Papers by Luciane M Perazzolo
Infectious diseases represent the most serious threat to shrimp farming worldwide. Understanding ... more Infectious diseases represent the most serious threat to shrimp farming worldwide. Understanding the molecular mechanisms driving shrimp-pathogen interactions is necessary for developing strategies to control disease outbreaks in shrimp production systems. In the current study, we experimentally reproduced mortality events using standardized infections to characterize the hemocyte transcriptome response of the shrimp Litopenaeus vannamei succumbing to infectious diseases. By using a high-throughput microfluidic RT-qPCR approach, we identified molecular signatures in shrimp during lethal infections caused by the White Spot Syndrome Virus (WSSV) or the filamentous fungus Fusarium solani. We successfully identified gene expression signatures shared by both infections but also pathogen-specific gene responses. Interestingly, whereas lethal WSSV infection induced the expression of antiviral-related genes, the transcript abundance of many antimicrobial effectors was reduced by lethal F. solani infection. To our knowledge, this is the first report of the immune-gene repertoire of infected shrimp at the brink of death.
Fibrinogen-related proteins (FREPs) comprise a large family of microbial recognition proteins inv... more Fibrinogen-related proteins (FREPs) comprise a large family of microbial recognition proteins involved in many biological functions in both vertebrate and invertebrate animals. By taking advantage of publicly accessible databases, we have identified a FREP-like homolog in the most cultivated penaeid shrimp, Litopenaeus vannamei (LvFrep). The obtained sequence showed a conserved fibrinogen-related domain (FReD) and displayed significant similarities to FREP-like proteins from other invertebrates and to ficolins from crustaceans. The expression of LvFrep appeared to be limited to circulating hemocytes. Interestingly , LvFrep gene expression was induced in shrimp hemocytes only in response to a Vibrio infection but not to the White spot syndrome virus (WSSV). Moreover, LvFrep transcript levels were detected early in fertilized eggs, suggesting the participation of this immune-related gene in the antimicrobial defenses during shrimp development.
Fibrinogen-related proteins (FREPs) comprise a large family of microbial recognition proteins inv... more Fibrinogen-related proteins (FREPs) comprise a large family of microbial recognition proteins involved in
many biological functions in both vertebrate and invertebrate animals. By taking advantage of publicly
accessible databases, we have identified a FREP-like homolog in the most cultivated penaeid shrimp,
Litopenaeus vannamei (LvFrep). The obtained sequence showed a conserved fibrinogen-related domain
(FReD) and displayed significant similarities to FREP-like proteins from other invertebrates and to ficolins
from crustaceans. The expression of LvFrep appeared to be limited to circulating hemocytes. Interestingly,
LvFrep gene expression was induced in shrimp hemocytes only in response to a Vibrio infection but not to the White spot syndrome virus (WSSV). Moreover, LvFrep transcript levels were detected early in fertilized eggs, suggesting the participation of this immune-related gene in the antimicrobial defenses during shrimp development
Journal of lipid research, 1998
Receptors that transport vitellogenin (VTG) into oocytes are of vital importance to egg-laying sp... more Receptors that transport vitellogenin (VTG) into oocytes are of vital importance to egg-laying species because they mediate a key step in oocyte development. Here we describe the cloning of the first piscine oocyte-specific receptor cDNA, i.e., that encoding the VTG receptor from the rainbow trout (Oncorhynchus mykiss). The receptor, a 826-residue type-I membrane protein, is a member of the low density lipoprotein receptor (LDLR) superfamily. It closely resembles the mammalian so-called very low density lipoprotein receptors, in that its aminoterminal ligand binding domain consists of a cluster of 8 cysteine-rich repeats. The short intracellular portion contains the internalization signal typical for the LDLR superfamily, Phe-Glu-Asn-Pro-Val-Tyr. Notably, the receptor lacks a domain with a high density of potential O-glycosylation sites often found in somatic cell-specific members of the LDLR family. A specific transcript of 3.9 kb is abundant in ovary, but undetectable in muscle an...
