extfieldsofstudy: Biology Medicine

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Transactivation of the Epidermal Growth Factor Receptor by Heat Shock Protein 90 via Toll-like Receptor 4 Contributes to the Migration of Glioblastoma Cells*

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca2+ appearing after 1–3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca2+ oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P2 inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca2+ rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca2+ signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca2+ oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca2+ increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.

Glioma ranges from slowly growing low grade tumors to rapidly growing high grade tumors, including anaplastic astrocytoma and glioblastoma (1,2). High grade gliomas include anaplastic tumor cells, necrotic foci, and rich vascularity, due largely to the aberrant expression of angiogenic factors by tumor cells (3). Tumor cells are highly proliferative and inva-sive within the brain. Despite the development of various treatments, the life expectancy of patients remains poor (4).
One of the molecules that could contribute to invasion is the stress or heat shock protein 90 (HSP90). In several tumor types, cell surface expression of HSP90 correlates with metastatic potential (5), and its inhibition with antibodies (6,7) or with cell-impermeable inhibitors (8) reduces cell migration in vitro. Of the two HSP90 isoforms, only HSP90␣ has been described extracellularly, which argues against cell lysis as the source of extracellular HSP90 (8). The metalloprotease MMP-2 can be associated with extracellular HSP90␣ (8), which is favored by acetylation of the stress protein (9). Surface HSP90␣ also participates in extracellular matrix proteininduced c-Src/integrin association and reorganization of the actin cytoskeleton (10). The cell-impermeable inhibitor of HSP90, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG)-N-oxide, displays anti-invasive and anti-metastatic activity in vitro and in vivo, respectively, probably through the inhibition of actin polymerization and focal adhesion formation (11). Actin polymerization can be activated by tyrosine kinase receptors of the EGFR 2 family (12). Cell surface HSP90␣ interacts specifically with the extracellular domain of HER-2 (also known as ErbB2), a ligand-less receptor, forming heterodimers with ligand-bound members ErbB1 (EGFR), ErbB3, and ErbB4. The HSP90␣/HER-2 interaction favors HER-2 heterodimerization with ErbB3 and signal transduction pathways via MAPK and PI3K-Akt, leading to actin rearrangement and cell motility (7). EGFR (ErbB1, or HER1) is overexpressed in 60% of multiform glioblastomas (13,14) and promotes invasion, proliferation, and metastasis (15)(16)(17) in response to several ligands known as EGF-related peptide growth factors (18). Binding of these ligands to the extracellular domain of EGFR leads to the formation of homo-and heterodimers, which triggers autophosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR, inducing signaling cascades. Ligand-independent EGFR activation, referred to as EGFR transactivation, can also be observed in response to various agents such as Toll-Like receptor (TLR) agonists and stress conditions (19,20). * This work was supported by grants from the CNRS, INSERM, and by Conseil Ré gional de Bourgogne (CRB, Faber Program) and by grants from are supported by the Ligue Nationale Contre le Cancer (to E. S. and C. G.). □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S8. 1 To whom correspondence should be addressed. Tel.: 33-3-80-39-34-17; Fax: 33-3-80-39-34-34; E-mail: dominique.thuringer@u-bourgogne.fr. 2 The abbreviations used are: EGFR, epidermal growth factor receptor; TLR, Toll-like receptor; TSG, thapsigargin; M␤CD, methyl-␤-cyclodextrin; HB, heparin binding; MMP, matrix metalloproteinase; ER, endoplasmic reticulum; P, phospho; HC, heavy chain; ADAM, a disintegrin and metalloproteinase.
The aim of this study was to investigate the role of extracellular HSP90␣ in signaling events triggered by EGFR in the human astrocytoma cell line (U87). We show that exogenously applied HSP90␣ mediates a cross communication between TLR4 and EGFR and accelerates cell migration.
Specific Cell Treatments-EGF was used at a unique dose of 10 ng/ml. To rule out the possible implication of HER2 in EGF-induced cell responses, 5 M gefitinib was added. For cholesterol depletion, cells were incubated for 30 -60 min at 37°C in serum-free medium containing 0.1% bovine serum albumin (BSA) and 7.5 mM methyl-␤-cyclodextrin (M␤CD), a mixture known to reduce plasma membrane cholesterol levels with no cell death (21). Cells were preincubated with Polymyxin B, a potent LPS inhibitor (10 M; 30 -60 min), to avoid bacterial contamination of HSP90␣ before activation. An additional control was to boil HSP90␣ at 100°C for 1 h to suppress the specific effect of HSP90␣ because LPSs are resistant to heat inactivation but not HSPs (22)(23)(24).
Immunofluorescence Experiments-Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were incubated with the primary antibody for 2 h and then secondary antibody (F(abЈ)2 fragment of IgG) conjugated with Alexa Fluor 488 nm or 568 nm (1:500) or with Alexa Fluor 488-conjugated phalloidin for 30 min. Substituting the primary antibody with preimmune serum demonstrates specificity of labeling. Counterstaining of nuclei was made using 40,6-diamidino-2-phenylindole (DAPI; 3 g/ml, 1-3 min). Images were collected on Axioplan 2 microscope using Axiovision Viewer 3 software (Zeiss). The intensity of labeled area (10 m 2 ) was measured with ImageJ analysis software. In some experiments, imaging was performed with a Leica SP2 RS confocal microscope, and Z-series of optical sections taken from 0.4-m intervals were collected using the scanning format 1024 ϫ 1024 pixels.
Cytosolic Free Ca 2ϩ Concentration-Changes in Fura-2 fluorescence were used to measure [Ca 2ϩ ] i . Briefly, confluent cells were incubated with 2 M fura-2-acetometoxymethyl ester in culture medium (40 min at 37°C) and then in HEPESbuffered saline solution containing in mM: 120 NaCl, 5.4 KCl, 0.8 MgCl 2 , 10 glucose, and 20 HEPES, pH 7.4. In nominally Ca 2ϩ -free conditions, no CaCl 2 addition was made. Dimethyl sulfoxide (DMSO) or ethanol, used to dissolve drug, was added in other solutions. Measurements were made on an inverted microscope (Nikon TS100) equipped with a 20ϫ objective (Nikon S Fluor, 0.75 NA) attached to a dual-excitation spectrofluorometer with excitation wavelengths alternating between 340 and 380 nm. Emission fluorescence (510 nm) was collected at a rate of 20/min. The 340/380 nm ratio was determined for each cell, and average values were reported as a function of time.
ATP Assay-Cells were exposed to HSP90␣ and/or EGF, and then the supernatants were collected to assess the amount of ATP release with luciferin-based ENLITEN ATP assay (Promega) at various time periods. Briefly, 100 l of luciferin-luciferase solution were added to supernatants, and light emission was recorded with a FLUOstar luminometer (BMG LABTECH). We calibrated light emission by using standard samples furnished by the manufacturer.
Internalization Assay-Cells were incubated with 200 g/ml anti-HSP90 for various time periods from 5 min to 2 h and then washed and fixed in paraformaldehyde. To detect possible internalization of anti-HSP90 antibody, permeabilized cells were incubated with Alexa Fluor 488-conjugated secondary antibody. For endocytosis analysis, cells were incubated 15 min in HEPES-buffered saline solution with CaCl 2 before experiment. The control solution containing 5 M BSA-Alexa Fluor 568 was applied 15 min to attest the absence of nonspecific endocytosis. EGF-BSA-Alexa Fluor 488 or BSA-Alexa Fluor 568 (1:20) were applied for 15 min (about 20 nM EGF) with or without HSP90␣. Cells were post-fixed and mounted on slides to be analyzed.
Immunodetection of Protein Phosphorylation-Cell monolayers were washed five times with ice-cold PBS and lysed (45 min on ice) using 1 ml of lysis buffer containing: 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM NaF, 100 M sodium orthovanadate, 100 M phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Roche Applied Science). Cells were scraped and centrifuged, and lysates were collected. Proteins were boiled for 5 min in 2 l of SDS buffer, fractionated using 8% SDS-PAGE, and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h with 5% BSA in Tris-buffered saline with Tween 20 (0.1%) (TBS-T) and incubated overnight with antibodies. For EGFR phosphorylation, proteins (20 -60 g/lane) were immunoblotted with anti-P-EGFR (1:50) and revealed with ECL reagents (Amersham Biosciences). Membrane was stripped and reprobed with antibody against EGFR to evaluate the total amount of EGFR.
Wound Healing Migration-A 1-mm-wide cell-free area was generated by gently scratching the cell monolayer with a sterile yellow Gilson pipette tip. The recording DMEM containing drugs or specific antibodies was applied (5% CO 2 in air at 37°C). Migration of astrocytes within the gap was monitored at given time intervals, using a Zeiss microscope equipped with a video camera. The extent of healing was defined as the ratio of denuded areas of the original wound and the wound after 11-22 h.
Bromodeoxyuridine Incorporation Assay-Serum-starved U87 cells cultured in the presence or absence of agents were exposed to bromodeoxyuridine at a concentration of 3 mM for 1 h. Cells were subsequently rinsed and fixed in 70% ethanol overnight, then washed and reincubated for another 10 min in 2 M HCl containing 0.1% Triton X-100 followed by a 20-min incubation in 0.1 M sodium borate. Cells were processed for immunofluorescence using an anti-bromodeoxyuridine antibody (Dako) and an appropriate Alexa Fluor 488-conjugated secondary antibody.
Statistical Analysis-One-way analysis of variance (Statview Software) was used to compare data groups. p values Ͻ 0.05 were significant.

