Evaluation of Likelihood of Co-Occurrence of Erwinia amylovora with Mature Fruit of Winter Pear

ABSTRACT Phytosanitary concerns about fire blight prohibit export of U.S.-grown pears to some countries without this disease. To examine these concerns, we evaluated the potential for co-occurrence of Erwinia amylovora with mature, symptomless winter pear fruit by inoculation experiments and by survey of commercial orchards. Immature pear and apple fruit were inoculated in orchards with E. amylovora strain 153N as resuspended lyophilized cells or as ooze from diseased tissues. Regardless of inoculum source, population size of Ea153N on fruit declined by an order of magnitude every 3 to 4 days during the first 2 weeks after inoculation; at 56 days after inoculation, Ea153N was not detected, except on 1 of 450 fruit with 4 colony forming units (CFU). After inoculation of flowers, calyx-end survival of Ea153N on pear and apple fruit declined from high populations at petal fall to a few cells at harvest, with no detection of the pathogen after a 7-week cold storage. Migration of Ea153N into symptomless pear fruit from diseased branches was evaluated by enrichment assay and nested polymerase chain reaction of internal fruit core tissues; these assays failed to detect the pathogen in healthy fruit from diseased trees. At harvest, E. amylovora could not be detected on 5,599 of 5,600 fruit of d'Anjou pear sampled from commercial orchards in major production areas of the Pacific Northwest; one fruit yielded 32 CFU of the pathogen. Postharvest, mature pear fruit contaminated with Ea153N and subsequently wounded required a dose of >10,000 cells at the wound site to allow for persistence of the pathogen through a 7-week-cold storage. We conclude that epiphytic E. amylovora shows similar survival characteristics on both pear and apple fruit, this pathogen is not an endophyte within mature symptomless pear fruit, its presence is exceptionally rare on commercially produced fruit, and that epiphytic survival of E. amylovora through a postharvest chilling period is unlikely given the unrealistically high population size required for persistence.     

Dietary Effects on Sperm Quality of Litopenaeus vannamei Broodstock

A 56‐d feeding trial was conducted to investigate the effect of diet on sperm quality of Pacific white shrimp Litopenaeus vannamei broodstock. Dietary treatments consisted of a combination of 75% dry maturation diet and 25% fresh‐frozen squid (dry‐weight basis). Supplemental nutrients of the maturation diet were selectively deleted and replaced with wheat starch to produce the following treatments: 1) 75% basal maturation diet plus 25% squid (control); 2) 75% maturation diet without supplemental vitamins plus 25% squid; 3) 75% maturation diet without supplemental cholesterol and phospholipids plus 25% squid; 4) 75% maturation diet without supplemental astaxanthin plus 25% squid; and 5) a fresh diet composed of 60% squid and 40% Maine bloodworms. Shrimp fed the control diet and the diet without supplemental astaxanthin had significantly higher mean (± SEM) change in sperm count (4.6 ± 3.2 million sperm cells and 2.9 ± 2.5 million sperm cells, respectively), with respect to baseline (8.7 ± 1.0, 6.4 ± 1.0, 9.0 ± 1.3, 6.6 ± 0.7, and 6.0 ± 0.8 million sperm cells for treatments 1, 2, 3, 4, and 5, respectively), than shrimp fed the diet without supplemental vitamins (‐1.7 ± 2.6 million sperm cells), but not significantly higher than those of shrimp receiving the diets without supplemental cholesterol‐phospholipids (1.2 ± 2.5 million sperm cells) and the fresh diet (1.3 ± 1.6 million sperm cells). Dietary deficiencies also were reflected in weight gain of shrimp fed the diet without supplemental vitamins (‐2.0 g) and the fresh diet (‐0.8 g). which were significantly lower than weight gain of shrimp fed the control diet (1.1 g) and the diet without supplemental cholesterol‐phospholipids (0.8 g). No significant differences were detected among treatments for percentage of abnormal sperm and survival data. Results demonstrated a significant effect of diet on reproductive quality of male L. vannamei and indicated that the typical combination of fresh‐food organisms used is not nutritionally optimal for male broodstock.     

