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  • 1.
    Angthong, Pacharaporn
    et al.
    Chulalongkorn Univ, Program Biotechnol, Fac Sci..
    Roytrakul, Sittiruk
    Natl Sci & Technol Dev Agcy, Natl Ctr Genet Engn & Biotechnol BIOTEC..
    Jarayabhand, Padermsak
    Chulalongkorn Univ, Grad Sch, Interdisciplinary Grad Program Maritime Adm..
    Jiravanichpaisal, Pikul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Characterization and function of a tachylectin 5-like immune molecule in Penaeus monodon2017In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 76, p. 120-131Article in journal (Refereed)
    Abstract [en]

    Tachylectin5A and its homolog, tachylectin5B both contain a fibrinogen-related domain (FReD) and have been studied in horseshoe crabs, Tachypleus tridentatus and Carcinoscorpius rotundicauda and shown to be involved in host defense. Here, we demonstrate the presence of tachylectin5-like genes in shrimp, Penaeus monodon, designated as Penlectin5-1 (PL5-1) and Penlectin5-2 (PL5-2), which both contain a signal peptide and a single FReD with an acetyl group and a calcium binding sites and they are both structurally similar to horseshoe crab tachylectin/carcinolectin5. The PL5-land PL5-2 transcript were expressed in various shrimp tissues in normal shrimp, and their expression was upregulated in tissues such as hemocytes and hindgut following challenge with pathogenic Vibrio harveyi. The PL5-2 protein was detected in various tissues as well as in cell-free hemolymph. The biological function of the PL5-2 protein is to recognize some Gram-positive and Gram-negative bacteria regardless whether they are non-pathogenic or pathogenic. They have hemagglutination activity on human erythrocyte and bacterial agglutination activity to both Gram negative and Gram positive bacteria. Possible binding sites of PL5-2 to bacteria could be at the N-acetyl moiety of the G1cNAc-MurNAc cell wall of the peptidoglycan since the binding could be inhibited by G1cNAc or GaINAC. The presence of PL5-2 protein in both circulating hemolymph and intestine, where host and microbes are usually interacting, may suggest that the physiological function of shrimp tachylectin-like proteins is to recognize and bind to invading bacteria to immobilize and entrap these microbes and subsequently clear them from circulation and the host body, and probably to control and maintain the normal flora in the intestine.

  • 2.
    Angthong, Pacharaporn
    et al.
    Program in Biotechnology, Faculty of Science, Chulalongkorn University..
    Roytrakul, Sittiruk
    National Center for Genetic Engineering and Biotechnology (BIOTEC); National Science and Technology Development Agency (NSTDA)..
    Jarayabhand, Padermsak
    Interdisciplinary Graduate Program on Maritime Administration, Graduate School, Chulalongkorn University..
    Jiravanichpaisal, Pikul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Involvement of a tachylectin-like gene and its protein in pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in the shrimp, Penaeus monodon2017In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 76, p. 229-237Article in journal (Refereed)
    Abstract [en]

    A shrimp disease, the so-called acute hepatopancreatic necrosis disease (AHPND) is caused by a specific strain of Vibrio parahaemolyticus (VP) and it has resulted in significant losses to the global shrimp farming industry. In our previous study, three of tachylectin-like genes were cloned and characterized from the intestine of Penaeus monodon, designated as Penlectin5-1 (PL5-1), Penlectin5-2 (PL5-2) and Penlectin5-3 (PL5-3). These three genes all contain fibrinogen-related domain (FReD). The expression level of PL5-1, PL5-2 and PL5-3 was elevated in the stomach after oral administration with AHPND-causing V. parahaemolyticus 3HP (VP3HP). A polyclonal antibody to PL5-2 was successfully produced in a rabbit using the purified recombinant P15-2 as an immunogen, and this because only the predominant protein PL5-2 could be successfully purified from shrimp plasma by affinity chromatography using a N-Acetyl-oglucosamine column allowed us to perform functional studies of this lectin. The native purified PL5-2 protein had binding and agglutination activities towards VP3HR To further understand the functions and the involvements of this lectin in response to AHPND in shrimp, RNAi-mediated knockdown of PL5-1, PL5-2 or PL5-3 was performed prior to an oral administration of VP3HP. As a result, Penlectin5-silencing in shrimp challenged with VP3HP showed higher mortality and resulted in more severe histopathological changes in the hepatopancreas with typical signs of AHPND. These results therefore suggest a role for crustacean fibrinogen-related proteins (FRePs) in innate immune response during the development of AHPND, and maybe also during other infections.

  • 3.
    Apitanyasai, Kantamas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology. Chulalongkorn Univ, Dept Biochem, Ctr Excellence Mol Biol & Genom Shrimp, Fac Sci, 254 Phayathai Rd, Bangkok 10330, Thailand..
    Noonin, Chadanat
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Tassanakajon, Anchalee
    Chulalongkorn Univ, Dept Biochem, Ctr Excellence Mol Biol & Genom Shrimp, Fac Sci, 254 Phayathai Rd, Bangkok 10330, Thailand..
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Characterization of a hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus2016In: Fish and Shellfish Immunology, ISSN 1050-4648, E-ISSN 1095-9947, Vol. 58, p. 429-435Article in journal (Refereed)
    Abstract [en]

    Hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus has a distinct role from that of its homolog PmHHAP in the shrimp Penaeus monodon. Knockdown of PIHHAP in vitro using double-stranded RNA (dsRNA) had no effect on the cell morphology of hematopoietic tissue (HPT) cells. The total hemocyte number and caspase activity were unchanged after PIHHAP knockdown in vivo, in contrast to the results found in shrimp. Moreover, suppression of PIHHAP both in vitro and in vivo did not change the mRNA levels of some genes involved in hematopoiesis and hemocyte homeostasis. Interestingly, bacterial count and scanning electron microscope revealed that depletion of PIHHAP in intestine by RNAi resulted in higher number of bacteria in the crayfish intestine. Together, these results suggest that PIHHAP is not involved in hemocyte homeostasis in the crayfish P. leniusculus but appears to affect the bacterial number in the intestine through an unknown mechanism. Since PIHHAP has different functions from PmHHAP, we therefore named it HHAP-like protein.