Extracellular HSP90␣ Accelerates Glioblastoma Cell Migration in
Vitro-Immunofluorescence analyses detected the HSP90␣ isoform at the cell surface of glioblastoma U87 cells (Fig. 1A). HSP90␣ isoform was mainly expressed at the front edge of migration of cells. The extracellular localization of HSP90␣ was confirmed by flow cytometry. In contrast, no HSP70 was detected at the cell surface (Fig. 1A, right panel). To analyze the cellular basis for the invasive effect of extracellular HSP90␣, we used a wound healing assay. The addition of recombinant HSP90␣ protein to serum-free medium enhanced cell invasion in a dose-dependent manner (i.e. in the range from 60 ng/ml to 6 g/ml). This resulted in higher rates of gap closure observed as early as the first hour of cell incubation and confirmed in the following hours, 6 and 12 h (Fig.  1B). It is worthy to note that the effect of HSP90␣ is directed more toward cell migration than toward cell proliferation because a lower percentage of bromodeoxyuridine incorporation was observed (Fig. 1B, table). For instance, cell proliferation and invasion were respectively increased by 5 Ϯ 1 and 15 Ϯ 2% from control values following 6 h of cell exposure to 6 g/ml HSP90␣. Such an increase in cell migration was also observed with 10 ng/ml EGF and amplified by the combination of the two molecules (Fig. 1C). An additive effect of the activation of cell invasion was observed when both agents were added together into the cell bath medium. These results suggested that extracellular HSP90␣ exerted an additive effect on the ability of EGF to promote U87 cell migration.
Extracellular HSP90␣ Increases [Ca 2ϩ ] i in U87 Cells-To determine the role of Ca 2ϩ store in U87 response to HSP90␣, we measured [Ca 2ϩ ] i , in the presence and absence of extracellular calcium, in fura-2-loaded cells. Recombinant HSP90␣ induced a transient increase in [Ca 2ϩ ] i within 1-3 min ( Fig.  2A, left panel), whereas EGF induced a more sustained biphasic calcium response with a rapid peak followed by a plateau phase ( Fig. 2A, right panel). In a few experiments, the agonist was applied in the absence of external Ca 2ϩ to reveal release from internal stores. Then, Ca 2ϩ was restored extracellularly to reveal increase in plasma membrane Ca 2ϩ permeability (Fig. 2B). A subsequent application of 2 M TSG was made at the end of some recordings to estimate the Ca 2ϩ content of intracellular endoplasmic reticulum (ER) pools. In nominally Ca 2ϩ -free conditions, 6 g/ml HSP90␣ induced a transient Ca 2ϩ release from intracellular pools (Fig. 2B, left panel). The addition of Ca 2ϩ to the bath medium did not induce a significant increase in [Ca 2ϩ ] i , whereas TSG drastically increased [Ca 2ϩ ] i in the absence of external Ca 2ϩ , demonstrating that HSP90␣ did not deplete completely internal stores. In Ca 2ϩfree conditions, the amplitude of Ca 2ϩ peak induced by EGF remained unchanged, but the duration of the plateau phase was decreased, and Ca 2ϩ readdition to the bath medium resulted in a transient Ca 2ϩ influx through plasma membrane channels due to capacitative Ca 2ϩ entry (Fig. 2B, right panel). Upon stimulation with EGF, the cells showed calcium oscillations in response to HSP90␣ (i.e. with a frequency of three spikes per 5 min; 98%, n ϭ 50), which were rapidly abolished by the removal of HSP90␣ (Fig. 2C) or the addition of the EGFR/ErbB1 inhibitor Tyrphostin (AG1478), whereas the addition of ErbB2 inhibitor Gefitinib had no effect (supplemental Fig. S2). These results suggested that HSP90␣ could promote calcium oscillation through EGFR/ErbB1. ADP/ATP Contributes to HSP90␣-induced [Ca 2ϩ ] i Rise in Glioblastoma Cells-To determine whether the calcium oscillatory process elicited by HSP90␣ and EGF may involve the release of ATP/ADP and their interaction with purinergic (P2) receptors (25,26), we pretreated U87 cells with the P2 receptor antagonist suramin (10 M for 10 min) and the extracellular ATP scavenger apyrase (40 units/ml for 10 min). Both compounds drastically attenuated Ca 2ϩ oscillations generated by exogenously added EGF plus HSP90␣ (Fig. 2C, right  panel). To assess the effect of HSP90␣ and EGF on the subsequent ATP release, cells were exposed to these agents for various periods of time (from 15 min to 1, 6, and 12 h), and supernatants were collected to evaluate amounts of ATP by bioluminescence assay (Fig. 2D). Clearly no significant release of ATP could be detected in the absence or presence of HSP90␣. In contrast, HSP90␣ plus EGF released ATP from U87 cells to a higher extent than did EGF alone within the first 15-30 min of cell stimulation. Altogether, EGF triggers Ca 2ϩ oscillations that are amplified by extracellular HSP90␣ through release of ATP acting on purinergic P2 receptors.
Extracellular HSP90␣ Induces EGFR Internalization and Tyrosine Phosphorylation-Cell exposure to HSP90␣ for 15 min markedly reduced EGFR surface expression (Fig. 3A). U87 cells were incubated in the presence of BSA-Alexa Fluor 568, a membrane-impermeable macromolecule, before adding HSP90␣ for 15 min and then staining permeabilized cells.
Green EGFR was identified at the cell periphery and in the juxtanuclear region (Fig. 3B). In HSP90␣-stimulated cells, BSA co-localized with EGFR in the juxtanuclear region, suggesting a rapid internalization (Fig. 3B). Pretreatment with M␤CD to deplete cholesterol and disrupt lipid raft organization prevented the calcium response to HSP90␣ in the absence or presence of EGF, suggesting that lipid raft integrity may be essential for HSP90␣-induced Ca 2ϩ signaling (Fig.  3C). Cell exposure to HSP90␣ did not change significantly the level of ErbB1 versus total EGFR proteins (not shown). Both EGF and HSP90␣ induced EGFR phosphorylation at Tyr-1068 within 15 min, which was attenuated by cell pretreatment with the PKC␦ inhibitor, rottlerin (Fig. 4A). Because PKC   isozymes do not directly phosphorylate proteins at tyrosine residues, we examined the role of c-Src as an intermediate kinase between PKC and EGFR (supplemental Fig. S3). We found that the c-Src inhibitor PP2 inhibited HSP90␣-induced EGFR phosphorylation, whereas its inactive form (PP3) did not. Because this EGFR transactivation was attenuated by the matrix metalloproteinase (MMP) inhibitor (GM6001) and the heparin-binding (HB)-EGF inhibitor (CRM197; see supplemental Fig. S4), MMP activation and proHB-EGF shedding slightly contribute to this process. Thus, HSP90␣ interacts with PKC␦, which in turn phosphorylates Src to activate EGFR.
HSP90␣-induced EGFR Activation and TLR4 Signaling in U87 Cells-Extracellular HSP90␣ can interact with a variety of membrane receptors, including ErbB2 and TLR4 (7), and FIGURE 3. Extracellular HSP90␣ induced internalization of a pool of EGFR. A, flow cytometry analysis of EGFR expression in unstimulated (basal) and stimulated cells with 6 g/ml HSP90␣ alone or with the combination of HSP90␣ and EGF (10 ng/ml) for 15 min. Gray filled histograms are isotype IgG controls. B, HSP90␣-induced sequestration of BSA-Alexa Fluor 568 in EGFR endosomes. Cells were incubated for 15 min in HEPES-buffered saline solution containing HSP90␣ plus 5 M BSA-Alexa Fluor 568 (room temperature). The merged panel indicates co-localization of EGFR and BSA (yellow; n ϭ 3). C, raft-disrupting drug inhibits HSP90␣-induced Ca 2ϩ signaling. Cells pretreated with 7.5 mM M␤CD for 60 min were exposed to HSP90␣ and/or 10 ng/ml EGF. (2 M TSG; average of 50 cells; n ϭ 3). FEBRUARY (19). As observed for HSP90␣, the bacterial LPS (1 g/ml), a representative TLR4 ligand, also induced EGFR phosphorylation at Tyr-1068 within 15 min (Fig. 4A). Flow cytometry analysis indicated that TLR4 was expressed, whereas TLR2 could not be detected at the cell surface of U87 cells (Fig. 4B). No significant cell expression of TLR2 was shown (supplemental Fig. S5). Following external application of HSP90␣, TLR4 totally disappeared at the cell surface within 15 min. Cell surface expression of EGFR was also significantly reduced on U87 cells treated with LPS. Confocal microscopy observations revealed distinct patterns of cell expression, i.e. EGFR was mainly located at the cell periphery, whereas TLR4 was found throughout the cell (Fig.  4C). However, the two receptors co-localized on the plasma membrane and concentrated in pseudopodia (or filopodia). Upon cell stimulation with HSP90␣, EGFR was redistributed from the cell membrane to the juxtanuclear region where it co-localized with a small pool of internalized TLR4 (Fig. 4C). (Internalized EGFR by HSP90␣ is not targeted for degradation; see supplemental Fig. S7.) To determine the downstream signaling events of both TLR4 and EGFR activation, cells were incubated with 6 g/ml HSP90␣ for 0, 5, 10, 30, 60, and 320 min (Fig. 4D). Phosphorylated and total AKT, p44/42 MAPK, SAPK/JNK, NFB p65, and IRF-3 were examined by Western blot analysis. The AKT phosphorylation was induced within 5-10 min and then decreased progressively with time. The phosphorylation of p44/42 MAPK observed within 5 min reached a peak at 10 min and then returned to basal level by 6 h. The phosphorylation of SAPK/JNK also occurred rapidly within 5 min and followed a similar time course as AKT but to a lesser extent. The transcription factors, NFB p65 and IRF-3, related to TLR4 activation, were phosphorylated within the first 10 min of HSP90␣ application and maintained for at least 6 h. (Intracellular calcium is required for NFB p65 phosphorylation; see supplemental Fig. S8.) Because clathrin-mediated endocytosis is the major pathway of EGFR internalization, we also examined the co-localization of internalized receptor with clathrin-HC (heavy chain). As shown in Fig. 4E, P-EGFR (Tyr-1068) was internalized with FIGURE 4. Tyrosine phosphorylation of EGFR and partial co-localization of TLR4 and EGFR in glioblastoma cells. A, extracellular HSP90␣ induces EGFR tyrosine phosphorylation. U87 cells were unstimulated (control (Cont.)) or stimulated with EGF or HSP90␣ and 1 g/ml LPS for 15 min; EGFR phosphorylation at Tyr-1068 was analyzed by Western blot. In a few cases, U87 cells were pretreated for 60 min with the PKC␦ inhibitor, rottlerin (10 M). The membrane was stripped and reprobed for total ErbB1 subtype. On the bottom of the figure, values indicate the relative optical density (OD) of P-EGFR versus ErbB1 analyzed by densitometry (mean Ϯ S.D., n ϭ 2). B, flow cytometry analysis of cell surface expression of EGFR (ErbB1), TLR4, and TLR2 in unstimulated (basal) and stimulated U87 cells with HSP90␣ or LPS (with EGF) for 15 min. Gray filled histograms are isotype IgG controls (n ϭ 3). C, U87 cells were unstimulated (control) or stimulated with HSP90␣ for 15 min and double-stained for TLR4 and EGFR (ErbB1). In the merged panel, yellow labeling indicates co-localization of both receptors. Confocal optical sections of 0.4 m thickness are shown (n ϭ 3). D, phosphorylation of Akt, p44/p42 MAPK, and SAPK/JNK (left panel) and of the transcription factors, NFB p65 and IRF-3 (right panel). U87 cells were exposed to 6 g/ml HSP90␣ for the indicated periods, and whole cell lysates were subjected to immunoblot analysis using the indicated specific antibodies recognizing phosphorylated or total forms of proteins (n ϭ 4). E, co-localization of HSP90␣-induced P-EGFR and clathrin-HC. Serum-starved U87 cells, stimulated with 6 g/ml HSP90␣ for 30 min, were fixed and double-stained for the phosphorylated form of EGFR (Tyr-1068) (red) and the clathrin-Alexa Fluor 488 (green). In the merged panel, yellow labeling indicates co-localization of clathrin and P-EGFR. Confocal optical sections of 0.4 m thickness are shown (n ϭ 3). DiC, differential interference contrast.