Diseases of Aquatic Organisms, 2015
Disease in Pacific white shrimp Litopenaeus vannamei caused by the infectious myonecrosis virus (... more Disease in Pacific white shrimp Litopenaeus vannamei caused by the infectious myonecrosis virus (IMNV) causes significant socioeconomic impacts in infection-prone shrimp aquaculture regions. The use of synthetic dsRNA to activate an RNA interference (RNAi) response is being explored as a means of disease prophylaxis in farmed shrimp. Here, survival was tracked in L. vannamei injected with long synthetic dsRNAs targeted to IMNV open reading frame (ORF) 1a, ORF1b, and ORF2 genome regions prior to injection challenge with IMNV, and real-time RT-PCR was used to track the progress of IMNV infection and mRNA expression levels of the host genes sid1, dicer2, and argonaute2. Injection of dsRNAs targeting the ORF1a and ORF1b genes but not the ORF2 gene strongly inhibited IMNV replication over a 3 wk period following IMNV challenge, and resulted in 90 and 83% shrimp survival, respectively. Host gene mRNA expression data indicated that the Sid1 protein, which forms a transmembrane channel involved in cellular import/export of dsRNA, increased in abundance most significantly in shrimp groups that were most highly protected by virus-specific dsRNA injection. Subclinical IMNV infections present in the experimental L. vannamei used increased markedly in the 2 d between injection of any of the 4 virus-specific or non-specific dsRNAs tested and IMNV challenge. While handling and injection stress are implicated in increasing IMNV replication levels, the underlying molecular factors that may have been involved remain to be elucidated.
PLoS ONE, 2013
Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a ... more Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded b-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPSbinding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family.
Pesquisa Agropecuária Brasileira, 2013
Biology of Reproduction, 1999
The expression and localization of vitellogenin (VTG) receptor (VTGR) mRNA were identified throug... more The expression and localization of vitellogenin (VTG) receptor (VTGR) mRNA were identified throughout ovarian development in the rainbow trout, Oncorhynchus mykiss. Northern blot confirmed the presence of a transcript (approximately 3.9 kilobases [kb]) that was specific to the ovary. The expression of VTGR mRNA varied throughout ovarian development and was highest in previtellogenic ovaries and in ovaries at the onset of vitellogenesis containing ovarian follicles (OF) from 35 to 600 microm in diameter. In situ hybridization using 35S riboprobes showed that the transcription of the VTGR gene was initiated in OF measuring 45-50 microm in diameter, with transcripts being exclusively localized in the ooplasm. A dramatic increase in mRNA synthesis occurred during previtellogenic growth (OF from 50 to 200 microm); this was followed by a gradual decrease during the vitellogenic growth phase. VTGR mRNA was not detected in OF greater than 1000 microm in diameter (oocytes actively sequestering VTG). Immunocytolocalization of yolk proteins derived from VTG demonstrated that oocytes started to sequester VTG when they were around 300 microm in diameter, shortly after the time of maximal density of VTGR mRNA in the ooplasm. The timing of transcription of the VTGR gene, predominantly during previtellogenesis, suggests that the VTGR is recycled to the oocyte surface during the vitellogenic growth phase.
Aquaculture, 2004
The main purpose of this study was to examine the modulation of some hemato-immunological paramet... more The main purpose of this study was to examine the modulation of some hemato-immunological parameters in female shrimp L. vannamei submitted to unilateral eyestalk ablation and whose diet was supplemented with superdoses of vitamin C, as a form of immunostimulation. The analyzed parameters were: total hemocyte counts (THC), protein concentration (PC), agglutinating and phenoloxidase (PO) activities in the serum, and levels of glucose and lactate in the plasma. Broodstock shrimp females were analyzed on the day before ablation and on days 1, 3, 7 and 14 after ablation. The diet was supplemented with superdoses of vitamin C, in the form L-ascorbyl-2polyphosphate (ApP). The females were divided into a control group (NS), not supplemented with superdoses of ApP, and two other groups (S1 and S2) supplemented with doses 10 and 20 times the recommended dose of 200 mg/kg, respectively. Surprisingly, the great majority of the hematoimmunological parameters analyzed did not exhibit any statistically significant change in relation to eyestalk ablation in NS group. There was only a significant reduction in the agglutinating activity on the third day following ablation. Similarly, in S1 and S2 groups, there were no significant changes in 0044-8486/$ -see front matter D (M.A. Barracco).
