Age estimation of the wild population of Japanese mantis shrimp <Emphasis Type="Italic">Oratosquilla oratoria</Emphasis> (Crustacea: Stomatopoda) in Tokyo Bay,Japan, using lipofuscin as an age marker

ABSTRACT:   In this study, the age composition of the Japanese mantis shrimp, Oratosquilla oratoria , in Tokyo Bay, central Japan, was investigated using lipofuscin, an autofluorescent pigment, as an age marker. Lipofuscin in a histological section of protocerebral bridge cell mass (PBCM) in the brain was identified by confocal microscopy, and its concentration was quantified by image analysis. Modal analysis of the lipofuscin concentration showed four regularly-spaced modes that could each be regarded as a distinct age group. This implied a constant lipofuscin accumulation in PBCM at a 6.5 × 10⁻²% volume fraction per year; it also implied the existence of individuals that are at least 4 years old. The lipofuscin concentration was found to be a more appropriate index than body length for estimating the age of O. oratoria , because the modal analysis on the body–length histogram failed to detect apparent age groups. This was probably a result of the declining growth rate with age and the individual variations in growth through molting leading to considerable overlap in the size between different age groups. The lipofuscin analysis suggested that fast-growing individuals in each cohort are recruited to the fishery, and most individuals attain the exploitable size between 2 and 3 years of age.     

Microbial communities in various waters used for fish larval rearing

A major concern in larvae production is a mass mortality caused by fish diseases. In larvae production, pumped‐up natural seawater filtered through a sand filter system is used for fish rearing, and microalgae and rotifer cultures. Here, we investigated the community structures of eukaryotic microbes, as well as total bacteria and vibrios, in various processed ‘waters’ used in a larvae production site. We observed that ultraviolet irradiation of seawater was effective to reduce not only total bacteria and vibrios but also eukaryotic microbes. Moreover, the community structures of total bacteria and vibrios in rearing waters for fish larvae were different from those in rotifer cultures fed with Chlorella, but rather similar to those in natural seawater and microalgae cultures. These results suggest that the bacterial community in rearing waters may originate mainly from natural seawater and then be selected by microalgae in rearing water. Overall, this study provides useful information for avoiding the risk of fish disease outbreaks in a larvae production site.     

Iron uptake in a bicarbonate-resistant cell line of tobacco

Abstract

In alkaline soils, plant growth is impaired mainly by high pH and high concentration of bicarbonates. The bicarbonate concentration increases the pH value, and causes deficiency of iron. A bicarbonate-resistant cell line (BR line) of tobacco (Nicotiana tabacum L. cv. Burley21) was selected by adding excess bicarbonate ions (20 mmol L^?1) to the culture medium. The pH of the medium was buffered 8.0 to 8.3. Under these conditions, about 80% of iron in the medium became insoluble. However, under such conditions, the BR line grew well. In this report, we examined some characteristics of the growth and iron uptake in the BR line under iron-deficient (i.e. high pH or no-iron) condition. At pH 5.8, the Fe³⁺ reduction activity was not significantly different between the non-selected line and the BR line. At pH 8.0, however, the Fe³⁺ reduction activity of the BR line was higher than that of the non-selected line. In no-iron condition, the growth of the non-selected line was markedly reduced after 2 weeks, while the BR line was not affected. The content of malic acid in both lines increased with the medium pH, and the content in the BR line was higher than that in the non-selected line. The BR line was able to adapt to the conditions, which restricted iron uptake, such as high bicarbonate concentration, high pH, and low iron conditions. The high ability of Fe³⁺ reduction was maintained at even high pH conditions. Further, the BR line may be able to improve the utilization of iron in the cells.     

