Molecular cloning and mRNA expression of two peptidoglycan recognition protein (PGRP) genes from mollusk Solen grandis

Peptidoglycan recognition proteins (PGRPs) play crucial role in innate immunity for both invertebrates and vertebrates, owing to their prominent ability in detecting and eliminating invading bacteria. In the present study, two short PGRPs from mollusk Solen grandis (designated as SgPGRP-S1 and SgPGRP-S2) were identified, and their expression patterns, both in tissues and toward three PAMPs stimulation, were then characterized. The full-length cDNA of SgPGRP-S1 and SgPGRP-S2 was 1672 and 1285 bp, containing an open reading frame (ORF) of 813 and 426 bp, respectively, and deduced amino acid sequences showed high similarity to other members of PGRP superfamily. Both SgPGRP-S1 and SgPGRP-S2 encoded a PGRP domain. The motif of Zn²⁺ binding sites and amidase catalytic sites were well conserved in SgPGRP-S1, but partially conserved in SgPGRP-S2. The two PGRPs exhibited different tissue expression pattern. SgPGRP-S1 was highly expressed in muscle and hepatopancreas, while SgPGRP-S2 was highly in gill and mantle. The mRNA expression of SgPGRP-S1 could be induced acutely by stimulation of PGN, and also moderately by β-1,3-glucan, but not by LPS, while expression of SgPGRP-S2 was significantly up-regulated (P < 0.01) when S. grandis was stimulated by all the three PAMPs, though the expression levels were relatively lower than SgPGRP-S1. Our results suggested SgPGRP-S1 and SgPGRP-S2 could serve as pattern recognition receptors (PRRs) involved in the immune recognition of S. grandis, and they might perform different functions in the immune defense against invaders.     

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca²⁺ binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln¹²²-Pro¹²³-Asn¹²⁴) in LvLectin-1, but QPD (Gln¹²⁸-Pro¹²⁹-Asp¹³⁰) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P < 0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P < 0.01) and 12 h (P < 0.05), but the expression level of LvLectin-1 down-regulated significantly (P < 0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus.     

Identification and transcriptional analysis of two types of lectins (SgCTL-1 and SgGal-1) from mollusk Solen grandis

C-type lectin and galectin are two types of animal carbohydrate-binding proteins which serve as pathogen recognition molecules and play crucial roles in the innate immunity of invertebrates. In the present study, a C-type lectin (designated as SgCTL-1) and galectin (designated as SgGal-1) were identified from mollusk Solen grandis, and their expression patterns, both in tissues and toward three pathogen-associated molecular patterns (PAMPs) stimulation were characterized. The full-length cDNA of SgCTL-1 and SgGal-1 was 1280 and 1466 bp, containing an open reading frame (ORF) of 519 and 1218 bp, respectively. Their deduced amino acid sequences showed high similarity to other members of C-type lectin and galectin superfamily, respectively. SgCTL-1 encoded a single carbohydrate-recognition domain (CRD), and the motif of Ca(2+)-binding site 2 was EPN (Glu(135)-Pro(136)-Asn(137)). While SgGal-1 encoded two CRDs, and the amino acid residues constituted the carbohydrate-binding motifs were well conserved in CRD1 but partially conserved in CRD2. Although SgCTL-1 and SgGal-1 exhibited different tissue expression pattern, they were both constitutively expressed in all tested tissues, including hemocytes, gonad, mantle, muscle, gill and hepatopancreas, and they were both highly expressed in hepatopancreas and gill. Furthermore, the mRNA expression of two lectins in hemocytes was significantly (P < 0.01) up-regulated with different levels after S. grandis were stimulated by lipopolysaccharide (LPS), peptidoglycan (PGN) or β-1,3-glucan. Our results suggested that SgCTL-1 and SgGal-1 from razor clam were two novel members of animal lectins, and they might function as pattern recognition receptors (PRRs) taking part in the process of pathogen recognition.     

Germinoma in razor clam Ensis arcuatus (Jeffreys, 1865) in Galicia (NW Spain)

Germinoma is a gonadal neoplasm originating from progenitor cells in germinal epithelium. Over the last four decades it has been diagnosed in several species of marine bivalve molluscs but most consistently in some populations of Mercenaria mercenaria and Mya arenaria in North America. Tissue sections of gonads from Ensis arcuatus (family Pharidae--superfamily Solenacea), collected in Ría de Vigo (Galicia-NW Spain), revealed germinoma in both males and females. Proliferating, undifferentiated, germ cells, with no evidence of maturation, had formed discrete masses in the walls and lumens of gonadal follicles. This is the first report of germinoma in superfamily Solenacea.     

