A re-evaluation of silk measurement by the cecropia caterpillar (Hyalophora cecropia) during cocoon construction reveals use of a silk odometer that is temporally regulated.

The late 5th instar caterpillar of the cecropia silk moth (Hyalophora cecropia) spins a silken cocoon with a distinct, multilayered architecture. The cocoon construction program, first described by the seminal work of Van der Kloot and Williams, consists of a highly ordered sequence of events. We perform behavioral experiments to re-evaluate the original cecropia work, which hypothesized that the length of silk that passes through the spinneret controls the orderly execution of each of the discrete events of cocoon spinning. We confirm and extend by three-dimensional scanning and quantitative measurements of silk weights that if cocoon construction is interrupted, upon re-spinning, the caterpillar continues the cocoon program from where it left off. We also confirm and extend by quantitative measurements of silk weights that cecropia caterpillars will not bypass any of the sections of the cocoon during the construction process, even if presented with a pre-spun section of a cocoon spun by another caterpillar. Blocking silk output inhibits caterpillars from performing normal spinning behaviors used for cocoon construction. Surprisingly, unblocking silk output 24-hr later did not restart the cocoon construction program, suggesting the involvement of a temporally-defined interval timer. We confirm with surgical reductions of the silk glands that it is the length of silk itself that matters, rather than the total amount of silk extracted by individuals. We used scanning electron microscopy to directly show that either mono- or dual-filament silk (i.e., equal silk lengths but which vary in their total amount of silk extracted) can be used to construct equivalent cocoons of normal size and that contain the relevant layers. We propose that our findings, taken together with the results of prior studies, strongly support the hypothesis that the caterpillar uses a silk "odometer" to measure the length of silk extracted during cocoon construction but does so in a temporally regulated manner. We further postulate that our examination of the anatomy of the silk spinning apparatus and ablating spinneret sensory output provides evidence that silk length measurement occurs upstream of output from the spinneret.

Body Shape and Coloration of Silkworm Larvae Are Influenced by a Novel Cuticular Protein.

The genetic basis of body shape and coloration patterns on caterpillars is often assumed to be regulated separately, but it is possible that common molecules affect both types of trait simultaneously. Here we examine the genetic basis of a spontaneous cuticle defect in silkworm, where larvae exhibit a bamboo-like body shape and decreased pigmentation. We performed linkage mapping and mutation screening to determine the gene product that affects body shape and coloration simultaneously. In these mutant larvae we identified a null mutation in BmorCPH24, a gene encoding a cuticular protein with low complexity sequence. Spatiotemporal expression analyses showed that BmorCPH24 is expressed in the larval epidermis postecdysis. RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout of BmorCPH24 produced the abnormal body shape and the inhibited pigment typical of the mutant phenotype. In addition, our results showed that BmorCPH24 may be involved in the synthesis of endocuticle and its disruption-induced apoptosis of epidermal cells that accompanied the reduced expression of R&R-type larval cuticle protein genes and pigmentation gene Wnt1 Strikingly, BmorCPH24, a fast-evolving gene, has evolved a new function responsible for the assembly of silkworm larval cuticle and has evolved to be an indispensable factor maintaining the larval body shape and its coloration pattern. This is the first study to identify a molecule whose pleiotropic function affects the development of body shape and color patterns in insect larvae.

Sequential steps of macroautophagy and chaperone-mediated autophagy are involved in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori.

To elucidate the degradation process of the posterior silk gland during metamorphosis of the silkworm ITALIC! Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP) and after cocoon formation (CO) were used to analyze macroautophagy, chaperone-mediated autophagy (CMA) and the adenosine triphosphate (ATP)-dependent ubiquitin proteasome. Immediately after entering metamorphosis stage PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form II at all stages. The levels of ubiquitinated proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the posterior silk gland during metamorphosis.

Protruding structures on caterpillars are controlled by ectopic Wnt1 expression.

Spine-like or protruding structures, which may be aposematic for predators, are often observed in multiple segments of lepidopteran larvae (caterpillars). For example, the larvae of the Chinese wheel butterfly, Byasa alcinous, display many protrusions on their backs as a warning that they are toxic. Although these protrusions are formed by an integument lined with single-layered epidermal cells, the molecular mechanisms underlying their formation have remained unclear. In this study, we focused on a spontaneous mutant of the silkworm, Bombyx mori, Knobbed, which shows similar protrusions to B. alcinous and demonstrates that Wnt1 plays a crucial role in the formation of protrusion structures. Using both transgene expression and RNAi-based knockdown approaches, we showed that Wnt1 designates the position where epidermal cells excessively proliferate, leading to the generation of knobbed structures. Furthermore, in the B. alcinous larvae, Wnt1 was also specifically expressed in association with the protrusions. Our results suggest that Wnt1 plays a role in the formation of protrusions on the larval body, and is conserved broadly among diverse species in Lepidoptera.

