Weatherly L.M.,University of Maine
Journal of visualized experiments : JoVE | Year: 2013
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells(3-5). Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells(6), can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies(1). Originally published by Naal et al.(1), we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here. Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease(7-11), although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function(2). In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280 = 4,200 L/M/cm)(12). This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
News Article | April 15, 2016
A wireless leak detection system created by University of Maine researchers is scheduled to board a SpaceX rocket bound for the International Space Station this summer.
News Article | August 24, 2016
For the last 30 years, Drummond, professor of insect ecology at the University of Maine, has studied the biology, ecology, disease susceptibility and pesticide exposure of Maine's 275 native species of bees, as well as the millions of commercial honey bees annually trucked into the state to aid in crop pollination.
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were carried out using the TR146 buccal epithelial squamous cell carcinoma line32 obtained from the European Collection of Authenticated Cell Cultures (ECACC) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were routinely tested for mycoplasma contamination using mycoplasma-specific primers and were found to be negative. Prior to stimulation, confluent TR146 cells were serum-starved overnight, and all experiments were carried out in serum-free DMEM. C. albicans wild-type strains included the autotrophic strain BWP17 + CIp30 (ref. 33) and the parental strain SC5314 (ref. 34). Other C. albicans strains used and their sources are listed in Extended Data Tables 1 and 2. C. albicans cultures were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30 °C overnight. Cultures were washed in sterile PBS and adjusted to the required cell density. Antibodies to phospho-MKP1 and c-Fos were from Cell Signalling Technologies (New England Biolabs UK), mouse anti-human α-actin was from Millipore (UK), and goat anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies were from Jackson Immunologicals (Stratech Scientific, UK). Ece1p peptides were synthesized commercially (Proteogenix (France) or Peptide Synthetics (UK). ECE1 deletion was performed as previously described35. Deletion cassettes were generated by PCR36. Primers ECE1-FG and ECE1-RG were used to amplify pFA-HIS1 and pFA-ARG4 -based markers. C. albicans BWP17 (ref. 37), was sequentially transformed38 with the ECE1-HIS1 and ECE1-ARG4 deletion cassettes and then transformed with CIp10 (ref. 39), yielding the ece1∆/Δ deletion strain. For complementation, the ECE1 gene plus upstream and downstream intergenic regions were amplified with primers ECE1-RecF3k and ECE1-RecR and cloned into plasmid CIp10 at MluI and SalI sites. This plasmid was transformed into the uridine auxotrophic ece1Δ/Δ strain, yielding the ece1∆/Δ + ECE1 complemented strain. For generation of the ece1Δ/Δ + ECE1 strain, the CIp10-ECE1 was amplified with primers Pep3-F1 and Pep3-R1, digested with ClaI and re-ligated, yielding the CIp10 + ECE1 plasmid. This plasmid was transformed into the uridine auxotrophic ece1Δ/Δ strain, yielding the ece1Δ/Δ + ECE1 strain. All integrations were confirmed by PCR/sequencing and at least two independent isogenic transformants were created to confirm results. KEX1 deletion was performed exactly as the ECE1 deletion but using primers KEX1-FG and KEX1-RG for creating the deletion cassette. Fluorescent strains of ece1Δ/Δ and BWP17 were constructed as previously described40. Briefly, the ece1Δ/Δ and BWP17 strains were transformed with the pENO1-dTom-NATr plasmid. Primers used to clone and construct the ECE1 genes and intragenic regions are listed in Extended Data Table 4. Strains are listed in Extended Data Table 2. ECE1 promoter (primers 5′ECE1prom–NarI / 3′ECE1prom–XhoI) and terminator (5′ECE1term–SacII / 5′ECE1term–SacI) were amplified and cloned into pADH1-GFP. Resulting pSK-pECE1-GFP was verified by sequencing. C. albicans SC5314 was transformed with the pECE1-GFP transformation cassette38. Resistance to nourseothricin was used as selective marker and correct integration of GFP into the ECE1 locus was verified by PCR. Primers for cloning and validation are listed in Extended Data Table 4. Strains are listed in Extended Data Table 