Cloning and expression analysis of Mx cDNA from Senegalese sole (Solea senegalensis)

Senegalese sole (Solea senegalensis) is a promising fish species of growing interest in European aquaculture. In fish farming, viral infections are a constant threat therefore, understanding fish defence mechanisms is a main priority to avoid economic losses. Mx proteins are involved in the innate antiviral response of fish. They are induced by type I interferons (alpha and beta) and are essential to investigate viral defence mechanisms in fish, due to the difficulty in tracking interferon activity in these species. In this study a full-length Senegalese sole Mx cDNA has been RT-PCR cloned, resulting in 2322bp coding for 623 amino acids. The sequence accounts for the main characteristics of Mx proteins but lacking nuclear localisation signal (NLS), which suggests cytoplasmic localisation. The alignments of Senegalese sole Mx sequence showed the highest identity with the flatfish species, 80.1% identity with flounder and 78.9% with halibut. The spatial and temporal expression pattern has been analysed in control and challenged fish by RT-PCR. In control fish a constitutive level of sole Mx expression has been obtained and a clear induction was observed after treatment with Poly[I:C], which supports a putative role for the Mx in Senegalese sole viral defence. These findings contribute to increasing the knowledge of the role of interferon pathway in fish innate immunity and to develop new tools to fight virus infections in the culture of this species.     

Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis)

The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.     

The sialic acid-containing lipopolysaccharides of Salmonella djakarta and Salmonella isaszeg (serogroup O: 48): Chemical characterization and reactivity with a sialic acid-binding lectin from Cepaea hortensis

Abstract Lipopolysaccharides (LPS) of Salmonella djakarta and Salmonella isaszeg , as well as of a spontaneous R-mutant of S. djakarta were investigated as to their content in neuraminic acid (Neu) and its individual linkage. The two Salmonella serovars both belong to the O:48 serogroup of Salmonella , but to two different subgroups. LPS of both S-forms contained high amounts of Neu, although in different quantities, whereas the R-form was completely devoid of it. Methylation analysis indicated that Neu is exclusively terminally linked in S. djakarta whereas both terminal and 4-linked Neu were recognized in S. isaszeg . Although terminally linked, a sialidase from Arthrobacter ureafaciens was unable to split Neu even after prolonged incubation from both S-type LPSs. When LPS was first treated by mild alkali, however, the total amount of Neu from S. djakarta LPS and about 50% from that of LPS of S. isaszeg could be removed. In contrast, alkali-treated LPS, but also the non-treated one, proved to be effective inhibitors for a sialic acid-binding lectin from Cepaea hortensis . The resistance of terminal Neu towards sialidase may be due to the presence of an O-acetyl group which would be removed during the methylation analysis but would, especially when linked to C-4, not interfere with the reactivity of the lectin.     

Presence and histopathological effects of the Parvatrema sp. (Digenea, Gymnophallidae) in the stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae)

The stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae) has a wide geographic distribution range, including the Brazilian coasts from the northeast (Alagoas) to the south (Santa Catarina). In March 2008, an episode of mass T. plebeius mortality (70%) occurred in an intertidal bed at The Pontal da Daniela, State of Santa Catarina, Brazil. We report here high prevalences (to 100%) of the trematode parasite Parvatrema sp. Cable, 1953 (Digenea, Gymnophallidae) infecting T. plebeius at high intensities. We describe the gymnophalid, echinostomatid and unidentified metacercariae parasites infecting the clam and the host reactions elicited by them. The use of special diagnostic techniques such as Ray’s fluid thioglycollate medium (RFTM) and PCR assays to detect Perkinsus sp. pathogens, hemolymph cytology, and histopathological examinations did not show Perkinsus sp. infections, microcell infections, or neoplastic conditions. However, neither infections or pathology caused by trematode parasites; nor any other pathological condition could be uniquely correlated with the mortality event. A coincident flash flood might have contributed to cause the mortality episode. This is the first report of the Parvatrema sp. metacercariae larvae infecting the stout razor clam T. plebeius from Brazil.     

