Drugs from bugs: the use of insects as a valuable source of transgenes with potential in modern plant protection strategies

Transgenic expression of antimicrobial peptides in crops has become a novel approach among the strategies to combat phytopathogens in modern plant protection measures. The first antimicrobial transgenes of insect origin, modified cecropins, have been demonstrated to confer resistance of several transgenic cultivars against both bacterial and fungal phytopathogens. Insects represent a promising reservoir for antimicrobial peptides to engineer disease resistant crops. The increasing knowledge about the potent insect innate immunity may help to develop a novel strategy in sustainable agriculture. Several approaches are presently under investigation to prevent evolution of phytopathogens that can overcome disease resistance in transgenic crops expressing an insect antimicrobial peptide. Pathogen-induced expression of insect antimicrobial peptides in crops and combined multiple expression of different antimicrobial peptides along with proteinase inhibitors from insects may prevent selection of resistant phytopathogens. The potential of insect antimicrobial peptides as transgenes to render disease resistant crops has just started to be explored and may provide tools to be ahead of the evolutionary adaptability of phytopathogens.     

Interactive effects of elevated CO2 concentration and nitrogen supply on partitioning of newly fixed 13C and 15N between shoot and roots of pedunculate oak seedlings (Quercus robur)

Pedunculate oak (Quercus robur L.) seedlings were grown for 3 or 4 months (second- and third-flush stages) in greenhouses at two atmospheric CO2 concentrations ([CO2]) (350 or 700 micromol mol(-1)) and two nitrogen fertilization regimes (6.1 or 0.61 mmol N l(-1) nutrient solution). Combined effects of [CO2] and nitrogen fertilization on partitioning of newly acquired carbon (C) and nitrogen (N) were assessed by dual 13C and 15N short-term labeling of seedlings at the second- or third-flush stage of development. In the low-N treatment, root growth, but not shoot growth, was stimulated by elevated [CO2], with the result that shoot/root biomass ratio declined. At the second-flush stage, overall seedling biomass growth was increased (13%) by elevated [CO2] regardless of N fertilization. At the third-flush stage, elevated [CO2] increased growth sharply (139%) in the high-N but not the low-N treatment. Root/shoot biomass ratios were threefold higher in the low-N treatment relative to the high-N treatment. At the second-flush stage, leaf area was 45-51% greater in the high-N treatment than in the low-N treatment. At the-third flush stage, there was a positive interaction between the effects of N fertilization and [CO2] on leaf area, which was 93% greater in the high-N/elevated [CO2] treatment than in the low-N/ambient [CO2] treatment. Specific leaf area was reduced (17-25%) by elevated [CO2], whereas C and N concentrations of seedlings increased significantly in response to either elevated [CO2] or high-N fertilization. At the third-flush stage, acquisition of C and N per unit dry mass of leaf and fine root was 51 and 77% greater, respectively, in the elevated [CO2]/high-N fertilization treatment than in the ambient [CO2]/low-N fertilization treatment. However, there was dilution of leaf N in response to elevated [CO2]. Partitioning of newly acquired C and N between shoot and roots was altered by N fertilization but not [CO2]. More newly acquired C and N were partitioned to roots in the low-N treatment than in the high-N treatment.     

Comment on "Productivity is a poor predictor of plant species richness"

Adler et al. (Reports, 23 September 2011, p. 1750) analyzed the standardized sampling data from 48 herbaceous-dominated plant communities and concluded that "Productivity is a poor predictor of plant species richness" at fine-scale. However, their method was biased toward site-number-dominated plant communities. They also failed to provide enough data for regional analysis and detailed information for within-site analysis.     

Effect of Several Antioxidants on Thawed Ram Spermatozoa Submitted to 37°C up to Four Hours

Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N‐acetyl‐cysteine (NAC) and rutin (RUT), at 0.1 and 1 mm , in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP‐Hepes with 1 mm or 0.1 mm of each antioxidant, performing a replicate with induced oxidative stress (Fe²⁺/ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4 h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4 h. Antioxidants, except DHA 0.1 mm , decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1 mm DHA, the antioxidants reduced ROS at 4 h. Moreover, NAC 1 mm , rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N‐acetyl‐cysteine, rutin 1 mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1 mm , DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous.     

