Skip to main content

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.

Corresponding author
*Corresponding author: Tel: +44 208 547 2466. Fax: +44 208 547 7562. E-mail:
Hide All
Abebe M., Ribeiro J. M. C., Cupp M. S. and Cupp E. W. (1996). Novel anticoagulant from salivary glands of Simulium vittatum (Diptera: Simuliidae) inhibits activity of coagulation factor V. Journal of Medical Entomology 33, 173176.
Agustí N., Shayler P., Harwood J. D., Vaughan I. P., Sunderland K. D. and Symondson W. O. C. (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.
Andreotti R., Gomes A., Malavazi-Piza K. C., Sasaki S. D., Sampaio C. A. M. and Tanaka A. S. (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.
Azzolini S. S. A., Sasaki S. D., Campos I. T. N., Torquato R. J. S., Juliano M. A. and Tanaka A. S. (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.
Azzolini S. S. A., Sasaki S. D., Torquato R. J. S., Andreotti R., Andreotti E. and Tanaka A. S. (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.
Blankenship L. E. and Yayanos A. A. (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.
Cadée G. C., Checa A. G. and Rodriguez-Tovar F. J. (2001). Burrows of Paragnathia (Crustacea: Isopoda) and Bledius (Arthropoda: Staphylinidae) enhance cliff erosion. International Journal for Plant and Animal Traces 8, 255260. doi: 10.1080/10420940109380193.
Campbell E. M., Pert C. C. and Bowman A. S. (2009). RNA-interference methods for gene-knockdown in the sea louse, Lepeophtheirus salmonis: studies on a putative prostaglandin E synthase. Parasitology 136, 867874. doi: 10.1017/S0031182009990357.
Cavaleiro F. I. and Santos M. J. (2009). Seasonality of metazoan ectoparasites in marine European flounder Platichthys flesus (Teleostei: Pleuronectidae). Parasitology 136, 855865. doi: 10.1017/S003118200900626X.
Ciprandi A., De Olivera S. K., Masuda A., Horn F. and Termignoni C. (2006). Boophilus microplus: its saliva contains microphilin, a small thrombin inhibitor. Experimental Parasitology 114, 4046. doi: 10.1016/j.exppara.2006.02.010.
Dahlbäck B. (2000). Blood coagulation. The Lancet 355, 16271632. doi: 10.1016/S0140-6736(00)02225-X.
Davies A. J. and Smit N. J. (2001). The life cycle of Haemogregarina bigemina (Adeleina: Haemogregarinidae) in South African hosts. Folia Parasitologica 48, 169177.
Doolittle R. F. (2011). Coagulation in vertebrates with a focus on evolution and inflammation. Journal of Innate Immunity 3, 916. doi: 10.1159/000321005.
Dreyer H. and Wägele J. W. (2001). Parasites of crustaceans (Isopoda: Bopyridae) evolved from fish parasites: molecular and morphological evidence. Zoology 103, 157178.
Ferreira M. L., Smit N. J., Grutter A. S. and Davies A. J. (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.
Ferreira M. L., Smit N. J., Grutter A. S. and Davies A. J. (2010). Gnathia grutterae sp. nov. (Crustacea: Isopoda) parasitising representatives of the teleost families Balistidae and Labridae from Lizard Island, Great Barrier Reef, Australia. Zootaxa 2718, 3950.
Hanspal J. S., Bushell G. R. and Ghosh P. (1983). Detection of protease inhibitors using substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analytical Biochemistry 132, 288293. doi: 10.1016/0003-2697(83)90010-6.
Hayes P. M., Smit N. J. and Davies A. J. (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.
Hayes P. M., Smit N. J., Grutter A. S. and Davies A. J. (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.
Horn F., Coutinho dos Santos P. and Termignoni C. (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.
Jones C. M., Nagel L., Hughes G. L., Cribb T. H. and Grutter A. S. (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.
Kanost M. R. (1999). Serine proteinase inhibitors in arthropod immunity. Developmental & Comparative Immunology 23, 291301. doi: 10.1016/S0145-305X(99)00012-9.
