Photoperiodism of Diapause Induction in the Silkworm, Bombyx mori.

The silkworm Bombyx mori exhibits a photoperiodic response (PR) for embryonic diapause induction. This article provides a comprehensive review of literature on the silkworm PR, starting from early works on population to recent studies uncovering the molecular mechanism. Makita Kogure (1933) conducted extensive research on the PR, presenting a pioneering paper on insect photoperiodism. In the 1970s and 80s, artificial diets were developed, and the influence of nutrition on PR was well documented. The photoperiodic photoreceptor has been investigated from organ to molecular level in the silkworm. Culture experiments demonstrated that the photoperiodic induction can be programmed in an isolated brain (Br)-subesophageal ganglion (SG) complex with corpora cardiaca (CC)-corpora allata (CA). The requirement of dietary vitamin A for PR suggests the involvement of opsin pigment in the photoperiodic reception, and a cDNA encoding an opsin (Boceropsin) was cloned from the brain. The effector system concerning the production and secretion of diapause hormone (DH) has also been extensively investigated in the silkworm. DH is produced in a pair of posterior cells of SG, transported to CC by nervi corporis cardiaci, and ultimately released into the hemolymph. Possible involvement of GABAergic and corazonin (Crz) signal pathways was suggested in the control of DH secretion. Knockout (KO) experiments of GABA transporter (GAT) and circadian clock genes demonstrated that GAT plays a crucial role in PR through circadian control. A model outlining the PR mechanism, from maternal photoperiodic light reception to DH secretion, has been proposed.

A new method for random mutagenesis by error-prone polymerase chain reaction using heavy water.

Error-prone polymerase chain reactions (epPCRs) are often used to introduce mutations in random mutagenesis, which has been used as a tool in protein engineering. Here, we developed a new method of epPCR using heavy water as a solvent instead of normal water (H(2)O). Rhodopsin cDNA of the Ayu fish (Plecoglossus altivelis) was used as a template and was amplified using five different conditions: (A) 100% H(2)O with no Mn(2+), (B) 100% H(2)O/0.6mM Mn(2+), (C) 99% D(2)O with no Mn(2+), (D) 99% D(2)O/0.6mM Mn(2+) and (E) 99% H(2)(18)O with no Mn(2+). The 13,960 (for each of the conditions A to D) and 33,504 (for condition E) base pairs were sequenced. A maximum error rate of 1.8×10(-3)errors/bp was detected in condition D, without any particular hot-spot mutations. A high preference for AT→GC transitions was observed in condition D, whereas a high preference for transitions over transversions was observed in condition C. All of the mutations observed in condition E were transversions. When conditions A and C were applied to another template, the honeybee actin gene, the results were comparable to those for Ayu rhodopsin. Based on these results, the use of heavy water, instead of H(2)O, as a solvent for epPCR can introduce random mutations without positional bias, template dependency or decreased yield. Our new epPCR method, and possibly combining the use of D(2)O and H(2)(18)O, may be a powerful random mutagenesis technique.

Apis cerana japonica discriminates between floral color phases of the oriental orchid, Cymbidium floribundum.

Foragers of the Japanese honeybee (Apis cerana japonica) were attracted by flowers of an oriental orchid (Cymbidium floribundum) and were observed to carry the pollinia on their scutella. After the removal of pollinia from the flowers, their labial color changed from white to reddish brown. Both artificial removal of pollinia and ethrel treatment of the flowers also induced this labial color change. Labia in color-changed flowers showed a decreased reflectance of wavelengths less than 670 nm compared to control intact flower. Both reflectance irradiance spectra and ultraviolet photographs showed that only the nectar guide in white (unchanged) flowers reflected ultraviolet light, and that this reflectance decreased with labial color change. Dual choice experiments showed that the honeybee foragers preferentially visited flowers having white labia rather than reddish brown. We suggest that Japanese honeybees discriminate between the floral phases of C. floribundum using color vision.

Sexual difference in color sense in a lycaenid butterfly, Narathura japonica.

