Hatching synchrony is controlled by a two-step mechanism in the migratory locust Locusta migratoria (Acrididae: Orthoptera): Roles of vibrational stimuli.

The eggs of the migratory locust, Locust migratoria, hatch in synchrony from their pod. In this study, we examined the mechanism controlling hatching synchrony. Two eggs obtained from the same pod hatched in synchrony when kept in contact with one another, whereas those separated by a few millimeters hatched less synchronously. When a screen separated the eggs, the hatching was even more sporadic, indicating that hatching synchrony might be controlled by a two-step mechanism. We hypothesize that in the first step the embryos shortly before hatching control the time to enter a standby stage using some signal from neighboring eggs. The eggs in the standby stage hatch promptly when an additional stimulus is received from neighboring eggs. Before this stage, eggs cannot respond to that stimulus by hatching but may spontaneously hatch later. Introduction of a newly hatched nymph to single eggs 1 or 2 days before hatching advanced hatching of these eggs, but hatching occurred only sporadically. Eggs kept in contact with other eggs that had been killed by freezing shortly before hatching hatched as if they had been kept singly in separate containers, providing no evidence for involvement of chemical stimuli in controlling hatching synchrony. By contrast, eggs separated by several millimeters hatched as synchronously as those kept in contact with one another when they were connected by a piece of wire. Furthermore, vibrational stimulation derived from music greatly advanced hatching of separately kept eggs; however, hatching synchrony was not achieved unless the music started shortly before hatching. These results are consistent with the two-step hypothesis and indicated that locust embryos used vibrational stimuli from neighboring eggs for synchronous hatching.

Fabrication Scheme for Obtaining Transparent, Flexible, and Water-Insoluble Silk Films from Apparently Dissolved Silk-Gland Fibroin of Bombyx mori Silkworm.

Films from silk fibroin protein are one of the most promising biomaterials because of their exquisite balance between mechanical properties and biocompatibility. Numerous schemes have been proposed for processing fibroin film, utilizing liquid silk fibroin (LSF) or regenerated silk fibroin (RSF). The films cast from LSF or RSF in the solution state are water-soluble, and therefore require postproduction treatment inducing ß-sheet formation, to render them insoluble in water. Many kinds of postproduction treatments, using alcohol-water solution, water vapor, or controlled temperature, have been developed. However, the tuning and reproducibility of such treatments are quite sensitive and frequently render the fibroin films less flexible or even brittle because of the formation of an over content of ß-sheet. To overcome this, we developed a novel scheme for fibroin processing using silk-gland fibroin (SGF). The essence of this scheme is to create a softly solidified fibroin-gel state of the silk glands with an imperfect ß-sheet structure, by treating them with an ethanol/water mixture. Such a fibroin gel was found to dissolve in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The SGF film cast from the HFIP solution shows a flexible and water-insoluble nature with high reproducibility. In addition to this improvement, the SGF film produced by this method contains a significantly low level of residual HFIP molecules compared to the traditional RSF films prepared from an HFIP solution. The mechanism underlying these advantageous characteristics was investigated from the structural viewpoint, by using techniques such as 13C solid-state NMR, differential scanning calorimetry, and wide-angle X-ray diffraction.

Comparison between the Amount of Environmental Change and the Amount of Transcriptome Change.

Cells must coordinate adjustments in genome expression to accommodate changes in their environment. We hypothesized that the amount of transcriptome change is proportional to the amount of environmental change. To capture the effects of environmental changes on the transcriptome, we compared transcriptome diversities (defined as the Shannon entropy of frequency distribution) of silkworm fat-body tissues cultured with several concentrations of phenobarbital. Although there was no proportional relationship, we did identify a drug concentration "tipping point" between 0.25 and 1.0 mM. Cells cultured in media containing lower drug concentrations than the tipping point showed uniformly high transcriptome diversities, while those cultured at higher drug concentrations than the tipping point showed uniformly low transcriptome diversities. The plasticity of transcriptome diversity was corroborated by cultivations of fat bodies in MGM-450 insect medium without phenobarbital and in 0.25 mM phenobarbital-supplemented MGM-450 insect medium after previous cultivation (cultivation for 80 hours in MGM-450 insect medium without phenobarbital, followed by cultivation for 10 hours in 1.0 mM phenobarbital-supplemented MGM-450 insect medium). Interestingly, the transcriptome diversities of cells cultured in media containing 0.25 mM phenobarbital after previous cultivation (cultivation for 80 hours in MGM-450 insect medium without phenobarbital, followed by cultivation for 10 hours in 1.0 mM phenobarbital-supplemented MGM-450 insect medium) were different from cells cultured in media containing 0.25 mM phenobarbital after previous cultivation (cultivation for 80 hours in MGM-450 insect medium without phenobarbital). This hysteretic phenomenon of transcriptome diversities indicates multi-stability of the genome expression system. Cellular memories were recorded in genome expression networks as in DNA/histone modifications.

K+ excretion: the other purpose for puddling behavior in Japanese Papilio butterflies.