Kazimírová M., Šulanová M., Kozánek M., Takáč P., Labuda M. and Nuttall P. A. (2001). Identification of anticoagulant activities in salivary gland extracts of four horsefly species (Diptera, Tabanidae). Haemostasis 31, 294305. doi: 10.1159/000048076.
Kazimírová M., Šulanová M., Trimnell A. R., Kozánek M., Vidlička L., Labuda M. and Nuttall P. A. (2002). Anticoagulant activities in salivary glands of tabanid flies. Medical and Veterinary Entomology 16, 301309. doi: 10.1046/j.1365-2915.2002.00379.X.
Kirkim F., Kocataş A., Katağan T. and Sezgin M. (2008). A report on parasitic isopods (Crustacea) from marine fishes and decapods collected from the Aegean Sea (Turkey). Türkiye Parazitoloji Dergisi 32, 382385.
Koh C. Y. and Kini R. M. (2009). Molecular diversity of anticoagulants from haematophagous animals. Thrombosis and Haemostasis 102, 437453. doi: 10.1160/TH09-04-0221.
Mann K. G. and Lorand L. (1993). Introduction: blood coagulation. Methods in Enzymology 222, 110. doi: 10.1016/0076-6879(93)22003-X.
Manship B. M. (2009). Aspects of development and feeding in Paragnathia formica (Hesse, 1864). Thesis (Ph.D.), School of Life Sciences, Kingston University, London.
Manship B. M., Walker A. J. and Davies A. J. (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.
Manship B. M., Walker A. J., Jones L. A. and Davies A. J. (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.
Marino F., Giannetto S., Paradiso M. L., Bottari T., Devico G. and Macri B. (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.
McGinnis S. and Madden T. L. (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.
Menezes J. (1984). Parasitose des mulets par les larves de Paragnathia. Fiches d'identification des maladies et parasites des poissons, crustacés et mollusques, 10. Conseil International pour l'Exploration de la Mer: Copenhagen, Denmark, 14.
Monod T. (1926). Les Gnathiidae. Essai monographique (Morphologie, Biologie, Systematique). Mémoires de la Société des Sciences Naturelles du Maroc 13, 1668.
Morris S. R. and Sakanari J. A. (1994). Characterization of the serine protease and serine protease inhibitor from the tissue-penetrating nematode Anisakis simplex. The Journal of Biological Chemistry 269, 2765027656.
Mugridge R. E. R. and Stallybrass H. G. (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.
Nagel L. and Grutter A. S. (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.
Nagel L. and Lougheed S. C. (2006). A simple molecular technique for identifying marine fish by sequencing bloodfeeding parasites. Journal of Parasitology 92, 665668. doi: 10.1645/GE-3541RN.1.
Pichon B., Egan D., Rogers M. and Gray J. (2003). Detection and identification of pathogens and host DNA in unfed host-seeking Ixodes ricinus L. (Acari: Ixodidae). Journal of Medical Entomology 40, 723731.
Raynard R. S., Bricknell I. A., Billingsley P. F., Nisbet A., Vigneau A. and Sommerville C. (2002). Development of vaccines against sea lice. Pest Management Science 58, 569575. doi: 10.1002/ps.474.
Ribeiro J. M. C. and Francischetti I. M. (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.
Ribeiro J. M. C., Makoul G. T., Levine J., Robinson D. R. and Spielman A. (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.
Ribeiro J. M. C., Schneider M. and Guimarães J. A. (1995). Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus. The Biochemical Journal 308, 243249.
Ricci C. G., Pinto A. F. M., Berger M. and Termignoni C. (2007). A thrombin inhibitor from the gut of Boophilus microplus ticks. Experimental and Applied Acarology 42, 291300. doi: 10.1007/s10493-007-9097-7.
Richards E. H., Jones B. and Bowman A. (2011). Salivary secretions from the honeybee mite, Varroa destructor: effects on insect haemocytes and preliminary biochemical characterization. Parasitology 138, 602608. doi: 10.1017/S0031182011000072.
Rimphanitchayakit V. and Tassaanakajon A. (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.
