Cell cycle analysis and synchronization of the TN-368 insect cell line

Summary A cell cycle analysis of theTrichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G₂, 8.5 hr; M, 0.5 hr; G₁, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50mm thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G₂. This work was supported in part by U.S. Environmental Protection Agency Grant R-802516.     

Migration of Gasterophilus intestinalis larvae (Diptera: Gasterophilidae) in the equine oral cavity

Cogley T. P., Anderson J. R. and Cooley L. J. 1982. Migration of Gasterophilus intestinalis larvae (Diptera: Gasterophilidae) in the equine oral cavity. International Journal for Parasitology12: 473–480. Larvae of G. intestinalis pursued a specific migratory pathway within the equine oral cavity en route to the stomach. The larval migration included the following sequence: burrowing in the tongue mucosa, invasion of the interdental spaces, transitory attachment at the root of the tongue and movement to the stomach. The molt from first to second instar did not occur in the tongue, as commonly believed, but between the interdental spaces. Ninety five percent of the larvae invading the interdental spaces were associated with gingiva of the upper molars. SEM analysis revealed further details of the oral migration: (1) air holes excavated in the epithelium which connect with deeper burrows; (2) an intimate association between air holes and posterior spiracles of larvae; (3) precise impressions of larvae in tissue immediately surrounding the most recently formed burrows; and (4) initial larval entry into the tongue through the use of natural disruptions or healing lesions. Factors influencing the development of the oral migration are discussed.     

Characterization of DNA from three nuclear polyhedrosis viruses pathogenic for Choristoneura sp

Three nuclear polyhedrosis viruses isolated from larvae of the insect genus Choristoneura showed polyhedrins of 28–30,000 daltons, genome sizes of 78–82 × 10⁶ daltons, and guanine plus cytosine contents of 47.9–49.4%. It was demonstrated by comparison of restriction endonuclease fragment patterns that two of the viruses are closely related genetically.     

Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria

A technique for the collection of stable hemolymph from larvae of Manduca sexta has been developed. The method avoids the cell clumping and melanization reactions commonly encountered with insect hemolymph by minimizing contact between hemocytes and surfaces which provoke defensive or repair responses. The circulating hemocyte population of second-day, fifth-instar larvae (2dL5) of M. sexta consisted of 4.5 ± 2.5 × 10⁶ cells/ml (n = 15, range 2–7 × 10⁶ cells/ml) and contained five cell types: prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. Two strains of Pseudomonas aeruginosa which differ in pathogenicity (P11-1 and 9027) and Escherichia coli D31 grew well at 26°C in cell-free hemolymph prepared from naive (nonimmunized) 2dL5 M. sexta. When viable cells of any of the three bacteria were injected into M. sexta larvae, changes in both the total hemocyte count (THC) and differential hemocyte count were observed. Viable bacteria were not required to produce these changes since formalin-killed cells of P. aeruginosa 9027 produced a qualitatively and quantitatively similar response. Following injection of bacteria, the THC increased, reaching a maximal level at 1 hr postinjection, and remained elevated for at least 4 hr after injection. While prohemocytes, plasmatocytes, granulocytes, and spherulocytes all increased in number, 80% of the increased cell population at 1 hr postinjection of bacteria were the latter two cell types. Granulocytes and spherulocytes are cells with recognized defensive capabilities. The increased numbers of these cells in circulation soon after injection of bacteria may confer an advantage on M. sexta larvae in dealing with bacterial infections. This could explain in part the unusual resistance of M. sexta to certain bacterial pathogens.     