  • 4.
    Backström, Tobias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organism Biology, Comparative Physiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Pettersson, Andreas
    Johansson, Viktoria
    Winberg, Svante
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    CRF and urotensin I effects on aggression and anxiety-like behavior in rainbow trout2011In: Journal of Experimental Biology, ISSN 0022-0949, E-ISSN 1477-9145, Vol. 214, no 6, p. 907-914Article in journal (Refereed)
    Abstract [en]

    Corticotropin-releasing factor (CRF) is central in the stress response but also modulates several behaviors including anxiety-related behaviors and aggression. In this study, juvenile rainbow trout (Oncorhynchus mykiss) were tested for competitive ability, determined during dyadic fights for dominance, after intracerebroventricular (i.c.v.) administration of CRF, urotensin I (UI), the non-specific CRF antagonist alpha-helical RF9-41 (ahCRF) or the CRF receptor subtype 1-specific antagonist antalarmin, when paired with a mass-matched con-specific injected with saline. In addition, isolated fish received the same substances. Plasma cortisol and brain monoamines were monitored in all fish. Most fish receiving CRF showed a conspicuous behavior consisting of flaring the opercula, opening the mouth and violent shaking of the head from side to side. When this occurred, the fish immediately forfeited the fight. Similar behavior was observed in most fish receiving UI but no effect on outcome of dyadic fights was noted. This behavior seems similar to non-ambulatory motor activity seen in rats and could be anxiety related. Furthermore, fish receiving CRF at a dose of 1000. ng became subordinate, whereas all other treatments had no effects on the outcome of dyadic fights. In addition, isolated fish receiving ahCRF had lower brain stem concentrations of 5-hydroxyindoleacetic acid, serotonin, 3,4-dihydroxyphenylacetic acid and dopamine. In conclusion, CRF seems to attenuate competitive ability, and both CRF and UI seem to induce anxiety-like behavior.

  • 5.
    Backström, Tobias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organism Biology, Comparative Physiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Schjolden, Joachim
    Overli, Oyvind
    Thörnqvist, Per-Ove
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Winberg, Svante
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness2011In: Hormones and Behavior, ISSN 0018-506X, E-ISSN 1095-6867, Vol. 59, no 1, p. 180-186Article in journal (Refereed)
    Abstract [en]

    The aim for this study was to examine whether the F4 generation of two strains of rainbow trout divergent in their plasma cortisol response to confinement stress (HR: high responder or LR: low responder) would also differ in stress-induced effects on forebrain concentrations of mRNA for corticotropin-releasing factor (CRF). arginine vasotocin (AVT). CRF receptor type 1 (CRF-R1). CRF receptor type 2 (CRF-R2) and AVT receptor (AVT-R). In addition, plasma cortisol concentrations, brainstem levels of monoamines and monoamine metabolites, and behaviour during confinement were monitored. The results confirm that HR and LR trout differ in their cortisol response to confinement and show that fish of these strains also differ in their behavioural response to confinement. The HR trout displayed significantly higher locomotor activity while in confinement than LR trout. Moreover, following 180 min of confinement HR fish showed significantly higher forebrain concentrations of CRF mRNA than LR fish. Also, when subjected to 30 min of confinement HR fish showed significantly lower CRF-R2 mRNA concentrations than LR fish, whereas there were no differences in CRF-R1. AVT or AVT-R mRNA expression between LR and HR fish either at 30 or 180 min of confinement. Differences in the expression of CRF and CRF-R2 mRNA may be related to the divergence in stress coping displayed by these rainbow trout strains.

  • 6.
    Bagchi, Sonchita
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Wallén-Mackenzie, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    In Situ Proximity Ligation Assay (PLA)2015In: ELISA: Methods and Protocols / [ed] Hnasko, R, Springer-Verlag New York, 2015, Vol. 1318, p. 149-159Chapter in book (Refereed)
    Abstract [en]

    In situ proximity ligation assay (PLA) is a method to identify physical closeness of proteins, where a signal will only be produced if the two proteins are closer than 40 nm, in tissue section or cell cultures. Modifications of the PLA method can also be used to increase specificity or sensitivity of standard immunohistochemistry protocols.

  • 7. Benton, Jeanne
    et al.
    Kery, Rachel
    Li, Jingjing
    Noonin, Chadanat
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Beltz, Barbara
    Cells from the Immune System Generate Adult-Born Neurons in Crayfish2014In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 30, no 3, p. 322-333Article in journal (Refereed)
    Abstract [en]

    Neurogenesis is an ongoing process in the brains of adult decapod crustaceans. However, the first-generation precursors that produce adult-born neurons, which reside in a neurogenic niche, are not self-renewing in crayfish and must be replenished. The source of these neuronal precursors is unknown. Here, we report that adult-born neurons in crayfish can be derived from hemocytes. Following adoptive transfer of 5-ethynyl-2′-deoxyuridine (EdU)-labeled hemocytes, labeled cells populate the neurogenic niche containing the first-generation neuronal precursors. Seven weeks after adoptive transfer, EdU-labeled cells are located in brain clusters 9 and 10 (where adult-born neurons differentiate) and express appropriate neurotransmitters. Moreover, the number of cells composing the neurogenic niche in crayfish is tightly correlated with total hemocyte counts (THCs) and can be manipulated by raising or lowering THC. These studies identify hemocytes as a source of adult-born neurons in crayfish and demonstrate that the immune system is a key contributor to adult neurogenesis.

  • 8.
    Benton, JL
    et al.
    Wellesley Coll, Neurosci. Program, Wellesley.
    Zhang, Y
    Wellesley Coll, Neurosci. Program, Wellesley.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Beltz, BS
    Wellesley Coll, Neurosci. Program, Wellesley, MA 02181 USA .
    The crustacean cytokine astakine-1: a link between adult neurogenesis and hematopoiesis?2012Conference paper (Refereed)
    Abstract [en]

    Adult neurogenesis occurs in the brains of vertebrate and invertebrate species. In the crayfish Procambarus clarkii, neurogenic niches located on the ventral surface of the brain contain the 1st-generation precursors that produce adult-born neurons. These cells divide symmetrically and both daughter cells migrate in streams to proliferation zones in interneuronal cell clusters, where they divide at least one more time. Double-nucleoside labeling has confirmed that the 1st-generation neuronal precursors are not self-renewing. However, previous studies have shown that the niche is not depleted and, further, that niche cell numbers increase throughout the animal’s lifetime (Zhang et al., 2009). Our goal, therefore, is to identify the source of niche cells and in particular the 1st-generation neuronal precursors. Experiments have revealed that hemocytes, and in particular semi-granular cells, are attracted to and incorporated into the niches of dissected intact brains in culture; cell types from other tissues show no affinity for the niche (Benton et al., 2011). Lin et al. (2010) demonstrated that a prokineticin family cytokine, astakine-1, promotes the differentiation and release of semi-granular cells from hematopoietic tissue. In the present studies, we have injected astakine-1 into crayfish and examined subsequent changes in niche cell numbers, as well as BrdU incorporation into cells in the niche, streams and proliferation zones. By 48 hrs following astakine-1 injection, three alterations in the neurogenic system have been demonstrated: (1) increased total numbers of cells in the neurogenic niche compared to saline-injected shams; (2) increased BrdU-incorporation into cells in the niche, streams and proliferation zone in cell cluster 10; (3) increased probability of observing cells within the vascular cavity in the niche, implying that the numbers of cells interacting with the niche is upregulated. These experiments exploring the relationship between the hematopoietic system and adult neurogenesis suggest that cells released from hematopoietic tissues following astakine-1 injection influence the cellular composition of the niche and the rate of BrdU-incorporation into niche cells.