The spectral sensitivity of a lycaenid butterfly, Narathura japonica, was investigated by electroretinography using an integrating sphere that could illuminate the compound eye from almost all directions. Samples were collected from three locations. Butterflies from different locations showed a similar pattern; the first, second, and third peaks (or a shoulder) were located at about 380, 460, and 560 nm, respectively. Males clearly showed the highest sensitivity at the first peak point. In contrast, females showed a higher relative sensitivity than males at the second and third peak points in all samples, and showed broad spectral sensitivity. This male-specific UV-sensitivity is discussed in terms of ecological factors.

Effects of temperature on circadian rhythm in the Japanese honeybee, Apis cerana japonica.

Temperature influences key aspects of insect circadian rhythms. The locomotor rhythm in foragers of the Japanese honeybee, Apis cerana japonica, was entrained to a skeleton temperature cycle. An initial warm temperature pulse was imposed at the beginning of subjective day and a second was applied at the end of the subjective day. A single warm pulse given every early subjective day in constant darkness (DD) entrained the locomotor rhythm without a second temperature pulse, but a single pulse given in late subjective day allowed a free-running rhythm. When honeybees were kept under a light-dark cycle, their body temperatures increased by 7-8 degrees C with locomotor activity. This temperature elevation remained during the photophase but followed the ambient environmental temperature at night. Body temperature oscillations continued to be circadian in DD, and temperature elevation occurred during the subjective day. In DD, the free-running period tau of locomotor activity increased when the ambient temperature increased from 27 to 37 degrees C, although these changes were within the range of temperature compensation for many organisms. Under continuous light conditions (LL), tau remained constant with more strict temperature compensation. Patterns of brain period mRNA levels of forager bees maintained at different temperatures in LL revealed that the free-running period of per mRNA rhythm was temperature compensated. In addition, temperature strongly influenced the amplitude of the circadian transcriptional rhythms during the free-run period in LL, which may confer temperature compensation. We also discuss the possibility that daily changes in forager body temperatures may act as an internal Zeitgeber by fluctuating hive temperature.

Molecular cloning of cone opsin genes and their expression in the retina of a smelt, Ayu (Plecoglossus altivelis, Teleostei).

Five cone opsin genes of landlocked ayu fish (Plecoglossus altivelis) were cloned, and the expression patterns of these genes were investigated. AYU-LWS, -RH2-1, -RH2-2, -SWS1-1, and -SWS1-2 were isolated and had high (more than 75%) identity with red, green, green, UV, and UV-sensitive opsin, respectively, genes of other fish reported previously. The results of Southern blotting experiments showed that each gene is present as a single copy. Gene expression was measured by RT-PCR using four populations collected from rivers and a lake in spring and summer. The results of the RT-PCR experiment showed that AYU-SWS1-2 was highly expressed, whereas AYU-SWS1-1 was scarce. Two RH2 opsins were expressed simultaneously in the same individual, and the expression ratio between these opsins changed among populations. In situ hybridization revealed that AYU-LWS and -RH2-1 were expressed in the double cones and that AYU-RH2-2 and -SWS1-2 were expressed in the long and short single cones (LSC and SSC), respectively. It was shown that an individual ayu expresses two RH2 opsins simultaneously in different types of cone cells.

Genetic diversity and structure of urban populations of Pieris butterflies assessed using amplified fragment length polymorphism.

Conservation programs in urban ecosystems need to determine the genetic background in populations of urban dwellers. We examined the genetic diversity and structure of Pieris rapae and P. melete using AFLP markers, and compared them between species and between urban and rural environments. As a result: (i). in both species, there was no reduction in genetic diversity within urban populations by direct comparison of diversity measurements, although the analysis of molecular variance suggested significant reductions in the variance within seasonal subpopulations in urban populations; (ii). P. rapae retained greater genetic diversity within species and populations; (iii). populations of both species showed significant genetic differentiation, and P. melete was more strongly subdivided; (iv). in both species, geographically close populations did not cluster with one another in the upgma analysis; (v). there was no genetic isolation due to geographical distance in either species; (vi). the genetic composition of seasonal subpopulations differed in urban populations of both species, and the genetic distances among subpopulations were correlated with seasonal differences in P. rapae and with temporal differences in P. melete. These results indicate that the genetic diversity in urban populations of both species was reduced at times, but was maintained by dispersal from genetically differentiated populations. Differences in the ability and mode of dispersal in the two species may be reflected in the degree of population subdivision and patterns of seasonal change in the genetic composition.

