Frass produced by the primary pest Rhyzopertha dominica supports the population growth of the secondary stored product pests Oryzaephilus surinamensis, Tribolium castaneum, and T. confusum.

Primary pests such as Rhyzoperta dominica may increase the contents of dockage, dust, and frass in grain mass. Although it has been suggested that frass can affect the population growth of stored product pests and ecological interactions among primary and secondary pests in stored grain, this has not been validated experimentally. Therefore, this work experimentally tested the hypothesis that R. dominica wheat frass may support population increases in secondary pests such as Tribolium confusum, T. castaneum, and Oryzaephilus surinamensis for the first time. The effect of frass on secondary pest performance was compared with the effects of various physical qualities of wheat grain (i.e., intact grain kernels, grain fragments, flour, grain + frass) and an artificially enriched control diet (milled wheat kernels, oat flakes, and yeast). The results showed that the clean intact grain kernels did not support the population growth of any tested species, and the nutrient-rich control diet provided the best support. Frass was a significantly better food medium for O. surinamensis and T. castaneum than flour or cracked grain, while T. confusum performed equally well on flour and frass. Our results showed that in terms of food quality and suitability for the tested species, frass occupied an intermediate position between the optimized breeding diet and simple uniform cereal diets such as cracked grain or flour. The results suggest that (i) the wheat frass of primary pest R. dominica is a riskier food source for the development of the tested secondary pests than intact or cracked wheat grain or flour; (ii) frass has the potential to positively influence interspecific interactions between R. dominica and the tested secondary pests; and (iii) wheat grain should be cleaned if increases in R. dominica populations and/or accumulated frass are detected.

Evaluation of contamination of packages containing cereal-fruit bars by eggs of the pest Indian meal moth (Plodia interpunctella, Lepidoptera) due to perforations in their polypropylene foil packaging.

The quality and type of food packaging affect the level of food protection against pests. This work first evaluated the effect of package perforations on the infestation of cereal-fruit bars by the eggs of the Indian meal moth (Plodia interpunctella). We measured the differential oviposition of moths on the unpackaged bars, empty packages, bars in packages and bars in perforated packages in choice and no-choice experiments. Almost 100% of the laid eggs were laid directly on the bars when they were placed in the enclosure without packaging. A low proportion of the eggs (0.4-3%) were laid on either the empty or non-perforated polypropylene foil packages. Plodia interpunctella efficiently located and infested the bars with eggs when 5 mm package perforations were present in no-choice test. In choice test P. interpunctella preferred to oviposit on open bars than on the packaged or perforated bars. After deposition on the bars the egg hatchability ranged between 40 and 74%. For the food industry, the most significant practical conclusions of this study are that non-perforated packages provide protection against oviposition of P. interpunctella, but small perforations enable the moth to infest the resource with eggs.

Microsatellite markers for Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) and other Cryptolestes species.

Cryptolestes ferrugineus (Stephens, 1831) is an important insect pest of stored products. Due to its broad host range, short life cycle, and high reproductive capacity, this species has rapidly colonized temperate and tropical regions around the world. In this study, we isolated 18 novel polymorphic microsatellite loci from an enriched genomic library based on a biotin/streptavidin capture protocol. These loci will be useful tool to better understand the genetic structure and migration patterns of C. ferrugineus throughout the world. The genetic parameters were estimated based on 80 individual C. ferrugineus from two natural populations. The results revealed that 18 loci were different polymorphic levels. The numbers of alleles ranged from 3 to 12, and eleven loci demonstrated polymorphic information contents greater than 0.5. The observed (H O) and expected (H E) heterozygosities ranged from 0.051 to 0.883 and 0.173 to 0.815, respectively. Five locus/population combinations significantly deviated from Hardy-Weinberg equilibrium. We also demonstrated the potential utility of the C. ferrugineus microsatellites as population and species markers for four additional Cryptolestes species.

Splenic lipidosis in intensively cultured perch, Perca fluviatilis L.

