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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

Published online by Cambridge University Press:  06 February 2012

B. M. MANSHIP ,
A. J. WALKER ,
L. A. JONES  and
A. J. DAVIES
B. M. MANSHIP
Affiliation:
School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK
A. J. WALKER*
Affiliation:
School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK
L. A. JONES
Affiliation:
School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK
A. J. DAVIES
Affiliation:
School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK
*
*Corresponding author: Tel: +44 208 547 2466. Fax: +44 208 547 7562. E-mail: t.walker@kingston.ac.uk
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Summary

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.

Keywords

Paragnathia formicafish ectoparasiteblood feedinghost-parasite interactionsserine protease inhibitoranticoagulants

Information

Type
Research Article
Information
Parasitology , Volume 139 , Issue 6 , May 2012 , pp. 744 - 754
Copyright
Copyright © Cambridge University Press 2012

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References

REFERENCES

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, 173–176.CrossRefGoogle ScholarPubMed
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, 3467–3475. doi: 10.1046/j.1365-294X.2003.02014.X.CrossRefGoogle ScholarPubMed
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, 557–563. doi: 10.1016/S1567-5769(01)00203-X.CrossRefGoogle ScholarPubMed
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, 30–36. doi: 10.1016/j.exppara.2005.03.013.CrossRefGoogle Scholar
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, 176–182. doi: 10.1016/S0003-9861(03)00344-8.CrossRefGoogle Scholar
Blankenship, L. E. and Yayanos, A. A. (2005). Universal primers and PCR gut contents to study marine invertebrate diets. Molecular Ecology 14, 891–899. doi: 10.1111/j.1365-294X.2005.02448.X.CrossRefGoogle ScholarPubMed
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, 255–260. doi: 10.1080/10420940109380193.Google Scholar
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, 867–874. doi: 10.1017/S0031182009990357.CrossRefGoogle ScholarPubMed
Cavaleiro, F. I. and Santos, M. J. (2009). Seasonality of metazoan ectoparasites in marine European flounder Platichthys flesus (Teleostei: Pleuronectidae). Parasitology 136, 855–865. doi: 10.1017/S003118200900626X.CrossRefGoogle ScholarPubMed
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, 40–46. doi: 10.1016/j.exppara.2006.02.010.CrossRefGoogle ScholarPubMed
Dahlbäck, B. (2000). Blood coagulation. The Lancet 355, 1627–1632. doi: 10.1016/S0140-6736(00)02225-X.CrossRefGoogle ScholarPubMed
Davies, A. J. and Smit, N. J. (2001). The life cycle of Haemogregarina bigemina (Adeleina: Haemogregarinidae) in South African hosts. Folia Parasitologica 48, 169–177.CrossRefGoogle Scholar
Doolittle, R. F. (2011). Coagulation in vertebrates with a focus on evolution and inflammation. Journal of Innate Immunity 3, 9–16. doi: 10.1159/000321005.Google ScholarPubMed
Dreyer, H. and Wägele, J. W. (2001). Parasites of crustaceans (Isopoda: Bopyridae) evolved from fish parasites: molecular and morphological evidence. Zoology 103, 157–178.Google Scholar
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, 1066–1075. doi: 10.1645/GE-1920.1.CrossRefGoogle Scholar
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, 39–50.CrossRefGoogle Scholar
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, 288–293. doi: 10.1016/0003-2697(83)90010-6.CrossRefGoogle ScholarPubMed
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, 55–58. doi: 10.1111/j.1365-2761.2007.00777.X.CrossRefGoogle ScholarPubMed
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, 563–566. doi: 10.1111/j.1365-2761.2011.01261.X.CrossRefGoogle ScholarPubMed
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, 68–73. doi: 10.1006/abbi.2000.2076.CrossRefGoogle ScholarPubMed
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, 927–935. doi: 10.1016/j.ijpara.2007.01.011.CrossRefGoogle ScholarPubMed
Kanost, M. R. (1999). Serine proteinase inhibitors in arthropod immunity. Developmental & Comparative Immunology 23, 291–301. doi: 10.1016/S0145-305X(99)00012-9.CrossRefGoogle ScholarPubMed
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, 294–305. doi: 10.1159/000048076.Google ScholarPubMed
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, 301–309. doi: 10.1046/j.1365-2915.2002.00379.X.Google ScholarPubMed
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, 382–385.Google ScholarPubMed
Koh, C. Y. and Kini, R. M. (2009). Molecular diversity of anticoagulants from haematophagous animals. Thrombosis and Haemostasis 102, 437–453. doi: 10.1160/TH09-04-0221.CrossRefGoogle ScholarPubMed
Mann, K. G. and Lorand, L. (1993). Introduction: blood coagulation. Methods in Enzymology 222, 1–10. doi: 10.1016/0076-6879(93)22003-X.CrossRefGoogle ScholarPubMed
Manship, B. M. (2009). Aspects of development and feeding in Paragnathia formica (Hesse, 1864). Thesis (Ph.D.), School of Life Sciences, Kingston University, London.Google Scholar
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, 135–145. doi: 10.1016/j.asd.2010.12.004.CrossRefGoogle ScholarPubMed
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, 473–482. doi: 10.1007/s00227-007-0834-6.CrossRefGoogle Scholar