a b s t r a c t -1,3-Glucan binding proteins (GBPs) are soluble pattern recognition proteins/re... more a b s t r a c t -1,3-Glucan binding proteins (GBPs) are soluble pattern recognition proteins/receptors that bind to -1,3-glucans from fungi cell walls. In crustaceans, GBPs are abundant plasmatic proteins produced by the hepatopancreas, and have been proved to play multiple biological functions. Here, we purified and characterized novel members of the GBP family from the hemolymph of two Brazilian shrimps, Farfantepenaeus paulensis (FpGBP) and Litopenaeus schmitti (LsGBP). As observed for other crustacean species, FpGBP and LsGBP are monomeric proteins (∼100 kDa) able to enhance the activation of the prophenoloxidase system, a potent antimicrobial defense conserved in arthropods. More interestingly, we provided here evidence for a novel biological activity for shrimp GBPs: the agglutination of fungal cells. Finally, we investigated the modulation of the ˇGBP gene in F. paulensis shrimps experimentally infected with a cognate fungal pathogen, Fusarium solani. From our expression data, ˇGBP gene is constitutively expressed in hepatopancreas and not modulated upon a non-lethal fungal infection. Herein, we have improved our knowledge about the GBP family by the characterization of a novel biological role for this multifunctional protein in shrimp.
Hybridoma, 2010
The monoclonal antibody (MAb) LITO-1 was produced from a stable hybridoma cell line generated by ... more The monoclonal antibody (MAb) LITO-1 was produced from a stable hybridoma cell line generated by the fusion of NS1 myeloma cells with spleen cells isolated from Balb=c mice immunized with a paraformaldehydefixed hemocyte suspension of Litopenaeus vannamei. This MAb reacted with all three hemocyte subtypes, but no reaction was observed with components of plasma. Immunohistochemistry assays demonstrated that LITO-1 was very effective in specifically distinguishing hemocytes infiltrated in several tissues such as striated muscle, brain, and hepatopancreas. Moreover, this antibody was able to recognize hemocytes from two shrimp species, Litopenaeus schmitti and Farfantepenaeus paulensis, as well as hemocytes of the oyster Crassostrea gigas. No reaction was observed against hemocytes from the terrestrial insect Triatoma klugi or with mammalian RAW cells. This novel MAb can be useful in revealing the presence and function of a conservative epitope in hemocytes of marine crustaceans and mollusks.
Fish & Shellfish Immunology, 2008
Alpha2-macroglobulin; Thioester domain; Crustacean immunity; Molecular cloning; Proteinase inhibi... more Alpha2-macroglobulin; Thioester domain; Crustacean immunity; Molecular cloning; Proteinase inhibitor Alpha2-macroglobulin (a2M) is a broad-spectrum proteinase inhibitor that belongs to the group of the thioester plasma protein superfamily, which includes also the complement components C3, C4 and C5 and the human's pregnancy zone protein. This protein superfamily contains in its structure a characteristic internal reactive thioester bond (except C5) that links cysteinyl and glutamyl amino acid residues [1,2]. The a2M inhibition mechanism starts with the proteolytic attack of its ''bait region'' by a proteinase. This bait region has target sites for all proteinases and after proteolysis, the native a2M molecule goes through a conformational change that culminates with the formation of a molecular cage that entraps the bound proteinase. As a consequence of this structural change, the a2M internal thioester bond is also cleaved and exposes its cysteinyl thiol and carbonyl groups of glutamic acid. The carbonyl groups are highly reactive and can form covalent crosslinks with 3-amino groups of the lysines of the encaged proteinase [2].