2. C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 μl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated for 5 min (65 °C) before subjected to 2× freeze-thawing. Lysates were clarified by centrifugation and the RNA precipitated with isopropyl alcohol/0.3 M sodium acetate by incubating for 1 h at −20 °C. Precipitated pellets were washed (2× 1 ml 70% ice-cold ethanol), resuspended in DEPC-treated water and stored at −80 °C. RNA integrity and concentration was confirmed using a Bioanalyzer (Agilent). RNA (500 ng) was treated with DNase (Epicentre) and cDNA synthesized using Reverse Transcriptase Superscript III (Invitrogen). cDNA samples were used for qPCR with EVAgreen mix (Bio&Sell). Primers (ACT1-F and ACT1-R for actin, ECE1-F and ECE1-R for ECE1 Extended Data Table 4) were used at a final concentration of 500 nM. qPCR amplifications were performed using a Biorad CFX96 thermocycler. Data was evaluated using Bio-Rad CFX Manager 3.1 (Bio-Rad) with ACT1 as the reference gene and t as the control sample. TR146 cells were lysed using a modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease (Sigma-Aldrich) and phosphatase (Perbio Science) inhibitors41, left on ice (30 min) and then clarified (10 min) in a refrigerated microfuge. Lysate total protein content was determined using the BCA protein quantitation kit (Perbio Science). 20 μg of total protein was separated on 12% SDS–PAGE gels before transfer to nitrocellulose membranes (GE Healthcare). After probing with primary (1:1,000) and secondary (1:10,000) antibodies, membranes were developed using Immobilon chemiluminescent substrate (Millipore) and exposed to X-ray film (Fuji film). Human α-actin was used as a loading control. DNA binding activity of transcription factors was assessed using the TransAM transcription factor ELISA system (Active Motif) as previously described41, 42. Serum-starved TR146 epithelial cells were treated for 3 h before being differentially lysed to recover nuclear proteins using a nuclear protein extraction kit (Active Motif) according to the manufacturer’s protocol. Protein concentration was determined (BCA protein quantitation kit (Perbio Science)) and 5 μg of nuclear extract was assayed in the TransAM system according to the manufacturer’s protocol. Data was expressed as fold-change in A relative to resting cells. Cytokine levels in cell culture supernatants were determined using the Performance magnetic Fluorokine MAP cytokine multiplex kit (Bio-techne) and a Bioplex 200 machine. The data were analysed using Bioplex Manager 6.1 software to determine analyte concentrations. Following incubation, culture supernatant was collected and assayed for lactate dehydrogenase (LDH) activity using the Cytox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) according to the manufacturer’s instructions. Recombinant porcine LDH (Sigma-Aldrich) was used to generate a standard curve. Quantification of C. albicans adherence to TR146 epithelial cells was performed as described previously43. Briefly, TR146 cells were grown to confluence on glass coverslips for 48 h in tissue culture plates in DMEM medium. C. albicans yeast cells (2 × 105) were added into 1 ml serum-free DMEM, incubated for 60 min (37 °C/5% CO ) and non-adherent C. albicans cells removed by aspiration. Following washing (3× 1 ml PBS), cells were fixed with 4% paraformaldehyde (Roth) and adherent C. albicans cells stained with Calcofluor White and quantified using fluorescence microscopy. The number of adherent cells was determined by counting 100 high-magnification fields of 200 μm × 200 μm size. Exact total cell numbers were calculated based on the quantified areas and the total size of the cover slip. C. albicans invasion of epithelial cells was determined as described previously43. Briefly, TR146 epithelial cells were grown to confluence on glass coverslips for 48 h and then infected with C. albicans yeast cells (1 × 105), for 3 h in a humidified incubator (37 °C/5% CO ). Following washing (3× PBS), the cells were fixed with 4% paraformaldehyde. All surface adherent fungal cells were stained for 1 h with a rabbit anti-Candida antibody and subsequently with a goat anti-rabbit-Alexa Fluor 488 antibody. After rinsing with PBS, epithelial cells were permeabilized (0.1% Triton X-100 in PBS for 15 min) and fungal cells (invading and non-invading) were stained with Calcofluor White. Following rinsing with water, coverslips were visualized using fluorescence microscopy. The percentage of invading C. albicans cells was determined by dividing the number of (partially) internalized cells by the total number of