Induction of antibacterial protein synthesis by soluble peptidoglycan in isolated fat body from larvae of the silkworm, Bombyx mori

Synthesis and secretion of bactericidal protein (cecropin) and lysozyme were induced by soluble peptidoglycan fragments (SPG) from Escherichia coli in a culture of fat body from Bombyx mori larvae. The rate of the secretion by fat body increased as a function of SPG concentration added to the culture medium. The induction of bactericidal activity was specific for peptidoglycan of a particular structure. Thus, SPG from Micrococcus luteus was 500-times less potent than E. coli SPG, and various glucans and peptides structurally related to peptidoglycan were all ineffective as elicitor. These results support the hypothesis that bacteria invading the haemocoel have to be partially degraded to generate peptidoglycan fragments as a signal molecule, which subsequently acts on a receptor on fat body cells and induces antibacterial protein synthesis.     

Fractionation of Different Life Cycle Stages of Microsporidia Nosema grylli from Crickets Gryllus bimaculatus by Centrifugation in Percoll Density Gradient for Biochemical Research

ABSTRACT. A new method of fractionation and purification of different life cycle stages of microsporidia Nosema grylli , parasitizing the fat body of cricket Gryllus bimaculatus , by centrifugation in Percoll density gradient is elaborated. The whole procedure can be summarized as: 1) infected fat body preparation, 2) homogenization in buffer and filtration through cotton wad and filter paper, 3) first centrifuging, resulting in the separation of the pellet into three layers containing different life cycle stages, 4) second centrifuging of the chosen layer in Percoll density gradient, 5) washing out the Percoll from the fraction under study. After centrifugation in Percoll density gradient, meronts and early sporonts form a band in the area corresponding to density 1.016 g/ml. Mature spores form the pellet at the bottom of centrifuge tube, while immature spores are distributed throughout the layer of 1.016 g/ml up to the bottom of the centrifuge tube, according to their buoyant densities. The offered technique is simple, it takes about one hour and may become a routine procedure for biochemical studies on microsporidia.     

Transmission and infectivity of spores of Burenella dimorpha (Microsporida: Burenellidae)

Burenella dimorpha infects the tropical fire ant, Solenopsis geminata, producing two morphologically distinct types of spores. A binucleate, nonpansporoblast membrane-bounded (NPMB) spore develops in and destroys the hypodermis, rupturing the cuticle in the pupal stage. A uninucleate, pansporoblast membrane-bounded (PMB) spore develops in the fat body. Adult ants cannibalize ruptured pupae but do not ingest spores. Instead, the spores and particulate foods are diverted to the infrabuccal cavity, formed into an infrabuccal pellet, and fed to fourth-instar larvae only. This larval instar is the only stage in the life cycle of S. geminata that is vulnerable to infection. NPMB spores are infective, but PMB spores do not extrude their polar filaments in the larval gut and are expelled in the meconium upon pupation.     

Light and electron microscope study of Thelohania solenopsae n. sp. (Microsporida: Protozoa) in the red imported fire ant,Solenopsis invicta

A new species of Microsporida, Thelohania solenopsae, is described from the red imported fire ant, Solenopsis invicta. The Thelohania infections are localized in the fat body of workers. Meronts causing infections of progeny are found in the ovaries of queens. Spores occur only in adult ants and only vegetative stages are present in larvae and pupae. Both uninucleate octospores (eight spores within a pansporoblast membrane) and binucleate free spores (spores developing in isolation) are formed.     

Evaluation of malacosporean life cycles through transmission studies

Myxozoans, belonging to the recently described Class Malacosporea, parasitise freshwater bryozoans during at least part of their life cycle, but no complete malacosporean life cycle is known to date. One of the 2 described malacosporeans is Tetracapsuloides bryosalmonae, the causative agent of salmonid proliferative kidney disease. The other is Buddenbrockia plumatellae, so far only found in freshwater bryozoans. Our investigations evaluated malacosporean life cycles, focusing on transmission from fish to bryozoan and from bryozoan to bryozoan. We exposed bryozoans to possible infection from: stages of T. bryosalmonae in fish kidney and released in fish urine; spores of T. bryosalmonae that had developed in bryozoan hosts; and spores and sac stages of B. plumatellae that had developed in bryozoans. Infections were never observed by microscopic examination of post-exposure, cultured bryozoans and none were detected by PCR after culture. Our consistent negative results are compelling: trials incorporated a broad range of parasite stages and potential hosts, and failure of transmission across trials cannot be ascribed to low spore concentrations or immature infective stages. The absence of evidence for bryozoan to bryozoan transmissions for both malacosporeans strongly indicates that such transmission is precluded in malacosporean life cycles. Overall, our results imply that there may be another malacosporean host which remains unidentified, although transmission from fish to bryozoans requires further investigation. However, the highly clonal life history of freshwater bryozoans is likely to allow both long-term persistence and spread of infection within bryozoan populations, precluding the requirement for regular transmission from an alternate host.     

