The present paper reviews current studies on various aspects of reproductive isolation in a series of closely related phytophagous ladybird beetles called the Epilachna vigintioctomaculata complex, which is composed of two groups and four species: group A comprising E. vigintioctomaculata, and group B comprising one northerly distributed species, E. pustulosa, and two southerly distributed species, E. niponica and E. yasutomii.

Being broadly sympatric from middle through northern Japan, the two groups are reproductively isolated by a combination of several factors, each of which functions as an incomplete barrier to gene flow: difference of host plants, weak sexual isolation, and low hatching rates of eggs produced by interspecific matings. Conspecific sperm precedence further lessens the probability of producing hybrids in mixed populations. By contrast, the two sympatric species of group B, E. niponica and E. yasutomii, are reproductively isolated from each other solely by fidelity to different host plants. However, there are no effective barriers to gene exchange between either of these two species and the northerly distributed E. pustulosa.

These situations have relevance to various controversial issues in evolutionary biology, covering the mode of speciation in host specific insects, role of postinsemination barriers to fertilization, reinforcement of reproductive isolation, and treatment of allopatric populations in speciation studies. The E. vigintioctomaculata complex thus offers a rare opportunity to extend our understanding of the nature of animal species and their origin.

Amiloride-blockable Na channels participate in active Na transport across adult, but not larval, bullfrog skin. Their development is induced in vitro by culturing the tadpole skin with aldosterone. When tadpoles were raised in aldosterone (5 × 10⁻⁷ M) for 2 weeks, however, neither development of such channels nor localization of antigen A, a marker of adult-type epidermis, was seen, the skin still being of the larval type. In contrast, aldosterone treatment did potentiate (by a factor of two) the activity of the acetylcholine receptor (ACh-receptor) channel, a functional marker of larval-type skin. The short-circuit current (SCC) across the skin, far from being inhibited by amiloride, was stimulated by both amiloride and ACh. The nystatin-stimulated SCC was about twice its control amplitude, suggesting that the aldosterone treatment also potentiated the activity of the Na pump.

The effects of 20-hydroxyecdysone (20-HE) on cyprid larval attachment and metamorphosis of the barnacle Balanus amphitrite were examined. When exposed to 10-100 μM 20-HE, cyprids failed to metamorphose, but attached to substrata. However, 0.001-0.1 μM 20-HE promoted attachment and metamorphosis. Moreover, effects of a combination of 20-HE and methyl farnesoate (MF) on cyprids were examined. The metamorphosis-inducing effect of MF was suppressed by pretreatment with 100 μM 20-HE. In contrast, after pretreatment with 10 μM MF, 100 μM 20-HE inhibited metamorphosis. High performance capillary electrophoresis (HPCE) revealed the presence of 20-HE in the extract from cyprid larvae of B. amphitrite, suggesting the intrinsic hormonal role of 20-HE in larval metamorphosis process.

Cannibalisms among larvae of a salamander Hynobius retardatus were affected by level of food supply, their density, body size and kinship. Kin recognition during cannibalisms among mixed-sibling larvae was examined using larvae with similar body size (within 10% differences in total body length at hatching stage) developed from different egg clutches. In order to distinguish larvae developed from an egg clutch from ones developed from different egg clutches, individual marking system by eye ball-removal operation was conducted. The eye ball-removal itself had no effect on the cannibalism, either to consume or to be comsumed. It was found that the larvae can recognize kinship without sence of sight and preferentially consume non-kins or avoid killing siblings, when larvae hatched from different egg clutches were mixed. The cannibalism, however, was either induced or not among larvae hatched from an egg clutch. The destiny of larvae from an egg clutch whether they became “cannibalistic” or “non-cannibalistic” was determined in every egg clutch by observing the cannibalism in larvae hatched from each egg clutch, respectively, during the first 10 days after hatching. Thus, it was possible to distinguish “cannibalistic” clutches which contained cannibalistic larvae from “non-cannibalistic” clutches which never showed the cannibalism. Even larvae of the “non-cannibalistic” clutch, however, had an ability to eat another individual when mixed with larvae hatched from different egg clutches.

