Kenji Kiguchi

Ecdysteroid levels and developmental events during larval moulting in the silkworm, Bombyx mori

Abstract In the silkworm, Bombyx mori, apolysis of the spiracle is the first visible sign of the initiation of a larval moult. After spiracular apolysis, the characteristic sequence of new spiracle formation can be recognized easily through the cuticle around the old spiracle. This sequence, called the spiracle index, was used as the basis for a precise developmental chronology of the larval moulting period. At certain spiracle-index stages, histological changes in the larval ventral abdominal integument were also examined. By allatectomy and abdominal ligations, the critical periods for the secretion of juvenile hormone and ecdysone respectively were found to be at the initiation of spiracular apolysis and at the time when general apolysis was complete. The haemolymph-ecdysteroid level as measured by radioimmunoassay was about 30–50 ng/ml during the first 2 days of the 4th instar, 60–70 ng/ml at the time of initiation of spiracular apolysis, and 200 ng/ml at the time of completion of apolysis of the general body surface. The maximal level of the hormone was about 290 ng/ml at the mid-moulting period. The relationship between the ecdysteroid titre and morphological events occurring during the larval moult was discussed.

Hormonal control of storage protein synthesis and uptake by the fat body in the silkworm, Bombyx mori

Abstract The female silkworm, Bombyx mori , rapidly accumulates two storage proteins, that are synthesized by the fat body, in the haemolymph during the feeding stage of the last-larval instar, and then sequesters them from the haemolymph into fat body during the larval-pupal transformation. The rapid synthesis and uptake of storage proteins by the fat body are shown to be induced by allatectomy in the early-penultimate larval instar. A juvenile hormone analogue, methoprene, is highly effective in inhibiting the allatectomy-induced synthesis, and, in a higher dosage, further blocks the uptake. Allatectomy in the late-penultimate larval instar shortly before moulting does not enhance the storage protein synthesis, but causes the uptake to occur two days earlier in the last-larval instar. Injection of 20-hydroxyecdysone is not stimulatory for synthesis of the proteins, but is effective to induce their uptake. Starvation during the early last-larval instar completely blocks the synthesis. From these results, it is suggested that storage protein synthesis is induced in the absence of juvenile hormone by some supplementary stimulus, possibly the supply of nutrient after feeding, and uptake is induced by ecdysteroids after a decline in the juvenile hormone level.

Utilization of silkworm cocoon waste as a sorbent for the removal of oil from water

The aim of this study is to investigate the utilization of silkworm cocoon waste, such as pierced or stained cocoons, as a sorbent material for the removal of motor and vegetable oils from water. The oil-sorption capacity, rate and reusability of the material were evaluated. The results show the high sorption capacity of the silkworm cocoon waste sorbent (42-52 g(oil)/g(sorbent) for motor oil and 37-60 g(oil)/g(sorbent) for vegetable oil). The oil sorbed onto the material could be recovered by squeezing the sorbent, and the squeezed material showed an oil-sorption capacity over 15 g(oil)/g(sorbent). We concluded that the material shows a high performance as a low cost and environmental friendly sorbent for the removal of oil from water.

Classification of larval circulating hemocytes of the silkworm, Bombyx mori, by acridine orange and propidium iodide staining

Circulating hemocytes of the silkworm can be classified by fluorescence microscopy following staining with acridine orange and propidium iodide. Based on their fluorescence characteristics, three groups of circulating hemocytes can be distinguished. The first group, granulocytes and spherulocytes, is positive for acridine orange and contain bright green fluorescent granules when observed by fluorescence microscopy. In granulocytes, these green granules are heterogeneous and relatively small. In contrast, in spherulocytes, the green granules appear more homogenous and larger. The second group of hemocytes consists of prohemocytes and plasmatocytes. These cells appear faint green following staining with acridine orange and do not contain any green fluorescent granules in the cytoplasm. Prohemocytes are round, and their nuclei are dark and clear within a background of faint green fluorescence. Inside the nucleus there are one or two small bright green fluorescent bodies. Plasmatocytes are irregularly shaped and their nuclei are invisible. Oenocytoids belong to the third group, and their nuclei are positive for propidium iodide. Therefore, all five types of circulating hemocytes of the silkworm, including many peculiar ones that are difficult to identify by light microscopy, can now be easily classified by fluorescence microscopy following staining with acridine orange and propidium iodide. In addition, we show that hemocytes positive for acridine orange and propidium iodide are in fact living cells based on assays for hemocyte composition, phagocytosis, and mitochondrial enzyme activity.

In Planta Transformation of Kenaf Plants (Hibiscus cannabinus var. aokawa No. 3) by Agrobacterium tumefaciens

Kenaf was transformed by inoculation of Agrobacterium tumefaciens onto the meristems of young plants in pots. The transformation was demonstrated by three lines of evidence: a phenotypic inheritance from T(0) to T(1) plants, detection of the transgene in both T(0) and T(1) plants, and rescue of plasmids composed of T-DNA of the binary vector and flanking plant genomic DNA from T(1) plants.

