Growth and gonadal development of gynogenetic diploid Scophthalmus maximus

The survival, growth, gonadal development and sex ratios of gynogenetic diploid turbot Scophthalmus maximus between 9 and 36 months of age were investigated. Gynogenesis was induced by activation of freshly collected eggs with diluted (1 : 10 with Ringer) and UV‐irradiated (30000 erg mm⁻²) sperm, followed by a cold shock at −1 to 0° C for 25 min, starting at 6·5 min after fertilization. The exclusive maternal inheritance of the resulting gynogenetic diploids was confirmed in all individuals used in the experiment by microsatellite markers. Mean total length and mass throughout the experiment was higher in the controls than in the gynogenetics. The gonadal development in gynogenetic males proceeded normally, and in gynogenetic females it was delayed during the first sexual maturation but was normal during the second one. The sex ratio was 1 male (M) : 1 female (F) in the controls, as expected, but 1 M : 3 F in the gynogenetics. In a second, independent experiment, carried out with fish originating from different broodstock, however, the sex ratio was 1 M : 1 F in the controls and 0 M : 1 F ( i.e . 100% females) in the gynogenetics. Together, these results suggested that the main sex‐determining genetic component in turbot fits well to female homogamety, in accordance with data from other flatfishes. These results indicate that it will be possible to obtain near or all‐female turbot stocks through induced gynogenesis or by crosses involving hormonally sex‐reversed fish.     

Artificial fertilization and induction of triploidy and meiogynogenesis in the European sea bass, Dicentrarchus labrax L.

The European sea bass, Dicentrarchus labrax L., was successfully subjected to chromosome manipulation. Triploidy was induced by cold-shocking eggs at 0–2°C for 20 min starting 5 min after fertilization in order to prevent the extrusion of the second polar body. Meiogynogenesis was also obtained by fertilizing eggs with UV-irradiated sperm (3300 and 6600 erg m⁻²) and subsequently doubling the chromosome set by meiotic block as above. The commercial advantages of culturing triploid and gynogenetic sea bass are discussed.     

Triploidy induction in the Caspian salmon, Salmo trutta caspius, by heat shock

Induction of triploidy was attempted in the Caspian salmon, Salmo trutta caspius, using heat shocks. The optimal temperature level (26, 28 and 30°C), initiation time [5, 10, 20 and 40 min post-fertilization (PF)] and duration of thermal shock (5, 10 and 20 min) required for effective induction of triploidy were investigated. Incidence of triploid fry was determined by surface and volume measurements of erythrocytes as well as from flow-cytometric analysis of some blood samples. Survival from fertilization to swim-up, triploid rates and triploid yields were in the range of 0–70%, 0–97% and 0–57%, respectively. The highest triploid yield was obtained with a shock treatment at 26°C for 10 min duration initiated 40 min PF.     

The production of gynogenetic Atlantic salmon,Salmo salar L.

The production of Atlantic salmon gynogenomes by the combined use of a novel method for sperm irradiation and differently timed high hydrostatic pressure shocks is described. Sperm solutions were exposed to UV irradiation in a temperature-controlled flow-through device. Eggs fertilised with such sperm were exposed to shocks of 9500 psi at 30 min or approximately 7 h after fertilisation in order to produce meiotic and mitotic gynogenomes respectively. Yields of meiotic gynogenomes were generally high (up to 95%); those of mitotic gynogenomes were lower (range 2–20%). Analyses of the offspring by ploidy status and fingerprinting confirmed their gynogenetic origin. Small numbers of mitotic gynogenetic fish were grown on for 2 years in fresh and salt water. S1/S2 ratios were lower in gynogenetic fish and mean age at maturity was greater. Of the presumptive gynogenetic fish subjected to destructive sampling (n = 87) all were female.     

Heat-resistant Bacteria in Pasteurized Whole Egg

Samples of egg melange taken from an egg packing station contained an average of 7·3 x 10⁴ organisms/ml which survived laboratory pasteurization at 65°C for 3 min. Many of the organisms surviving pasteurization were found to be coryneform bacteria related to Microbacterium lacticum which could be differentiated into several groups. The remainder were a miscellaneous collection of unidentified cocci and coccobacilli and some Bacillus spp. The coryneform bacteria were shown to be the most heat-resistant isolates with negligible loss of viability after 60 min at 65°C, At least two of the representative strains were very heat-resistant, 0·01% surviving 20 and 38 min at 80°C in phosphate buffer at pH 7·1. Growth tests showed that none of the isolates grew at 5°C after 10 d incubation but those capable of growing most rapidly at 10° and 15°C were also the most heat-resistant. Such strains had a doubling time at 15°C of between 6 and 8 h in whole egg. Freezing the coryneform bacteria in liquid whole egg at –18°C had negligible effect on viability or heat-resistance at 65°C.     