Macroscopically visible lipid deposition varying in size from pinpoint to 8-mm diameter was found in spleens of a population of intensively farmed perch, Perca fluviatilis L. over a 24-month rearing period. Large agglomerates of adipocytes distinguishable from surrounding normal tissue occurred in all individuals with spleen lipidosis. Several affected fish presented total dystrophy of large clusters of hepatocytes. Prevalence of lipidosis was 5.0% at 12 months and 16.6% at 24 months. There was no significant difference between fatty acid profiles of liver or perivisceral fat of perch with and without lipidosis except for linoleic, myristic, γ-linoleic, cis-eicosatrienic, palmitooleic acid. Body weight and hepatosomatic, perivisceral fat and splenosomatic indices were not associated with lipidosis. There was no significant effect of lipidosis on mortality or growth.

COI barcode based species-specific primers for identification of five species of stored-product pests from genus Cryptolestes (Coleoptera: Laemophloeidae).

Flat grain beetles of the genus Cryptolestes (Coleoptera: Laemophloeidae) are one of the economically most important stored-product pests which feed on many kinds of agricultural products, especially grains. Nine of more than 40 described Cryptolestes species are recognized as stored-product pests and two of the pest species have a cosmopolitan distribution. Given the rapid growth in global trade of food products, ecological barriers to the spread of pests are easily overcome. Therefore, development of reliable systems for routine quarantine inspection and early infestation detection is vital. In the present study, we established a new rapid and accurate cytochrome c oxidase subunit I-based system for molecular identification of five common stored-product Cryptolestes species, namely, Cryptolestes capensis, Cryptolestes ferrugineus, Cryptolestes pusilloides, Cryptolestes pusillus and Cryptolestes turcicus. Five species-specific primer pairs for traditional uniplex polymerase chain reaction assay are described and their specificity and sensitivity for the identification process is evaluated using larval samples of 12 different populations from three continents (Asia, Europe and North America).

Susceptibility of Two Strains of the Confused Flour Beetle (Coleoptera: Tenebrionidae) Following Phosphine Structural Mill Fumigation: Effects of Concentration, Temperature, and Flour Deposits.

In this study, we evaluated phosphine efficacy against two strains of the confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), one laboratory strain, with no previous exposure to phosphine, and one field strain, originated from the flour mill on which we performed the fumigation. The standard Detia Degesh Phosphine Resistance Kit showed that the adults of the field strain of T. confusum required 4.4 × longer time to be knocked down than the laboratory strain. In order to assess the efficacy of phosphine in the field against these strains, aluminium phosphide (AlP) was applied in a flour mill in Czech Republic, in 2014. In this application, temperature among the six floors of the flour mill varied between 20 and 30°C, relative humidity (RH) between 44 and 78%, and phosphine concentration-time-products (CtP) between 24 and 38 g.m(-3).h(-1). Moreover, the insects were bioassayed in dishes that contained either no commodity or 1, 3, and 5 cm of flour. Nevertheless, despite these variations, all adults and larvae from both strains were dead, regardless of the floor, the quantity of the commodity, and the conditions prevailing. However, larval emergence from eggs that were used in the bioassays for both strains was recorded in some of the locations tested. In addition, larval emergence was negatively correlated with both temperature and RH. At the same time, emergence was generally similar between strains. The results of the present study illustrate that highly visible dead adults and larvae after the application of phosphine falsely imply good fumigation efficacy, given that a considerable number of eggs are very likely to survive in a wider range of conditions, and the concomitant larval emergence may result in rapid population grown right after the fumigation.

DNA barcoding of five common stored-product pest species of genus Cryptolestes (Coleoptera: Laemophloeidae).

Several species of the genus Cryptolestes Ganglbauer, 1899 (Coleoptera: Laemophloeidae) are commonly found in stored products. In this study, five species of Cryptolestes, with almost worldwide distribution, were obtained from laboratories in China, Czech Republic and the USA: Cryptolestes ferrugineus (Stephens, 1831), Cryptolestes pusillus (Schönherr, 1817), Cryptolestes turcicus (Grouvelle, 1876), Cryptolestes pusilloides (Steel & Howe, 1952) and Cryptolestes capensis (Waltl, 1834). Molecular identification based on a 658 bp fragment from the mitochondrial DNA cytochrome c oxidase subunit I (COI) was adopted to overcome some problems of morphological identification of Cryptolestes species. The utility of COI sequences as DNA barcodes in discriminating the five Cryptolestes species was evaluated on adults and larvae by analysing Kimura 2-parameter distances, phylogenetic tree and haplotype networks. The results showed that molecular approaches based on DNA barcodes were able to accurately identify these species. This is the first study using DNA barcoding to identify Cryptolestes species and the gathered DNA sequences will complement the biological barcode database.