adherent cells. At least 100 fungal cells were counted on each coverslip. TR146 cells (105 per ml) seeded on glass coverslips in DMEM/10% FBS were infected with C. albicans (2.5 × 104 cfu per ml) in DMEM and incubated for 6 h (37 °C/5% CO ). Cells were washed with PBS, fixed overnight (4 °C in 4% paraformaldehyde) and stained with Concanavalin A-Alexa Fluor 647 in PBS (10 μg ml−1) for 45 min at room temperature in the dark with gentle shaking (70 r.p.m.) to stain the fungal cell wall. Epithelial cells were permeabilized with 0.1% Triton X-100 for 15 min at 37 °C in the dark, then washed and stained with 10 μg ml−1 Calcofluor White (0.1 M Tris-HCl pH 9.5) for 20 min at room temperature in the dark with gentle shaking. Cells were rinsed in water and mounted on slides with 6 μl of ProLong Gold anti-fade reagent, before air drying for 2 h in the dark. Fluorescence microscopy was performed on a Zeiss Axio Observer Z1 microscope, and 5 phase images were taken per picture. For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of C. albicans incubation on epithelial cells at an MOI of 0.01, cell media was removed and samples were fixed overnight at 4 °C with 2.5% (v/v) glutaraldehyde in 0.05 M HEPES buffer (pH 7.2) and post-fixed in 1% (w/v) osmium tetroxide for 1 h at room temperature. After washing, samples were dehydrated through a graded ethanol series before being critical point dried (Polaron E3000, Quorum Technologies). Dried samples were mounted using carbon double side sticky discs (TAAB) on aluminium pins (TAAB) and gold coated in an Emitech K550X sputter coater (Quorum Technologies Ltd). Samples were examined and images recorded using a FEI Quanta 200 field emission scanning electron microscope operated at 3.5 kV in high vacuum mode. Zebrafish infections were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01 at the University of Maine. To determine sample size, a power calculation was done for all experiments based on two-tailed t-tests in order to detect a minimum effect size of 0.8, with an alpha error probability of 0.05 and a power (1 – beta error probability) of 0.95. This gave a minimum number of 42 fish for each group. The fish selected for the experiments were randomly assigned to the different groups by picking them from a pool without bias and the groups were injected in different orders. No blinding was used to read the results. Ten to twenty zebrafish per group per experiment were maintained at 33 °C in E3 + PTU and used as previously described40. Briefly, 4 days post-fertilization (dpf) larvae were treated with 20 μg ml−1 dexamethasone dissolved in 0.1% DMSO 1 h before infection and thereafter. For tissue damage and neutrophil recruitment, individual AB or mpo:GFP fish (respectively) were injected into the swimbladder with 4 nl of PBS with or without 25–40 C. albicans yeast cells of ece1Δ/Δ-dTomato, ece1Δ/Δ + ECE1 + dTomato, ece1Δ/Δ + ECE1 + dTomato or BWP17-dTomato. For tissue damage, 1 nl of Sytox green (0.05 mM in 1% DMSO) was injected at 20 h post-infection into the swimbladder and fish were imaged by confocal microscopy at 24 h post-infection. For neutrophil recruitment, fish were imaged at 24 h post-injection. For synthetic peptide damage, AB or α-catenin:citrine44 fish were injected with 2 nl of peptide (9 ng or 1.25 ng per fish) or vehicle (40% DMSO or 5% DMSO) + SytoxGreen (0.05 mM in 1% DMSO) or SytoxOrange (0.5 mM in 10% DMSO) and the fish imaged by confocal microscopy 4 h later. Numbers of neutrophils and damaged cells observed were counted and tabulated for each fish. Live zebrafish imaging was carried out as previously described40. Briefly, fish were anaesthetized in Tris-buffered Tricaine (200 μg ml−1, Western Chemicals) and further immobilized in a solution of 0.4% low-melting-point agarose (LMA, Lonza) in E3 + Tricaine in a 96-well plate glass-bottom imaging dish (Greiner Bio-On). Confocal imaging was carried out using an Olympus IX-81 inverted microscope with an FV-1000 laser scanning confocal system (Olympus). Images were collected and processed using Fluoview (Olympus) and Photoshop (Adobe Systems). Panels are either a single slice for the differential interference contrast channel (DIC) with maximum projection overlays of fluorescence image channels (red-green), or maximum projection overlays of fluorescence channels. The number of slices for each maximum projection is specified in the legend of individual figures. Murine infections were performed under UK Home Office Project Licence PPL 70/7598 in dedicated animal facilities at King’s College London. No statistical method was used