Normal and cocoon-forming peritrophic membrane in larvae of the beetle Gibbium psylloides

Larvae of Gibbium psylloides secrete a peritrophic membrane (PM) which has a mainly orthogonal fibrillar structure, but this merges freely with hexagonal and random arrangements of microfibres. The random arrangement predominates in PM used for cocoons. Shortly before the cocoon is constructed the PM no longer forms a tube but collapses and is compressed in the gut and emerges from the anus as a flat thread. This thread is wound around the larva and forms the cocoon ‘silk’. Both normal and cocoon-forming PMs are produced mainly in the posterior midgut and their constituent microfibres appear first at the tips of the microvilli which form the brush border of the midgut cells.     

The fine structure of peritrophic membranes of the blowfly, Calliphora erythrocephala, grown in vitro under different conditions.

The formation of peritrophic membranes (PM) in vitro was studied in a flow chamber in order to avoid the accumulation of metabolic substances during prolonged incubation. During the first 8 to 10 hr of incubation the production of PM was nearly constant—3·5 ± 1·4 mm PM/hr. After about 10 hr it decreased and stopped after about 35 hr. Between 1 and 8 hr after the beginning of incubation the width of the periodic crossband pattern reached or nearly reached the values found in PM which had formed in vivo; afterwards it decreased more and more. During the first 20 min of incubation a ‘disturbed zone’ of PM without any regular crossband pattern is formed.In the cardia of adult Calliphora erythrocephala there are three formation zones forming three PM of different fine structures. The fine structure of PM 1 to 3 formed in vitro during the first 6 to 8 hr of incubation in Leloup's medium 1, with an osmolarity of 340 mOsmol, a pH of 6·8, and a temperature of 27°C, does not differ from the PM grown in vivo. PM 1–3 grown in vitro in Tyrode's solution with added glutamine or in Leloup's medium with added β-ecdysone show a considerable increase in thickness and a disturbed formation of the electron dense layer of PM 1.     

Presence of Culturable Bacteria in Cocoons of the Earthworm Eisenia fetida

Viable bacteria were found to coexist with developing embryos in egg capsules (cocoons) of the earthworm Eisenia fetida. Earthworms were reared under standardized conditions, and bacterial densities were measured in distinct batches of cocoons collected weekly for 10 weeks. Cocoons weighing 12 mg contained a mean viable bacterial population of approximately 10⁸ CFU/g of cocoons. No difference was found in viable counts obtained from cocoons incubated at 15°C and cocoons incubated at 24°C. Viable bacterial numbers increased with cocoon age, while acridine orange direct counts of microbial cells were stable at approximately 10⁹ cells per g of cocoons. Bacteria isolated from cocoons were used to develop antisera in rabbits for the production of strain-specific fluorescent antibodies. Fluorescent antibody and selective plating techniques were used to monitor populations of these bacteria in earthworm bedding and to determine whether cocoons acquire bacteria from the environment in which they are formed. Cocoon isolates were readily recovered from cocoons formed in inoculated bedding at densities of 10⁸ CFU/g of cocoons. Bradyrhizobium japonicum USDA 110 and UMR 161 added to bedding were also recovered from cocoons, but at lower densities than cocoon isolates. Escherichia coli K-12(pJP4) inoculum was recovered from bedding but not from cocoons. The bacterial complement of Eisenia fetida cocoons is affected by inoculation of selected bacterial isolates in the worm growth environment.     