Investigation of the Role of Androstenedione-19-oic Acid in the Presence of 19-Norandrostenedione in Intact Male Horse Plasma Using Liquid Chromatography–Tandem Mass Spectrometry

A liquid chromatography–tandem mass spectrometry method was developed to confirm the presence of androstenedione-19-oic acid in intact male equine plasma and to show the source of 19-norandrostenedione in equine plasma. Androstenedione-19-oic acid was recovered from acidified plasma by liquid–liquid extraction using methyl tert-butyl ether and separated on an Ace 5 C₈ column. A triple quadrupole mass spectrometer was used to detect the analytes in negative electrospray ionization mode. Limits of detection, quantification, and confirmation of the method were 0.1, 0.5, and 1.0 ng/mL, respectively. The linear dynamic range of quantification was 0.5–50 ng/mL. The presence of androstenedione-19-oic acid was confirmed in all plasma samples obtained from intact male horses but not those from gelded and female horses; the average concentration was 3.1 ± 1.6 ng/mL, suggesting androstenedione-19-oic acid is an endogenous compound only in intact male horse plasma samples. The conversion of androstenedione-19-oic acid to 19-norandrostenedione in equine plasma was demonstrated by spiking androstenedione-19-oic acid into blank plasma and monitoring the generation of 19-norandrostenedione and its increase in concentration during storage. Results indicated that androstenedione-19-oic acid was readily converted into 19-norandrostenedione; the higher the storage temperature, the faster the conversion. The conversion was not affected by the types of plasma samples collected from gelded and female horses or by anticoagulants used in blood collection to harvest plasma. Compared with other matrices such as water, methanol, and phosphate-buffered saline, the conversion of androstenedione-19-oic to 19-norandrostenedione in equine plasma was faster, suggesting that there is an unknown factor(s) in equine plasma that enhances the conversion.     

Digestive Characteristics of the Sea-Urchin Lytechinus variegatus (Lamarck) (Echinodermata: Echinoidea) Fed Prepared Feeds

Lytechinus variegatus were fed two prepared feeds identical in composition except one contained only soybean protein and the other contained equal concentrations of fish and soybean protein. Feeding rate, defecation rate, passage time through the gut, apparent dry matter digestibility of protein and carbohydrate, and the production of gut tissue did not differ significantly ( P > 0.05) with feed type. Individuals about 50 mm in diameter and 60% wet body-weight consumed about 3-g wet weight of feed per day. Feeding rate decreased during the experiment, but defecation rate did not. The throughput rate was two days with considerable mixing of feed in the gut. Fecal production ceased two days after cessation of feeding. Only trace amounts of feed remained in the gut three days after feed was not available. The apparent dry matter digestibility was 62% for soluble protein and 35% for soluble carbohydrate for both feeds. The apparent dry matter digestibility of ash was 19% for the feed with soybean meal alone, and 11% for soybean and fish meal. The apparent dry matter digestibility calculated by the use of chromic oxide is greater than that calculated by the use of ash.     

Practical Molt Staging of Penaeus setiferus and Penaeus stylirostris

Molt staging has many applications in penaeid aquaculture as a routine procedure. Using criteria developed for various crustaceans, a method of rapid molt staging was applied to Penaeus stylirostris and Penaeus setiferus adults. The most useful characters were the degree of epidermal retraction from the setal bases and epidermal retraction from the cuticle coupled with the development of new setae. The duration of the molt cycle of P. stylirostris was 11.5 ± 1.0 days ( N = 5) at 27–29 C. The early postmolt, late postmolt, intermolt, early premolt and late premolt stages were readily determined. For P. stylirostris , the duration of postmolt was 27% of the cycle; intermolt, 17%; and premolt, 56%. The length of the molt cycle of P. setiferus at 27–29 C was 13.6 ± 1.0 days ( N = 21) with postmolt occupying 22% of the cycle; intermolt, 19%; and premolt, 59%. Unilaterally eyestalk enucleated P. setiferus had a significantly shorter molt cycle than intact P. setiferus (11.7 ± 1.21 days vs. 13.6 ± 1.02 days, P < 0.05). There were no differences in molt cycle duration between the sexes, whether enucleated or intact ( P > 0.05). A series of illustrations identifies the key characteristics for determining stage of molt in these penaeids.     