Effects of gonadotropin-releasing hormone agonist administered in microparticles on sperm quality and quantity, and plasma sex steroid levels in northern pike.

Artificial reproduction of northern pike Esox lucius is impeded by the likelihood of obtaining only a small volume of sperm of inconsistent quality. A controlled-release hormone delivery system has the potential to enhance sperm production while avoiding multiple injections The objective of this study was to investigate the effects of mammalian gonadotropin-releasing hormone agonist (mGnRHa) incorporated into poly(lactic-co-glycolic acid) (PLGA) microparticles on milt production, spermatozoon characteristics, and secretion of 17ß-estradiol (E2), 11-keto testosterone (11-KT), and testosterone in northern pike. Fish were divided into four groups and injected with 2 mg/kg BW carp pituitary extract (CPE), 20 µg/kg BW mGnRHa in PLGA microparticles, or 20 µg/kg BW mGnRHa plus 20 mg/kg BW metoclopramide (MET) in PLGA microparticles (PLGA + MET), along with a control group injected with 1 ml/kg 0.9% NaCl. At 48 h postinjection, the volume of milt produced was significantly greater in groups treated with CPE and PLGA + MET than in other groups. At 96 h postinjection, all hormone-treated groups exhibited significantly higher spermatozoon average velocity than recorded in the control group. Spermatozoon motility was significantly increased (P < 0.05) in the CPE and PLGA groups compared to baseline values. All treated groups showed significantly lower levels of 11-KT after the hormone injection compared to baseline values and to controls. Plasma testosterone levels increased in all hormone-treated groups. The use of PLGA microparticles, with or without metoclopramide, is suitable for use as a carrier of hormone treatments to regulate spermiation in mature northern pike.

Sensory and textural attributes and fatty acid profiles of fillets of extensively and intensively farmed Eurasian perch (Percafluviatilis L.).

Sensory attributes, texture and fatty acid profiles of fillets of Eurasian perch (Percafluviatilis L.) reared under two conditions were compared. Perch were reared either in an extensive pond-based (EC) system in polyculture with carp, or intensively cultured (IC) in a recirculation system. Attributes of raw and cooked fillets of marketable perch (120-150g) were compared. No significant differences were found between groups for odour, flavour, aftertaste, or consistency in subjective evaluation of cooked fillets. The texture profile analysis (TPA) showed raw fillets from the EC group to exhibit higher values of hardness, springiness, cohesiveness, and gumminess than the IC group. Fish from the IC group had a lower content of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) and a higher content of monounsaturated fatty acids (MUFA) in comparison to EC perch. The proportion of iso- and anteiso-SFAs was 2.6% in the EC group and 0.75% in the IC group. The content of n-3 PUFA was lower in IC than in EC, while the content of n-6 PUFA was higher in IC than in EC. The ratio of n-3:n-6 PUFA was 1.42 for the IC group and 2.85 for the EC group.

Lymphoid cells mediating tumor-specific cytotoxicity to carcinoma of the urinary bladder. Separation of the effector population using a surface marker.

Peripheral lymphocytes from patients with urinary bladder carcinoma and controls have been separated on the basis of rosette formation with sheep erythrocytes. The fractions were tested for tumor-specific cytotoxicity. The E rosette-forming cells of purity 90% respond well in PHA-induced cytotoxicity but are totally inactive in the tumor assay. The non-E rosette-forming cells (purity 91%) give enhanced activity in the tumor-specific cytotoxicity as well as in antibody-mediated target cell lysis in a model system. These data support the notion that the effector cells in cell-mediated immunity to carcinoma of the urinary bladder are members of the nonthymus-derived population of peripheral lymphocytes.

Lymphocyte transformation test for diagnosis of isothiazolinone allergy in man.

