I studied effects of UV-B radiation on egg and larval golden shiner (Notemigonus crysoleucas) in Castle Lake, California, USA. To compare egg hatching and survival of larval golden shiner, I exposed eggs and larval fish to three different UV-B treatments, low UV-B (4% UV-B), high UV-B (92% UV-B), and ambient (100% UV-B). The egg experiment was conducted in lake and pond water, whereas proto- and mesolarval stage experiments were conducted in lake water only. High and ambient UV-B radiation affected the hatching rate of golden shiner eggs. The mean hatching rate was 13% lower in high UV-B, and 9% lower in ambient UV-B compared to the low UV-B treatments. The hatching rate, however, was not significantly different between lake and pond treatments. The survival of egg to hatched larval stage was reduced under high and ambient UV- 8 radiation compared to low UV-8 treatment in the lake water experiment. In the pond water experiment, however, the survival of newly hatched larvae was not different among UV-8 treatments. During the experimental periods, no survival difference among treatments was observed for larval stages of golden shiners. Mean amount of UV-8 radiation penetrating an oligo-mesotrophic lake differed from that in an adjoining pond during the summer of 1997. UV-8 radiation in pond water was highly absorbed compared to absorbency in lake water. High concentrations of dissolved organic carbon in pond water were likely responsible for the difference in absorbency of UV-8 radiation.

Belonging to the diverse order Araneae, the black widow spider Latrodectus 4 hesperus produces high-performance silks with a broad range ofbiological functions and mechanical properties. The cob weaver spider spins different fibers by using seven specialized glands located in its abdomen. Egg case silk originates from the tubuliforrn gland and to date, no proteins that participate in the assembly process of egg case silk proteins have been identified. The goal of this project was the expression, purification, and characterization of such protein products. De novo sequencing of peptides from in-solution tryptic digestion of black widow spider dragline silk, the most studied type of silk, identified a novel cysteine-rich nonfibroin- like peptide that we named cysteine-rich component or CRC- 1. Further analysis of a large pool of nucleic acid sequences deposited in our custom eDNA database revealed 4 additional sequences with similarities to each other at the amino acid level called CRC-2, CRC-3, CRC-4, and CRC-5, suggesting a new family of proteins. Specifically, Q-PCR analysis revealed that the CRC-5 mRNAs were predominantly expressed in the tubuliform and aggregate glands. Since the aggregate gland manufactures a more complex aqueous solution compared to the tubuliforrn gland, we focused these studies on the tubuliform gland and resultant egg case fibers. Westem blot analysis using a cross-reactive polyclonal anti-CRC-1 antiserum conoborated the presence of CRC-5 in the tubulifmm gland and egg case silk, supporting the colocalization ofTuSpl, a tubuliform gland-specific protein, and CRC-5. Thus, we have demonstrated that these two proteins are present within tubuliform silks. In vitro studies suggested that recombinant CRC-5 displayed enzymatic activity similar to a sulfhydryl oxidase. Collectively, our findings provide new insights into novel proteins that have a potential role in the silk assembly and extrusion pathway of egg case silk fibers.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19018号 / 農博第2096号 / 新制||農||1029(附属図書館) / 学位論文||H27||N4900(農学部図書室) / 31969 / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 近藤 直, 教授 飯田 訓久, 准教授 小川 雄一 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

