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Blood feeding in juvenile Paragnathia formica (Isopoda: Gnathiidae): biochemical characterization of trypsin inhibitors, detection of anticoagulants, and molecular identification of fish hosts

  • B. M. MANSHIP (a1), A. J. WALKER (a1), L. A. JONES (a1) and A. J. DAVIES (a1)

The 3 post-marsupial juvenile stages of the gnathiid isopod, Paragnathia formica, are haematophagous ectoparasites of fishes that may, in heavy infestations, cause host mortality. Protein digestion in fed stage 3 juveniles is accomplished by cysteine proteinases, but what bioactive compounds attenuate host haemostatic, inflammatory and immunological responses during feeding is unknown. Trypsin inhibitory activity and anticoagulant activity were detected in crude extracts of unfed P. formica stage 1 juveniles; fractionation of stage 1 crude extracts by ion exchange chromatography resulted in 3 preparations each displaying these bioactivities. Further characterization revealed anti-thrombin activity in 2 of these preparations, whilst the third displayed the strongest anticoagulant activity that targeted a factor of the intrinsic coagulation pathway. Three trypsin inhibitors (18 kDa, 21 kDa, and 22 kDa) were also detected using reverse zymography. In parallel, homogenates of fed stage 2 and 3 juveniles were used to identify their fish hosts by amplifying the 16S mitochondrial rDNA and 18S genomic rDNA vertebrate gene regions. Blood from at least 4 fish families had been ingested by separate individuals during feeding. This study demonstrates that trypsin inhibitors and anticoagulants are present in P. formica juveniles which could suppress host haemostatic, inflammatory and immunological responses during feeding, and that juveniles are not host specific.

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M. Abebe , J. M. C. Ribeiro , M. S. Cupp and E. W. Cupp (1996). Novel anticoagulant from salivary glands of Simulium vittatum (Diptera: Simuliidae) inhibits activity of coagulation factor V. Journal of Medical Entomology 33, 173176.

N. Agustí , P. Shayler , J. D. Harwood , I. P. Vaughan , K. D. Sunderland and W. O. C. Symondson (2003). Collembola as alternative prey sustaining spiders in arable ecosystems: prey detection within predators using molecular markers. Molecular Ecology 12, 34673475. doi: 10.1046/j.1365-294X.2003.02014.X.

R. Andreotti , A. Gomes , K. C. Malavazi-Piza , S. D. Sasaki , C. A. M. Sampaio and A. S. Tanaka (2002). BmTI antigens induce a bovine protective immune response against Boophilus microplus tick. International Immunopharmacology 2, 557563. doi: 10.1016/S1567-5769(01)00203-X.

S. S. A. Azzolini , S. D. Sasaki , I. T. N. Campos , R. J. S. Torquato , M. A. Juliano and A. S. Tanaka (2005). The role of HiTI, a serine protease inhibitor from Haematobia irritans irritans (Diptera: Muscidae) in the control of fly and bacterial proteases. Experimental Parasitology 111, 3036. doi: 10.1016/j.exppara.2005.03.013.

S. S. A. Azzolini , S. D. Sasaki , R. J. S. Torquato , R. Andreotti , E. Andreotti and A. S. Tanaka (2003). Rhipicephalus sanguineus trypsin inhibitors present in the tick larvae: isolation, characterization, and partial primary structure determination. Archives of Biochemistry and Biophysics 417, 176182. doi: 10.1016/S0003-9861(03)00344-8.

L. E. Blankenship and A. A. Yayanos (2005). Universal primers and PCR gut contents to study marine invertebrate diets. Molecular Ecology 14, 891899. doi: 10.1111/j.1365-294X.2005.02448.X.

A. Ciprandi , S. K. De Olivera , A. Masuda , F. Horn and C. Termignoni (2006). Boophilus microplus: its saliva contains microphilin, a small thrombin inhibitor. Experimental Parasitology 114, 4046. doi: 10.1016/j.exppara.2006.02.010.

B. Dahlbäck (2000). Blood coagulation. The Lancet 355, 16271632. doi: 10.1016/S0140-6736(00)02225-X.

A. J. Davies and N. J. Smit (2001). The life cycle of Haemogregarina bigemina (Adeleina: Haemogregarinidae) in South African hosts. Folia Parasitologica 48, 169177.

M. L. Ferreira , N. J. Smit , A. S. Grutter and A. J. Davies (2009). A new species Gnathia aureamaculosa n. sp. (Crustacea. Isopoda. Gnathiidae) parasitising teleosts from Lizard Island, Great Barrier Reef. Journal of Parasitology 95, 10661075. doi: 10.1645/GE-1920.1.

