Phase contrast and electron microscopy of the mycetocytes and symbiotes of the pea aphid, Acyrthosiphon pisum

Phase contrast, scanning electron, and transmission electron microscopy of the symbiotes of Acyrthosiphon pisum was undertaken. Some staining properties of the symbiotes were also studied.The symbiotes of the pea aphid were found to be coccoid bodies 2 to 5 μ in diameter, gram negative, stained slightly by Fuelgen's, and stained blue by Machiavello's. The symbiotes appear to be surrounded by three membranes. Ribosomes may occur within the cytoplasm of the symbiotes. The cytoplasm of the mycetocytes contains large numbers of mitochondria, endoplasmic reticulum, ribosomes, and a large nucleus, and nucleolus.A discussion of the classification of the symbiotes is also presented.     

Intracellular symbiotes of the pea aphid,Acyrthosiphon pisum

Histological and ultrastructural studies on the mycetome of the pea aphid, Acyrthosiphon pisum, disclosed two types of symbiotes. The more common primary symbiotes were oval in shape and were found in large mycetocytes making up the bulk of the mycetome. The secondary symbiotes were smaller, rod-shaped, and were restricted to an apparently syncytial sheath partially enclosing the primary mycetocytes. Extensive rough endoplasmic reticulum and Golgi occurred in the sheath but not in the primary mycetocytes. Lysosomal breakdown occurred in both primary and secondary symbiotes but the two processes differed markedly. In the primary mycetocytes, a small number of symbiotes were broken down individually to form small, compact residual bodies. In the sheath, breakdown of secondary symbiotes was more extensive: large numbers were broken down within cytolysomes.     

Linoleic acid biosynthesis in the pea aphid,Acyrthosiphon pisum (Harris)

The pea aphid Acyrthosiphon pisum (Harris) incorporated [1-¹⁴C]acetate into a phospholipid dienoic fatty acid in a time-dependent manner. In 2-h incubations, the incorporation of radioactivity into the 18:2 fraction was minimal, whereas after 45 h 18:2 was the major fatty acid labeled. Ozonolysis of the isolated dienoic fatty acid methyl ester followed by radio-gas-liquid chromatography showed that radioactivity was associated with fragments containing carbons 1–9 and 13–18. These data established the location of the double bonds in the 9,12 positions and indicated that the entire molecule was labeled from [1-¹⁴C]acetate. Tetracycline-treated aphids synthesized linoleic acid in the same proportions as untreated controls. Scanning electron microscopy showed that over 50% of the treated insects had greatly reduced numbers of intracellular symbiotes or lacked them or most of the existing symbiotes had an abnormal appearance. Therefore, we conclude that intracellular symbiotes are not involved in the biosynthesis of linoleic acid in the pea aphid.     

Isolation and characterization of symbiotes from the Rocky Mountain wood tick, Dermacentor andersoni

Wolbachia-like symbiotes in the Rocky Mountain wood tick, Dermacentor andersoni, were isolated repeatedly by injection of ovarial tissues into 5-day-old chick embryos. In Giemsa-stained smears of infected embryo tissues, the organisms appeared as blueish or pinkstained coccal bodies indistinguishable from those seen in the ovaries of ticks, where they are located in the luminal epithelium and funicle cells, as well as in oocytes.Electron microscopy revealed that these symbiotes are highly pleomorphic and vary in size from 0.6 to 3.4 μm in diameter. Their fine structure in tissue cells is differentiated into a granular, cortical region, which contains densely stained ribosomes, and a medullary region consisting of a diffuse reticulum partially or completely devoid of granular material or ribosomes. Multiplication is by binary fission. Each organism is delimited by a distinct plasmalemma; a cell wall as in bacterial and rickettsial agents was not observed in organisms from ovarial tissues.Symbiotes cultivated in chick embryos and then injected intracoelomically into adult D. andersoni, developed rapidly and produced massive infestations in hemocytes, hypodermal tissues, salivary glands, and in connective tissues surrounding midgut, Malpighian tubules, and ovary. In hypodermal tissue, organisms with a distinct bilayered cell envelope were occasionally detected. The massive invasion of tissues by injected symbiotes invariably proved fatal for ticks.Results of complement-fixation tests and of fluorescent antibody staining indicated that symbiotes in D. andersoni are closely related to Wolbachia persica, previously isolated from Argas arboreus.     

