title: Characterization of hemolymph phenoloxidase activity in two Biomphalaria snail species and impact of Schistosoma mansoni infection.
abstract: BACKGROUND
  Biomphalaria snails are the intermediate host of the blood fluke Schistosoma mansoni, which infect more than 67 million people in tropical areas. Phenoloxidase enzymes (POs), including tyrosinases, catecholases, and laccases, are known to play a role in the immune defenses of arthropods, but the PO activity present in Biomphalaria spp. hemolymph has not been characterized. This study was designed to characterize substrate specificity and reaction optima of PO activity in Biomphalaria spp. hemolymph as a starting point to understand the role of this important invertebrate enzyme activity in snail biology and snail-schistosome interactions.
  
  
  METHODS
  We used spectrophotometric assays with 3 specific substrates (L-tyrosine for tyrosinase, L-DOPA for catecholase, and PPD for laccase) and diethylthiocarbarmate (DETC) as specific PO inhibitor to characterize PO activity in the hemolymph of uninfected snails from two Biomphalaria species, and to determine the impact of the parasite Schistosoma mansoni on the PO activity of its B. glabrata vector.
  
  
  RESULTS
  We identified laccase activity in hemolymph from uninfected B. glabrata and B. alexandrina. For both species, the activity was optimal at 45 °C and pH 8.5, and located in the plasma. The K m and V max of PO enzymes are 1.45 mM and 0.024 OD.min(-1) for B. glabrata, and 1.19 mM and 0.025 OD.min(-1) for B. alexandrina. When the snail vector is parasitized by S. mansoni, we observed a sharp reduction in laccase activity seven weeks after snail infection.
  
  
  CONCLUSIONS
  We employed a highly specific spectrophotometric assay using PPD substrate which allows accurate measurement of laccase activity in Biomphalaria spp. hemolymph. We also demonstrated a strong impact of the parasite S. mansoni on laccase activity in the snail host.
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, P.O. Box 760549, San Antonio, TX, 78245, USA. winkal@txbiomed.org.
authors.forename: Winka
authors.initials: W
authors.lastname: Le Clec'h
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, P.O. Box 760549, San Antonio, TX, 78245, USA. tanderso@txbiomed.org.
authors.forename: Timothy J C
authors.initials: TJ
authors.lastname: Anderson
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, P.O. Box 760549, San Antonio, TX, 78245, USA. fcheval@txbiomed.org.
authors.forename: Frédéric D
authors.initials: FD
authors.lastname: Chevalier
mesh_terms: D000818:Animals; D001700:Biomphalaria / Q000201:enzymology* / Q000469:parasitology; D004050:Ditiocarb / Q000494:pharmacology; D006458:Hemolymph / Q000201:enzymology*; D006790:Host-Parasite Interactions; D006801:Humans; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010655:Phenylenediamines / Q000378:metabolism; D012550:Schistosoma mansoni / Q000502:physiology*; D012555:Schistosomiasis mansoni / Q000469:parasitology*; D013053:Spectrophotometry; D013696:Temperature
chemical_list: D010655:Phenylenediamines; D004050:Ditiocarb; D014442:Monophenol Monooxygenase; C029728:4-phenylenediamine