatment had an additive effect on both HA and EY, as well as a synergistic effect on collagen content. This group demonstrated a 164% increase in HA, a 231% increase in EY, an 85% increase in GAG/wet weight (WW), and a 173% increase in collagen/WW, relative to control.The objective of this study was to determine the effects of hydrostatic pressure (HP), with and without growth factors, on the biomechanical and biochemical properties of engineered articular cartilage constructs, using a two-phased approach. In phase I, a 3×3 full-factorial design of HP magnitude  and frequency  was used, and the best two treatments were selected for use in phase II. Static HP at 5 MPa and 10 MPa resulted in significant 95% and 96% increases, respectively, in aggregate modulus (HTo our knowledge, this is the first study to demonstrate increases in the biomechanical properties of tissue from pure HP application, using a cartilage model. Furthermore, it is the only study to demonstrate additive or synergistic effects between HP and growth factors on tissue functional properties. These findings are exciting as coupling HP stimulation with growth factor application has allowed for the formation of tissue engineered constructs with biomechanical and biochemical properties spanning native tissue values. Hydrostatic pressure plays an important role in the mechanoregulation of several tissues; including cartilage Cartilage is typically exposed to pressures in the physiologic range of 3–18 MPa Several prior studies have also demonstrated the benefits of growth factors, including BMP-2, IGF-I, and TGF-β1, on construct functional properties Though several studies have been performed in assessing the role of HP in tissue biochemistry, to the best of our knowledge, no studies have demonstrated enhanced biomechanical properties from HP application in any tissue. Furthermore, studies that systematically assess the effects of multiple HP magnitudes and frequencies on construct functional properties are lacking. Additionally, there is a dearth of studies demonstrating synergistic effects on tissue functionality from combining hydrostatic pressure and growth factors.Using a scaffoldless cartilage tissue engineering model 6 cells were added in 100 μl of culture medium; t = 0 was defined as 24 hrs after seeding.Cartilage from the distal femur of wk-old male calves was obtained At t = 10 days, self-assembled constructs (n = 6/group) were removed from confinement in 5 mm dia. agarose wells and exposed to HP for 1 h/day, for 5 days. The study employed a 3×3 full-factorial design of magnitude  and frequency , for a total of 9 treatment groups. The constructs were then placed in individual agarose-coated wells of 48-well culture plates for the remainder of the study. A control (CC) consisted of constructs removed from confinement in 5 mm dia. agarose wells at 10 days, and cultured in individual wells of 48-well culture plates coated with 2% agarose for the remainder of the study. Per construct, 500 μl of medium was changed daily, and all constructs were assessed at t = 4 wks.This study employed a 3×3 full-factorial design of HP  and growth factor application  for a total of nine treatment groups. The hydrostatic pressure regimens were selected in phase I  at 4°C for 72 hrs. Next, 0.1 mL of 10× TBS was added along with 0.1 mL pancreatic elastase and mixed at 4°C overnight. From this digest, total DNA content was measured by Picogreen® Cell Proliferation Assay Kit . Total sulfated GAG was quantified using the Blyscan Glycosaminoglycan Assay kit (Biocolor) A), permeability, and Poisson's ratio of the samples were then determined using the linear biphasic theory Y) was calculated from the linear region of each stress-strain curve. Construct thickness was measured using digital calipers.To obtain salient compressive properties, samples were evaluated under conditions of creep indentation Biochemical and biomechanical assessments were performed on all constructs (n = 6 or 7). In each phase, a single factor ANOVA was used to analyze the samples, and a Fisher LSD post hoc test was used when warranted. Significance was defined as p<0.05. Additionally, in phase II, the interaction term of a two factor ANOVA was used to test for synergism, as described previously All constructs reached a diameter of approximately 6 mm at t = 4 wks . In phasIn both studies, positive staining for collagen  and GAG In phase I, all values of cells/construct, GAG/WW, and collagen/WW are found in In phase II, all values of cells/construct, GAG/WW, and collagen/WW are found in A and EY are found in A relative to the control group  demonstrate increases in the biomechanical properties of tissue from pure HP application, using a cartilage model, 2) demonstrate additive or synergistic effects between HP and growth factors on tissue functional properties, and 3) systematically assess the effects of varying physiologic magnitudes and frequencies of HP on tissue functional properties.A and EY with parallel increases in GAG and collagen content. These results support our hypothesis, as static hydrostatic pressure was found to have the greatest effect on construct biochemical and biomechanical properties. Since 5 MPa and 10 MPa static HP were the only regimens to significantly increase the compressive and tensile stiffness as well as GAG/WW and collagen/WW, these two regimens were selected for use in phase II.In phase I, 5 MPa and 10 MPa static HP were the only regimens that increased both HA and EY, as the increases in compressive and tensile stiffness for the combined treatment were equal to the sum of the effects of the two individual treatments. Additionally, the combined treatment exhibited a synergistic increase in collagen/WW, as the effect of the combined treatment was statistically greater than the sum of the effects of each individual treatment. Excitingly, the collagen/WW of this group, at 15.3%, spanned reported values for native articular cartilage In phase II, the combination of 10 MPa static HP and TGF-β1 treatment had significant effects on construct biomechanical and biochemical properties, thus supporting the hypothesis that combined HP and growth factor treatment would have additive and synergistic effects on construct functional properties. The combined treatment of 10 MPa static HP and TGF-β1 had an additive effect on both HHowever, although 5 MPa and 10 MPa static HP have similar effects on construct properties when applied alone, 5 MPa static HP did not exhibit the same