In vivo sterol biosynthesis by pea aphid symbiotes as determined by digitonin and electron microscopic autoradiography

Summary Pea aphid primary symbiotes have previously been shown to synthesize cholesterol in vitro. Two electron microscopic techniques were used here to determine whether the symbiotes also synthesize cholesterol in vivo and whether this cholesterol is made available to the aphid. We also inquired into a possible role of secondary symbiotes in cholesterol biosynthesis. Treatment of aphids with digitonin resulted in significant alteration of ultrastructural sites in primary and secondary symbiote membranes. We concluded that these sites are areas of high cholesterol concentration in the symbiotes.Electron microscopic autoradiography with ³H-mevalonate precursor indicated that both primary and secondary symbiotes synthesize cholesterol; in both cases, the majority of grains were associated with the symbiote membranes. While the frequency of grains on the symbiotes remained constant, irrespective of incubation time in labelled media, the frequency of grains over surrounding tissues increased exponentially as the time of incubation was increased from 30 min to 8 h, indicating that symbiote cholesterol is transported to other tissues. High voltage electron microscopic autoradiography permitted thick section autoradiography, reducing the time of emulsion exposure from 54 days (thin section) to 12 days (0.5 m sections).Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by a research grant (PCM 74-2401 A01) from The National Science FoundationThe authors wish to thank Dr. G.A. DeZoeten for his invaluable advice and assistance with the autoradiographic techniques, Mr. Gary Gaard for his help with electron microscopy, and Dr. Dale Johnston and Dr. Damien Neuberger for their generous help in the use of the high voltage EM     

Ovulation and the mechanism of follicle rupture

Summary The effects of penicillin and chlortetracyline HCl on the fine structure of the intracellular symbiotes of the pea aphid were studied in an attempt to remove the symbiote population. High penicillin concentrations, 1% and 0.1%, caused symbiote breakdown but were toxic and/or repellent to the aphids; at 0.1% specific effects were observed on the symbiotes' cell walls. After the use of 0.01% penicillin in the aphid diet, the symbiotes had abnormal cell walls and were abnormally dilated; however, symbiote division and transmission from one aphid generation to the next seemed unaffected and the aphids appeared normal. Aphids fed 0.1% chlortetracycline failed to reproduce. After 7 days, their symbiotes were found to break down at a high rate but aphid mitochondria were also adversely affected at this stage. Following 0.002% chlortetracycline, the aphids produced aposymbiotic progeny with apparently normal mitochondrial populations; these larvae failed to develop.Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by a research grant (GB 31904 X) from the National Science Foundation.     

Some effects on the aphid Myzus persicae of ingesting antibiotics incorporated into artificial diets

Ten antibiotics were incorporated individually at different concentrations into artificial diets fed to the aphid Myzus persicae. The survival and larviposition of apterous adult aphids, and the growth and development of larvae were severely affected by albamycin (sodium novobiocin), chloromycetin (chloramphenicol), ilotycin glucoheptonate (erythromycin glucoheptonate), and the tetracyclines aureomycin (chlortetracycline HCl), oxytetracycline HCl, and tetracycline HCl. Alata-production by the apterous adults that were maintained in groups on the diets was dramatically curtailed by these antibiotics, some at concentrations as low as 0·0001%. Mandelamine (methenamine mandelate), penicillin-G, streptomycin sulphate, and viomycin sulphate had a much lesser effect on larval growth, even when these antibiotics were incorporated into the diet at concentrations as high at 0·1%. The results are discussed in relation to the probable functions of the aphid's symbiotes in the nutrition of the aphid.     

Artificially Induced Symbiosis Between Marine Flagellates and Vertebrate Tissues in Culture*

SYNOPSIS. Algal symbiotes isolated from marine invertebrate hosts, and grown axenically in culture, have been successfully combined in artificial associations with chick fibroblasts and grunt (Haemulon sp.) fin explants. Growth (measured by cell numbers and chlorophyll a) is stimulated in both symbiotes and host cells. Studies of nitrogen uptake and exchange suggest that these artificial associations are similar to other, natural symbioses involving invertebrate hosts, e.g. the hermatypic corals.     

The susceptibility of the pea aphid to intrahemocoelic infection by Serratia marcescens

The bacterium Serratia marcescens is pathogenic for the pea aphid, Acyrthosiphon pisum. When S. marcescens cells are inoculated into the hemocoel of the aphid, there is a variable lag period of 2–3 hr duration. This phase is followed by a rapid increase in pathogen numbers causing a general septicemia and the death of the aphid. The LD₅₀ for fourth-instar pea aphid nymphs was estimated as 190 viable S. marcescens cells (95% confidence limits, 123–288). It is suggested that being sucking insects aphids are protected against infection by bacterial pathogens in the field.     

