Evolutionary dynamics of mushroom body Kenyon cell types in hymenopteran brains from multifunctional type to functionally specialized types.

Evolutionary dynamics of diversification of brain neuronal cell types that have underlain behavioral evolution remain largely unknown. Here, we compared transcriptomes and functions of Kenyon cell (KC) types that compose the mushroom bodies between the honey bee and sawfly, a primitive hymenopteran insect whose KCs likely have the ancestral properties. Transcriptome analyses show that the sawfly KC type shares some of the gene expression profile with each honey bee KC type, although unique gene expression profiles have also been acquired in each honey bee KC type. In addition, functional analysis of two sawfly genes suggested that the functions in learning and memory of the ancestral KC type were heterogeneously inherited among the KC types in the honey bee. Our findings strongly suggest that the functional evolution of KCs in Hymenoptera involved two previously hypothesized processes for evolution of cell function: functional segregation and divergence.

Effects of Functional Depletion of Doublesex on Male Development in the Sawfly, Athalia rosae.

The doublesex (dsx) gene, which encodes a transcription factor, regulates sexual differentiation in insects. Sex-specific splicing of dsx occurs to yield male- and female-specific isoforms, which promote male and female development, respectively. Thus, functional disruption of dsx leads to an intersexual phenotype in both sexes. We previously identified a dsx ortholog in the sawfly, Athalia rosae. Similar to dsx in other insects, dsx in the sawfly yields different isoforms in males and females as a result of alternative splicing. The sawfly exploits a haplodiploid mode of reproduction, in which fertilized eggs develop into diploid females, whereas unfertilized eggs parthenogenetically develop into haploid males. In the present study, we knocked down the A. rosae ortholog of dsx (Ardsx) during several developmental stages with repeated double-stranded RNA (dsRNA) injections. Knockdown of Ardsx via parental RNA interference (RNAi), which enables knockdown of genes in offspring embryos, led to a lack of internal and external genitalia in haploid male progeny. Additional injection of dsRNA targeting Ardsx in these animals caused almost complete male-to-female sex reversal, but the resulting eggs were infertile. Notably, the same knockdown approach using diploid males obtained by sib-crossing caused complete male-to-female sex reversal; they were morphologically and behaviorally females. The same RNAi treatment did not affect female differentiation. These results indicate that dsx in the sawfly is essential for male development and its depletion caused complete male-to-female sex reversal. This is the first demonstration of functional depletion of dsx not causing intersexuality but inducing total sex reversal in males instead.

A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis.

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace's insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

Correction to: Establishment and characterization of novel cell lines derived from six lepidopteran insects collected in the field.

This article was originally published with the final word of the title, "field", omitted.

Establishment and characterization of novel cell lines derived from six lepidopteran insects collected in the field.

Insect cell lines are used to study cellular interactions and gene functions in vitro in several research areas. However, suitable cell lines for experiments are not always available, especially in non-model species. Here, we established novel cell lines derived from fat bodies of six lepidopteran insects: Cydia kurokoi (named NARO-Cyku), Cephonodes hylas (NARO-Cehy), Haritalodes basipunctalis (NARO-Haba), Theretra oldenlandiae (NARO-Thol), Lymantria dispar (NARO-Lydi), and Hyphantria cunea (NARO-Hycu) collected in the field. The larval fat body was a promising tissue for the starting material when samples were limited due to field collection. It was critical that the medium volume was kept to a minimum for primary culture to maintain adherence of the fat body cells to the flask. The flask was coated with poly-L-lysine for effective induction of adherence and cell division. The identities of cell lines were confirmed using DNA barcoding with the mitochondrial cytochrome c oxidase I gene after cultures were passaged over 50 times. All lines except for NARO-Lydi and NARO-Hycu are adherent cells, and population doubling time of six cell lines ranged from 1.03 to 2.49. Induction of gene expression was practicable in the four adherent cell lines as revealed by transfection of expression vectors and found the immediate early 2 and the Bombyx actin 3 were effective gene promoters. The results suggest that these cell lines are capable of gene functional analysis. Thus, establishments of cell line using our methods for non-model lepidopterans could make a practical contribution to pest management and insect utilization.

Ontogeny of Cheliped Laterality and Mechanisms of Reversal of Handedness in the Durophagous Gazami Crab, Portunus trituberculatus.

