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Clonorchis sinensis is major fish-borne trematode, endemic in North Vietnam. Risk factors described so far include individual eating behaviors and environmental factors. Here, additional to conventional risk factors, we report on socially influenced liver fluke transmission in endemic communities. A cross-sectional study on risk factors and fish sharing networks was conducted in 4 villages of Gia Thinh Commune, Ninh Binh Province. A total of 510 residents in 272 households were recruited for risk factor analysis while 220 households, 28 fishermen and 10 fish-sellers were enrolled for social network study. Fecal examination for C. sinensis eggs was performed. Average C. sinensis infection rate at Gia Thinh commune was 16.5% (range 2% to 34.4%). Higher infection rates were significantly associated with males, lower educational levels, eating raw fish, and location of the villages. Social network analysis (SNA) showed a strong positive correlation between ego network size (number of households in fish sharing network) and quantity of raw fish consumed (r=0.603, P< 0.05). The infection rate in people who ate raw-fish caught from a nearby river was significantly higher than those who consumed fish taken from farmed ponds (P

Opisthorchis viverrini (OV) infection is endemic to the Northeast Thailand where the prevalence of Type 2 Diabetes mellitus (T2DM) is higher whilst the incidence of cardiovascular diseases (CVDs) is lower than the rest of Thailand. Helminth infection has both nutritional and immunological impact on their definitive hosts. Thus, a cross-sectional study was performed to see the effects of OV infection on glucose and lipid profiles. For this purpose, 200 each of OV infected and uninfected residents were recruited and their glycated hemoglobin (HbA1c), total cholesterol, triglycerides, low- and high-density lipoproteins (LDL and HDL) levels and anthropometric measurements, including BMI were examined. Then, as the prospective follow- up study, changes of those metabolic parameters of OV positive subjects (n = 120) before and after Praziquantel (PZQ) treatment were monitored for six months. The results showed that OV infection has a protective effect against hyperglycemia (OR 0.482 and p = .04) and metabolic disease risk group (OR 0.478 and p = .03). OV positive participants had lower HbA1c (5.5% Vs. 6.01%, p = .001) but higher HDL (54.07 Vs. 49.46 mg/dL, p = .001) than OV negative participants that are statistically significant. After PZQ treatment for OV-positive subjects, their serum levels of HbA1c (p

A survey of twospotted spider mite, Tetranychus urticae Koch, hosts was conducted during the spring months of 2007, 2008, and 2009 in Mississippi. Uncultivated field borders that supported a diverse flora of herbaceous plants and adjacent in-field weeds were sampled from late-March through May for the presence of twospotted spider mite. Twospotted spider mite preference for each plant species was determined and ranked in a 0 - 3 scale. Henbit, Lamium amplexicaule L., was the most consistent and preferred host of twospotted spider mite in Mississippi. Carolina geranium, Geranium carolinianum L., cutleaf geranium, Geranium dissectum L., vetch, Vicia spp., volunteer soybean, Glycine max L., purple deadnettle, Lamium purpureum L., and spiny sowthistle, Sonchus asper (L.) Hill, were other frequently infested dicotyledonous species. Of the monocotyledonous species sampled, only rescuegrass, Bromus catharticus Vahl, john-songrass, Sorghum halepense (L.) Pers., and volunteer corn, Zea mays L., appeared t...

Opisthorchis viverrini (Ov) is a fish-borne parasite endemic in parts of Lao PDR, Cambodia, southern Vietnam and Northeast Thailand (Isaan) where an estimated 10 million people are infected. Human Ov infection, associated with hepatobiliary complications, including cholangiocarcinoma (CCA), occurs when infected fish are consumed raw or undercooked, a longstanding cultural tradition in the region. This mixed- methods descriptive study was carried out in Isaan villages around Lawa Lake, Khon Kaen Province, known for their Ov endemicity. Focus group discussions (FGDs) and in depth interviews (IDIs) were used to explore socio-cultural determinants underlying raw fish consumption practices, and global positioning system (GPS) devices to map local fish distribution networks. Qualitative data affirmed major socio-cultural and dietary lifestyle transitions occurring consequent on recent decades of modernization policies and practices, but also the persistence of Isaan traditional raw-fish eating practices and incorrect beliefs about infection risk avoidance. Fish traders/middlemen purchase most of the catch at the lakeshore and play the dominant role in district market fish distribution networks, at least for the larger and less likely infected, fish species. The lower economic value of the small potentially-infected cyprinid fish means local fishermen typically distribute them free, or sell cheaply, to family and friends, effectively concentrating infection risk in already highly Ov infected villages. Our study confirmed the persistence of traditional Isaan raw-fish meal practices, despite major ongoing socio-cultural lifestyle transitions and decades of Ov infection health education programs. We contend that diffuse socio-cultural drivers underpin this practice, including its role as a valued cultural identity marker. A "fish economics" factor was also evident in the concentration of more likely infected fish back into local villages due to their low economic value at district market level. The complexity of factors supporting "risky" fish-eating traditions in Isaan underscores the importance of integrated liver fluke infection control strategies to draw on transdisciplinary knowledge beyond biomedicine and also embrace participatory protocols for engaging communities in developing, implementing and evaluating interventions.