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Eggs are a popular food widely consumed worldwide, due to their nutrient density and shelf life even at room temperature. This study aimed to investigate if eggs keep better in the refrigerator than at room temperature post-boiling. The shelf life of boiled eggs stored at room temperature and refrigerated was analyzed regarding lipid oxidation and microbiological growth. Lipid oxidation was determined by measuring thiobarbituric acid reaction substances (TBARS) at 532 nm, formed as the oxidation product malondialdehyde and thiobarbituric acid (TBA). Microbiological presence and growth were analyzed on egg yolk and egg white determining total counts of aerobic microorganisms on agar incubated for three days at room temperature. No difference in counts of aerobic microorganisms between boiled eggs stored at room temperature or in the refrigerator was detected for either lipid oxidation or microbiological growth. After 14 days, a difference in lipid oxidation was noted as compared to 7 days. The study found that boiled eggs have a long shelf life and can be stored at room temperature for 7 days. / Ägg är ett omtyckt livsmedel som konsumeras flitigt i hela världen, och det är inte förvånande på grund av dess näringstäthet samt dess hållbarhet, även i rumstemperatur. Syftet med denna studie var att undersöka hur bra ägg håller i rumstemperatur efter kokning jämfört med i kylskåp. Hållbarheten på kokta ägg förvarade i rumstemperatur och kyl har analyserades med avseende på lipidoxidation och mikrobiologisk tillväxt. Lipidoxidation bestämdes genom att mäta thiobarbituric acid reactive substances (TBARS), vid 532nm, som bildas genom att oxidationsprodukten malondialdehyd reagerar med thiobarbituric acid (TBA). Mikrobiell förekomst och tillväxt i äggulan och äggvitan bestämdes på plate count agar efter inkubation i tre dagar i rumstemperatur. Ingen skillnad mellan kokta ägg förvarade i rumstemperatur eller kyl observerades för vare sig lipidoxidation eller mikrobiologisk tillväxt. Efter 14 dagar noterades en skillnad avseende lipidoxidationen i äggula jämfört med 7 dagar. Studien visade att kokta ägg har en lång hållbarhet och kan förvaras i rumstemperatur i upp till 7 dagar.

The ion, energy, lipid, nitrogen and fat-soluble vitamin contents of freshly ovulated eggs and neonates of the viviparous lizard Niveoscincus metallicus were measured to quantify uptake of nutrients across the placenta. This species is particularly interesting because it has a chorio-allantoic placenta that is intermediate in complexity compared to viviparous species that have been the focus of other studies. Newly ovulated eggs have a wet mass of 79.6±4.6 mg and a dry mass of 41.8±2.8 mg, compared to the neonates that have a wet mass of 224.2±8.2 mg and dry mass of 37.9±1.2 mg. Thus, there is no significant net uptake of dry matter across the placenta. Neonates have significantly less lipid (6.2±0.4 mg) than eggs (12.7±0.5 mg) but no significant difference in nitrogen (4.1±0.3 mg) compared to eggs (4.5±0.2 mg). Energy densities reflect the protein and lipid composition and the relative dry masses of the eggs and neonates. There is significantly more energy (1029.1±80.0 J) in the egg than in the neonate (858.2±36.6 J). The increase in the ash content of the neonates (2.9±0.2 mg) compared to fresh eggs (2.1±0.3 mg) was not significant, even though there was an approximately threefold increase in the amount of sodium (0.11±0.01 mg in neonates, 0.34±0.01 mg in eggs) and potassium (0.12±0.017 in neonates, 0.40±0.01 mg in eggs) in neonates compared to eggs. There was no significant uptake of calcium and magnesium during development. The egg lipids consisted of triacylglycerol (66.7±2.3%), phospholipid (18.9±0.7%), cholesteryl ester (4.9±1.6%) and free cholesterol (5.6±1.5%). The egg phospholipid contained comparatively high proportions of arachidonic and eicosapentanoic acids but low levels of docosahexaenoic acid (DHA), whereas the phospholipid of the neonate was greatly enriched in DHA. In the egg, the predominant vitamin E was α-tocopherol (62.6±3.4 mg g-1), although there was some γ-tocotrienol (3.5±0.3 mg g-1), and vitamin A was present (1.5±0.2 mg g-1). The ratio of neonate dry mass to egg dry mass of N. metallicus (0.91) lies between that of species with type I (0.78) and type III (1.70) chorio-allantoic placentae, confirming our conclusion that the placenta of N. metallicus is functionally intermediate, as well as intermediate in complexity, between these other two types.

