Artificial insemination using cryopreserved sperm in the silkworm,Bombyx mori

We report in this paper that female moths artificially inseminated with cryopreserved sperm (-196 degrees C) could oviposit eggs when the sperm was preserved for 356days, and that the fertilization rate and the number of eggs laid were almost equivalent to those obtained in normally mated moths. The optimal cooling rate for sperm freezing was 5-65 degrees C/min for maintaining a high fertility of sperm. The simple and reliable method of cryopreservation was to put the semen first in a deep freezer at -80 degrees C and thereafter put them in liquid nitrogen. When female moths of 'white 2' egg-color mutant strain were inseminated with a mixture of frozen-thawed sperm from males of normal-colored egg strain and non-frozen sperm from males of the 'white 2', female moths deposited a majority of 'white 2' eggs and a very small number of eggs of normal color. The result shows that there was a competitive fertilization of sperm between the two strains of the silkworm, and that sperm fertility was reduced to a considerable extent by freezing at -196 degrees C. These results may contribute not only to basic studies on fertilization in Lepidoptera but also to the development of long-term preservation procedure of genetic resources by using cryopreserved sperm of Bombyx mori.     

BmPMFBP1 regulates the development of eupyrene sperm in the silkworm,Bombyx mori

Sperm deliver the male complement of DNA to the ovum, and thus play a key role in sexual reproduction. Accordingly, spermatogenesis has outstanding significance in fields as disparate as infertility treatments and pest-control, making it a broadly interesting and important focus for molecular genetics research in a wide range of species. Here we investigate spermatogenesis in the model lepidopteran insect Bombyx mori (silkworm moth), with particular focus on the gene PMFBP1 (polyamine modulated factor 1 binding protein 1). In humans and mouse, PMFBP1 is essential for spermatogenesis, and mutations of this gene are associated with acephalic spermatozoa, which cause infertility. We identified a B. mori gene labeled as “PMFBP1” in GenBank’s RefSeq database and sought to assess its role in spermatogenesis. Like in mammals, the silkworm version of this gene (BmPMFBP1) is specifically expressed in testes. We subsequently generated BmPMFBP1 mutants using a transgenic CRISPR/Cas9 system. Mutant males were sterile while the fertility of mutant females was comparable to wildtype females. In B. mori, spermatogenesis yields two types of sperm, the nucleated fertile eupyrene sperm, and anucleated unfertile apyrene sperm. Mutant males produced abnormal eupyrene sperm bundles but normal apyrene sperm bundles. For eupyrene sperm, nuclei were mislocated and disordered inside the bundles. We also found the BmPMFBP1 deficiency blocked the release of eupyrene sperm bundles from testes to ejaculatory seminalis. We found no obvious abnormalities in the production of apyrene sperm in mutant males, and double-matings with apyrene-deficient sex-lethal mutants rescued the ΔBmPMFBP1 infertility phenotype. These results indicate BmPMFBP1 functions only in eupyrene spermatogenesis, and highlight that distinct genes underlie the development of the two sperm morphs commonly found in Lepidoptera. Bioinformatic analyses suggest PMFBP1 may have evolved independently in lepidoptera and mammals, and that despite the shared name, are likely not homologous genes.     

Storage proteins in the silkworm,Bombyx mori

In the silkworm, Bombyx mori, two storage proteins named SP-1 and SP-2 were shown to decline in concentration in the haemolymph and increase in the fat body during the larval-pupal transformation, when protein granules are formed in the fat body at the same time as the degeneration of mitochondria and endoplasmic reticulum. At the larval-pupal ecdysis, in females the two proteins account for 60% of total fat body protein (80% of the soluble protein), while males have very little SP-1 and SP-2 comprises only 20% of the total fat body protein. The concentration of protein granules in the fat body cytoplasm is much greater in females than in males, and the granules in females have partially crystalline inner zones. This is different from males where granules with non-crystalline structure are most numerous.The properties of these proteins purified from pupal fat body are similar to those of Cecropia storage proteins and calliphorin, all of which have molecular weights of around 500,000 and are composed of subunits of mol. wt about 85,000. SP-1 differs from SP-2 by having an exceptionally high content of methionine, but much less glutamate, phenylalanine and tyrosine. SP-1 resembles another female-specific protein, vitellogenin and SP-2 resembles calliphorin in amino acid composition.From these results, it is concluded that SP-1 and SP-2 have storage roles and are deposited in protein granules.     

Functional analysis of Ultrabithorax in the silkworm, Bombyx mori, using RNAi

The formation of abdominal appendages in insects is suppressed by the Hox genes Ultrabithorax (Ubx) and abdominal-A (abd-A), but mechanisms of the suppression can differ among species. As the function of Ubx and abd-A has been described in only a few species, more data from various insects are necessary to elucidate the evolutionary transition of regulation on abdominal appendages. We examined the function of Ubx in the silkworm Bombyx mori (Bm-Ubx) by embryonic RNA interference (RNAi). This is the first case in which functional analysis for Ubx is performed in lepidopteran insects. Larvae treated with Bm-Ubx dsRNA displayed an additional pair of thoracic leg-like protuberances in A1, whereas the other abdominal segments had no transformation. Our results suggest that Bm-Ubx is a suppressor of leg development in A1.     