Semen cryopreservation of salmonid fishes: influence of handling parameters on the postthaw fertilization rate

Using the cryopreservation method of Lahnsteiner, Berger, Weismann & Patzner (1995, Aquaculture Research 26 , 801-807) the influence of allowable variations of methodical parameters (storage of semen before cryopreservation, dilution ratios in the extender, equilibration in the extender, cooling rates, storage of deep-frozen semen in liquid nitrogen, storage of frozen/thawed semen, minimal sperm/egg ratio) was investigated under the aspect of routine utilization. Under optimized experimental conditions, fertilization rates were 90-100% of controls in Oncorhynchus mykiss (Walbaum), Salmo trutta L. f. lacustrisSalmo truttaL.f. fario and Salvelinus fontinalis (Mitchill). The following results were obtained: 1. Storage of untreated semen for more than 1 h before cryopreservation decreased the postthaw fertility. 2. Equilibration of semen up to 20 min in the extender did not affect the postthaw fertility. 3. Optimal dilution ratio of semen in the extender was threefold in Oncorhynchus mykiss and Salvelinus fontinalis. Lower dilution ratios decreased the postthaw fertility, higher dilution ratios did not affect the postthaw fertility. In Salmo trutta f. lacustris and Salmo trutta f. fario, which have a higher sperm density, optimal dilution ratio of semen in the extender was fivefold to sevenfold. 4. In Oncorhynchus mykiss, as in Salmo trutta f lacustris and Salmo trutta f. fario, the optimal freezing height was at 1.5 cm above the level of liquid nitrogen (-110 ± 2^oC); in Salvelinus fontinalis it was 2.5 cm above the level of liquid nitrogen (-92 ± 2^oC). Changes in the freezing height of 0.5 cm (about 10^oC) resulted in a significant decrease of postthaw fertility. 5. Storage of deep-frozen semen for up to 370 days in liquid nitrogen had no influence on its postthaw fertilization rate. 6. Storage of frozen/thawed semen for 30 s before insemination significantly decreased its postthaw fertility. 7. Reliable minimal sperm:egg ratio to obtain fertilization rates of 90-100% of control was 3-5 X 10⁵ spermatozoa egg^-1.     

Spermatophore Generation Times in Penaeus setiferus, P. vannamei, and P. stylirostris

Spermatophore development times were studied in three species of penaeid shrimp, Penueus setifcrus, P. vannumei , and P. stylirosrris . In the terminal ampoule, the progression of spermatophore formation occurred in 4 stages (I-IV). The duration of each stage varied with species and ablation state. In unablated males, with spermatophores manually or electrically removed, structnrally complete spermatophores containing high numbers of morphologically normal sperm were formed in 2–4 days for P. vannamei , 4–6 days for P. stylirostris and 5–7 days for P. setiferus . Unilateral eyestalk ablation of P. setiferus accelerated spermatophore production time by approximately 2 days, and significantly increased spermatophore weight and sperm count without affecting sperm quality.
Spermatophore removal techniques had little effect on development time in P. vannamei . However, in P. setiferus , new spermatophores were produced faster in males mating naturally than in those males whose spermatophores were removed electrically.     