additive and synergistic effects when combined with TGF-β1 treatment. This result suggests that there are different cellular responses to varying HP magnitudes; for example, it can be speculated that increasing HP from 5 MPa to 10 MPa in the presence of TGF-β1 may activate additional intracellular pathways that lead to enhanced production of ECM proteins and increased biomechanical properties. Interestingly, a similar effect has been observed previously in work on chondrogenic differentiation of human mesenchymal stem cells (MSCs) It is also interesting to note that combining BMP-2+IGF-I treatment with either of the HP treatments did not lead to further enhancement of construct properties, and actually negated the beneficial effects of HP alone on construct properties. It has previously been shown that HP modulates the level of TGF-β mRNA By demonstrating the beneficial effects of static HP over cyclic HP application on construct biomechanical and biochemical properties, this study contradicts several prior studies that have shown positive effects from cyclic HP When assessing the effects of combined HP and growth factor treatment on cartilage properties, the results presented here agree with prior studies that have combined these treatments as differentiation agents for mesenchymal stem cells Physiologic HP does not deform cartilage The beneficial effects of HP on tissue biochemical properties are not confined merely to cartilage, and it is possible that the approach of this study, namely combining optimized HP and growth factor treatments, may be applicable to several other tissues. For example, Stover et al. Multiple studies have assessed the effects of both static and intermittent HP regimens on gene expression and protein production. This study, which investigated the effects of multiple HP magnitudes and frequencies on construct functional properties, demonstrated enhanced biomechanical and biochemical tissue properties. Additionally, it systematically assessed the effects of combining HP and growth factors on construct functional properties, and indicated synergistic and additive effects. Future studies should determine the effects of temporal HP application during construct development, as well as examine the immediate and long-term effects of HP application on construct properties.


Carduelis chloris and chaffinch Fringilla coelebs, two of the most common birds in Britain. Morphological and molecular analyses showed this to be due to Trichomonas gallinae. Trichomonosis emerged as a novel fatal disease of finches in Britain in 2005 and rapidly became epidemic within greenfinch, and to a lesser extent chaffinch, populations in 2006. By 2007, breeding populations of greenfinches and chaffinches in the geographic region of highest disease incidence had decreased by 35% and 21% respectively, representing mortality in excess of half a million birds. In contrast, declines were less pronounced or absent in these species in regions where the disease was found in intermediate or low incidence. Also, populations of dunnock Prunella modularis, which similarly feeds in gardens, but in which T. gallinae was rarely recorded, did not decline. This is the first trichomonosis epidemic reported in the scientific literature to negatively impact populations of free-ranging non-columbiform species, and such levels of mortality and decline due to an emerging infectious disease are unprecedented in British wild bird populations. This disease emergence event demonstrates the potential for a protozoan parasite to jump avian host taxonomic groups with dramatic effect over a short time period.Emerging infectious diseases are increasingly cited as threats to wildlife, livestock and humans alike. They can threaten geographically isolated or critically endangered wildlife populations; however, relatively few studies have clearly demonstrated the extent to which emerging diseases can impact populations of common wildlife species. Here, we report the impact of an emerging protozoal disease on British populations of greenfinch  Emerging infectious diseases (EIDs) are increasingly cited as threats to wildlife, livestock and humans alike Trichomonas gallinae is a common protozoan parasite of pigeons (Columbiformes) which principally infects the upper alimentary tract where it can cause the disease, necrotic ingluvitis Ectopistes migratoriusNesoenas mayeriHieraaetus fasciatusCarduelis chloris, chaffinch Fringilla coelebs and dunnock Prunella modularis; (ii) demonstrate a spatially contemporaneous decline in the occurrence of frequently affected bird species in gardens; and (iii) combine this with national monitoring of bird abundance to show that this decline was followed by significant reductions in regional breeding populations. Public reporting of wild bird carcasses has been utilised as a surveillance tool for West Nile virus and Usutu virus elsewhere Carpodacus mexicanusOpportunistic surveillance of bird deaths in Britain since 2000 has shown a seasonal pattern in finch mortality driven primarily by low-levels of salmonellosis . In autuUnpublished data); post mortem examinations indicated that this seasonal peak was largely due to salmonellosis in Fringillidae and Passeridae species (Unpublished data). Between January 1st 2000 and December 31st 2004, we examined 750 garden birds post mortem, of which 67% of greenfinch deaths (168/252 birds) were due to salmonellosis and no cases of finch trichomonosis were confirmed.Opportunistic monitoring of garden bird mortality by the Royal Society for the Protection of Birds (RSPB) between 2001 and 2004 showed an annual seasonal peak in mid-winter (Dec/Jan), with 37–76% of reports per annum occurring in these two months  was diagnosed in 24 (13%), while salmonellosis was confirmed in only 4 (2%).Of 241 finch carcasses examined during the period 1 April 2006 to 30 September 2006, 179 were diagnosed as having died of infectious disease. Of these 179 birds, trichomonosis accounted for 144 (80%) of the deaths , Pyrrhula pyrrhula, 4 goldfinch Carduelis carduelis, 2 brambling Fringilla montifringilla and 2 siskin Carduelis spinus). Columbidae species accounted for 11% (37/347) of cases . The only other species in which the disease was diagnosed were house sparrow Passer domesticus (9 cases), yellowhammer Emberiza citrinella (4 cases), dunnock (3 cases) and great tit Parus major (2 cases). Concurrent soiling of the beak and facial plumage with food and saliva was frequently present in affected