The lymphocyte transformation test (LTT) has been used for evaluation of in vitro lymphocyte responses in 18 patients with dermatitis and positive patch tests to 200 ppm of a combination of 5-chloro-2-methylisothiazolinone and 28methylisothiazolinone (MCI) in nine patients with dermatitis unrelated to MCI and in seven subjects without skin diseases. Two workers sensitized by occupational exposure to a formulation containing 1,2-benzisothiazolin-3-one (BIT) were also studied. Lymphocytes from nine patch-test-positive patients proliferated vigorously to MCI in vitro. Lymphocytes from the remaining nine patients were not stimulated. Lymphocytes from two BIT-sensitized workers responded to BIT in vitro. The lymphocyte proliferation to isothiazolinones indicates the presence of memory cells in the patients' blood and confirms immunologic reaction to the inducing agent. To establish clinical relevance of LTT results, 12 MCI patch-test-positive patients underwent "use test" with lotion containing 15 ppm MCI. Four of five LTT-positive patients were use-test-positive, whereas seven of seven LTT-negative patients were use-test-negative. LTT-positive and lotion-positive patients responded to 100 ppm or lower concentrations of MCI on patch testing, whereas seven of eight LTT-negative and lotion-negative patients responded to 200 ppm only. In the case of MCI, proliferation was due to the chlorinated component, indicating that this part contains an allergenic epitope. Finally, MCI-specific lymphocyte proliferation was observed only in patients with MCI-positive skin test, but not in nine patients with dermatitis induced by other agents, or in seven subjects without skin diseases. Thus, the lymphocyte transformation test is able to distinguish between irritant and allergic skin responses. It may also be valuable in establishing the clinically relevant patch-test concentration of allergens with irritative properties.

HLA-association in patients with intolerance to mercury and other metals in dental materials.

A group of selected 25 patients with serious intolerance to heavy metals used for dental restoration were examined for HLA antigens. A significant increase for HLA -- B37, B47 and DR4 was found. The value of the relative risk is not significant after correction for the number of antigens tested and therefore further studies of more patients are needed.

DNA binding proteins in canine sera. A method for removal of nonspecific DNA binding in the Farr assay.

A panel of canine sera, the majority of which were collected from clinically healthy dogs, were investigated for antibodies against double stranded (dsDNA) by the Farr radioimmunoassay technique. Non-specific DNA binding agents interfering with the Farr assay were detected in all sera. Heat inactivation at 60 degrees C or treatment with dextran sulphate was shown to eliminate this kind of unspecific DNA binding while not affecting true antibodies to dsDNA. Canine sera positive in the Farr assay after inactivation at 60 degrees C were positive also in immunofluorescence for anti-nuclear antibody on rat liver sections and for dsDNA with Chrithidia luciliae as antigen preparation. IgG or glycoprotein nature of the non-specific DNA binding could be excluded by means of affinity chromatography on protein A and the lectin lentil.

MELISA-an in vitro tool for the study of metal allergy.

The sensitizing properties of metals widely used in medical and dental care have been studied with the help of an optimized lymphocyte proliferative assay, MELISA. MELISA (memory lymphocyte immuno-stimulation assay) was originally developed for the screening of allergenic epitopes of drugs and other chemicals of low molecular weight, but has recently been adapted for the study of metal-induced sensitization. The patients studied suffered from various oral mucosal problems which were suspected to be caused by the release of metal ions from dental restorations. They were also troubled by chronic fatigue persisting over many years. One patient was also occupationally exposed to metals while working in a dental practice. Healthy subjects without any discomfort due to metal devices served as controls. In addition to metals used in dentistry, lymphocyte responses to organic mercurials used widely as preservatives in vaccines, eye/nose drops and contact lense fluids were studied. The results indicated that mercurials, as well as other metals such as gold or palladium, induce strong lymphocyte proliferative responses in patients with oral or systemic symptoms, but not in similarly exposed unaffected subjects. The results of MELISA performed with a pair of identical twins with chronic fatigue syndrome (CFS) indicated that metal-specific responses may be dependent on the genetics of the patient. Thus, many metals that are today accepted for use in medicine and dentistry carry a definite sensitizing risk for certain genetically predisposed individuals. Therefore, the use of these metals should be limited in the future.

Mites and fungi in heavily infested stores in the Czech Republic.