The paired claws in Gazami crabs, Portunus trituberculatus, are bilaterally asymmetrical, and asymmetry is remarkable on the distal two segments of the first pereiopod, that is, the dactylus and propodus. Shells are exclusively cracked by use of the right chela, representing handedness. In Gazami crabs, handedness is reversed after autotomy of the right chela. Our study focused on the ontogeny of handedness and the mechanism of handedness reversal. Morphologically, asymmetry was first detected in megalopa larvae where the right propodus was significantly larger than the left, as was the canine at the base of the right dactylus. Presumably, the rate of chelagenesis differed between the left and right chelae. With these morphological features, the right chela functioned as a crusher. The crusher exerted a closing force two to three times that of the cutter. With loss of the right crusher, the left chela was bigger than the regenerated right chela and was converted to the crusher. In contrast, the performance of the regenerated right chela deteriorated compared to that of the original right crusher, and exertion of full closing force was inhibited by the more active left chela. Furthermore, crabs with two crusher chelae did not clearly show handedness. A decrease in size and performance of the regenerated right chela can be explained by a default program hypothesis. In conclusion, a difference in the chelagenesis rate results in bilateral asymmetry of the two chelipeds, and then handedness is generated by neural regulation in the thoracic ganglion innervating these claws. Since handedness is reversed after autotomy, the thoracic ganglion would not be lateralized in Gazami crabs. A default program hypothesis is proposed to explain the ontogeny of bilateral chela asymmetry and handedness reversal.

Diversity and function of multicopper oxidase genes in the stinkbug Plautia stali.

Multicopper oxidase (MCO) genes comprise multigene families in bacteria, fungi, plants and animals. Two families of MCO genes, MCO1 (laccase1) and MCO2 (laccase2), are conserved among diverse insects and relatively well-characterized, whereas additional MCO genes, whose biological functions have been poorly understood, are also found in some insects. Previous studies reported that MCO1 participates in gut immunity and MCO2 plays important roles in cuticle sclerotization and pigmentation of insects. In mosquitoes, MCO2 was reported to be involved in eggshell sclerotization and pigmentation, on the ground that knockdown of MCO2 caused deformity and fragility of the eggshell. Here we identified a total of 7 MCO genes, including PsMCO1 and PsMCO2, and investigated their expression and function in the brown-winged green stinkbug Plautia stali. RNA interference (RNAi) knockdown of MCO genes by injecting double-stranded RNA (dsRNA) into nymphs revealed that MCO2, but not the other 6 MCOs, is required for cuticle sclerotization and pigmentation, and also for survival of P. stali. Trans-generational knockdown of MCO2 by injecting dsRNA into adult females (maternal RNAi) resulted in the production of unhatched eggs despite the absence of deformity or fragility of the eggshell. These results suggested that MCO2 plays an important role in sclerotization and pigmentation of the cuticle but not in eggshell integrity in P. stali. Maternal RNAi of any of the other 6 MCO genes and 3 tyrosinase genes affected neither survival nor eggshell integrity of P. stali. Contrary to the observations in the red flour beetle and the brown rice planthopper, RNAi knockdown of MCO6 (MCORP; Multicopper oxidase related protein) exhibited no lethal effects on P. stali. Taken together, our findings provide insight into the functional diversity and commonality of MCOs across hemipteran and other insect groups.

Gene content evolution in the arthropods.

BACKGROUND: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. RESULTS: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. CONCLUSIONS: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

Wing vein development in the sawfly Athalia rosae is regulated by spatial transcription of Dpp/BMP signaling components.

Wing venation among insects serves as an excellent model to address how diversified patterns are produced. Previous studies suggest that evolutionarily conserved Decapentaplegic (Dpp)/Bone Morphogenetic Protein (BMP) signal plays a critical role in wing vein development in the dipteran Drosophila melanogaster and the hymenopteran sawfly Athalia rosae. In sawfly, dpp is ubiquitously expressed in the wing during prepupal stages, but Dpp/BMP signal is localized in the future vein cells. Since localized BMP signaling involves BMP binding protein Crossveinless (Cv), redistribution of BMP ligands appears to be crucial for sawfly wing vein formation. However, how ubiquitously expressed ligands lead to a localized signal remains to be addressed. Here, we found that BMP binding protein short gastrulation (Sog) is highly expressed in the intervein cells. Our data also reveal that BMP type I receptors thickveins (Tkv) and saxophone (Sax) are highly expressed in intervein cells and at lower levels in the vein progenitor cells. RNAi knockdown of Ar-tkv or Ar-sax indicates that both receptors are required for localized BMP signaling in the wing vein progenitor cells. Taken together, our data suggest that spatial transcription of core- and co-factors of the BMP pathway sustain localized BMP signaling during sawfly wing vein development.