P. M. Hayes , N. J. Smit and A. J. Davies (2007). Pathology associated with parasitic juvenile gnathiids feeding on the puffadder shyshark, Haploblepharus edwardsii (Voight). Journal of Fish Diseases 30, 5558. doi: 10.1111/j.1365-2761.2007.00777.X.

P. M. Hayes , N. J. Smit , A. S. Grutter and A. J. Davies (2011). Unexpected response of a captive blackeye thicklip, Hemigymnus melapterus (Bloch), from Lizard Island, Australia, exposed to juvenile isopods Gnathia aureamaculosa Ferreira & Smit. Journal of Fish Diseases 34, 563566. doi: 10.1111/j.1365-2761.2011.01261.X.

F. Horn , P. Coutinho dos Santos and C. Termignoni (2000). Boophilus microplus anticoagulant protein: an antithrombin inhibitor isolated from the cattle tick saliva. Archives of Biochemistry and Biophysics 384, 6873. doi: 10.1006/abbi.2000.2076.

C. M. Jones , L. Nagel , G. L. Hughes , T. H. Cribb and A. S. Grutter (2007). Host specificity of two species of Gnathia (Isopoda) determined by DNA sequencing blood meals. International Journal for Parasitology 37, 927935. doi: 10.1016/j.ijpara.2007.01.011.

M. R. Kanost (1999). Serine proteinase inhibitors in arthropod immunity. Developmental & Comparative Immunology 23, 291301. doi: 10.1016/S0145-305X(99)00012-9.

M. Kazimírová , M. Šulanová , A. R. Trimnell , M. Kozánek , L. Vidlička , M. Labuda and P. A. Nuttall (2002). Anticoagulant activities in salivary glands of tabanid flies. Medical and Veterinary Entomology 16, 301309. doi: 10.1046/j.1365-2915.2002.00379.X.

K. G. Mann and L. Lorand (1993). Introduction: blood coagulation. Methods in Enzymology 222, 110. doi: 10.1016/0076-6879(93)22003-X.

B. M. Manship , A. J. Walker and A. J. Davies (2011). Brooding and embryonic development in the crustacean Paragnathia formica (Hesse, 1864) (Peracarida: Isopoda: Gnathiidae). Arthropod Structure & Development 40, 135145. doi: 10.1016/j.asd.2010.12.004.

B. M. Manship , A. J. Walker , L. A. Jones and A. J. Davies (2008). Characterisation of cysteine proteinase activities in the digestive tract of juvenile Paragnathia formica isopods, ectoparasites of estuarine fish. Marine Biology 153, 473482. doi: 10.1007/s00227-007-0834-6.

F. Marino , S. Giannetto , M. L. Paradiso , T. Bottari , G. Devico and B. Macri (2004). Tissue damage and haematophagia due to praniza larvae (Isopoda, Gnathiidae) in some aquarium seawater teleosts. Diseases of Aquatic Organisms 59, 4347. doi: 10.3354/dao059043.

S. McGinnis and T. L. Madden (2004). BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acid Research 32, (WEB SERVER ISSUE):W20-W25. doi: 10.1093/nar/gkh435.

R. E. R. Mugridge and H. G. Stallybrass (1983). A mortality of eels, Anguilla anguilla L., attributed to Gnathiidae. Journal of Fish Diseases 6, 8182. doi: 10.1111/j.1365-2761.1983.tb00054.X.

L. Nagel and A. S. Grutter (2007). Host preference and specialization in Gnathia sp., a common parasitic isopod of coral reef fishes. Journal of Fish Biology 70, 497508. doi: 10.1111/j.1095-8649.2007.01320.X.

L. Nagel and S. C. Lougheed (2006). A simple molecular technique for identifying marine fish by sequencing bloodfeeding parasites. Journal of Parasitology 92, 665668. doi: 10.1645/GE-3541RN.1.

B. Pichon , D. Egan , M. Rogers and J. Gray (2003). Detection and identification of pathogens and host DNA in unfed host-seeking Ixodes ricinus L. (Acari: Ixodidae). Journal of Medical Entomology 40, 723731.

R. S. Raynard , I. A. Bricknell , P. F. Billingsley , A. Nisbet , A. Vigneau and C. Sommerville (2002). Development of vaccines against sea lice. Pest Management Science 58, 569575. doi: 10.1002/ps.474.

J. M. C. Ribeiro and I. M. Francischetti (2003). Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annual Review of Entomology 48, 7388. doi: 10.1146/annurev.ento.48.060402.102812.

J. M. C. Ribeiro , G. T. Makoul , J. Levine , D. R. Robinson and A. Spielman (1985). Antihemostatic, anti-inflammatory, and immunosuppressive properties of the saliva of a tick, Ixodes dammini. Journal of Experimental Medicine 161, 332344. doi: 10.1084/jem.161.2.332.