Toxigenic and allergen-producing fungi represent a serious hazard to human food and animal feed safety. Ninety-four fungal species were isolated from mite-infested samples of seeds taken from Czech seed stores. Fungi were isolated from the surface of four kinds of seeds (wheat, poppy, lettuce, and mustard) and from the gut and external surface of five species of mites (i.e., Acarus siro L., 1758, Caloglyphus rhizoglyphoides (Zachvatkin, 1973), Lepidoglyphus destructor (Schrank, 1781), Tyrophagus putrescentnae (Schrank, 1781) and Cheyletus malaccensis Oudemans 1903) separately. Multivariate analysis of fungi complex composition showed that the frequency of fungal was species significantly influenced by the kind of seed. Fungal frequencies differed between mites gut and exoskeleton surface and between the surfaces of mites and seeds. Three groups of fungal species were recognized: 1) mite surface-associated fungi: Penicillium brevicompactum, Alternaria alternata, and Aspergillus versicolor; 2) mite surface- and seed-associated fungi: Aspergillus niger, Penicillium crustosum, Penicillium aurantiogriseum, Penicillium chrysogenum, and Aspergillus flavus; and 3) seed-associated fungi: Cladosporium herbarum, Mucor dimorphosporus f. dimorphosporus, Botrytis cinerea, Penicillium griseofulvum, and Eurotium repens. Mite-carried species of microfungi are known to produce serious mycotoxins (e.g., aflatoxin B1, cyclopiazonic acid, sterigmatocystin, ochratoxin A, and nephrotoxic glycopeptides) as well as allergen producers (e.g., A. alternata and P. brevicompactum). Storage mites may play an important role in the spread of some medically hazardous micromycetes. In addition, these mite-fungi associations may heighten the risk of occurrence of mycotoxins in food and feed stuffs and cause mixed contamination by fungal and mite allergens.

[Effect of heavy metals on immune reactions in patients with infertility]. / Vliv tezkých kovu na imunitní reakci u pacientu s prokázanou neplodností.

BACKGROUND: Heavy metals can negatively influence reproduction because in sensitive persons they are able to alter the immune reactions including autoantibodies production. The altered immune reaction can then cause infertility. METHODS AND RESULTS: The in vitro lymphocyte reaction after stimulation with metals, the production of interferon (IFN-gamma) and antisperm antibodies in supernatants after lymphocyte stimulation in patients with infertility and with the antisperm antibodies present in their serum were investigated. The cause of antisperm antibodies presence was not determined. The diagnosis of metal intolerance was performed by the proliferation method modified for metals (Melisa). In supernatants of tissue cultures of lymphocytes without the antigen stimulation and after stimulation with mercury chloride, the in vitro production of gamma interferon and antisperm antibodies was studied by Elisa. More than 50% of patients did not tolerate mercury, iron, aluminium and silver. When the lymphocyte reaction was compared in patients with and without mercury intolerance we found that lymphocytes of patients with mercury intolerance produced less gamma interferon and more antisperm antibodies in supernatants after mercury stimulation of lymphocytes. CONCLUSIONS: In patients with metal intolerance diagnosed by the Melisa test, metal ions released from the dental materials can represent a factor, that does not cause infertility but is able to influence it negatively.

Lymphocyte proliferative response to Helicobacter pylori antigens in H. pylori-infected patients.

Helicobacter pylori (Hp) contributes to the development of gastric and extra-gastric diseases such as autoimmune thyroiditis (AT), and causes persistent life-long infection despite local and systemic immune response. We determined the specific cellular immune response to Hp antigens and PWM (control mitogen) in two groups of Hp infected patients--group A (n = 21), involving patients with autoimmune thyroiditis and group B (n = 13) of patients without AT--using modified lymphocyte transformation test before and after eradication therapy in comparison with healthy controls (group C, n = 15). Immune reactivity to the majority of Hp antigens (aHp, hHp, HpAg, CagA) was significantly lower in group B before eradication therapy in comparison with healthy Hp negative controls. A significant increase in immune reactivity was observed in group B to certain Hp antigens after successful eradication. The same levels (but insignificant) of immune reactivity were shown in group A. Our results indicate that Hp can cause the inhibition of the specific cellular immune response in Hp infected patients with or without autoimmune diseases such as AT, which can be abrogated by successful eradication of Hp. Lymphocyte transformation test appears to be a good tool for detection of immune memory cellular response in patients with Hp infection.