Life cycle of Amblyospora indicola (Microspora: Amblyosporidae), a parasite of the mosquito Culex sitiens and of Apocyclops sp. Copepods

The life cycle of Amblyospora indicola, a parasite of the mosquito Culex sitiens, was revealed by field observations and laboratory infection experiments conducted in Australia. In northern Queensland, infected C. sitiens larvae were often found breeding in association with two cyclopoid copepods: Apocyclops dengizicus and an undescribed species of the same genus. The latter species was found to be an intermediate copepod host of this microsporidium whereas A. dengizicus was not. One complete cycle of the parasite extends over two mosquito generations (by transovarial transmission from females with binucleate spores to their eggs) and by horizontal transmission between mosquitoes and copepods. The latter involves horizontal transmission from mosquitoes to copepods via meiospores produced in larval fat body infections and horizontal transmission from copepods to mosquitoes via uninucleate spores produced within infected copepods. Uninucleate clavate spores were formed in Apocyclops sp. nov. copepods 7-10 days after exposure to larval meiospores and were infectious to larvae of a microsporidian-free colony of C. sitiens. The development of A. indicola within mosquito larvae exposed to infected copepods is similar to that of A. dyxenoides infecting C. annulirostris. It proceeds from stages with a single nucleus to diplokaryotic binucleate cells in oenocytes. These stages persist through pupation to adult emergence after which time a proportion of male mosquitoes and female mosquitoes may develop binucleate spores without the need for a blood meal. A proportion of both male and female larval progeny of infected females with binucleate spores develop patent fat body infections via transovarial transmission and die in the fourth larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune function differences between two color morphs of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) at different life stages

Several studies demonstrated that in insects cuticle melanism is interrelated with pathogen resistance, as melanin‐based coloration and innate immunity possess similar physiological pathways. For some insects, higher pathogen resistance was observed in darker individuals than in individuals with lighter cuticular coloration. Here, we investigated the difference in immune response between two color morphs (black and red) and between the life stages (pupa and adult) of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae). Here in this study, cuticle thickness, microbial test (antimicrobial activity, phenoloxidase activity, and hemocyte density), and immune‐related gene expression were evaluated at different stages of RPW. Study results revealed that cuticle thickness of black phenotype was thicker than red phenotype at old‐pupa stage, while no significant difference found at adult stage. These results may relate to the development processes of epidermis in different stages of RPW. The results of antimicrobial activity, phenoloxidase (PO) activity, and hemocyte density analyses showed that adults with a red phenotype had stronger pathogen resistance than those with a black phenotype. In addition to antimicrobial activity and PO activity, we tested relative gene expression in the fat body of old pupae. The results of hemolymph antimicrobial analysis showed that old pupae with a red phenotype were significantly different from those with a black phenotype at 12 hr after Staphylococcus aureus injection, suggesting that red phenotype pupae were more sensitive to S. aureus. Examination of gene expression in the fat body also revealed that the red phenotype had a higher immune response than the black phenotype. Our results were inconsistent with the previous conclusion that dark insects had increased immune function, suggesting that the relationship between cuticle pigmentation and immune function in insects was not a direct link. Additional possible factors that are associated with the immune response, such as life‐history, developmental, physiological factors also need to be considered.     