Mechanism of fluorescent cocoon sex identification for silkworms Bombyx mori

By using silkworms, Bombyx mori, fluorescent cocoon sex identification (FCSI) as an experimental material, direct fluorescence spectrometry of the cocoon surface indicates that the fluorescent color of silkworm cocoons is made up of two peaks of yellow and blue-purple fluorescence emission. The fluorescent difference between male and female cocoons is attributed to the differential absorption of yellow fluorescent substances by the midgut tissue of 5th instar female silkworms. Thin layer chromatography (TLC) and fluorescent spectra indicate that blue-purple fluorescent substances are composed of at least five blue-purple fluorescent pigments, and yellow fluorescent substances are made up of at least three. UV spectra and AlCl₃ color reaction show that the three fluorescent yellow pigments are flavonoids or their glycosides. Silkworm FCSI is due to selective absorption or accumulation of the yellow fluorescent pigments by the posterior midgut cells of female 5th instar larvae. The cells of the FCSI silkworm midgut, especially the cylinder intestinal cells of the posterior midgut have a component which is a yellow fluorescent pigment-specific binding protein that may be vigorously expressed in the 5th instar larvae.     

Metabolic Engineering of a Glycerol-Oxidative Pathway in Lactobacillus panis PM1 for Utilization of Bioethanol Thin Stillage: Potential To Produce Platform Chemicals from Glycerol

Lactobacillus panis PM1 has the ability to produce 1,3-propanediol (1,3-PDO) from thin stillage (TS), which is the major waste material after bioethanol production, and is therefore of significance. However, the fact that L. panis PM1 cannot use glycerol as a sole carbon source presents a considerable problem in terms of utilization of this strain in a wide range of industrial applications. Accordingly, L. panis PM1 was genetically engineered to directly utilize TS as a fermentable substrate for the production of valuable platform chemicals without the need for exogenous nutrient supplementation (e.g., sugars and nitrogen sources). An artificial glycerol-oxidative pathway, comprised of glycerol facilitator, glycerol kinase, glycerol 3-phosphate dehydrogenase, triosephosphate isomerase, and NADPH-dependent aldehyde reductase genes of Escherichia coli, was introduced into L. panis PM1 in order to directly utilize glycerol for the production of energy for growth and value-added chemicals. A pH 6.5 culture converted glycerol to mainly lactic acid (85.43 mM), whereas a significant amount of 1,3-propanediol (59.96 mM) was formed at pH 7.5. Regardless of the pH, ethanol (82.16 to 83.22 mM) was produced from TS fermentations, confirming that the artificial pathway metabolized glycerol for energy production and converted it into lactic acid or 1,3-PDO and ethanol in a pH-dependent manner. This study demonstrates the cost-effective conversion of TS to value-added chemicals by the engineered PM1 strain cultured under industrial conditions. Thus, application of this strain or these research findings can contribute to reduced costs of bioethanol production.     

Rate of cocoon production by the larva of the fly Rhynchosciara americana and storage sites of precursors

A chemical analysis of Rhynchosciara americana cocoons at four stages of spinning was performed and the amount and rate of the cocoon production was measured. These results, together with the amount and distribution of the nutrient reserves in the larva during spinning, were used to identify the possible storage sites of cocoon precursors. The physiology, mechanics, and regulation of spinning by R. americana are discussed.     

Differentiation of mid-gut in adults and over-wintering copepodids of Calanus finmarchicus (Gunnerus) and C. helgolandicus Claus

The digestive enzyme content and the fine structure of the mid-gut in different developmental stages and generations of Calanus finmarchicus (Gunnerus) and C. helgolandicus Claus have been investigated. A reduced epithelium and low digestive enzyme activities were found in the over-wintering copepodids and males collected in the spring, whereas the corresponding females, and especially the summer adults, had higher enzyme activities. This is discussed in respect to the special physiological condition of the over-wintering stage. The enzyme content can be correlated with the structural characteristics of the glandular part of the mid-gut: high enzyme activities are accompanied by a more developed mid-gut epithelium, which is expressed in a larger cellular volume and in the possession of a large number of vacuolar cells (B-cells). In addition, more cell types are found in the glandular part of the mid-gut in the stages that display higher enzyme activities.     