Ras1(CA) overexpression in the posterior silk gland improves silk yield.

Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm, Bombyx mori, but has reached a plateau during the last decades. For the first time, we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%, while increasing food consumption by only 20%. Ras activation by Ras1(CA) overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins, up-regulated fibroin mRNA levels, increased total DNA content, and stimulated endoreplication. Moreover, Ras1 activation increased cell and nuclei sizes, enriched subcellular organelles related to protein synthesis, and stimulated ribosome biogenesis for mRNA translation. We conclude that Ras1 activation increases cell size and protein synthesis in the posterior silk gland, leading to silk yield improvement.

Influência da alimentação na morfologia da glândula sericígena de lagartas no 5º ínstar de Bombyx mori L. utilizando diferentes cultivares de amoreira / Influencia de la alimentación en la morfología de la glándula sericígena de lagartas en el 5º instar de Bómbix mori L utilizando diferentes cultivos de morera / Influence of the feeding in the morphology of the silk-producing gland of insects in 5º period of Bombyx mori L. using different mulberry cultivars

O bicho-da-seda, Bombyx mori L. (Lepidoptera: Bombycidae), é o inseto produtor de casulos de seda, destinados à fiação industrial têxtil, para posterior produção de tecidos de seda. Os filamentos de seda, expelidos pelas glândulas sericígenas do inseto, se solidificam ao entrar em contato com o ar. A sericicultura abrange a criação das lagartas de B. mori e a cultura da amoreira, principal fonte de alimentação da lagarta. Com o objetivo de avaliar a influência da alimentação na morfologia da glândula sericígena de lagartas de B. mori, realizou-se experimento em que foram fornecidos aos insetos quatro cultivares de amoreira, sendo estes a variedade Korin e os híbridos comerciais FM86, SK4 e IZ40. Os dados obtidos revelaram que, quanto ao peso da glândula sericígena, as lagartas alimentadas com SK4 e Korin apresentaram maior peso quando comparadas com os cultivares FM86 e IZ40. O híbrido SK4 revelou diferença significativa no comprimento da glândula posterior. Porém, os dados gerais apresentaram um certo equilíbrio entre os cultivares, perdendo em alguns aspectos e superando outros, quando comparados entre si.

El gusano de seda, Bombyx mori L. (Lepidoptera: Bombycidae), es el insecto productor de los capullos de seda, destinado a la hilatura industrial textil, para posterior producción de tejidos de seda. Los filamentos de seda, expulsados por las glándulas sericígenas del insecto, se solidifican al ponerse en contacto con el aire. La sericicultura abarca la creación de lagartas de B. mori y el cultivo de la morera, principal fuente de alimentación de la lagarta. Con el fin de evaluar la influencia de la alimentación en la morfología de la glándula sericígena de lagartas de B. mori, se realizó experimento en que fueron suministrados a los insectos cuatro cultivares de morera, siendo estos la variedad Korin y los híbridos comerciales FM86, SK4 y IZ40. Los datos obtenidos revelaron que, cuanto al peso de la glándula sericígena, las lagartas alimentadas con SK4 y Korin presentaron mayor peso en comparación con los cultivares FM86 y IZ40. El híbrido SK4 reveló diferencia significativa a lo largo de la glándula posterior. Sin embargo, los datos generales muestran un cierto equilibrio entre los cultivares, perdiendo en algunos aspectos y superando en otros, cuando comparados entre ellos.

The silkworm, Bombyx mori L. (Lepidoptera:Bombycidae), is the insect that produces cocoons of silk - the more important raw material for the silk industry. Silk filaments, which are expelled by the insect’s silk-producing gland, become solid in contact with air. Silk culture encloses the creation of silkworm and mulberry plant growing – its major source of food. With the objective of evaluating the influence of feeding in the morphology of the silk-producing gland of the B. mori worms, an experiment was conducted with four mulberry cultivars: Korin , FM86, SK4 e IZ40. The results revealed that with respect to wight of the silk-producing gland, the silkworms fed with SK4 and Korin presented higher weight when compared to the FM86 and IZ40 cultivars. SK4 presented significant difference with respect to the posterior gland length. However, general data presented certain balance among the cultivars when compared mutually.