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16–20 h old). Toxicity tests showed that levels of 10–15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40–60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min−1 to −10 °C, holding for a further 5 min, then cooling at 0.5 °C min−1 to −35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.

The effect of cryopreservation on aspects of sperm function was investigated for sperm of the Greenshell™ mussel ( Perna canaliculus ). Fertility, DNA integrity, viability and motility were measured before and after cryopreservation. Cryopreservation had a significant effect on fertility with many cryopreserved samples failing to achieve 50% oocyte fertilisation at the highest sperm concentration measured. Sperm DNA integrity, measured using the Sperm Chromatin Structure Assay, showed a small but significant increase in DNA damage as a result of cryopreservation. Sperm viability, measured using propidium iodide/SYBR-14 dual staining, decreased significantly following cryopreservation (69.9 ± 3.5% for fresh sperm versus 25.3 ± 2.9% for cryopreserved sperm). Aspects of motility were similarly affected. The number of sperm tracked, curvilinear velocity (VCL), average path velocity (VAP), variation of straight line velocity (PFT), mean angular displacement (MAD), beat cross frequency (BCF), amplitude of lateral head displacement (ALH) and dance mean (DMN) all decreased following cryopreservation. Correlation analyses between the various aspects of sperm function were carried out. However, no single aspect of sperm function was significantly correlated with sperm fertility following cryopreservation. In addition, the technique for fertility measurement is relatively quicker and less dependent on high tech equipment than the other parameters studied. Fertility therefore remains the most important and relevant end point to measure. Further optimisation of the cryopreservation protocol for Greenshell™ mussel sperm is needed to improve post-thaw fertility.

A series of eight experiments was conducted to determine the most suitable cryoprotectants (CPAs), dilution rates and methods of CPA addition as well as freezing method for the cryopreservation of Greenshell™ mussel ( Perna canaliculus ) sperm. The experiments utilized a fertilization assay to determine the treatments that gave the highest post-thaw fertility. The assays revealed that dimethyl sulphoxide (DMSO) gave the best results of the seven CPAs tested. A concentration of DMSO of approximately 12% [volume: volume] in Milli-Q water provided the best results. A dilution rate of 1:1 (sperm: CPA solution) was superior to 1:3 indicating that more concentrated sperm had higher post-thaw fertility. The time that sperm were exposed to the CPA solution prior to freezing had no effect over the range tested which gives some flexibility in the amount of time sperm can be exposed to CPA prior to freezing. The addition of the CPA in either one step or in 4 equal volume steps over two min had no significant effect on fertility and the two different freezing methods (programmable freezer or rack floating above liquid nitrogen) produced similar results. The post-thaw fertility of mussel sperm using the final protocol developed (12% DMSO plus 0.2 M trehalose, sperm diluted 1:1, loaded into 0.5 mL straws and cooled on a rack 3 cm above liquid nitrogen followed by immersion in liquid nitrogen) was significantly reduced in comparison to fresh sperm. On average, approximately 363 x more sperm were required to achieve an equivalent level of fertilization as that of fresh sperm. The method is being used in mussel selective breeding, however, there is considerable between-animal variability. Further refinement to the method may help to improve post-thaw fertility.

BACKGROUND: Pyrethroid insecticides have historically been effective for controlling bean leaf beetles in soybeans. Perceived field efficacy in the Mississippi Delta region has been lower over the past several years. Laboratory and field data were collected to determine whether there has been a reduction in pyrethroid insecticide susceptibility. RESULTS: Laboratory and field data both confirmed that control of bean leaf beetles with pyrethroid insecticides is poorer in the Delta regions of Mississippi and Louisiana than in all other regions tested. However, it was not possible to show that this reduced susceptibility changed over time. Furthermore, insect movement appeared to make insecticide susceptibility regional, so that localized insecticide applications had little impact on the susceptibility of successive generations. CONCLUSION: Lack of field control owing to low efficacy of pyrethroid insecticides against bean leaf beetle populations in the Delta region should be expected. Selection pressure on a regional basis may further decrease efficacy. Carbamate insecticides generally still provide high levels of control. Copyright © 2011 Society of Chemical Industry