  • 9.
    Berg, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
    Backström, Tobias
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Winberg, Svante
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Lindberg, Richard
    Brandt, Ingvar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
    Developmental Exposure to Fluoxetine Modulates the Serotonin System in Hypothalamus2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 1, p. e55053-Article in journal (Refereed)
    Abstract [en]

    The selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLU, Prozac (R)) is commonly prescribed for depression in pregnant women. This results in SSRI exposure of the developing fetus. However, there are knowledge gaps regarding the impact of SSRI exposure during development. Given the role of serotonin in brain development and its cross-talk with sex hormone function, we investigated effects of developmental exposure to pharmacologically relevant concentrations of FLU (3 and 30 nM (measured)) on brain neurotransmitter levels, gonadal differentiation, aromatase activity in brain and gonads, and the thyroid system, using the Xenopus tropicalis model. Tadpoles were chronically exposed (8 weeks) until metamorphosis. At metamorphosis brains were cryosectioned and levels of serotonin, dopamine, norepinephrine, and their metabolites 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were measured in discrete regions (telencephalon, hypothalamus and the reticular formation) of the cryosections using high-performance liquid chromatography. Exposure to 30 nM FLU increased the concentration of 5-hydroxyindoleacetic acid in hypothalamus compared with controls. FLU exposure did not affect survival, time to metamorphosis, thyroid histology, gonadal sex differentiation, or aromatase activity implying that the effect on the serotonergic neurotransmitter system in the hypothalamus region was specific. The FLU concentration that impacted the serotonin system is lower than the concentration measured in umbilical cord serum, suggesting that the serotonin system of the developing brain is highly sensitive to in utero exposure to FLU. To our knowledge this is the first study showing effects of developmental FLU exposure on brain neurochemistry. Given that SSRIs are present in the aquatic environment the current results warrant further investigation into the neurobehavioral effects of SSRIs in aquatic wildlife.

  • 10.
    Cerenius, Lage
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Andersson, M. Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Aphanomyces astaci and crustaceans.2009In: Oomycete Genetics and Genomics.: Diversity, Interactions, and Research Tools. / [ed] Kurt Lamour and Sophien Kamoun, Hoboken, New Jersey: John Wiley & Sons, Inc. , 2009, p. 425-433Chapter in book (Other academic)
  • 11.
    Cerenius, Lage
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Jiravanichpaisal, Pikul
    Liu, Hai-peng
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Crustacean Immunity2010In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 708, p. 239-259Article in journal (Refereed)
    Abstract [en]

    This chapter provides a review of recent progress in the elucidation of innate immune mechanisms in crustaceans. Mainly due to the importance of crustacean aquaculture interest in this field is large and the subject for extensive research efforts. Here, we provide detailed data on the molecular characterisation of lectins, antiviral reactions, hemocyte formation and differentiation and on the regulation of innate immune pathways.

  • 12.
    Cerenius, Lage
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Kawabata, Shun-ichiro
    Lee, Bok Luel
    Nonaka, Masaru
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Proteolytic cascades and their involvement in invertebrate immunity2010In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 35, no 10, p. 575-583Article, review/survey (Refereed)
    Abstract [en]

    Bacteria and other potential pathogens are cleared rapidly from the body fluids of invertebrates by the immediate response of the innate immune system. Proteolytic cascades, following their initiation by pattern recognition proteins, control several such reactions, notably coagulation, melanisation, activation of the Toll receptor and complement-like reactions. However, there is considerable variation among invertebrates and these cascades, although widespread, are not present in all phyla. In recent years, significant progress has been made in identifying and characterizing these cascades in insects. Notably, recent work has identified several connections and shared principles among the different pathways, suggesting that cross-talk between them may be common.

  • 13.
    Cerenius, Lage
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Kawabata, Shun-ichiro
    Kyushu University, Fukuoka, Japan.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Biological and Immunological Aspects of Innate Defence Mechanisms Activated by (1,3)- β -Glucans and Related Polysaccharides in Invertebrates2009In: Chemistry, Biochemistry and Biology of (1-›3)-β-Glucans and Related Polysaccharides. / [ed] Antony Bacic, Geoffrey B. Fincher & Bruce A. Stone, Burlington, MA: Academic Press , 2009, p. 563-577Chapter in book (Other academic)
    Abstract [en]

    (1,3)- β -glucans are powerful stimulants of a wide variety of innate defence reactions in invertebrates. These polysaccharides exert a great influence on reactions such as induction of antimicrobial peptides, cellular defence such as encapsulation and phagocytosis, and on the melanization and coagulation cascades. In most cases, these reactions set up an effective defence against microorganisms containing (1,3)- β -glucans or (1,3;1,6)- β -glucans in their outer structures (i.e. mainly fungi and oomycetes).

     

  • 14.
    Cerenius, Lage
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Arthropoda: Pattern recognition proteins in crustacean immunity.2018Book (Other academic)
  • 15.
    Cerenius, Lage
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Coagulation in invertebrates2011In: Journal of Innate Immunity, ISSN 1662-811X, Vol. 3, no 1, p. 3-8Article, review/survey (Refereed)
    Abstract [en]

    In most animals there is a need to quickly prevent the loss of blood or equivalent fluids through inflicted injuries. In invertebrates with an open circulatory system (and sometimes a hydroskeleton as well) these losses may otherwise soon be fatal. Also, there is a need to prevent microbes that have gained access to the body through the wound from disseminating throughout the open circulatory system. Therefore, many invertebrates possess a coagulation system to prevent such accidents from having too serious consequences. In this review we discuss recent developments in a few animals - mainly arthropods - where more detailed data are available. It is likely, however, that corresponding systems are present in most phyla, but this is still unchartered territory.

  • 16.
    Cerenius, Lage
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Commentary: variable immune molecules in invertebrates2013In: Journal of Experimental Biology, ISSN 0022-0949, E-ISSN 1477-9145, Vol. 216, no 23, p. 4313-4319Article in journal (Other academic)
    Abstract [en]

    Recently it has become evident that invertebrates may mount a highly variable immune response that is dependent on which pathogen is involved. The molecular mechanisms behind this diversity are beginning to be unravelled and in several invertebrate taxa immune proteins exhibiting a broad range of diversity have been found. In some cases, evidence has been gathered suggesting that this molecular diversity translates into the ability of an affected invertebrate to mount a defence that is specifically aimed at a particular pathogen.