The effect of lectins on the attachment and invasion of Enteromyxum scophthalmi (Myxozoa) in turbot (Psetta maxima L.) intestinal epithelium in vitro

The involvement of the lectin/carbohydrate interaction in the invasion of the turbot intestinal epithelium by Enteromyxum scophthalmi was studied in vitro using explants of turbot intestine and pre-treatment of parasite stages with the plant lectins of Canavalia ensiformis (Con A) and Glycine max (SBA). Both lectins inhibited the attachment and invasion of E. scophthalmi stages to the intestinal epithelium, though the inhibitory effect was higher for SBA than for Con A. Such results point to the involvement of N-acetyl-galactosamine (GalNAc) and galactose (Gal) residues and also of mannose/glucose residues in the E. scophthalmi-intestinal epithelium interaction. The inhibitory effect of both lectins on the parasite adhesion and penetration points to the interest of further studies to confirm the presence of putative lectins recognising GalNAc-Gal and mannose/glucose residues in turbot intestine. The obtained results demonstrated also the adequacy of turbot intestinal explants as an in vitro model to study the interaction with E. scophthalmi.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.     

Isolation of two Locust protein targets of a protein tyrosine phosphatase from Metarhizium anisopliae strain CQMa102

In entomopathogenic fungi, secretory protein phosphatases might function in the utilization of phosphoproteins from the environment. But if secreted into the host, secretory protein phosphatases might play a role in pathogenesis by dephosphorylation of host phosphoproteins. Our group purified a novel phosphatase from entomopathogenic fungi, Metarhizium anisopliae. The substrate specificity and inhibitor sensitivity indicate that the phosphatase is a protein tyrosine phosphatase (PTPase). In order to analyze the targets of the PTPase in Locusta migratoria hemolymph, two-dimensional electrophoresis and mass spectrometry were used. The results indicated that the PTPase could specifically dephosphorylate two phosphoproteins from L. migratoria hemolymph. One phosphoprotein was identified as trans-Golgi p230. Previous studies have shown that trans-Golgi p230 participates in vesicular transport of functional proteins from the distal Golgi compartment. trans-Golgi p230 can be inactivated by dephosphorylation, which implies that M. anisopliae could interfere with the correct transportation of functional proteins by secreting extracellular PTPase into the hemolymph. There are some secretion proteins, such as transferrin, have been thought to participate in the insect innate immune against microbial infection, therefor M. anisopliae could interfere with immune defenses of L. migratoria by secreting extracellular PTPase into the hemolymph.     

Cloning, characterization and promoter analysis of S-RNase gene promoter from Chinese pear (Pyrus pyrifolia)

The 5'-flanking region of the S(12)-, S(13)-, S(21)-RNase with a length of 854 bp, 1448 bp and 1137 bp were successfully isolated by TAIL-PCR from genomic DNA from 'Jinhua', 'Maogong' (Pyrus pyrifolia) and 'Yali' (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S(13)-, S(12)-, S(21)-RNase gene promoter sequences with S(2)-, S(3)-, S(4)-, S(5)-RNase 5'-flanking sequences indicated that a homology region of about 240 bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5'-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S(12)-pro-(1 to 5)-GUS-pBll01.2 transgenic pistils and progressively decreased from S(12)-pro-1-GUS-pBI l01.2 to S(12)-pro-5-GUS-pBll01.2. No GUS activity was detected in S(12)-pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S(12)-RNase promoter is pistil specific expression promoter.     

Nosema ceranae in drone honey bees (Apis mellifera)

Nosema ceranae is a microsporidian intracellular parasite of honey bees, Apis mellifera. Previously Nosema apis was thought to be the only cause of nosemosis, but it has recently been proposed that N. ceranae is displacing N. apis. The rapid spread of N. ceranae could be due to additional transmission mechanisms, as well as higher infectivity. We analyzed drones for N. ceranae infections using duplex qPCR with species specific primers and probes. We found that both immature and mature drones are infected with N. ceranae at low levels. This is the first report detecting N. ceranae in immature bees. Our data suggest that because drones are known to drift from their parent hives to other hives, they could provide a means for disease spread within and between apiaries.     

Cloning and Structural Analysis of a Haemocyanin from the Stonefly Perla grandis

We characterized two subunits of a putative haemocyanin from the stonefly species Perla grandis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression in different insect stages. Moreover, using the deduced amino acid sequences, homology studies were performed both on their primary and tertiary structures. 3-D molecular modelling data showed that the residues involved in the oxygen transport and subunits contacts were located in spatial positions preserving the functionality of the molecule. Despite it was paradigmatically affirmed that insects do not have respiratory proteins, our data suggest that the haemocyanin could be involved in the respiratory mechanisms of P. grandis. As far as we know, this is the first haemocyanin 3-D structure described and analyzed in insects.