Cumacean Dimorphostylis asiatica exhibits an endogenous swimming rhythm, free-running period of which spontaneously changes from circa 12-hr to circa 24-hr in the laboratory. Administration of a 4-hr pulse of hydrostatic pressure stimulus and a 4-hr light pulse confirmed that the circa 12-hr rhythm is circatidal and the circa 24-hr rhythm circadian, with respect to the phase setting characteristics. The activity records provide evidence of a “splitting” phenomenon, which suggests that these two types of rhythms are governed by (an) identical pacemaker(s). This species appears to have acquired tidal synchrony by making use of flexibly coupled circadian pacemakers, accompanied by expansion of their subtidal habitat to shallow, tide-affected area.

The ultrastructural features of the cardia from fourth instar larvae of the midge Chironomus tentans are consistent with it being the source of the peritrophic membrane. The group of anterior epithelial cells display short basal membrane infoldings, abundant rough endoplasmic reticulum, Golgi complexes, mitochondria, free ribosomes, vesicles and electron-dense secretory granules. The secretory material is secreted into the narrow luminal cleft between the evenly spaced microvilli and the apposing non-secretory esophageal valve. The valve and apices of secretory cells act as a press to mold the secretory product into the definitive peritrophic membrane. The single 255–488 nm thick uniform peritrophic membrane is devoid of holes and is composed of three layers in the posterior cardia, two layers in the rectum but only one 110 nm thick discernable layer in the midgut. Since the inner loosely arranged third layer is absent in the midgut and the rectum, it is postulated that it is incorporated into the electron-dense second layer. The second or middle layer which represents the whole peritrophic membrane in the midgut displays unique vertical striations that enclose 6.5 nm diameter channels. It is suggested that these newly described open-ended channels facilitate passage of water, salts, digestive enzymes and digested food material in either direction.

KIF3A is a member of the kinesin superfamily proteins (KIFs), but its gene has been cloned only in mouse and sea urchin. We have cloned a homolog of KIF3A from the frog, Rana rugosa (rrKIF3A). The sequence encoded a 699 amino acid protein that shares 93% similarity with mouse KIF3A (mKIF3A) and 69% with sea urchin kinesin-related protein (SpKRP85). The putative ATP-binding domain was completely identical to that of mKIF3A and SpKRP85. The level of rrKIF3A mRNA appeared to be high in the brain and testis of adult frogs, but low in the heart, lung and kidney. The results suggest that the rrKIF3A gene is expressed in the brain and testis more than other tissues of adult frogs examined, and that KIF3A is widely distributed in eukaryotic organisms.

Leech (Whitmania edentula, Haemadipsa zeylanica var. japonica and Erpobdella lineata) extracellular hemoglobins are basically composed of three constituent subunits, a dimer (D₁ and D₂ chains) and two monomers (M1 and M2 chains). We isolated these four chains from respective species by a combination of reversed-phase chromatography on a Resource RPC column and gel-filtration on a Superdex 75 column. The apparent molecular masses of the four globin chains were estimated by SDS-PAGE analysis to be 13 kDa (M1), 16 kDa (M2; 19 kDa in its reduced form) and about 27 kDa for the dimer subunit (13 kDa for D₁;15 kDa for D₂), regardless of the source. The amino (N)-terminal segments (21–30 residues) from twelve globin chains of the above three species were determined and aligned. It was found that the twelve sequences could be separated into two distinct globin groups A and B. This finding supports the original idea of “two globin strains in annelid hemoglobin”, which was proposed without any evidence for leech hemoglobins. Comparing the sequences in the three classes of Annelida, Hirudinea, Oligochaeta and Polychaeta, we found two invariant amino acids, Cys and Trp, which are interposed by eleven amino acid residues. Furthermore, the globin chains belonging to strain A were readily discernible as they had three more invariants, Ser-13, Asp-16 and Trp-28, while the globin chains of strain B had two more invariants, Lys-12 and Arg-27. Consequently, we propose that each of the three classes of Annelida have two distinct groups of globin chains that are akin to vertebrate hemoglobin α and β chains.