Changes in Testicular Development, Ultrasonographic and Histological Appearance of the Testis in Buck Kids Immunized Against LHRH Using Recombinant LHRH Fusion Protein

This study was designed to evaluate the effectiveness of recombinant Ovalbumin-LHRL (OL) immunization on changes in testicular size, histological appearance and testosterone production in buck kids. Thirty native buck kids at 18 weeks of age were divided into three groups, control (n = 10), immunization (n = 10) and castration (n = 10) groups. Immunized animals received OL protein generated by recombinant DNA technology. Ultrasonographic and histological examinations of the testes were performed. Animals were slaughtered at 44 weeks of age. Semen and epididymides were evaluated for the presence of sperm cells. Immunized animals generated anti-LHRH antibodies. Testosterone production, testicular and accessory glands development and sperm production were suppressed in the immunized animals (p < 0.01). Semineferous tubule diameters decreased (p < 0.01), basal membrane of the tubule was thickened and hyalinized in immunized kids. Immunization affected ultrasonographic appearance of the testes drastically. While testes of control animals gained their normal ultrasonographic appearance as the age increased, immunized animals had uniform hypoechogenic testicular structure as observed at 18 weeks of age until slaughter. Simultaneous histological and ultrasonographic evaluations indicated that the changes in testicular histology could partly be monitored via ultrasonographic imaging; nevertheless, it is difficult to claim that ultrasonographic image reflects the exact changes in such instances. In conclusion, these results indicate that recombinant OL fusion protein is effective in immunocastration in buck kids and has a potential to be used as an alternative to physical castration. Further researches should be conducted to help assessing reproductive status of testes from ultrasound images.     

Refrigerated Storage of Red Deer Epididymal Spermatozoa in the Epididymis, Diluted and with Vitamin C Supplementation

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 × 10⁶ sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 m m vitamin C. The remaining epididymides and the diluted samples were stored at 5°C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.     

Reproductive Performance of Rabbit Does Artificially Inseminated via Intravaginal Administration of [des-Gly 10, d-Ala6]-LHRH Ethylamide as Ovulation Inductor

This study was aimed to evaluate the reproductive performance of rabbit does artificially inseminated (AI) with a GnRH analogue [des‐Gly10, d ‐Ala6]‐LHRH. ethylamide to induce ovulation by intravaginal administration, delivered in the seminal dose. In a preliminary experiment, 39 does were divided into three groups (n = 13) that, at the time of AI, received the following ovulation induction treatments: (i) control group: 20 μg of gonadorelin administered intramuscularly; (ii) 25 μg of the GnRH analogue added to the seminal dose; (iii) 30 μg of the GnRH analogue added to the seminal dose. Fertility did not differ between the three groups (control: 80.6%, group 2: 82.8%, group 3: 73.3%). In a second experiment, a large‐scale field trial was conducted to test the use of 25 μg of the GnRH analogue [des‐Gly10, d ‐Ala6]‐LHRH ethylamide delivered in the seminal dose (n = 270) against 20 μg of gonadorelin administered intramuscularly. Fertility was higher (p < 0.05) when ovulation was induced by intravaginal administration of the GnRH agonist (91.1% vs 85.6%). Prolificacy or mortality at birth was never affected by the ovulation induction treatments. In a third experiment, two groups of does [control group (n = 39): ovulation was induced using 20 μg of gonadorelin administered intramuscularly; treatment group (n = 40): ovulation was induced using 25 μg of [(des‐Gly10, d ‐Ala6)‐LHRH ethylamide added to the seminal dose] were inseminated at 42‐day intervals for five successive AI cycles, to test the response to the GnRH agonist after repeated intravaginal administration to the same animals. Fertility and prolificacy were not influenced by the ovulation induction treatment neither there was an interaction between treatment and parity. The last experiment was aimed to determine whether it could be possible to add the GnRH agonist to the semen in the AI Center, just after semen collection and dilution, or it would have to be added in the farm, immediately before AI. Kindling rates did not significantly differ when ovulation was induced by intramuscular injection of gonadorelin (84.5%) or when the GnRH agonist was added to the seminal dose just at the moment (93.8 %) or 24 h before AI (90.4 %), but it was significantly lower when the hormone was added to the semen 32 h before AI (76.3 %). Prolificacy, however, was not influenced by the ovulation induction treatment.     