to pre-determine sample size. No method of randomization was used to allocate animals to experimental groups. Mice in the same cage were part of the same treatment. The investigators were not blinded during outcome assessment. A previously described murine model of oropharyngeal candidiasis using female BALB/c mice45 was modified to use for investigating early infection events. Briefly, mice were treated subcutaneously with 3 mg per mouse (in 200 μl PBS with 0.5% Tween 80) of cortisone acetate on days −1 and +1 post-infection. On day 0, mice were sedated for ~75 min with an intra-peritoneal injection of 110 mg per kg ketamine and 8 mg per kg xylazine, and a swab soaked in a 107 cfu per ml of C. albicans yeast culture in sterile saline was placed sublingually for 75 min. After 2 days, mice were euthanized, the tongue excised and divided longitudinally in half. One half was weighed, homogenized and cultured to derive quantitative Candida counts. The other half was processed for histopathology and immunohistochemistry. C. albicans-infected murine tongues were fixed in 10% (v/v) formal-saline before being embedded and processed in paraffin wax using standard protocols. For each tongue, 5-μm sections were prepared using a Leica RM2055 microtome and silane coated slides. Sections were dewaxed using xylene, before C. albicans and infiltrating inflammatory cells were visualized by staining using Periodic Acid-Schiff (PAS) stain and counterstaining with haematoxylin. Sections were then examined by light microscopy. Histological quantification of infection was undertaken by measuring the area of infected epithelium and expressed as a percentage relative to the entire epithelial area. TR146 epithelial cells were grown in 35-mm Petri dishes (Nunc) for 48 h before recordings at low cell density (10–30% confluence). Cells were superfused with a modified Krebs solution (120 mM NaCl, 3 mM KCl, 2.5 mM CaCl , 1.2 mM MgCl , 22.6 mM NaHCO , 11.1 mM glucose, 5 mM HEPES pH 7.4). Isolated cells were recorded at room temperature (21–23 °C) in whole cell mode using microelectrodes (5–7 MΩ) containing 90 mM potassium acetate, 20 mM KCl, 40 mM HEPES, 3 mM EGTA, 3 mM MgCl , 1 mM CaCl (free Ca2+ 40 nM), pH 7.4. Cells were voltage clamped at −60 mV using an Axopatch 200A amplifier (Axon Instruments) and current/voltage curves were generated by 1 s steps between −100 to +50 mV. Treatments were applied to the superfusate to produce the final required concentration, with vehicle controls similarly applied. Data was recorded using Clampex software (PClamp 6, Axon Instrument) and analysed with Clampfit 10. TR146 cells were grown in a 96-well plate overnight until confluent. The medium was removed and 50 μl of a Fura-2 solution (5 μl Fura-2 (Life Technologies) (2.5 mM in 50% Pluronic F-127 (Life Technologies):50% DMSO), 5 μl probenecid (Sigma) in 5 ml saline solution (NaCl (140 mM), KCl (5 mM), MgCl (1 mM), CaCl (2 mM), glucose (10 mM) and HEPES (10 mM), adjusted to pH 7.4)) was added and the plate incubated for 1 h at 37 °C/5% CO . The Fura-2 solution was replaced with 50 μl saline solution and baseline fluorescence readings (excitation 340 nm/emission 520 nm) taken for 10 min using a FlexStation 3 (Molecular Devices). Ece1 peptides were added at different concentrations and readings immediately taken for up to 3 h. The data was analysed using Softmax Pro software to determine calcium present in the cell cytosol and expressed as the ratio between excitation and emission spectra. tBLMs with 10% tethering lipids and 90% spacer lipids (T10 slides) were formed using the solvent exchange technique46, 47 according to the manufacturer’s instructions (SDx Tethered Membranes Pty Ltd, Sydney, Australia). Briefly, 8 μl of 3 mM lipid solutions in ethanol were added, incubated for 2 min and then 93.4 μl buffer (100 mM KCl, 5 mM HEPES, pH 7.0) was added. After rinsing 3× with 100 μl buffer the conductance and capacitance of the membranes were measured for 20 min before injection of Ece1 peptides at different concentrations. All experiments were performed at room temperature. Signals were measured using the tethaPod (SDx Tethered Membranes Pty, Sydney, Australia). Intercalation of Ece1 peptides into phospholipid liposomes was determined by FRET spectroscopy applied as a probe-dilution assay48. Phospholipids mixed with each 1% (mol/mol) of the donor dye NBD-phosphatidylethanolamine (NBD-PE) and of the acceptor dye rhodamine-PE, were dissolved in chloroform, dried, solubilized in 1 ml buffer (100 mM KCl, 5 mM HEPES, pH 7.0) by vortexing, sonicated with a titan tip (30 W, Branson