External HSP90␣ Transactivates EGFR
clathrin after 30 min of cell stimulation with HSP90␣. No co-localization with P-EGFR and caveolin-1 was observed (data not shown). Furthermore, chlorpromazine, a well known inhibitor of clathrin-mediated endocytosis, significantly blocked the internalization of P-EGFR and the MAPK signaling induced by HSP90␣ (see supplemental Fig. S6). Thus, clathrin-mediated internalization is essen-tial for a sustained HSP90␣-mediated EGFR signaling. Because cholesterol depletion by M␤CD also blocks calcium signaling and endocytic processes, our findings support the view that HSP90␣-induced endocytosis recruits at least two kinds of membrane rafts: one including TLR4 and the second including EGFR. Altogether, our data suggest that signaling via the PKC␦/c-Src pathway is involved in FIGURE 5. shRNA-mediated depletion of TLR4 inhibits HSP90␣-induced Ca 2؉ signaling in U87 cells. A, protein lysates from both normal human astrocytes (NHA) and glioblastoma U87 cells were probed with anti-TLR4 (HSC70 as loading control). TLR4 expression in normal human astrocytes and U87 cells was shown, and cell stimulation with 1 g/ml LPS for 60 min was performed. Flow cytometry analysis of TLR4 expression in transfected U87 cells (white histogram) when compared with IgG control (gray filled histogram; n ϭ 3) as performed. B, effects of HSP90␣, LPS (1 g/ml), TSG (2 M), and HSP90␣ plus EGF on [Ca 2ϩ ] i in TLR4 shRNA (grey curves) and control shRNA (black curves) transfected U87 cells (average of 6 cells; n ϭ 3). C, consequences of shRNA-mediated depletion of TLR4 and EGFR (ErbB1) onto the cell invasion. The quantitative effect of HSP90␣ (6 g/ml) and/or EGF (10 ng/ml) on the closure of wound after a 12-h treatment is depicted in the table (mean Ϯ S.D., n ϭ 2; *, p valuesϽ0.05 versus control). The following cells were used as control in this specific experiment; cultures of U87 cells and normal human astrocytes (Lonza Walkersville, Inc.) were grown to confluency in DMEM plus 10% fetal bovine serum (Lonza). For lentiviral transduction particles and transfections, human TLR4 shRNA, EGFR (ErbB) shRNA and control lentiviral particles were used according to the manufacturer's recommendations (Santa Cruz Biotechnology). FEBRUARY (Fig. 5A) abolished the Ca 2ϩ response to HSP90␣ and LPS but did not affect the TSG-induced increase in [Ca 2ϩ ] i (Fig. 5B). This down-regulation induced a significant reduction in both the release of internal Ca 2ϩ stores and the secondary external Ca 2ϩ influx in response to EGF plus HSP90␣. Thus, TLR4 may be required for HSP90␣-induced [Ca 2ϩ ] i increase in U87 cells. The contribution of TLR4 and EGFR (ErB1) to HSP90␣-mediated cell invasion is shown in Fig. 5C. In shRNA-mediated knockdown of TLR4 or EGFR, the cell migration was severely impaired in response to HSP90␣. Thus, in agreement with our previous results, TLR4 is required for HSP90␣ effects. To rule out a role of contamination of recombinant HSP90␣ with LPS, cells were preincubated with Polymyxin B, an endotoxin-binding agent (10 M; 30 -60 min). HSP90␣ without contaminant LPS was always able to amplify the EGF-induced cell migration (Fig. 6A). This cell pretreatment reduced but did not inhibit HSP90␣-induced transient rise in [Ca 2ϩ ] i (Fig. 6B, left panels). Preincubation of U87 cells with anti-TLR4-blocking antibody (27) abolished the increase in [Ca 2ϩ ] i and the cell migration induced by HSP90␣ (Fig. 6, right panels) and decreased EGFinduced cell responses (Fig. 6, right panels). Altogether, HSP90␣, through a direct or indirect activation of TLR-4, upregulates EGF-induced cell responses.

DISCUSSION
Cell surface expression of HSP90␣ has been associated in various tumor types to their migration capabilities and metastatic potential (5)(6)(7)(8). Here, we show in glioblastoma U87 cell line that extracellularly applied HSP90␣ activates a signal transduction pathway involving TLR4 to amplify the effects of EGF on cell shape and cell migration. More specifically, extracellular HSP90␣ triggers EGFR phosphorylation at Tyr-1068 through a pathway that can be inhibited by rottlerin and PP2, suggesting a role of PKC␦ and c-Src (Fig. 7). Extracellular HSP90␣ also transiently increases cytosolic [Ca 2ϩ ] in U87 cells and converts calcium response induced by EGF from a transient to an oscillatory pattern. Ca 2ϩ mobilization induces ATP release that amplifies the cell response through a suramin-inhibited pathway. Altogether, these results identify the additive effect of extracellular HSP90␣ and EGF on glioma cell migration capabilities in human brain tumors.
TLR4-mediated transactivation of EGFR has been identified in epithelial cells (8,19). For example, this receptor crosstalk has been observed in response to Helicobacter pylori protein in gastric cells (19). Here, we show that extracellular HSP90␣ can induce TLR4-mediated EGFR transactivation and its downstream effects, i.e. cell migration and Ca 2ϩ signaling, in U87 cells. Furthermore, shRNA-mediated downregulation of TLR4 or its neutralization with a specific antibody prevents HSP90␣-induced activation of EGFR. Co-localization of EGFR, TLR4-ligand complexes, and extra-cellular HSP90␣ in specific membrane domains could favor an interaction between TLR4 and EGFR that would allow EGFR activation. Depletion of cholesterol by methyl-␤-cyclodextrin disrupted this co-localization and impaired HSP90␣induced Ca 2ϩ rise, even in the presence of EGF. M␤CD could prevent the activation of PKC␦, Src, and Lyn kinases, as demonstrated in other cell types (19,28,29). Interestingly, EGFR and TLR4 were observed to co-localize in pseudopodia or filopodia of non-stimulated glioblastoma cells whose polarization and stabilization determines cell motility (30). Upon cell exposure to HSP90␣, EGFR and TLR-4 were rapidly internalized with kinetics similar to that induced by their respective ligands, EGF and LPS.
HSP90␣ appears to trigger EGFR transactivation through a pathway that is sensitive to both PKC␦ and c-Src inhibitors (Fig. 7). Because PKC isozymes do not directly phosphorylate proteins at tyrosine residues, c-Src would be an intermediate kinase between PKC␦ and EGFR (35,38). The expression and activity of PKC isozymes are highly elevated in gliomas and glioma cell lines (31)(32)(33), and PKC inhibitors markedly reduce glioma cell proliferation (34). PKC␦ isoform was shown to phosphorylate several kinases including c-Src (35), ADAM, and Lyn (36). Cellular Src and EGFR act synergically to facilitate the progression of human brain tumors (35,37,38), whereas ADAM and Lyn kinases regulate activation of MMPs, proHB-EGF shedding, and EGFR tyrosine phosphorylation (20,36). Inhibition of extracellular HSP90␣ was shown to decrease MMP-2 activity, blocking invasiveness (8). The EGFR (ErbB1) kinase inhibitor, AG1478, only attenuated HSP90␣induced transactivation of EGFR at Tyr-1068 in U87 cells, at a dose known to completely abrogate EGF-induced phosphorylation of EGFR. This suggests that HSP90␣-induced EGFR Tyr-1068 phosphorylation through PKC␦ and c-Src is upstream of EGFR kinase activity. This result agrees with similar studies that used phorbol 12-myristate 13-acetate to transactivate the EGFR (35). Intracellular Ca 2ϩ modulates the proliferative effects of EGF (16,39,40), and complex Ca 2ϩ oscillations are associated with human astrocytoma cell migration (25,41,42). A major component of Ca 2ϩ homeostasis is the ER, which stores micromolar levels of Ca 2ϩ under control of Ca 2ϩ -ATPases known as sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCAs), which maintain a low cytosolic [Ca 2ϩ ]. Specific inhibition of sarcoplasmic reticulum Ca 2ϩ ATPase activity with TSG results in ER Ca 2ϩ depletion and consequent opening of storeoperated calcium channels located in the plasma membrane. These channels elevate cytosolic [Ca 2ϩ ], and under physiological conditions, permit refilling of the ER Ca 2ϩ pool. The magnitude of the increase in [Ca 2ϩ ] i evoked by an agonist is thus dependent on the size of stores, leakiness of the ER membrane for Ca 2ϩ , and activity of store-operated calcium channels. Here, we show that extracellular HSP90␣ transiently increases cytosolic [Ca 2ϩ ] in glioblastoma U87 cells and converts calcium response induced by EGF from a transient to an oscillatory pattern. Similar calcium oscillations induced by EGF have been previously reported in astrocytes in response to glutamate (42), pro-inflammatory cytokines (41), or thimerosal, which directly activates the inositol 1,4,5- Our data are summarized in the box and linked to pathways (outside the box) previously proposed by other authors for cell migration. Specific inhibitors used in this work are written in grey. Upon HSP90␣ stimulation of TLR4, PKC␦ phosphorylates c-Src to activate EGFR in rafts. Assemblage of EGFR, c-Src, and phospholipase C␥ (PLC␥) might form a platform for the EGFR, resulting in the release of ATP from U87 cells. The released ATP, through its P2 receptors, triggers intracellular Ca 2ϩ waves, leading to the activation of ADAM protein(s), which cleaves pro-HB-EGF at the cell surface. HB-EGF in an autocrine and/or paracrine fashion binds EGFR, amplifying signals into the intracellular signaling network. The initial activation of PKC␦ could also involve Lyn, MMP2/9, and HB-EGF release. FEBRUARY 4, 2011 • VOLUME 286 • NUMBER 5 triphosphate receptor and induces calcium release from internal stores (43). These stimuli also induce release of ATP by astrocytes. Elevation of cytosolic Ca 2ϩ is a major regulator of nucleotide export in astrocytes, either by channel-mediated influx or through vesicle exocytosis. Rho activation and other changes in cytoskeletal organization have also been implicated in ATP release (44). Here, we show an elevated cytosolic Ca 2ϩ and an increase in F-actin content by stimulation of EGFR in U87 cells (supplemental Fig. S1). Stimulation of EGFR was reported to activate RhoA/Rho-associated protein kinase signaling, which could synergize with Ca 2ϩ mobilization to induce ATP release in response to HSP90␣ plus EGF.