Factors affecting entomophilic activity of Neoaplectana feltiae in mushroom compost

The host-searching ability of Neoaplectana feltiae Filipjev (= S. bibionis Bovien) (Nematoda: Steinernematidae) in response to larvae of a mushroom fly, Lycoriella solani Winn. was examined in a mushroom substrate. Individuals of L. solani were less attractive for the parasite than larvae of Galleria mellonella L. The nematode juveniles penetrated a 22 cm layer of casing mixture within 2–4 days. In the casing alone nematode effectiveness was better than in mushroom compost or in compost and casing together. In the casing mixture parasite dosages of 20 and 100 juveniles per cm² led to 22% and 45% parasitization of L. solani respectively, while all G. mellonella larvae were parasitized at both dosages. The prevalence of nematode infection depended on the content of water in the mushroom substrate. The highest N. feltiae infectivity was observed, when the ratio of the dry casing weight to the weight of water content was 1: 2.5. The practical aspects of the observed phenomena, essential for the use of N. feltiae in the protection of commercial mushroom cultivation are discussed.     

Influence of phenolglucosides and trichome density on the distribution of insects herbivores on willows

The effects of both trichome density and phenolglucoside content of leaves of 76 willow hybrids (Salix alba x fragilis) were measured to estimate their influence on the distribution of Phratora vitellinae (L.), Plagiodera versicolora Baly (Coleoptera: Chrysomelidae) and Pontania proxima (Lepeletier 1823) (Hymenoptera: Tenthredinidae) in a nursery at Gramont, Belgium.The willows showed differences in their phenolglucoside content and pilosity of leaves and are classified on these basis into four groups by a clustering method. Correlations and multiple regressions showed that these chemical and physical characteristics are good predictors of the abundance of insects. First, the abundance of larvae of Ph. vitellinae, adults of Pl. versicolora and galls of P. proxima is correlated positively with a high phenolglucoside content and a low pilosity of the leaves. Secondly, the distribution of adults of Ph. vitellinae and of larvae of Pl. versicolora is influenced by neither the chemical nor the physical leaves characteristics studied.     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

The influence of nutritional and genetic factors on larval performance of the cinnabar moth, Tyria jacobaeae

In a laboratory experiment using full-sibs, 60% of the variation in pupal weight of the monophage Tyria jacobaeae L. (Lepidoptera, Arctiidae) could be explained by variation in the nitrogen concentration of the food plant, Senecio jacobaea L. and only 4% by variation in sugar concentration. Larval weight and growth rates of young and old larvae were also positively correlated with nitrogen and sugar concentration. Developmental time was negatively correlated with nitrogen concentration. In a second experiment full-sib families differed significantly in larval weight at day 7, mortality, growth rate and developmental time. Pupal weight did not differ significantly among families, but was positively correlated with nitrogen concentration of Senecio. Larval performance was not significantly influenced by concentrations of sugars or alkaloids. We conclude that larval performance of Tyria during most of the larval period is mainly determined by genetic factors, but pupal weights are primarily determined by nitrogen concentration of the food plant.     

Labelling the blueberry leaftier, Croesia curvalana with foliar sprays of rubidium chloride

The feasibility of labelling blueberry leaftier by rearing the larvae on blueberry plants treated with foliar sprays of rubidium chloride (RbCl) at concentrations of 1000, 5000, 10 000 and 20 000 ppm were assessed. RbCl sprays above 5000 ppm significantly reduced survivorship to adult stage. The adult longevity, fecundity and mating were not affected when the larvae were reared on foliage treated with 5000 ppm RbCl solution. Reciprocal matings of 5000 ppm treated moths with untreated moths revealed transfer of label above the 0.1 µg Rb/insect threshold level from treated males to untreated females (in 8 out of 13 pairs) and vice versa (in 1 out of 9 pairs). Considerable loss of Rb (56–64%) occurred from the leaves over a 15 day period. All of the moths and pupae collected from the RbCl treated plots in 1989 and 1990 respectively, had a Rb content higher than the threshold level. In a preliminary dispersal study, marked male and female moths were found in sweepnet samples collected 20 and 60 m from the centre of the treated field. Labelled male moths were also captured in pheromone traps arranged in a circle, 40 m from the treated plot.