Romestand B. and Trilles J. P. (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.
Romestand B. and Trilles J. P. (1976 b). Production d'une substance anticoagulante par les glandes exocrines céphalothoraciques des Isopodes Cymothoidae Meinertia oestroides (Risso, 1826) et Anilocra physodes (L., 1758) (Isopoda, Flabellifera, Cymothoidae). Comptes rendus hebdomadaires des Séances de l'Académie des Sciences 282, 663665.
Sicard M., Desmarais E. and Lambert A. (2001). Molecular characterisation of Diplozoidae populations on five Cyprinidae species: consequences for host specificity. Comptes Redus de l'Academie des Sciences – Series III – Sciences de la Vie 342, 709717. doi: 10.1016/S0764-4469(01)01352-X.
Silva G., Costa J. L., Raposo de Almeida P. and Costa M. J. (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.
Simon C. (1991). Appendix 3. In Molecular Techniques in Taxonomy (ed. Hewitt G. M., Johnston A. and Young J. P.), pp. 345355. Springer-Verlag, Berlin, Germany.
Singh S. K. and Girschick H. J. (2003). Tick-host interactions and their immunological implications in tick-borne diseases. Current Science 85, 12841298.
Smit N. J. and Davies A. J. (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.
Smit N. J., Grutter A. S., Adlard R. D. and Davies A. J. (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.
Stark K. R. and James A. A. (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.
Stark K. R. and James A. A. (1996). Anticoagulants in vector arthropods. Parasitology Today 12, 430437. doi: 10.1016/0169-4758(96)10064-8.
Stoll C. (1962). Cycle évolutif de Paragnathia formica (Hesse) (Isopoda: Gnathiidae). Cahiers de Biologie Marine 3, 401416.
Tanaka A. S., Andreotti R., Gomes A., Torquato R. J. S., Sampaio M. U. and Sampiao C. A. M. (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.
Tanaka-Azevedo A. M., Morais-Zani K., Torquato R. J. S. and Tanaka A. S. (2010). Thrombin inhibitors from different animals. Journal of Biomedicine and Biotechnology 2010, Article ID 641025. doi: 10.1155/2010/641025.
Tanaka K. (2007). Life history of gnathiid isopods-current knowledge and future directions. Plankton & Benthos Research 2, 111. doi: 10.3800/pbr.2.1.
Tavares-Dias M. and Oliveira S. R. (2009). A review of the blood coagulation system of fish. Revista Brasileira de Biociências 7, 205224.
Thompson J. D., Higgins D. G. and Gibson T. J. (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.
Tinsley M. C. and Reilly S. (2002). Reproductive ecology of the saltmarsh-dwelling marine ectoparasite Paragnathia formica (Crustacea, Isopoda). Journal of the Marine Biological Association of the United Kingdom 82, 7984. doi: 10.1017/S0025315402005192.
Upton N. P. D. (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.
Valenzuela J. G. (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.
Wagner G. N., Fast M. D. and Johnson S. C. (2008). Physiology and immunology of Lepeophtheirus salmonis infections of salmonids. Trends in Parasitology 24, 176183. doi: 10.1016/
Watanabe R. M. O., Soares T. S., Morais-Zani K., Tanaka-Azevedo A. M., Maciel C., Capurro M. L., Torquato R. J. S. and Tanaka A. S. (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.
Willadsen P. (2006). Vaccination against ectoparasites. Parasitology 133 (Suppl.) S9S25. doi: 10.1017/S0031182006001788.
Zaidi R. H., Jaal Z., Hawkes N. J., Hemingway J. and Symondson O. C. (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.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 0031-1820
  • EISSN: 1469-8161
  • URL: /core/journals/parasitology
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Full text views

Total number of HTML views: 2
Total number of PDF views: 16 *
Loading metrics...

Abstract views

Total abstract views: 104 *
Loading metrics...

* Views captured on Cambridge Core between September 2016 - 23rd January 2018. This data will be updated every 24 hours.