Immersion heat treatments for inactivation of Salmonella enteritidis with intact eggs

The effects of water-bath immersion heat treatments on the inactivation of Salmonellaenteritidis within intact shell eggs were evaluated. Six pooled strains of Salm. enteritidis ( ca 3×10⁸ cfu, inoculated near the centre of the yolk) were completelyinactivated within 50–57·5 min at a bath temperature of 58°C and within 65–75min at 57°C (an 8·4 to 8·5- D process per egg). Following the initial 24 to35-min come-up period, semilogarithmic survivor curves obtained at 58 and 57°C yieldedapparent decimal reduction times ( D -values) of 4·5 and 6·0 min, respectively.Haugh unit values increased during heating, while yolk index and albumen pH values wereunaffected. Albumen clarity and functionality were affected by the thermal treatments; therefore,extended whip times would be required for meringue preparation using immersion-heated eggwhites. Immersion-heated shell eggs could provide Salmonella -free ingredients for thepreparation of a variety of minimally-cooked foods of interest to consumers and foodserviceoperators.     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

Effect of chilling, heat shock, and vigor on the growth of cucumber (Cucumis sativus) radicles

Chilling at 2.5°C reduced the subsequent growth of cucumber ( Cucumis sativus L.) radicles at 25°C. The reduction in radicle growth was linear for 1–3 days of chilling at ≈10% per day of treatment, but then it increased in a non-linear pattern until subsequent radicle growth was all but eliminated by 6 days of chilling. A heat shock of 40°C for 4–12 min increased chilling tolerance such that 4 days of chilling caused only a 36% decrease in radicle growth, compared to 66% for seedlings not heat shocked. Heat shocks were only able to protect that part of radicle growth that was in excess of the linear decrease in radicle growth projected from 0–3 days. There appear to be two effects of chilling on radicle growth. The first inhibition of subsequent growth was linear and was not affected by heat shocks. The second inhibition was much more severe; it appeared after 3 days of chilling and could be prevented by heat shock. Seeds classified with different levels of vigor (i.e., different initial rates of growth) did not respond significantly different to chilling stresses following heat-shock treatments.     

Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating

H umphrey , T.J. 1990. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating. Journal of Applied Bacteriology 69 493–497.
Storage of cultures of Salmonella enteritidis PT4 at either 4° or 8°C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4–7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60° rather than 55°C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37°C prior to heating enabled the organisms to recover heat resistance.     

Diploid gynogenesis induced by hydrostatic pressure in rainbow trout, Salmo gairdneri Richardson

Diploid gynogenetic fry of rainbow trout, Salmo gairdneri , were produced by treating eggs activated by UV-irradiated sperm with hydrostatic pressure. Treatments for l0 min at 40 min after activation with 55 000 kPa (8000 psi) pressure resulted in high hatching rates and yields of heterozygous diploid gynogenomes of up to 81%. Attempts to produce homo-zygous diploid gynogenomes by the suppression of the first mitotic division ofthe eggs failed. Ether treatment alone did not induce diploid gynogenesis.     

Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis

Phase intensity changes of individual germinating spores of Bacillus subtilis were determined by phase-contrast light microscopy and image analysis. Two germination phases were investigated. The length of the time period before a change in phase brightness was evident and the duration of the phase intensity change until a constant greylevel was maintained. The incubation temperature (37 and 20 °C) and heat activation (10 min at 65 °C) had a distinct effect on both phases. At 37 °C, spores of B. subtilis 604 started to show a decrease in brightness in l -alanine buffer after 3–39 min and needed 10–39 min to complete the phase change. At 20 °C, lag times of 10–100 min were observed and the spores needed 30–100 min to reach a constant greylevel. Heat activation and subsequently exposure to l -alanine buffer at 20 °C reduced the lag phase to 6–90 min and the phase change was finished after 30–60 min. Our results indicate enzymatic involvement before and during the phase intensity change of germinating spores.     

Effects of high hydrostatic pressure on Clostridium sporogenes spores

Spores of Clostridium sporogenes were found to be resistant to ultra high pressure, with treatments of 600 MPa for 30 min at 20 °C causing no significant inactivation. Combination treatments including heat and pressure applied simultaneously (e.g. 400 MPa at 60 °C for 30 min) or sequentially (e.g. 80 °C for 10 min followed by 400 MPa for 30 min) proved more effective at inactivating spores. Pressure cycling (e.g. 60 MPa followed by 400 MPa at 60 °C) also reduced spore numbers. Overall, these pressure treatments resulted in less than a 3 log reduction, and it was concluded that the spores could not be inactivated by pressure alone. This could indicate that for the effective inactivation of bacterial spores, high pressure technology may have to be used in combination with other preservation methods.     