External HSP90␣ Transactivates EGFR
Altogether, our data indicate that the ability of extracellular HSP90␣ to increase cytosolic [Ca 2ϩ ] in tumoral astrocytes and to induce complex Ca 2ϩ oscillations in the concomitant presence of EGF probably facilitates the migration of these cells. It remains to be demonstrated whether amplification of this pathway in tumor cells accounts for the relationship established between extracellular HSP90␣ and tumor cell invasion and metastasis formation.


s2fieldsofstudy:
Biology
Agricultural And Food Sciences
Medicine

text:
Characterization of 13 multi-drug resistant Salmonella serovars from different broiler chickens associated with those of human isolates

Background Salmonella are frequently isolated from chickens and their products. Prevalent serogroups and serovars of Salmonella as well as their genotypes and antibiograms were determined for cloacal samples from 1595 chickens. To understand the possible serovar and H antigens for transmission between chicken and human, serovars and their H antigens of 164 chicken and 5314 human isolates were compared. Results Prevalence of Salmonella differed among chicken lines and ages. Chicken and human isolates belonged mainly to serogroup B, C1, C2-C3, D, and E. 13 serovars and 66 serovars were identified for chicken and human isolates respectively. The common serovars for chicken and human isolates were S. Typhimurium, S. Enteritidis, S. Albany, S. Derby, and S. Anatum and shared common H1 antigens "g complex; i; e,h; and z4,z24" and H2 antigens "1 complex and -". In human isolates, H1 antigen "i" and H2 antigen "-" were common in all serogroups. In chicken, antimicrobial susceptibility differed among serogroups, serovars and three counties. All isolates were susceptible to cefazolin and ceftriaxone, but highly resistant to ampicillin, chloramphenicol, flumequine, streptomycin, sulfamethoxazole-trimethoprim, and tetracycline. Except those isolates of serogroup C1 of Chick group and serogroup G, all isolates were multi-drug resistance. Only S. Kubacha, S. Typhimurium, S. Grampian, and S. Mons were resistant to ciprofloxacin and/or enrofloxacin. Conclusion In chicken, prevalent serogroups and serovars were associated with chicken ages, lines and regions; and flouroquinolone-resistant and MDR isolates emerged. H1 antigens "g complex and i" and H2 antigens "1 complex and -" might be important for transmission of Salmonella between chicken and human.

Background
S. Enteritidis and S. Typhimurium, as two main zoonotic and broad-host-range pathogens that cause human salmonellosis, have been frequently isolated from poultry and their products [1][2][3][4][5][6][7][8]. Prevalence of Salmonella differs between layers and broilers [9,10]. Factors influencing the prevalence of chicken-associated Salmonella are feeds and growth environment [11], transportation process [12,13], and chick sources [14]. Moreover, age-associated prevalence has been reported in layers, maximal prevalence at 18 weeks before egg production and gradually decreases with aging [15]. In broiler the prevalence differed depending on sale sites from 17.9% in slaughterhouses [16] and up to nearly 100% in the open markets and supermarkets [17].
Appearance of monophasic variants such as in S. Typhimurium [4,5,12:1:-] [18,19] increases the problem in serotyping. Therefore, molecular methods have been developed to differentiate the serovars based on the nucleotide sequence variations in flagellar structural genes fliC and fljB [20][21][22] and PFGE analysis [15,23,24]. Prevalent serovars differ between chickens and ducks [25] and are associated with chicken lines and geographic area [15,[25][26][27]. In Taiwan, we reported that Salmonella serogroup C1 and B, especially S. Typhimurium, were predominant Salmonella in duck and geese [7,8]. In another study of duck, the prevalence of Salmonella was 4.6% and S. Potsdam, S. Dusseldorf, and S. Indiana were the predominant serovars [28]. Therefore, we analyzed the prevalence of Salmonellae among different chicken sources and determined serotypes by PFGE analysis first, followed by traditional agglutination test of each genotype. After characterizing antibiograms and genomic variations in chromosome and plasmid of chicken isolates, flagellar antigens of chicken and human isolates were compared to understand the common antigens possibly for transmission of Salmonella between human and chicken.

Sample collection and enrichment
Totally 1595 chickens of 1-year-old broiler breeder, 1-day-old chicks (Chick) and 9-week-old chickens (NHC) of Taiwan broiler chicken, 1-year-old layers and 3-week-old broiler were sampled by 108C Amies Agar Gel -Single plastic swab (Copan Diagnostic Inc. Murrieta CA 92562 USA) from cloaca of each chicken fed at different farms in Chiayi of Taiwan from 2002 to 2003. Layers and broilers were fed in commercial cage and house farm respectively. The sampled swabs were grown in 9 mL of gram-negative broth (GN, Difco 0486) at 37°C for 24 h. Over-night GN bacterial broth was streaked on xylose lysine deoxycholate (XLD, Difco 0788) plates, which were incubated at 37°C for 24 h. Black colonies were further examined by biochemical tests including triple sugar iron agar (TSI), Christensen's urea agar (URE), Simmons' citrate agar (CIT), sulfide-indole-motility medium (SIM), Voges-Proskauer medium (VP), Moller's ornithine decarboxylase medium (ORN), lysine iron agar (LIA) and mobility-indole-ornithine agar (MIO) purchased from Merck (Taiwan). At least two positive isolates from each plate were maintained on brain heart infusion agar (BHIA). In addition, Salmonellae from 9-week-old NHC in Tainan (36 isolates) and Pintung (30 isolates) at same period were also analyzed.

Serogroup and serotype identification
Salmonella-positive isolates were further serogrouped by the slide agglutination test with the use of O-antigen antiserum and serotyped by the tube agglutination test with the use of H-antigen antisera. Both antisera were purchased from Difco (Becton Dickinson Co., Franklin Lakes, NJ, USA). In addition, 5314 Salmonellae were collected from 19 medical centers and district hospitals located throughout the countries from 2003 to 2005 and serotyped in the Salmonella Reference Laboratory of Centers for Disease Control (CDC), Department of Health, Taiwan, with antisera purchased from S&A Reagents Lab (Bangkok, Thailand), Denka Seiken (Tokyo, Japan), Statens Serum Institut (Copenhagen, Denmark), and a local biotech company, LTK Biolaboratories (Taoyuan, Taiwan). Phase induction was performed using a paper-bridged method developed in the laboratory of Taiwan CDC [29].

Plasmid and genotype analysis
Plasmid DNA pattern was determined by Kado and Liu method [31] and purified plasmid DNA was subjected to gel electrophoresis with 0.6% SeaKem GTG agarose (Cambrex Bio Science Rockland, Inc, Rockland, ME, USA) at 50 V for 2.5 hrs. Genotypes of all isolates were determined by PFGE analysis with restriction endonuclease XbaI digestion. The procedure of PFGE analysis was described earlier [32]. The digested DNA was separated by CHEF Mapper XA system (BioRad, Hercules, California, USA) in 0.5 × TBE at 14°C for 22 h with Auto-Algorithm model of 30-600 kb, 6 V/cm, switching interval 4.0-70.0 sec. The genotypes were defined as 3 band differences between two isolates [33].

Prevalent serogroups and serovars among chicken lines and locations
Prevalence of Salmonella differed between chicken lines (0% for layer vs 0.3% for breeder broiler and 11.3% for broiler) and ages from 10.3% for Chick and 3.8% for NHC of Taiwan broiler chicken (Table 1). 164 Salmonella isolates belonged to serogroup C1, B, D, C2-C3, E, and G in the decreasing order and the number of serogroups differed among 3 counties. Further, region-specific serogroups were identified as serogroup G in Chiayi, serogroup D in Tainan, and serogroup C2-C3 and E in Pintung (Table 1). In Chiayi, age-associated serogroups were found for serogroup C1 Salmonella in Chick group and serogroup B and G in NHC group (Table 1).
164 Salmonella isolates were firstly examined for their genotypes by XbaI-PFGE analysis ( Figure 1) and further isolates of each genotype were serotyped by traditional agglutination method. In total, 18 PFGE patterns belonged to 13 serovars (Table 2)     Antibiogram of each isolate was determined by the resistance to antimicrobials ampicillin (A), chloramphenicol (C), ciprofloxacin (Ci), ceftriaxone (Cr), cefazolin (Cz), enrofloxacin (En), flumequine (Ub), streptomycin (S), sulfamethoxazole-trimethopriem (Sxt), tetracycline (T). The association of resistance type with antibiogram was the followings: resistance type A for antibiogram S, B for Ub, C for UbS, D for ST, E for SxtUb, F for CSTUb, G for ASSxtTUb, H for ACSSxtT, I for CSSxtTUb, J for ACSSxtTUb, K for ACEnSSxtTUb, L for ACCiEnSxtTUb, and M for ACCiEnSSxtTUb. b PFGE genotypes was determined by 3 band differences between two isolates [ Figure 1, [32]].  Table 2). d NT: non-typable Chick and S. Grampian from NHC in serogroup C1, and S. Albany in serogroup C2-C3 (Table 2).

Antimicrobial susceptibility
All isolates were susceptible to CZ and Cro. In contrast to resistance only to streptomycin for 77 S. Choleraesuis isolates in Chick group and two isolates of serogroup G, all isolates were MDR (Table 3). Serogroup B, C2-C3 and E were highly resistance to A, C, S, Sxt, T and Ub. However, serogroup D was relatively low in resistance to above antimicrobials. Serogroup and serovars isolated from broiler and NHC group differed in resistance to three quinolone antimicrobials. Except serogroups E and G, all serogroups, were nearly 100% resistance to Ub and only serogroups B and C1 were resistant to En and Ci (Table 3). Among 164 isolates, we only found 4 Enresistant S. Mons and 13 En and Ci-resistant isolates including 2 S. Kubacha isolates, 2 S. Typhimurium isolates, and 1 S. Typhimurium var. Copenhagen isolates of serogroup B and 8 S. Grampian isolates of serogroup C1 (Table 2). Importantly, near 40% of isolates from Pintaung were resistant to En and Ci. According to resistance to 9 antimicrobials tested, 13 antibiograms differed among serogroups and serovars (Table 2 and 3). Highest drug-resistant types L with antibiogram ACCiEnSxtTUb and M with antibiogram ACCiEnSSxt-TUb were only found in serogroup B and C1 of NHC group from Pintung mostly and Tainan. Salmonella genomic island (SGI) related ACSSuT resistance was found in serogroup B, C2 and E. Resistance to antimicrobials tested varied among 3 counties (Table 3 and Additional file 1: Table S1). Highest resistance was found in isolates from Pintung, followed by Tainan, and Chiayi and lowest Sxt resistance rate was observed in isolates from Tainan.