A culture collection of freshwater planktonic and benthic cyanobacteria collected from sites across New Zealand has been established at the Cawthron Institute, Nelson, New Zealand. Limited resources led to uncertainty regarding the long‐term maintenance of this collection. The present study demonstrates cryopreservation to be a viable method for long‐term storage of cyanobacteria. Seventeen of 20 strains evaluated were successfully cryopreserved using the permeating cryopreservation agent dimethyl sulfoxide (Me2SO), at a final concentration of 15% (v/v). Cyanotoxin analysis was undertaken on selected strains known to produce microcystins, nodularin, anatoxin‐a, and saxitoxins. All strains retained their ability to produce these toxins following cryopreservation.

A 3-yr study was conducted to determine seasonal abundance of major pentatomid, noctuid, and mirid pests in a multicrop environment in south Arkansas. Replicated field plots (~0.1 ha) containing cotton, Gossypium hirsutum (L.), genetically engineered to express the Cry 1Ac toxin of Bacillus thuringiensis (Bt), nonBt cotton, Bt corn, Zea mays (L.), genetically engineered to express the Cry 1Ab toxin of B. thuringiensis, nonBt corn, maturity group (MG) III, IV, V, and VI soybean, Glycine max (L.) Merrill, and grain sorghum, Sorghum bicolor (L.) Moench, were planted each year. Crop preferences of 3 stink bug species [southern green stink bug, Nezara viridula (L.), green stink bug, Acrosternum hilare (Say), and brown stink bug, Euschistus servus (Say)] were recorded and observations also were made on the relative abundance of corn earworm, Helicoverpa zea (Boddie), tobacco budworm, Heliothis virescens (F.), and tarnished plant bug, Lygus lineolaris (Palisot de Beavois). Significantly higher densities of stink bugs were present in MG IV soybean than in all other crops in July and August during all 3 yrs of study. Densities of heliothines were significantly greater in grain sorghum and corn than in other crops during July. Tarnished plant bugs were most commonly observed in cotton. Preferences of these polyphagous pests for different crops at specific stages of phenological development appear to be predictable aspects of insect behavior that may be exploited in trap crops or other landscape management systems.

This paper describes a simple method for cryopreserving sperm of the Pacific oyster ( Crassostrea gigas Thunberg) in quantities suitable for commercial spat production. Experiments to refine the cryoprotectant mixtures demonstrated the key role of trehalose. Trehalose alone (at 0.45 M final concentration) was an effective cryoprotectant. The addition of 2.5–15% dimethyl sulphoxide (DMSO) in combination with 0.45 M trehalose gave only modest improvement in fertility over trehalose alone ( p =0.056). There was no significant difference in fertility among DMSO concentrations ( p =0.611). Seawater (SW) without cryoprotectant gave very poor results, but yielded some fertilization at very high sperm concentrations (7±1% at 10 7 sperm mL −1 , 21±2% at 3.2×10 7 sperm mL −1 , mean±S.E., n =3). The fertility of unfrozen sperm was 30- to 100-fold higher than that of sperm cryopreserved with DMSO and/or trehalose. For sperm cryopreserved in 4.5-mL cryovials, two simplified freezing methods gave fertilization rates equivalent to sperm cryopreserved by controlled rate freezing ( p =0.386). These methods involved securing the cryovials to aluminium canes and then either placing them into a bath of methanol chilled with dry ice, or holding them on a floating rack 3 cm above liquid nitrogen. A third technique of plunging the cryovials directly into liquid nitrogen gave reduced and variable fertility relative to the methanol/dry ice bath method ( p =0.032). The commercial applicability of the protocols was demonstrated on a batch of 30 million eggs. Fertilization with cryopreserved sperm yielded 81% fertilization, and larval rearing by normal commercial practises yielded 3.7 million settled spat, which was comparable to the 2.5 million spat from a parallel batch fertilized with unfrozen sperm.

Cryopreservation is a valuable technique for aquaculture as it enables a library or bank of genetically valuable animals to be maintained in a cost-effective manner. Here, we describe a method to cryopreserve the sperm of the Greenshell™ mussel (Perna canaliculus) and how to use the sperm post-thawing to maximize larval production from thawed sperm in selective breeding.