THE EFFECT OF TEMPERATURE ON EGGS AND IMMATURE STAGES OF CULEX ANNULIROSTRIS SKUSE (DIPTERA: CULICIDAE)

Abstract
No immature stages of Culex annulirostris were found during field sampling in 1979–1980 when the average water temperature was < 17 °C; they reappeared when the average water temperature was 19 °C and reached the peak density (mean 107 immatures/cylinder) at 26.5 °C.
The effect of 6 temperatures (15–40°C) on egg hatching, development and survival of the immature stages of Cx annulirostris in the laboratory showed that at 15 and 40°C, eggs failed to hatch and larvae died in the first instars. The optimum temperatures for egg hatching and the survival of immature stages were 25 and 30°C. At these temperatures, 85 and 82% respectively of egg rafts hatched, the mean number of larvae per raft was 258 ± 9.8 and 260 ± 11.4 with immature survival of 83.5 and 79.0% respectively. Mean time to hatch at 20–35°C ranged from 1.2 d (35°C) to 2.9 d (20 °C). Developmental times from first instar to adult ranged from 7.1 d (35 °C) to 25.2 d (20 °C). The threshold for development of the immatures was 15.6 ± 2.5°C and the thermal constant was 142.9 ± 26.5 day—degrees (incubation temperatures 20–35°C). At less suitable temperatures of 20 and 35 °C, hatching (57.5 and 45%), number larvae per raft (mean 139.8 ± 9.8 and 102.6 ± 14.2) and survival were low.     

Effect of temperature on the biology of Creontiades dilutus (Stål) (Heteroptera: Miridae)

The egg and nymphal development, fecundity and survival of the green mirid, Creontiades dilutus were examined at a range of temperatures and a modified day-degree model fitted to the data. Day degree (DD) requirements for egg and nymphal development, and threshold temperatures were calculated from the fitted lines. Female fecundity and longevity, egg and nymphal development, and survival of C. dilutus were significantly influenced by temperature. Eggs and nymphs failed to complete development at temperatures below 17 and at 38°C. Females also failed to produce any eggs at 11 and 38°C. The optimum temperature range for female fecundity was found to be 26–32°C. The optimum temperature for the development of eggs was calculated from the model as 30.5°C and for nymphs as 31.5°C. The threshold temperature for development was 15.8°C for egg and 15.1°C for nymph; 69.4 and 156.7 DD were required for completing the egg and the nymphal development, respectively. At the optimum temperature, it was estimated that development from egg to adult took 15 days. Survival was highest at 26°C for eggs and at 30–32°C for nymphs.     

Shock-induced triploidy and its effect on growth and gonad development of the European catfish, Silurus glanis L.

Triloidy was induced in European catfish, Silurus glanis L., by cold-shocking eggs at 4°C for 30 and 40 min respectively, starting 5 min after fertilization. The hatching success of cold-shocked eggs was 25–30%. Cold shocks longer than 1 h caused total mortality. The triploid character of the cold-shocked European catfish was proved by karyological and red blood cell size analyses. The gonads of the triploid fish were significantly smaller than those of the diploids, while the growth rate values of the triploids were significantly higher than those of the diploids.     

Note: Influence of different heating media on thermal resistance of Neosartorya fischeri isolated from papaya fruit

E. RAJASHEKHARA, E.R. SURESH AND S. ETHIRAJ. 1996. A heat-resistant mold identified as a strain of Neosartorya fischeri was isolated from microbiologically spoiled papaya fruits. The optimum heat activation temperature and time for the ascospores of the test mold was found to be 80°C for 15–30 min. The decimal reduction times ( D -values) at 85°, 87° and 89°C in phosphate buffer (pH 7·0) as heating medium were 35·25, 11·1 and 3·90 min respectively and hence the calculated z -value was 4·0°C. In grape and mango juices as heating media, the D _80°C and the D _85°C values were increased as the °Brix level raised from 10 to 45. In commercial fruit juices of mango, orange, pineapple and mango-pineapple blend as heating media D _85°C values were greater than those observed for phosphate buffer.     

Effect of Iysozyme concentration, heating at 90°C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10⁶ spores per tube. Hen egg white lysozyme (0–50 μg ml^-1) was added, and the tubes were given a heat treatment equivalent to 19·8 min at 90°C, cooled, and incubated at 8°, 12°, 16° and 25°C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6–D inactivation was therefore achieved. In tubes to which lysozyme (5–50 μg ml^-1) had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 μg ml^-1, growth was first observed after 68 d at 8°C, 31 d at 12°C, 24 d at 16°C, and 9 d at 25°C. Thus, in these circumstances, a heat treatment equivalent to 19·8 min at 90°C was not sufficient, on its own, to give a 6–D inactivation. A combination of the heat treatment, maintenance at less than 12°C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10⁶.     