Effects of 6-hydroxydopamine on the development of the immune system in chickens

It has been suggested that the sympathetic nervous system communicates with lymphocytes expressing cell surface receptors for neurotransmitters such as norepinephrine (NE), on the basis of the finding that neurotransmitters modify immune responses in mammalian species. We confirmed that chicken lymphocytes in the brusa of Fabricius, thymus and spleen expressed beta-adrenergic receptor (beta-AR) mRNA from embryonic day (E) 10 and that intracellular cAMP level was elevated by NE, suggesting that lymphocytes express functional beta-AR on their surface at an early embryonal stage. To clarify whether the nervous system is involved in the development of the immune system, the effects of 6-hydroxydopamine (6-OHDA), one of sympathectomizing agents, on chicken lymphocytes was investigated. A single injection of 6-OHDA at a dose of 400 microg into a chicken embryo was carried out at E7 or 14 (as referred to E7 group and E14 group, respectively). NE level and the relative proportion of Bu-1a(+), CD4(+) and CD8(+) cells in the spleen of 3-week-old chickens were not altered by 6-OHDA treatment. However, the proliferative responses and expression of IL-2 mRNA in spleen cells cultured with pokeweed mitogen were reduced in E7 group compared with those of control. Furthermore, in CD8(+) spleen cells of E14 group of 3-week-old chickens, the expression of beta-AR mRNA and the relative increase of intracellular cAMP stimulated with NE were significantly decreased. These results suggest that the sympathetic nervous system affects the development of the immune system.     

Protective effects of sugar cane extracts (SCE) on Eimeria tenella infection in chickens

The effects of oral administration of sugar cane extracts (SCE) on Eimeria tenella oocysts infection in chickens were studied with 2 different experiments. In Experiment 1, 3-week-old inbred chickens (MHC; H.B15) were inoculated into the crop with SCE (500 mg/kg/day) for 1 day or 3 consecutive days, and then challenged with E. tenella sporulated oocysts (2 x 10(4) cells/chicken). In Experiment 2, 1-week-old chickens were orally administered SCE at the same dose for 3 consecutive days, and then initially infected with E. tenella sporulated oocysts (2 x 10(3) cells/chicken). At 2 and 3 weeks of age, these chickens were immunized intravenously with the mixed antigens of sheep red blood cells (SRBC) and Brucella abortus (BA). At 4 weeks of age, chickens were challenged with E. tenella sporulated oocysts (1 x 10(5)/chicken). Challenged chickens with E. tenella oocysts showed markedly decreased body weight gain/day, severe hemorrhage and great number of shedding oocysts in feces and high lesion scores. Oral administration of SCE and initial infection with oocysts (2 x 10 (3)/chicken) resulted in a remarkable improvement in body weight gain/day, hemorrhage, the number of shedding oocysts and lesion score, compare to other infected groups. In addition, SCE-inoculated chickens with the initial infection showed a significant increase in antibody responses against SRBC and BA and also improvement in decreased relative proportions of Bu-1a(+) and CD4( )cells in cecal tonsil lymphocytes of E. tenella-challenged chickens. Cecal tissues of chickens administered SCE and initially infected with E. tenella oocysts showed lower numbers of schizonts, gametocytes and oocysts than those of infected control chickens. These results suggest that SCE have immunostimulating and protective effects against E. tenella infection in chickens.     

Effects of a high-protein diet versus dietary supplementation with ammonium chloride on struvite crystal formation in urine of clinically normal cats

OBJECTIVE: To evaluate the effects of a high-protein diet versus dietary supplementation with ammonium chloride (NH4Cl) on struvite crystal formation in the urine of clinically normal cats by measuring the urine concentration of hydrochloric acid (HCl)-insoluble sediment, urine pH, struvite activity product (SAP), number of struvite crystals in urine, and urine volume. ANIMALS: 23 healthy adult cats. PROCEDURE: Urine was fractionated by centrifugation with subsequent extraction of the sediment with 1 N HCl (study 1). Diets containing either 29% crude protein or 55% crude protein were fed to cats in a crossover trial of 3 weeks/period (study 2). Diets supplemented with either sodium chloride (NaCl) or NH4Cl were fed, by use of a 3 x 3 Latin-square design with 3 wk/period (study 3). In studies 2 and 3, urine samples were collected for the last 7 days of each period. RESULTS: The HCl-insoluble sediment contained Tamm-Horsfall glycoprotein (THP; study 1). The high-protein diet (study 2) and dietary supplementation with NH4Cl (study 3) resulted in a decrease in urine pH, SAP, and the number of struvite crystals in urine. However, the high-protein diet decreased urine concentrations of HCl-insoluble sediment containing THP (study 2), in contrast to the NH4Cl supplementation that increased urine volume without a significant effect on the urine concentration of the HCl-insoluble sediment (study 3). CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that compared with dietary supplementation with NH4Cl, the high-protein diet is preferable as a urine acidifier for the prevention of struvite crystal formation in clinically normal cats.     