finches and such birds were typically thin or emaciated. Histopathological examination of the crop confirmed focally extensive moderate to severe mucosal ulceration and submucosal necrosis with infiltration by moderate numbers of degenerate and viable heterophils, lymphocytes and macrophages. Superficially, there was often a layer of necrotic crop epithelial tissue within which groups of 10–20 µm diameter round cells  and numerous clusters of mixed bacterial colonies were seen. Autolysis of the alimentary tract precluded meaningful histological examination in many cases; consequently histopathology was not used as a routine diagnostic test for confirmation of trichomonosis.Fringillidae species accounted for 84% (292/347) of trichomonosis cases diagnosed in 2005 and 2006, including greenfinch (173 cases), chaffinch (106 cases) and four other finch species  typical of a trichomonad parasite with a single nucleus and axostyle, anterior flagella and an undulating membrane. Scanning and transmission electron microscopy  confirmeTrichomonas gallinae (EU215369 (multiple columbid and hawk species from the USA), EU290649 (house finch Carpodacus mexicanus/corvid species from the USA), EF208019 (Mauritian columbid species), AY349182 ). Thus the organism infecting the British finches was identified as T. gallinae.Amplification of the ITS1/5.8S/ITS2 ribosomal region was performed on DNA extracted from oesophageal lesions from nine greenfinches and nine chaffinches  that died of trichomonosis in 2005 or 2006. An identical consensus sequence of 214 nucleotides was identified for all PCR products , EU215373.1 (rock pigeon Columba livia), EU215374.1 (collared dove), and EU215375.1 (broad-winged hawk Buteo platypterus); all from the USA) with 100% sequence identity and 100% query coverage and five Trichomonas sp. reports with 100% sequence identity and 97% query coverage. Multiple Tetratrichomonas gallinarum and Tetratrichomonas sp. reports had 98% or less sequence identity with 100% query coverage. The specificity of the nested PCR for a range of species within the Trichomonadidae was not assessed in this study, therefore this technique cannot currently be used in isolation as a diagnostic test for T. gallinae. However, use of the nested PCR appears robust within the case definition for finch trichomonosis that we have employed.Sequencing of second stage products of a nested PCR for the detection of trichomonads from oesophageal lesion extracts from seven greenfinches and one chaffinch examined in 2005 or 2006 with trichomonosis identified a consensus sequence of 149 nucleotides in all eight cases . As these rates may have been influenced by local publicity, we quantified incidence in a network of garden sites where diseased birds were searched for systematically according to a defined protocol throughout the study period. Trichomonosis (according to our incident definition) was identified at 39 sites (5.2%) but the incidence varied spatially in a manner similar to the opportunistic reports, with 10.4% (n = 115) of participants in the High region reporting an incident, 7.8% (n = 154) in the Intermediate region and 0.0% (n = 164) in the Low region .Rates of opportunistic reports of trichomonosis (identified according to our incident definition) varied greatly among counties, with rates in excess of 0.20 incidents per thousand households (ipth) found in Gloucestershire, Powys and Warwickshire. We used the opportunistic reports to define regions of High, Intermediate and Low disease incidence . Overall9,23 = 24.23, p<0.0001). Analysis of a subset of these sites which recorded counts of individual birds (rather than presence) showed a similar reduction in mean abundance in gardens reporting greenfinches in the latter half of 2006.The weekly reporting rates of greenfinch occurrence in all gardens contributing to the British Trust for Ornithology's (BTO) Garden BirdWatch survey β = −1.32±0.12, p<0.001), but less so in the region with Intermediate  or Low mortality levels   and dunnock  in the region of High trichomonosis-associated mortality were lower, and there were no significant reductions in occurrence of either species in the regions of Intermediate or Low incidence.The reporting rate for greenfinches in the following spring (2007) was significantly reduced in the area of High trichomonosis-associated mortality ( p<0.01) . ReductiThese observed reductions in occurrence were reflected in changes in the size of wider regional breeding populations obtained from the independently-derived Breeding Bird Survey (BBS) T. gallinae infection in columbiform species Trichomonas sp. protists, including T. gallinaeT. gallinae, confirming the parasite species identification We diagnosed trichomonosis as an emerging and widespread cause of death in British finches in 2005 and 2006. The gross and histopathological findings are consistent with upper alimentary tract lesions caused by T. gallinae in white-winged dove Zenaida asiatica carcasses found that most reliable diagnostic results were obtained on sampling within 8 hours of death and that parasite culture from T. gallinae-positive birds was successful in only 44% of carcasses sampled at 48 hours following death Experimental assessment of the survival of Consideration of two independent data sets of bird mortality in Britain (i.e. (1) surveillance based opportunistically on unsolicited reports from members of the public and (2) systematic monitoring according to a defined protocol by our volunteer network) in combination enables us to maximise their relative benefits. The large-scale, but ad hoc, sampling from the opportunistic survey provided indication of the commencement of the epidemic and samples to determine its epidemiology. The systematic sampling using a pre-existing network provided a quantitative measure of incidence which will be relatively robust to, for example, increases in reporting frequency in response to media coverage. Such data could not realistically be collected in any other way and should, over time, provide much needed detail on the background complement of disease agents in wild bird populations.unpublished data).The winter of 2006 was relatively mild  from the mid 1980s to 2006, but decreased (significantly) by 15% in 2007 compared to the previous year T. gallinae infection in finches is currently unknown but columbiform species are considered the most likely source. Future research priorities include molecular studies to identify the origin of T. gallinae in