To elucidate the purpose of butterfly puddling, we measured the amounts of Na+, K+, Ca2+, and Mg2+ that were absorbed or excreted during puddling by male Japanese Papilio butterflies through a urine test. All of the butterflies that sipped water with a Na+ concentration of 13 mM absorbed Na+ and excreted K+, although certain butterflies that sipped solutions with high concentrations of Na+ excreted Na+. According to the Na+ concentrations observed in naturally occurring water sources, water with a Na+ concentration of up to 10 mM appears to be optimal for the health of male Japanese Papilio butterflies. The molar ratio of K+ to Na+ observed in leaves was 43.94 and that observed in flower nectars was 10.93. The Na+ amount in 100 g of host plant leaves ranged from 2.11 to 16.40 mg, and the amount in 100 g of flower nectar ranged from 1.24 to 108.21 mg. Differences in host plants did not explain the differences in the frequency of puddling observed for different Japanese Papilio species. The amounts of Na+, K+, Ca2+, and Mg2+ in the meconium of both male and female butterflies were also measured, and both males and females excreted more K+ than the other three ions. Thus, the fluid that was excreted by butterflies at emergence also had a role in the excretion of the excessive K+ in their bodies. The quantities of Na+ and K+ observed in butterfly eggs were approximately 0.50 µg and 4.15 µg, respectively; thus, female butterflies required more K+ than male butterflies. Therefore, female butterflies did not puddle to excrete K+. In conclusion, the purpose of puddling for male Papilio butterflies is not only to absorb Na+ to correct deficiencies but also to excrete excessive K+.

Japanese Papilio butterflies puddle using Na+ detected by contact chemosensilla in the proboscis.

Many butterflies acquire nutrients from non-nectar sources such as puddles. To better understand how male Papilio butterflies identify suitable sites for puddling, we used behavioral and electrophysiological methods to examine the responses of Japanese Papilio butterflies to Na(+), K(+), Ca(2+), and Mg(2+). Based on behavioral analyses, these butterflies preferred a 10-mM Na(+) solution to K(+), Ca(2+), and Mg(2+) solutions of the same concentration and among a tested range of 1 mM to 1 M NaCl. We also measured the ion concentrations of solutions sampled from puddling sites in the field. Na(+) concentrations of the samples were up to 6 mM, slightly lower than that preferred by butterflies in the behavioral experiments. Butterflies that sipped the 10 mM Na(+) solution from the experimental trays did not continue to puddle on the ground. Additionally, butterflies puddled at sites where the concentrations of K(+), Ca(2+), and/or Mg(2+) were higher than that of Na(+). This suggests that K(+), Ca(2+), and Mg(2+) do not interfere with the detection of Na(+) by the Papilio butterfly. Using an electrophysiological method, tip recordings, receptor neurons in contact chemosensilla inside the proboscis evoked regularly firing impulses to 1, 10, and 100 mM NaCl solutions but not to CaCl(2) or MgCl(2). The dose-response patterns to the NaCl solutions were different among the neurons, which were classified into three types. These results showed that Japanese Papilio butterflies puddle using Na(+) detected by the contact chemosensilla in the proboscis, which measure its concentration.

Mice lacking two sperm serine proteases, ACR and PRSS21, are subfertile, but the mutant sperm are infertile in vitro.

Although sperm serine protease and proteasome have long been believed to play an important role in the fertilization process, the molecular mechanism is still controversial. In this study, we have produced double-knockout mice lacking two sperm serine proteases, ACR and PRSS21, to uncover the functional role of the trypsinlike activity in fertilization. The double-knockout male mice were subfertile, likely owing to the incompleteness of fertilization in the oviductal ampulla. Despite male subfertility, the mutant epididymal sperm exhibited the inability to undergo acrosomal exocytosis on the zona pellucida (ZP) surface and to traverse the ZP, thus resulting in the failure of fertilization in vitro. The double-knockout epididymal sperm were also defective in penetration through the cumulus matrix to reach the ZP. When epididymal sperm were artificially injected into the uterus of wild-type mice, the 2-cell embryos, which had previously been fertilized by double-knockout sperm, were recovered at a low but significant level. The mutant epididymal sperm were also capable of fertilizing the oocytes in the presence of uterine fluids in vitro. These data demonstrate that the trypsinlike protease activity of ACR and PRSS21 is essential for the process of sperm penetration through the cumulus matrix and ZP in vitro, and suggest that the female reproductive tract partially compensates for the loss of the sperm function. We therefore conclude that the sperm trypsinlike activity is still important but not essential for fertilization in vivo in the mouse.

Identification of Ser2 proteins as major sericin components in the non-cocoon silk of Bombyx mori.

Sericins are glue proteins of Bombyx mori silk fibers. They are produced in the middle silk gland (MSG) cells, stored in the lumen, and pushed out from the spinneret surrounding the fibroin fibers. The Ser2 gene was partly cloned from the anterior region of the MSG more than 20 years ago and is regarded as a sericin-encoding gene; however, Ser2 proteins do not appear to be major components of cocoon proteins. We used northern blotting to analyze the expression of three sericin genes--Ser1, Ser2, and Ser3--in the MSG of third to fifth instar larvae, and measured the corresponding cDNA levels by competitive PCR. The results revealed that Ser2 gene expression dominated until the middle period of the fifth instar, while the expression of the other genes was mainly observed after the middle fifth instar. Protein analysis demonstrated that the two Ser2 proteins produced by alternative splicing were major coating proteins of larval silk threads spun during the growing stages. The molecular components of larval silk sericin were completely different from those of cocoon sericin, and the difference may be related to the functions of the two kinds of silk fibers.