Detection of mitogen-induced lymphocyte proliferation by bromodeoxyuridine (BrdU) incorporation in the chicken

3H-thymidine (3H-TdR) incorporation assay has been generally used to measure lymphocyte proliferation in the chicken. Disadvantages of this assay are that radioisotope is biological hazard to the person and environment and that it can not measure which subset of lymphocytes proliferates. In this study, bromodeoxyuridine (BrdU) incorporation assay by flow cytometry was compared with 3H-TdR incorporation assay. As a result, BrdU incorporation assay showed a strong correlation with 3H-TdR incorporation assay, and it could be applied simultaneously to detect BrdU incorporation and expression of cell surface marker antigens. These results suggest that the BrdU incorporation assay by flow cytometry is useful to analyze lymphocyte proliferation in detail.     

Immunostimulating and growth-promoting effects of sugar cane extract (SCE) in chickens

Polymorphonuclear cells of the peripheral blood in the chicken significantly increased their phagocytosis when cultured with sugar cane extract (SCE; 250-1,000 microg/ml) for 24 hr. Chickens orally administered SCE (500 mg/kg/day) for 3 or 6 consecutive days at 1 week of age showed significantly higher body weight and gain in body weight/day and a lower food conversion ratio within the growing period of 6 weeks than physiological saline-administered control chickens. Furthermore, oral administration of SCE also resulted in significantly higher immune responses against sheep red blood cells and Brucella abortus. These results suggest that SCE has immunostimulating and growth promoting effects in chickens.     

Comparative functional morphology of the masticatory apparatus in the long-snouted crocodiles

The masticatory muscles and their related structures of the skull were observed in the Indian gavial (Gavialis gangeticus), the false gavial (Tomistoma schlegelii), and the African slender-snouted crocodile (Mecistops cataphractus) to detail some morphological differences in comparison with the other crocodile species, and to compare and elucidate the functional strategy of themasticatory apparatus in these long-snouted species. The Musculus pterygoideus posterior was relatively smaller in the three species compared with many short-snouted crocodiles. It suggests that the masticatory power in fish-eating long-snouted species is not so high as in the short-snouted crocodiles, while the masticatory muscles were morphologically different among the three long-snouted species as follows. The M. pterygoideus posterior of the false gavial was extended in the lateral side of the lower jaw unlike the Indian gavial. The M. pseudotemporalis and the Fenestra supratemporalis were largely developed in the Indian gavial, however we suggest that the other two species possess the weak bundles in this muscle. The false gavial and the African slender-snouted crocodile have the pterygoid bone well-developed extending dorso-ventrally and it is suggested that the M. adductor mandibulae posterior attached to the pterygoid bone may be much larger than the Indian gavial. These data morphologically clarify the masticatory mechanism in the long-snouted crocodiles different from the short-snouted species, and demonstrate that the evolutional strategy to share the functional role in the masticatory muscles have been differently established between the Indian gavial and the other two species. We also obtained the morphological data in the fossil skull of the Machikane crocodile (Toyotamaphymeia machikanense) and concluded from the fossil characters that the considerable developments of the M.pterygoideus posterior and the M.pseudotemporalis in this species had not morphologically been consistent with both the Indian and false gavials.     

In vitro but not in planta encapsidation of Rice gall dwarf virus core particles by the outer capsid P8 protein of Rice dwarf virus expressed in transgenic rice plants

Transencapsidation of the Rice gall dwarf virus (RGDV) inner core by the Rice dwarf virus (RDV) outer capsid P8 protein was examined in vitro and in planta. When RGDV core particles were incubated with an extract from RDV P8-transgenic rice leaf tissue, RDV P8 encapsidated the RGDV core particles to form double-shelled virus-like particles in vitro. In contrast, when RDV P8-transgenic rice plants were inoculated with RGDV, progeny RGDV particles contained RGDV P8 but RDV P8 was not detectable in the virions. No significant differences were found in acquisition by the vector insects and subsequent transmission rates between RGDV infecting nontransgenic rice plants and those infecting RDV P8-transgenic rice plants. These results indicate that mechanisms of and/or requirements for interactions between P8 and the inner core particles of phytoreoviruses differ between in vitro and in planta.