finches, for example through comparing parasite isolates derived from finch, columbiform and other wild bird species, and to assess T. gallinae strain diversity in Great Britain on the basis of geography, species and virulence. Retrospective surveys using the nested PCR methodology on archived garden bird tissues, collected prior to 2005, could identify presence of the parasite in non-columbiform populations prior to the onset of epidemic finch mortality, although the chance of detecting a low prevalence from the relatively small number of archived carcasses would be low.The origin of T. gallinae infection in garden habitats (and chaffinches somewhat less so), the reasons for this are not clear. The greenfinch is one of the species most frequently affected by other infectious diseases that are commonly diagnosed in garden birds, such as salmonellosis and colibacillosis . The gregarious and granivorous habits of finch species sharing food and water at feeding stations with high contact rates are likely to facilitate pathogen spread. Trichomonosis, however, was confirmed only rarely in members of the Paridae, which also commonly flock to garden bird feeders, so feeding behaviour is unlikely to be the sole driver of greenfinch susceptibility. Investigations of wildlife species mortality which rely on the reporting of sick and dead birds by members of the public have an inherent risk of bias due to variation in observer effort. Also, some species, such as large or brightly coloured ones, are more likely to be detected. Systematic sampling by volunteers in our network provides a relatively consistent level of observer effort and will have reduced variability in detection bias between gardens so should provide a robust measure of relative disease incidence. Although we have quantified the occurrence of trichomonosis in dead birds, the overall prevalence of T. gallinae infection in wild bird populations remains unknown. Prospective studies to screen multiple species of live birds for trichomonad parasites T. gallinae.Although greenfinches appear to be the species most frequently affected by Trichomonas gallinae can be transmitted through direct contact between birds, for example courtship and feeding of young, and through indirect routes including shared food and water sources T. gallinae is a pathogen of potential significance to the racing pigeon, aviculture, game bird and poultry industries and the implications for finch trichomonosis to these industries remain poorly understood. Continued monitoring of diseases in wild bird populations is required to better quantify and understand their impact on population dynamics Land-use change and habitat degradation have led to an increased national focus on garden habitats as a useful refuge for British wildlife No live animals were used for this research, however, the project was reviewed and approved by the Zoological Society of London's Ethics CommitteeSince 2000, opportunistic nationwide monitoring of the causes of garden bird mortality in Great Britain has been carried out by the Institute of Zoology , the Wildlife Veterinary Investigation Laboratory  and the Scottish Agricultural Colleges (Ayrshire). In 2005, these organisations, together with the BTO, the RSPB, the Department of Veterinary Pathology, University of Liverpool and the Universities Federation for Animal Welfare, established a coordinated surveillance network as part of the Garden Bird Health initiative (GBHi). The GBHi was established before the emergence of trichomonosis in British finches was identified and has always included wide-ranging investigations to identify pathogens responsible for garden bird disease.The GBHi surveillance of garden bird morbidity and mortality takes place via opportunistic reports obtained from the general public and weekly reports from identified volunteers who form a systematic reporting network. As opportunistic reports are vulnerable to temporal or spatial observer bias, for example following regional media reports, the systematic surveillance network was established in April 2005 in order to quantify disease prevalence rates. This network utilised the BTO's Garden BirdWatch (GBW) volunteer network of approximately 15,000 households throughout Britain Post mortem examinations using a standardised protocol were performed on carcasses submitted from a subset of reported garden bird mortality incidents. Cases were selected for post-mortem investigation based on fresh carcass availability. Birds thought to have died as a result of trauma, predation or infectious disease were examined; our selection criteria did not specifically or solely target finch species or suspected cases of trichomonosis, but rather aimed to achieve a representative cross-section of species and aetiologies. In 2005 and 2006, a combined total of 995 garden birds of 42 species were examined, of which Fringillidae species accounted for 64% of submissions.Fresh carcasses were submitted by post or were hand-delivered and were refrigerated at 4°C and examined fresh within 48 hours of submission where possible, or were frozen at −20°C on submission and examined at a later date. Each submitted carcass was assigned a unique post mortem reference code and the species, age, sex and body weight were recorded for each bird examined. Systematic external and internal examinations of body systems were performed and any gross lesions described. Where indicated, and where the state of carcass decomposition permitted, samples were taken for microbiological, parasitological and histopathological investigations.2 conditions. Small intestinal contents were plated directly onto the following media: (1) Xylose-Lysine Deoxycholate (XLD) agar (QCM laboratories), or MacConkey agar without salt (E&O laboratories) and Brilliant green agar (E&O laboratories), incubated under aerobic conditions; (2) CBA, incubated under aerobic conditions; (3) Campylobacter Blood Free Selective medium (modified CCDA-Preston) (QCM laboratories), incubated under microaerophilic conditions, and (4) immersed into selenite Salmonella-selective enrichment broth (QCM laboratories or E&O laboratories) under aerobic conditions for 24 h followed by subculture onto XLD agar aerobically. At the Institute of Zoology, liver also was plated onto CBA and incubated anaerobically. The same bacteriology protocol was used for examination of necrotic ingluvitis lesions as for the intestinal contents with the exception of the modified CCDA-Preston media. Bacterial isolates were identified using colony and Gram's staining morphology, followed by biochemical properties which were determined using the API biochemical test strip method .Liver and contents of the mid-small intestinal loop were sampled aseptically from the majority of cases, as were any lesions found, and were examined for the presence of pathogenic bacteria. Briefly, liver was plated directly onto the following media: Colombia blood agar supplemented with 5% horse or sheep blood (CBA), incubated under aerobic conditions, and Chocolate blood agar (CCBA) (QCM laboratories), incubated under 5–10% CO3) from cases with necrotic ingluvitis were incubated at 30°C in Trichomonas Media No. 2.  