  • 17.
    Cerenius, Lage
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Crayfish immunity: Recent findings2018In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 80, p. 94-98Article in journal (Refereed)
    Abstract [en]

    Freshwater crayfish is an important commodity as well as a successful model for studies on crustacean immunity. Due to the ease with which they are kept and the available methods for hemocyte separation and culture they have proven to be very useful. Here, recent progress regarding pattern recognition, immune effector production and antiviral mechanisms are discussed. Several cases of functional resemblance between vertebrate complement and the crayfish immune reactions are highlighted.

  • 18.
    Cerenius, Lage
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Crustacean immune responses and their implications for disease control2012In: Infectious disease in aquaculture: prevention and control / [ed] Austin, B., Cambridge: Woodhead Publishing Limited, 2012, p. 69-87Chapter in book (Refereed)
    Abstract [en]

    This chapter reviews recent advances in our knowledge of crustacean immunity. Emphasis is given to shrimp due to their importance in aquaculture and trade and to freshwater crayfish since they serve as model organisms for research in crustacean immunology. Crustaceans lack antibodies, interferon and some other components from the mammalian immune arsenal but can still mount an efficient defence against many potential pathogens. Crustacean innate immunity relies on a combination of efficient hemocyte and humoral reactions carried out by plasma proteins.

  • 19. Diao, Yupu
    et al.
    Lu, Anrui
    Yang, Bing
    Hu, Wenli
    Peng, Qing
    Ling, Qing-Zhi
    Beerntsen, Brenda T.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Ling, Erjun
    Existence of Prophenoloxidase in Wing Discs: A Source of Plasma Prophenoloxidase in the Silkworm, Bombyx mori2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, p. e41416-Article in journal (Refereed)
    Abstract [en]

    In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.

  • 20.
    Donpudsa, Suchao
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Rimphanitchayakit, Vichien
    Tassanakajon, Anchalee
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus2010In: Journal of Invertebrate Pathology, ISSN 0022-2011, E-ISSN 1096-0805, Vol. 104, no 3, p. 234-238Article in journal (Refereed)
    Abstract [en]

    The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coil expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 mu M and 3.5-8 mu M, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.

  • 21.
    Ericsson, Lena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Astakines in arthropods-phylogeny and gene structure2018In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 81, p. 141-151Article in journal (Refereed)
    Abstract [en]

    Astakinel was isolated as a hematopoietic cytokine in the freshwater crayfish Pacifastacus leniusculus. In this study we detect and compare 79 sequences in GenBank, which we consider to be possible astakine orthologs, among which eleven are crustacean, sixteen are chelicerate and 52 are from insect species. Available arthropod genomes are searched for astakines, and in conclusion all astakine sequences in the current study have a similar exon containing CCXX(X), thus potentially indicating that they are homologous genes with the structure of this exon highly conserved. Two motifs, RYS and YP(N), are also conserved among the arthropod astakines. A phylogenetic analysis reveals that astakinel and astakine2 from P. leniusculus and Procambarus clarkii are distantly related, and may have been derived from a gene duplication occurring early in crustacean evolution. Moreover, a structural comparison using the Mamba intestinal toxin (MITI) from Dendroaspis polylepis as template indicates that the overall folds are similar in all crustacean astakines investigated.

  • 22.
    Guo, Enen
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Korkut, Gül Gizem
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Jaree, Phattarunda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    A Pacifastacus leniusculus serine protease interacts with WSSV2017In: Fish and Shellfish Immunology, ISSN 1050-4648, E-ISSN 1095-9947, Vol. 68, p. 211-219Article in journal (Refereed)
    Abstract [en]

    Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PIcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S)motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PIcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells.

  • 23.
    Isaksson, Amanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Optimization of PCR protocols used for genotyping transgenic mice & Evaluation of a method for co-detecting mRNA and protein2017Independent thesis Basic level (professional degree), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    The aim of the current study was divided into two separate goals, (i) optimization of a number of PCR-based protocols employed for genotyping transgenic mouse lines and (ii) evaluating a protocol for co-detection of mRNA and its correlated protein in the mouse midbrain.

    The optimization was performed on PCR protocols for genotyping the following transgenic mouse lines; Dat-Cre, Vglut2-Lox, Vglut2-Cre and Vmat2-Lox. Also, two different polymerases were evaluated parallel to each other – KAPA and Maxima Hot Start. One of the main findings from the PCR optimizations were that for the Vglut2-Lox protocol. By decreasing the annealing temp and increasing the MgCl2 the bands appeared brighter. 

    For the second part of the project, in-situ hybridization (ISH) was used to detect the mRNA expression with a `non-radioactive in situ hybridization´ protocol, using digoxigenin or fluorescein labelled riboprobes (mRNA probes). To detect the correlated protein a basic immunohistochemistry (IHC) protocol with the use of primary and secondary antibodies was implemented. The combined protocol was tested with Nd6 and Grp markers. Before testing to combined the protocols the ISH protocol was performed alone with riboprobes for Girk2, Lpl and Fst. The combined protocol detected mRNA and protein for both the control marker Th and the Nd6 marker.

    In conclusions, the optimized PCR protocols were optimal when used with the Maxima Hot Start polymerase and the new combined ISH and IHC protocol worked for markers Th and Nd6.

  • 24.
    Jearaphunt, Miti
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Noonin, Chadanat
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Jiravanichpaisal, Pikul
    Nakamura, Seiko
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Tassanakajon, Anchalee
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity2014In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 10, no 4, p. e1004059-Article in journal (Refereed)
    Abstract [en]

    Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.

  • 25.
    Jiravanichpaisal, Pikul
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Inflammation in Arthropods2010In: Current pharmaceutical design, ISSN 1381-6128, E-ISSN 1873-4286, Vol. 16, no 38, p. 4166-4174Article, review/survey (Refereed)
    Abstract [en]

    The inflammatory process in arthropods includes primarily the recruitment of circulating hemocytes to wounds or sites of microbial infections. Melanization, capsule formation and clotting reactions will finally result in the sealing of wounds. In this review we will focus on recent research about hemolymph clotting and melanization reactions, and the recruitment of hemocytes to wounds and infections. We further describe in more detail new knowledge about crustacean hematopoiesis that is crucial for hemocyte recruitment to the site of an infection and there develop an inflammatory response Moreover, we pay special attention to the gut as an important route of infection in arthropods. Since the gastrointestinal tract provides a first line of defense and regulation of the indigenous bacteria and the intestine often harbors loads of potential pathogenic microorganisms. Therefore the integrity of intestinal epithelium and to maintain the correct flora is crucial to animal health.