Molecular cloning and characterization of rhodopsin in a teleost (Plecoglossus altivelis, Osmeridae).

Amplified fragments encoding exon-4 of opsin cDNAs were cloned from the retina of landlocked ayu (Plecoglossus altivelis), and sequenced. On the basis of the sequence homology to previously characterized fish visual pigments, one clone was identified as rod opsin (AYU-Rh), and two clones as green (AYU-G1, -G2), one as red (AYU-R) and two as ultraviolet (AYU-UV1, -UV2) cone opsins. The 335-amino acid sequence deduced from the full-length cDNA of AYU-Rh included residues highly conserved in vertebrate rhodopsins and showed the greatest degree (88%) of similarity with salmon rhodopsin. Southern blotting analysis indicated that ayu possess two rhodopsin genes, one encoding visual rhodopsin (AYU-Rh) and the other non-visual extra-ocular rhodopsin (AYU-ExoRh). RT-PCR experiments revealed that AYU-Rh was expressed in the retina and AYU-ExoRh in the pineal gland. In situ hybridization experiments showed that the mRNA of AYU-Rh was localized only in rod cells not in cone cells. Lake and river type landlocked ayu having different amounts of retinal and 3-hydroxyretinal in their retinas expressed a rhodopsin (AYU-Rh) of identical amino acid sequence.

Molecular cloning of lysozyme-encoding cDNAs expressed in the salivary gland of a wood-feeding termite, Reticulitermes speratus.

Two kinds of PCR-product cDNAs that encode premature lysozyme peptides (Rs-Lys1 and Rs-Lys2) were cloned from workers of a Japanese damp-wood termite, Reticulitermes speratus. The Rs-Lys1 and Rs-Lys2 cDNAs encoded deduced sequences of 170 and 164 amino acids, respectively. Alignment of these sequences with those of other insect lysozymes showed that the cDNAs encode lysozyme homologues with putative signal peptides, insertions eight amino acids long, and a relatively long C-terminus (13-17 amino acids). A maximum likelihood tree, constructed using the cDNA sequences, indicated that the termite lysozymes are related to those of mosquitoes and lepidopterans. Southern-blotting analysis identified single copies of these lysozyme genes in the termite. Reverse transcript (RT)-PCR and in situ hybridization experiments showed that Rs-Lys1 and Rs-Lys2 are expressed in the salivary glands of worker termites. Here, we discuss the possible digestive function of these lysozymes.

A novel isoform of vertebrate ancient opsin in a smelt fish, Plecoglossus altivelis.

Vertebrate ancient (VA) opsin of nonvisual pigment in fishes was reported to exist in two isoforms, i.e., short and long variants with an unusual predicted amino acid sequence length compared to vertebrate visual opsins. Here we cloned an isoform (Pal-VAM) of VA opsin showing the usual opsin length in addition to the long type isoform (Pal-VAL) from a smelt fish, Plecoglossus altivelis. Pal-VAM and Pal-VAL were composed of 346 and 387 amino acids, respectively. The deduced amino acid sequences of these variants were identical to each other within the first 342 residues, but they showed divergence in the carboxyl-terminal sequence. Pal-VAL corresponded to the long isoform found in zebrafish and carp, and Pal-VAM was identified as a new type of VA opsin variant. Southern blotting experiments indicated that the VA opsin gene of the smelt is present as a single copy, and RT-PCR analysis revealed that Pal-VAM and Pal-VAL mRNA were expressed in both the eyes and brain. In situ hybridization showed that Pal-VAM and Pal-VAL mRNA are expressed in amacrine cells in the retina. Pal-VAM is a new probably functional nonvisual photoreceptive molecule in fish.