Female-biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori.

Olfaction plays an important role in the life history of insects, including key behaviours such as host selection, oviposition and mate recognition. Odour perception by insects is primarily mediated by the large diverse family of odourant receptors (Ors) that are expressed on the dendrites of olfactory neurones housed within chemosensilla. However, few Or sequences have been identified from the Lepidoptera, an insect order that includes some of the most important pest species worldwide. We have identified 41 Or gene sequences from the silkworm (Bombyx mori) genome, more than double the number of published Or sequences from the Lepidoptera. Many silkworm Ors appear to be orthologs of the 17 published tobacco budworm (Heliothis virescens) Ors indicating that many Or lineages may be conserved within the Lepidoptera. The majority of the Or genes are expressed in adult female and male antennae (determined by quantitative real-time PCR analysis), supporting their probable roles in adult olfaction. Several Or genes are expressed at high levels in both male and female antennae, suggesting they mediate the perception of common host or conspecific volatiles important to both sexes. BmOrs 45-47 group together in the same phylogenetic branch and all three are expressed at moderate female-biased ratios, six to eight times higher in female compared to male moth antennae. Interestingly, BmOrs19 and 30 appear to be expressed predominantly in female antennae, opposite to that of the published silkworm pheromone receptors BmOrs 1 and 3 that are specific to male antennae. These results suggest that BmOr19 and 30 may detect odours critical to female behaviour, such as oviposition cues or male-produced courtship pheromones.

Bombyx Y-box protein BYB facilitates specific DNA interaction of various DNA binding proteins independently of the cold shock domain.

A new member of the Y-box protein family of the silkworm Bombyx mori (BYB) was co-purified with the fibroin gene enhancer-binding protein FMBP-1, and stimulated the binding of FMBP-1 to its cognate DNA element. However, the stimulatory effect was not specific to FMBP-1, BYB also enhancing the binding of mammalian transcription factors OTF2, SP1 and AP2 to their specific binding elements. Besides the above transcription regulatory factors, BYB facilitated the binding of basal transcription factor TBP, and enhanced transcription from the adenovirus 2 major late promoter in a reconstituted transcription system. Moreover, BYB stimulated the reactions of some restriction endonucleases under cold conditions. The C-terminal region of BYB was sufficient for these stimulatory effects, and the highly conserved cold shock domain (CSD) in the N-terminal region was dispensable. GST-pull down experiments showed that the C-terminal region could interact with DNA independently of the CSD. The above results suggest that the C-terminal region of BYB causes the active interaction of various DNA binding proteins with their targets. Such a function of the C-terminal region of BYB may partly explain the functional diversity of Y-box proteins.

Roles of actin networks in peristaltic squeezing of sperm bundles in Bombyx mori.

Two types of sperm are produced in the silkworm, Bombyx mori. Nucleate eupyrene sperm is an ordinary sperm that contributes to fertilization, while anucleate apyrene sperm is considered to play important roles in assisting eupyrene sperm. At the very late stage of spermatogenesis, a phenomenon called "peristaltic squeezing" occurs in both types of sperm, whereby cytoplasm of the eupyrene and nuclei of the apyrene sperm are discarded from the posterior end, forming matured sperm. In this study, rhodamine-phalloidin staining for actin was applied to sperm bundles. Before the start of peristaltic squeezing, actin filament networks are spread on the cyst cells and constrictions by the networks appear in several places of the bundles. Actin particles, which are later recognized as circlets, are localized within the bundles. Squeezing action by the networks occurs from the anterior region and transfers toward the posterior, eliminating cytoplasm together with circlets from the posterior end. It seems that actin filaments contribute to the peristaltic squeezing of the sperm bundles in Bombyx mori.

Spatio-temporal expression of wnt-1 during embryonic-, wing- and silkgland development in Bombyx mori.