J. M. C. Ribeiro , M. Schneider and J. A. Guimarães (1995). Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus. The Biochemical Journal 308, 243249.

C. G. Ricci , A. F. M. Pinto , M. Berger and C. Termignoni (2007). A thrombin inhibitor from the gut of Boophilus microplus ticks. Experimental and Applied Acarology 42, 291300. doi: 10.1007/s10493-007-9097-7.

V. Rimphanitchayakit and A. Tassaanakajon (2010). Structure and function of invertebrate Kazal-type serine proteinase inhibitors. Developmental and Comparative Immunology 34, 377386. doi: 10.1016/j.dci.2009.12.004.

B. Romestand and J. P. Trilles (1976 a). Au sujet d'une substance antithrombinique mise en évidence dans les glandes latéro-oesophagiennes de Meinertia oestroides (Risso, 1826) (Isopoda, Flabellifera, Cymothoidae: parasite de poissons). Zeitschrift für Parasitenkunde 50, 8792.

G. Silva , J. L. Costa , P. Raposo de Almeida and M. J. Costa (2006). Structure and dynamics of a benthic invertebrate community in an intertidal area of the Tagus estuary, western Portugal: a six year data series. Hydrobiologia 555, 115128. doi: 10.1007/s10750-005-1110-8.

N. J. Smit and A. J. Davies (2004). The curious life-style of the parasitic stages of gnathiid isopods. Advances in Parasitology 58, 289392. doi: 10.1016/S0065-308X(04)58005-3.

N. J. Smit , A. S. Grutter , R. D. Adlard and A. J. Davies (2006). Hematozoa of teleosts from Lizard Island, Australia, with some comments on their possible mode of transmission and the description of a new hemogregarine species. Journal of Parasitology 92, 778788. doi: 10.1645/GE-756R.1.

K. R. Stark and A. A. James (1995). A factor Xa-directed anticoagulant from the salivary glands of the Yellow Fever mosquito Aedes aegypti. Experimental Parasitology 81, 321331. doi: 10.1006/expr.1995.1123.

K. R. Stark and A. A. James (1996). Anticoagulants in vector arthropods. Parasitology Today 12, 430437. doi: 10.1016/0169-4758(96)10064-8.

A. S. Tanaka , R. Andreotti , A. Gomes , R. J. S. Torquato , M. U. Sampaio and C. A. M. Sampiao (1999). A double headed serine proteinase inhibitor - human plasma kallikrein and elastase inhibitor - from Boophilus microplus larvae. Immunopharmacology 45, 171177. doi: 10.1016/S0162-3109(99)00074-0.

A. M. Tanaka-Azevedo , K. Morais-Zani , R. J. S. Torquato and A. S. Tanaka (2010). Thrombin inhibitors from different animals. Journal of Biomedicine and Biotechnology 2010, Article ID 641025. doi: 10.1155/2010/641025.

K. Tanaka (2007). Life history of gnathiid isopods-current knowledge and future directions. Plankton & Benthos Research 2, 111. doi: 10.3800/pbr.2.1.

J. D. Thompson , D. G. Higgins and T. J. Gibson (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 46734680. doi: 10.1093/nar/22.22.4673.

N. P. D. Upton (1987). Asynchronous male and female life cycles in the sexually dimorphic, harem-forming isopod Paragnathia formica (Crustacea, Isopoda). Journal of Zoology (London) 212, 677690. doi: 10.1111/j.1469-7998.1987.tb05964.X.

J. G. Valenzuela (2002). High-throughput approaches to study salivary proteins and genes from vectors of disease. Insect Biochemistry and Molecular Biology 32, 11991209. doi: 10.1016/S0965-1748(02)00083-8.

G. N. Wagner , M. D. Fast and S. C. Johnson (2008). Physiology and immunology of Lepeophtheirus salmonis infections of salmonids. Trends in Parasitology 24, 176183. doi: 10.1016/

R. M. O. Watanabe , T. S. Soares , K. Morais-Zani , A. M. Tanaka-Azevedo , C. Maciel , M. L. Capurro , R. J. S. Torquato and A. S. Tanaka (2010). A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity. Biochimie 92, 933939. doi: 10.1016/j.biochi.2010.03.024.

R. H. Zaidi , Z. Jaal , N. J. Hawkes , J. Hemingway and O. C. Symondson (1999). Can multiple-copy sequences of prey DNA be detected amongst the gut contents of invertebrate predators? Molecular Ecology 8, 20812087. doi: 10.1046/j.1365-294x. 1999.00823.X.

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