Early queen infection shapes developmental dynamics and induces long-term disease protection in incipient ant colonies

Infections early in life can have enduring effects on an organism's development and immunity. In this study, we show that this equally applies to developing ‘superorganisms’––incipient social insect colonies. When we exposed newly mated Lasius niger ant queens to a low pathogen dose, their colonies grew more slowly than controls before winter, but reached similar sizes afterwards. Independent of exposure, queen hibernation survival improved when the ratio of pupae to workers was small. Queens that reared fewer pupae before worker emergence exhibited lower pathogen levels, indicating that high brood rearing efforts interfere with the ability of the queen's immune system to suppress pathogen proliferation. Early-life queen pathogen exposure also improved the immunocompetence of her worker offspring, as demonstrated by challenging the workers to the same pathogen a year later. Transgenerational transfer of the queen's pathogen experience to her workforce can hence durably reduce the disease susceptibility of the whole superorganism.     

Evidence of intracolony transmission of Thelohania solenopsae (Microsporidia: Thelohaniidae) in red imported fire ants (Hymenoptera: Formicidae) and the first report of spores from pupae.

Red imported fire ant, Solenopsis invicta, colonies were infected horizontally by introducing live brood (mainly larvae and pupae) infected with Thelohania solenopsae. Live, infected brood introduced into uninfected colonies were adopted and raised to adulthood instead of being executed by the recipient colony. Introductions of infected larvae with uninfected pupae, which eclose into adult worker caste fire ants, resulted in an 80% infection rate of the inoculated colonies. Infections from introductions of infected pupae with uninfected larvae resulted in a 37.5% infection of inoculated colonies. Infections were also detected in 11.6 and 3.7% of the adult worker caste ants that eclosed from uninfected large larvae and pupae, respectively, that were held with infected adult workers. Microscopic examination of infected brood revealed sporoblasts and large numbers of spores of T. solenopsae in S. invicta pupae.     

Antibacterial Immune Competence of Honey Bees (Apis mellifera) Is Adapted to Different Life Stages and Environmental Risks

The development of all honey bee castes proceeds through three different life stages all of which encounter microbial infections to a various extent. We have examined the immune strength of honey bees across all developmental stages with emphasis on the temporal expression of cellular and humoral immune responses upon artificial challenge with viable Escherichia coli bacteria. We employed a broad array of methods to investigate defence strategies of infected individuals: (a) fate of bacteria in the haemocoel; (b) nodule formation and (c) induction of antimicrobial peptides (AMPs). Newly emerged adult worker bees and drones were able to activate efficiently all examined immune reactions. The number of viable bacteria circulating in the haemocoel of infected bees declined rapidly by more than two orders of magnitude within the first 4–6 h post-injection (p.i.), coinciding with the occurrence of melanised nodules. Antimicrobial activity, on the other hand, became detectable only after the initial bacterial clearance. These two temporal patterns of defence reactions very likely represent the constitutive cellular and the induced humoral immune response. A unique feature of honey bees is that a fraction of worker bees survives the winter season in a cluster mostly engaged in thermoregulation. We show here that the overall immune strength of winter bees matches that of young summer bees although nodulation reactions are not initiated at all. As expected, high doses of injected viable E.coli bacteria caused no mortality in larvae or adults of each age. However, drone and worker pupae succumbed to challenge with E.coli even at low doses, accompanied by a premature darkening of the pupal body. In contrast to larvae and adults, we observed no fast clearance of viable bacteria and no induction of AMPs but a rapid proliferation of E.coli bacteria in the haemocoel of bee pupae ultimately leading to their death.     

Life cycle,ultrastructure and molecular phylogeny of Hyalinocysta chapmani (Microsporidia: Thelohaniidae), a parasite of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae)