Characterisation of an anaerobic symbiont and the associated aerobic bacterial flora of Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

A Gram-positive anaerobic bacterium was regularly found in great numbers in histological sections and anaerobic cultures of the third bulbous mid-gut portion of Pyrrhocoris apterus (Linné 1758). It formed long chains of irregular pear-shaped cells with large spherical involutions. The fermentation products were L-lactic acid, ethanol and acetic acid, CO₂ and H₂. The peptidoglycan belongs to the Lys-Asp type. The G + C content was determined to be around 61 mol%. The organism is supposed to be a symbiont of P. apterus and to be a species of a new genus.In the aerobic cultures Streptococcus lactis, an as yet not classified Streptococcus of the serological group D and Hafnia alvei were isolated from the third part of the mid-gut.     

Regulatory properties of 6-phosphofructo-1-kinase of the mid-gut of the grasshopper,Poekilocerus bufonius

The fine structure of the mid-gut of Poekilocerus bufonius has been examined and three types of epithelial cells were identified; normal epithelial cells with their apical part possessing well developed microvilli, goblet-like cells containing myelin-like figures and the small basal cells with small and round nuclei, nidi. The regulation of 6-phosphofructo-1-kinase (PFK-1) prepared from the mid-gut of the grasshopper, Poekilocerus bufonius, was studied. Mid-gut PFK-1 displayed cooperativity with respect to fructose-6-phosphate at pH 7.0, and the enzyme was inhibited by high concentrations of ATP. The affinity of the enzyme for fructose-6-phosphate was increased by fru-2,6-P₂ whereas the inhibition of the enzyme by high concentrations of ATP was relieved by fru-2,6-P₂. The activity of mid-gut PFK-1 was highly stimulated in a simultaneous presence of low concentrations of fru-2,6-P₂ and AMP. ADP, AMP and c-AMP were all shown to be activators of the mid-gut PFK-1 with AMP being the greatest effector. The enzyme was not inhibited by citrate either in the presence of low or high concentrations of ATP. These results suggest that the PFK-1 of the mid-gut of the grasshopper is highly regulated with positive stimulators, specially fru-2,6-P₂, whereas the enzyme is not regulated by citrate or glucose-1,6-bisphosphate.     

The larval silk of Hypera spp. (Coleoptera: Curculionidae). A new example of the cross-β protein conformation in an insect silk

X-ray diffraction studies on the larval cocoon silk of the weevils Hypera postica and H. rumicis (Coleoptera: Curculionidae) indicate that it is a new example of the cross-β conformation in proteins. This silk is secreted by Malpighian tubules and stored in the rectum. Chemically it is more complex, with a greater range of constituent amino acids, than Chrysopa (Neuroptera) silk (the first well-documented example of a naturally occurring cross-β protein). There is a relatively high proline content, an unusual feature for an arthropod silk. X-ray data, and direct measurement of dispersed ribbons, suggest that the micellar width is about 3.0 compared to 2.5 nm in Chrysopa silk. This latter silk is the ‘type structure’ for naturally occurring cross-β protein conformations. Hypera silk can be considered the ‘next one up’ in a possible hierarchy of cross-β structures.     

Chitin is a necessary component to maintain the barrier function of the peritrophic matrix in the insect midgut