Culture of fat body of Periplaneta americana: Tissue development and establishment of cell lines

Long-term culture of larval fat body from Periplaneta americana was carried out. The cultures in a chemically defined medium show a strict dependance of the fat body on oxygen. The cultures with serum supplementation give rise to numerous cell migrations. We have studied the development of the different cell types and especially that of the mycetocytes and adipocytes. The mycetocytes can be cultured provided that they remain associated with the adipocytes. In these mycetocytes, the progressive loss of the symbiotes is related to the lipid load. The adipocytes continue to store lipids and glycogen for over 6 months. Then, the selection of different phenotypes gives rise to a homogeneous and adipocyte-like cell population. After 7 months, these cells can be subcultured regularly and a new cell line from P. americana has been established which is the only one isolated from explants of the insect fat body.     

An Experimental Test of whether the Defensive Phenotype of an Aphid Facultative Symbiont Can Respond to Selection within a Host Lineage

An experiment was conducted to test whether parasitoid resistance within a single clonal line of pea aphid (Acyrthosiphon pisum) might increase after exposure to the parasitoid wasp Aphidius ervi. Any change in resistance was expected to occur through an increase in the density of protective symbiotic bacteria rather than genetic change within the aphid or the bacterial symbiont. Six aphid lineages were exposed to high parasitoid attack rates over nine generations, each line being propagated from individuals that had survived attack; a further six lineages were maintained without parasitoids as a control. At the end of the experiment the strength of resistance of aphids from treatment and control lines were compared. No differences in resistance were found.     

The control of Monomorium pharaonis (Hymenoptera: Formicidae) with Bacillus thuringiensis

In this study, the effect of different preparations made from Bacillus thuringiensis var. thuringiensis (strains: CCEB 555 and CCEB 058) on ants, Monomorium pharaonis, under laboratory conditions is reported. The different preparations tested consisted of (1) a liquid culture of the strain B. thuringiensis CCEB 555 (containing spores and exotoxin), (2) the supernatant of the culture broth of strain CCEB 555 (containing exotoxin), and (3) the biological preparation “Bathurin” prepared from the strain B. thuringiensis CCEB 058 (containing spores and inclusions, without exotoxin). The preparations were used either pure or in alternation with borax, i.e., 1 wk borax, 3 wk the respective preparation for several months. All preparations were found to be toxic to M. pharaonis and their effect was characterized by a slow extinction of the ant colony. Administration of “Bathurin” (1.3%) yielded a 100% mortality after 20 wk. Using a liquid culture of B. thuringiensis var. thuringiensis, 100% mortality was recorded after 21 wk, a period of time which did not differ from that obtained with the supernatant of the culture containing exotoxin. The alternation with borax was found to accelerate ant mortality by 9–10 wk after administration. In all experiments, the worker ants died first, the queen ants surviving them by 1–3 wk.In experiments employing worker ants only, a 100 and 98% mortality, respectively, occurred within 3 wk after administration of a liquid culture of B. thuringiensis and “Bathurin” supplemented with borax.     

Rapid Multiplication and Induction of Early In Vitro Flowering in Dendrobium Primulinum Lindl

Dendrobium primulinum is an important epiphytic orchid. A successful protocol for mass multiplication and early in vitro flowering was developed. Immature embryos of 4 week after pollination exhibited about 96% germination within 30 days of culture on MS medium containing sucrose (3%) (w/v), NAA and BA (6 and 9 μM) in combination. Protocorm-like bodies (PLBs) formed from the germinating seeds on the germination medium. Rooted plantlets were formed within 2-3 wk on MS medium containing sucrose (3%), NAA and BA (3 and 12 μM in combination) where about 29 shoot/buds produced per cycle of 4 wk interval. The well rooted plantlets produced 4-5 floral buds per spike when they were maintained on MS medium containing sucrose (3%), fresh apple juice (10%) (v/v) for four wk followed by on MS medium freed of apple juice but enriched with NAA and BA (3 and 12 μM respectively). The hardened plantlets were transferred to community potting mix where the about 80% transplants survived after two months of transfer.     

Sterols in Laodelphax striatellus with special reference to the intracellular yeastlike symbiotes as a sterol source

Sterols were analysed to investigate the sterol source in Laodelphax striatellus and three other rice plant-sucking homopterous insects. In L. striatellus, cholesterol, 24-methylenecholesterol and β-sitosterol were detected. The host plant (rice) contained campesterol, stigmasterol and β-sitosterol. From the honeydew excreted by L. striatellus, cholesterol, β-sitosterol and negligible amounts of campesterol were recovered. Laodelphax striatellus possesses yeastlike symbiotes which can be destroyed by high temperature. Fifth instar nymphs, which have been exposed to 35°C for 3 days in their 1 st instar, showed lower cholesterol concentration and markedly reduced amounts of 24-methylenecholesterol. From the results it is concluded that L. striatellus has two sterol sources: one from the host plant and the other from the yeastlike symbiotes which appear to provide 24-methylenecholesterol.     