Exogastrula-inducing peptides (EGIPs) are intrinsic factors that are present in eggs and embryos of the sea urchin Anthocidaris crassispina. They induce exogastrulation when added exogenously to the embryos. In the present study, we isolated an EGIP-D-binding protein (EBP) from a homogenate of mesenchyme blastulae. EBP had an apparent molecular weight of 33,000. The N-terminal amino acid sequence of EBP had a sequence homology to HLC-32 and bep4 identified in other sea urchin embryos. In addition to its ability of binding to EGIP-D, EBP also inhibited exogastrulation induced by EGIP-D. These results suggest that EBP plays an essential role in EGIP-D-induced exogastrulation.

We studied immunohistologically the distribution of a Ca² -binding protein, calreticulin (CLT), at different stages of growing oocytes in the frog, Rana rugosa. Northern blot analysis showed that the CLT gene expression in gonads of metamorphosing tadpoles was low, but was extremely strong in the ovary, but not in the testis, of 2-month post-metamorphosis frogs, followed by decline to a lower level in adult frogs. On the contrary, the β-actin gene expression did not increase in the same ovary. As for the distribution of CLT protein, a weak immunostaining was observed in indifferent gonads of tadpoles at stage I. The CLT distribution in oocytes from stage A to F was stage-dependent. In addition, Western blot analysis revealed that the CLT level was low in gonads of tadpoles at stage I, but increased at stage XVI. It still increased in the ovary of frogs 2 months after metamorphosis, and then dropped to a lower level in adult frogs. The results indicate that CLT gene expression occurs in the early stage of growing oocytes, and that CLT is synthesized actively in oocytes in the ovary of frogs after metamorphosis. Based on these findings, the role of CLT is discussed.

In order to know the function of protein tyrosine kinases (PTKs) in the development of sea urchin embryos, we performed reverse transcription-polymerase chain reaction (RT-PCR) to obtain partial cDNA fragments for PTK genes using primers to highly conserved regions of the PTK family. A total of seven PTK sequences were identified, two of which represented receptor PTK (RTK1 and RTK2), and five of which were non-receptor PTKs (NRTK1-5). RTK1 was highly similar to FGF receptor and Ret kinase, while RTK2 showed features of the insulin receptor family. NRTK1 and 2 belonged to the Src family and could be involved in egg activation at fertilization. NRTK3 showed the features of the Btk family kinases, while NRTK4 seemed to be a member of the Syk/ZAP70 family. NRTK5 is the Csk-type kinase of the sea urchin, which is known to negatively regulate the Src family kinases. RTK1 was not detected in unfertilized eggs and was activated after blastula stage. All the other PTK genes were expressed both maternally in unfertilized eggs and zygotically after fertilization, though each gene showed distinct temporal patterns.

We isolated two cDNAs, termed D7 and C2 in the present study, from a cDNA library of the 16-cell embryo of the sea urchin Anthocidaris crassispina. The nucleotide sequence was determined completely for D7, and partially for C2. D7 does not have any significant open reading frames. Both D7 and C2 contain a common sequence that is 62% homologous to the sea urchin retroposon family 1 (SURF1). The SURF1 is a short interspersed repetitive element identified from the sea urchin Strongylocentrotus purpuratus, and is reported to be transcribed by RNA polymerase III. The structural feature of D7 and C2, however, suggests that they may be transcribed by RNA polymerase II. RT-PCR analyses revealed that (1) both D7 and C2 transcripts exist as a maternal RNA in the egg, (2) they appear evenly distributed in the 16-cell embryo, and (3) C2 transcripts are present throughout the development up to the gastrula, while D7 transcripts decrease in amount after the early cleavage stage.