and screened for motile trichomonads at 24, 48, 72 hrs and 5 days. Wet mount preparations of small intestinal contents were examined in a subset of cases for evidence of nematode, cestode and protozoan parasites.In addition, oesophageal lesions , were fixed in neutral-buffered 10% formalin and processed for histopathological examination using routine methods. Duplicate samples of organs and diseased tissues were stored frozen at −20°C or −80°C for future analyses.A combination of morphological and molecular techniques was used to identify the trichomonad species. Giemsa-stained preparations of trichomonad cultures were examined using light microscopy to assess parasite morphology. These were prepared by placing a drop of active trichomonas culture onto a standard glass microscope slide; this was then air dried, alcohol-fixed and stained using routine methods. Transmission and scanning electron microscopy was performed on trichomonad cultures fixed in 2.5% buffered gluteraldehyde and post-fixed in 1% osmium tetroxide  at the University College Medical School, Royal Free Campus, using Philips 201 and 501 microscopes.2 (Qiagen), 0.5 µL of 5 U/µL HotStar Taq Plus DNA Polymerase (Qiagen), 2 µL template DNA, 0.4 µL of 100 mM dNTP mix , 3 µL of 10 µM forward and reverse primer and molecular grade water to complete the 50 µL per reaction. Oligonucleotide primers were supplied by Operon Biotechnologies, Germany. After an initial 15 min denaturation at 94°C, 35 cycles of 94°C for 1 min, 65°C for 30 sec and 72°C for 1 min were carried out, followed by a 5 min extension at 72°C using a thermal cycler . Each PCR run contained a negative control of water and a positive control of purified trichomonad DNA obtained from parasites cultured from an affected greenfinch found dead as part of this study.DNA was extracted from frozen/thawed necrotic ingluvitis lesions collected from finches using the Biosprint 15 DNA Blood Kit  for purification of DNA from tissue according to the manufacturer's instructions. DNA was extracted from trichomonad cultures using the same technique. PCR was used to amplify the ITS1/5.8S/ITS2 ribosomal region using published TFR1 and TFR2 primers www.synthesisgene.com). Sequences from the forward TFR1 primer and the reverse complement of the TFR2 primer PCR product were aligned in both directions for each sample using MEGA 4.1 software and ClustalW (www.megasoftware.net). Sequences were compared with available gene sequences within NCBI Genbank using the BLAST search function to determine species identification within the Trichomonadidae.The PCR products, consisting of a clear single band, were visualised under UV light after ethidium bromide staining of a 1% agarose gel and the expected product size (circa 400 bps) was confirmed using Ready-Load 100 bp DNA ladder . PCR products were purified using the QIAquick PCR purification kit (Qiagen) and submitted for sequencing at the John Innes Genome Laboratory, UK, using the Applied Biosystems 3730xl with POP7 polymer and the TFR1 and TFR2 primers. Chromatograph profiles were inspected using Chromas 2 software  rRNA primers (forward -TACTTGGTTGATCCTGCC and reverse - TCACCTACCGTTACCTTG) from ATAGGACTGCAAAGCCGAGA) and TN4 reverse (TGATTTCACCGAGTCATCCA), were then designed using Prime3 online software A nested PCR protocol was designed to increase the sensitivity of detection of trichomonad parasite DNA in template DNA extracted from lesions sampled post mortem and to provide a diagnostic tool for cases where autolysed carcass condition precluded The first stage used 2 µL of 10X PCR buffer (Qiagen), 0.1 µL of 5 U/µL HotStar Taq Plus DNA Polymerase (Qiagen), 2 µL template DNA, 0.4 µL of 10 mM each dNTP mix (Qiagen), 0.4 µL of 100 µM forward and reverse primer and molecular grade water to complete the 20 µL reaction. After an initial 5 min denaturation at 94°C, 40 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 mins were carried out, followed by a 5 min extension at 72°C using a thermal cycler .. The sequence from the forward TN3 primer and the reverse complement of the TN4 primer PCR product were aligned in both directions for each sample using MEGA 4.1 software and ClustalW. Sequences were compared with available gene sequences within NCBI Genbank using the BLAST search function.The reaction mixture for the second amplification was the same as for the first, except for the use of the nested primers. The amplification mix comprised 19 µl of mix and 1 µl of PCR product template from the first amplification round. After an initial 5 min denaturation at 94°C, 35 cycles of 94°C for 45 sec, 50°C for 45 sec and 72°C for 45 sec were carried out, followed by a 7 min extension at 72°C. Each amplification contained a negative control, consisting of water and a positive control of purified DNA obtained from cultured trichomonad parasites from a greenfinch. Amplified PCR products were visualised under UV light after ethidium bromide staining of a 3% agarose gel and the expected product size (circa 200 bps) was confirmed using Easy Ladder I (Bioline). PCR products were submitted for sequencing at Cogenics (UK) using the ABI 3730 xl platform with the TN3 forward and TN4 reverse primers. Chromatograph profiles were inspected using Chromas 2 softwareSalmonella Typhimurium Definitive Type (DT)40 and DT56 variant (v), lesions from 56 greenfinches and six chaffinches confirmed to have died due to salmonellosis by microbiological examination in 2005 and 2006  were screened. The majority of cases were negative on nested PCR (54/62 cases) and all cases examined during the study period 1 April 2006 to 30 September 2006 were negative. This indicates that 8 cases examined outside the study period might have had concurrent infection with Trichomonas sp. and Salmonella sp.. Carcass condition precluded