sonifier, cell disruptor B15), and subjected to three cycles of heating to 60 °C and cooling down to 4 °C, each for 30 min. Lipid samples were stored at 4 °C for at least 12 h before use. Ece1 peptide was added to liposomes and intercalation was monitored as the increase of the quotient between the donor fluorescence intensity I at 531 nm and the acceptor intensity I at 593 nm (FRET signal) independent of time. CD measurements were performed using a Jasco J-720 spectropolarimeter (Japan Spectroscopic Co., Japan), calibrated as described previously49. CD spectra represent the average of four scans obtained by collecting data at 1 nm intervals with a bandwidth of 2 nm. The measurements were performed in 100 mM KCl, 5 mM HEPES, pH 7.0 at 25 °C and 40 °C in a 1.0 mm quartz cuvette. The Ece1-III concentration was 15 μM. Planar lipid bilayers were prepared using the Montal-Mueller technique50 as described previously51. All measurements were performed in 5 mM HEPES, 100 mM KCl, pH 7.0 (specific electrical conductivity 17.2 mS per cm) at 37 °C. Candida strains were cultured for 18 h in hyphae inducing conditions (YNB medium containing 2% sucrose, 75 mM MOPSO buffer pH 7.2, 5 mM N-acetyl-d-glucosamine, 37 °C). Hyphal supernatants were collected by filtering through a 0.2 μm PES filter, and peptides were enriched by solid phase extraction (SPE) using first C4 and subsequently C18 columns on the C4 flowthrough. After drying in a vacuum centrifuge, samples were resolubilized in loading solution (0.2% formic acid in 71:27:2 ACN/H O/DMSO (v/v/v)) and filtered through a 10 kDa MWCO filter. The filtrate was transferred into HPLC vials and injected into the LC-MS/MS system. LC-MS/MS analysis was carried out on an Ultimate 3000 nano RSLC system coupled to a QExactive Plus mass spectrometer (ThermoFisher Scientific). Peptide separation was performed based on a direct injection setup without peptide trapping using an Accucore C4 column as stationary phase and a column oven temperature of 50 °C. The binary mobile phase consisting of A) 0.2% (v/v) formic acid in 95:5 H O/DMSO (v/v) and B) 0.2% (v/v) formic acid in 85:10:5 ACN/H O/DMSO (v/v/v) was applied for a 60 min gradient elution: 0–1.5 min at 60% B, 35–45 min at 96% B, 45.1–60 min at 60% B. The Nanospray Flex Ion Source (ThermoFisher Scientific) provided with a stainless steel emitter was used to generate positively charged ions at 2.2 kV spray voltage. Precursor ions were measured in full scan mode within a mass range of m/z 300–1600 at a resolution of 70k FWHM using a maximum injection time of 120 ms and an automatic gain control target of 1e6. For data-dependent acquisition, up to 10 most abundant precursor ions per scan cycle with an assigned charge state of z = 2–6 were selected in the quadrupole for further fragmentation using an isolation width of m/z 2.0. Fragment ions were generated in the HCD cell at a normalized collision energy of 30 V using nitrogen gas. Dynamic exclusion of precursor ions was set to 20 s. Fragment ions were monitored at a resolution of 17.5k (FWHM) using a maximum injection time of 120 ms and an AGC target of 2e5. Thermo raw files were processed by the Proteome Discoverer (PD) software v220.127.116.118 (Thermo). Tandem mass spectra were searched against the Candida Genome Database (http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly22/current/C_albicans_SC5314_A22_current_orf_trans_all.fasta.gz; status: 2015/05/03) using the Sequest HT search algorithm. Mass spectra were searched for both unspecific cleavages (no enzyme) and tryptic peptides with up to 4 missed cleavages. The precursor mass tolerance was set to 10 p.p.m. and the fragment mass tolerance to 0.02 Da. Target Decoy PSM Validator node and a reverse decoy database was used for (q value) validation of the peptide spectral matches (PSMs) using a strict target false discovery (FDR) rate of <1%. Furthermore, we used the score versus charge state function of the Sequest engine to filter out insignificant peptide hits (xcorr of 2.0 for z = 2, 2.25 for z = 3, 2.5 for z = 4, 2.75 for z = 5, 3.0 for z = 6). At least two unique peptides per protein were required for positive protein hits. TransAM and patch clamp data were analysed using a paired t-test while cytokines, LDH and calcium influx data were analysed using one-way ANOVA with all compared groups passing an equal variance test. Murine in vivo data was analysed using the Mann–Whitney test. Zebrafish data was analysed using the Kruskal–Wallis test with Dunn’s multiple comparison correction. In all cases, P < 0.05 was taken to be significant.