Arrow worms (the phylum Chaetognatha), one of the major marine planktonic animals, exhibit features characteristic to both deuterostomes and protostomes, and their ancestry therefore remains unknown. As the first step to elucidate the molecular bases of arrow worm phytogeny, physiology and embryology, we isolated cDNA clones for three different actin genes (PgAct1, PgAct2 and PgAct3) from the benthic species Paraspadella gotoi, and examined their expression patterns in adults and juveniles. The amino acid sequences of the three actins resembled each other, with identities ranging from 86% to 92%. However, the patterns of the spatial expression of the genes were independent. The PgAct1 gene might encode a cytoplasmic actin and was expressed in oogenic cells, spermatogenic cells, and cells in the ventral ganglion. The PgAct2 and PgAct3 genes encoded actins of divergent types. The former was expressed in well-developed muscle of the head (gnathic) region and trunk muscle cells, whereas the latter was expressed in muscle of the trunk and tail regions and oogenic cells. These results suggest that, similarly to other metazoans, the chaetognath contains multiple forms of actins, which are expressed in various manners in the adult and juvenile arrow worm.

Tubifex eggs individually inherit a single maternal centrosome during the first mitosis (mitosis I), and thereby form an asymmetrically organized monastral mitotic apparatus for the unequal cleavage. This maternal centrosome does not duplicate itself at the transition to mitosis I but does so at the transition to the second mitosis (Shimizu T, 1996, Roux's Arch Dev Biol 205: 290-299). To examine whether the maternal centrosome duplication is under cytoplasmic control, we produced syncytial eggs by electric fusion of an early meiosis II egg with an early mitosis I egg in an equator-to-equator fashion. As suggested from the diffusion rate of microinjected Texas Red-dextran (10,000 MW), diffusible ooplasmic components in syncytial eggs appear to be intermingled within 20 min following fusion. At ∼120 min after fusion, the meiosis II spindle and the mitosis I nucleus in each syncytial egg complete respective meiosis and mitosis almost simultaneously. Shortly after completion of the meiosis/mitosis, syncytial eggs individually exhibit three centrosomes, two of which are associated with a nucleus derived from the mitosis I spindle, and the remaining one with a nucleus derived from the meiosis II spindle. These results suggest that maternal (meiosis ll) centrosomes fail to duplicate under cytoplasmic conditions that favor the duplication of mitotic centrosomes and that meiosis II centrosomes are intrinsically distinct from mitotic centrosomes. We propose that inhibitory factors that block centrosome duplication are associated with meiotic centrosomes and that their release during mitosis I gives rise to reproduction-competent maternal centrosomes.

Apical ectodermal ridge (AER)-mesoderm interaction is important for morphogenesis in the developing chick limb bud. Genes whose expression is dependent upon the presence of AER, are likely to play important roles in the AER-mesoderm interaction. We report here the gene expression pattern of the chick homolog of the 67 kDa laminin binding protein (LBP), which is a non-integrin laminin receptor whose function relates to cell attachment, spreading, and polarization. Northern analysis showed that a single 1.4 kb transcript exists in stage 20 limb buds and which is dramatically reduced 24 hr after removal of AER. In situ hybridization analysis revealed that the chick 67 kDa laminin binding protein gene (cLbp) was expressed in the mesodermal region overlapping the Msx1-expressing domain and in the AER in early stage limb buds. Expression in the mesoderm was gradually restricted to the distal region underneath the AER as development proceeds. The expression in the limb mesoderm could be induced by local application of FGF-2 which could thus mimic the AER functions. These results indicated that the expression of cLbp depends on AER signals and that the 67 kDa non-integrin receptor binding to laminin plays a role in the AER-mesoderm interaction.