The Effects on Temperature on Growth in vitro of Pseudomonas syringae and Xanthomonas pruni

Growth rates in vitro of Pseudomonas syringae and Xanthomonas pruni were measured over the temperature range 0–36 °C. The estimated temperature optimum for X. pruni was 31 °C, with a doubling time of 1.53 h. The estimated temperature optimum for P. syringae was 28 °C with a doubling time of 1.27 h, although analysis showed no significant difference in the doubling times over the range 23–33 °C, indicating an unusual plateau at the maximum rate of growth of this organism. P. syringae and related plant pathogenic Pseudomonas spp. grew well at low temperatures, but X. pruni did not. Cultures of P. syringae and X. pruni had a very short lag phase after their incubation temperature was changed from 4 °C to a temperature close to their optimum (29 °C). When the incubation temperature of these organisms was changed from 11.5–29 °C, X. pruni grew without a lag phase at the rate expected for the higher temperature. However, the initial growth rate of P. syringae at the higher temperature was significantly greater than that at which the organism subsequently developed. The ecological significance of these points is discussed. The usefulness of the Arrhenius coefficients as characteristics of these organisms is discussed.     

High efficiency of meiotic gynogenesis in sea lamprey Petromyzon marinus

Induction of androgenesis and gynogenesis by applying a pressure (PS) or heat shock (HS) to double the haploid chromosomal set results in progenies possessing only chromosomes from a single parent. This has never been accomplished in representatives of Agnatha. The objective of this study was to induce gynogenesis and androgenesis in sea lamprey Petromyzon marinus. For gynogenesis experiments, ultraviolet (UV)-irradiated sperm was used to activate sea lamprey eggs and HS or PS were applied to inhibit the second meiotic division and consequently induce diploidy in the embryos. The UV irradiation of immobilized sperm was performed for 1 min at 1,719 J m(-2). HS of 35+/-1 degrees C for 2 min and PS of 9,000 psi for 4 min were applied at different times after egg activation (8, 12, 20, and 24 min or 8, 16, and 24 min for HS or PS, respectively). Regardless of the induction time of the HS, survivals at pre-hatching stage were similar. In contrast, PS applied 8 min after activation appears to increase survival rate of pre-hatched embryos in comparison to 16 and 24 min after activation. In control groups, without shock treatment (no diploidization), there were no survivors. All deformed, gynogenetic embryos were confirmed to be haploids and died prior to burying themselves in the sand. We confirmed by flow cytometry that progenies produced using both shock methods surviving to the next stage, burying in the substrate, were diploid gynogenetic. For the androgenesis experiments, UV-irradiated eggs (1,719 J m(-2) for 1 min) were fertilized with non-treated sperm and HS was applied to restore diploidy of the eggs. Several attempts have been made to optimize the parameters used. HS of 35+/-1 degrees C was applied 110, 140, 170, 200, and 230 min after activation for 2 min. Low yields of androgens were obtained and all animals died within a week after hatching. These techniques will allow to establish meiotic gynogenetic lines of sea lamprey for determining sex differentiation in this species and to analyze its hormonal and environmental regulation.     

Sensitivity of wild and genetic sexing strain Ceratitis capitata to high-temperature disinfestation of Valencia oranges

Abstract:  The purpose of this research was to investigate post-harvest heat treatment of Valencia oranges as an effective disinfestation protocol (fast, no fruit damage) for the Mediterranean fruit fly Ceratitis capitata (Wiedemann). Forced high-temperature air was applied under the following conditions: (a) exposure to air temperature of 56°C, for fast increase of temperature in the interior of the fruit, (b) reduction in air temperature to 47°C when the fruit centre reached 47°C and (c) maintenance of fruits in the chamber for another 30 min. Relative humidity in the treatment chamber was kept between 50% and 65% during treatment. Forced air at 47°C applied for 30 min on eggs before hatch or late third instar larvae (the most heat-tolerant stages) resulted in complete kill. Egg and larval sensitivity to high temperature differed between a wild strain and a laboratory genetic-sexing strain based on white pupa mutation. In this strain males emerge from brown pupae and females from white pupae. In particular, mature eggs from the wild strain were significantly more temperature resistant than eggs from the laboratory strain. Exposure of Valencia oranges of a diameter of 7–7.5 cm to 56°C forced air for about 86 and 99 min was required to increase temperature to 47°C at 1.5 cm depth and the fruit centre respectively. Treated oranges showed no substantial peel or interior deterioration, or change in colour and taste when kept at 25°C and 50–60% RH for a period of up to 1 month following treatment. Treatment in 1% O₂ atmosphere, produced by flushing of CO₂ into the treatment chamber, resulted in about 1°C reduction in killing temperature and faster increase in temperature inside the fruit to a lethal level.