In vitro cultivation of spermatocysts to matured sperm in the silkworm Bombyx mori

Bombyx spermatogonia are bipotential, producing nucleate eupyrene sperm and anucleate apyrene sperm. An in vitro cultivation of spermatocysts of Bombyx mori from spermatocytes to matured sperm was established. The present experiment made clear that: (i) spermatocysts must be isolated; (ii) constant shaking at 45 r.p.m. was necessary; and (iii) the addition of Bombyx hemolymph (BH) was indispensable for successful cultivation. In the absence of BH, spermatogenesis proceeded normally for 2 or 3 days and, thereafter, spermatocytes and sperm bundles began to degenerate. The best results for normal eupyrene spermatogenesis were obtained when culture medium containing BH of the corresponding stage was used in every exchange of the medium at 72 h intervals. None or only a small number of apyrene sperm bundles was produced by this culture system when spermatocysts from larval testes were used, although eupyrene spermatogenesis proceeded normally to form matured, or squeezed, sperm bundles.     

Dietary sterol preference in the silkworm, Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that beta-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: beta-sitosterol > ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouth part might be different from the sterol recognition mechanism present in sterol metabolism.     

家蚕拟浆细胞具有吞噬功能

目的：通过观察家蚕Bombyx mori吞噬细胞的微细结构,来确定拟绛色细胞是否也具有吞噬功能。方法：用荧光小球微量注射家蚕pnd pS品系的幼虫,经荧光染色剂丫啶橙和碘化丙啶染色循环血细胞后,在荧光显微镜下观察并扫描拍摄。结果：观察发现除颗粒细胞和浆血细胞外,一些原血球细胞（血干细胞）和拟绛色细胞（多酚氧化酶）也能吞噬荧光小球。在拟绛色细胞里还发现许多和颗粒细胞一样的能被丫啶橙染色的颗粒。尽管在小球细胞中没有发现被吞噬的荧光小球,但该类血球有比较多的能被丫啶橙染色的大颗粒,这表明它们可能是已经被吞噬的凋亡小体。结论：除颗粒细胞和浆血细胞外,一些原血球细胞和拟绛色细胞也能吞噬荧光小球。说明拟绛色细胞也具有吞噬功能。     

家蚕Hox基因研究进展

Hox基因（homeobox genes）在昆虫躯体模式（body plan）的发育调控机制中扮演着重要角色,其表达具有严格的组织特异性和胚胎发育的程序性。家蚕Bombyx mori作为鳞翅目昆虫的代表,其Hox基因也陆续得到鉴定。在家蚕中存在一个拟复等位基因群--E群基因,其突变表型均与过剩斑纹和过剩附肢有关,这可能与Hox基因有着密切联系。家蚕全基因组测序完成后,发现其Hox基因簇中存在12个特有的homeobox基因（Bmshx1~Bmshx12）, 说明家蚕Hox基因可能具有独特的生物学意义。我们还利用家蚕基因芯片数据分析了Bmlab与Bmpb基因的组织表达特征。通过对家蚕Hox基因的研究,探索家蚕躯体模式建立机制,可望为解析其他鳞翅目昆虫的躯体模式的建立机制提供理论依据。本文就家蚕Hox基因的表达、功能及其与E群突变的关系等方面进行了综述。     

The G protein-coupled receptors in the silkworm,Bombyx mori

G protein-coupled receptors (GPCRs) are the largest and most versatile family of transmembrane receptors in the cell, occupying the highest hierarchical positions in the regulation of many physiological processes. Although they have been extensively studied in a number of model insects, there have been few investigations of GPCRs in large Lepidopterans, such as Bombyx mori, an organism that provides a means to perform detailed tissue expression analyses, which may help to characterize GPCRs and their ligands. In addition, B. mori, also known as the silkworm, is an insect of substantial economic importance, due to its use in silk production and traditional medicines. In this work, we computationally identified 90 putative GPCRs in B. mori, 33 of which represent novel proteins. These GPCRs were annotated and compared with their homologs in Drosophila melanogaster and Anopheles gambiae. Phylogenetics analyses of the GPCRs from these three insects showed that GPCRs may easily duplicate or disappear during insect evolution, especially in the neuropeptide and protein hormone receptor subfamily. Interestingly, we observed a decrease in the quantity and diversity of the stress-tolerance gene, Methuselah, in B. mori, which may be related to its long history of domestication. Moreover, the presence of many Bombyx-specific GPCRs suggests that neither Drosophila nor Anopheles is good representatives for the GPCRs in the Class Insecta.     