Plasmid profile analysis
Based on plasmid number and size determined by gel electrophoresis and plasmid size marker 50 kb and 90 kb of OU7526, in total 19 plasmid profiles were identified and the plasmid profiles and their number differed among serogroups and serovars (Additional file 2: Table  S2; Additional file 3: Figure S1). Among 13 serovars, S. Albany, S. Blockley, S. Havana, and S. Redba as well as few isolates of S. Choleraesuis, S. Enteritidis, and S. Typhimurium lacked plasmid. All other serovars harbored at least one plasmid and differed in plasmid profile.

Serovar association between chicken and human isolates
S. Albany, S. Anatum, S. Choleraesuis, S. Derby, S. Enteritidis, and S. Typhimurium were in common for 13 chicken serovars and 66 human serovars and other 7 serovars of chicken isolates were not or barely observed in human (Table 2, 4 and 5). Total serovar number of each serogroup decreased from serogroup C1, B, C2, E to D for human isolates ( Table 4). Despite of the presence of 66 serovars, there were only presence of 11 H1 antigens including b, c, d, j, k, r, y, eh, g-complex, and z-complex and 5 H2 antigens including -, z 6 , lw, 1-complex, and en-complex (Table 4). Common antigens in all serogroups were "i" for H1 antigen: and "-" for H2 antigen. In compared the chicken and human isolates from Taiwan, United Kingdom and United States, the common serovars were S. Typhimurium, S. Enteritidis, S. Anatum, and S. Derby with common antigens of . "g complex; i; z 4 ,z 24 ; and e,h" for H1 antigen and "and 1 complex" for H2 antigen (Table 5).

Discussion
As one of main pathogen to cause foodborne diseases, Salmonella has been frequently reported among different animal sources, especially more divergent Salmonella serovars found in chickens [34]. With the limited serovars in 164 chicken isolates, serogroups C2, D, E and G were restricted in one county and serogroup B and C1 were found in all three counties (Table 2), suggesting possibly that serogroup B and C1 isolates may be more adapted to chicken. In human isolates, we found that the serovar number in each serogroup were not associated positively with the serogroup prevalence, such as highest serovar number in low prevalent serogroup C1 vs lower serovar number in high prevalent serogroup B and serogroup D (Table 4). These results imply that serogroup C1 may occasionally infect human isolates. Further, serovars are determined by flagellins: H1 and H2 antigens encoded by fliC and fljB. As one of the most important immunogens, flagellin interacts with the toll-like receptor 5 (TLR5) to activate NFB pathway and proinflammatory genes to regulate innate and adaptive immune system [35][36][37][38]. However, aflagellar serovars S. Pullorum and S. Gallinarum cause more severe infection than flagellar serovars in chicken because of aflagellar S. Typhimurium could avoid the TLR5 regulation of IL-1β expression and polymorphonuclear cell infiltration in gut [39]. Such evasion of TLR5 is critical for survival of flagellar bacteria at muscos [40]. [In the present study, we found that i of H1 antigen and lack of H2 antigen were the common antigens for all serogroups in human isolates (Table 4). However, in comparing 13 chicken serovars and 66 human serovars of this study with serovars of chicken and human isolates from UK and USA, only S. Enteritidis, S. Typhimurium, S. Albany, S. Derby, S. Anatum and S. Havana were common in both hosts (Table 5). However, these serovars shares same antigens: g complex; i; and z 4 ,z 24 of H1 antigen and 1 complex and -of H2 antigens (Table 5), implying these antigens may be important for Salmonella transmission between chicken and human. Prevalent serogroups and serovars are related to chicken lines (Table 1) [9,10] and ages [15]. In layer, agerelated prevalence was reported earlier [15] and no Salmonella was isolated from 1-year-old layers in the present study (Table 1). Such age-associated clearance may be due to stronger antigen-specific T-cell response in older chicken [41] and not related to B-cell response [42]. Age-related serovars were also identified in Taiwan broiler chickens (Table 2). Almost all isolates were S. Choleraesuis and non-typable Salmonella (possibly monophasic S. Choleraesuis) of serogroup C1 in Chick group and S. Mons of serogroup B in NHC group ( Table 2). As swine-adapted pathogen, S. Cholearesuis has seldom reported from chicken. However, S. Choleraesuis in 1-day-old chicks may be contaminated from  the hatchery, particular from eggshell membrane; in which S. Typhimurium, not S. Choleraesuis, is main serovar [43]. If highly invasive S. Choleraesuis could infect chicks and use the chicken as reservoir, it will lead to a public problem of circulating such high invasive serovar in animals. In broiler, prevalence of Salmonella differed between chicken parts (2.36% for legs and 4.25% for breasts of broiler) [19]. Further, prevalent serovars differ between sampling sources e.g. the S. Anatum and S. Rissen in chicken meat [44] and S. Blockley, S. Hadar and S. Bredeney in the cecal samples (24). Several methods have been developed to differentiate clinical isolates. In this study, PFGE patterns almost matched serotypes, although S. Albany and S. Havana appeared multiple genotypes with highly similar banding patterns (Table 2). Therefore, PFGE typing is a useful tool to assist serotyping of Salmonella isolates before doing traditional serotypes [2,27]. In contrast to PFGE type, plasmid analysis is the most convenient method for subtyping [15,45]. In this study, plasmid variations were more diverse than genomic variations; however, S. Albany and S. Havana with highly genomic variations lacked plasmid (Table 2). These results may imply that recent evolution of Salmonella might be mainly through plasmid acquisition to introduce beneficial genes for host serovar to survival.
Antimicrobial susceptibility of Salmonella can be used to monitor drug abuse in different regions (Table 2) [46] and animal sources [44,47]. Early study reported that Salmonella from chicken, not from human, pig and cattle, was less resistance to A, C, and Sxt [47]. Nevertheless, resistance to T was frequently found in chicken isolates [48]. Since discovery of ACSSuT-resistant region in SGI of S. Typhimurium DT104 [49], variations within SGI and complex integron In104 change the antimicrobial resistance [50]. In this study, our chicken isolates were highly resistant to antimicrobials A, C, S, Sxt, T and Ub (Table 3). These results imply that S. Albany, S. Anatum, S. Grmpian, S. Hissar, S. Kubacha, S. Mons, and S. Typhimurium with resistance types from H to M may be derived from misuse of antimicrobials or due to presence of SGI and/or integron [51]. Mechanism to develop En and Ci resistance is due to mutation in quinolone-resistance determining region or expression of   efflux pump [52]. Earlier, fluoroquinolone-resistant Salmonella was seldom reported in poultry's isolates worldwide [10,44,47,48]. Until recently, resistance to similar fluoroquinolones: En and Ci has been reported from chicken in Spain [16]. In contrast to same prevalence of resistance to En and Ci in swine and human isolates [32], we found that resistance rate to En was higher than that of Ci (Table 2). However, En and Ci resistant isolates were only found in few serovars of serogroups B and C1 and mainly in Pintung area (Table 3). These results indicate that possibly En was misuse in Pintung county to induce resistance in prevalent serovars.

Conclusion
13 chicken serovars were identified and differed in drug resistance and prevalence associated with chicken lines, ages and regions. Five serovars were common between these chicken serovars and 66 human serovars Additional file 1: Table S1. Association of antibiograms with serogroups among three counties. Antibiograms differed among three counties and serogroups.
Additional file 2: Table S2. Plasmid profiles of serovars in each serogroup. Plasmid profiles determined by size and number was associated with serotypes.
Additional file 3: Figure S1. Representative plasmid profiles of Salmonella isolates collected from chickens. Plasmid size and number of each representative plasmid profile was determined by Kado-Liu method and standard plasmid size of 50 kb and 90 kb plasmid of OU7526.


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Prevalence of the Intestinal Flukes Haplorchis taichui and H. yokogawai in a Mountainous Area of Phongsaly Province, Lao PDR

Phongsaly Province, located in the northernmost area of Lao PDR, was previously suggested to be endemic for the liver fluke Opisthorchis viverrini infection. To confirm, or rule out, this suggestion, the Phonxay village in the Khoua District, Phongsaly Province, was selected for a survey. Ten volunteers (8 men and 2 women aged 31-57 years) who consumed raw freshwater fish and had gastrointestinal troubles were treated with a single dose of praziquantel (40 mg/kg) and pyrantel pamoate (10 mg/kg) and purged with magnesium sulfate to recover any worm parasites. Eight of the 10 volunteers expelled 1 or more species of trematodes, nematodes, or cestodes (worm positive rate; 80%). The worms were morphologically identified as H. taichui (861 worms from 8 people), H. yokogawai (59 from 6 people), Phaneropsolus bonnei (1 from 1 person), Trichostrongylus sp. (2 from 2 people), Ascaris lumbricoides (2 from 1 person), Enterobius vermicularis (11 from 3 people), and Taenia saginata (1 strobila with scolex from 1 person). The results indicate that the mountainous area of Phongsaly Province, Lao PDR, is not endemic for the liver fluke but endemic for intestinal flukes, in particular, Haplorchis taichui and H. yokogawai.