Fish & Shellfish Immunology, 2011
In this study, we report on the isolation and characterization of an alpha2-macroglobulin (a2M) f... more In this study, we report on the isolation and characterization of an alpha2-macroglobulin (a2M) from the plasma of the pink shrimp Farfantepenaeus paulensis, its sub-cellular localization and transcriptional changes after infection by fungi. The molecular mass of the a2M was estimated at 389 kDa by gel filtration and 197 kDa by SDS-PAGE, under reducing conditions, suggesting that a2M from F. paulensis consists of two identical sub-units, covalently linked by disulphide bonds. The N-terminal amino acid sequence of the a2M from F. paulensis was very similar to those of other penaeid shrimps, crayfish and lobster (70e90% identity) and to a less extent with that of freshwater prawn (40% identity). A monoclonal antibody raised against the Marsupenaeus japonicus a2M made it possible to demonstrate that a2M of F. paulensis is stored in the vesicles of the shrimp granular hemocytes (through immunogold assay).
Developmental & Comparative Immunology, 1997
q Abstract-In the present study we investigated the prop0 activating system of the penaeid Penaeu... more q Abstract-In the present study we investigated the prop0 activating system of the penaeid Penaeus paulensis, focusing on its role in the shrimp immune system. The great majority of PO activity (more than 90%) was found in the shrimp hemocytes. The enzyme activity was greatly enhanced by components of microorganism cell walls, such as LPS and f&1,3-glucans, suggesting its involvement in non-self recognition.
Aquaculture Research, 2009
The capacity of reactive oxygen intermediates production upon haemocyte stimulation is one of the... more The capacity of reactive oxygen intermediates production upon haemocyte stimulation is one of the most important immunoparameter utilized to assess the health status in cultivated shrimps. In the present study, we compared oxidative stress potential, by measuring the superoxide anion production in three penaeid shrimps: two wild Atlantic species, the pink shrimp Farfantepenaeus paulensis and the white shrimp Litopenaeus schmitti and one cultivated Paci-¢c species, the white shrimp, Litopenaeus vannamei, through the nitro-blue-tetrazolium-reduction assay. We also proposed an optimized experimental protocol for this assay, that produces rapid and consistent results with low levels of basal superoxide anion (O 2 À ) production by unstimulated haemocytes and high levels of this oxygen radical after cell stimulation. Among the di¡erent cell elicitors used (zymosan, laminarin, lipopolysaccharide and phorbol myristate acetate), laminarin (b-1,3-glucans^2 mg mL À 1 ) was the most potent cell activator for the haemocytes of all three penaeids and we recommend this immunostimulant to routinely evaluate shrimp respiratory burst. In general terms, the most elevated levels of O 2 À production, after cell stimulation with microbial components, were detected in L. schmitti. Interestingly, the stimulation pro¢le of the haemocytes of L. vannamei was more similar to F. paulensis, than to L. schmitti, which is more phylogenetically related.
Aquaculture, 2002
The main purpose of this study was to examine the modulation of some hemato-immunological paramet... more The main purpose of this study was to examine the modulation of some hemato-immunological parameters in female shrimp L. vannamei submitted to unilateral eyestalk ablation and whose diet was supplemented with superdoses of vitamin C, as a form of immunostimulation. The analyzed parameters were: total hemocyte counts (THC), protein concentration (PC), agglutinating and phenoloxidase (PO) activities in the serum, and levels of glucose and lactate in the plasma. Broodstock shrimp females were analyzed on the day before ablation and on days 1, 3, 7 and 14 after ablation. The diet was supplemented with superdoses of vitamin C, in the form L-ascorbyl-2polyphosphate (ApP). The females were divided into a control group (NS), not supplemented with superdoses of ApP, and two other groups (S1 and S2) supplemented with doses 10 and 20 times the recommended dose of 200 mg/kg, respectively. Surprisingly, the great majority of the hematoimmunological parameters analyzed did not exhibit any statistically significant change in relation to eyestalk ablation in NS group. There was only a significant reduction in the agglutinating activity on the third day following ablation. Similarly, in S1 and S2 groups, there were no significant changes in 0044-8486/$ -see front matter D (M.A. Barracco).