  • 26.
    Johansson, Karin C
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Lind, Maria I
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Pefabloc: A sulfonyl fluoride serine protease inhibitor blocks induction of Diptericin in Drosophila l(2)mbn cells2012In: Insect Science, ISSN 1672-9609, E-ISSN 1744-7917, Vol. 19, no 4, p. 472-476Article in journal (Refereed)
    Abstract [en]

    Insects protect themselves against microbial infection by an efficient innate immune system that is activated by recognition of invariant microbial surface molecules. In the fruit fly Drosophila melanogaster the presence of bacteria is associated with expression of antimicrobial peptides in host immune-competent tissues. Host receptors detect infection and relay the signal to mount the appropriate immune response. In Drosophila hemocyte-like l(2)mbn cells pre-infection treatment with Pefabloc, a commonly used serine protease inhibitor, induced two major effects: it blocked expression of the antibacterial peptide Diptericin in response to live Gram-negative bacteria and bacterial surface molecules (crude lipopolysaccharide contaminated by peptidoglycans) and it induced morphological changes.

  • 27. Ju, Jin Sung
    et al.
    Cho, Mi Hyang
    Brade, Lore
    Kim, Jung Hyun
    Park, Ji Won
    Ha, Nam-Chul
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Brade, Helmut
    Lee, Bok Luel
    A novel 40-kDa protein containing six repeats of an epidermal growth factor-like domain functions as a pattern recognition protein for lipopolysaccharide.2006In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 177, no 3Article in journal (Refereed)
    Abstract [en]

    Determination of structures and functions of pattern recognition proteins are important for understanding pathogen recognition mechanisms in host defense and for elucidating the activation mechanism of innate immune reactions. In this study, a novel 40-kDa protein, named LPS recognition protein (LRP), was purified to homogeneity from the cell-free plasma of larvae of the large beetle, Holotrichia diomphalia. LRP exhibited agglutinating activities on Escherichia coli, but not on Staphylococcus aureus and Candida albicans. This E. coli-agglutinating activity was preferentially inhibited by the rough-type LPS with a complete core oligosaccharide. LRP consists of 317 aa residues and six repeats of an epidermal growth factor-like domain. Recombinant LRP expressed in a baculovirus system also showed E. coli agglutination activity in vitro and was able to neutralize LPS by inhibition of LPS-induced IL-6 production in mouse bone marrow mast cells. Furthermore, E. coli coated with the purified LRP were more rapidly cleared in the Holotrichia larvae than only E. coli, indicating that this protein participates in the clearance of E. coli in vivo. The three amino-terminal epidermal growth factor-like domains of LRP, but not the three carboxyl epidermal growth factor-like domains, are involved in the LPS-binding activity. Taken together, this LRP functions as a pattern recognition protein for LPS and plays a role as an innate immune protein.

  • 28.
    Junkunlo, Kingkamon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    A comparative global proteomic analysis of the hematopoietic lineages in the crustacean Pacifastacus leniusculus.2018In: Dev Comp Immunol.Article in journal (Refereed)
    Abstract [en]

    In crustaceans as in other arthropods, the circulating hemocytes are vital for protecting the animal against attacking microorganisms. As many hemocytes are destroyed early during an infection, new hemocytes must fast get in place to prevent disperse of a pathogenic microbe, In order to understand the hematopoietic process in more detail we here report a complete proteomic analysis from purified cell types from the APC of the hematopoietic tissue, via the remaining parts of the HPT to the mature semigranular and granular hemocytes. Several possible cell type specific proteins are detected and new putative biomarkers within the crayfish hematopoietic lineage that can be used to increase the understanding of how the differentiation process is regulated is described.

  • 29.
    Junkunlo, Kingkamon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Regulation of hematopoiesis in the freshwater crayfish, Pacifastacus leniusculus: role of transglutaminase2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The freshwater crayfish, Pacifastacus leniusculus, has been used as a model for studying hematopoiesis or blood cell production or hematopoiesis and immunity. The work of this thesis aims to investigate the impact of factors such as ROS signaling, Ast1, and the PVF/PVR signaling pathway in controlling stem cell behavior during hematopoiesis and specifically the role of the crosslinking enzyme transglutaminase (TGase) in regulation of hematopoiesis.

    The role of ROS in crayfish hematopoiesis was characterized by using the antioxidant named NAC to inhibit ROS production. Low ROS level resulted in a prolonged decrease in hemocyte numbers and a combined injection of LPS and NAC caused a slower rate of new hemocyte production. A low ROS level in cell cultures supplemented with crude Ast1 was found to inhibit cell spreading and a high extracellular TGase activity was detected on the surfaces of APC and HPT cells. We suggest that ROS serves as a prime signal to control proliferation and differentiation of progenitor cells by affecting extracellular TGase activity. We reported an inhibitory effect of Ast1 on TGase enzyme activity and on its crosslinking activity and consequently Ast1 affects the clot formation and thus coagulation by inhibiting the crosslinking activity of the TGase enzyme. Secretion of the clot protein (CP) and the production of CP filament network between spreading cells were observed in HPT cell cultures in vitro. In the presence of CP together with Ast1 in 3D-collagen-I cultures, HPT cells were found to be more elongated and they formed chains of cells throughout the surrounding matrix. In the HPT tissue, CP was located around the HPT cells or around the lobules of HPT, and thus, CP was demonstrated to be a part of ECM and to possibly function together with collagen in generating a suitable environment for HPT progenitor cells. The inhibition of PVF/PVR downstream signaling pathway by Sunitinib malate resulted in a dramatic change of cell morphology and induction of an increase cell surface area during cell culture. The addition of crude Ast1 into the cell cultures in vitro enhanced this effect. Consequently, cell migration was stimulated and a high extracellular TGase activity on HPT cell surface was found after this inhibition. In conclusion, the work in this thesis provides new insight in understanding the role of the extracellular matrix (ECM) and extracellular TGase activity in controlling stem cell activity.

    List of papers
    1. Reactive oxygen species affect transglutaminase activity and regulate hematopoiesis in a crustacean
    Open this publication in new window or tab >>Reactive oxygen species affect transglutaminase activity and regulate hematopoiesis in a crustacean
    2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 34, p. 17593-17601Article in journal (Refereed) Published
    Abstract [en]

    Reactive oxygen species (ROS) serve as a prime signal in the commitment to hematopoiesis in both mammals and Drosophila. In this study, the potential function of ROS during hematopoiesis in the crayfish Pacifastacus leniusculus was examined. The antioxidant N-acetylcysteine (NAC) was used to decrease ROS in both in vivo and in vitro experiments. An increase in ROS was observed in the anterior proliferation center (APC) after LPS injection. In the absence of NAC, the LPS-induced increase in ROS levels resulted in the rapid restoration of the circulating hemocyte number. In the presence of NAC, a delay in the recovery rate of the hemocyte number was observed. NAC treatment also blocked the spread of APC and other hematopoietic tissue (HPT) cells, maintaining these cells at an undifferentiated stage. Extracellular transglutaminase (TGase) has been shown previously to play a role in maintaining HPT cells in an undifferentiated form. In this study, we show that extracellular TGase activity increased when the ROS level in HPT or APC cells was reduced after NAC treatment. In addition, collagen, a major component of the extracellular matrix and a TGase substrate were co-localized on the HPT cell surface. Taken together, the results of this study show that ROS are involved in crayfish hematopoiesis, in which a low ROS level is required to maintain hematopoietic progenitor cells in the tissue and to reduce hemocyte release. The potential roles of TGase in this process are investigated and discussed.