In a preliminary experiment, male Oreochromis mossambicus which received silastic implants of oestradiol (E₂; 10, 50 or 125 μg/g body weight) had elevated serum E₂ levels 14 days later, compared with those receiving blank implants. In two subsequent experiments, groups of 15 males received either blank implants or one of these three doses of E₂, and were then transferred to 33% seawater; they were either maintained in this medium for the subsequent 10 days, or they were transferred back to freshwater on the day after implantation and maintained in the latter for the remaining nine days. There was evidence for a dose-independent increase in the proportion of black males in E₂-treated groups, regardless of salinity, implying increased territorial aggression. Subsequent to the 10-day exposure, polyacrylamide gel electrophoresis of their pituitaries was used to quantify levels of growth hormone (GH) and the large and small forms of prolactin (PRL). When comparing groups receiving blank pellets, levels of GH and both PRLs were greater in fish maintained for most of the post-operative period in freshwater. There was a dose-dependent increase in the pituitary content of all three hormones in fish receiving implants of E₂; this was most marked for the smaller of the two forms of PRL in fish adapted to freshwater.

In Japanese flounder (Paralichthys olivaceus), metamorphic period involves not only transformation from larva to juvenile but also migration from offshore areas to estuaries. In the present study, the role of endocrine systems in low-salinity adaptation was examined during early development and metamorphosis of the flounder. Survival rate 48 hr after transfer to 1/8 SW was relatively high in yolk-sac larvae, decreased gradually to 0% at premetamorphosis, and increased to 100% at metamorphic climax. The ratio of prolactin (PRL)-immunoreactive part to whole pituitary increased gradually during larval stages and reached a constant level during metamorphosis. When the larvae at premetamorphosis and metamorphic climax and the benthic juveniles were transferred from SW to 1/4 SW, PRL-immunoreactive part increased significantly 48 hr after the transfer at all stages examined. Whole-body concentration of Cortisol was measured with a modified extraction method which is much robuster to lipid-rich sample than the ordinary method, but no significant difference was observed after the transfer. Whole-body concentrations of thyroid hormones decreased slightly but significantly at premetamorphosis and metamorphic climax. These results suggest possible involvement of PRL and thyroid hormones in low-salinity adaptation of the flounder during metamorphosis and inshore migration.

A complementary DNA (cDNA) encoding eel atrial natriuretic peptide (ANP) precursor was specifically amplified from eel atrial mRNAs by rapid-amplification polymerase chain reaction. The sequence analysis of the cDNA using multiple clones revealed that the preproANP consists of 140 amino acid residues carrying a signal sequence at its N-terminus and a mature ANP at its C-terminus. An additional glycine residue was attached to the C-terminus of previously isolated eel ANP. The glycine residue may be used for amidation of the C-terminus or removed after processing. The cleavage site of a signal peptide with 22 amino acid residues was confirmed by isolation of proANP protein from eel atria. The proANP sequence deduced from the cDNA was also confirmed for 71% of the isolated protein. Sequence comparison with other natriuretic peptides revealed that eel ANP is more similar to mammalian ANP than to B-type natriuretic peptide (BNP) at both amino acid and nucleotide sequence levels. The eel ANP gene was a single copy gene as shown by Southern blot analysis. Northern blot analysis showed that eel ANP mRNA is approximately 0.8 kb in size and exclusively detected in the atrium. Thus, eel ANP is a true atrial hormone judging from both the sequence and the site of production. However, reverse transcription-polymerase chain reaction detected ANP message in the brain, gill, cardiac ventricle, red body of swim bladder (rete mirabilis), intestine, head kidney (including interrenal and chromaffin tissues) and kidney. Most of these tissues are involved in ion and/or gas exchange in fishes.