Malaria infectivity of xanthurenic acid-deficient anopheline mosquitoes produced by TALEN-mediated targeted mutagenesis.

Anopheline mosquitoes are major vectors of malaria parasites. When the gametocytes of the malaria parasite are transferred from a vertebrate to mosquitoes, they differentiate into gametes, and are fertilized in the midguts of mosquitoes. Xanthurenic acid (XA), a waste product of the ommochrome synthesis pathway, has been shown to induce exflagellation during microgametogenesis in vitro; however, it currently remains unclear whether endogenous XA affects the infectivity of anopheline mosquitoes to malaria parasites in vivo due to the lack of appropriate experimental systems such as a XA-deficient line. In the present study, we produced a XA-deficient line in Anopheles stephensi using transcription activator-like effector nuclease (TALEN)-mediated gene targeting (knockout) of the kynurenine 3-monooxygenase (kmo) gene, which encodes an enzyme that participates in the ommochrome synthesis pathway. The knockout of kmo resulted in the absence of XA, and oocyst formation was inhibited in the midguts of these XA-deficient mosquitoes, which, in turn, reduced sporozoite numbers in their salivary glands. These results suggest that endogenous XA stimulates exflagellation, and enhances the infectivity of anopheline mosquitoes to malaria parasites in vivo. The XA-deficient line of the anopheline mosquito provides a useful system for analyzing and understanding the associated factors of malaria gametogenesis in the mosquito midgut.

Wolbachia infections of the butterfly Eurema mandarina interfere with embryonic development of the sawfly Athalia rosae.

Although maternally transmitted microorganisms such as Wolbachia are well known to have a variety of effects on the reproduction of diverse insect species, little is known about the underlying mechanisms of actions. Artificial transfer of Wolbachia between taxonomically distant host species may provide insights into Wolbachia-induced manipulations of hosts. Here we performed a cross-order transfer of feminizing Wolbachia derived from a butterfly, Eurema mandarina. The Wolbachia were propagated in the Eurema hecabe cell line, called NTU-YB, and then used to inject prepupal/pupal females of a Wolbachia-free hymenopteran sawfly, Athalia rosae. The 14 females that emerged as adults looked morphologically and behaviorally healthy, and ovarian development appeared normal on dissection. However, in contrast to the control, none of the 333 eggs harbored by the seven Wolbachia-injected females developed successfully. Similarly, none of the 140 eggs laid on host plant by the four Wolbachia-injected females, which were mated with males, showed any signs of development. Wolbachia infection was detected from whole-body samples of the inoculated individuals, but not from the eggs they produced. Disruption of embryonic development despite the absence of Wolbachia in the egg cytoplasm may represent a new phenotype involving maternal effects that result in female sterility.

Identification and functional characterization of the sex-determining gene doublesex in the sawfly, Athalia rosae (Hymenoptera: Tenthredinidae).

Sexual fate of the sawfly, Athalia rosae (Hymenoptera: Tenthredinidae) is determined by the complementary sex determination (CSD) mechanism as is the case in honeybees. However, to date, genes involved in sex determination have not been identified in this species. In this study, we attempted to identify orthologs of complementary sex-determiner (csd), feminizer (fem), and doublesex (dsx) from the A. rosae genome, all of which are crucial components of the sex determination cascade in the honeybee. As a result, we identified a sawfly ortholog of dsx (designated as Ardsx). Rapid amplification of cDNA ends (RACE) using total RNA extracted from male and female larvae identified three male-specific variants and three female-specific variants. Comparison between the full-length Ardsx cDNAs and the genomic sequence revealed that exon 5 was differentially spliced between the male- and female-specific variants. RT-PCR analysis demonstrated that Ardsx pre-mRNA was spliced alternatively in a sex-dependent manner at almost all the developmental stages. RNAi-mediated knockdown of Ardsx in males caused severe defects in the reproductive organs and, notably, induced development of the ovipository apparatus containing the dorsal pair of blades and the sheath. These males also showed abnormalities in testes and seminal vesicles and lacked mature sperm. The present study provides the first direct evidence that dsx is essential for sexual development in hymenopteran species.