    Keywords
    extracellular matrix, hematopoiesis, invertebrate, reactive oxygen species (ROS), transglutaminase
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biology with specialization in Comparative Physiology
    Identifiers
    urn:nbn:se:uu:diva-305568 (URN)10.1074/jbc.M116.741348 (DOI)000383241300011 ()27339892 (PubMedID)
    Funder
    Swedish Research Council, VR 20114797, VR 621-2012-2418
    Available from: 2016-10-19 Created: 2016-10-19 Last updated: 2017-08-22
    2. Role of astakine1 in regulating transglutaminase activity
    Open this publication in new window or tab >>Role of astakine1 in regulating transglutaminase activity
    2017 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 76, p. 77-82Article in journal (Refereed) Published
    Abstract [en]

    Transglutaminase (TGase) has been implicated in maintaining the undifferentiated stage of hematopoietic stem cells (HSC) in the crayfish Pacifastacus leniusculus. TGase activity has been reported to be regulated by astakine1, an essential crayfish cytokine for inducing new hemocyte synthesis in hematopoietic tissue (HPT). Here, the role of astakine1 in TGase activity regulation and clotting protein (CP) cross-linking was characterized. A reduction in TGase activity was observed by the addition of purified astakine1 in vitro for both endogenous crayfish TGase and a commercial purified guinea pig liver TGase. As a result, we observed that astakine1 inhibits TGase enzyme activity and acts as a non-competitive inhibitor for the TGase enzyme. Additionally, the clotting reaction was impaired in the presence of astakine1. A decrease in TGase-mediated crosslinking of ε(γ-glutamyl)-lysine bonds was also observed in the presence of astakine1. In conclusion, this study shows that astakine1 acts as an inhibitor of TGase activity and that it also affects CP cross-linking during crayfish hematopoiesis.

    Place, publisher, year, edition, pages
    Elsevier, 2017
    Keywords
    Astakine1, Clotting protein, Hematopoiesis, Transglutaminase activity
    National Category
    Developmental Biology Immunology Zoology
    Research subject
    Biology with specialization in Comparative Physiology
    Identifiers
    urn:nbn:se:uu:diva-327217 (URN)10.1016/j.dci.2017.05.015 (DOI)000407985100009 ()28528959 (PubMedID)
    Funder
    Swedish Research Council, VR 621-2012-2418
    Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2017-10-09Bibliographically approved
    3. PDGF/VEGF-related receptor affects transglutaminase activity to control cell migration during crustacean hematopoiesis
    Open this publication in new window or tab >>PDGF/VEGF-related receptor affects transglutaminase activity to control cell migration during crustacean hematopoiesis
    2017 (English)In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 26, no 20, p. 1449-1459Article in journal (Refereed) Published
    Abstract [en]

    The platelet-derived growth factor (PDGF) receptor, a tyrosine kinase (TK) receptor whose ligand is PDGF, is crucial in the transduction of extracellular signals into cells and mediates numerous processes, such as cell proliferation, differentiation, survival, and migration. We demonstrate the important roles of a receptor TK related to the PDGF/VEGF family protein (PVR) in controlling hematopoietic progenitor cell migration by affecting extracellular transglutaminase (TGase) activity. Pl_PVR1, GenBank accession No. KY444650, is highly expressed in hemocytes and the hematopoietic tissue (HPT). Sunitinib malate was used to block the PVF/PVR downstream pathway in HPT cell culture. The addition of Sunitinib also caused the HPT cells to increase in size and begin spreading. An increase in extracellular TGase activity on the HPT cell membrane was observed in a dose-dependent manner after treatment with Sunitinib malate. The presence of crude Ast1 provided a combinatorial beneficial effect that enhanced the number of spreading cells after inhibition of the Pl_PVR downstream signaling cascade. In addition, an increased immunoreactivity for beta-tubulin and elongation of beta-tubulin filaments were found in Pl_PVR signaling-inhibited cells. The potential roles of PVF/PVR signaling in controlling progenitor cell activity during hematopoiesis in crayfish were investigated and discussed.

    Keywords
    PDGF/VEGF, hematopoiesis, Transglutaminase, Ast1, crayfish
    National Category
    Developmental Biology Cell Biology Immunology
    Research subject
    Biology with specialization in Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-327243 (URN)10.1089/scd.2017.0086 (DOI)000412919700001 ()28805145 (PubMedID)
    Funder
    Swedish Research Council, VR 2011-4797, VR 621-2012-2418
    Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2018-01-03Bibliographically approved
    4. Clotting protein - an extracellular matrix (ECM) protein involved in crustacean hematopoiesis
    Open this publication in new window or tab >>Clotting protein - an extracellular matrix (ECM) protein involved in crustacean hematopoiesis
    (English)In: ISSN 0145-305XArticle in journal (Refereed) Submitted
    Keywords
    clotting protein, ECM, hematopoiesis, crustacean
    National Category
    Immunology Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-327248 (URN)
    Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2017-08-14
  • 30.
    Junkunlo, Kingkamon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Noonin, Chadanat
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    PDGF/VEGF-related receptor affects transglutaminase activity to control cell migration during crustacean hematopoiesis2017In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 26, no 20, p. 1449-1459Article in journal (Refereed)
    Abstract [en]

    The platelet-derived growth factor (PDGF) receptor, a tyrosine kinase (TK) receptor whose ligand is PDGF, is crucial in the transduction of extracellular signals into cells and mediates numerous processes, such as cell proliferation, differentiation, survival, and migration. We demonstrate the important roles of a receptor TK related to the PDGF/VEGF family protein (PVR) in controlling hematopoietic progenitor cell migration by affecting extracellular transglutaminase (TGase) activity. Pl_PVR1, GenBank accession No. KY444650, is highly expressed in hemocytes and the hematopoietic tissue (HPT). Sunitinib malate was used to block the PVF/PVR downstream pathway in HPT cell culture. The addition of Sunitinib also caused the HPT cells to increase in size and begin spreading. An increase in extracellular TGase activity on the HPT cell membrane was observed in a dose-dependent manner after treatment with Sunitinib malate. The presence of crude Ast1 provided a combinatorial beneficial effect that enhanced the number of spreading cells after inhibition of the Pl_PVR downstream signaling cascade. In addition, an increased immunoreactivity for beta-tubulin and elongation of beta-tubulin filaments were found in Pl_PVR signaling-inhibited cells. The potential roles of PVF/PVR signaling in controlling progenitor cell activity during hematopoiesis in crayfish were investigated and discussed.