dsabane, Nathoune, and Phoxayneau) of the Khoua District, Phongsaly Province, the northernmost area of Lao PDR bordering southern China and northwest Vietnam (Fig. 1). The fecal examination results for 269 schoolchildren are shown in Table 1 (not shown in [5]). The prevalence of small trematode eggs was 27.6-57.4% (av. 48.7%) in addition to the high prevalence of soil-transmitted nematodes ( Table 1). The egg positive rates were not significantly different (P > 0.01) between men and women, or between different age groups (data not shown). Unless adult worms are recovered, it is not possible to determine whether the eggs are of the liver fluke, intestinal fluke, or both, because their eggs are very similar in size and shape. Therefore, in the present study, we attempted to determine the kinds of flukes through recovery of adult worms from residents of Phonxay village, a mountainous area of Khoua District, which is located in the southern part of Phongsaly Province, Lao PDR ( Fig. 1).
We visited Phonxay village, Khoua District, in December 2008 and explained our purpose of visit to the village leader. Ten volunteers, consisting of 8 men and 2 women aged 31-57 years who frequently consumed raw freshwater fish and experienced at times gastrointestinal troubles such as diarrhea and abdominal discomfort, were recruited randomly. After obtaining informed consent from each person, without preliminary fecal examination for helminth eggs, they were treated with a single oral dose of 40 mg/kg praziquantel (Distocide � , Shinpoong Pharm. Co., Seoul, Korea) and 10 mg/kg pyrantel pamoate (Hangzhou Minsheng Pharm. Group, Hangzhou, China), and then purged with 30 g magnesium sulfate. Whole diarrheic stools passed 4-5 times were pooled individually and processed as previously described [7]. Worms were collected using a glass pipette and then washed in water. Some were fixed in 10% formalin under cover slip pressure, acetocarmine-stained, and morphologically identified using a light microscope. This study was performed under the agreement of Korea-Laos International Collaboration Project on Parasite Control in Lao PDR (2007)(2008)(2009)(2010)(2011).
These results suggest that mountainous areas of Phongsaly Province, Laos are not endemic for the liver fluke but are endemic for intestinal flukes, in particular H. taichui and H. yokogawai.
The possibility for failure of praziquantel treatment and magnesium salt purgation to expel O. viverrini worms within a short time (5-6 hr) after treatment may be raised. However, we think that such possibility is low because, in our previous studies, the same protocol was applied and substantial numbers of O. viverrini worms were expelled in the diarrheic stools [4,[7][8][9]. In the present study, we could not collect even a single specimen of O. viverrini. We regret that we were unable to examine freshwater fish caught from the streams nearby the village. To confirm that there is no O. viverrini in this area, surveys of snails and fish are essential. In addition, surveys of H. taichui and H. yokogawai infections in intermediate hosts in this area (in particular, freshwater fish, the source of human infections) are urgently needed to establish preventive and control strategies against these intestinal fluke infections.
The results of the present study again clearly demonstrated that fecal examination is not enough to understand the real status of trematode infections or to determine the species of trematodes endemic in certain areas. This issue has been pointed out in Vientiane Municipality and Saravane Province [7], Savannakhet Province [8], and Khammouane Province [9], Laos. In Vientiane Municipality, where small trematode egg positive rate of some riverside people was 53.3% [7], the worms expelled after chemotherapy and purgation were a mixture of several different species of trematodes, including O. viverrini, H. taichui, H. pumilio, H. yokogawai, Centrocestus caninus, P. molenkampi, and P. bonnei. Of the 1,656 fluke specimens recovered from 18 persons [7], O. viverrini comprised of 62.9% (1,041 worms) and 6 other species of trematodes comprised 37.1% (615 worms). Similar results were reported in Savannakhet Province, where people were mixed-infected with approximately 50% O. viverrini and 50% minute intestinal flukes [8,10].
The term 'small trematode eggs' should be used when trematode eggs of 20-30 mm in length are detected in fecal examinations of people in southeast Asia, including Laos.  [11][12][13][14] should be included among the list of differential diagnosis. Worm collection after chemotherapy and purgation is essential to draw a precise diagnosis. However, it is tedious, not feasible, and impracticable as a routine procedure. Therefore, other diagnostic techniques, using ultrastructural, molecular, or genetic characteristics, should be developed for differential diagnosis of small trematode eggs in fecal samples.  57M  305  28  0  1  0  1  0  335  42M  302  9  0  0  0  7  0  318  56M  95  15  0  0  0  3  0  113  48M  78  4


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Molecular evolution of the crustacean hyperglycemic hormone family in ecdysozoans

Background Crustacean Hyperglycemic Hormone (CHH) family peptides are neurohormones known to regulate several important functions in decapod crustaceans such as ionic and energetic metabolism, molting and reproduction. The structural conservation of these peptides, together with the variety of functions they display, led us to investigate their evolutionary history. CHH family peptides exist in insects (Ion Transport Peptides) and may be present in all ecdysozoans as well. In order to extend the evolutionary study to the entire family, CHH family peptides were thus searched in taxa outside decapods, where they have been, to date, poorly investigated. Results CHH family peptides were characterized by molecular cloning in a branchiopod crustacean, Daphnia magna, and in a collembolan, Folsomia candida. Genes encoding such peptides were also rebuilt in silico from genomic sequences of another branchiopod, a chelicerate and two nematodes. These sequences were included in updated datasets to build phylogenies of the CHH family in pancrustaceans. These phylogenies suggest that peptides found in Branchiopoda and Collembola are more closely related to insect ITPs than to crustacean CHHs. Datasets were also used to support a phylogenetic hypothesis about pancrustacean relationships, which, in addition to gene structures, allowed us to propose two evolutionary scenarios of this multigenic family in ecdysozoans. Conclusions Evolutionary scenarios suggest that CHH family genes of ecdysozoans originate from an ancestral two-exon gene, and genes of arthropods from a three-exon one. In malacostracans, the evolution of the CHH family has involved several duplication, insertion or deletion events, leading to neuropeptides with a wide variety of functions, as observed in decapods. This family could thus constitute a promising model to investigate the links between gene duplications and functional divergence.

Background
The study of the evolutionary history of arthropods is a challenging field of research, as they constitute the large majority of known metazoan species and exhibit an extreme diversity of body plans and physiology. Nowadays, evo-devo approaches largely contribute to this research area [reviewed in [1]], but the evolution of peptide hormone families directly involved in physiological adaptations remains poorly investigated in arthropods, especially when compared to vertebrates. The Crustacean Hyperglycemic Hormone (CHH) family could constitute a model of choice to address this question, as CHH family peptides are well known in decapod crustaceans, where they do play major roles in many physiological processes. Since decapods were the first invertebrates where a neuroendocrine mechanism was discovered, they have been extensively used in comparative endocrinology studies, and CHH has become the archetype of a neuropeptide family including around 150 members to date [reviewed in [2][3][4]]. CHH family peptides are 72 to 78 amino acids long and are, based on structural features, divided into two sub-families [5]. Type I peptides, the CHHs sensu stricto, are pleiotropic hormones involved in the regulation of energetic and ionic metabolism and, in addition, can also exert an inhibitory effect on molting and reproduction [reviewed in [6]]. By a tissue-specific alternative splicing mechanism, CHH genes produce a second peptide, devoid of hyperglycemic activity, named CHH-L (for CHH long isoform), which may be involved in osmoregulation [7][8][9]. Type II peptides, namely the molt-inhibiting hormones (MIHs), the vitellogenesis-inhibiting hormones (VIHs) and the mandibular organ-inhibiting hormones (MOIHs), are functionally more specialized than CHHs. Characterized through their inhibitory actions on molting and reproduction, they never elicit hyperglycemia [reviewed in [2,10]].
The evolution of CHHs has been recently discussed in decapods [11] and we have intended here to extend the evolutionary study to the entire family, by including data from non-decapod taxa. Little is known about the CHH family outside decapods, but it may be present in the entire Arthropoda, and even in all Ecdysozoa. Indeed, a CHH and a VIH were isolated from an isopod crustacean [12,13], and neuropeptides sharing the same structural signature, the ion transport peptides (ITPs), were also characterized in several insect species [reviewed in [14]]. As for decapod CHHs, ITP genes produce an ITP-L isoform by alternative splicing, whose function remains elusive. To go further, heterologous immunostainings revealed the occurrence of CHH-like peptides in two branchiopods [15], a myriapod [16] and a chelicerate [17], and a gene was found in the genome of the nematode Caenorhabditis elegans, which encodes a putative peptide exhibiting a CHH family structural signature [3]. To increase our knowledge on the CHH family in the taxa cited above, we have conducted an in silico study on available genomic sequences from arthropods and nematodes. Exon-intron patterns of the genes were determined and sequences of putative CHH family peptides were deciphered. In addition, CHH family sequences were obtained by molecular cloning in a collembolan and in a branchiopod, whose phylogenetic positions are currently uncertain. Indeed, the grouping of crustaceans and hexapods in a Pancrustacea clade is widely accepted but the relationships inside Pancrustacea remain controversial. Recent molecular phylogenies based on mitochondrial genes ( Figure 1A) notably yield a paraphyletic Hexapoda, with Collembola separated from Insecta, and Branchiopoda as a sister-group to Malacostraca and Cephalocarida [18][19][20]. These phylogenies largely differ from those based on nuclear genes ( Figure 1B), where Hexapoda appears monophyletic, and Branchiopoda are generally sister-group to Hexapods [21][22][23].
Newly characterized sequences were included in CHH family sequence datasets, which were then used to build phylogenies of this family and to investigate its evolutionary history. Because of the reduced size of these datasets, a test was conducted to assess which of the two phylogenetic hypotheses presented Figure 1 was more strongly supported by our data, and could be further used as a basis to elaborate evolutionary scenarios of the CHH family genes.

CHH family peptides characterized in Daphnia magna and Folsomia candida
In the branchiopod Daphnia magna, RT-PCRs conducted with degenerate primers designed from insect ITP transcripts and followed by 3'-5'RACE produced two complete cDNAs. Their length was 1053 and 1198 bp, respectively, and they encoded peptide precursors composed of a signal peptide, a precursor-related peptide and a mature hormone. The two cDNA sequences were identical except for a 145 bp stretch (position 383 to 527) present in only one of them, suggesting the  [19]. (B) Phylogeny built from the analysis of nuclear protein-encoding genes EF-1a, EF-2 and POLII [23].
occurrence of an alternative splicing during mRNA processing. The short cDNA encoded a 72-residue peptide with a C-terminus end putatively amidated, which was named ion transport peptide (ITP) as it appeared to be related to insect ITPs (Figure 2A). On the other hand, the long cDNA encoded a non-amidated peptide of 79 amino acids, only differing from ITP after the fortieth residue, which was named ITP-L ( Figure 2B).
The two complete cDNA sequences obtained from the collembolan Folsomia candida led to similar results: two transcripts of different lengths (1307 and 2018 bp) encoded prepro-peptides which only differed after the fortieth residue of the mature peptide. The 72 residue amidated peptide, translated from the short cDNA, was named ITP and the 82 residue non-amidated peptide, translated from the longest cDNA, was named ITP-L ( Figure 2A and 2B).