Human liver fluke, Opisthorchis viverrini, a food-borne trematode is a significant public health problem in Southeast Asia, particularly in Thailand. Despite a long history of control programmes in Thailand and a nationwide reduction, O. viverrini infection prevalence remains high in the northeastern provinces. Therefore, a new strategy for controlling the liver fluke infection using the EcoHealth/One Health approach was introduced into the Lawa Lake area in Khon Kaen province where the liver fluke is endemic. A programme has been carried using anthelminthic treatment, novel intensive health education methods both in the communities and in schools, ecosystem monitoring and active community participation. As a result, the infection rate in the more than 10 villages surrounding the lake has declined to approximate one third of the average of 50% as estimated by a baseline survey. Strikingly, the Cyprinoid fish species in the lake, which are the intermediate host, now showed less than 1% prevalence compared to a maximum of 70% at baseline. This liver fluke control programme, named “Lawa model,” is now recognised nationally and internationally, and being expanding to other parts of Thailand and neighbouring Mekong countries. Challenges to O. viverrini disease control, and lessons learned in developing an integrative control programme using a community-based, ecosystem approach, and scaling-up regionally based on Lawa as a model are described.

One hundred and four, 5to 10-month-old Holstein heifers and steers were blocked by age within gender and randomly assigned to treatments. Calves were vaccinated with 5way modified-live respiratory viral vaccine, Mannheimia hemolytic bacterin/toxoid, and 5way Leptospira bacterin, administered via either needle-free or conventional needle-andsyringe injection techniques. Blood samples were collected from all calves at the time of vaccination and 21 days later. Blood sera were analyzed for antibody titers to infectious bovine rhinotracheitis (IBR) virus as the indicator of serological response to the 5-way viral vaccine, to Leptospira pomona (LP) as the indicator of serological response to the 5-way Leptospira bacterin, and to Mannheimia hemolytica (MH) leukotoxoid. Responses of heifers on day 21 to the IBR fraction of the 5way viral vaccine, MH bacterin, and LP fraction of the 5-way Lepto bacterin did not differ between methods of administration. Responses of steers on day 21 to the IBR fraction of the 5-way viral vaccine and MH bacterin were greater for the needle-free method of administration, whereas serological response to the LP fraction did not differ between methods of administration. We conclude that needle-free injections can eliminate broken needles in the carcass, reduce needle-borne transmission of disease, and possibly produce greater serological responses to various antigens, compared with those obtained with conventional needle-and-syringe injection systems.

Twelve field experiments and one laboratory experiment were conducted to determine the effects of furrow applied aldicarb and seed treatments of thiamethoxam, imidacloprid, Avicta (thiamethoxam + abamectin), Aeris (imidacloprid + thiodicarb), and acephate on twospotted spider mite, Tetranychus urticae Koch, on cotton, Cossypium hirsutum L. For the field experiments, data were pooled across all experiments for analysis. Aeris, thiamethoxam, and imidacloprid treatments resulted in twospotted spider mite densities greater than those in the untreated check, aldicarb, and acephate treatments. However, cotton treated with Avicta (thiamethoxam + abamectin) had 34% fewer mites than other neonicotinoid seed treatments when infestations occurred near cotyledon stage. Untreated check and aldicarb treatments had the lowest mite densities. Only aldicarb reduced mite densities below that in the untreated check. In a laboratory trial, the fecundity of twospotted spider mite was measured. While neonicotinoid seed treatments increased mite densities in the field, they did not increase fecundity in the laboratory experiment. Foliar applied thiamethoxam slightly elevated average fecundity in the laboratory experiment. Increased use of neonicotinoid seed treatments instead of furrow applied aldicarb is likely at least partly responsible for recent increased twospotted spider mite infestations in seedling cotton across the mid-south.

Cryopreservation is a powerful tool for selective breeding in aquaculture as it enables genetic material from selected stock to be stored and crossed at will. The aim of this study was to develop a method for cryopreserving oocytes of the Greenshelltrade mark mussel (Perna canaliculus), New Zealand's main aquaculture species. The ability of oocytes to be fertilized post-thawing was used as the criterion for success in initial experiments and then subsequently, the ability of frozen oocytes to develop further to D-stage larvae was assessed. Ethylene glycol, propylene glycol, dimethyl sulphoxide and glycerol were evaluated at a range of concentrations with and without the addition of 0.2M trehalose using post-thaw fertilization as the endpoint. Ethylene glycol was most effective, particularly when used in combination with trehalose. A more detailed investigation revealed that ethylene glycol at 9% or 10% in the presence of 0.2-0.4M trehalose afforded the best protection. In experiments varying sperm to egg ratio and egg density in post-thaw fertilization procedures, D-larval yield averaged less than 1%. Following these results, a detailed experiment was conducted to determine the damaging steps in the cryopreservation process. Fertilization losses occurred at each step whereas D-larval yield approximately halved following CPA addition and was almost zero following cooling to -10 degrees C. Cryomicroscopy studies and fertilization results suggest that the inability of oocytes to develop to D-larvae stage after cooling to -10 degrees C and beyond are most likely related to some form of chilling injury rather than extracellular ice triggering intracellular ice formation. Further research is needed to determine the causes of this injury and to reduce CPA toxicity and/or osmotic effects.