Peristaltic squeezing of sperm bundles at the late stage of spermatogenesis in the silkworm, Bombyx mori

Silkworm (Lepidoptera) males produce dimorphic sperm, nucleate eupyrene sperm, and anucleate apyrene sperm. The eupyrene sperm is the ordinary sperm fertilizing eggs, while the function of the apyrene sperm, which are about four times as numerous as the eupyrene sperm, is still uncertain. We found the peristaltic phenomenon at the very late stage of spermatogenesis. Peristalsis occurs in both eupyrene and apyrene sperm bundles. Through peristaltic action, cytoplasm of the eupyrene sperm and both cytoplasm and nuclei of the apyrene sperm are discarded from the posterior end of the sperm bundles. Peristaltic squeezing seems to be a process to eliminate the irregular nuclei of apyrene sperm while preserving the nuclei of eupyrene sperm.     

Sterol composition in larvae of the silkworm, Bombyx mori

Sterols in silkworm larvae were analyzed. Cholesterol was predominantly detected in all tissues examined. Dietary phytosterols and desmosterol, a putative biosynthetic intermediate from phytosterols to cholesterol, were also detected, indicating that imperfect intestinal conversion from phytosterols to cholesterol influences the sterol composition in larval tissues.     

The DNA content of sperm and hemocyte nuclei of the silkworm,Bombyx mori L.

To estimate the size of the haploid genome of the silkworm, Bombyx mori (Lepidoptera), amounts of Feulgen-DNA staining in individual nuclei of primary spermatocytes, spermatids, maturing sperm, and larval or pupal hemocytes were determined with an integrating microdensitometer and compared with the Feulgen-DNA levels found for chicken erythrocyte nuclei, or the sperm and erythrocyte nuclei of Xenopus laevis that were included with each Bombyx preparation as empirical reference standards of 2.5, 3.15, and 6.3×10^?12 g DNA per cell, respectively. Under these conditions, the haploid male genome of B. mori was estimated as 0.52±0.01 (S.E.)×10^?12 g DNA, corresponding to a molecular weight of roughly 3.1×10¹¹ daltons. From similar measurements of Feulgen-stained hemocyte nuclei, approximately 1.0±0.05 (S.E.)×10^?12 g DNA was estimated for the diploid or 2C male genome of Bombyx. These values compare favorably with estimates of genome size based upon analysis of the kinetics of reassociation of DNA isolated from B. mori and provide an independent basis for assessing the degree of polyploidy achieved by the giant nuclei in the posterior silk gland prior to its secretion of fibroin at the end of the fifth larval instar.     

Characterization of Tudor-sn-containing granules in the silkworm,Bombyx mori

The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs.     

D-Glutamic acid-induced muscle contraction in the silkworm, Bombyx mori

Agonists for muscle contraction in silkworms were screened by injecting test solutions into the hemolymph of decapitated silkworm larvae. Kainic acid, a glutamate receptor agonist, and D-glutamic acid induced muscle contractions, and D-aspartic acid was partially effective, whereas NMDA and AMPA, representative mammalian glutamate receptor agonists, did not induce contraction. L-Glutamic acid inhibited the kainic acid or D-glutamic acid-induced contraction. Amino acid analysis revealed that 3% of the total glutamic acid in the silkworm hemolymph is D-glutamic acid. These results suggest that d-glutamic acid acts physiologically as an agonist for muscle contraction in silkworms, and that L-glutamic acid functions as an inhibitor.     

Sperm-mediated gene transfer in the silkworm Bombyx mori

Plasmid DNA containing the CAT reporter gene was injected into the testis of V instar silkworm larvae. The persistence, expression, and transmission of the injected DNA were monitored in the injected individuals till eclosion as well as in the progeny. The DNA injected into the testis persisted extrachromosomally during the entire period of metamorphosis and was also transferred into the egg via sperm during fertilization. Injected plasmids were rescued from the moths that emerged from the injected larvae and also from the eggs laid by the moths that copulated with injected males. Positive signals for CAT assay in the experimental samples suggested that the injected DNA was internalized in the testicular cells and sperm. The persistence, expression, and transmission of the DNA injected into the testes indicate that sperm-mediated gene transfer is possible in the silkworm, Bombyx mori. Arch. Insect Biochem. Physiol. 37:168–177, 1998. © 1998 Wiley-Liss, Inc.     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

Dosage analysis of Z chromosome genes using microarray in silkworm,Bombyx mori

In many organisms, dosage compensation is needed to equalize sex-chromosome gene expression in males and females. Several genes on silkworm Z chromosome were previously detected to show a higher expression level in males and lacked dosage compensation. Whether silkworm lacks global dosage compensation still remains poorly known. Here, we analyzed male:female (M:F) ratios of expression of chromosome-wide Z-linked genes in the silkworm using microarray data. The expression levels of genes on Z chromosome in each tissue were significantly higher in males compared to females, which indicates no global dosage compensation in silkworm. Interestingly, we also found some genes with no bias (M:F ratio: 0.8–1.2) on the Z chromosome. Comparison of male-biased (M:F ratio more than 1.5) and unbiased genes indicated that the two sets of the genes have functional differences. Analysis of gene expression by sex showed that M:F ratios were, to some extent, associated with their expression levels. These results provide useful clues to further understanding roles of dosage of Z chromosome and some Z-linked sexual differences in silkworms.