The bambooleaf wrasse, Pseudolabrus japonicus, exhibits diandric protogyny, and the secondary male performs pair spawning with a female who enters his territory. We examined diurnal spermatogenesis and spawning in the secondary male of a protogynous wrasse. In captivity, a single secondary male spawned daily over one month between 06:00 and 09:00 from October to November. Number of B-type spermatogonia and spermatocytes showed the lowest level at 00:00, increased gradually thereafter, peaked at 15:00, and decreased rapidly from 21:00 to 00:00. Spermatid number did not change significantly throughout the day. The number of spermatozoa increased gradually from 18:00, reached a maximum at 06:00, just prior to spawning, and thereafter decreased markedly at 09:00, after spawning. These results clearly showed that spermatogonial proliferation and meiosis occurred between 00:00 and 15:00, and spermiation occurred between 18:00 and 06:00. Thus, the secondary male of bambooleaf wrasse exhibits a diurnal rhythm of spermatogenesis and spermiation.

Adult sockeye salmon (Oncorhynchus nerka) in Lake Shikotsu were captured in September, October and November adjacent to their natal hatchery prior to spawning. They were sampled for hormones, tagged and released in the center of lake. Fish were again sampled at recapture to characterize changes in steroid hormone levels in individual migrants as well as homing percentage and duration in each month. All males returned faster than females early in the breeding season, although a half of the tagged males did not return to the natal site late in the season (November). A high percentage of females always returned, and homing duration shortened late in the season. In males, the shortening of homing duration coincided with an increase in serum testosterone (T) and 11-ketotestosterone levels. In females, the shortening of homing duration corresponded to an elevation of serum T and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) levels, and a drop in serum estradiol-17β levels. Sustained administration of gonadotropin-releasing hormone analog (GnRHa, via implants) in September greatly shortened homing duration, especially in females. GnRHa treatment caused a dramatic increase in serum DHP levels in both sexes on average. Individual GnRHa-treated males which rapidly returned, however, showed higher serum T levels and lower serum DHP levels than slower returning males. The present study indicates sexual differences in homing profiles as well as shortening of homing duration following GnRHa implantation in lacustrine sockeye salmon in Lake Shikotsu which may be reflective of changes in serum steroid hormone levels.

The incidence of embryonic diapause in the band-legged ground cricket, Dianemobius nigrofasciatus was examined in regard to egg production by individual females both in the laboratory and in the field in Osaka, Japan. (1) Females were classified into three types: diapause egg-producers (laid only diapause eggs), nondiapause egg-producers (laid only nondiapause eggs) and mixed egg-producers (laid both diapause and nondiapause eggs). (2) The longer the photophase, the smaller was the incidence of diapause egg-producers, and the larger was that of the nondiapause egg- and mixed egg-producers. The critical day-length was between 13 and 14 hr. (3) The longer the photophase, the smaller was the incidence of diapause eggs laid by mixed egg-producers. (4) In the field, both nondiapause egg-producers and mixed egg-producers were observed from June to August. In September and October, most of the females were diapause egg-producers. The present findings revealed that adults of the overwintered generation occur from June to August and those of the first generation occur in September and October. The former adult females become mixed egg-producers or nondiapause egg-producers under long-day photoperiods, and the latter females become diapause egg-producers under short-day photoperiods.

Environmental factors that regulate the induction, maintenance and termination of adult diapause and postdiapause oviposition were examined in a phytophagous insect, Dybowskyia reticulata (Heteroptera: Pentatomidae). When insects were reared from eggs at 25°C in the laboratory, all adults under a short-day photoperiod of 12:12 (L:D) hr and most adults under a long-day photoperiod of 16:8 (L:D) hr entered diapause. Overwintering adults started oviposition after being transferred to the long-day or short-day photoperiod at 25°C in March. Under the short-day photoperiod, however, they entered diapause again after a short oviposition period. Transferral to a long-day photoperiod after a long exposure to a short-day photoperiod synchronously terminated diapause. After exposure to a low temperature during diapause, the adults began to lay eggs under both long-day and short-day photoperiods. Low temperature in winter probably plays a dominant role in the termination of diapause in nature. In addition, the females started oviposition only after feeding when transferred from outdoor conditions to the laboratory in spring, and the onset of oviposition after starvation in the females was more synchronous than that without starvation. The regulation of the seasonal life cycle by various environmental factors in D. reticulata is discussed as an adaptation to the phenology of host plants.