Developmental and Comparative Immunology, 2014
Infectious diseases represent the most serious threat to shrimp farming worldwide. Understanding ... more Infectious diseases represent the most serious threat to shrimp farming worldwide. Understanding the molecular mechanisms driving shrimp-pathogen interactions is necessary for developing strategies to control disease outbreaks in shrimp production systems. In the current study, we experimentally reproduced mortality events using standardized infections to characterize the hemocyte transcriptome response of the shrimp Litopenaeus vannamei succumbing to infectious diseases. By using a high-throughput microfluidic RT-qPCR approach, we identified molecular signatures in shrimp during lethal infections caused by the White Spot Syndrome Virus (WSSV) or the filamentous fungus Fusarium solani. We successfully identified gene expression signatures shared by both infections but also pathogen-specific gene responses. Interestingly, whereas lethal WSSV infection induced the expression of antiviral-related genes, the transcript abundance of many antimicrobial effectors was reduced by lethal F. solani infection. To our knowledge, this is the first report of the immune-gene repertoire of infected shrimp at the brink of death.
Comparative Biochemistry and Physiology B-biochemistry & Molecular Biology - COMP BIOCHEM PHYSIOL PT B, 2005
A clotting protein (CP) was purified from the plasma of the pink shrimp Farfantepenaeus paulensis... more A clotting protein (CP) was purified from the plasma of the pink shrimp Farfantepenaeus paulensis by sequential anion-exchange chromatography. The shrimp CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+, suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase present in shrimp hemocytes. Dansylcadaverine was incorporated into the shrimp CP in the presence of endogenous transglutaminase (hemocyte lysate), confirming that the shrimp purified CP is the substrate for the transglutaminase enzyme. The molecular mass of the CP was determined by gel filtration to be 341 kDa and 170 kDa by SDS-PAGE under reducing conditions. These results suggest that the shrimp CP consists of two identical subunits, covalently linked by disulphide bonds. The amino acid sequence at the N-terminus was 100% identical to that of the penaeids Litopenaeus vannamei and Penaeus monodon and 66% to 80% identical to the CPs of other decapods. This is...
Books by Luciane M Perazzolo
Marine Shrimp (penaeids) Pathology and Immunology. Spanish.
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Papers by Luciane M Perazzolo
many biological functions in both vertebrate and invertebrate animals. By taking advantage of publicly
accessible databases, we have identified a FREP-like homolog in the most cultivated penaeid shrimp,
Litopenaeus vannamei (LvFrep). The obtained sequence showed a conserved fibrinogen-related domain
(FReD) and displayed significant similarities to FREP-like proteins from other invertebrates and to ficolins
from crustaceans. The expression of LvFrep appeared to be limited to circulating hemocytes. Interestingly,
LvFrep gene expression was induced in shrimp hemocytes only in response to a Vibrio infection but not to the White spot syndrome virus (WSSV). Moreover, LvFrep transcript levels were detected early in fertilized eggs, suggesting the participation of this immune-related gene in the antimicrobial defenses during shrimp development
Books by Luciane M Perazzolo
many biological functions in both vertebrate and invertebrate animals. By taking advantage of publicly
accessible databases, we have identified a FREP-like homolog in the most cultivated penaeid shrimp,
Litopenaeus vannamei (LvFrep). The obtained sequence showed a conserved fibrinogen-related domain
(FReD) and displayed significant similarities to FREP-like proteins from other invertebrates and to ficolins
from crustaceans. The expression of LvFrep appeared to be limited to circulating hemocytes. Interestingly,
LvFrep gene expression was induced in shrimp hemocytes only in response to a Vibrio infection but not to the White spot syndrome virus (WSSV). Moreover, LvFrep transcript levels were detected early in fertilized eggs, suggesting the participation of this immune-related gene in the antimicrobial defenses during shrimp development