The boule gene is essential for spermatogenesis of haploid insect male.

boule (bol), a member of the Deleted in Azoospermia (DAZ) gene family plays an important role in meiosis (reductional maturation divisions) in a spermatogenesis-specific manner in animals by regulating translation of the downstream cell division cycle 25 (cdc25) phosphatase mRNA. Orthologues of bol are conserved among animals and found in the genomes of hymenopteran insects, in which the general mode of reproduction is haplodiploidy: female is diploid and male is haploid. In this mode of reproduction, haploid males produce haploid sperm through non-reductional maturation divisions. The question thus arises of whether the bol gene actually functions during spermatogenesis in these haploid males. In this study, we identified two transcriptional isoforms of bol orthologue (Ar bol and Ar bol-2), and one cdc25 orthologue (Ar cdc25) in the hymenopteran sawfly, Athalia rosae. Ar bol was expressed exclusively in the testis when maturation divisions occurred, while Ar bol-2 was expressed ubiquitously. Knockdown of all bol transcripts (both Ar bol and Ar bol-2) resulted in a lack of mature sperm, whereas males with sole knockdown of Ar bol-2 were able to produce a small number of mature sperm. The cell cycle was arrested before maturation divisions in the testis in which all bol transcripts were knocked down, as revealed by flow cytometry. Although no mature sperm was produced, sperm elongation was partially observed when Ar cdc25 alone was knocked down. These results indicate that Ar bol is essential for the entry and progression of maturation divisions and sperm differentiation in haploid males.

Insights into the molecular mechanisms underlying diversified wing venation among insects.

Insect wings are great resources for studying morphological diversities in nature as well as in fossil records. Among them, variation in wing venation is one of the most characteristic features of insect species. Venation is therefore, undeniably a key factor of species-specific functional traits of the wings; however, the mechanism underlying wing vein formation among insects largely remains unexplored. Our knowledge of the genetic basis of wing development is solely restricted to Drosophila melanogaster. A critical step in wing vein development in Drosophila is the activation of the decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signalling pathway during pupal stages. A key mechanism is the directional transport of Dpp from the longitudinal veins into the posterior crossvein by BMP-binding proteins, resulting in redistribution of Dpp that reflects wing vein patterns. Recent works on the sawfly Athalia rosae, of the order Hymenoptera, also suggested that the Dpp transport system is required to specify fore- and hindwing vein patterns. Given that Dpp redistribution via transport is likely to be a key mechanism for establishing wing vein patterns, this raises the interesting possibility that distinct wing vein patterns are generated, based on where Dpp is transported. Experimental evidence in Drosophila suggests that the direction of Dpp transport is regulated by prepatterned positional information. These observations lead to the postulation that Dpp generates diversified insect wing vein patterns through species-specific positional information of its directional transport. Extension of these observations in some winged insects will provide further insights into the mechanisms underlying diversified wing venation among insects.

Artificial activation of mature unfertilized eggs in the malaria vector mosquito, Anopheles stephensi (Diptera, Culicidae).

In the past decade, many transgenic lines of mosquitoes have been generated and analyzed, whereas the maintenance of a large number of transgenic lines requires a great deal of effort and cost. In vitro fertilization by an injection of cryopreserved sperm into eggs has been proven to be effective for the maintenance of strains in mammals. The technique of artificial egg activation is a prerequisite for the establishment of in vitro fertilization by sperm injection. We demonstrated that artificial egg activation is feasible in the malaria vector mosquito, Anopheles stephensi (Diptera, Culicidae). Nearly 100% of eggs dissected from virgin females immersed in distilled water darkened, similar to normally oviposited fertilized eggs. It was revealed by the cytological examination of chromosomes that meiotic arrest was relieved in these eggs approximately 20 min after incubation in water. Biochemical examinations revealed that MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated protein kinase) and MEK (MAPK/ERK kinase) were dephosphorylated similar to that in fertilized eggs. These results indicate that dissected unfertilized eggs were activated in distilled water and started development. Injection of distilled water into body cavity of the virgin blood-fed females also induced activation of a portion of eggs in the ovaries. The technique of artificial egg activation is expected to contribute to the success of in vitro fertilization in A. stephensi.

Dpp/BMP transport mechanism is required for wing venation in the sawfly Athalia rosae.

The pattern of wing venation varies considerably among different groups of insects and has been used as a means of species-specific identification. However, little is known about how wing venation is established and diversified among insects. The decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling pathway plays a critical role in wing vein formation during the pupal stages in Drosophila melanogaster. A key mechanism is BMP transport from the longitudinal veins (LVs) to the posterior crossvein (PCV) by the BMP-binding proteins, short gastrulation (Sog) and twisted gastrulation2/crossveinless (Tsg2/Cv). To investigate whether the BMP transport mechanism is utilized to specify insect wing vein patterns in other than Drosophila, we used the sawfly Athalia rosae as a model, which has distinct venation patterns in the fore- and hindwings. Here, we show that Ar-dpp is ubiquitously expressed in both the fore- and hindwings, but is required for localized BMP signaling that reflects distinct wing vein patterns between the fore- and hindwings. By isolating Ar-tsg/cv in the sawfly, we found that Ar-Tsg/Cv is also required for BMP signaling in wing vein formation and retains the ability to transport Dpp. These data suggest that the BMP transport system is widely used to redistribute Dpp to specify wing venation and may be a basal mechanism underlying diversified wing vein patterns among insects.