  • 31.
    Junkunlo, Kingkamon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Clotting protein - an extracellular matrix (ECM) protein involved in crustacean hematopoiesisIn: ISSN 0145-305XArticle in journal (Refereed)
  • 32.
    Junkunlo, Kingkamon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Clotting protein: An extracellular matrix (ECM) protein involved in crustacean hematopoiesis2018In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 78, p. 132-140Article in journal (Refereed)
    Abstract [en]

    Hematopoietic progenitor cells in crustaceans are organized in lobule-like structures surrounded by different types of cells and extracellular matrix (ECM) proteins in a Hematopoietic tissue (HPT). Here we show that the clotting protein (CP) is part of the ECM in HPT and is secreted during HPT cell culture. The formation of a filamentous network of CP was observed in HPT cell culture. A high amount of CP protein was detected at the surfaces of undifferentiated cells (round-shaped) compared with migrating cells (spindle shaped). Co-localization of the CP protein and TGase activity was observed on the cell surface and filamentous network between cells. A role for CP together with collagen was revealed in a 3D culture in which a collagen-I matrix was immobilized with CP or supplemented with CP. The results showed possible functions of CP, collagen, TGase and the cytokine Ast1 in the regulation of HPT progenitor cell behavior. This is the first study to provide insight into the role of CP, which probably not only participates in clot formation but also functions as an ECM component protein controlling hematopoietic stem cell behavior.

  • 33.
    Junkunlo, Kingkamon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Noonin, Chadanat
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Reactive oxygen species affect transglutaminase activity and regulate hematopoiesis in a crustacean2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 34, p. 17593-17601Article in journal (Refereed)
    Abstract [en]

    Reactive oxygen species (ROS) serve as a prime signal in the commitment to hematopoiesis in both mammals and Drosophila. In this study, the potential function of ROS during hematopoiesis in the crayfish Pacifastacus leniusculus was examined. The antioxidant N-acetylcysteine (NAC) was used to decrease ROS in both in vivo and in vitro experiments. An increase in ROS was observed in the anterior proliferation center (APC) after LPS injection. In the absence of NAC, the LPS-induced increase in ROS levels resulted in the rapid restoration of the circulating hemocyte number. In the presence of NAC, a delay in the recovery rate of the hemocyte number was observed. NAC treatment also blocked the spread of APC and other hematopoietic tissue (HPT) cells, maintaining these cells at an undifferentiated stage. Extracellular transglutaminase (TGase) has been shown previously to play a role in maintaining HPT cells in an undifferentiated form. In this study, we show that extracellular TGase activity increased when the ROS level in HPT or APC cells was reduced after NAC treatment. In addition, collagen, a major component of the extracellular matrix and a TGase substrate were co-localized on the HPT cell surface. Taken together, the results of this study show that ROS are involved in crayfish hematopoiesis, in which a low ROS level is required to maintain hematopoietic progenitor cells in the tissue and to reduce hemocyte release. The potential roles of TGase in this process are investigated and discussed.

  • 34.
    Kery, R
    et al.
    Neurosci. Program, Wellesley Col., Wellesley.
    Beltz, BS
    Neurosci. Program, Wellesley Col., Wellesley .
    Benton, JL
    Neurosci. Program, Wellesley Col., Wellesley .
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Hematopoietic-derived cells as potential neural precursors in adult neurogenesis2012Conference paper (Other academic)
    Abstract [en]

    Adult neurogenesis occurs in many vertebrate and invertebrate species, including decapod crustaceans. In the crayfish Procambarus clarkii, new neurons are integrated into interneuronal cell clusters innervating the olfactory and accessory lobes. The 1st-generation precursor cells reside in a niche, where they divide symmetrically; both daughter cells migrate along streams to proliferation zones in the cell clusters, where additional divisions and neuronal differentiation occur. Although divisions of existing niche cells do not replenish the niche, niche cell numbers increase with age. Niche precursor cells must, therefore, originate from an extrinsic source. It has been hypothesized that circulating stem cells of possible hematopoietic origin migrate into the niche from the hemolymph. Previous studies in crayfish have suggested that circulating semi-granular cells are attracted to the niche (Benton et al., 2011). Astakine-1, a prokineticin, has been shown to promote the differentiation and release of semi-granular cells from the hematopoietic tissue (Lin and Soderhall, 2011). Astakine-1 injection also results in increased numbers of cells in the neurogenic niche. In this study, hematopoietic tissue (HPT) was ablated in order to better define the relationship between the hematopoietic system and adult neurogenesis. The most dorsal regions of HPT were removed, leaving some ventral blood-generating tissue. Ten days post-surgery, ablated animals had fewer niche cells relative to sham controls (p<0.001). There were no differences in numbers of dividing (BrdU-labeled) cells in the niche or cell clusters where the adult-born neurons reside. If astakine-1 is injected into HPT-ablated animals 48 hours prior to sacrifice, this deficit in niche cells is ameliorated. A strong positive correlation was found between the numbers of circulating hemocytes immediately before sacrifice and the numbers of cells in the neurogenic niche. Therefore, following HPT ablation the numbers of niche cells are reduced, but can be restored by astakine-1 injection after ablation. Taken together, these data demonstrate a close and dynamic relationship between hematopoiesis and adult neurogenesis in the crayfish brain.

  • 35. Kong, Lulu
    et al.
    Lu, Anrui
    Guan, Jingmin
    Yang, Bing
    Li, Muwang
    Hillyer, Julian F.
    Ramarao, Nalini
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Liu, Chaoliang
    Ling, Erjun
    Thermolysin Damages Animal Life Through Degradation of Plasma Proteins Enhanced by Rapid Cleavage of Serpins and Activation of Proteases2015In: Archives of Insect Biochemistry and Physiology, ISSN 0739-4462, E-ISSN 1520-6327, Vol. 88, no 1, p. 64-84Article in journal (Refereed)
    Abstract [en]

    Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.

  • 36.
    Korkut, Gül Gizem
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Interaction between crayfish and some microorganisms; Effect of temperature2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Innate immunity, which constitutes the first line of defense in vertebrates, is the only immune system that invertebrates rely on to protect themselves from pathogens. The invertebrate immune system is composed of cellular and humoral components. Cellular immunity is phagocytosis, opsonization and encapsulation. The humoral part is mainly composed of the events taking place upon secretion of granules and the enzymes within that lead to the lysis of the pathogen by antimicrobial peptides (AMPs) and the melanization cascade. The Prophenoloxidase (proPO) activating system is an important pathway that is stored in the granules of semigranular and granular hemocytes (blood cells). These cells will degranulate and release the proPO system when activated upon pathogen recognition. This cascade results in the melanization reaction and to trap and eliminate pathogens. 

