MDNR-NOAA trawl standardization study

Long-term living resource monitoring programs are commonly conducted globally to evaluate trends and impacts of environmental change and management actions. For example, the Woods Hole bottom trawl survey has been conducted since 1963 providing critical information on the biology and distribution of finfish and shellfish in the North Atlantic (Despres-Patango et al. 1988). Similarly in the Chesapeake Bay, the Maryland Department of Natural Resources (MDNR) Summer Blue Crab Trawl survey has been conducted continuously since 1977 providing management-relevant information on the abundance of this important commercial and recreational species. A key component of monitoring program design is standardization of methods over time to allow for a continuous, unbiased data set. However, complete standardization is not always possible where multiple vessels, captains, and crews are required to cover large geographic areas (Tyson et al. 2006). Of equal issue is technological advancement of gear which serves to increase capture efficiency or ease of use. Thus, to maintain consistency and facilitate interpretation of reported data in long-term datasets, it is imperative to understand and quantify the impacts of changes in gear and vessels on catch per unit of effort (CPUE). While vessel changes are inevitable due to ageing fleets and other factors, gear changes often reflect a decision to exploit technological advances. A prime example of this is the otter trawl, a common tool for fisheries monitoring and research worldwide. Historically, trawl nets were constructed of natural materials such as cotton and linen. However modern net construction consists of synthetic materials such as polyamide, polyester, polyethylene, and polypropylene (Nielson et. al. 1983). Over the past several decades, polyamide materials which will be referred to as nylon, has been a standard material used in otter trawl construction. These trawls are typically dipped into a latex coating for increased abrasion resistance, a process that is referred to as “green dipped.” More recently, polyethylene netting has become popular among living resource monitoring agencies. Polyethylene netting, commonly known as sapphire netting, consists of braided filaments that form a very durable material more resistant to abrasion than nylon. Additionally, sapphire netting allows for stronger knot strength during construction of the net further increasing the net’s durability and longevity. Also, sapphire absorbs less water with a specific gravity near 0.91 allowing the material to float as compared to nylon with specific gravity of 1.14 (Nielson et. al. 1983). This same property results in a light weight net which is more efficient in deployment, retrieval and fishing of the net, particularly when towing from small vessels. While there are many advantages to the sapphire netting, no comparative efficiency data is available for these two trawl net types. Traditional nylon netting has been used consistently for decades by the MDDNR to generate long term living resource data sets of great value. However, there is much interest in switching to the advanced materials. In addition, recent collaborative efforts between MDNR and NOAA’s Cooperative Oxford Laboratory (NOAA-COL) require using different vessels for trawling in support of joint projects. In order to continue collaborative programs, or change to more innovative netting materials, the influence of these changes must be demonstrated to be negligible or correction factors determined. Thus, the objective of this study was to examine the influence of trawl net type, vessel type, and their interaction on capture efficiency.

Development, validation, and utilization of a competitive enzyme-linked immunosorbent assay for the detection of antibodies against Brucella species in marine mammals

A competitive enzyme-linked immunosorbent assay (cELISA) was developed by using a whole-cell antigen from a marine Brucella sp. isolated from a harbor seal (Phoca vitulina). The assay was designed to screen sera from multiple marine mammal species for the presence of antibodies against marine-origin Brucella. Based on comparisons with culture-confirmed cases, specificity and sensitivity for cetacean samples tested were 73% and 100%, respectively. For pinniped samples, specificity and sensitivity values were 77% and 67%, respectively. Hawaiian monk seal (Monachus schauinslandi; n = 28) and bottlenose dolphin (Tursiops truncatus; n = 48) serum samples were tested, and the results were compared with several other assays designed to detect Brucella abortus antibodies. The comparison testing revealed the marine-origin cELISA to be more sensitive than the B. abortus tests by the detection of additional positive serum samples. The newly developed cELISA is an effective serologic method for detection of the presence of antibodies against marine-origin Brucella sp. in marine mammals.

PCR-based assay for detection of four coral pathogens

Several microorganisms have been identified as pathogenic agents responsible for various outbreaks of coral disease. Little has been learned about the exclusivity of a pathogen to given disease signs. Most pathogens have only been implicated within a subset of corals, leaving gaps in our knowledge of the host range and geographic extent of a given pathogen. PCR-based assays provide a rapid and inexpensive route for detection of pathogens. Pathogen-specific 16S rDNA primer sets were designed to target four identified coral pathogens: Aurantimonas coralicida, Serratia marcescens, Vibrio shilonii, and Vibrio coralliilyticus. Assays detected the presence of targets at concentrations of less than one cell per microliter. The assay was applied to 142 coral samples from the Florida Keys, Puerto Rico, and U.S. Virgin Islands as an in situ specificity test. Assays displayed a high-level of specificity, seemingly limited only by the resolution of the 16S rDNA.

Standardization and frozen storage of fish cutlet from bleached and unbleached mackerel mincemeat

In the present study, fish cutlets were prepared from bleached and unbleached mackerel mincemeat. Fish cutlets prepared from bleached meat had scored higher values for taste, flavour and overall acceptability as compared to those from unbleached mincemeat. Fish cutlets prepared with corn flour at the rate of 15% of fish mincemeat had scored higher values for all attributes as compared to other levels. Between the bleached and unbleached mincemeat, the scores for cutlet prepared with bleached mincemeat had higher score than that for the latter. There were no cracks in cutlets prepared with 15% and above corn flour levels as compared to those with lower levels. Fish cutlets prepared from bleached and unbleached mincemeat with spice mixture at 20 and 30% of the fish mince, respectively, had higher scores for taste, flavour, texture and overall acceptability as compared to those with other levels. Organoleptic quality of cutlet prepared from bleached and unbleached mackerel mince did not show changes in the appearance, colour and texture during storage. Changes were more prominent in flavour, taste and overall acceptability. Fish cutlets prepared from bleached mincemeat were acceptable for two months and those from unbleached mincemeat were acceptable up to one month from the point of view of organoleptic and biochemical qualities.

