Marion A. Brooks

INTRACELLULAR SYMBIOSIS IN COCKROACHES. I. PRODUCTION OF APOSYMBIOTIC COCKROACHES

1. The trans-ovarial inheritance of fat-body intracellular symbiotes in the German cockroach was prevented by subjecting the parent insects to high temperature or by feeding the parents aureomycin or sulfathiazole.2. The most certain and simple method of obtaining aposymbiotic nymphs is by feeding the parents ground dog biscuit plus 0.1% aureomycin all of their lives.3. Aposymbiotic nymphs are practically incapable of growth on a natural diet which is adequate for symbiotic nymphs.4. The addition of large amounts of dried brewers yeast to the diet enabled the aposymbiotic nymphs to grow to maturity in two to three times the period required by normal nymphs.5. Adult aposymbiotic cockroaches suffered impaired reproductive ability. The males were affected as well as the females.6. The second and third generations of aposymbiotic roaches are similar to the first in both histology and behavior.

THE DISSOCIATION OF INSECT EMBRYOS FOR CELL CULTURE

SummaryProcedures and solutions were developed for dissociating embryos ofBlattella germanica in preparation for primary cell culture. Trypsin solutions were maximally effective at 0.01% for germ bands but higher concentrations, 0.05 to 0.1% were needed for embryos in later stages.

Isolation of cell lines from embryos of the cockroach, Blattella germanica.

SummaryCell lines were isolated from three stages of embryos ofBlattella germanica dissociated with trypsin. The lines have been subcultured 50 to 134 times in 3 years. Line UM-BGE-1 was isolated from germ band embryos at stages of segmentation and limb-bud formation (5 days old). Line UM-BGE-2 was derived from embryos at dorsal closure (7 days old). Line UM-BGE-4 arose from embryos in the germ band and dorsal closure stages (5 and 7 days old); these cells colonize as hollow spheres or vesicles. Line UM-BGE-5, isolated during organogenesis (10 days old), developed into two distinct sublines. Subline α is composed of round cells that do not attach to the flask. Subline β grows as an attached monolayer; the cells can be removed with a saline solution containing 20mM disodium dihydrogen Versenate®. Most of the cells of these lines have the diploid chromosome number (23 or 24) excepting line UM-BGE-1 in which the tetraploid number predominates.

Growth-retarding effect of carbon-dioxide anaesthesia on the German cockroach

Abstract German cockroach nymphs exposed to a high concentration of carbon dioxide for 3 minutes weekly until maturity were retarded in growth by 14% to 53% compared to nonanaesthetized controls, as determined by the time required to moult to adults. There was no apparent correlation between diet and the carbon-dioxide effect. The adult weight of roaches subjected to weekly anaesthesia during the nymphal period was less than that of nonanaesthetized roaches. Nymphs reared as isolated individuals were retarded by carbon dioxide relatively as much as nymphs reared in groups. Nymphs kept under constant carbon-dioxide anaesthesia for 2 1 2 hours became greatly distended by gas, remained paralysed, and died within several days. The retardation and lethal factors seem to be carbon-dioxide specific rather than a general immobilization, nervous, or oxygen-deficit effect inasmuch as nitrogen was relatively innocuous. Consideration was given to the possibility of the formation of a toxic carbon-dioxide compound which is retained by the blood.

The rate of development of a microsporidan in moth cell culture.

A microsporidan parasite of the forest tent caterpillar Malacosoma disstria infected cells and replicated in vitro in a line from the moth Heliothis zea. After spore germination, the incidence of infected cells increased with time until leveling off with sporulation. During the first 24 hr, there was a static number of parasites, followed by a 2-day logarithmic growth phase during which the population doubled five to six times. The growth rate was 9 to 11 hr per population doubling. Sporulation commenced on day 3, and 40 to 50 spores were recovered from each infected cell. The life cycle was completed within 6 days, culminating in spores that were infectious for cultured cells. The antibiotic fumagillin at a dose of 1 ppm in the culture medium was microsporida-static.

Intracellular bacteroids: Electron microscopy of Periplaneta americana injected with lysozyme☆

Abstract The intracellular bacteroids of Periplaneta americana formed protoplasts in vitro following injections of egg-white lysozyme into the host. The sequence of ultrastructural events resembled protoplast formation in free-living bacteria. Cockroaches given injections of lysozyme exhibited behavioral anomalies and high mortality. Sublethal pathologic conditions in the host were indicated by swollen mitochondria, lysosomes, vacuolation and membrane fragmentation. Lysozyme which had been heated at 90 C for 30 min had the same effect in vivo as did the natural enzyme. It was concluded that lysozyme probably disrupts the integrity of the symbiotic association by altering a number of cytological structures, in both the host and the microorganism.

Continuous rearing of the aster leafhopper, Macrosteles fascifrons, on a chemically defined diet

Abstract A holidic diet for feeding the aster leafhopper, Macrosteles fascifrons , was formulated. The amino acids, B-vitamins, and sucrose are less concentrated than in aphid diets. Cholesterol, at 5 mg/ml, is required for the last ecdysis. Although leafhoppers reared on this diet have poorer survival and shorter life span than those reared on plants, they produce more progeny. Leafhoppers reared on this diet have completed the ninth generation and the culture is still thriving.

Influence of physical factors on the growth of insect cells in vitro. I. Effect of osmotic pressure on growth rate of a moth cell line.

SummaryThe influence of osmotic pressure on the growth rate of a cell line (IMC-HZ-1) from a moth,Heliothis zea, was studied. The cells grew at approximately the same rate in media ranging from 290 to 360mOsm per kg. On either side of these osmolalities the growth rate fell off considerably. The osmotic pressure tolerances of the cells were similar to those reported for other animal cells. Upon transfer to media of higher osmolality, the moth cells effected further increases in the osmolality of the medium. The relationship between the logarithm of the change in osmolality effected per cell and the initial osmolality of the medium was linear.

Growth of lepidopteran epithelial cells and hemocytes in primary cultures

Abstract Cells from four species of Lepidoptera larvae known to be susceptible to several intracellular pathogens were cultured in vitro. Undifferentiated cells had the greatest potential for growth and survival. Imaginal wing disc explants formed extensive monolayers as a result of cell migration and mitosis. Mitoses were abundant in prohemocytes but not in other types of hemocytes. Differentiated larval tissues were unsuitable sources of cells for continuous culture. Migration of cells from these tissue explants was limited, and those cells which isolated themselves from the explant did not divide.

THE EFFECTS OF REPEATED ANESTHESIA ON THE BIOLOGY OF BLATTELLA GERMANICA (LINNAEUS)1

Short exposures of cockroach nymphs once each week to anesthetic gases (nitrogen, nitrous oxide, cyclopropane, carbon dioxide, and ethyl ether) retarded the growth rate, decreased the adult weight, reduced the number of offspring produced, and prolonged adult life. Certain of these effects were more pronounced with some gases than with others, so that no one gas can be considered safest.