parasite culture in all but one of these cases, but this case yielded trichomonad parasites confirming the existence of dual infection. These findings suggest that the nested PCR does not cross react non-specifically with Salmonella Typhimurium DT40 and DT56(v) and can be used within our case definition for the diagnosis of trichomonosis in finches.Reliable discrimination between necrotic ingluvitis due to salmonellosis or trichomonosis in finches is not possible based on gross examination alone. In order to evaluate whether our nested PCR cross-reacted non-specifically with Salmonella sp. on culture. No cases of dual infection were identified during the study period.Cases of trichomonosis were diagnosed on the basis of the presence of necrotic ingluvitis lesions in combination with positive culture of motile trichomonads and/or positive nested PCR amplification. Salmonellosis also causes necrotic ingluvitis and this was diagnosed by lesions being positive for i surveyed all or part of their garden systematically in a consistent manner each week to record the number, and putative cause, of dead or sick birds found. The clinical signs of ill health in birds affected by trichomonosis typically included non-specific malaise, although dysphagia was noted in a large proportion of reported incidents. This contrasts with the clearly recognisable external signs of conjunctivitis caused by Mycoplasma gallisepticum in the house finch and which were used to monitor spatial spread of mycoplasmosis Garden Bird Watch participants (c. 9000) record the presence of a range of bird and other wildlife species encountered each week throughout the year; a subset (c. 30%) submit actual counts of each species seen using an online recording form. Participants maintain a consistent level of observational effort from one week to the next; data from weeks that are under- or over-observed are discarded. Variation in observer effort and competence is inevitable, but this can be controlled for by introducing a site effect into the models used to examine the data unpublished data), have shown the disease to be seasonal, occurring almost exclusively within the period 1st October to 31st March.In order to evaluate the geographical distribution of the trichomonosis epidemic, data from the opportunistic and systematic reporting schemes were examined for the period between 1 April and 30 September 2006. This interval was selected to minimise the likelihood of confounding these data with mortality due to salmonellosis, outbreaks of which occur during the winter months and which also result in non-specific signs of malaise in finches. Previous studies of salmonellosis in Great Britain As resources and logistics did not allow all dead birds found to be submitted for post mortem examination, we established specific criteria for determining if a mortality incident should be classified as likely being due to trichomonosis. For the purposes of this analysis, a trichomonosis incident was defined if, within the six month period of the study (between 1 April and 30 September 2006), mortality included two or more dead finches (greenfinch or chaffinch), one or more sick finch(es) with typical signs of disease, or if trichomonosis was confirmed post mortem. To give a measure of incidence for the opportunistic reports, we expressed the total number of trichomonosis incidents reported in each county per thousand households according to the 2001 UK National Census mgcv for R 2.6.0 Each week, GBW participants recorded the presence of each bird species in their garden and, optionally, the peak number of birds counted each week To exclude the possibility that changes in other environmental factors, such as climate, between years might have caused changes in greenfinch numbers, we also modelled reporting rates of chaffinch, which has a similar body size and ecology to the greenfinch, and was the second most frequently affected species in which trichomonosis was recorded, and of dunnock, which also feeds around garden feeders Abundance of birds in gardens varies seasonally in a non-linear fashion, with peak numbers occurring in late winter or spring and lowest counts in late summer/autumn, when many birds are moulting To quantify impacts on the breeding season following the epidemic of trichomonosis in autumn 2006, we constructed a model to compare reporting rate at the start of the 2007 breeding season  with the average reporting rate for the same period in the previous two years . We restricted the analysis to the previous two years to avoid potential confounding effects of long-term trends in bird numbers and reporting rates. As we were considering a relatively short time-span within a year, it was not necessary to fit a smoothed term of week; rather we modelled reporting rate as a function of garden size, week (and its square to account for any non-linearity) and a two-level dummy variable, year, with weeks in 2005/06 having year = 0 and those in 2007 year = 1. The estimate of this latter term then gives the change in reporting rate in 2007 relative to that in the previous two years. As before, we included garden identifier as a random effect and fitted an auto-correlated binomial error structure.Mortality due to trichomonosis varied spatially throughout the country, so we defined three regions representing areas of High, Intermediate and Low incidence of trichomonosis, based on the results of opportunistic sampling. We restricted these analyses to England south of a line from the Mersey to the Humber (approx. 53° 30′N) as it is in this region that gardens participating in GBW are most representative of the landscape as a whole . Greenfinch, chaffinch and dunnock populations occur widely across this region, being observed in 70–80% of gardens in the GBW scheme. For convenience, we defined these three regions in terms of administrative county boundaries with areas of High , Intermediate  and Low  incidence of trichomonosis. To test for differences in the change in reporting rate in spring 2007 among areas, we included an interaction term between area and the dummy year variable described above.Numbers of birds present across Britain during the breeding season are monitored using line-transect counts in a sample of c. 3,000 randomly selected 1×1 km squares by the Breeding Bird Survey (BBS)