Research in vocal vibrato has established that vocal tract filtering is primarily responsible for the amplitude modulation (AM) present in Western classical vibrato. Using electroglottography (EGG) and the EGG speed quotient, which is sensitive to fluctuations in the amplitude of vocal fold vibration, AM was detected at the laryngeal (source) level, in addition to the subsequent AM which results from vocal tract filtering. Seventeen classically-trained opera singers sang vowels in three pitch and loudness conditions. EGG and microphone measurements of FM and AM and their rates, extents, and periodicity were made. Airflow was also measured, and the samples were rated by voice professors for vibrato consistency, speed, and width. Physiologic and acoustic data revealed that AM from vocal tract filtering, or the resonance-harmonics interaction (RHI) described by Horii and associates, was present throughout the vibrato samples. Laryngeal-level AM was also present throughout, with soft conditions having the highest mean extents. Singers with lower degrees of laryngeal-level AM were also those rated highest for vibrato consistency. Vibrato rate increased as pitch increased, and, to a lesser extent, as intensity increased. These findings document, in addition to the AM resulting from the RHI, the concurrent presence of laryngeal-level AM in a group of singers representing a range of training and experience.

For many organisms, embryonic development begins directly after an egg (ovum) has been fertilized by sperm; however, some organisms delay the onset of embryonic development until conditions are favorable for raising young. This delayed onset of development could occur by delaying implantation of fertilized ovum on the uterine wall, as seen in many mammals. Birds delay embryonic development by laying a set of fertilized ova over a period of consecutive days. These fertilized ova are protected from the ambient environment by an exterior shell, and it is in this shell outside of the female s body that embryonic development occurs, but only when females initiate incubation. The number of fertilized ova (eggs) that can be laid by a single female in a single clutch varies among and within bird species, and understanding this variation remains a vital, unanswered question in ornithology. A latitudinal gradient in clutch size is widely recognized, but the reason for this pattern is unclear. Some birds lay relatively large clutches over many days, thus we should expect that eggs could withstand fairly long exposure to ambient temperature and remain viable. However, recent evidence suggests that egg viability declines with increased exposure to ambient temperatures. The egg viability hypothesis predicts that eggs will fail to hatch if exposed to warm ambient temperatures for prolonged periods. I conducted a natural experiment to determine whether egg viability can explain site-specific variation in hatching failure. Hatching failure is higher in a suburban population of Florida Scrub-Jays than it is in a wildland population, possibly because suburban scrub-jays lay larger clutches. Scrub-jays, like many bird species, lay one egg per day and begin incubation with the last-laid egg, thus first-laid eggs in the larger suburban clutches should be exposed to the warm ambient temperatures of sub-tropical Florida longer than first-laid eggs in the smaller clutches typical of the wildland population. As predicted, I found hatching failure is higher in first-laid eggs in the suburbs, and these eggs experience increased exposure to warm ambient temperatures. At both sites, females appear to begin incubation earlier in the laying period as ambient temperatures increase seasonally, possibly to minimize exposure to warm ambient temperatures and minimize hatching failure in first-laid eggs. However, early onset of incubation causes eggs to hatch asynchronously ( > 24 hours between the first and last-hatched egg), and hatching asynchrony increases within-brood size-asymmetries, which leads to an increased frequency of brood reduction (the nonrandom loss of last-hatched young because of starvation). Thus, a tradeoff may exist between beginning incubation earlier in the laying period to minimize hatching failure in first-laid eggs and delaying the onset of incubation to minimize hatching asynchrony and brood reduction. This tradeoff can have profound effects on avian clutch sizes, and may potentially explain the widely known negative relationship between latitude and clutch size.