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, 43–47. doi: 10.3354/dao059043.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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, 1–4.Google Scholar
Monod, T. (1926). Les Gnathiidae. Essai monographique (Morphologie, Biologie, Systematique). Mémoires de la Société des Sciences Naturelles du Maroc 13, 1–668.Google Scholar
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, 27650–27656.CrossRefGoogle ScholarPubMed
Mugridge, R. E. R. and Stallybrass, H. G. (1983). A mortality of eels, Anguilla anguilla L., attributed to Gnathiidae. Journal of Fish Diseases 6, 81–82. doi: 10.1111/j.1365-2761.1983.tb00054.X.CrossRefGoogle Scholar
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, 497–508. doi: 10.1111/j.1095-8649.2007.01320.X.CrossRefGoogle Scholar
Nagel, L. and Lougheed, S. C. (2006). A simple molecular technique for identifying marine fish by sequencing bloodfeeding parasites. Journal of Parasitology 92, 665–668. doi: 10.1645/GE-3541RN.1.CrossRefGoogle ScholarPubMed
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, 723–731.Google ScholarPubMed
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, 569–575. doi: 10.1002/ps.474.CrossRefGoogle ScholarPubMed
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, 73–88. doi: 10.1146/annurev.ento.48.060402.102812.CrossRefGoogle ScholarPubMed
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, 332–344. doi: 10.1084/jem.161.2.332.CrossRefGoogle ScholarPubMed
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, 243–249.CrossRefGoogle ScholarPubMed
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, 291–300. doi: 10.1007/s10493-007-9097-7.CrossRefGoogle ScholarPubMed
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, 602–608. doi: 10.1017/S0031182011000072.CrossRefGoogle ScholarPubMed
Rimphanitchayakit, V. and Tassaanakajon, A. (2010). Structure and function of invertebrate Kazal-type serine proteinase inhibitors. Developmental and Comparative Immunology 34, 377–386. doi: 10.1016/j.dci.2009.12.004.CrossRefGoogle ScholarPubMed
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, 87–92.CrossRefGoogle ScholarPubMed
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, 663–665.Google ScholarPubMed
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, 709–717. doi: 10.1016/S0764-4469(01)01352-X.Google Scholar
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, 115–128. doi: 10.1007/s10750-005-1110-8.CrossRefGoogle Scholar
Simon, C. (1991). Appendix 3. In Molecular Techniques in Taxonomy (ed. Hewitt, G. M., Johnston, A. and Young, J. P.), pp. 345–355. Springer-Verlag, Berlin, Germany.Google Scholar
Singh, S. K. and Girschick, H. J. (2003). Tick-host interactions and their immunological implications in tick-borne diseases. Current Science 85, 1284–1298.Google Scholar
Smit, N. J. and Davies, A. J. (2004). The curious life-style of the parasitic stages of gnathiid isopods. Advances in Parasitology 58, 289–392. doi: 10.1016/S0065-308X(04)58005-3.CrossRefGoogle ScholarPubMed
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, 778–788. doi: 10.1645/GE-756R.1.CrossRefGoogle ScholarPubMed
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, 321–331. doi: 10.1006/expr.1995.1123.CrossRefGoogle ScholarPubMed
Stark, K. R. and James, A. A. (1996). Anticoagulants in vector arthropods. Parasitology Today 12, 430–437. doi: 10.1016/0169-4758(96)10064-8.CrossRefGoogle ScholarPubMed
Stoll, C. (1962). Cycle évolutif de Paragnathia formica (Hesse) (Isopoda: Gnathiidae). Cahiers de Biologie Marine 3, 401–416.Google Scholar
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, 171–177. doi: 10.1016/S0162-3109(99)00074-0.Google ScholarPubMed
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.CrossRefGoogle ScholarPubMed
Tanaka, K. (2007). Life history of gnathiid isopods-current knowledge and future directions. Plankton & Benthos Research 2, 1–11. doi: 10.3800/pbr.2.1.CrossRefGoogle Scholar
Tavares-Dias, M. and Oliveira, S. R. (2009). A review of the blood coagulation system of fish. Revista Brasileira de Biociências 7, 205–224.Google Scholar
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, 4673–4680. doi: 10.1093/nar/22.22.4673.CrossRefGoogle ScholarPubMed
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, 79–84. doi: 10.1017/S0025315402005192.CrossRefGoogle Scholar
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, 677–690. doi: 10.1111/j.1469-7998.1987.tb05964.X.CrossRefGoogle Scholar
Valenzuela, J. G. (2002). High-throughput approaches to study salivary proteins and genes from vectors of disease. Insect Biochemistry and Molecular Biology 32, 1199–1209. doi: 10.1016/S0965-1748(02)00083-8.CrossRefGoogle ScholarPubMed
Wagner, G. N., Fast, M. D. and Johnson, S. C. (2008). Physiology and immunology of Lepeophtheirus salmonis infections of salmonids. Trends in Parasitology 24, 176–183. doi: 10.1016/j.pt.2007.12.010.CrossRefGoogle ScholarPubMed
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, 933–939. doi: 10.1016/j.biochi.2010.03.024.CrossRefGoogle ScholarPubMed
Willadsen, P. (2006). Vaccination against ectoparasites. Parasitology 133 (Suppl.) S9–S25. doi: 10.1017/S0031182006001788.CrossRefGoogle ScholarPubMed
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, 2081–2087. doi: 10.1046/j.1365-294x. 1999.00823.X.CrossRefGoogle ScholarPubMed