Effects of anti-juvenile hormone “ETB” on the development and metamorphosis of the silkworm, Bombyx mori

Abstract As in the tobacco hornworm Manduca sexta , the synthetic juvenile hormone analogue ETB (ethyl 4-[2-(tert-buthylcarbonyloxy)butoxy]benzoate) showed both juvenile hormone-like and anti-juvenile hormone activities in the silkworm, Bombyx mori . When ETB was topically applied to allatectomized 4th-instar larvae, the compound counteracted the effects of allatectomy, such as induction of precocious metamorphosis and black pigmentation in the larval markings. Therefore, ETB had juvenile hormone activity, but it could neither induce brown pigmentation in the markings nor induce an extra-larval moult as can juvenile hormone. When intact 3rd-instar larvae were treated with the compound, the majority underwent precocious metamorphosis in the 4th-instar, and later formed fertile miniature adults. Some moulted into larval-pupal intermediates or 5th-instar larvae with darkened larval markings and/or with abnormality of specific regions of the silk-gland. The optimal dose for such anti-juvenile effects was about 1–10 μg/larva, and higher doses showed less activity. Such anti-juvenile hormone effects of ETB were counteracted by administration of the juvenile hormone analogue, methoprene, before a certain critical time in the 4th-instar. The corpora allata of treated larvae appeared cytologically normal, and the corpora allata from ETB-induced miniature moths secreted juvenile hormone when implanted into allatectomized 4th-instar larvae.

A physiological saline for Lepidopterous insects: Effects of ionic composition on heart beat and neuromuscular transmission

Abstract Three species of Lepidoptera, Bombyx mori, Agrius convolvuli and Antheraea yamamai were used to prepare a suitable physiological saline for maintaining heart beat and junctional potentials of somatic muscle cells. A variety of salines designed for Lepidoptera were reexamined. Heart beat in the three species could not be maintained in salines used previously as physiological or culture solutions for Lepidoptera. Ionic compositions were determined for physiological salines that were suitable for maintaining the normal heart beat and normal neuromuscular transmission in body muscle cells. It was found that salines should contain 12–28 mM NaCl, 32-16 mM KCl ([Na+] + [K+ ] = 44) and at least 9 mM CaCl2. In the salines, stimuli to motor nerves evoke action potentials of more than 40 mV in amplitude in ventral muscle cells. The ionic composition of a new physiological saline for Lepidoptera is as follows: NaCl, 12–28; KCl, 32-16([Na+] + [K+] = 44); CaCl2, 9; NaH2PO4, 1.5; Na2HPO4, 1.5; MgCl2, 18; sucrose, 175 (mM), pH 6.5.

Application of the powder of porous titanium carbide ceramics to a reusable adsorbent for environmental pollutants.

The aim of this study is to investigate the utilization of the powder of porous titanium carbide (TiC) ceramics as a novel adsorbent or a material for solid-phase extraction (SPE). The adsorption and elution of inorganic and organic pollutants, Pb(II), 2,4,6-trichlorophenol (TCP), perfluorooctane sulfonate (PFOS), and perfluorooctanoic acid (PFOA), to the material were evaluated. The cartridge packed with TiC ceramics powder was used for the extraction test of pollutants. The solution containing pollutants at 1.0 μg mL(-1) was passed through the TiC cartridge, and the substances were almost quantitatively removed. Furthermore, the pollutants retained in the cartridge were eluted with 3N HCl for Pb(II) and with methanol for organic pollutants. The recoveries of pollutants were over 80%. In addition, we used the TiC cartridge for the solid-phase extraction of water samples (500 mL each of the distilled water and the river water) by adding pollutants at determined concentrations. Every pollutant was adsorbed almost quantitatively, and eluted by 3N HCl or methanol. From these results, we concluded that the powder of porous TiC ceramics is a useful reusable adsorbent for the water cleanup and solid-phase extraction.

Radiosurgery using heavy ion microbeams for biological study: Fate mapping of the cellular blastoderm-stage egg of the silkworm, Bombyx mori

We investigated the effects of heavy ions on embryogenesis of the silkworm, Bombyx mori using a collimated heavy ion microbeam from the vertical beam line of an AVF-cyclotron. Eggs were exposed to carbon ions at the cellular blastoderm stage. Microbeams were found to be extremely useful for radio-microsurgical inactivation of nuclei or cells in the target site. Spot irradiation caused abnormal embryos, which showed localized defects such as deletion, duplication and fusion, depending on dose, beam size and site of irradiation. The location and frequency of defects on the resultant embryos were closely correlated to the irradiation site. Based on this correlation, a fate map was established for the Bombyx egg at the cellular blastoderm stage.

Cocoon Spinning Behavior in the Silkworm, Bombyx mori : Comparison of Three Strains Constructing Different Cocoons in Shape

Abstract The spinning behavior of the silkworm, Bombyx mori, was recorded on videotapes from two angles and analysed by three dimentional computer graphics using the Japanese (J. 124), Chinese (C.124) and their hybrid (J.124 × C.124) strains. These strains constructed typical peanut-shaped, spherical and ellipsoidal cocoons, respectively. Linear representation of the spinning posture revealed that larvae fixed the posterior half of the larval body (6th to 13th segment) and spun silk moving their anterior half (1st to 5th segment) for the most spinning period in all strains used. Little difference was observed in the average spinning speed among them. The Japanese strain spun primarily in a S-letter posture and changed its direction frequently. The larva of Chinese strain often assumed a C-letter posture and showed directionchanging behavior with comparatively lower frequency. The hybrid larva threw the head back largely in an U-letter shape during most of the spinning period and showed cocoon expansion behaviors most frequently. The cocoon expansion behavior occurred mainly at both ends of the peanut-shaped cocoon (J.124), at the center part of the spherical cocoon (C.124) and at both shoulders in the ellipsoidal cocoon of the hybrid strain. Thus, there exist strain-specific features in the spinning behavior, and it is suggested that the main behavioral factors affecting cocoon shape formation are the spinning posture and the cocoon expansion behavior during spinning.