Research has shown Spirulina to be a viable feedstuff in poultry production. Other species of microalgae are being studied to determine their effects on poultry production. This project compared four microalgaes: Hydrodictyon, Vaucheria, Uronema, and Spirulina and was broken down into three trials. The first experiment investigated the digestibility of an algae species, Spirulina, in broiler chicken diets. Eighty, 25-day-old Ross 708 broiler chicks were sorted into 20 metabolism cages and received one of two diets: Control Diet or Test Diet (75% basal diet + 25% Spirulina). The apparent ileal amino acid digestibility (AIAAD) of lysine, methionine, cysteine, threonine, isoleucine, valine and arginine in Spirulina was 94.5%, 91.3%, 56.1%, 71.4%, 76.8%, 69.8% and 90.4% respectively. Apparent metabolizable energy (AME) of Spirulina was 2279 kcal/kg. The second experiment focused on digestibility of Spirulina and novel algae species in laying hens. Sixty, 80-week-old Lohman LSL-Lite laying hens were randomly assigned to Control Diet, Hydrodictyon Test Diet (87.5% basal diet + 12.5% hydrodictyon), 12.5% or one of three other test diets (75% basal diet + 25% Spirulina, Uronema, and Vaucheria). Spirulina diets contained the highest crude protein (25.5%) and methionine levels (0.68%). Uronema had the highest gross energy content of 3880.5 kcal/kg and lysine levels (1.12%). Hydrodictyon diet was the most nutritionally similar to the control. Crude protein was at 15.3% and 16.6% for control and Hydrodictyon respectively. Gross energy was 3429.6 kcal/g in control versus 3316.1 kcal/kg in Hydrodictyon. Lastly, crude fat was 2.7% compared to Hydrodictyon at 2.4%. The last trial was to determine the effects of the addition of microalgae in the diet on laying hen production. Seventy-eight, 23-month-old laying hens were assigned to 1 of 3 of the following experimental diets: control, control + 1% microalgae, or control + 2% microalgae. Microalgae, regardless of inclusion level, had no significant effect on egg weight in this study. Increasing level of inclusion of Spirulina, Hydrodictyon, and Uronema from 0 to 2 % significantly increased yolk color. Vaucheria influenced yolk color at the 2% level. No significance differences shown from any of the algae species regardless of inclusion level for egg weight, egg mass, shell thickness, shell strength, shell weight, albumen weight, yolk weight, and albumen height. In addition, there was no significant difference in feed conversion ratio, feed intake, body weight, or tibial strength between the treatments. However, as these are novel algaes, further studies are required to determine the true usability in poultry production.

Wolbachia are widespread obligate intracellular bacteria that are maternally transmitted and modulate reproduction of their invertebrate host. Mosquitoes transinfected with Wolbachia have reduced capacity for transmitting vector borne diseases and can replace native populations in the field because of a reproductive advantage. The cellular mechanisms of how reproduction is altered by Wolbachia are poorly understood. In this work Wolbachia-induced reproductive changes in the model organism Drosophila were used to pinpoint underlying cellular processes affected by the bacteria. Specifically, egg production (or fecundity) of Wolbachia-infected Drosophila mauritiana was compared to non-infected flies that had been generated by antibiotic treatment of infected flies. Immediately before the fecundity experiment backcrossing of both fly lines ensured an equivalent nuclear genetic background. Initially egg production in Wolbachia-infected flies was increased by 4-fold but in less than 30 generations this changed to a 0.84 fold decrease with a slight advantage for the non-infected line. Additional backcrossing experiments determined that selection on the host nuclear genome is one of the factors underlying this reversion of fecundity gains. Other non-Mendelian factors, such as the microbiota, may also play a role in this rapid change. Wolbachia alterations in egg production were always linked to Wolbachia induced changes in programmed cell death (PCD) in the germarium during oogenesis and germline stem cell (GSC) division. Germline stem cells are maintained and regulated through their interaction with the germline stem cell niche (GSCN). Interestingly, these cells are both frequently infected with Wolbachia and possess a high bacterial titer. A developmental time course revealed the mechanism of how Wolbachia accumulate in the niche cells. The data suggest that the bacteria actually coordinate their replication with the differentiation of the niche cells. Future work on understanding the cellular and molecular basis of Wolbachia - host interaction will not only give insight into novel mechanisms of host manipulation by a pathogen, but will also expand our current understanding of stem cell niche morphogenesis and modulation of stem cell proliferation. / 2018-06-05T00:00:00Z