The complete life cycle of the microsporidium Hyalinocysta chapmani is described from the primary mosquito host Culiseta melanura and the intermediate copepod host Orthocyclops modestus. Infections are initiated in larval C. melanura following the oral ingestion of uninucleate spores from infected copepods. Spores germinate within the lumen of the midgut and directly invade fat body tissue where all development occurs. Uninucleated schizonts undergo binary division (schizogony) followed by karyokinesis (nuclear division) to form diplokaryotic meronts. Merogony is by synchronous binary division. The onset of sporogony is characterized by the simultaneous secretion of a sporophorous vesicle and meiotic division of the diplokaryon resulting in the formation of eight ovoid meiospores enclosed within a sporophorous vesicle. Most infected larvae die during the fourth stadium and there is no evidence of a developmental sequence leading to vertical transmission. Hyalinocysta chapmani is horizontally transmitted to O. modestus via oral ingestion of meiospores. Infections become established within ovarian tissue of females and all parasite development is haplophasic. Uninucleate schizonts divide by binary division during an initial schizogonic cycle. Newly formed uninucleate cells produce a thin sporophorous vesicle and undergo repeated nuclear division during sporogony to produce a rosette-shaped, multinucleated sporogonial plasmodium with up to 18 nuclei. This is followed by cytoplasmic cleavage, sporogenesis, and disintegration of the sporophorous vesicle to form membrane-free uninucleate spores. Infected females eventually die and there is no egg development. The small subunit rDNA sequence of H. chapmani isolated from meiospores from C. melanura was identical to the small subunit rDNA sequence obtained from spores from O. modestus, corroborating the laboratory transmission studies and confirming the intermediary role of O. modestus in the life cycle. Phylogenetic analysis was conducted with closely related microsporidia from mosquitoes. Hyalinocysta chapmani did not cluster within described Amblyospora species and can be considered a sister group, warranting separate genus status.     

The ontogeny of immunity: development of innate immune strength in the honey bee (Apis mellifera)

Honey bees (Apis mellifera) are of vital economic and ecological importance. These eusocial animals display temporal polyethism, which is an age-driven division of labor. Younger adult bees remain in the hive and tend to developing brood, while older adult bees forage for pollen and nectar to feed the colony. As honey bees mature, the types of pathogens they experience also change. As such, pathogen pressure may affect bees differently throughout their lifespan. We provide the first direct tests of honey bee innate immune strength across developmental stages. We investigated immune strength across four developmental stages: larvae, pupae, nurses (1-day-old adults), and foragers (22-30 days old adults). The immune strength of honey bees was quantified using standard immunocompetence assays: total hemocyte count, encapsulation response, fat body quantification, and phenoloxidase activity. Larvae and pupae had the highest total hemocyte counts, while there was no difference in encapsulation response between developmental stages. Nurses had more fat body mass than foragers, while phenoloxidase activity increased directly with honey bee development. Immune strength was most vigorous in older, foraging bees and weakest in young bees. Importantly, we found that adult honey bees do not abandon cellular immunocompetence as has recently been proposed. Induced shifts in behavioral roles may increase a colony's susceptibility to disease if nurses begin foraging activity prematurely.     

Evaluation of hydrogen peroxide vapour as a method for the decontamination of surfaces contaminated with Clostridium botulinum spores

The aim of this study was to evaluate the efficacy of hydrogen peroxide vapour (HPV) against spores of Clostridium botulinum, for use as a method for decontaminating environments where this pathogen has been handled. Spores were dried onto stainless steel slides and exposed to HPV in a sealed glovebox enclosure, transferred to a quenching agent at timed intervals during the exposure period, before survivors were cultured and enumerated. D-values were calculated from graphs of log₁₀ survivors plotted against time and were found to range from 1.41 to 4.38 min. HPV was found to be effective at deactivating spores of toxigenic Cl. botulinum, non-toxigenic Clostridium spp. and Geobacillus stearothermophilus dried onto stainless steel surfaces. HPV could be used to decontaminate cabinets and rooms where Cl. botulinum has been handled. The cycle parameters should be based on studies carried out with relevant spores of this organism, rather than based on inactivation data for G. stearothermophilus spores, which have been used in the past as a standard biological challenge for disinfection and sterilisation procedures. HPV could provide an attractive alternative to other decontamination methods, as it was rapid, residue-free and did not give rise to the health and safety concerns associated with other gaseous decontamination systems.     

The nature of Thelohania solenopsae (Microsporidia) cysts in abdomens of red imported fire ants, Solenopsis invicta

Sixty four percent of Solenopsis invicta workers infected with Thelohania solenopsis contained 1-6 "cysts" ranging from 70 to 260 microm in diameter. Light and electron microscope analyses showed that cysts are hypertrophied adipocytes transformed by the parasites, each cyst presumably forming from a single cell. In the first step of the pathogenesis, Nosema-like spores functioning in autoinfection are produced; a diplokaryotic sequence leading to their formation causes fat body hypertrophy. When meiosis occurs, it switches parasite development to production of octospores and/or megaspores. Adipocytes become 2-4xlarger than normal in conjunction with intensive parasite multiplication and octospore maturation. Infected cells eventually lose their cellular organization and are converted into reservoirs for spores. There were no manifestations of cellular immunity, such as encapsulation or nodule formation. Similarly, there were no signs of specialized host-parasite interaction that might be interpreted as xenoma-like complexes. The role of the cysts in the parasite's life cycle is unclear. They may represent a defensive reaction of the host sacrificing the infected cells to segregate the infection. Alternatively, the cyst may help protect spores from environmental hazards and provide a concentrated infectious dose to aid horizontal transmission of the microsporidium. We propose to refer to hypertrophied adipocytes filled with T. solenospsae spores as "sporocytosacs", not "cysts."     