    White spot syndrome virus (WSSV) is a deadly pathogen mainly targeting crustaceans and causing huge economic losses since its first emergence in 1992 in Taiwan. It is known that WSSV disables the immune system of the host by interfering with the proPO cascade. Temperature is a restricting factor for the WSSV infections however it is not known if its affects are on host immunity or on the virus itself.

    With the aim of elucidating WSSV infection, we studied the virus entry mechanisms. By crosslinking WSSV with the hemocytes we showed that a new clip-domain serine protease (PlcSP) plays an important role during the WSSV infection in crayfish by means of interacting with WSSV envelope protein VP28. Moreover, we have shown that the viral entry is inhibited at cold temperatures due to temperature’s inhibitory effect on PlcSP expression. We also showed that by slowing down of the host’s metabolism hence proliferation in host tissue either by low temperature or cell cycle inhibitors, we could inhibit WSSV replication once it has entered the host cell. We tested if the temperature effects host or pathogen, or both, we investigated the mortalities, phagocytosis, bacterial clearance, total hemocyte counts, degranulation and melanization rate of crayfish under a cold and warm temperature by using two strains of gram-negative bacteria and LPS. It is apparent that the cellular immunity is more effective at low temperature while the humoral immunity can become overactivated and toxic for the host at higher temperature. Furthermore, we aimed to study the cleavage specificity for PlcSP since it is predicted to be secreted from hemocytes and takes part in the serine protease cascade during melanization reaction.

    List of papers
    1. A Pacifastacus leniusculus serine protease interacts with WSSV
    Open this publication in new window or tab >>A Pacifastacus leniusculus serine protease interacts with WSSV
    Show others...
    2017 (English)In: Fish and Shellfish Immunology, ISSN 1050-4648, E-ISSN 1095-9947, Vol. 68, p. 211-219Article in journal (Refereed) Published
    Abstract [en]

    Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PIcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S)motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PIcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells.

    Keywords
    Hematopoietic tissue, Invertebrate, Serine protease, Virus, WSSV
    National Category
    Immunology
    Identifiers
    urn:nbn:se:uu:diva-335858 (URN)10.1016/j.fsi.2017.07.026 (DOI)000411299500022 ()28705723 (PubMedID)
    Funder
    Swedish Research Council, 621-2012-2418
    Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-06-26Bibliographically approved
    2. The effect of temperature on White Spot Disease progression in a crustacean, Pacifastacus leniusculus
    Open this publication in new window or tab >>The effect of temperature on White Spot Disease progression in a crustacean, Pacifastacus leniusculus
    2018 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 89, p. 7-13Article in journal (Refereed) Published
    Abstract [en]

    The effects of temperature on the progression of White Spot Disease (WSD) have been studied in the freshwater crayfish Pacifastacus leniusculus. In this study, we aimed to understand the reason for previously observed low mortalities with white spot syndrome virus (WSSV) infected crayfish at low temperatures. The susceptibility of freshwater crayfish to WSSV was studied at different temperatures. The mortality rate at 6°C was zero, meanwhile the animals kept at 22°C developed WSD symptoms and died in a few days after WSSV injections, however upon transfer of animals from 6°C to 22°C the mortality reached 100% indicating that the virus is not cleared. Moreover, the VP28 expression at 6°C was significantly lower compared to animals kept at 22°C. We injected animals with demecolcine, an inhibitor that arrests the cell cycle in metaphase, and observed a delayed mortality. Furthermore, the VP28 expression was found to be lower in these animals receiving both injections with WSSV and demecolcine since cell proliferation was inhibited by demecolcine. We quantified WSSV copy numbers and found that virus entry was blocked at 6°C, but not in demecolcine treatments. We supported this result by quantifying the expression of a clip domain serine protease (PlcSP) which plays an important role for WSSV binding, and we found that the PlcSP expression was inhibited at 6°C. Therefore, our hypothesis is that the WSSV needs proliferating cells to replicate, and an optimum temperature to enter the host hematopoietic stem cells successfully. 

    Keywords
    White spot syndrome virus, temperature, innate immunity, crustacean, serine protease
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-356485 (URN)10.1016/j.dci.2018.07.026 (DOI)
    Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2018-11-27Bibliographically approved
    3. Expression of an active and inactive clip-domain Serine Protease Isolated from Signal Crayfish in an insect cell line
    Open this publication in new window or tab >>Expression of an active and inactive clip-domain Serine Protease Isolated from Signal Crayfish in an insect cell line
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    In this study, we aimed to elucidate the cleavage specificity and endogenous substrate of a clip-domain serine protease from the crayfish Pacifastacus leniusculus (PlcSP). This serine protease can bind to white spot syndrome virus (WSSV) and is important for the entry of WSSV into the hematopoietic tissue (HPT). In order to understand its intrinsic role in the serine protease cascade, we wanted to use substrate phage display method to clarify cleavage specificity of PlcSP. Firstly, we aimed to make recombinant active and inactive forms of this PlcSP using human embryonic kidney cell line (HEK293) however PlcSP appeared to be toxic for mammalian cells and we were not able to express this crustacean protein. However, we were able to express these recombinantPlcSP both in their zymogenic and active forms in Sf9 insect cell line.

    Keywords
    Serine protease, Pacifastacus leniusculus, invertebrate immunity, Sf9, substrate phage display
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-356482 (URN)
    Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2018-08-02
  • 37.
    Korkut, Gül Gizem
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Noonin, Chadanat
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The effect of temperature on White Spot Disease progression in a crustacean, Pacifastacus leniusculus2018In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 89, p. 7-13Article in journal (Refereed)
    Abstract [en]

    The effects of temperature on the progression of White Spot Disease (WSD) have been studied in the freshwater crayfish Pacifastacus leniusculus. In this study, we aimed to understand the reason for previously observed low mortalities with white spot syndrome virus (WSSV) infected crayfish at low temperatures. The susceptibility of freshwater crayfish to WSSV was studied at different temperatures. The mortality rate at 6°C was zero, meanwhile the animals kept at 22°C developed WSD symptoms and died in a few days after WSSV injections, however upon transfer of animals from 6°C to 22°C the mortality reached 100% indicating that the virus is not cleared. Moreover, the VP28 expression at 6°C was significantly lower compared to animals kept at 22°C. We injected animals with demecolcine, an inhibitor that arrests the cell cycle in metaphase, and observed a delayed mortality. Furthermore, the VP28 expression was found to be lower in these animals receiving both injections with WSSV and demecolcine since cell proliferation was inhibited by demecolcine. We quantified WSSV copy numbers and found that virus entry was blocked at 6°C, but not in demecolcine treatments. We supported this result by quantifying the expression of a clip domain serine protease (PlcSP) which plays an important role for WSSV binding, and we found that the PlcSP expression was inhibited at 6°C. Therefore, our hypothesis is that the WSSV needs proliferating cells to replicate, and an optimum temperature to enter the host hematopoietic stem cells successfully.