In most insects, the peritrophic matrix (PM) partitions the midgut into different digestive compartments, and functions as a protective barrier against abrasive particles and microbial infections. In a previous study we demonstrated that certain PM proteins are essential in maintaining the PM's barrier function and establishing a gradient of PM permeability from the anterior to the posterior part of the midgut which facilitates digestion (Agrawal et al., 2014). In this study, we focused on the effects of a reduction in chitin content on PM permeability in larvae of the red flour beetle, Tribolium castaneum. Oral administration of the chitin synthesis inhibitor diflubenzuron (DFB) only partially reduced chitin content of the larval PM even at high concentrations. We observed no nutritional effects, as larval growth was unaffected and neutral lipids were not depleted from the fat body. However, the metamorphic molt was disrupted and the insects died at the pharate pupal stage, presumably due to DFB's effect on cuticle formation. RNAi to knock-down expression of the gene encoding chitin synthase 2 in T. castaneum (TcCHS-2) caused a complete loss of chitin in the PM. Larval growth was significantly reduced, and the fat body was depleted of neutral lipids. In situ PM permeability assays monitoring the distribution of FITC dextrans after DFB exposure or RNAi for TcCHS-2 revealed that PM permeability was increased in both cases. RNAi for TcCHS-2, however, led to a higher permeation of the PM by FITC dextrans than DFB treatment even at high doses. Similar effects were observed when the chitin content was reduced by feeding DFB to adult yellow fever mosquitos, Aedes aegypti. We demonstrate that the presence of chitin is necessary for maintaining the PM's barrier function in insects. It seems that the insecticidal effects of DFB are mediated by the disruption of cuticle synthesis during the metamorphic molt rather than by interfering with larval nutrition. However, as DFB clearly affects PM permeability, it may be suitable to increase the efficiency of pesticides targeting the midgut.     

Reproductive biology of the invasive Asian freshwater leech Barbronia weberi (Blanchard, 1897)

Barbronia weberi were collected from Hydrilla verticillata purchased from aquarium suppliers in Melbourne, Australia and additional B. weberi were obtained from aquatic plants in Canberra, ACT, Australia. Adult leeches fed Tubifex sp. ad libitum successfully reproduced under laboratory conditions (21 ± 5?°C ). Reproductively mature B. weberi produced cocoons every week for up to three months at which time the adults began to senesce. Cocoons contained one to five (barx=2.41±0.78 SD, N=58) eggs with juveniles leaving the cocoon within 27 ± 3.4 SD days (N=13) of cocoon production. Juvenile B. weberi reached reproductive maturity within four months (N=7) of hatching and had a maximum growth rate of 1.10 mm² d^?1 once they left the cocoon at around 30 days. Individual egg volume (r=0.63, scaling exponent = 1.21) and cocoon volume (r=0.65, scaling exponent = 1.24) showed a significant (P<0.001) and nearly isometric relationship when scaled with maternal body size. Because this species can grow rapidly and produce a large number of eggs over a short period of time and can piggyback with plant species and travel through the aquarium trade, there is potential for B. weberi to rapidly invade new localities.     

Polymerization of tubulin in the presence of colchicine or podophyllotoxin. Formation of a ribbon structure induced by guanylyl-5'-methylene diphosphonate

GTP-dependent in vitro polymerization of rat brain microtubular protein is inhibited to 50% by substoichiometric concentrations of the antimitotic drugs colchicine (0.12 mol/mol of tubulin) and podophyllotoxin (0.14 mol/mol of tubulin). Substitution of pp(CH₂)pG² for GTP, however, results in an extensive microtubular protein polymerization at such concentrations. In the presence of pp(CH₂)pG, suprastoichiometric concentrations of podophyllotoxin (19 mol/mol of tubulin) are required to inhibit the polymerization process by 50%. Colchicine is very ineffective since 3 × 10⁵ moles/mole of tubulin are required to give a 50% inhibition. Electron microscopical analysis shows that the polymers formed by microtubular protein in the presence of suprastoichiometric concentrations of drugs are not the normal short microtubules typical of pp(CH₂)pG-driven polymerization, but are ribbons with three or four protofilaments. The colchicine content of the harvested ribbons has been measured directly and found to be approximately 0.8 moles colchicine/mole of tubulin. Treatment of microtubular protein with substoichiometric concentrations of drugs results in an increase in the number of protofilaments forming the ribbons. Many of the ribbons can close into morphologically normal microtubules when microtubular protein is treated with only 0.05 moles of either colchicine or podophyllotoxin per mole of tubulin.     