Molecular characterization of an inhibitor of apoptosis in the Egyptian armyworm, Spodoptera littoralis, and midgut cell death during metamorphosis

The cDNA corresponding to an inhibitor of apoptosis (IAP) from the Egyptian armyworm, Spodoptera littoralis, was cloned by RT-PCR. Sequence analysis showed that the IAP of S. littoralis (SlIAP) contains two baculoviral IAP repeat (BIR) motifs, followed by a RING finger, an organization which is very similar to that of other lepidopteran IAPs. SlIAP mRNA was detected in ovary, testis, salivary gland, fat body, epidermis, brain and midgut of S. littoralis. During the last larval instar, prepupal and pupal stages, brain mRNA levels remained approximately constant, whereas those of midgut showed a large peak centred in the prepupal stage. Midgut morphology changed during metamorphosis from a semi-transparent, cylindrical structure in last instar larvae to a brownish globular mass in pupae. TUNEL assays, LysoTracker staining and caspase-3 immunohistochemistry, indicated that programmed cell death in midgut starts actively at the onset of pupation process, coinciding with the dramatic decrease of SlIAP mRNA levels observed at the same time.     

家蚕(Bombyx mori)主要血浆蛋白质及其基因表达的研究

本文用SDS-PAGE法观察不同发育阶段蚕血液中主要血浆蛋白质sp、30KP浓度的变化;从不同发育阶段的蚕脂肪体提取RNA和poly(A)~+-RNA,在兔网织红细胞系作体外翻译并检测翻译产物。结果表明,5龄蚕脂肪体mRNA合成蛋白质的速率为初蛹的2倍;5龄及初蛹脂肪体30KP mRNA活性的发育变化与其相应蛋白质在血液中的浓度变化一致;sp-1在5龄幼虫脂肪体内的表达及卵黄原蛋白(Vg)在蚕蛹脂肪体内的表达具有雌特异性,其表达和性特异性大体是在前翻译水平被调节的。     

A Multiple-Pathogen System for Bioherbicidal Control of Several Weeds

A novel system using four host-specific fungal plant pathogens applied in a single, postemergent spray to control pigweed, sicklepod, and showy crotalaria was tested under greenhouse conditions. The four pathogens were Phomopsis amaranthicola (a pathogen of pigweed species), Alternaria cassiae (a pathogen of sicklepod and showy crotalaria), Colletotrichum dematium f. sp. crotalariae and Fusarium udum f. sp. crotalariae (pathogens of showy crotalaria). Spore suspensions of each pathogen alone (10⁶ spores mL^-1) or a mixture of the four pathogens (1:1:1:1, v/v, 2.5×10⁵ spores mL^-1 of each pathogen, total 10⁶ spores mL^-1) were tested on four- to six-leaf stage seedlings of the three weed species grown together in pots. One week after inoculation (WAI), all sicklepod and showy crotalaria seedlings were killed, and all pigweed seedlings were killed by 6 WAI when inoculated with their respective pathogen(s) alone or a mixture of the pathogens. None of the weeds inoculated with the root-infecting pathogen F. udum developed wilt disease by the time the experiment was completed (6WAI). The results demonstrate the feasibility to control three weeds simultaneously with different fungi without loss of efficacy or alterations in host-specificity of each fungus in the given mixture. Scanning electron microscopy showed visual differences in the appearance or germination and further development of conidia of each pathogen on its respective host leaf surface compared to nonhost leaf surfaces, whether the pathogen was applied alone or in a mixture with the other pathogens studied. Application of several host-specific fungal pathogens in a bioherbicide mixture as a multi-component bioherbicide system may be advantageous for further development of simultaneous, broad-spectrum weed control.