Characterization of intake and effluent waters from intensive and semi-intensive shrimp farms in Texas

Two commercial shrimp farms in south Texas were evaluated for influent and effluent water quality from June to October 1994. The intensive farm, Taiwan Shrimp Village Association (TSV) had an average annual yield of 4630 kg ha^?1 while the semi‐intensive farm, Harlingen Shrimp Farm (HSF), had a yield of 1777 kg ha^?1. The study had three objectives: (1) to compare influent and effluent water from the intensive and semi‐intensive shrimp farms, (2) to show which effluent water‐quality indicators exceeded allowable limits, (3) to indicate inherent problems in farms operated with water exchange and summarize how findings from this study led to changes in farms' management that limited potential negative impact on receiving streams. Water samples were collected and analysed twice a week for the TSV farm and once a week for the HSF farm. Samples were analysed for dissolved oxygen (DO), salinity, pH, ammonia‐nitrogen (NH₃‐N), nitrite‐nitrogen (NO₂‐N), nitrate‐nitrogen (NO₃‐N), total phosphorus (TP), total reactive phosphorus (TRP), five‐day carbonaceous biochemical oxygen demand (cBOD₅), total suspended solids (TSS) and settleable solids (SettSols). Most of the effluent constituents showed fluctuations throughout the sampling period often related to harvest activity. Effluent pH at TSV was lower than influent values but within the regulatory requirements set by Texas Commission of Environmental Quality (TCEQ), formerly known as Texas Natural Resource Conservation Commission (TNRCC). HSF effluent pH values were higher than its influent, but still within TCEQ limits. Effluent DO mean levels were generally below the regulatory daily mean requirement, with values at TSV often below those for influent. Effluent nutrient concentrations and net loads were generally higher at the intensive shrimp farm, with NH₃‐N mean concentrations above the daily mean set by the TCEQ on several occasions. Effluent TSS concentrations were higher than influent for both farms, with daily mean values above the TCEQ limit. The two farms presented similar TSS concentrations despite their different stocking densities. However, TSS total net load and net load per hectare were higher at the intensive farm. The semi‐intensive farm presented higher cBOD₅ concentrations and net loads despite its lower stocking density, with daily mean values above the TCEQ limit. The cBOD₅ net load at TSV presented negative values indicating higher load at the influent than at the effluent. Analyses showed no evidence of self‐pollution between influent and effluent at the two farms. The high feed conversion ratio (FCR) values (2.3 and 2.7 for the intensive and the semi‐intensive farm respectively) suggest that better feed management is needed to reduce nutrient and solid net loads release from the two farms. The data obtained from this study resulted in several modifications in design and management of the two farms that reduced the potential negative impact on receiving streams. A brief summary of the improvement in selected effluent water‐quality indicators at the intensive shrimp farm is provided.     

The Effect of Controlled Soil Sulfur Concentration on Growth and Survival of Litopenaeus vannamei

Abstract.— In a microcosm study, sodium sulfate was mixed with a controlled composition soil to yield sulfur concentrations of 100 to 3,000 mg/kg and the mixture was inundated to a depth of 35 cm with 18-ppt salinity water. One juvenile Litopenaeus vannamei with initial weight averaging 0.3 g was placed in each soil-water microcosm and fed for 96 d. Dissolved oxygen concentration was maintained at approximately 70% air saturation. Calculated interstitial hydrogen sulfide concentration 0.5 cm below the soil-water interface increased with time, reaching maximum values ranging from 57 to 113 mg/L. Interstitial sulfide concentrations 0.5 cm below the soil-water interface increased as a function of soil S. Average weight gain of shrimp ranged from 7.4 to 8.7 g across treatment groups. Differences in shrimp weight gain and survival among treatments were not significant. A higher dissolved oxygen demand due to sulfide oxidation implies greater risk of shrimp mortality if the soil-water boundary is disturbed. Further, this study suggests that shrimp growth is not adversely affected by high soil sulfide concentration provided the sediment surface remains oxidized and that dissolved oxygen concentration in the overlying water is maintained at 70% saturation.