A homologue of the segment polarity gene wnt-1 from Bombyx mori (Bmwnt-1) has been characterized. The segmentally reiterated pattern of Bmwnt-1 transcrip9t distribution in B. mori embryos suggested its segment polarity function. Maximal levels of Bmwnt-1 RNA during embryonic development were reached by stage 21A. In the larval stages, Bmwnt-1 was expressed in the fore- and hindwing discs, ovaries, testes and gut, reminiscent of the expression domains in Drosophila. Bmwnt-1 was expressed in the wing-margin area of both the fore- and hindwing discs. The pattern of wnt-1 expression in the hindwing discs was similar to that in the butterfly Precis coenia but subtle differences existed in forewing discs of the two species, which correlated well with the absence of proximal bands of pigmentation in the adult Bombyx wings. In addition, Bmwnt-1 was expressed in the silkglands and the expression was confined to the anterior sub-compartment within the middle silkglands throughout development from the embryonic to late larval stages. This domain of Bmwnt-1 expression overlapped with those of Cubitus interruptus (BmCi) and sericin-2 but excluded the Engrailed expression domain viz. the middle and posterior sub-compartments of middle silkglands. Bmwnt-1 expression was detected only during the intermoults and not in the moulting periods.

Ultrastructural studies on the pheromone-producing cells in the silkmoth, Bombyx mori: formation of cytoplasmic lipid droplets before adult eclosion.

In Bombyx mori, pheromone-producing cells accumulate a number of lipid droplets in the cytoplasm preceding the production of the sex pheromone, bombykol. The process of lipid droplet formation in the pheromone-producing cells was investigated by using light and electron microscopy. Light microscopy revealed that the lipid droplets appeared from 2 days before adult eclosion and dramatic accumulation took place between 2 days and 1 day before eclosion. Electron microscopical studies revealed that smooth endoplasmic reticulum and numerous vesicles, their sizes being less than 1 microm, were detectable 2 days before eclosion, and some vesicles were fused with mitochondria at this stage. These characteristic changes in the pheromone-producing cells suggest that fatty acyl-CoA synthesis following de novo fatty acid synthesis takes place at this time. Involutions in the basal plasma membrane of the cells occurred throughout the observed period, which were extensive on the day before adult eclosion. Besides extensive basal involutions, immature lipid droplets appeared and then mature fully electron-dense lipid droplets were observed on the day of adult eclosion. These ultrastructural observations, combined with recent physiological studies suggest, that the basal involutions presumably reflect the uptake of lipidic components required for the construction of lipid droplets, the function of which is to store the bombykol precursor and to provide it for bombykol biosynthesis in response to pheromonotropic stimuli by pheromone biosynthesis activating neuropeptide (PBAN).

The wings of Bombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report.

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm, Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development in B. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike in Drosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adult Drosophila wings. Partially excised wing discs did not show in situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early in B. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades in B. mori unlike evagination of only the pouch region as wing blades seen in Drosophila.

Effect of estradiol-17beta on cell area, lumen area and trehalase activity of posterior silk gland of Bombyx mori L.

Estradiol-17beta (E2) at the dose of 1 microg/g caused an increase in cell area, lumen area and the total (cell + lumen) area of posterior silk gland (PSG) in Bombyx mori indicating that exogenously applied estradiol-17beta has a regulatory influence on silk gland activity. A dose-dependent variation in trehalase activity of PSG was found on the 5th day after topical administration of estradiol on 1st and 2nd day of the fifth larval instar. Of all the doses of E2 used, 1 microg/g dose had maximum stimulatory effect on trehalase activity. Co-administration of each of a specific receptor antagonist for estradiol, the ICI-182780 and a protein biosynthetic blocker, cycloheximide with E2 suppressed the E2-induced increase in silk gland activity. The results suggest some specific metabolic action of E2 on silk gland and offer a promising way for future investigations regarding the physiological significance of vertebrate steroids in insects.

Heterogeneity of odorant-binding proteins in the antennae of Bombyx mori.

Different odorant-binding proteins (OBPs) were isolated from total antennal homogenates of male and female Bombyx mori. Proteins were separated according to their isoelectric point by using preparative fast-flow isoelectrofocusing. Odorant-binding proteins were identified in immunoblots by antisera raised against the pheromone-binding protein (anti-PBP) and the general odorant-binding protein (anti-GOBP2) of Antheraea polyphemus. Four proteins cross-reacting with anti-PBP were detected in males and two in females, while three proteins cross-reacting with anti-GOBP2 were found in males and five in females. Both anti-PBP and anti-GOBP2 cross-reacting proteins had an apparent molecular weight of 15-16 kDa. In parallel, the same two antisera were used in immunocytochemical studies in order to determine the distribution of these proteins within the various subtypes of olfactory sensilla. The presence of multiple odorant-binding proteins within one moth species as well as their complex distribution pattern support the suggestion that soluble OBPs might have a function in odorant discrimination.