Initiation and orientation of the symbiote migration in the human body louse Pediculus humanus L.

Bacterial symbiotes in the human body louse Pediculus humanus migrate from the mycetome to the lateral oviducts during the adult moult. Experimental results are presented suggesting that the symbiote migration is initiated by a humoral factor associated with the adult moult; that the factor is present in both sexes but the male mycetome is unable to respond to it; and that the symbiotes are attracted by the female reproductive tract.     

The etiological agent of cytoplasmic incompatibility in Culex pipiens

All individuals of Culex pipiens (wide sense) which have been examined were infected with Wolbachia pipientis. Larvae reared in tetracycline were freed of these symbiotes and remained free (aposymbiotic) in future generations. When males were freed of their symbiotes, they no longer displayed incompatibility. Aposymbiotic males were compatible with all females, whether infected or not. Aposymbiotic females, on the other hand, laid fertile eggs after mating with aposymbiotic males, but not after mating with normal males. Most eggs laid by aposymbiotic females after mating with normal males showed no development at all, even though the females had been inseminated.     

Diversity of Bacteria Associated with Natural Aphid Populations

The bacterial communities of aphids were investigated by terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments generated by PCR with general eubacterial primers. By both methods, the γ-proteobacterium Buchnera was detected in laboratory cultures of six parthenogenetic lines of the pea aphid Acyrthosiphon pisum and one line of the black bean aphid Aphis fabae, and one or more of four previously described bacterial taxa were also detected in all aphid lines except one of A. pisum. These latter bacteria, collectively known as secondary symbionts or accessory bacteria, comprised three taxa of γ-proteobacteria (R-type [PASS], T-type [PABS], and U-type [PAUS]) and a rickettsia (S-type [PAR]). Complementary analysis of aphids from natural populations of four aphid species (A. pisum [n = 74], Amphorophora rubi [n = 109], Aphis sarothamni [n = 42], and Microlophium carnosum [n = 101]) from a single geographical location revealed Buchnera and up to three taxa of accessory bacteria, but no other bacterial taxa, in each aphid. The prevalence of accessory bacterial taxa varied significantly among aphid species but not with the sampling month (between June and August 2000). These results indicate that the accessory bacterial taxa are distributed across multiple aphid species, although with variable prevalence, and that laboratory culture does not generally result in a shift in the bacterial community in aphids. Both the transmission patterns of the accessory bacteria between individual aphids and their impact on aphid fitness are suggested to influence the prevalence of accessory bacterial taxa in natural aphid populations.     

Ultrastructure of Symbiotic Bacteria in Normal and Antibiotic-Treated Blastocrithidia culicis and Crithidia oncopelti*

SYNOPSIS. An electron microscope study of diplosomes in Blastocrithidia culicis and bipolar bodies in Crithidia oncopelti has shown that both entities appear to be intracellular symbiotes and have a similar fine structure. They are enclosed by 2 unit membranes which are separated by a large space of very low density. The outer membrane is derived probably from the host cell. The matrix of the symbiotes is composed of dense ribosome-like particles and of areas of low density containing fine fibrillae. The particles are of the same size as ribosomes in bacteria and the fibrils have the characteristics of bacterial DNA. Thus, the lucid areas with fibrillae correspond to the nucleoids in bacteria. These observations suggest that the symbiotes are bacteria. The effect of chloramphenicol (CAP) and penicillin G (PCL) on these symbiotic bacteria was studied by culturing the host flagellates in media containing the antibiotics. The effect was analyzed at different intervals after the treatment by electron microscopy. After single treatment in the blood broth containing 0.08% (w/v) CAP, symbiotes appeared to have enlarged nucleoids, became deformed and eventually degenerated. In Grace's medium (supplemented with 10% fetal bovine serum) containing 0.6 or 2.4% (w/v) PCL, symbiotes of C. oncopelti remained unaltered, whereas some symbiotes of B. culicis became pleomorphic. Symbiotes of both species persisted after repeated transfers in PCL media and reverted to normal forms when transferred to PCL-free media. Sensitivity of symbiotes to CAP provides further evidence of their bacterial nature. The effect of PCL on the symbiotes of B. culicis suggests the presence in their cell envelopes of mucopeptide, which probably provides rigidity for maintaining the bacterial shape of the symbiotes.     

Effects of elevated temperature on the mycetome and symbiotes of the bed bug Cimex lectularius (Heteroptera)

High temperature treatment significantly reduced the reproductive rate of Cimex lectularius and simultaneously rendered their mycetomes nearly symbiote-free. The possibility that the symbiotes may affect host reproductive potential is discussed.