The ability to routinely cryopreserve micro-algal species reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. Cryopreservation is also a useful adjunct in aquaculture hatcheries for strains of micro-algae where the nutritional status may change as a result of continuous sub-culture. In this study, cryopreservation of isolates from seven micro-algal classes was investigated. Successful candidates included the marine dinoflagellates Amphidinium carterae, Amphidinium trulla, and Gymnodinium simplex, and the haptophytes Chrysochromulina simplex, Prymnesium parvum, Prymnesium parvum f. patelliferum, Isochrysis galbana, and Pavlova lutheri. Also successfully cryopreserved were the planktonic diatoms Chaetoceros calcitrans, Chaetoceros muelleri, Chaetoceros sp., and the benthic Nitzschia ovalis, the chlorophyte Chlamydomonas coccoides, the rhodophyte Porphyridium purpureum, the prasinophytes Tetraselmis chuii, and Tetraselmis suecica, and the cyanophytes Raphidiopsis sp., and Aphanizomenon flos-aquae. All species were successfully cryopreserved using 15% Me2SO.

Cryopreservation can be a powerful tool in shellfish aquaculture for selective breeding programmes and hatchery production. Most species that are commercially farmed are broadcast spawners, releasing sperm and eggs into the environment where they fertilize and develop. However, some species are brooders (e.g. flat oysters (Ostrea chilensis) ), which retain and rear embryos until they reach later developmental stages. Sperm cryopreservation has been successfully achieved for several farmed species including the Pacific oyster ( Crassostrea gigas ), greenshell™ mussel ( Perna canaliculus ) and abalone ( Haliotis iris ). In general, dimethyl sulphoxide and trehalose are the preferred cryoprotectants (CPAs) and simple methods to control cooling rate (e.g. floating rack on liquid nitrogen; methanol/dry ice bath) have worked well for our main species of interest (Pacific oyster, greenshell™ mussel and abalone). Interestingly, sperm perform better in CPAs that are prepared in Milli-Q water as opposed to seawater, indicating that a lower salt concentration during freezing is beneficial. Recent studies with flat oysters and geoducks ( Panopea zelandica ) have also resulted in successful sperm cryopreservation. Shellfish oocyte and larvae cryopreservation has proven more challenging, however, successful cryopreservation of Pacific oyster oocytes has been achieved. Pacific oyster oocytes are able to be fertilized and develop to normal spat post-thawing. However, for other species (e.g. greenshell™ mussel) and for larvae, cryo-injuries are not always immediately apparent post-thawing but manifest during development and are often lethal. greenshell™ mussel oocytes are irretrievably damaged when they reach -6 to -8°C and attempts to overcome this sub-zero chilling injury by vitrification have been unsuccessful. Towards understanding these injuries and their effects, we investigated the effect of cryopreservation on the meiotic spindle of greenshell™ mussel oocytes. Results show that the meiotic spindle undergoes major changes during both CPA addition and cooling. However, the spindle is able to re-form normally in most instances given sufficient time post thawing. For a proportion of oocytes, cryopreservation appears to trigger oocyte activation. The effect of cryopreservation on oxidative stress in greenshell™ mussel oocytes has also been investigated. Although, there are changes in oxidative stress parameters as a result of cryopreservation and strong correlations between fertilization, development and oxidative stress parameters (lipid hydroperoxides, superoxide dismutase, protein carbonyls, total glutathione and glutathione state), preliminary attempts to mitigate oxidative stress using antioxidants have so far failed to enable normal development following cooling, suggesting that oxidative stress may be a secondary rather than primary stress event. Research on shellfish larval cryopreservation utilizing SEM and confocal microscopy has also been carried out for greenshell™ mussel. Later larval stages (D-larvae) appear more resilient to cryopreservation than trochophore larvae, but abnormalities are still apparent (e.g.shell abnormalities and delayed organogenesis). Future studies are aimed toward elucidating mechanisms of injury using new tools such as proteomics and then targeting susceptible cellular processes for protection during cryopreservation. Source of funding: This research was funded by the New Zealand Ministry of Business Innovation and Employment (CAWX0802). Conflict of interest: None declared. serean.adams@cawthron.org.nz