A survey of the effectiveness of non-cell autonomous RNAi throughout development in the sawfly, Athalia rosae (Hymenoptera).

RNA interference (RNAi) is a powerful and convenient tool not only for functional analysis of specific genes, but also for large-scale screening of gene function in insects; however, reports on its efficiency throughout development in a single species are limited. We demonstrate here that non-cell autonomous RNAi by injection of double-stranded RNA (dsRNA) knocks down targeting genes in most developmental stages in the sawfly, Athalia rosae. Injection of dsRNA targeting the green fluorescence protein (gfp) gene into eggs of a transgenic strain carrying the constitutively expressing gfp gene resulted in the absence of GFP fluorescence during embryogenesis, while a portion of the gfp dsRNA-injected embryos began exhibiting GFP fluorescence at late embryogenesis. When gfp dsRNA was injected into parental female pupae, the RNAi effect was carried over to all embryos of the next generation and the effect lasted until mid-larval stages. Parental injection of dsRNA was more efficient than embryonic injection in terms of penetrance of the effect and the survival rate. After injection of gfp dsRNA into last instar larvae, the RNAi effect was sustained during prepupal and pupal stages and in adults. The gfp gene transcript markedly decreased in these knockdown phenotypes. It was revealed by employing fluorescence-labeled dsRNA that injected dsRNA was taken up in internal organs. Knockdown of an endogenous gene, Distal-less (Dll), resulted in typical phenotypes represented by the lack and malformation of Dll-expressing organs, such as distal parts of the appendages and wing edges without showing off-target effects. In contrast, RNAi by dsRNA injection seems to be hardly effective in mid- to late-larval stages.

Characterization of abdominal appendages in the sawfly, Athalia rosae (Hymenoptera), by morphological and gene expression analyses.

Larvae of the sawfly, Athalia rosae, have remarkable abdominal prolegs. We analyzed the morphogenesis of appendages and the expression of decapentaplegic and Distal-less genes during embryonic development to characterize the origin of prolegs. Proleg primordia in abdominal segments A1-A9 appeared shortly after the inner lobes (endites) of gnathal appendages were formed. These were located on the ventral plates, medioventral to the appendages of the other segments in light of serial homology. Nothing was seen where the main axis of the appendage should develop in abdominal segments. The primordia in A1 and A9 disappeared before larval hatching. Anal prolegs appeared separate from cerci, the main axes of appendages, which were formed temporarily in A11. The expression of decapentaplegic, which reflects the primary determination of appendages, was detected in the lateral juxtaposition with the prolegs. Distal-less was expressed in the main axes of appendages, protruding endites and the cerci, but not in prolegs and anal prolegs or the gnathal endites which do not protrude. These findings suggest a possibility that the abdominal and anal prolegs of A. rosae are outgrowths of ventral plates which derived from coxopodal elements, but not main axes of appendages.

Involvement of Mos-MEK-MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae.

Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK-MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule.

Expression pattern of Bombyx vasa-like (BmVLG) protein and its implications in germ cell development.

Germ cell development in the silkworm Bombyx mori is interesting in that the species has no recognizable germ plasm, and its germ cells appear first on the ventral side of the embryo, not on the posterior pole as in Drosophila melanogaster. We previously reported the isolation of a vasa homologue (BmVLG) from B. mori and revealed the specific expression of transcript in the germ cells. In this paper, we describe the embryonic expression pattern of BmVLG protein. Consistent with the lack of recognizable germ plasm, the protein is not localized in freshly laid eggs, and its specific expression is first detectable several hours after energids penetrate the periplasm. This is in contrast to D. melanogaster, where germ cell lineage can be traced with anti-vasa antibody just after the formation of pole cells as they sequester vasa-positive germ (pole) plasm during cellularization. It is also revealed that, within the first few hours of their appearance when extensive cell movement does not seem to occur, stained cells are sometimes widely dispersed along the midline, which eventually may lead to the formation of ectopic germ cells. The implications of these results for germ cell development are discussed.