Standardization of polyphosphate treatment for tiny prawns (Parapenaeopsis stylifera) with special reference to its rehydration capacity and organoleptic quality

In the present study, an attempt was made to explore the benefits of polyphosphate for enhancement of dried prawn (Parapenaeopsis stylifera) quality commercially known as "sode" in Maharashtra coast. Dip treatment in polyphosphate solution at different concentrations (viz., 3, 5, 7 and 10%) was given to pealed and no deveined P. stylifera for different durations (viz., 1, 2, 3, 4 and 5 min). Treated prawns were dried and subjected to rehydration capacity test and organoleptic evaluation. Among the different treatments, rehydration capacity was found to increase with the increased duration and concentration of treatment. Tiny prawns treated with sodium tripolyphosphate solution at the rate of 5% concentration for 5 minutes showed an increase in pH from acidic to alkaline, and had better quality with respect to rehydration capacity and textural attributes as compared to other concentrations and durations of polyphosphate treatment.

Detection of usual and atypical aldehyde dehydrogenase alleles by mismatch amplification mutation assay

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical ALDH(2)(2) allele have higher levels of acetaldehyde in their plasma. The atypical ALDH(2)(2) allele has a nucleotide base transition (G-->A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual ALDH(2)(1) allele and the atypical ALDH(2)(2) allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical ALDH(2)(2) allele was found to be 12%.

Standardization of setting temperature and time for fish meat

Meat to water ratio used for washing was 1:3 for oil sardine and mackerel; but for pink perch and croaker, it was 1:2. Again the washing process was repeated three times for oil sardine and mackerel; but two times for pink perch and croaker. The washed meat was mixed with 2.5% NaC1 and set at +5°C and +40°C for 1, 2 and 3hrs. The gel strength and expressible water content was measured. Basing on this study, setting temperature at +40°C was selected and with respect to time 1hr for sardine and mackerel and 3hrs for pink perch and croaker was selected.

Biomonitoring of polycyclic aromatic hydrocarbons (PAH) and heavy metals Ni, V in sediments and anodonts of the Anzali Lagoon and applicability of netural red retention assay (NRR) as biomarkers of these pollutants

Biochemical ecotoxicology and biomarkers using are a new sciences that are used for biomonitoring in aquatic environment. Biomonitoring plays a vital role in strategies to identify, assess, and control contaminants. On the other hands in recent year's attention to polycyclic Aromatic Hydrocarbons (PAHs) and heavy metals increased in aquatic environments because of their carcinogenic and mutagenic properties combined with their nearly ubiquitous distribution in depositional environments by oil pollution or industrial waste waters. The present research aimed to assess PAHs and Ni, V levels in surface sediments and bivalves (Anodonta cygnea)and the effects of PAHs and heavy metals (Ni,V) on the hemocyte of the Anodonta cygnea were investigated in 2 stations (Mahrozeh, Selke in Anzali Lagoon, North of Iran). Samples were collected during at 2 different periods of the year, Dry and rain seasons, (June & September) and to confirm our first observations, Cage station is added. The bivalves hemocytes were monitored for membrane injury by NRR methods (neutral red retention assay). Heavy metal (Ni, V) concentrations were determined by Atomic Absorption in Anodonta cygnea and the sediments in Anzali Lagoon. The vanadium concentration in bivalves and sediments was ND(not detect )-0.4231 μg/g and 1.4381-306.9603 μg/g dry weight respectively. Nickel concentration in bivalves and sediments was 0.0231-1.3351, 0.4024-19.3561 μg/g dry weight respectively. PAHs concentrations were determined by GC-Mass in Anodonta cygnea and the sediments. Average concentration of PAHs is 115-373.788 ng/g dry weight in bivalves and average concentration of PAHs is 34.85-1339.839 ng/g dry weight in sediments. Bioaccumulation sediments factor(BASF) is high about PAHs (>1) and BASF is low for Ni, V (<1) . Internal Damage mechanisms of bivalves hemocytes (cell mortality, dye leakage, decreased membrane stability, are observed (Lowe Methods). Statistical analysis was used to explore the relationship between altered cellular and above contaminants. There are power and negative correlations between PAHs and NRR method for hemocytes in Anodonta cygnea (P<0.0005), but good correlation is not observed between Ni, V and NRR method for hemocytes in every time. This research indicates that the NRR assay is a useful screening technique able to discriminate polluted sites and at first we announce that Anodonta cygnea hemocytes are efficient biomarker for PAHs pollutants in fresh water.

CORRELATION OF ZONA-BINDING WITH OOCYTE MATURATION AND SPERM MOTILITY IN RHESUS-MONKEYS BY HEMIZONA ASSAY

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PB1). The mean numbers of sperm bound to hemizona for PBI, PVS, GV, and MC groups were 132.9 +/- 12.0, 71.5 +/- 10.1, 36.1 +/- 4.0, and 20.1 +/- 2.9 (Mean +/- SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 +/- 2.2 and 25.3 +/- 2.9 (Mean +/- SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1)The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the ''zona maturation'' hypothesis. (C) 1994 Wiley-Liss, Inc.

An improved microtiter assay for evaluating anti-HIV-I neutralizing antibodies from sera or plasma

Background: The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay. Methods: In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor( s), the tested sera or plasma was removed by a washout procedure after initial 3 - 6 h culture in the assay. Result: This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method. Conclusion: The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma.

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.