The course and mortality of a hematopoietic neoplasm in the soft-shell clam,Mya arenaria

Results from two experiments containing approximately 280 Mya arenaria indicated that significantly higher (P < 0.05) mortality occurred within the neoplastic clam population than in the nonneoplastic clam population. Using an in vivo blood cytological technique, five levels of severity were established. The levels were based on the number of neoplastic cells in the circulation with level 1 as the lowest severity and level 5 as the highest severity. Neoplastic clams that were diagnosed as the lowest level had higher survival rates (60%) than those clams diagnosed as the highest level (0%). The hematopoietic neoplasm in M. arenaria followed one of three courses: (1) in approximately 50% of the neoplastic clams the disease progressed to a higher severity and resulted in death; (2) in approximately 40% of the neoplastic clams the disease was chronic, i.e., remained at a stable level; and (3) in approximately 10% of the neoplastic clams the disease diminished in severity or disappeared entirely. In addition, 10% of the clams diagnosed as nonneoplastic at the beginning of the experiments were neoplastic by the termination of the experiment. The contraction of the disease may have been de novo or by stimulation of latent infections.The prevalence of the neoplasm in clams collected from an epizootic area followed a biphasic seasonal pattern. The highest prevalences occurred in October, November, and in May. The hematopoietic neoplasm in M. arenaria was also age and species specific.     

Predation on soft-shell clams (Mya arenaria) by the nemertean Cerebratulus lacteus in Atlantic Canada: implications for control measures

The nemertean, Cerebratulus lacteus Verrill (Nemertinea: Heteronemertini), has been identified as an important threat to soft-shell clam (Mya arenariaL.) populations in Atlantic Canada. The biology of this species, however, is still largely unknown. Field and laboratory studies were undertaken in 1998 and 1999 in Prince Edward Island, Canada, to test certain control measures to reduce predation on soft-shell clam populations and to better describe the relationship between C. lacteus and M. arenaria. Field abundance of C. lacteus, M. arenaria and Nereis virens Sars were evaluated in relation to particular habitat modifications that were used as control measures. Sediment manipulations tested were: (1) addition of shells and (2) use of a hydraulic rake. No difference was observed on the abundance of C. lacteus, M. arenaria and N. virens after treatments were applied. In the laboratory, C. lacteus was shown to be an efficient predator of M. arenaria. Clam mortalities reached 100% in the presence of C. lacteus while 0% mortality was observed in its absence. A complementary set of experiments was carried out to see if the sympatric polychaetes N. virens and Glycera dibranchiata Ehlers had any impact on the relationship between C. lacteus and M. arenaria. N. virens showed no impact on C. lacteus predation on clams. The presence of G. dibranchiata significantly reduced the nemertean predation rate. Analysis of clam size selection revealed no significant preference by C. lacteus. Other experimental studies revealed that high predator densities did not impede predation on clams and that C. lacteus preferred soft-shell clams among other commercial bivalve species presented (Mercenaria mercenariaL., Crassostrea virginica Gmelin and Mytilus edulisL.). This study should provide a better understanding of the relationship between C. lacteus and M. arenaria and lead to the development of improved nemertean control measures.     

Isolation of a viral agent causing hematopoietic neoplasia in the soft-shell clam,Mya arenaria

A virus from neoplastic soft-shell clams, Mya arenaria, has been isolated. Its morphological and physical properties are similar to those of B-type retroviruses. It is enveloped, pleomorphic, averaging 120 nm in size with an eccentric nucleoid. Possible A-type particles have also been observed. The bouyant density of the particle ranged from 1.17 to 1.18 g/cm³ and the ultraviolet absorption $\text{260}\text{280}\text{-}\text{nm}$ ratio was 1.23. The virus has been shown to cause a hematopoietic neoplasm in Mya. Purified virus from neoplastic clams was innoculated into nonneoplastic clams. Most of these clams developed tumors within 2 months. Virus isolated from inoculated clams which developed neoplasia was inoculated into nonneoplastic clams. Most of these clams also developed neoplasia thus completing Koch's postulates.     