More than 127 million pounds of lobsters were caught in 2012—an increase of approximately 18 million pounds over 2011, but prices for the larger-than-expected catch were the second-lowest on record—second only to the Great Depression year of 1939. The price drop caused hardship and anguish in the lobstering community and beyond. The price collapse occurred, in part, because frequent molting increased the portion of lobsters with soft- or new-shells compared to hard-shells on the market, and the increased supply was out of balance with processing availability and market demand. Maine lobsterman Dave Cousens states flatly, "We can't have another year like 2012 and not be prepared for it." A taste of things to come? Lobstering is big business in Maine. Preliminary numbers from Maine's 2014 commercial lobster harvest indicate that the industry brought more than $450 million into the state. Much of that value remains in the state, moving among individuals and businesses, supporting local economies. In general, lobstering communities are doing well. Since 1985, annual income from lobster fishing has increased by nearly 400 percent. In a changing climate, however, the industry's future may not be as rosy as its past. While Maine's lobster fishery has enjoyed success in recent decades, lobster abundance in southern New England—which includes Massachusetts, Connecticut, and Rhode Island—has declined by more than 70 percent. This population collapse has been attributed to disease and stresses related to rising ocean temperatures. Problems down the coast have been so severe that fishery managers have proposed closing the southern New England fishery. A collapse of the same magnitude in Maine would be devastating. Esperanza Stancioff, Extension Associate Professor with the University of Maine's Cooperative Extension and Maine Sea Grant, and her associates worked with lobstermen and women in South Thomaston to explore the impacts of changing fishing conditions on their bottom lines. Using the Vulnerability, Consequences, and Adaptation Planning Scenarios (VCAPS) participatory planning process (see link in sidebar, under Tools), the community discussed their vulnerability to future climate threats and the potential consequences of changes in the system. Exploring the group's combined knowledge helped them think strategically about ways to improve the fishery in order to build resilience—for example, if a future heat wave threatens a similar crash in the lobster trade. Researchers used the fishing community's specific experiences to create a social-ecological model that links together equations describing economics, biology, and fishing effort, so that fishermen could anticipate changes in prices and costs as the fishing season progressed. The model simulates what could happen as climate influences lobster biology, market forces vary in response to lobster availability, and the possibility for expenses to outweigh income in some circumstances. One scenario surprised lobstermen and women: it indicated the potential for a pronounced positive economic impact if the community would reduce their fishing efforts during times when waters are extremely warm and when early-molting lobsters are present. Reducing fishing effort in that circumstance, if implemented across the community, might be enough to help them avoid repeating the situation they experienced in 2012. The researchers helped the lobstermen better understand the economics of their fishing efforts under distinct climate conditions. In turn, the lobstermen helped researchers understand that an accurate valuation of resources is essential to gain their trust, especially if the goal is to influence harvesting schedules and intensities. The effort can also help fishery managers understand that they need an accurate view of fishermen's fixed and fluctuating costs and income in order to adapt management and policy instruments to address issues of a particular fishery. In the face of climate variability and change, and the tight linkage between ocean conditions and the financial health of the lobster industry, system dynamics models can be valuable as a discussion tool to further policy decisions that support the industry in becoming more flexible and adaptive to warming conditions. After seeing the direct effect of climate-related changes in the environment on their businesses, many lobstermen and women were eager to find strategies that could improve their resilience and help inform fisheries management policy. These strategies include activities such as engaging in marketing campaigns and collaborating with fishery managers. The VCAPS approach, combined with qualitative system dynamics modeling, provides strong support as they make these important environmental and economic decisions. Other fishing communities may also benefit from similar methods. More information: R. S. STENECK et al. Creation of a Gilded Trap by the High Economic Value of the Maine Lobster Fishery, Conservation Biology (2011). DOI: 10.1111/j.1523-1739.2011.01717.x