Inhaled carbon monoxide concentration during halothane or isoflurane anesthesia in horses

OBJECTIVE: The purpose of this study was to assess carbon monoxide (CO) exposure during equine anesthesia with either halothane (H) or isoflurane (I) delivered in a circle rebreathing system. STUDY DESIGN: Prospective clinical investigation. ANIMALS: Fifty client-owned horses. METHODS: Horses were randomly assigned for anesthetic maintenance with H (n = 26) or I (n = 24). Two large animal anesthetic machines were used and assigned to a single agent for 2-4 weeks at a time. Machines were disassembled and soda lime changed prior to switching anesthetic agents. Inhalant anesthetic concentration and CO concentration were measured in gas samples obtained from the inspiratory limb of the anesthetic circuit. Values were recorded at 15 minute intervals for 90 minutes. Soda lime status (new or used) and mode of ventilation (spontaneous or mechanical) were also recorded. Data were analyzed using a five-factor ANCOVA with repeated measures. RESULTS: Inspired CO concentration for H and I increased from 1 +/- 3 and 6 +/- 11 ppm at baseline to 54 +/- 33 and 21 +/- 18 ppm at 90 min, respectively (mean +/- sd). H was associated with significantly greater CO concentrations than I at 30 to 90 min, although baseline CO was significantly greater in the I group than the H group. Oxygen flow rates were 9.9 +/- 0.5 L/min at baseline for H and I, and 5.0 +/- 0.4 and 5.0 +/- 0.7 L/min at 90 min for H and I, respectively. There were no significant differences between groups for O2 flow at any time point. Neither mechanical ventilation nor new versus used soda lime affected CO concentration. CONCLUSIONS: Significantly higher concentrations of CO were recorded during the administration of H than I. CLINICAL RELEVANCE: Levels of CO observed during the administration of either H or I for 90 minutes to horses were not clinically significant.     

Evaluation of forelimb horseshoe characteristics of thoroughbreds racing on dirt surfaces

OBJECTIVE: To describe forelimb horseshoe characteristics of horses racing on dirt surfaces and determine whether these characteristics vary with region of California, season, horse characteristics, and race-related factors. ANIMALS: 5,730 Thoroughbred racehorses. PROCEDURE: From June 17, 2000, to June 16, 2001, the characteristics of 1 forelimb horseshoe of horses that raced on dirt surfaces at 5 major racetracks in California were recorded. These characteristics included shoe type; toe grab height; and presence of a rim, pad, and heel traction devices (jar caulks, heel stickers, heel blocks, and special nails). Horse and race information was obtained from commercial records. One race/horse was randomly selected. RESULTS: 99% of forelimb horseshoes were aluminum racing plates, 35% had a pad, 23% had a rim, and 8% had a heel traction device. A toe grab was observed on 75% of forelimb horseshoes (14% very low [< or = 2 mm], 30% low [> 2 and < or = 4 mm], 30% regular [> 4 and < or = 6 mm], and 1% high [> 6 and < or = 8 mm]). Forelimb horseshoe characteristics varied with region of California, season, age and sex of the horse, race purse and distance, and track surface condition. Log-linear modeling revealed that all of these factors were significantly interrelated. CONCLUSIONS AND CLINICAL RELEVANCE: Complex interrelationships among forelimb horseshoe characteristics and region, season, age and sex of the horse, and race-related factors need to be considered when evaluating the relationships between injury and horseshoe characteristics in Thoroughbred racehorses.