Sequences characterized in silico
For the insects Anopheles gambiae, Apis mellifera, Culex pipiens, Nasonia vitripennis and Pediculus humanus, nucleotide sequences encoding for ITP or ITP-L, which have not been recovered by automatic annotation programs were deduced from the gene sequence following alternative splicing rules. A gene encoding putative ITP and ITP-L isoforms was also found in the genome of another water flea, Daphnia pulex. At the present time, both ITP (72 or 73 residue amidated peptides) and ITP-L (79 to 90 residue non-amidated peptides) sequences are available from 12 insect and two branchiopod species (Figure 2A and 2B).
In the genome of the deer tick Ixodes scapularis (Chelicerata), a gene was found that encoded only one mature ITP-like peptide. In addition, cDNA and peptide sequences of another ITP-like peptide were found,  Table 2.
during database mining, in the American dog tick Dermacentor variabilis. These peptides are 74 and 75 residue long, respectively, and are not amidated on their C-terminal end (Figure 2A).
In the Caenorhabditis elegans genome, the gene ZC168.2 has been described as putatively encoding a CHH family peptide of 83 amino acid residues [3]. In the present study, a second gene (C05E11.6) which may encode another putative CHH family peptide was found in the C. elegans genome: this gene encodes a mature peptide of 97 amino acids containing six cysteyl residues whose positions are similar to those found in CHH family hormones ( Figure 3). The corresponding peptides ZC168.2 and C05E11.6 were named ITP 1 and ITP 2, respectively, based on their similarity with insect ITPs and without predicting any physiological function. Similar ITP 1 and ITP 2 peptides were also deduced from the genomes of Caenorhabditis briggsae and Caenorhabditis remanei. Finally, single CHH family peptides were deduced from the genomes of the Malaysian filarial worm Brugia malayi and the whipworm Trichinella spiralis. These putative peptides were 102 and 60 amino acids long, respectively ( Figure 3).
For each of the genes described here, exon-intron structures were determined and compared to those already described ( Figure 4). These data were further used, in addition to phylogenetic information, to infer evolutionary scenarios of the CHH family.

Phylogeny of CHH family peptides in Pancrustacea
In the maximum-likelihood (ML) phylogeny of pancrustacean CHH family peptides ( Figure 5), type I and type II peptides of decapods were both arranged in monophyletic sub-groups. Type I and II peptide clades were also found in the phylogeny built by Bayesian inference (BI) based on the DNA dataset. The "type I" clade, supported by a bootstrap value of 95% in the ML analysis (posterior probability, or pp, of 1.00 in BI), contained all CHHs from decapods, with the CHH from the isopod Armadillidium vulgare at their base. Similarly, the "type II" clade, supported by a bootstrap value of 100% (pp of 1.00 in BI), contained all MIHs, VIHs and MOIHs from decapods, with the isopod VIH at their base. This topology suggests that the type I and type II peptide genes constitute paralogous lineages, which have appeared by duplication of an ancestral gene before the radiation of malacostracans (taxa that contains decapods and isopods). Inside the type I clade, the clade of Dendrobranchiata (peneid shrimps) CHHs encoded by three-exon genes is at the base of the other decapod CHHs, encoded by four-exon genes, found both in Dendrobranchiata and Pleocyemata (the remaining decapods). It suggests that a duplication of a CHH gene has occurred before the radiation of decapods and the split between Dendrobranchiata and Pleocyemata, but this node was strongly supported only in the BI analysis (pp of 1.00, bootstrap value of 45% in the ML analysis).
A clade supported by a bootstrap value of 76% (pp of 0.99 in BI) was positioned at the base of the type I and type II clades and contained all ITPs from insects, together with the peptides characterized in the collembolan Folsomia candida and the branchiopods Daphnia magna and Daphnia pulex, which were located at the base. The type III sub-family was thus created to designate peptides contained in this clade.
Since the topology of this clade was only weakly supported (notably with insects appearing paraphyletic), amino acid and DNA datasets containing less taxa but more characters were used for phylogenetic inference. When an outgroup (DNA or amino acid sequences    Table 2. For taxa in which the genes have been sequenced, the number of exons (three, four or five) is indicated in a black circle after the name of the species. A four-exon pattern was assigned for taxa in which two peptides arising by alternative splicing have been described from Ixodes scapularis) was included in these datasets, a structure of two monophyletic groups was found within the ingroup: a "type I" clade (supported by a bootstrap value of 100% in the ML analysis based on the amino acid dataset and by posterior probabilities of 1.00 in BI analyses using either amino acid or DNA dataset) and a "type III" clade (bootstrap value of 48% in the ML analysis of the amino acid dataset, posterior probabilities of 0.79 and 0.98 in BI analyses based on the amino acid and the DNA datasets, respectively). Since the four exons used in these datasets exist only in the ingroup species, the inclusion of the outgroup resulted in a loss of phylogenetic signal. Then, the outgroup was removed for subsequent analyses and the resulting trees were rooted using the structure in two clades previously found.
Phylogenies built from these 23-taxa datasets using ML and BI methods were similar ( Figure 6). Insects appeared monophyletic, albeit with moderate support, and the relationships within the insect clade were not resolved. Yet, the collembolan Folsomia candida was positioned at the base of the insects, thus forming a moderately supported monophyletic hexapod clade, and branchiopods were a sister group to hexapods, with high support.

Evolutionary scenarios of the CHH family genes
Using the 23-taxa sequence datasets, a KH test was conducted to decide which of the two competing phylogenetic hypotheses for arthropods presented in Figure 1 could be used to build an evolutionary scenario of the CHH family. This test clearly supported (P < 0.0001) the tree based on nuclear genes, with a monophyletic Hexapoda sister-group to Branchiopoda ( Figure 1B), versus the tree based on mitochondrial genes ( Figure 1A). Then, the ML phylogeny of CHH family peptides ( Figure 5) was superimposed on a species tree in which Branchiopoda were sister-group to Hexapoda, to create a topology accounting for gene duplications in Pancrustacea ( Figure 7A). In nematodes, a gene duplication was also inferred in the Caenorhabditis phylum, as two paralogs were found in genomes of Caenorhabditis species. In the most parsimonious scenario of  gene structure evolution based on this topology, CHH family genes of Ecdysozoa evolved from an ancestral twoexon gene, with nine partial insertion or deletion events ( Figure 7A).In this scenario, CHH family genes of arthropods have all evolved from a three-exon gene, and the four-exon gene structure would have appeared independently twice, in a common ancestor of decapods and in a common ancestor of insects and branchiopods.
Because of the weak support, in the ML phylogeny (Figure 5), at the node indicating the duplication which led to the two paralogous CHH genes found in Dendrobranchiata (with three or four exons), an alternative topology was considered for pancrustacean CHH family genes, where the CHH gene duplication would have occurred only in the Dendrobranchiata phylum ( Figure 7B). In that case, the most parsimonious scenario implied eight intron or exon insertion/deletion events, and the exon duplication leading to the four-exon gene structure would have occurred only once.
Regardless of the evolutionary scenario considered, the putative ancestral gene was likely composed of two exons and a phase 2 intron interrupting the codon of the amino acid residue following the fourth cysteine of the mature peptide. Such an intron was found in every known CHH family gene, except the ITP2 genes in Caenorhabditis species and the ITP gene of Trichinella spiralis ( Figure 4).
Dibasic processing sites used for precursor maturation by prohormone convertase then likely appeared independently twice, in the common ancestor to arthropods on one hand and in the common ancestor to the Caenorhabditis genus on the other hand, leading to the occurrence in these lineages of precursor related peptides (blue boxes in Figures 4 and 7), such as the CHHprecursor related peptides (CPRPs) of decapods.

Evolution of the CHH family in decapods and functional implications
Since the discovery of neuroendocrine factors within the eyestalks of decapods seventy years ago [24][25][26], structural and functional data about CHH family peptides in these crustaceans have been accumulated. While our knowledge increased, it became more and more difficult to propose a model of endocrine regulation applying to all decapods. Indeed, CHH family peptides are not identical in all decapod groups, and their function varies with the species. This diversity prompted us to investigate the evolutionary history of these peptides.
In decapods, many gene duplications have occurred, leading to two main paralogous lineages (type I and type II peptides) and to a large polymorphism of CHH family peptides inside these lineages (Figure 7). For example, two clusters containing each at least 7 different CHH genes were identified in the shrimp Metapenaeus ensis [27]. A paradigm in evolutionary biology is that gene duplication represents the major mechanism for emergence of new functions, as one of the two copies is potentially freed from selective pressure and will accumulate more mutations during evolution [28,29]. In the CHH family, the functional divergence observed between the two paralogous lineages could result from a subfunctionalization [30,31], rather than from a neofunctionalization. In fact, the ancestral gene may have encoded a pleiotropic hormone, like current CHHs, and after duplication of this gene the type II paralogous lineage may have evolved faster to peptides with a CHH-like structure, but devoid of hyperglycemic activity and conversely exhibiting more specialized activities, such as inhibition of molting or vitellogenesis (for MIHs and VIHs, respectively). The faster evolution rate of type II peptide genes may explain the loss of phylogenetic signal observed in MIHs, VIHs and MOIHs sequences. Indeed, the topology of the "type II" clade ( Figure 5) is not congruent with the phylogeny of decapods, whereas it is for the "type I" clade, as shown in earlier work [11]. This clearly impedes the elucidation of evolutionary relationships between type II peptides. The only paralogous lineages clearly identified so far are the MIHs and MOIHs of Cancer crabs, as the MOIH genes seem to have appeared from a duplicated MIH gene only in this genus [32]. In lobsters, there is no MIH identified so far but a VIH instead, whose gene may have evolved from a MIH gene subjected to a reduced selective pressure, as molt inhibition is exerted in this taxa by a CHH isoform [33].

Evolutionary history "outside decapods"
A high number of gene duplications were observed in decapods only. So far, the only paralogous lineages identified outside decapods are the "ITP1" and "ITP2" genes found in Caenorhabditis species (Figure 7), and only one CHH family peptide gene could be found in genomes of the other nematodes, the insects and the deer tick. In the water flea Daphnia pulex, one ITP gene was also rebuilt from whole genome shotgun (WGS) sequences but other WGS sequences sharing sequence identity with the ITP gene have been found. However, it is unclear whether these sequences correspond to another CHH family peptide coding gene or to a pseudo-gene: this gene would have a peculiar structure, with exon III lacking, and the prepro-peptide putatively encoded would lack the dibasic processing site found in every CHH or ITP precursors and the 6 following residues of the mature peptide as well. Yet, although only one ITP and one ITP-L cDNA have been cloned in Daphnia magna, recently published EST data suggest that both genes may be expressed in Daphnia pulex [34], but EST and genomic sequences do not match (only 85% identity between deduced amino acid sequences). To include these data in an evolutionary scenario, genomic and mRNA sequences should be characterized by molecular cloning.
This apparent lack of paralogous lineages outside decapods raises the question of a possible conservation of function between the ITPs of insects and the related peptides found in branchiopods, chelicerates and nematodes. Yet, functional studies are too scarce to address this question. In insects, the effect of ITP on ionic and water balance was only demonstrated in locusts [35,36] and nothing is known about the function of ITP-like peptides in chelicerates or nematodes. In branchiopods, only one gene expression study on sex determination in Daphnia magna has indicated that CHH family peptides may inhibit methyl farnesoate synthesis by the mandibular organ [37]. It is noteworthy that several CHH family peptides are known to play such a role in decapods [38][39][40]. It will be informative to determine whether Daphnia peptides exhibit other typical "decapod" functions such as hyperglycemia or ecdysteroid synthesis inhibition, or if they also possess an anti-diuretic activity, as shown for insect ITPs. A similar question about another neuropeptide family, the RPCH/AKH family, has recently found a partial answer. Like for the CHH family, the putative RPCH (red pigment-concentrating hormone) of Daphnia magna shares more sequence similarity with AKHs (adipo-kinetic hormones) of insects than with RPCH of decapod crustaceans [41]. In a heterologous bioassay, this peptide was able to trigger a mobilization of lipid reserves when injected into the green shield bug Nezara viridula whereas it did not provoke pigment migration when injected into the shrimp Palaemon pacificus [42]. Although heterologous bioassays must be considered cautiously, such results suggest that this peptide could have an adipokinetic function in water fleas (like in insects) rather than a pigment-concentration function (like in decapods).