Sex alteration in soft-shell clams (Mya arenaria) in an intertidal zone of the Saint Lawrence river (Quebec,Canada)

The purpose of this study was to verify whether any changes in sex ratio might occur in soft-shell clams (Mya arenaria) located in an intertidal harbor zone located at the mouth of the Saguenay Fjord in the Saint Lawrence estuary (Baie Sainte-Catherine (BSC), Québec, Canada) likely to be contaminated by organotin compounds. Bivalves were harvested at the BSC harbor site and from two reference sites. Condition index (weight to length ratio), gonado-somatic index, sex ratio, vitellin-like proteins, organotin concentrations in gonad tissue, maturation stages of the gonads, the number of estradiol-17beta binding sites and the capacity of female gonad extracts to produce estradiol-17beta were determined in collected animals. Results showed that sex ratio in clams was significantly skewed toward males. Moreover, the condition and gonad-somatic indices, vitellin-like proteins in female gonads and the capacity of female gonads to produce estradiol-17beta were significantly reduced at the harbor site with respect to the reference sites. Maturation status of male gonads was clearly delayed at the harbor site. Additionally, gonad tissue contained tributyltin (TBT) at an average level of 109+/-18 ngSn/gdry wt. at the harbor site while organotins were not detected from the reference sites. Finally, female gonads had a higher number of unoccupied estradiol binding sites at the harbor site indicating low levels of this steroid in this tissue. Overall, this paper is first to report that clams collected in the vicinity of a TBT contaminated harbor are subject to masculinizing effects which seems to be consistent with biological effects that organotins are known to exert toward some other marine invertebrates.     

Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

Background  

Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used.     

Two epizootic diseases in Chesapeake Bay commercial clams,Mya arenaria and Tagelus plebeius

Declining Chesapeake Bay harvests of softshell clams, together with historical and emerging reports of epizootic diseases in Mya arenaria, prompted a survey in summer 2000 of the health status of selected commercial clam populations. All sampled populations (8 M arenaria softshell clam, 2 Tagelus plebeius razor clam) were infected by Perkinsus sp. protozoans at prevalences ranging from 30 to 100% of sampled clams. Nucleotide sequences for the internal transcribed spacer (ITS) region of the rRNA gene complex were determined for clonal in vitro Perkinsus sp. isolates propagated from both M. arenaria and T plebeius. Multiple polymorphic sequences were amplified from each isolate, but phylogenetic analysis placed all sequences into 2 clades of a monophyletic group, which included both recently described clam parasites P. chesapeaki and P. andrewsi. Sequences amplified from each clonal isolate were found in both sister clades, one containing P. andrewsi and the other P. chesapeaki. Most (7 of 8) M. arenaria samples were also affected with disseminated neoplasia (DN), at prevalences of 3 to 37%, but neither T. plebeius sample showed DN disease. Disease mortalities projected for sampled clam populations, especially those affected by both diseases, may further deplete subtidal commercial clam populations in mesohaline portions of Chesapeake Bay.     

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of European eel,Anguilla anguilla

Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (β-actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real-time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue-dependent and treatment-dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV-challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.     

5-bromodeoxyuridine induction of hematopoietic neoplasia and retrovirus activation in the soft-shell clam, Mya arenaria

Hematopoietic neoplasia and virus replication were induced in shoft-shell clams, Mya arenaria, by 5-bromodeoxyuridine (5-BrdUrd). Eighty clams were found to be nonneoplastic by the in vivo bleeding method. These clams were randomly distributed into four aquaria and maintained in seawater at 1°C. 5-BrdUrd was added to aquaria in the following concentrations: 0, 20, 100, and 200 μg/ml. After 4 days of treatment, the aquaria were drained, rinsed, and fresh sea water without 5-BrdUrd was added. Sea water was changed on a weekly basis thereafter. The clams were diagnosed for neoplasia by the in vivo method at days 4, 9, 16, and 23 of the experiment. Results showed that on day 23, neoplasia was not found in the aquarium without 5-BrdUrd, but in the aquaria containing 20, 100, and 200 μg/ml of 5-BrdUrd the incidences of neoplasia were 37, 79, and 35%, respectively. 5-BrdUrd-induced neoplastic tissue was homogenized and subjected to differential ultracentrifugation. Retrovirus-like particles resembling ones previously shown to induce neoplasia were isolated from neoplastic clams which were induced by 5-BrdUrd. When these particles were inoculated into healthy clams, the clams developed neoplasia. Neoplastic hemocytes, cultured in sea water containing 50 μg/ml of 5-BrdUrd, formed pseudopodia after 4 days. Pseudopod formation is a trait of normal hemocytes. This indicates that differentiation of neoplastic hemocytes may be induced by the drug.     

Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR

For accurate and reliable gene expression results, normalization of real-time PCR data is required against a control gene, which displays highly uniform expression in living organisms during various phases of development and under different environmental conditions. We assessed the gene expression of 10 frequently used housekeeping genes, including 18S rRNA, 25S rRNA, UBC, UBQ5, UBQ10, ACT11, GAPDH, eEF-1alpha, eIF-4a, and beta-TUB, in a diverse set of 25 rice samples. Their expression varied considerably in different tissue samples analyzed. The expression of UBQ5 and eEF-1alpha was most stable across all the tissue samples examined. However, 18S and 25S rRNA exhibited most stable expression in plants grown under various environmental conditions. Also, a set of two genes was found to be better as control for normalization of the data. The expression of these genes (with more uniform expression) can be used for normalization of real-time PCR results for gene expression studies in a wide variety of samples in rice.     

Different proteins in the hemolymph sera from sarcomatous and healthy soft shell clams, Mya arenaria L.

1. Serum proteins showed quantitative and qualitative differences between sarcomatous and healthy soft shell clams, Mya arenaria L. 2. Total protein concentration was significantly higher in the serum of sarcomatous clams than of healthy clams. 3. According to SDS-PAGE, more serum proteins with more variability distinguished sarcomatous clams from healthy ones. 4. Sarcomatous clams had unique serum proteins of M(r)23,000, 45,000 and 54,000. Healthy clams had unique serum proteins of M(r) 24,000, 103,000 and 105,000. 5. During disease progression, sarcoma-specific proteins appeared while normal proteins disappeared. 6. We propose that some sarcoma-associated proteins may have tumor promoting and/or cytotoxic functions and that some normal proteins which disappear during disease progression may be involved in the humoral defense system.     

Field and laboratory comparisons of mortality in normal and neoplastic Mya arenaria

The results of a 6-month mark and recapture experiment involving approximately 900 adult Mya arenaria demonstrated that under natural conditions, significantly higher (P much less than .001, chi 2 test) mortality occurred among animals with neoplasia than those diagnosed as normal. Using a blood screening technique, the clams were diagnosed and placed in one of three diagnostic groups based on the severity of the disease (the percentage neoplastic cells per total number of blood cells): Nonneoplastic (NN), 0%; low severity neoplastic (LSN), less than 50%; and high severity neoplastic (HSN), greater than 50%. Fifty-one percent of those clams initially diagnosed as HSN died by the end of the test period as compared to 8% of the LSN clams and only 3% of the normals. Both progression and remission of the disease were also evident. Approximately 10% of the clams in the NN and LSN groups progressed to a LSN or HSN condition, whereas 16% of those clams initially identified as LSN, and that were recovered alive, underwent complete remission during the test period. Comparison of the field results with those of an 18-week laboratory study suggests that studies of mortality done under laboratory conditions may not provide useful data for the interpretation of the quantitative effects of a disease process, such as molluscan neoplasia, on the natural population of the animal studied.     

Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

Background  

Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples.     

Evaluation of reference genes for real-time quantitative PCR studies in

ABSTRACT: BACKGROUND: The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. RESULTS: The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1a, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-[increment][increment]CT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-[increment][increment]CT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments. CONCLUSIONS: We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.     

Patterns of p53, p73 and mortalin gene expression associated with haemocyte polyploidy in the soft-shell clam, Mya arenaria

The molecular mechanisms by which haemocytes of clams are transformed in the course of haemic neoplasia remain by far unknown. The aim of this study was to quantify the expression of p53/p73 and mortalin genes, in relation with the ploidy status of clam haemocytes and to correlate the p53 expression with mortalin expression. For this purpose, soft-shell clams, Mya arenaria, were collected from an endemic zone for neoplasia. The ploidy of haemocytes was assessed for each individual clam by flow cytometry using a propidium iodide protocol, while p53/p73 and mortalin gene expressions were quantified by real-time RT-PCR. Results show that haemocytes of some clams with a moderate percentage (15-50%) of tetraploid cells have a significantly high level of p53 and p73 in comparison with clams belonging to categories with low (<15%) or high levels (>50%) of tetraploid cells, where low levels of expression of these genes were observed. Furthermore, mortalin gene expression is strongly correlated (r² = 0.68, p < 0.01) with p53 gene expression level. This reinforces the hypothesis of a cytoplasmic p53 sequestration mechanism in clam haemic neoplasia. Further studies are needed to confirm these preliminary results and further unravel the molecular pathways involved in this process. Our results are believed to provide phenotypic foundation for such studies to be undertaken.     

Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR

Background  

Flatfish metamorphosis involves major physiological and morphological changes. Due to its importance in aquaculture and as a model for developmental studies, some gene expression studies have focused on the understanding of this process using quantitative real-time PCR (qRT-PCR) technique. Therefore, adequate reference genes for accurate normalization are required.