Ultrastructure of posterior silk gland cells and liquid silk in Indian tasar silkworm,Antheraea mylitta drury (Lepidoptera : Saturniidae)

The ultrastructural characteristics of the posterior silk glands of the mature Antheraea mylitta (Lepidoptera : Saturniidae) larvae were clarified. Fibroin globules containing a small dense mass of fibroin fibers are produced in Golgi vacuoles, and released from the apical surface into the lumen by exocytosis. Bundles of microfilaments, which serve as a dynamic skeleton, are well developed. Numerous autophagosomes originating; from mitochondria accumulate in both basal and apical ends of the gland cell; the degenerated materials are released in the basement membrane and into the gland lumen, respectively. These materials invade the central fibroin column, leaving digested vacuoles in the fibroin cocoon filament. Ours may be the first finding of this rare phenomenon in which the degenerated lysosomes originated from mitochondria in both liquid silk in the lumen and cocoon filament.     

Agro-industrial wastes as carriers for bacterial inoculants

Peat is an ideal carrier for Rhizobium and Azotobacter, but high-quality peat is not abundantly and readily available in India. The experiments have shown the usefulness of indigenously available agro-industrial wastes such as dehydrated powdered bagasse with traces of mahua (Madhuca latifolia) cake and silk cocoon waste as substrates for the production of bioinoculants.     

Proteins in the Cocoon of Silkworm Inhibit the Growth of Beauveria bassiana

Silk cocoons are composed of fiber proteins (fibroins) and adhesive glue proteins (sericins), which provide a physical barrier to protect the inside pupa. Moreover, other proteins were identified in the cocoon silk, many of which are immune related proteins. In this study, we extracted proteins from the silkworm cocoon by Tris-HCl buffer (pH7.5), and found that they had a strong inhibitory activity against fungal proteases and they had higher abundance in the outer cocoon layers than in the inner cocoon layers. Moreover, we found that extracted cocoon proteins can inhibit the germination of Beauveria bassiana spores. Consistent with the distribution of protease inhibitors, we found that proteins from the outer cocoon layers showed better inhibitory effects against B. bassiana spores than proteins from the inner layers. Liquid chromatography-tandem mass spectrometry was used to reveal the extracted components in the scaffold silk, the outermost cocoon layer. A total of 129 proteins were identified, 30 of which were annotated as protease inhibitors. Protease inhibitors accounted for 89.1% in abundance among extracted proteins. These protease inhibitors have many intramolecular disulfide bonds to maintain their stable structure, and remained active after being boiled. This study added a new understanding to the antimicrobial function of the cocoon.     

Effects of relative humidity on cocoon formation and survival in the braconid wasp Cotesia glomerata

Abstract The effects of relative humidity (RH) on cocoon formation and survival in the braconid parasitoid wasp Cotesia glomerata (L.) (Hymenoptera: Braconidae) are investigated under various humidity conditions (50, 75, 90, 95 and 100% RH) at 20 °C and under an LD 16 : 8 h photoperiod. The mortality rate at the time of egression from hosts under 100% RH is significantly higher than for other RHs. Cocoon clusters formed at 100% RH spread significantly more than those formed at 50, 75, or 90% RH. Developmental periods differ significantly among RHs under which wasps developed. The mean period from the egression from hosts to adult emergence is 8.7 days when developed at 50–95% RHs, and 8.0 days at 100% RH. The emergence rates of C. glomerata that are maintained under the same humidity conditions after egression from hosts are not significantly different among RHs. However, emergence rates from cocoons that are transferred from 100% RH to 50 and 75% RH are < 70%, although the rates are > 90% in most cases. Some wasps do not emerge from cocoons: more than 60% die after adult eclosion at all RHs; the relative frequency of adult deaths is approximately 90% at 50% RH. Relative humidity influences the cluster and cocoon status strongly: both good clusters and cocoons are formed at low RHs. Emergence rates from cocoons of different ranks are significantly different: the rates of low‐rank cocoons are low at low RHs. The survival of C. glomerata is affected strongly by RH through cocoon formation.