Emergence of the four-exon genes
One important step in the evolution of the CHH family is the emergence of the four-exon genes found in decapods, branchiopods and insects. In these genes, exons III and IV encode peptide sequences of similar lengths, with two cystein residues in the same positions, thus suggesting that the supplementary exon has appeared by tandem duplication of another exon. At first glance, it seems more likely that this duplication has occurred only once, in a common ancestor of Pancrustacea ( Figure 7B). In all these taxa, two peptides are produced by alternative splicing; one peptide (CHH or ITP) is 72 or 73 amino acids long and is always amidated on its C-terminal end, and the other peptide (CHH-L or ITP-L) is slightly longer and is never amidated. Moreover, the expression of these two isoforms seems to be similar in decapods and insects; in the crab Carcinus maenas and the caridean shrimp Macrobrachium rosenbergii, CHH transcripts were mainly found in neurons of the central nervous system (more precisely in the X-organ located in the eyestalk) whereas CHH-L transcripts were found in neurons of the peripheral nervous system [7,8]; in the moths Manduca sexta and Bombyx mori and the locust Schistocerca americana, ITP-immunoreactive neurons were only found in the brain, whereas ITP-L-immunoreactive ones were found along the ventral nervous chain. ITP-L immunoreactivity was also detected in the brain, but was weak and restricted to cell bodies, thus suggesting that ITP-L was not secreted from this tissue [43].
Yet, even if the hypothesis of an independent emergence of the fourth exon ( Figure 7A) appears unlikely, it should not be rejected, as a similar exon duplication also occurred in a Drosophila ancestor, leading to the five-exon gene found in Drosophila melanogaster [44].

Controversy about pancrustacean phylogeny
Given the relative short length of the sequences used, the objective of this study was not to build a phylogeny of pancrustaceans using CHH family peptides, but to choose the appropriate phylogeny to infer evolutionary scenarios of the CHH family. The KH test carried out with our datasets indicated a congruence of the data with recent molecular phylogenies of pancrustaceans based on nuclear gene sequences [21][22][23] and not with those based on mitochondrial genes [18][19][20]. Moreover, the robustness and quality of the signal contained in mitochondrial gene sequences for arthropod phylogenetics have recently been questioned [45,46]. Supplementary taxa will have to be represented within CHH family datasets to confirm this result, but studying the evolutionary history of this family may help to select synapomorphies to support concurrent phylogenetic hypotheses about pancrustacean relationships. For example, the presence of paralogous type I and type II peptides could constitute a trait shared by malacostracans and their sister-groups.

Conclusion
The evolutionary scenarios proposed in this study suggest that genes encoding Crustacean Hyperglycemic Hormone family peptides of Ecdysozoa evolved from an ancestral two-exon gene. In the malacostracan lineage (including decapods), the evolution of the CHH family has involved numerous duplications, insertions and deletions of exons or entire genes, leading to the wide variety of functions displayed by the encoded neurohormones in decapods. This neuropeptide family could thus constitute a promising model to investigate the evolutionary forces at the root of the functional divergence of duplicated genes. Outside malacostracans, the number of gene duplications seems to have been lower, which may reflect different evolutionary pathways. At present, CHH family peptides characterized outside decapods are still scarce, and this work constitutes a first step in a wider quest. In the future, the evolutionary scenarios elaborated here will be completed and amended, as CHH family peptides will probably be characterized in new taxa. The 38 genome sequencing projects currently under way in ecdysozoan species will be of valuable help as soon as they are completed, to enlarge our view on the evolution of this multigenic family.

Biological material and RNA extraction
Daphnia magna and Folsomia candida (strain TO) specimens were supplied by the BIOEMCO laboratory and the Ecology & Evolution laboratory, respectively, both located at the Ecole Normale Supérieure (Paris, France). Total RNA was extracted from 10 mg of whole animals, using SV Total RNA Isolation System (Promega, Madison, WI). cDNA was synthesized from 300 ng of total RNA using 200 U of M-MLV Reverse Transcriptase (Promega), 20 pmoles of each dNTP and 30 pmoles of an Oligo(dT) primer. This synthesis was performed at 42°C for 1 hour, followed by an inactivation step at 70°C for 15 min.

PCR amplification and cloning
First, a central region of putative ITP precursor cDNAs was amplified using degenerate primers DgnITP-Up (5'-CTTCGAYATCMAKTGYAARGG-3') and DgnITP-Down (5'-RAGRCAWCCTTTGAAGWATG-3') designed from the alignment of known insect ITP cDNAs. The reaction mixture contained 1.5 U of GoTaq® DNA Polymerase (Promega), 5 pmoles of each dNTP and 10 pmoles of each degenerate primer. Amplifications were conducted with a denaturation step at 94°C for 3 min, followed by 40 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, and a final extension step at 72°C for 10 min. After their purification with the Wizard® SV Gel and PCR Clean-Up System (Promega), products were cloned using pGEM®-T Easy Vector and JM109 Competent Cells (Promega). Plasmids were purified using the Wizard® Plus SV Minipreps DNA Purification System (Promega), and their sequencing was performed by Cogenics -Genome Express (Meylan, France).

3' and 5'RACE
To create cDNAs which contain a synthetic adaptor either at the 5' or 3' end, total RNA was reverse-transcribed using the SMART™ RACE cDNA Amplification Kit (Clontech, Moutain View, CA). For PCR amplifications, two sets of primers, specific of Daphnia magna and Folsomia candida, were designed from the partial cDNA sequences obtained by RT-PCR. These primers are listed in Table 1. First amplifications were performed using the adaptor primer supplied with the kit (Universal Primer Mix) and specific primers Up1 (for 3'RACE) or Down1 (for 5'RACE). Then, nested PCRs were performed on 1/150 th of the first PCR products, between the Nested Universal Primer from the kit and specific primers Up2 or Down2. All reverse-transcription and PCR amplification steps were carried out following the protocol supplied in the kit. RACE products were cloned and sequenced as described above.

Database mining
A search for CHH family peptide sequences from arthropods and nematodes was performed with the BLAST program at the NCBI website http://blast.ncbi. nlm.nih.gov/Blast.cgi. The non-redundant protein sequences (nr) database was mined by blastp algorithm using Schistocerca gregaria ITP or the putative peptide ZC168.2 of Caenorhabditis elegans as query sequences for arthropods and for nematodes, respectively. Corresponding mRNA sequences were also collected, and aligned with genome sequences to determine the exonintron structure of the genes. Sequences of genes encoding putative CHH family peptides of Daphnia pulex (Branchiopoda), Ixodes scapularis (Chelicerata), Caenorhabditis remanei and Brugia malayi (Nematoda) were assembled from whole genome shotgun (WGS) sequences available in the NCBI Trace Archives database, by assembling trace reads with CLC Genomics Workbench software (CLCbio, Aarhus, Denmark).

Phylogenetic analyses
Datasets containing mature CHH family peptide sequences and the corresponding DNA sequences  Table 2). Alignments were performed manually with Se-Al v2.0a11 http://tree.bio.ed. ac.uk/software/seal/, and after removal of N-terminal and C-terminal unconserved residues, the dataset contained 56 taxa and 73 characters. The DNA dataset contained 49 taxa and 219 characters. DNA and amino acid datasets including CHH-L and ITP-L sequences were also created. For the 23 taxa in which such peptides are known, the amino acid sequence of the mature CHH-L or ITP-L (encoded by exons II and III of the gene) was concatenated with the C-terminal sequence of CHH or ITP (encoded by exon IV of the gene). Alignments were performed manually and 109 unambiguously aligned residues were conserved in the dataset. The DNA dataset contained 327 characters.
At first, DNA and translated amino acid sequences of Ixodes scapularis ITP-like were included in these datasets, to be used as an outgroup in order to determine the global branching order in the ingroup. Since the gene encoding this peptide lacks exon III in the outgroup taxon, corresponding characters were replaced with "?" symbols in the alignments. Because of the loss of phylogenetic signal brought by the inclusion of the outgroup, it was removed from datasets for subsequent phylogenetic analyses, but the global structure previously found was kept.
Phylogenetic reconstructions were carried out using Bayesian inference and maximum likelihood. Bayesian analyses were performed with MrBayes 3.1.2 with four chains of 10 6 generations, trees sampled every 100 generations and burn-in value set to 20% of the sampled trees. We checked that standard deviation of the split frequencies fell below 0.01 to insure convergence in tree search. Protein sequences were analyzed with a mixed amino acid model [47], and DNA sequences were considered with an evolutionary model designed for coding sequences and taking the genetic code into account [48][49][50].
Maximum likelihood reconstructions were carried out only on amino acid sequences. For both datasets, the WAG+I+G substitution model [51] was determined as the best-fit model of protein evolution by ProtTest 1.3 [52]http://darwin.uvigo.es/software/prottest_server.html, following Akaike Information Criterion. Rate heterogeneity was set at four categories. The gamma distribution parameter and the proportion of invariable sites were

Evolutionary scenarios
The two phylogenetic trees shown in Figure 1 were used as competing hypotheses to assess if one was more strongly supported by the 23-taxa sequence datasets. This was done via a KH test using a maximum likelihood criterion [54]. This allowed us to choose a "species tree" on which the "gene tree" could be super-imposed. The evolution of CHH family gene structures was analyzed using Mesquite 2.6 [55], under a parsimony framework. For this analysis, two alternate topologies depicting the evolution of the CHH family genes were created. All the known intron locations within CHH family genes were considered as characters and the presence/absence of intron in each location was coded 0 (absence) or 1 (presence) to create a character matrix.

CHH-L ABP88270
(1) Sequences deduced from genomic sequences or from the alternatively spliced product