Intrathecal (IT) morphine provides excellent post-operative analgesia, but causes multiple side effects including nausea and vomiting (PONV), pruritus and respiratory depression, particularly at higher doses. The lowest effective dose of spinal morphine in patients undergoing total knee arthroplasty is not known.We evaluated the analgesic efficacy and side effect profile of 100 – 300 μg IT morphine in patients undergoing elective total knee replacement in this prospective, randomized, controlled, double-blind study. Sixty patients over the age of 60 undergoing elective knee arthroplasty were enrolled. Patients were randomized to receive spinal anaesthesia with 15 mg Bupivacaine and IT morphine in three groups: (i) 100 μg; (ii) 200 μg; and (iii) 300 μg.Both 200 μg and 300 μg IT morphine provided comparable levels of postoperative analgesia. However, patients that received 100 μg had greater pain postoperatively, with higher pain scores and a greater requirement for supplemental morphine. There were no differences between groups with regard to PONV, pruritus, sedation, respiratory depression or urinary retention.Both 200 μg and 300 μg provided comparable postoperative analgesia, which was superior to that provided by 100 μg IT morphine in patients undergoing total knee arthroplasty. Based on these findings, we recommend that 200 μg IT morphine be used in these patients.ClinicalTrials.gov Identifier NCT00695045 The provision of high quality postoperative analgesia after total knee arthroplasty continues to present a challenge. Systemic opioids can cause post operative nausea and vomiting, sedation, and respiratory depression, which may be especially undesirable in older patients with co-existing respiratory disease, such as patients undergoing major joint replacement surgery. Intrathecal (IT) opiates are a useful option in patients undergoing knee arthroplasty, and can markedly reduce postoperative opioid requirements -3.However, the use of IT morphine may be associated with a number of distressing side effects e.g. pruritus, urinary retention, nausea and vomiting -7, and pThe efficacy of doses of IT Morphine of 300 μg or greater, in patients undergoing total knee arthroplasty, has been well demonstrated ,3. HowevThe aim of this study was to determine the analgesic efficacy and side-effects profile of 100 μg and 200 μg of IT morphine, in comparison to 300 μg, in patients undergoing elective total knee arthroplasty.After obtaining approval by the Hospital Ethics Committee, and written informed patient consent, we enrolled ASA physical status I-III patients scheduled for elective total knee arthroplasty in a prospective, randomized, double blind, controlled clinical trial. Exclusion criteria included non-suitability for spinal anaesthesia as deemed by the anaesthetist providing care for the patient; history of allergy or severe nausea with opioid analgesics or local anesthetic agents; or any medical condition resulting in ASA status greater than III. All patients received spinal anesthesia. The allocation sequence was generated by random number tables, and the allocation concealed in sealed envelopes, which were not opened until patient consent had been obtained. Patients were randomized to receive one of three IT doses of preservative free morphine: 100 μg, 200 μg, and 300 μg.®, AstraZeneca Group, Luton, UK), and either 100 μg, 200 μg or 300 μg of preservative free morphine into the intrathecal space, depending on group allocation. A total volume of 4 ml was administered to each patient, with the balance of the IT injectate composed of 0.9% NaCl. The dose of IT morphine administered was not noted in the patients anesthesia record; however this was documented in a sealed envelope attached to the patients anesthesia record which could be opened in case of an adverse event. All patients received 40% oxygen via face mask for the duration of the procedure. Standard monitoring, including non-invasive blood pressure, electrocardiogram, and oxygen saturations were used in all patients for the duration of surgery.No patient received premedication. Spinal anesthesia was performed with the patient in the sitting position at the L3/4 intervertebral space utilizing a 25 gauge Whitacre needle. Patients were administered 15 mg of hyperbaric bupivacaine  for a period of 24 hours post-operatively. All patients received nurse administered 2 mg intravenous morphine bolus on demand. Each administration was initiated by patient request and a minimal interval of ten minutes was required between of each morphine bolus. First line treatment of PONV consisted of prochlorperazine 12.5 mg by intramuscular injection. If this proved ineffective second line therapy consisted of 4 mg of ondansetron by intravenous injection. Each administration was initiated by patient request and a minimal interval of one hour was required between administration of first and second line agents. Treatment for pruritus consisted of promethazine 20 mg by intramuscular injection six hourly and treatment in all cases was initiated upon patient request. Naloxone was reserved for treatment of pruritus that was resistant to promethazine therapy.Patients were monitored for 24 hours by the HDU nursing staff, who were unaware of the patient allocation. Assessment parameters included severity of postoperative pain, and the presence and severity of postoperative nausea and vomiting (PONV), pruritus, sedation, and respiratory depression. The primary outcome variable, the severity of pain in the first 24 h postoperatively, was assessed every 4 hours utilizing a Visual Analogue Scale (VAS) and by the amount of supplemental morphine required in the first 24 hours. In addition, the time to first request for supplemental analgesics was recorded.The presence and severity of PONV was assessed every 4 hours using an ordinal scale . In addition, the time to first request for antiemetic therapy, and total amount of supplemental antiemetic administered, was recorded. The presence and severity of pruritus was assessed every 4 hours using an ordinal scale . The time to first request for therapy for pruritus, and total amount of promethazine administered, were recorded.2 < 94% on room air) moderate (SaO2 < 90% on room air) and severe (SaO2 < 85% on room air) arterial hypoxemia were recorded. At each 4 hourly data collection point, the lowest respiratory rate and lowest arterial oxygen saturation in the previous 4 hours were recorded.Respiratory rate and arterial oxygen saturation were assessed on a continuous basis over the 24 hour post-operative period. The incidence of (i) reduced respiratory rate, defined as respiratory rate < 12 breaths per minute; and (ii) mild  and the need for urethral catheterization (defined as absence of spontaneous voiding 8 h after surgery and urine volume at catheterization of >400 ml) was recorded.We based our sample size estimation on the postoperative morphine requirement. We considered that a clinically important reduction would be a one third decrease in postoperative morphine requirements. Based on pilot studies we projected this reduction to be 5 mg, with a projected standard deviation of 5 mg. Based on these figures, using an α = 0.05 and β= 0.2, for an experimental design incorporating three equal sized groups, we estimated that 20 patients would be required per group. We therefore aimed to enroll 60 patients into this study.2 analysis where applicable. For VAS data, group comparisons were performed at each time point using one way ANOVA with post hoc comparisons using Student-Newman-Keuls. Area under the VAS-time curve (AUC) was calculated by the trapezoidal method for each patient and compared using one way ANOVA. Ordinal data were analyzed using ANOVA on ranks with post hoc comparisons performed using Mann Whitney U test with the Bonferroni correction for multiple comparisons. The incidence of side-effects such as pruritus was compared using the Chi squared test for multiple variables.All analyses were performed on an intention-to-treat basis. The analysis was preformed using Sigmastat 3.5 . Demographic data were analyzed using one way analysis of variance (ANOVA), ANOVA on ranks, or χContinuous data are presented as means ± standard error of the mean (SEM), ordinal data are presented as medians ± quartiles (interquartile range), and categorical data are presented as raw data or as frequencies. The α level for all analyses was set as P < 0.05.Eighty patients were assessed for eligibility to participate in the study [See Additional file Both 200 μg and 300 μg of IT morphine provided comparable analgesia, in terms of postoperative VAS scores Figure , median 2 fell below 94% responded to 40% O2 via facemask  in the first 24 postoperative hours in any group (Table k Figure ; Table 2There were no significant between group differences in the incidence or severity of PONV Figure , 3. SpecThere were no significant between group differences in the incidence or severity of pruritus Figure , 3. SpecThere were no between group differences in sedation scores Table , or in tThis study demonstrates several important points. First, doses of 200 μg and 300 μg IT morphine provide comparable and effective postoperative analgesia in patients undergoing elective total knee arthroplasty. Second, 100 μg of IT morphine was not as effective in providing analgesia compared to the higher doses, and constitutes an inadequate dose of IT morphine in this patient group. Third, the incidence of respiratory depression and significant postoperative hypoxemia is low. No patient that received 200 μg of IT morphine developed severe arterial hypoxaemia in this study, attesting to the safety of this dose of IT morphine in this patient group. In summary, 200 μg IT morphine appears to provide comparable analgesia to 300 μg, and appears to be the best balance between analgesic efficacy and side-effect profile in this patient group.Our study clearly demonstrates that both 200 μg and 300 μg of IT morphine provide comparable analgesic efficacy in patients undergoing elective knee arthroplasty. Both regimens provided excellent analgesia and with a low requirement for rescue analgesia in the first 24 hours postoperatively. There was no difference in postoperative VAS scores at any time point between patients receiving 200 μg and 300 μg of IT morphine. In addition, there was no difference in the duration of analgesia provided by these doses, or in the need for or amount of supplemental analgesic therapy required. We have demonstrated that 100 μg does not provide a comparable level of analgesia to that provided by 200 μg or 300 μg of IT morphine, suggesting that this constitutes an inadequate dose, and therefore should not be used in this patient group.The finding that 300 μg is an effective dose of IT morphine in patients undergoing elective total knee arthroplasty has been demonstrated in previous studies . We demoRespiratory depression, the most serious side-effect of IT morphine, is more likely in the older patient, such as this patient group . VolunteThe incidence and severity of PONV is a major concern regarding the use of IT morphine ,6. In thPruritus has been identified as a major side effect associated of IT opioids, and may contribute significantly to patient discomfort ,6. It haAn important limitation in regard to our data for the side-effect profiles of the different doses of intrathecal morphine is that the study was powered to detect differences in analgesic efficacy, rather than in the incidence of side effects. Therefore, it remains possible that between group differences in more subtle, but nevertheless clinically important side effects, were not detected in this study. Caution is therefore warranted, particularly given the fact that doses of IT morphine as low as 200 μg has been demonstrated to produce significant respiratory depression in other studies . These fWe conclude that administration of 200 μg provides equally effective post operative analgesia as 300 μg of IT morphine. Conversely, 100 μg of IT morphine provided less effective postoperative analgesia in these patients. We therefore recommend that 200 μg IT morphine be used for postoperative analgesia in patients undergoing knee arthroplasty.2: Arterial oxygen saturation; SEM: standard error of the mean; VAS: Visual analogue scale.ANOVA: analysis of variance; AUC: Area under the curve; HDU: High Dependency Unit; IT: Intrathecal; L3/4: Lumbar 3/4 intervertebral space; mg: milligrams; mL: mililitres; μg: micrograms; NaCl: Sodium Chloride; PONV: postoperative nausea and vomiting; SaOThe authors declare that they have no competing interests.PH conceived of the study, and participated in its design and execution and helped to draft the manuscript. BA, PG and BK participated in the study, recruited patients, and helped to draft the manuscript. JL participated in the design and coordination of the study, performed the statistical analysis, and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Study Flowchart Hassett et al. This is a flow chart which provides full details of the patients screened, consented and enrolled in the study, and subjected to statistical analysis.Click here for file