Labial glands of the sweet potato hornworm Agrius convolvuli are homologous organs with silk glands of the silkworm Bombyx mori. Previously, we indicated that the lumen proteins from the glands were different in molecular size between the 5th instar feeding stage and the wandering stage. To explain why this change occurs, we examined in vivo and in vitro incorporation of [³⁵S] methionine in these proteins, and also examined the amino acid composition of typical proteins from each developmental stage.
By SDS-PAGE, three major proteins (67, 70, and 116kDa) were detected in the lumen of the gland during the 5th instar. These proteins disappeared within 12 hr after one set of the wandering stage, followed by the appearance of two high molecular mass proteins (180 and 220kDa). Sealing the spinneret of the gland had no effect on the change observed in protein composition, and labeling experiments revealed that [³⁵S] methionine was incorporated in each stage specific protein. In addition, the amino acid composition of the two major proteins at each stage (67 and 180kDa) was specific to the respective stage. These results suggest that the switchover of protein synthesis occurs at the transcriptional level, accompanied by a behavioral change from feeding to wandering.

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To elucidate the physiological role of _D-serine, an optical isomer of the common _L-serine, in invertebrates, we investigated in detail changes in the _D-serine level (concentration) occurring with growth and development in the silkworm, Bombyx mori, since the silkworm is known for containing high concentrations of _D-serine. The body d-serine level increased with age until the late pupal stage. The total amount of _D-serine in the body also increased with age and formed two peaks at the pre-spinning to early spinning stage and the late pupal stage, suggesting that the larvae may require _D-serine to become pupae and imagoes, respectively. Contrary to the _D-serine level, the _L-serine level decreased with age after the second instar, with increase-decrease repeats until the pupal stage. High concentrations of _D-serine were excreted in feces, gut purge, meconium, and exuvium, suggesting that the body d-serine is maintained at a proper level for growth and development by excluding the amino acid from the body.

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Silkworms contain high concentrations of _D-serine, an optical isomer of _L-serine. Serine racemase catalyzes conversion of free _L-serine to _D-serine and vice versa. To elucidate the physiological role of the _D-amino acid, we examined effects of O-phospho-_L-serine (OPLS) on silkworm growth and development by adding it to the silkworms’ diet because we had found that OPLS competitively inhibits serine racemase prepared from the pupae. The result clarified OPLS effects: larval development was delayed by approximately 6 days, and the larval survival rate was significantly decreased. These effects were reversed toward the control levels by _D-serine administered together with OPLS. Body _D-serine level was lower in the OPLS-administered larvae than in the control ones right up until the spinning stage. _D-Serine levels in _D-serine- and _L-serine-administered larvae were markedly higher than in the control larvae, but the growth rate and the survival rate were lower than those of the control group. The _L-serine may be converted to _D-serine by the catalytic function of serine racemase in the body. The _D-serine concentration in feces excreted from these larvae was markedly high suggesting contribution of feces in maintaining a proper body _D-serine level. All these results together suggest that _D-serine is necessary for normal growth and development of the silkworm, while excess amino acid reversed its function.

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Two different cDNAs similar to Bombyx mori aquaporins (Bommo AQPs) were identified and characterized from the larval alimentary canal of the oriental fruit moth, Grapholita molesta. The first cDNA (AQP-Gra1) encodes a 28 380 Da protein similar to the water-specific AQPs isolated from several liquid-feeding insects such as Haematobia irritans exigua, Aedes aegypti and Cicadella viridis, and from Drosophila melanogaster (DRIP) as well as one of Bommo AQPs (AQP-Bom1). The second cDNA (AQP-Gra2) encodes a 27 929Da protein similar to the other Bommo AQP (AQP-Bom2) and other putative AQPs from D. melanogaster (Aqp17662 and Aqp17664). Functional analysis in Xenopus oocytes microinjected with the capped RNA of G. molesta AQPs revealed that both stimulated osmotic water permeability in a mercury-sensitive manner. The water-specific AQP property in AQP-Gra1 and its higher identity of the deduced amino acid sequence (>70%) with AQP-Bom1 suggests that it is a hindgut-type AQP, likely involved in water-recycling functioning of G. molesta larvae. The AQP-Gra2, most similar (~40% identity) to AQP-Bom2, is considered as a midgut-type and it also increased glycerol and urea permeability. This is the second to demonstrate a member of the aquaglyceroporin family in insects after the first identification of insect aquaglyceroporin from B. mori by our group. The occurrence of two types of AQP, namely aquaporin (water channel) and aquaglyceroporin, appears to be characteristic of fluid-transpoting epithelia in lepidopteran larvae.

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The evaluation of the knots pattern that appeared on the fabric woven by the yarn with the knots as the doupion silk was examined. Variances of the number of knots for various areas were theoretically obtained by knots appearing model and the woven fabric model, which are defined in this report. The model to which the variance for each area can be estimated by using the knots appearing interval distribution of weft was made. The knots pattern on the fabric can be predictable because the variance for each area shows the scatter of the knots on the fabric before being actually woven. This method can be applied not only to the doupion silk but also to the spinning yarn.

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Ametabolous insects are important for understanding the mechanism of insect evolution based on their phylogenetic position. Thus, the development and application of an effective gene functional analysis using the RNA interference (RNAi) method is an important step in research on ametabolous insects. We tested RNAi utility in the firebrat, Thermobia domestica (Zygentoma, Lepismatidae) by focusing on the homeobox gene, Distal-less (Dll), based on its conserved sequence and obvious loss-of-function phenotype. Thermobia nymphs that were injected with Dll double-stranded RNA at an early embryonic stage displayed truncated appendages, and thus, we concluded that the RNAi method is useful for analyzing gene function in Thermobia. Remarkably, Dll RNAi induced truncation of the caudal appendage, cerci and the caudal non-appendage outgrowth, caudal filament. It is known that although these two caudal structures look similar, they have different origins. Our data suggests that these two types of outgrowths may be formed by similar developmental program, at least with respect to Dll, despite their different origins and that Dll even plays a role in a non-appendage structure.

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Larval labial glands of the sweet potato hornworm, Agrius convolvuli, secrete feeding- and wandering-stage specific proteins, which differ depending on the stage of development. The purpose of this study was to clarify the characteristics of feeding-stage specific proteins acquired from feeding-stage larvae.
Using two-dimensional (2D) gel electrophoresis, a total of 11 protein spots were detected in proteins collected from the labial gland lumens of 5th instar day 2 larvae. N-terminal amino acid sequencing revealed that the 67, 70 and 74kDa proteins had similar sequences, and were Asn and Ala rich. The 33 and 34kDa proteins were also similar, with both characterized as having characteristic Gly-X repeats. In addition, a number of similar mass (m/z) peaks were detected in the 67, 70 and 74kDa proteins and 33 and 34kDa proteins by use of MS spectrometry. These results suggest that some of the feeding-stage specific proteins are derived from homologous genes have similar functions.

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