Antennal-specific pheromone-degrading aldehyde oxidases from the moths Antheraea polyphemus and Bombyx mori.

Female moths produce blends of odorant chemicals, called pheromones. These precise chemical mixtures both attract males and elicit appropriate mating behaviors. To locate females, male moths must rapidly detect changes in environmental pheromone concentration. Therefore, the regulation of pheromone concentration within antennae, their chief organ of smell, is important. We describe antennal-specific aldehyde oxidases from the moths Antheraea polyphemus and Bombyx mori that are capable of catabolizing long chain, unsaturated aldehydes such as their aldehyde pheromones. These soluble enzymes are associated uniquely with male and female antennae and have molecular masses of 175 and 130 kDa, respectively. The A. polyphemus aldehyde oxidase has been localized to the olfactory sensilla which contain the pheromone receptor cell dendrites. These same sensilla contain a previously described sensilla-specific esterase that degrades the acetate ester component of A. polyphemus pheromone. We propose that sensillar pheromone-degrading enzymes modulate pheromone concentration in the receptor space and hence play a dynamic role in the pheromone-mediated reproductive behaviors of these animals.

An accessory olfactory pathway in Lepidoptera: the labial pit organ and its central projections in Manduca sexta and certain other sphinx moths and silk moths.

In the hawkmoth, Manduca sexta, the third segment of each labial palp contains a pit, which houses a densely packed array of sensilla. We have named this structure the labial pit organ (LPO). The sensilla within the pit are typical of olfactory receptors, characterized by a grooved surface, wall pores, and pore tubules. Axons arising from receptor cells that innervate these sensilla project bilaterally to a single glomerulus in each antennal lobe. We have compared this central projection with that in three other species of Manduca (M. quinquemaculata, M. dilucida, and M. lanuginosa) and in the silkmoths Antheraea polyphemus and Bombyx mori. A bilateral projection to a single glomerulus in each antennal lobe is present in all cases. We suggest that the LPO serves as an accessory olfactory organ in adult Lepidoptera.

Pheromone binding and inactivation by moth antennae.

The antennae of male silk moths are extremely sensitive to the female sex pheromone such that a male moth can find a female up to 4.5 km away. This remarkable sensitivity is due to both the morphological and biochemical design of these antennae. Along the branches of the plumose antennae are the sensilla trichodea, each consisting of a hollow cuticular hair containing two unbranched dendrites bathed in a fluid, the receptor lymph ,3. The dendrites and receptor lymph are isolated from the haemolymph by a barrier of epidermal cells which secreted the cuticular hair. Pheromone molecules are thought to diffuse down 100 A-wide pore tubules through the cuticular wall and across the receptor lymph space to receptors located in the dendritic membrane. To prevent the accumulation of residual stimulant and hence sensory adaptation, the pheromone molecules are subsequently inactivated in an apparent two-step process of rapid 'early inactivation' followed by much slower enzymatic degradation. The biochemistry involved in this sequence of events is largely unknown. We report here the identification of three proteins which interact with the pheromone of the wild silk moth Antheraea polyphemus: a pheromone-binding protein and a pheromone-degrading esterase, both uniquely located in the pheromone-sensitive sensilla; and a second esterase common to all cuticular tissues except the sensilla.

Initiation of fibroin biosynthesis. I. Isolation of nascent fibroin peptides from the posterior silk gland of Bombyx mori.

1. Peptidyl-tRNA was prepared from the posterior silk gland ribosomes of Bombyx mori on the fourth to fifth days of the fifth instar to explore the initiation process in fibroin biosynthesis. 2. The peptidyl-tRNA was hydrolyzed at an alkaline pH and the resulting nascent peptides were fractionated on Sephadex G-75 and Sephadex G-200 columns into twelve fractions. Each fraction was analyzed for amino acid composition. 3. The nascent peptides of smaller molecular size were rather rich in glutamic and aspartic acids. However, the amino acid composition of the nascent peptides gradually approached that of fibroin as their molecular size increased. 4. A comparison between the nascent peptides of smaller molecular size and the small subunit of fibroin was made in respect to amino acid composition and tryptic peptide map. Considerable similarity between these two proteins was observed. The implications of these results in relation to the initiation process in fibroin biosynthesis are discussed.